Journal of shellfish research

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JOURNAL OF SHELLFISH RESEARCH

VOLUME 21, NUMBER 1

JUNE 2002

The Journal of Shellfish Research

(formerly Proceedings of the National Shellfisheries Association)

is the offlcial publication of the National Shellfisheries Association

Editor

Sandra E. Shumway

Department of Marine Sciences

University of Connecticut

Groton, CT 06340

Standish K. Allen, Jr. (2002)

Aquaculture Genetics and Breeding

Technology Center

Virginia Institute of Marine Science

College of William and Mary

P.O. Box 1346

Gloucester Point, Virginia 23062

Shirley Baker (2004)

University of Florida

Department of Fisheries and Aquatic Sciences

7922 NW 71- Street

Gainesville, Florida 32653-3071

Brian Beal (2004)

University of Maine

9 O'Brien Avenue . .»v..^«v-^---- " ■

Machias, Maine 04654 , .

Peter Beninger (2003)

Laboratoire de Biologic Marine

Faculte des Sciences

Universite de Nantes :

BP 92208 ■ • .

44322 Nantes Cedex 3, France

Andrew Boghen (2003)
Department of Biology
University of Moncton
Moncton, New Brunswick
Canada EIA 3E9

Neil Bourne (2003)
Fisheries and Oceans
Pacific Biological Station
Nanaimo, British Columbia
Canada V9T 6N7

Andrew R. Brand (2003)
University of Liverpool
Port Erin Marine Laboratory
Port Erin, Isle of Man IM9 6JA
United Kingdom

EDITORIAL BOARD

Eugene Burreson (2003)

Virginia Institute of Marine Science

P.O. Box 1346

Rt. 1208 Create Road

College of William and Mary

Gloucester Point, Virginia 23062

Peter Cook (2002)
Department of Zoology
University of Cape Town
Rondebosch 7700
Cape Town, South Africa

Simon Cragg (2002)
Institute of Marine Sciences
University of Portsmouth
Ferry Road
Portsmouth P04 9LY
United Kingdom

Leroy Creswell (2003)
University of Florida/Sea Grant
8400 Picos Road, Suite 101
Fort Pierce, Florida 34945-3045

Lou D'Abramo (2002)
Mississippi State University
Department of Wildlife and Fisheries
Box 9690
Mississippi State, Mississippi 39762

Christopher V. Davis (2004)
Pemaquid Oyster Company, Inc.
P.O. Box 302
1957 Friendship Road
Waldoboro, Maine 04572

Ralph Elston (2003)

Aqua Technics/Pacific Shellfish Institute

455 West Bell Street

Sequim, Washington 98382

Susan E. Ford (2002)

Rutgers University

Haskin Shellfish Research Laboratory

6959 Miller Avenue

Port Norris, New Jersey 08349

Journal of Shellfish Research

Volume 21, Number 1

ISSN: 0730-8000

June 2002

www.shellfish.org/pubs/jsr.htm

Raymond Grizzle (2003)
Jackson Estuarine Laboratory
Durham, New Hampshire 03824

Karolyn Mueller Hansen (2004)
1524 Barley Circle
Knoxville, TN 37922

Mark Luckenbach (2003)

Virginia Institute of Marine Science

Eastern Shore Lab

P.O. Box 350

Wachapreague, Virginia 23480

Bruce MacDonald (2002)
Department of Biology
University of New Brunswick
Saint John, New Brunswick
Canada E2L 4L5

Roger Mann (2002)

Virginia Institute of Marine Science

Gloucester Point, Virginia 23062

Islay D. Marsden (2002)
Department of Zoology
Canterbury University
Christchurch, New Zealand

Tom Soniat (2002)
Biology Department
Nicholls State University
Thibodaux, Louisiana 70310

J. Evan Ward (2002)
Department of Marine Sciences
University of Connecticut
1080 Shennecossett Road
Groton, Connecticut 06340-6097

Gary Wikfors (2002)

NOAA/NMFS

Rogers Avenue

Milford, Connecticut 06460

Joiinuil of Slwllfiih Rfst'iirch. Vol, 21, No. 1. 1-2. 2002.

Honored Life Member
John B. Glude

. . -lOlocficgi rar)Ofa(o--v '

VVooas HoO Oceanograprnc Insiiiution

Libr.3ry

JUL 1 8 2002

John Glude was born August 2. 1918 to William and Florence Glud of Silverdale. Washington. William Glud worked at the
Bremerton Naval Shipyard and Florence worked as an elementary school teacher. Growing up in the Silverdale area with his brother
Clarence, John got his first taste of aquaculture when his father had two ponds on the property where trout were raised. The fact that
he was able to be involved with raising trout got him interested in aquaculture, which he continued to pursue and later became known
for.

Along with his skills related to farming, building, and raising fish, he helped his father create an innovative system for generating
electricity by taking advantage of the changing elevation of the stream entering their property. A wooden sluice carried water from the
stream to the pond. There the water was released, falling into cups on the paddle wheel they had constructed. Through a series of axles
and gears, enough force was created to generate all the electricity they needed. They were the first in the area to have electric lights.

John attended grade school and high school in Silverdale from which he graduated in 1935 as class salutatorian at the age of 16. He
then entered the University of Washington received his Bachelor of Science degree in Fisheries in 1939, with a major in Fisheries and
a minor in Engineering. He then got his first job with the Washington Department of Fisheries (WDF). but left briefly during Worid War
II. He put his education in engineering to work by taking a job as a naval architect draftsman at the Tacoma Naval Shipyard. His work
was considered vital to the war effort. After the war was over, he returned to his position as a Fishery Biologist with the WDF. He was
then involved with research to determine the effects of polluted waters from pulp mills on oysters. This work formed the basis for
regulatory actions to reduce sulfite liquor waste from pulp mills, which set the stage for assisting in the preservation of the valuable oyster
resources in the state of Washington.

After the war. John was sent to Japan to inspect sea oysters for export to the United States to prevent contamination and/or
introduction of undesirable organisms to the west coast oyster growing areas. Cedric Lindsay was a colleague of John's with the WDF
who also went along with him on some of the early trips to Japan to inspect seed oysters for importation to the United States. These trips
ultimately led to major seed importation to the Pacific coast of the United States and thus maintained the major oyster fisheries until
recent years when seed shipment from Japan was no longer necessary. There John learned a great deal about the various Japanese
methods for culturing oysters and other species which he brought back to the United States to share with growers.

In 1948, John was offered a position at the Woods Hole Oceanographic Institute in Woods Hole. Massachusetts. The main emphasis
during that time was research on the abundance and survival of soft shell clams throughout the entire U.S. east coast. The research project
was moved the following year to a former fish hatchery at Boothbay Harbor, Maine. He continued his research on sofishell clam
populations and other species found in the regiim. During this time, he became lab director of the Boothbay Harbor facility and instituted

2 Honored Life Member: John B. Glude

further research on artificial propagation of chinis and other species. Some of ihe earher efforts were relati\e to wild catches of soft shell
clams, but were also concerned with hatchery setting. He also studied the effects of green crab predation and their control. The research
during those years on the soft shell clam was ground-breaking and is still refened to extensively.

John continued his career in the federal government with the U.S. Fish and Wildlife Service and the subsequent National Marine
Fisheries Services (NMFS) under the National Oceanographic and Atmospheric Administration (NOAA). He later became director of
the NMFS Laboratory in Annapolis. Maryland where he moved in 1956 with his wife Jean, daughter Nancy and son Terry. This
laboratory was primarily engaged in research to develop methods for farming shellfish (main emphasis on oysters). With the lab's
closure. John was offered a position in the national headquarters of NMFS in Washington. D.C. While there, he was in charge of the
shellfish research branch of the NMFS and responsible for seven regional laboratories. At that time, he developed the first National
Aquaculture Plan through NOAA.

During President Kennedy's Administration. John was appointed to lead a team of fisheries experts to assist Ireland in improving their
fisheries resources. He spent one year on this project and recommended many changes to the Irish Department of Fisheries which were
implemented to improve the economic situation.

Yearning to return to the northwest and the state of Washington. John accepted a position of Assistant Regional Director of the
Northwest Region of the National Marine Fisheries Service, which was headquartered in Seattle, Washington. His family mined back
to the state where John not only oversaw federal fisheries research in the area but also pushed to promote and implement the NOAA
National Aquaculture Plan.

Upon retirement in Seattle, John started the Glude Aquaculture Consultants. Much of his work involved aquaculture. and he was a
primary consultant for numerous aquaculture projects in various countries. He continued to encourage aquaculture enterprises and
eventually organized a program in Puerto Rico to test the applicability of known culture methods for fresh water prawns. John also acted
as a consultant for the United Nations Food and Agriculture Organization where he headed a team of scientists to determine how to
increase fishery resources and revenues in a number of developing countries. One project was entitled "The South Pacific Fisheries
Investigation" through which recommendations were provided for best approaches to increa.se fisheries activities for the regions.

John has been a life member of several professional organizations. He served as Vice President and President of the World
Aquaculture Society in 1977 and 1978. respectively. He also served for two years as president of the National Shellfisheries Association
in 1963-65. Along with this service to the societies, he has published over 100 scientific papers related to his many areas of shellfish
research. John is retired now. but he is most certainly a pioneer in the fields of aquaculture and fisheries, best known for his research
on clam and oyster culture. An avid sportsman, his love for fly-fishing and duck hunting is well recognized. I have been on many duck
hunting trips with John and his brother-in-law Dick Steele in Dabob Bay. Hood canal in Washington State and know of his obsession
with bird hunting. John now resides at 6101 River Crescent Drive. Annapolis. MD 21401.

Dr. Kenneth Chew

College of Ocean and Fishery Sciences

University of Washington,

Seattle. Washington

Jniiriuil of Shellfish Resi-aivli. Vol. 21, No. 1. 3-12. 2002.

GROWTH OF THE NORTHERN QUAHOG, MERCENARIA MERCENARIA, IN AN
EXPERIMENTAL-SCALE UPWELLER

CRAIG L. APPLEYARD AND JOSEPH T. DEALTERIS

Depariineut of Fisheries. Aiiinicil unci VeteriiuiiY Scieiue, Universitx of Rliode Island,
Kiuiiston, Rhode Island 02HH1

ABSTRACT Upwellers have proven to be extremely effective as bivalve nursery units and their use is steadily increasing in North
America. The re-analysis of previous work by others suggests an asymptotic relationship between growth (9c volume increase per day)
and chlorophyll-(i effective flow rate (the amount food flowing past a unit biomass of northern quahogs. |jLg per minute per liter of
northern quahog volume). An experiment field study was conducted to define the relationship between food flow and bivalve stocking
density. Furthermore, this study was designed to investigate other significant environmental parameters influencing bivalve grovMh in
an experimental-scale upweller system.

Northern quahog, Merceimria mercenuria (Linnel, seed were grown from -2 (longest axis) to -13 mm in an experimental-scale
floating upweller from June 21 to August 19, 1999 (four separate experimental periods) in Point Judith Pond, Wakefield. Rhode Island.
Flow rates and stocking densities were varied in order to produce a chlorophyll-t; effective flow rate range of 360 to 1.500
jjLg ■ niin ' • r', and growth and environmental parameters were measured semiweekly. During the first two-week experiment (June 21
to July 7) an asymptotic relationship was observed between growth (% increase/day) and chlorophyll-^ effective flow rate. A
significant difference in growth was found between the treatments. The difference in the functional relationship between experiments
1 and 3 was possibly related to lower DO values, which reduced differential growth in experiment 3. In experiment 1. the low-biomass
treatments grew faster than the high-biomass treatments. A significant difference in growth between treatments was also observed in
experiment 3. although the asymptotic relationship was less pronounced. In experiment 3. the high-biomass replicates grew faster than
the low-biomass replicates. Experiments 1 and 3 both experienced similar environmental conditions; however, experiment 1 encoun-
tered higher morning dissolved oxygen (DO) levels. In addition, the within experiment variability in experiment 3 was much less than
the variability in experiment 1 ; therefore, accentuating growth differences in experiment 3. In both experiments 1 and 3 maximum
growth occurred near treatment 2 in a range of chlorophyll-o effective tlow rates of 550 to 650 jig ■ min"' ■ T'. In experiments. 2 and
4 there were no significant differences in growth between treatments.

Growth appeared to be limited by low oxygen. In order to eliminate the effect of food limitation on growth, the upper third of the
replicates (the fastest growing animals) were u.sed to calculate the relative growth rate (RGR) during the two-month experiment.
Growth was linearly correlated with morning-dissolved oxygen (R- = 0.42) and with chlorophyll-d (R- = 0.35). The critical DO
threshold for growth in upwellers appears to be 5 ppm. below which growth is adversely affected. During this study, morning DO levels
were less than 50 % saturated, indicating the potential for DO levels to be increased. Future research should investigate methods for
elevating DO levels in upwellers.

KEY WORDS: northern quahog. Mercenariu mercenarm. upweller. growth

INTRODUCTION

Over the last decade, the use of tipvvellers as bivalve nursery
units has increased dramatically in North America (Man/.i &
Castagna 1989). A number of studies have explored the relation-
ships between tlow rate, stocking density, and growth in upwellers
(Hadley et al. 1999; Baldwin et al. 1995; Malinowski & Siddall
1989; Malinowski 1988; Manzi & Hadley 1988; Manzi et al. 1986:
Manzi 1985; Hadley & Man/i 1984; Manzi et al. 1984; Baye.s
1981; Claus 1981; Manzi & Whetstone 1981; Rodhouse &
O' Kelly 1981 ). The majority of research on upwellers has focused
on the northern quahog. Mercenaria mercenaria, because of its
significant aquaculture potential. In particular, the northern quahog
grows well at high densities, has adapted to a variety of geographic
sites along the northeast coast, and has a lucrative market.

Manzi et al. (1986) described a qualitative relationship between
flow rate, stocking density, and growth in an experimental-scale
upweller. In their experiment, stocking densities were varied while
flow rates were held constant. The tlow rate was converted to an
effective flow rate by multiplying the amount of food (|jLg/l of
chlorophyll-fl) by the flow rate (l/min). The amount of food pass-
ing by a unit biomass of clams was defined as the chlorophyll-(/
effective flow rate (p,g ■ min"' ■ kg"' ). During a period of optimal
northern quahog growth in the fall 1982 the authors found that a
maximum biomass increase of 267% (over 30 days) occurred at the

highest chlorophyll-a effective flow rate of 1.929 |xg ■ min"' ■ kg"'
and the most efficient growth (213%) occuired at an intermediate
chlorophyll-a effective flow rate of 476 |a.g • min" ' • kg" ' . If growth
(% increase/day) is plotted as a function of chlorophyll-u effective
tlow rate, the data is represented by an asymptotic relationship; in
particular, as the chlorophyll-a effective flow rate increases,
growth increases steeply and then levels off with increasing chlo-
rophyll-o effective flow rates (Fig. 1). Efficiency in this upweller
system refers to economically optimizing both upweller space
(density) and pumping capacity (tlow). Theoretically, growth will
be optimized at some percentage of the maximum growth rate; as
indicated in Figure 1, 80 to 90% of the maximum growth rate
equates to a chlorophyll-n effective tlow rate range of 470 to 700
pg ■ min"' • kg"'.

Manzi et al. ( 1986) concluded that tood supply was the primary
limitation in their upweller system. Their data suggests that to
obtain unlimited growth, northern quahog seed needed to remove
approximately 150 p.g ■ min"' kg"'. The investigators deduce
thai northern quahog growth was reduced if more than 20% of the
ambient chlorophyll-^; concentration (p-g/l) was removed as water
passed by the bivalves. Consequently, to supply the necessary
ration of 150 p.g • min"' ■ kg"' without exceeding 20% removal, food
must be supplied to the bivalves at a rate of 750 |jLg • min"' • kg"'.
Malinowski and Siddall (1989) confirmed that ambient chloro-
phyll-(( concentrations were reduced by -20% through an initial

Appleyard and Dealteris

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90%

80% ^^

/

D 500 1000 1500

Chlorophyll-a Effective Flow Rate (ng«min ' '<r')

2000

Figure 1. Growth (% increase/davl of northern quahogs as a function of chlorophyll-n effective flow rate re-plotted from Manzi et al. (19861.

silo of northern quuhogs al similar stocking densities. However,
they I'ouiid that after water passed through an initial group of
northern quahogs it could then support an additional equivalent
biomass of northern quahogs at the same growth rate. They con-
clude that to achieve maximum growth of northern quahogs in an
upweller it is necessary to pass more water through the animals
than can actually be filtered; therefore, the low rate of chloro-
phyll-a removal reported by Man/i et al. ( I486) may reflect a large
amount of unused water through the system. The authors hypoth-
esize that this surplus water may be a physical requirement of the
system where minimum flow rates are required to create uniform
flows through the seedbed, remove waste products, maintain water
quality, and maintain a minimum concentration of chlorophyll-K.

Man/i et al. (1986) found that food was the primary limitation
in their upweller system, while Malinowski and Siddall (1989)
concluded that flow rate was the primaiy limitation. Malinowski
and Siddall (1989) also speculate on the importance of environ-
mental conditions, specifically water quality, but they fail to char-
acterize these parameters in their system. Growth and survival of
the northern quahog is clearly influenced by the surrounding en-
vironment. Northern quahog adults and juveniles can survive in
water temperatures from 1 to .WC. but grow optimally at 23"C
(Stanley 1985; Stanley & Dewitt 1983). Northern quahogs can
tolerate salinities between 10 and 35'^f (Stanley & Dewitt 1983)
for short periods, but prefer to inhabit waters greater than 20%o
(Ca.stagna & Kraeuter 1981 ). Northern quahogs have been known
to endure oxygen concentrations below 1 mg 0-J\ (Stanley &
Dewitt 1983) for more than three weeks; however, growth is sig-
nificantly reduced and an oxygen debt is incuired when oxygen
concentrations fall below ."> mg 0,/l (Stanley & Dewitt 1983;
Hamwi 1969).

Although there is a general disagreement as to the limiting
parameter for growth in upwellers. in the literature growth is
clearly related to both system operating parameters (flow rate and
stocking density) and environmental conditions at the site (tem-
perature and dissolved oxygen).

Given the environmental conditions m the study area, an ex-
perimental field study was conducted as to:

(1) Define a relationship between food flow, bivalve stocking
density, and growth so as to determine the chlorophyll-o
effective flow rate that optimizes growth; and

(2) Determine the most significant limiting parameter for bi-
valve growth in the upweller system.

The experiment monitored growth of northern quahog seed
in an experimental-scale floating upweller at three ranges of nomi-
nal chlorophyll-(/ effective flow rates of 350. 600 and 1.400
p.g ■ min"' r'. In addition, environmental conditions were moni-
tored at the site.

MATERIALS AND METHODS

Experimental Detign

Growth of northern quahog seed was studied over an 8-week
period in an experimental-scale floating upweller system located in
a nutrient-rich estuary. At the beginning of the experiment the
ambient chlorophyll-^ concentration (|jLg/l) was measured at the
site, and flow rates and stocking densities were adjusted to achieve
three nominal ranges of chlorophyll-t; effective flow rates, includ-

ing a low (-350 iJLg • min

), medium (-600 jjLg ■ min I ),

and high range (-1,200 p.g ■ min"' • P' ). Each combination of
effective flow rate (|j.g/min) and northern quahog biomass (1) or
chlorophyll-(( effective flow rate (p-g • min"' P' ) represents a
treatment, as shown in Table 1. The average chlorophyll-c; con-
centration during the time period was 1 1 .70 ± 2.06 jjig/1 (S.E.) and
the flow rates were set at 4 1/min, 6 1/min, and 8 1/min resulting in
three effective flow rates of 38.36 (ig/min, 57.84 p.g/min, and
77.12 |jLg/min. The northern quahog seed were initially slocked at
a biomass of 0.055 1 (density of 0.3 I/cm") and 0.109 1 (density of
0.6 1/cnr) resulting in the desired range of chlorophyll-o effective
flow rates. The experiment was a two (density) by three (effective
flow rate) factorial design with six treatments of chlorophyll-a
effective flow rates. Each treatment was replicated in triplicate
resultina in 18 observations (silos).

Growth of M. mercenaria in an Experimental Upweller

TABLE 1.

Chlorophyll-fl effective flow rates and their
corresponding treatment.

Initial

Stocking

Biomass

Effective Flow Rate

((ig/min)

(liter)

Low

High

Low

Treatment 4

Treatment 1

Medium

Treatmem S

Treatment 2

High

Treatment 6

Treatment 3

Silc Location

The experiment was conducted iit Rum Point Marina. Inc.. at
the head of Point Judith Pond. Wakefield. Rhode Island
(4I°25.57'N; 7r'2Q..S7'W) (Fig. 2). Ram Point Marina. Inc is

located on a spit between Silver Spring Cove and the Upper Pond.
The site was selected to take advantage of the relatively high and
consistent phytoplankton biomass (chlorophyll-^ concentrations
>10 M-g/l. Rheault 1993) during the summer months. The experi-
mental-scale floating upweller was situated at the northernmost
comer of the marina to ensure a water depth >1.4 meters at mean
low tide.

Upweller System

An experitriental-scale floating upweller was designed and con-
structed to ensure the control of flow through each silo. The up-
weller unit was 4.27-m long, 1.22-tii wide, and 1.35 m deep. Two
25 cm inside diameter (ID) polyvinyl chloride (PVC) pipes were
positioned at the base and ran the length of the unit forming a
tnanifold. Ten 15-cm (ID) silos were plutnbed into the top of each
manifold. Flow was provided by a half horsepower axial flow

41''25'

] Ram Point Marine

Silver Sprir g Cove //

Upper Pond

^ ^

m A

r-

Figure 2. Location of the experimental-scale upweller at Ram Point Marina, Point Judith Pond. Wakeneld. Rhode Island.

Appleyard and Dealteris

pump (Ice Eater. Power House) mounted in each manifold. Water
was pumped along the manifold, tlowed up each 15-cm silo, and
exited through an S-cm (ID) ball valve plumbed into the top of
each silo. The seed were placed on a Nytex screen 0.5 m above the
silo"s base. When the unit was in operation, each ball valve lay
approximately 8 cm above the water line. Flow through each silo
was manipulated with the ball valve and was measured volumetn-
cally with a graduated cylinder and a stopwatch.

Northern quahog seed (300,000 at 0.6 mm) were purchased
from Bluepoints Company. Inc., West Sayville, New York. The
seed were held in the upweller until they reached >2 mm (longest
axis).

Data Collection

At the beginning of each experiment the seed were pulled from
the unit, sieved, and randomly distributed throughout the 18 rep-
licates at a biomass of 0.055 1 (wet volume) and 0.109 1. In addi-
tion, the valve length of a random sample (/; = 75) of seed was
measured to the nearest O.Oi mm with vernier calipers. Five sub-
samples of northern quahogs were also taken to develop a rela-
tionship between wet volume (1) and wet weight (kg). Each ex-
periment was terminated when the biomass in the slowest growing
replicate doubled. This occurred approximately every two weeks
during the summer. At the termination of each two-week experi-
ment, the valve length of a random sample (/; = 25) of northern
quahogs from each replicate was determined. Four two-week ex-
periments were completed during the summer 1999.

The change in volume of each silo was measured semiweekly
resulting in 3- to 4-day growth intervals. Semiweekly flow rates to
each silo were also measured in the morning or late at night to
minimize wave activity. Care was taken to ensure that the upweller
unit was not altered during measurements and flows were adjusted
accordingly.

Chlorophyll-c( (Chl-((). particulate organic matter (POM), tem-
perature, salinity, and dissolved oxygen (DO) were measured
semiweekly from an empty silo. With the start of the second two-
week experiment (July 7) all environmental parameters were taken
in the morning, midday, and evening to quantify daily fluctuations
at the .site. Discrete chlorophyll-<7 samples (» = 3) were taken with
a syringe. Samples were pre-filtered with a 150 |jim Nytex screen
to remove particulates that bivalves are unable to filter (Defossez
& Hawkins 1997). Samples (10 ml) were forced through a 25-mm
diameter Whatman GF/F filter contained in a 25 mm Swinnex
fdter holder. The procedure for chlorophyll-a analysis is slightly
modified from the standard procedure outlined in Strickland and
Parsons ( 1972). Filters were dissolved in acetone for 24 hours and
read on a Turner Designs tluorometer (Model 10-005R, Turner
Designs. Inc., Sunnyvale, CA). All samples were conected for
phaeophytin-a. One-liter samples were also taken (;i = 2) for
POM analysis. The samples were pre-filtered on a 150 p.m Nytex
screen and later analyzed in the laboratory. In the laboratory,
samples were vacuum pumped through a pre-ashed 47 mm What-
man GF/F filter (normal pore size O.I |xm), rinsed with isotonic
ammonium formate, and dried in an oven at 11()°C for 24 to 48
hours. Filters were then ashed for >6 hours at 450"C in a muffle
furnace. Filters were weighed on an Ohaus electronic balance
(Model AS 120) to the nearest 0.1 mg. Temperature, salinity were
measured with an YSI (Model 30) probe and oxygen was mea-
sured with an YSI (Model 55) probe. The oxygen probe was cali-
brated prior to each measurement.

When measuring the change in volume of northern quahog
seed, each silo and screen was cleaned with freshwater. Once a
week the remainder of the upweller manifold was cleaned by a
di\er to ensure consistent flow through the svsteni.

Data Analysis

The chlorophyll-K effective flow rate (p-g • min~' ■ 1"') for each
replicate was calculated as the product of the average chloro-
phyll-K concentration ((j,g/l) during the period and the flow rate
(1/min) to the replicate all divided by the average biomass (1) of the
replicate during the same period. This study characterized growth
as the relative growth rate (RGR) and was calculated as:

RGR = {[(Volume,,-,,,,, - Volume, „„„,,|,)/Volume, „„,,_,,, ] x
1001/#ofdays

where volume is measured in liters. RGR is expressed as a per-
centage increase per day (9f increase/day). POM (mg/1) was cal-
culated as the difference between total suspended particulate mat-
ter (SPM) and particulate inorganic matter (PIM).

The Effecl of Food Limilalion on GroHtli

To elucidate differences in growth between treatments the total
RGR C^ volume increase) was divided by the longest period avail-
able, the length of each experiment. Since the RGR (% increase/
day) measures the change in volume over each two- week experi-
ment, the average chlorophyll-^ concentration and average treat-
ment biomass during the time was used to calculate treatment
chlorophyll-(( effective flow rates. Prior to ANOVA analysis, the
RGR (Vc increase/day) was arcsine transformed (Sokal & Rohlf
1995). Within each experiment, one-way ANOVAs were per-
formed for each two-week experiment with the average RGR (%
increase/day) as the dependent variable and treatment as the inde-
pendent variable. Differences between treatment means were elu-
cidated with the Tukey Honestly Significant Difference (HSD)
test. When the one-way ANOVA proved significant, a within ex-
periment two-way ANOVA was performed to funher investigate
the effective flow rate and density as independent variables. Again,
the Tukey HSD test was used to verify differences in means. The
strength of the relationship was characterized by the standard
omega-squared (w"), when appropriate. The to" was calculated as

0)= = [SSt;„eu - dfs,,x (MSs,,Ol/SS-r -I- MSs,a

where SS^,,,.^,, is the sum of squares of the effect, dfs,^ is the
degrees of freedom for the eiTor tenn, MS^/.^ is the mean squares
for the error term, and SS,- is the sum of .squares total.

A between experiment one-way ANOVA was performed to
elucidate growth differences between the two-week experiments.
The Tukey HSD test quantified differences between means.

The Effecl of Environituiital Characteristics on Growth

In order to illustrate the effect of environmental parameters on
northern quahog growth, growth was characterized at the finest
possible scale. In particular, RGR was calculated for each 3 to 4
day period (growth inter\al) between semiweekly volume deter-
minations. The daily value of each environmental parameter was
averaged over the concurrent growth interval.

To eliminate the effect of food limitation on growth, RGR of
the upper third of the replicates (upper 1/3 RGR) was calculated.
A linear regression analysis (S.AS Institute, Inc.) of the upper
one-third RGR was performed with temperature, salinity, chloro-

Growth of M. mercenaria in an Experimental Upweller

phyll-o. and dissolved oxygen to determine which independent
variables were significant in determining growth. Dissolved oxy-
gen concentrations were converted to percent saturation based on
temperature and salinity measurements (Benson & Krause 1984).
A step-wise linear regression analysis was also performed to elu-
cidate the most significant parameter(s) for predicting growth in
the experiment.

RESULTS

The Effect of Food Limitation on Growth

There was no observed mortality during the course of the two-
month experiment. Calculated mortality was extremely variable in
experiments 1 and 2 because counts per ml were not replicated. In
addition, counts were not made before and after sieving. In ex-
periments 3 and 4, counts per ml were replicated (n = 3) and
counts were made before and after sieving. Mortality was calcu-
lated to be 1 11 ± 3<7f (S.E.) and 99 ± 2<7(- (S.E.) respectively. The
first experiment began on June 21 and ended on July 7. 1999 ( 16
days) and the northern quahogs grew from 3.1 1 ± 0.06 mm (S.E.)
to 3.95 ± 0.05 mm (S.E.). The average chlorophyll-(7 concentration
was 16.42 ± 2.25 p,g/l (S.E.) and the average treatment biomass
ranged from 165.8 to 85.5 ml. The chlorophyll-a effective flow
rate ranged from 420 to 1,445 |ji.g • min ' • P' roughly correlating
with a RGR from 4.76 to 9.32"* increase/day. As the chlorophyll-i;/
effecti\e flow rate increased, the RGR increased until -650
IJLS ■ inin"' ■ r' at which point growth leveled off (Fig. 3a).
Growth, as measured by RGR. was subjected to a one-way
ANOVA with six levels of treatment. This was found to be sta-
tistically significant (f (5, 1 1) = 5.48, P < 0.05). The strength of
the relationship was 0.57 as indexed by the standard omega-
squared (aj~). The Tukey HSD test indicated that the mean RGR
for treatment 1 (M = 5.54) was significantly lower than the means
for treatment 4 (M = 8.14), 5 (M = 8,41), and 6 (A/ = 9.26). To
investigate the effect of effective flow rate and biomass on growth,
a two-way ANOVA was performed with three levels of seston flux

and two levels of volume. Both effective flow rate (f (2, 11) =
5.13. P < 0.05) and biomass (F (\, II) = 13.36, P < 0.05) were
statistically significant. The strength of the relationship (io~) was
0.21 and 0.31. respectively. The interaction between effective flow
rate and biomass was found ordinal; therefore, the main effects
were examined by the Tukey HSD test. The Tukey HSD test
indicated that the low-biomass treatments (M = 8.60) grew faster
than the high-biomass treatments (M = 6.99).

The second experiment began on July 7 and ended on July 22,
1999 ( 15 days) and the northern quahogs grew from 3,46 ± 0.1 1
mm (S.E.) to 6.28 ± 0.07 mm (S.E.). The average chlorophyll-fl
concentration was 1 1.83 ± 1.16 \i.°l\ (S.E.) and the average treat-
ment biomass ranged from 205.6 ml to 91 .6 ml. The chlorophyll-a
effective flow rate ranged from 231 to 977 jjig • min~' • 1"' roughly
correlating with a RGR from 9.58 to 12.73 % increase/day (Fig.
3b). RGR was consistently high within the chlorophyll-a effective
flow rate range specified. RGR was subjected to a one-way
ANOVA and there was no statistical difference between treat-
ments (f (5, 11) = 1.48. P > 0.05).

The third experiment began on July 22 and ended on August 5.
1999 (14 days) and the northern quahogs grew from 7.04 ± 0. 1 1
mm (S.E.) to 9.96 ± 0.07 mm (S.E.). The average chlorophyll-^^
concentration was 18.55 ± 2.12 |j,g/l (S.E.) and the average treat-
ment biomass ranged from 184.8 to 84.6 ml. The chlorophyll-c(
effective flow rate ranged from 411 to 1,720 p.g ■ min"' ■ P'
roughly corresponding to a RGR from 7.79 to 10.09 % increase/
day (Fig. 3c). The RGR increased slightly with an increase in the
chlorophyll-(7 effective flow rate until -610 |a.g ■ min~' 1"', at
which point growth decreased and leveled off RGR was subjected
to a one-way ANOVA and was found to be statistically significant
(f (5, 11) = 7.13, P< 0.05). The strength of the relationship was
0.64 as indexed by the u)". The Tukey HSD test indicated that the
mean RGR for treatment 2 (M = 9.76) was significantly higher
than the means for treatment 4 (M = 7.97), 5 (M = 8.62), and 6
(M = 8,38). In addition, the mean RGR for treatment 4 (A/ =
7.97) was significantly lower than the mean for treatment 3 {M =

14 -

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Figure 3. Growth ( 'H increase/day) as a function of chlorophyll-o effective flow rate for (a) experiment 1 (June 21 to July 7, 1999 with an average
chlorophyll-a concentration of 16.42 ± 2.25 (ig/l S.E.): (b) experiment 2 (Juh 7 to July 22, 1999 with an average chlorophyll-a concentration of
11.83 ± 1.16 (ig/l S.E.): (c) experiment 3 (July 22 to August 5, 1999 with an average chlorophyll-u concentration of 18.55 ± 2.12 fig/l S.E.); and,
(d) experiment 4 (.\ugust 5 to ,\ugust 19, 1999 with an average chlorophjil-a concentration of 17.91 ± 3.17 pg/1 S.E.).

Appleyard and Dealteris

9.20). A two-way ANOVA found both effective flow rate (F (2.
11) = 5.99, /'< 0.05) and bioniass(F(l, II) = 22.33. P < 0.05)
were statistically significant. The strength of the relationship (to")
was 0.21 and 0.45. respectively. The interaction between effective
flow rate and biomass was found ordinal; therefore, the main ef-
fects were examined by the Tukey HSD test. The Tukey HSD test
indicated that the high-biomass treatments (M = 9.25) grew faster
than the low-biomass treatments (W = 8.32). The Tukey HSD test
also found that the replicates with an effecli\e flow rate of 1 1 1.3
jig/min [M = 9.19) grew faster than the replicates with an effec-
tive flow rate of 74.2 |xg/min (M = 8.87).

The fourth experiment began on August 5 and ended on August
19, 1999 (14 days) and the northern quahogs grew from 9.37 ±
0.12 mm (S.E.) to 11.47 ± 0.08 mm (S.E.). The average chloro-
phyll-fi concentration was 17.91 ± 3.17 ^ig/l (S.E.) and the average
treatment biomass ranged from 147.4 to 73.4 nil. The chloro-
phyll-(( effective flow rate ranged from 491 to 1.905 |jLg ■ min^' ■ P'
roughly conesponding to a RGR from 4.98 to 5.96% increase/day
(Fig. 3d). RGR was consistently low within the chlorophyll-o ef-
fective flow-rate range specified. RGR was subjected to a one-way
ANOVA and there was no statistical difference between treat-
ments (f (5. 11) = 0.76. P > 0.05).

A one-way ANOVA was performed to compare the RGR be-
tween the two-week experiments. This was statistically significant
(f (3.64) = 135.34./'<0.05)with an a)-of0.80. The Tukey HSD
test indicated that there was a significant difference between all the
mean RGRs. with growth highest in experiment 2 (M = 11.89)
and decreasing in experiments 3 (M = 8.81). 1 (M = 7.71). and
4 (M = 5.57).

The Effect of Einironmeittal Charcuteristics on Growth

The upper one-third RGR varied considerably during the
course of the experiment from a high of 10.37 ± 0.43% increase/

day (S.E.) on June :

low of 5.03 ± 0.41% increase/dav (S.E.

on June 29 (Fig. 4a). During the course of the two-month experi-
ment. RGR decreased sharply (June 22 to June 29). then increased
(June 29 to July 17), and then gradually decreased (July 17 to
August 19).

Temperature during the experiment varied from 21.4 to 27.3°C
(Fig. 4h). Other than a brief drop in temperature in mid July due to
a rainstorm, temperature was consistent during the experiment. A
linear regression analysis indicated that temperature was not sig-
nificant in determining growth as indicated by the upper one-third
RGR (F (I. 15) = 0.58. P > 0.05). Similariy, salinity during the
experiment was relatively consistent ranging from 21.4 to 29.9%t
(Fig. 4c). Linear regression analysis determined that salinity was
not significant in predicting the upper one-third RGR (F(l, 15) =
0.70. P > 0.05). The seston concentration, as indexed by the chlo-
rophyll-(( concentration, varied substantially during the course of
the experiment from peaks of 21.2 ± 1.23 jxg/l (S.E.) on June 28
and 22.8 ± 0.86 |xg/l (S.E.) on July 3. to a nadir of 9.5 ± 0.09 p.g/1
(S.E.) on July 13 (Fig. 4d). Generally, as chlorophyll-(7 concentra-
tions increased growth decreased. Similariy. as chlorophyll-o con-
centrations decreased growth increased. Linear regression analysis
indicated that this trend was significant (f (1. 15) = 7.46. P <
0.05) with a correlation coefficient (R-) of 0.35 (Fig. 5a). There
was a minimal amount of variability between chlorophyll-</ mea-
surements as exemplified by the low standard errors between mea-
surements. The chlorophyll-(( concentration during the day. how-
ever, varied considerably as indicated by the high standard eiTors

between morning, midday, and evening chlorophyll-a determina-
tions. Morning DO was used to quantify the effect of limiting
dissolved oxygen on growth as inoming DO values were consis-
tently the lowest oxygen values experienced by the northern qua-
hogs. Morning DO values ranged from a high of 8.00 ppm on June
22 to a low of 3.37 ppm on July 8 (Fig. 4e). Morning DO values
correlate with the upper one-third RGR during the two-month
experiment. As morning DO values decreased growth decreased:
conversely, as morning DO values increased growth increased. A
linear regression analysis indicated that this trend was significant
(f (I. 15) = 10.13. P<0.05) withacorreiation coefficient (R-)of
0.42 (Fig. 5b). Morning dissolved oxygen (ppm) tracked the morn-
ing percent saturation of DO. During periods of low morning DO
(4-3 ppm), 7f saturation approached 50% and during periods of
high morning DO (7-8 ppm). % saturation ranged from 80 to
100%.

Growth was best characterized with morning DO and chloro-
phy!l-«(F(2. 15) = 8.22. P < 0.05) resulting in an R" of 0.56. As
the chlorophyll-(( concentration increased morning DO decreased
and as the chlorophyll-(( concentration decreased morning DO in-
creased (Figs. 4d and e).

DISCUSSION

The Effect of Food I.iinilalion on Growlli

The one-way ANOVAs found significant differences in growth
between treatments in experiinents I and 3. but none in experi-
ments 2 and 4. Experiments I and 3 were characterized by rela-
tively high morning DO values, while experiments 2 and 4 expe-
rienced relatively low morning DO values. According to the re-
evaluation of the data (Fig. 1) presented in Manzi et al. (1986).
growth as a function of the chlorophyll-;; effective flow rate (the
amount of food passing by a unit biomass of clams) should follow
an asymptotic function. Specifically, growth should increase from
the origin (zero growth and zero chlorophyll-a effective flow rate)
with increasing chlorophyll-o effective flow rates until a particular
point where growth asymptotes or even decreases.

In the first experiment, growth (% increase/day) followed the
relationship presented in Figure 1. Growth increased as the chlo-
rophyll-fl effective flow rate increased until -650 |a.g ■ min"' ■ P'
at which point growth reached an asymptote. The one-way
ANOVA found a significant difference in growth between the
treatments with a relatively strong relationship as indexed by the
standard omega-squared (oj- = 0.57). Furthermore, treatment I
grew significantly slower than treatments 4, 5. and 6. Treatments
4, 5. and 6 represent the asymptote of the function where growth
asymptotes regardless of an increase in the chlorophyll-a effective
flow rate. In addition, treatments 4. 5. and 6 were those with a low
initial stocking density of 0.3 I/cm". In order to further investigate
the effect of effective flow rate and biomass on growth a two-way
ANOVA was performed. There were significant differences be-
tween growth with the levels of effective flow rate and biomass. In
particular, the low-biomass replicates (treatments 4, 5, and 6) grew
faster than the high-biomass replicates (treatments I, 2, and 3).

In the third experiment, the one-way ANOVA also indicated a
significant difference in growth between the treatments with an
even stronger relationship (co- = 0.64). The functional relation-
ship between growth and chlorophyll-« effective flow rate was
different from that postulated in Figure I. Growth increased
slightly with increasing chlorophyll-a effecti\'e flow rate, but then
decrea.sed slightly, reaching an asymptote above l,(J(X) (j,g • min"' ■ T'.

Growth of M. mercenaria in an Experimental Upweller

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Figure 4. Relative growth rate (% increase/day) for the upper third of
rephcates (a) from June 21 through August 19, 1W» with (b) morning
temperature ( Cl; (cl morning saHnity ['it); (dl morning chlorophyll-a
l(ig/ll; and, (el morning dissolved oxygen (ppml.

This trend is supported by the Tukey HSD test, which indicated
that treatment 2 grew significantly faster than treatments 4. 5.
and 6. In addition, treatment 3 grew significantly faster than treat-
ment 4. Since treatment 3 and treatment 4 have nearly the same
chlorophyll-a effective flow rate, a significant difference in growth
indicates an effect of biomass on growth, with the higher bio-
mass treatment growing faster that the lower biomass treatment.
The two-way ANOVA also found a significant effect of biomass
on growth with the high-biomass replicates growing faster that the
low-biomass replicates. The two-way ANOVA also indicated a
significant effect of seston flux on growth with the replicates with
the intermediate effective flow rate of 1 1 1.3 pig/min growing faster
that the replicates with the lower effective flow rate of 74.2 p.g/
min.

Experiments 1 and 3 both experienced initially high morning
DO values that decreased during the course of the experiment.
Experiment 1 had higher initial morning DO values (8-7 ppni)
than experiment 3 (5.5-6 ppm). The chlorophyll-o concentration
also increased substantially during both experiments, with values
peaking at 21.19 ± 1.23 |jLg/l (S.E.) in experiment 1 and 22.83 ±
0.86 jjig/1 (S.E.) in experiment 3. In addition, both experiments
experienced the same range of chlorophyll-(v effective flow rates.
The difference in the relationship between growth and chloro-
phyll-(7 effective flow rate in experiment 1 and experiment 3 is
probably the result of a number of factors. First, since experiment
3 did not experience the initially high morning DO levels observed
in experiment 1, the treatments might not have had a chance to
separate or grow differentially. Second, the spread in replicates in
experiment 3 was considerably smaller than that in experiment 1 ;
therefore, small growth differences between treatments in experi-
ment 3 are essentially accentuated. In other words, the statistical
difference between treatments in experiment 3 is a result of the
relatively small within replicate variability. In both experiments 1
and 3. maximuin growth occurred near treatment 2 in a range of
chlorophyll-a effective flow rates of 550 to 650 (j-g ■ min"' ■ I"'. In
order to verify this result, growth should be investigated within the
chlorophyll-a effective flow rate range of 0 to 500 p.g ■ min"' ■ 1"'.

The benefit of defining a relationship between growth and the
amount of food passing by a unit biomass of animals (chlorophyll-
a effective flow rate) is apparent in the application to other grow-
ers. The relationship can be easily applied to upwellers in a variety
of locations, provided optimal environmental conditions persist.
An aquaculture extension agent could characterize the water con-
ditions at a site to determine that the minimum water quality stan-
dards are met, such as temperature, salinity, and dissolved oxygen.
The agent could then measure the amount of chlorophyll-a and
estimate the average food concentration at the site. With this es-
timate, the grower could determine the biomass and effective flow
rate needed to optimize growth in the upweller.

The Effect of EnviroiimeiUal Characleristics on Growth

When environmental conditions were suitable for northern qua-
hog growth, especially in the beginning of experiment 1, the effect
of food limitation on growth was apparent. When environmental
conditions were less than optimal, as in experiments 2 and 4,
growth appears constant over a wide range of chlorophyll-a effec-
tive tlow rates. In other words, growth was not controlled by food
limitation, but some other factor. To quantify the effect of envi-
ronmental conditions on growth, the upper one-third of replicates,
the fastest growing northern quahogs, were used to determine

10

Applevard and Dealteris

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Figure 5. Linear regression of the relative growth rate (% increase/dayl for the upper third of replicates from June 21 through August 19, 1999
versus lal chlorophyll-o ((ig/l) and (b) morning dissolved oxygen Ippnil.

growth. By eliminating the slowest two-thirds replicates, the effect
of food limitation on growth was minimized; therefore, differences
in growth were constrained by the en\ironmental conditions at the
time.

Manzi et al. (1986) concluded that food limited growth in their
expenmental-scale upweller. Although there were signs of food
limitation on growth in experiments 1 and 3. growth in experi-
ments 2 and 4 were controlled by other factors. Malinowski and
Siddall (1989) detemiined that the How rate limited growth in their
upweller system. They surmise that flow through the upweller had
to be above a critical threshold in order to create a uniform flow
(distribute food evenly among the clam seed), maintain water qual-

ity, remove wastes, and provide a sufficient chlorophyll-^ concen-
tration to the northern quahogs. Although Malinowski and Siddall
(1989) were unable to quantify the effect of water quality on
growth, they eluded to the importance of environmental conditions
on growth.

0\er the course of the iwo-month experiment, growth was
positively correlated with morning DO and negatively correlated
with chlorophyll-((. In late June and early July, the experimental
site at Point Judith Pond experienced a pronounced algae bloom.
The bloom was evident as an increase and peak in the chloro-
phyll-o concentration (Fig. 4d). There was a clear relationship
between chlorophyll-i; and morning DO. specifically as the chlo-

Growth of M. mercenaria in an Experimental Upweller

rophyll-<; concentration increased, morning DO levels decreased
(Figs. 4d and e). The decrease in morning DO was a result of a
combination of algae decomposition and algae respiration. At
night, the algae were constantly respiring, converting captured
energy into simple sugars, an o.xygen consuming and carbon di-
oxide producing process. The algae were also continually dying off
and decomposing, again an oxygen consuming process. A second
algae bloom in the upper pond was apparent in mid August. Again,
the same relationship between chlorophyll-i; and morning DO was
apparent. In late July, the chlorophyll-i; concentration decreased
substantially and morning DO levels increased. This decrease in
chlorophyll-(( was most likely a result of zooplankton grazing de-
scribed by Bengtson (1982). Alternatively, the decrease in chlo-
rophyll-a could have been caused by a crash or die off of a par-
ticular species of algae. The cychc pattern of algae in the upper
pond could be further verified by quantifying the species of algae
present as well as the amount of zooplankton at the study site.

The relationship between primary productivity (chlorophyll-o
concentrations) and low dissolved oxygen in shallow coastal and
estuarine areas has received considerable attention, as this phe-
nomenon appears to be increasing on a global scale. Diaz and
Rosenberg (1995) reviewed the diversity of research characteriz-
ing low DO events in the Chesapeake Bay. They found that the
cause and ecological consequences of these events varied from
tributary to tributary.

Regardless of the specific controlling mechanisms, an increase
in algae bioinass caused a distinct decrease in morning DO (<5
ppm) resulting in depressed clam growth. The relationship be-
tween low DO and depressed clam growth has not been charac-
terized in the literature: however, a number of researchers have
successfully characterized changes in stress levels (Sparks &
Strayer 1998) and predator behavior (Taylor & Eggleston 2000;
Tallqvist 2001 ) of bivalves exposed to low oxygen concentrations.
Hamwi (1969) determined that Mercenaria mercenaria were able
to maintain a constant rate of respiration with decreasing oxygen
levels until 5 ppm. The northern quahog is a classic oxygen regu-
lator (Hamwi 1969), As the oxygen concentration decreases, bi-
valves can increase their rate of oxygen consumption through two
mechanisms: (1) increasing their pumping rate: or (2) increasing
their percentage of oxygen utilization. Hamwi (1969) determined
that the pumping rate of northern quahogs remained constant with
decreasing oxygen concentrations; however, northern quahogs
were able to regulate O, consumption by increasing the percentage
of oxygen utilized. When oxygen levels reached 5 ppm or below,
Hamwi (1969) found that oxygen uptake in northern quahogs de-
creased continuously and an oxygen debt was incurred. Once con-
ditions were favorable, the oxygen debt was rapidly repaid in a

matter of hours and northern quahogs were able to function nor-
mally.

Although juvenile northern quahogs can survive in oxygen con-
centrations below 1 ppm for up to three weeks (Stanley & Dewitt
1989). 5 ppin is the critical threshold for northern quahog growth.
There have been a number of studies that have investigated the
effect of low oxygen levels on survival and tolerance, yet none
have investigated the effect of low oxygen levels on growth. Based
on the work completed by Hamwi (1969). 5 ppm is the critical
threshold for northern quahog growth. When oxygen concentra-
tions fall below 5 ppm. the northern quahogs cannot maintain
sufficient oxygen uptake and incur an oxygen debt. In essence, the
northern quahogs shut down and stop growing until oxygen levels
rise above this critical threshold.

The results of this study stress the importance of sufficient
oxygen concentrations for northern quahog growth in upweller
systems. A number of methods could be used to ensure optimal
oxygen levels in an upweller. The upweller could be moved to a
site that experiences lower chlorophyll-(( values and higher morn-
ing DO values, but food for the northern quahog would be com-
promised. Alternatively, the oxygen concentration in the upweller
could be increased. During periods of low morning DO (<4 ppm).
the '7c saturation was below 60: therefore, during periods of low
morning DO. oxygen concentrations have the potential of being
increased. Future research should investigate the most cost effec-
tive and efficient method of increasing dissolved oxygen levels in
this upweller as well as in the more traditional passive flow up-
wellers. With optimal DO levels, the effect of food limitation on
growth can be further defined and replicated.

SUMMARY AND CONCLUSIONS

The hypothesized relationship between growth and chloro-
phyll-fl effective flow rate was only apparent during the first two-
week experiment (experiment 1). Although there were significant
differences in growth between treatments in the third two-week
experiment (experiment 3). these differences were most likely the
result of small within sample variability. For the remainder of the
experiment, northern quahog growth was limited by environmental
conditions. Specifically, the relative growth rate of the upper one-
third of the replicates was positively correlated with morning-
dissolved oxygen (R" = 0.42) and negatively coirelated with chlo-
rophyll-d (R" = O..^.^). The critical dissolved oxygen threshold for
northern quahog growth in the experimental-scale upweller ap-
peared to be 5 ppm. below which growth was adversely affected.
Future research should investigate the most effective method for
elevating DO levels in commercial floating upwellers.

LITERATURE CITED

Baldwin. R. B.. W. Mook. N, H. Hadley. R. J- Rhodes, & M. R. DeVoe.
1995. Construction and operations manual for a tidal-powered up-
welling system. Charleston. SC: SC Sea Grant Consortium.

Hayes. J. C. 1981. Forced upwelling nurseries for oysters and clams using
impounded water systems. In: C. Claus. N. DePauw. & E. Jaspers,
editors. Nursery culturing of bivalve molluscs. Eur. Maricull. Soc.
Spec. Publ, Vol. 7. Belgium: Bredene. pp. 73-83.

Bengtson. D. A. 1982. Resource partitioning by Menidia menidia (L.) and
Menidia berylUna (Cope) in two Rhode Island estuaries. PhD disser-
tation. Kingston: University of Rhode Island. 214 pp.

Benson. B. B. & D, Krause. Jr, 1984. The concentration and isotopic

fractionation of oxygen dissolved in freshwater and seawater in equi-
librium with the atmosphere. Limnol. Oceanogr. 29:620-632.

Castagna, M. & J. N. Kraeuter. 1981. Manual for growing the hard clam.
Mercenaria. Special Report in Applied Marine Science and Ocean En-
gineering No. 249. Gloucester Point. Virginia: Virginia Institute of
Marine Science.

Claus. C. 1 98 1. Trends in nursery rearing of bivalve molluscs. In: C. Claus.
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Journal of Shellfish Research. Vol. 21. No. 1. 13-14. 2(102.

FLOW CYTOMETRIC MEASUREMENT OF HEMOCYTE VIABILITY AND PHAGOCYTIC
ACTIVITY IN THE CLAM, RUDITAPES PHILIPPINARUM

BASSEM ALLAM, KATHRYN A. ASHTON-ALCOX, AND SUSAN E. FORD*

Haskin Shellfish Research Laboratory, Rutgers University, 6959 Miller Avenue.
Port Norris. New Jersey 08349

ABSTRACT The assessment of blood cell viability and defense abilities is a major concern in the study ol pathological processes.
In this work, we devised and validated flow cytometric assays to measure viability and phagocytic activity of hemocytes from the clam
Ruditapes pliilippinanim. a species susceptible to the bactena-caused Brown Ring Disease (BRD). Validated assays were subsequently
used to measure hemocyte parameters following experimental contamination with BRD's etiologic agent Vibrio tapetis. Results show
that clams that developed BRD symptoms had lower phagocytic rates and a higher percentage of dead hemocytes than those that did
not. Ill vitro interactions between hemocytes and V. lapetis demonstrated that clam hemocytes are able to phagocytose formalin-fixed
I', tapetis. but at lower rates than latex beads. Live V. nipetis were able to kill clam hemocytes in vitro. The in vitro assay also showed
that phagocytosis increased with increasing temperature from 8' to 21°C. This work demonstrated the efficiency of flow cytometry for
measuring inolUiscan blood cell activities during host/pathogen interactions and points the way lor further experiments using this
analytical tool.

KEY WORDS: bivalve, bacteria, phagocytosis, hemocyte viability, flow cytometry. Ruditapes plulippiiiaruiii

INTRODUCTION

Changes in overall hemocyte activity have been observed in
parasitized bivalves or those experimentally exposed to pathogens
(Nottage & Birkbeck 1990; La Peyre et al. 1995; Anderson 1996;
Garreis et aL 1996; Allam el al. 2000u, 2000b). Techniques used to
investigate these changes generally have serious drawbacks. Mi-
croscopy is subjective and involves a considerable expenditure of
time, especially when large numbers of samples need to be evalu-
ated. Spectrophotometric inethods are based on the measurement
of activity in the whole sample and not on a cell-by-cell basis.
Flow cytometry, however, has proven to be extremely useful in
overcoming these problems. In the field of molluscan research,
flow cytometry has been used to quantify phagocytosis (Alvarez et
al. 1989; Brousseau et al. 2000; Allam et al. 2001; Foumier et al.
2001) and hemocyte viability (Ashton-Alcox & Ford 1998; Ash-
ton-Alcox el al. 2000; Fournier et al. 2001 ).

In flow cytometry, particles are passed single-file through a
laser beam. The light scattered by the particles indicates their size
and internal complexity. Fluorescence, whether aulofluorescence
or from a fluorescent tag, is measured by specific detectors. Flow
cytometric phagocytosis ineasurements usually involve the use of
fluorescent particles that are detectable by the flow cytometer even
after phagocytosis has taken place (Alvarez et al. 1989; Brousseau
et al. 2000). Specific fluorescent dyes that indicate membrane
integrity and permeability, intracellular redox potential, or enzy-
matic activity are available and can indicate cell viability using
flow cytometry (Coinbrier et al. 1989).

Although molluscan hemocytes have been studied by flow cy-
tometry before, validation of the results is rarely reported. In this
work, we refined and verified by microscopy, two flow cytometry-
based assays to measure the phagocytic activity and the viability of
hemocytes in the clam Ruditapes philippiminim. These allowed us
to investigate hemocyte activity in clams experimentally infected
with Vibrio tapetis. the bacterial agent of Brown Ring Disease
(BRD) in Europe (Paillard et al. 1994). We further studied the

♦Corresponding Author. Phone: -h1-8.^6-78.')-0074; Fax: -I- 1-856-785- 1.^44;
E-mail: susan@hsrl.rutgers.edu

effect of in vitro contact between the pathogenic bacterium and
hemocytes on hemocyte viability, and compared the uptake of V.
tapetis with that of similarly sized fluorescent beads. We also
investigated the effect of temperature on phagocytosis because
temperature is strongly associated with the prevalence and inten-
sity of BRD in nature and under experimental conditions (Paillard
et al. 1994; Allam 1998).

MATERIALS AND METHODS

Experimental Animals

Ruditapes philippinaruin were obtained from 2 locations:
southern Puget Sound. Washington. USA (length = .39.4 ± 0.46
mm, mean ± SEM). and the Bay of Brest, Brittany. France (length
= 37.7 ± 0.6.'i mm). Clains were shipped overnight to the labo-
ratory where they were immediately placed in quarantine, aerated
35-L standing-water tanks (about 35 clams per tank al 1 3°C and 34
ppt), in which they were kept during the remainder of the study.
Clams were fed daily throughout the experiments using a mixture
of cultured algae. All the experiments reported here were con-
ducted using clams from the USA, where BRD has never been
reported, except for the infection experitiient. which was done
using French clams.

Clams affected with BRD were obtained by challenging them
with V. tapetis as previously described (Allam et al. 2000a).
Briefly, a 0.5 ml (5 x 10^ bacteria) aliquot of a suspension made
with exponentially growing V. tapetis (ATCC 4600, strain PI 6)
was inoculated into the pallial cavity of each experimental clam.
Control clams were inoculated with the same volutne of sterile
seawater. After 4 weeks of incubation under the conditions de-
scribed above, hemolymph was collected and processed for phago-
cytosis and viability assays. The clams were then shucked and
BRD development on shells was recorded (Paillard & Maes 1994;
Allam et al. 2()(IOa).

Hemolymph Sampling

Animals and hemolymph samples were kept on ice during pro-
cessing to avoid hemocyte clumping. Hemolymph was withdrawn
from the posterior adductor muscle as described by Auffret and

14

Allam et al.

Oiihella (1995). Samples were immediately divided into aliquots
and diluted in ditfercnt solutions as described below.

Design and Validalion of the Phagocytic Assay

In vitro Incubation of Heniocytes Hith Beads

Fluorescent latex beads, 2.02 (im in diameter (Fluoresbrite
Calibration grade, Polysciences, USA), were dispersed by expel-
ling them through a 26-gauge needle and diluting them with fil-
tered seawater (FSW) to give a final concentration of 6 x 10"^ beads
ml"'. Two hundred microliters of this suspension were placed in
each well of a 24- well microplate, which was centrifuged for 10
min at 200 x g at room temperature (-21°C) to form a uniform
monolayer of beads on the bottom of each well. Hemolymph
samples were immediately diluted with ice-cold sterile seawater
(SSW) to give a final concentration of 5 to 7 x 10^ cells ml"' and
200-|xl aliquots of these suspensions were added to each well to
give approximately a 1:10, cell:bead ratio. Following a 30-minute
incubation in thermostatic chambers (Minifrige II®, Boekel Indus-
tries, Inc., Philadelphia. PA) adjusted to 2I°C, conditions deter-
mined to be optimal during preliminary assays, the cytoskeleton-
inhibitor Cytochalasin B (10 (xg mP', final concentration) was
added to each experimental well to stop hemocyte activity. At-
tached cells were released by trypsinization (0A7c trypsin in 1%
EDTA-saline solution for 10 min) followed by gentle sonication
for one minute at room temperature (RT). Microscopic observation
verified that this procedure detached cells from wells and also
released non-ingested particles from the surfaces of hemocytes.
Fomialin was then added for a final concentration of 2<'^i to fix the
sample, which was transferred to a microfuge tube where it was
held on ice until processed, within an hour, by flow cytometry.
Because the flow cytometric profile for hemocytes from each clam
was unique (see later), it was necessary to have a control (i.e.. no
phagocytosis) profile for each individual. Thus, a control well was
established for each clam in which Cytochalasin B was added at
the beginning of the incubation period to prevent phagocytosis.
After the incubation, the control wells received the trypsin and
formalin treatments as described above.

Flow Cytometry

Flow cytometry was performed on a Coulter EPICS C equipped
with an argon laser and operated at a wavelength of 488 nm. Gains
and photomultiplier high voltage settings were adjusted to include
all cell and bead particles. Forward light scatter (FLS) and green
fluorescence (GFL) list mode data were collected. A total of
1 0,000 particles were counted for each sample. The percent phago-
cytosis was calculated for each clam by bitmapping (electronic
outlining) each of three particle types: (i) free beads; (2) non-
bead-associated cells; and (3) bead-associated cells (Fig. 1). The
bitmaps had been established previously by running beads alone,
then non-bead-associated cells plus free beads, and then samples
with phagocytosed beads. For each clam, the sum of counts in
bitmaps 2 and 3 represented the total hemocytes in the flow cy-
tometry sample. In each sample, the percent phagocytosis was
computed as the ratio of bead-associated hemocytes to total
hemocytes x 100. For each clam, the percent phagocytosis was
calculated as the difference between the percent phagocytosis in
the test wells and the percent phagocytosis in the control well. The
percent phagocytosis was always below 0.5% in the control wells.
For each sample, mean fluorescence intensity (channel number)
was calculated within each bitmap.

Assay Validation

Epifluorescence microscopy was used to compare and correlate
the percent phagocytosis results obtained from flow cytometry.
Bead-associated and non-bead-associated hemocytes were counted
using a Zeiss ICM 405 microscope equipped with a standard FITC
filter set. A minimuin of 250 cells was counted in each sample.
This comparison included control samples with added Cytochala-
sin B. In addition, the reproducibility of the assay was tested by
establishing duplicate experimental wells for each clam and com-
paring flow cytometric results for the replicates using correlation
analysis. Finally, to determine the effect of storage on the percent
phagocytosis measurement, samples were collected as described
above, processed by flow cytometry, stored in glass tubes at 4'C,
and re-analyzed after 3 and 7 days.

O

(J
G
<u

u
o

a

o

A

■'Vi¥i¥*Vi'i-A-MlWI'li!4! i 1 1 u i >

Forward light scatter (FLS)

Figure I. Flow cytometry bivariate plots sboMiny the hitniups used to caliiiiate iHad-associated and non-head-associated cells in control (.\) and
test (B) mixtures. Bitmap 1: beads alone, Bitmap 2: non-bead-associated cells. Bitmap i: bead-associated cells.

Hemocyte Viability and Phagocytic Activity in Clam

15

Design and \'alidatioii of the Mabilily Assay

III vitro Incubation of Hemoc) tes with the Fluorescent Vital Stain

The percentage of dead cell^. was assessed using the fluorescent
nucleic acid stain ethidium homodimer-1 (EHD) previously used
by Ashton-Alcox and Ford ( 1998). EHD binds to nucleic acids by
intercalation. It does not permeate cells with intact membranes;
thus only dead cells, or those with damaged membranes, become
fluorescent. Hemolymph samples were diluted in cold Alsever's
solution (1:10. v:v) immediately after collection. Then, 2 |jlM EHD
(Molecular Probes, Eugene. Oregon. USA), dissolved in DMSO
according to manufacturer's directions, was added. The mixture
was incubated at room temperature for 30 minutes. An unstained
control sample was made for each clam.

Flow Cytometry

Flow cytometry was used to collect light scatter parameters and
log red fluorescence (LRFL) signals for at least 5000 cells. The
percentage of dead cells was determined by setting a cursor at the
upper limit of the LRFL signal for the unstained control, which
was used as the "zero" channel for the stained cells in the parallel
treated sample (Fig. 2). The percentage of dead cells was calcu-
lated as the ratio of cells above the "zero" channel to total
hemocytes x 100.

Assay Validation

Fluorescence microscopy was used to compare and coirelate
the hemocyte viability results obtained from flow cytometry. Fluo-
rescent and non-fluorescent hemocytes were counted using an epi-
fluorescence microscope as described above. A minimum of 200
cells was counted in each sample. The reliability of the assay was
also tested by using hemocytes that had been killed by immersion
in boiling water for 5 min. Hemocyte mortality was confirmed
microscopically, after EHD uptake, to be lOO'Tf . whereas untreated
cells were more than 96'7f viable. Five mixtures of untreated and

heat-killed hemocytes were made using Q9c, 25%, 50%, 75%. and
100% heat-killed hemocytes. The percentage of viable and non-
viable hemocytes was measured flow cytometrically as described
above.

Effect of Temperature on Phagocytosis

Because BRD development is partially controlled by tempera-
ture, the phagocytosis assay was used to quantify the effect of
temperature on phagocytosis by R. philippinarum hemocytes /;;
vitro. Hemolymph was withdrawn from clams and immediately
diluted with ice-cold SSW to give a flnal concentration of about 5
to 7 X lO*" cells ml"'. For each clam, 6 test wells and one control
well of hemocytes and beads were established. Two replicates
were incubated at 8°C. two at 13°C, and two at 2rC in the Mini-
frige 11'"' temperature chambers. These temperatures were selected
because they are associated with the development of significantly
different BRD prevalences and intensities in experimentally chal-
lenged clams (Allam 1998). The single control well was incubated
at 21°C since preliminary studies showed no evidence of phago-
cytosis in controls at any tested temperature. All samples were
processed for flow cytometry as described above.

Ill vitro Interactions Between Hemocytes and I', tapetis

The first experiment was devised to stud\ the uptake of fluo-
rescently labeled V. tapetis by hemocytes. Exponentially growing
V. tapetis, cultured on marine agar were suspended in phosphate-
buffered saline (PBS) to obtain about 10'' cfu ml"'. The bacteria
were then fixed in 2% formalin, washed 3 times with PBS. and
resuspended in 1 ml PBS (pH 7.4) containing 1 mg FITC (Sigma).
The mixture was incubated for 30 min at RT, washed twice in
PBS. and finally resuspended in sterile seawater. Labeled bacteria
were then placed in each well of a 24-well microplate. centrifuged
to form a uniform layer on the bottom of each well ( 10 min. 500
X g, 2I°C) and used for measuring the phagocytic activity of
hemocvtes as described above. Six wells were established for each

a>

192 256 64 128

Log red fluorescence (channel)

256

Figure 2. Distribution of log red fluorescence (LRFL) in unstained control and sample stained with ethidium homodimer. A cursor was set at
the upper limit of the I^RFL signal for unstained control, which was used as the "zero" channel for the stained cells within the sample. PDC:
Percent Dead Cells.

16

Allam et al.

clam using the same hemocyte:test particle ratio (1:10): 3 incu-
bated with labeled bacteria (2 test, and 1 control well with Cyto-
chalasin B added), and 3 incubated with standard lluorescent beads
(2 test, and 1 control well).

The second experiment investigated the elTect of live V. tapelis
on hemocyte viability. Hemolymph samples were diluted with cold
Alsever's solution (1:10, v:v) immediately after collection. Expo-
nentially growing V. tapelis were suspended and diluted in sterile
Alsever's solution to obtain about 10^ cfu ml"'. One milliliter of
this suspension was transferred to a sterile plastic tube and mixed
with I ml of diluted hemolymph to give approximately a 1:50,
celhbacteria ratio. Ten (xg mP' of Cytochalasin B was immedi-
ately added to the mixtures to prevent phagocytosis. They were
then incubated with mild agitation on a rocker plate for 2 h at room
teinperature. A duplicate tube containing only sterile Alsever's
solution and Cytochalasin B was prepared for each clam and was
used as a control preparation. After incubation, EHD was added
and the percentage of dead hemocytes measured using the flow
cytometer as described previously.

Statistics

Percent phagocytosis and viability values were arcsine trans-
formed before the use of statistical tests, however tables show
means and standard errors of non-transformed values. Correlation
analysis was used in the validation of the phagocytosis and viabil-
ity assays. Correlation analysis, as well as repeated measures
ANOVA, was also used to test the effect of storage time on the
percent phagocytosis. Mean values in clams with (symptomatic)
and without (asymptomatic) BRD symptoms were compared using
a Student's /-test. This test was also used to compare the /;; vitro
effect of V. tapetis on hemocyte viability. Differences were con-
sidered significant at a = 0.05.

RESULTS

Assay Validation

Phagocytosis

The percent phagocytosis of beads calculated by flow cytom-
etry (19.9 ± 2.5) was significantly {P = 0.039) lower than that
calculated by fluorescent microscopy (26.4 ± 2.6), but the two
methods were highly correlated (N = 24, r = O.Sl, P < 0.0001 ).
Microscopic observation confirmed that the beads associated with
hemocytes were internalized. The percent phagocytosis in dupli-
cate wells was also highly and significantly correlated (N = 72, r"
= 0.67, P < 0.0001 ); consequently, two wells per individual was
considered sufficient replication in all subsequent experiments.
Although there appeared to be a slight decrease in the percent
phagocytosis in samples stored for seven days at 4°C (Table 1 ), a
repeated measures ANOVA showed no statistically significant ef-
fect of time. Nevertheless, all further flow cytometric samples
were processed within 24 hours because there was some micro-
scopic evidence of an increase in broken cells and membrane
fragments in stored samples. Fluorescence intensity (channel num-
ber) was equal to 5.7 ± 0.3 in non-phagocytic cells (mean ± SEM),
12.6 ±4.1 in free beads, and 27.4 ± 7.3 in phagocytic cells. No
significant changes in fluorescence intensity were observed in pre-
served samples.

TABLE I.

Effects of storanc on flow cytometric counts of percent pliagocytosis

In K. philippinanim. .Samples were processed al the day of collection

and re-anal>zed after 3 and 7 da>s of storane at 4 C. Means,

standard errors of the means, and correlation coefficients are

presented (.V = 24 clams with 2 replicates eachl. No significant

differences were observed among means (repeated measures

ANOVA) which were highly correlated iP < 0.0001).

Dav 0

Dav 3

Dav 7

Mean ± SEM

22.3 ± 1.3

lS.S + 0.9

14.9 ± 1.1

Dav 0

1

0.81

0.S5

Dav 3

1

n.77

Da} 7

1

Viability

The percentage of dead hemocytes calculated by flow cytom-
etry (3.7 ± 1.1) was not significantly different from values mea-
sured microscopically (4.2 ± 1.0, P = 0.69). and the two methods
were highly correlated (N = 10. r^ = 0.92. P < 0.001). About
99.97f of heat-killed hemocytes were located within the upper
channels on the LRFL histograms, and thus detected by the flow
cytometer as dead cells. Flow cytometric estimates indicated that
the hemocyte mixture made without addition of heat-killed
hemocytes contained 3.89;- dead cells, while those containing 25,
50 and 75'7f heat-killed cells contained 28. 1, 52.0 and 75.6% dead
hemocytes, respectively.

Disease Effects on Phagocytosis and Hemocyte Viability

Flow cytometric measurements showed a somewhat lower per-
centage of phagocytic hemocytes (7.2 ± 1.07f, N = 21 ) compared
with those from asymptomatic animals (10.4 ± 1.3%. N = 23. P
= 0.044) and no difference in fluorescence intensity among
phagocytic hemocytes (33.2 ± 9.0 for diseased and 25.1 ± 5.1 for
asymptomatic clams. P = 0.420). Symptomatic clams had a sig-
nificantly higher (P = 0.0006) percentage of dead hemocytes
( 13.4 ± 1.0%. N = 54) when compared with asymptomatic clams
(9.2 ± 0.5%. N = 20).

Temperature Effects on Phagocytosis

The percent phagocytosis was clearly related to the temperature
at which the hemocyte-bead mixture was held (Table 2). This

TABLE 2.

Effect of temperature on in vitro phagocjtosis of heads bv

hemocv tes from R. philippinarum (/V = 24 clams with 2 replicates

each). For each parameter, letters (a. b and c; or x and yl represent

significant differences among different temperatures (ANOVA, P <

0.05). Fluorescence intensity refers to the peak fluorescent channel

of those hemocytes that had phagocv tosed heads.

8 C

13 C

2VC

% Phagocytosis

Mean ± SEM

7.9 ±0.7"

14,9 ± l.lf

21.1 ± 1,2'

Range

2.7-19.3

4.4-25.5

12.5-41.6

Fluorescence Intensity

Mean + SEM

L'i.l ±3.0^

24.0 ± 4.4^

29.6 ± 8.3>

Range

10-21

1 8-33

19-38

Hemocyte Viability and Phagocytic Activity in Clam

percentage at 2rC was about 2lVc. at 13°C. it was 15%, and at
8°C, it was 8%. Similarly, fluorescence intensity within phagocyt-
ic cells was also dependent upon the incubation temperature. This
was about 30 and 24 in mixtures incubated at 21 and 13°C. re-
spectively, and only 15 in those held at 8°C (Table 2).

Uptake of V. tapetis versus Beads

Clam hemocytes phagocytosed fluorescent latex beads at a sig-
nificantly higher rate (22.3 ± 1.3%) than they did FlTC-labeled V.
tapelis (12.8 ± 2.9%, P = 0.002, N = 24).

Hemocyte Killing by V. tapetis

The percentage of dead cells increased significantly after incu-
bation of hemocytes with the pathogenic bacterium, V. tapetis. In
control mixtures, the percentage of dead hemocytes was 6.8 ± 2.2
while it reached 20.4 ± 5.9% in mixtures with V. tapetis added
(Student's f-test, P = 0.029, N = 11).

DISCUSSION

Flow cytometry has been used in several previous studies to
quantify phagocytosis and hemocyte viability in marine bivalves
(Alvarez et al. 1989; Ashton-Alcox & Ford 1998; Brousseau et al.
2000; Fournier et al. 2001 ). However, none of these studies re-
ported validating the methodology by comparing the flow cyto-
metric results against standard microscopic measurements. In this
study, we quantified phagocytosis and hemocyte viability in indi-
vidual clams by both flow cytometry and microscopy. The high
correlation between the two methods shows that flow cytometry is
not only a rapid and versatile method for analyzing these two
important parameters, but that the results are very comparable to
more traditional methods for assaying marine bivalve hemocytes.

Notwithstanding the good correlation between flow cytometry
and microscopy, differences in means between the two methods
may be significant. Some of this disparity undoubtedly comes from
uncertainty in drawing bitmaps to delineate presumed flow cytom-
eter particle groupings. In our study, the fact that microscopy
estimated a higher percent phagocytosis may be the result of the
inclusion of some cell debris in samples analyzed by flow cytom-
etry. Large, bead-free debris could fall above the noise discrimi-
nator for forward light scatter and be counted as non-
phagocytosing cells, which would decrease the calculated percent
phagocytosis. Indeed, microscopic observation revealed the pres-
ence of a limited quantity of such cell debris, which increased
when samples were stored for several days. Such debris was not
generated in the viability assay since the experimental protocol did
not involve the addition of trypsin and the mild sonication used in
the phagocytosis assay to detach adhered cells. Probably for this
reason, the percentages of dead cells obtained using flow cytom-
etry were not different from those measured microscopically.

Critical to phagocytic assays is the detachment of cells from the
incubation vials and the separation of non-ingested particles from
the phagocytic cells. Trypsinization is generally used to detach
cells (Alvarez et al. 1989) while a variety of methods have been
developed to discriminate non-ingested particles (De Boer et al.
1996; Mortensen & Glette 1996; Lopez-Cortes et al. 1999). We
found that a single procedure, trypsinization in the presence of
EDTA followed by gentle sonication, was quite effective in both
detachment and separation steps. The centrifugation step over a
sucrose gradient (Alvarez et al. 1989) is not required here to sepa-
rate non-bound beads from phagocytic cells since the forward light

scatter allows easy differentiation between these particles based on
size.

In this study, fluorescent latex beads were used as standard
experimental particles for phagocytosis, as in the work of Alvarez
et al. (1989) and Brousseau et al. (2000) who studied phagocytosis
by hemocytes in Crassostrea virginica and Mya arenaria. respec-
tively. These commercially available, fluorescent beads are ex-
tremely convenient for reproducible flow cytometric work due to
their standardized sizes and fluorescence, qualities that are difficult
to establish by labeling natural microorganisms. It must be recog-
nized, of course, that latex beads may not e\ oke the same response
from hemocytes as would foreign cells. Indeed, the present work
demonstrated that the uptake of labeled V. tapetis was significantly
lower than that of beads, despite precautions taken to ensure the
same experimental conditions and hemocyte-to-pailicle ratios. Dif-
ferences between the tw o test particles may be the result of specific
interactions between hemocytes and beads or bacteria involving
recognition factors that retard the uptake of formalin-fixed bacte-
ria. Using microscopy, Lopez-Cortes et al. (1999) studied the
phagocytic activity of /?. pliilippinanim against V. tapetis and
noted that the uptake of bacteria depended on the V. tapetis strain
used. They also noted that viable V. tapetis were more efficiently
phagocytosed than were the formalin-fixed bacteria and concluded
that this was related to the presence of "specific recognition mol-
ecules" on the outer membrane of V. tapetis that combine with
hemocyte receptors and that might be altered during fixation. Most
work using labeled bacteria as test particles has been done after
fixation of the microorganisms, which stabilizes the tag intensity
because it prevents cell division or other processes that could alter
intensity. Nevertheless, with appropriate controls, the use of live
tagged bacteria should be included in cytometric assays to inves-
tigate the role of bacterial epitopes in recognition processes.

The development of BRD was associated with a significant
decrease in phagocytic activity by hemocytes and an increase in
the percentage of dead hemocytes. The lower phagocytic activity
in diseased clams could be related to the percentage of dead
hemocytes, since dead or moribund hemocytes are not capable of
phagocytosis. Previous work has shown that clams with BRD have
a high percentage of dead cells in the hemolymph compared to
healthy animals and that this percentage increases with the devel-
opment of the disease (AUam et al. 2000a, 2000b). The loss of
phagocytic capacity and the death of hemocytes may result from
deterioration of the physiological condition in severely infected
clams (Plana et al. 1996), or from direct killing of hemocytes by V.
tapetis. Indeed, results presented here demonstrate that V. tapetis is
able to kill clam hemocytes /;; vitro.

It is often of interest to know the number of particles a phago-
cyte has ingested (phagocytic index) as well as the proportion of
phagocytosing cells. The peak fluorescence intensity in the
hemocytes that did ingest beads provided an index for the relative
number of beads ingested by each cell. From this, it can be con-
cluded that although clams with BRD symptoms had a smaller
proportion of phagocytic hemocytes compared to asymptomatic
clams, there was no difference in the number of beads each cell
ingested.

Our results show a positive correlation between temperature
and both percent phagocytosis and the phagocytic index of latex
beads and agree with previous studies of other marine bivalves
(Feng & Feng 1974; Foley & Cheng 1975; Alvarez et al. 1989;
Tripp 1992; Chu & La Peyre 1993). It is relevant that the devel-
opment of BRD seems to be at least partially controlled by water

18

Allam et al.

temperature (Paillard el al. 1^97; Allam I99S). Laboratory experi-
ments have shown high prevalence and intensity of the disease
when clams are incubated at 8°C and 13°C compared to clams
incubated at 21°C (Allam 1998). Conversely, there are improved
repair processes (recalcit'ication) at 2I'-'C as compared to the lower
temperatures. It is possible that the low prevalence of BRD at 2 1 "C
is related to better performance of the clam's defense system,
including phagocytosis. The pathogen may also be less virulent at
this temperature, although growth of V. lupctis is not inhibited al
21°C (Maes 1992; Paillard et al.. unpublished).

In summary, we have described and validated flow cytometric
methods to measure the phagocytic activity and \iability of
hemocytes from the clam, Riulirapes pltilippiuaiiim. Additional
types of cytometric measurements will undoubtedly be adapted
from vertebrate systems for use with molluscs and other inverte-
brates. For example, reactive oxygen species (ROS) generated by
phagocytic cells represent a current concern of bivalve pathobi-

ologisls and ecotoxicologisls (Winston cl al. 1996; Bramble &
Anderson 1997; Lambert & Nicolas 1998). The successful use of
How cytometry for ROS measurements in aquatic mammals (De
Guise et al. 1995) and fish (Verburg van Kemenade et al. 1994)
indicate that flow cytometric methods can also be adapted for this
purpose in bivalves. As new cytometric assays come into use, the
developmental protocol should include some type of verit'ication
against a method that is considered the standard for that assay.

ACKNOWLEDGMENTS

The first author was supported by a fellowship from the French
Government. The authors thank Dr. Joth Davis (Taylor United.
Inc.) for providing us with clams from Puget Sound. We also thank
Dr. Christine Paillard for valuable discussions. This paper is contri-
bution No. 2002-9 from the Institute of Marine and Coastal Sciences
at Rutgers University and New Jersey Agricultural Experiment
Station Publication No. D-324(J5-2-02. supported by state funds.

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Shellfish Immunol. 9:543-555.

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tale.

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maximus) haemocytes maintained in vitro. Fish Shellfish Immunol. 6:
111-121.

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Mvtilus edulis. J. Invertehr Pathol. 56:15-19.

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1997. Epidemiological survey of the brown ring disease in clams ot
Atlantic coast: role ol Icmpeiaturc in variations of prevalence. Pro-

Hemocyte Viability and Phagocytic Activity in Clam

19

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Ann. Rev. Fish Dis. 4:219-240.

Paillard, C. & P. Maes. 1994. Brown ring disease in the Manila clam
Ruditapes philippinanim: establishment of a classification system. Dis.
Aquatic Org. 19:137-146.

Plana, S., G. Sinquin. P. Maes, C. Paillard & M. Le Pennec. 1996. Varia-
tions in biochemical composition of juvenile Ruditapes philippinanim
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Tripp. M. R. 1992, Phagocytosis by heinocytes of the hard clam. Merce-
naria mercenaria. J. Inverlebr. Pathol. 59:222-227.

Verburg van Kemenade, B. M. L., A. Groeneveld. B. T. Vanrens & J. H.
Rombout. 1994. Characterization of macrophages and neutrophilic
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Biol. 187:143-158.

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Journal of Shellfish Research. Vol. 21, No. 1. 21-27. 2002.

TRANSPLANTS OF INTERTIDAL SHELLFISH FOR ENHANCEMENT OF DEPLETED
POPULATIONS: PRELIMINARY TRIALS WITH THE NEW ZEALAND LITTLE NECK CLAM

M. J. STEWART* AND R. G. CREESE

Leigh Marine Lalwnitory. University of Auckland. P.O. Box 349. Warkworth. New Zealand

ABSTRACT The New Zealand little neck clam. Ausrrovenus stutchburyi (Wood 1828), is widely distributed in sheltered intertidal
habitats around New Zealand and has long been harvested by recreational and traditional fishers. Clam abundances have declined on
many beaches due to excessive harvesting and habitat change, such as sedimentation and pollution.

The feasibility of transplanting clams as a method of shellfish enhancement was tested. Manipulative field experiments using tagged
clams examined parameters likely to affect growth and survival of transplanted clams, including the size of seed used, density and shore
level at which seed is planted out, and the season in which the transplant is undertaken.

Juvenile clams (10-18 mm) had a mean recovery rate of 30'7r after 1 y. Growth was highest for clams transplanted to low on the
shore, but monality was also highest for these clams. Optimal placement of juvenile seed for enhancement would be at mid-shore
levels, where more clams were retained and reasonable growth still occurred. A much higher recovery rate ( 6(.1<7f-W9f ) was achieved
for adult clams (25-32 mm), and they were more likely to remain in the new area. These pilot trials demonstrate that transplant is
feasible and would be particularly successful for adult clams.

A'£l' WORDS: New Zealand Aiistroveiws stiitchbiini. clam, enhancement, transplant, growth, season, survival

INTRODUCTION

The commercial exploitation of soft shore bivalves in New
Zealand has been at low levels, with the exception of the surf clam.
Paphies ventricosa (Stace 1991). Other species, however, tradi-
tionally played an important part in recreational fisheries and in
traditional Maori food gathering (Dobbinson et al. 1989; Turner
1997; Stace 1991). Although considered nationally abundant,
many populations have become locally depleted (Browne & Paw-
ley 1995; Pawley et al. 1996; Pawley et al. 1997; Morrison et al.
1999. and considerable anecdotal evidence), Austrovenits stutcli-
buryi, also known as the New Zealand little neck clam (Belton
1986). is one of the most abundant of these soft shore bivalves, but
populations are experiencing declines in some areas. In the Auck-
land region in particular, temporary closures have been put in place
in an attempt to counter declining stocks. The causes of these
declines and the subsequent need for stringent countermeasures
have not been adequately identified or addressed, but have com-
monly been hypothesized as increased sedimentation, high levels
of contaminants in sediment and water, and overharvesting. These
causes are all associated with urban development in coastal areas.

Current management of shellfish populations in New Zealand
is largely restricted to the imposition of daily bag limits and local
bans, although these are often not well enforced (Kearney 1999),
Natural recruitment may lead to the recovery of clam beds if
harvesting bans continue and are enforced, but recovery may be
unacceptably slow. This appears to be the case in Auckland, where
beds have not returned to historical densities despite a 7-y har-
vesting ban (Morrison et al. 1999).

There are two main commercial approaches to shellfish en-
hancement which have been used in coastal environments around
the world: ( I) the spawning and rearing of spat in a hatchery, and
(2) the collection of spat from the wild (Malouf 1989; Peterson et
al, 1995; Marelli & Arnold 1996). Both are then followed by a
period of on-growing (usually in a land-based facility) before
transplantation to the site of enhancement. Another, more tradi-

*Corresponding author.

tional technique is the use of "spawner transplants," which in-
volves transplanting naturally established shellfish from locations
where the species is abundant to "enhancement" sites (usually sites
where numbers are very low). In Japan, clams and oysters have
been transplanted to enhance stocks for centuries (Wada 1993). It
is also a traditional management practice for hard clam (Merce-
naha mercenaria) fisheries in the eastern states of the United
States (Kassner & Malouf 1982). There are a number of earlier
studies where European ciams (.Cardium ediile) were taken from
areas either of high spat fall or of high density and transplanted to
areas of low density (Hancock 1969; Mason 1969), Recent trans-
plants of adult scallops in the western Bogue Sound. North Caro-
lina, resulted in enhanced recruitment of 568% compared to 3% for
control sites (Peterson et al. 1996).

In New Zealand, enhancement techniques are not developed to
the same level as those widely used overseas (Creese & Cole
1995). Although it is technically possible to rear New Zealand
clams in the laboratory (Stephenson & Chanley 1979). reliable
production of large numbers of spat has proved difficult, and no
commercial or sizeable hatchery has yet been established to pro-
duce the quantities needed. Enhancement using adult clams had
been suggested but not tested in New Zealand prior to the com-
mencement of this research (Dobbinson et al, 1989).

Manipulation of several factors, shown separately to influence
the survival of seed (juvenile or adult shellfish), can greatly im-
prove the success of enhancement (Peterson et al. 1995), The
literature on New Zealand clams and on international enhancement
attempts identified several factors that might influence the trans-
plant success of infaunal bivalves. The transplants of clams de-
scribed in this paper manipulated not just one. but several factors
that the literature suggested might be important in successful trans-
plant of shellfish. Although the literature suggests that separately
these factors might be important in restoration success, the inter-
actions among them may be just as important as the individual
factors themselves, A multifactorial design was used to delect all
possible effects of density, size of seed, season, and shore level,
including the interactions of these factors. Predation could not be
feasibly incorporated into this already large experimental design,
so this was investigated separately (authors' unpublished data).

In the long term, it would be hoped that transplanting clams

21

22

Stewart and Creese

would increase success of spawners and thereby recruitment. How-
ever, the primary objective of this experimental transplant was to
assess the feasibility of the technique as a method for future en-
hancement, and to determine what factors might affect the success
of transplants. In the long term, increased recruitment of clams or
changes in overall community structure would be important vari-
ables by which to measure the outcome of enhancement. However,
for this 1-y, small-scale project, it was decided that growth and
survival of transplanted clams would he the most direct measures
of the outcome of transplant.

METHODS AND MATERIALS

Experiments were carried out in the Whangateau Harbour near
Leigh in North Eastern New Zealand, with clams transplanted
from Lews Bay to Point Wells (Fig. 1 ). Clams were collected from
Lews Bay at low tide over 2 d. taken to the Leigh Marine Labo-
ratory, and held in a saltwater tlow-through system. Clams were
double tagged, with aluminum pieces glued to the shell (Stewart &
Creese 2000) and also with colored enamel paint to denote treat-
ments.

At each of the control and experimental sites, the experiment
was set up in a grid pattern with treatments (three replicates of
each) assigned to plots (each 0.25 m") within the grid using an
incomplete block design. There were a total of 15 plots at each site.
Each plot was 1 m from any other plot and shared no border.
Ausrroveniis sliitchbiiryi are relatively sedentary and throughout
the experiment no clams were observed to move between plots.
When the clams were planted out, the sediment from each 0.25-m"
plot was excavated and sieved through a 2-mm mesh sieve to
remove any resident macrofauna and thereby standardize the initial
state of sediments in the plots. To prevent desiccation and preda-
tion of newly transplanted clams before arrival of the incoming
tide, clams were placed in the excavated holes and partially cov-
ered with sieved sediment. It was observed that, despite being
covered initially, most clams would emerge and rebury themselves
fully by the next low tide.

Lews Bay, from where clams were originally collected, served
as a control site to enable the effect of translocating clams and site
effects to be distinguished. Control clams were subjected to the

same stress as those transplanted to Point Wells. Because clams are
of a small size high on the shore, and are larger lower on the shore
(Larcombe 1971 ), controls for each size class were only set up in
the area from which those clams were collected. That is, small
clams collected from high on the shore were returned to high on
the shore, and large clams collected from low on the shore were
returned to low on the shore (Fig. 2).

To investigate the effect of shore level on growth and survival
of transplanted clams, experiments at Point Wells were set up at
two shore levels, mid (150 m froin mean high water spring
[MHWS]) and low (350 m from MHWS) (Fig. 2). Preliminary
surveys of the site revealed a low abundance of clams at the high
shore, consistent with other studies of clam distribution in the
region (Larcombe 1971; Kearney 1999).

Within the transplant experiments, the effect of size was inves-
tigated by using two sizes of seed: "small" (10-18 mm shell
length), which represented nonspawning clams, and "large" (25-
.'^2 mm), which represented an adult spawning size (Larcombe
1971 ). Two densities were compared to investigate whether it is
better to space clams out or to pack them into smaller, more easily
managed plots. The two densities compared were 200 clams/0.25
nr for the packed treatment and 50 clams/0.25 m' for the spaced
treatment. Two transplant experiments were conducted, one com-
mencing in March 1998 (summer transplant experiment) and one
in September 1998 (winter transplant experiment), allowing com-
parison between two planting seasons.

A third trial (disturbance experiment) was established to assess
the effect of disturbance from the bimonthly excavation of plots
used in the two transplant experiinents. At the low shore site at
Point Wells, an additional three replicates of large clams at the
50/0.25 m" density were .set up at the start of each experiment.
These were excavated only at the end of the experiment, and
growth and survival of clams were compared to those in plots
disturbed every 2 mo.

]

^ews Bay

1

Point Wells

1 1

y

Mid Point Wells
IsOni from MHWS

High on
shore

^

1

^

[small

A

1

<r ( large

^ \

Low Point Wells
.\Mlni from MHWS

Low on
Shore

^^

Figure 1. Location of study sites, Lews Bay and Point Wells in Whan-
gateau Harbour, near Leigh in northeastern New /Aaland.

Figure 2. Small cockles were collected from high on the shore at Lews
Bay and transplanted to mid and low shore levels at Point Wells. As a
control, some small cockles were also relumed to high on the shore at
Lews Bay. Large cockles were collected from low <m the shore at Lews
Bay and transplanted to mid and low shore le>els at Point Wells. .As a
control, some large cockles were also returned to low on the shore at
Lews Bay.

Transplants of the New Zealand Little Neck Clam

23

Analysis

The effect of shore level was essentially a separate investiga-
tion at Point Wells. If those clams returned to Lews Bay had been
planted out at mid-shore and low-shore levels as at Point Wells,
this would not have served as a control, because clams would had
to have been transplanted to different levels from which they were
collected at Lews Bay. Because the "shore level"" treatment was
not balanced between Point Wells and Lews Bay, the two shore
levels at Point Wells were treated as separate sites, additional to
Lews Bay, for statistical analysis. The names "Mid Point Wells"
and "Low Point Wells" continued to be used in order to retain their
site identity.

Mortality

Weekly visits were made to each site and visual searches un-
dertaken for any dead shells (cluckers), which remained on the
surface. This was used as an estimate of apparent mortality (Ar-
nold 1984; Dobbinson et al. 1989), to minimize disturbance from
frequent digging.

Upon completion of all experiments (January 1999). plots were
identified using the metal detector to locate the aluminum-tagged
cockles (Stewart & Creese 2()()()). Plots had no external markings,
which avoided interference with plots on public beaches. All clams
were excavated and brought back to the laboratory. This enabled a
direct measure of the number of clams remaining. Sediment in
each plot was washed through a 2-mm sie\e and tagged individu-
als were retained. A number of tagged cluckers were also retrieved
during this process. The area (~ 1 m) immediately surrounding each
plot was also sieved until no further tagged clams were retrieved in
two successive sieves. In some instances, ""finger ploughing""
(James & Fairweather 1995) of the sediment was also used. When
no additional clams were retrieved either through finger ploughing
or sieving, the metal detector was used to locate any missed indi-
viduals. The detector was passed over the surface for a distance of
up to 5 m away and when a "hit"" was detected, the sediment was
finger ploughed to retrieve the clam. This was repeated until no
more clams were detected over the entire plot and surrounding
area.

For each replicate plot, the number of clams in each of four
categories was recorded; alive, missing, dead as undamaged empty
valves, and dead as empty valves with observable shell damage.

Because the "summer transplant experiment"" ran for 1 1 mo and
the "winter transplant experiment"" ran for 5 mo, a direct compari-
son of mortality could not be made between seasons. However,
data for the first 5 mo of the summer transplant experiment were
analyzed to provide a legitimate comparison between seasons. For
mortality, this was done using only the number of shells retrieved
during visual searches, and therefore it is a comparison of apparent
rather than actual mortality. At the end of the experiment, survival
(estimated from the number of live clams retrieved) was analyzed
separately for the two seasons. Data were tested for normality and
homogeneity (using Cochran"s test) prior to performing analysis of
variance (ANOVA).

Growth

Because clams were not numbered individually, growth was
assessed by following changes in the modal length of size cohorts
over time. Every 2 mo. a subsample of 20 clams was remeasured
from each 0.25-m- plot. Subsampling avoided undue disturbance
to plots. Plots and clams were relocated using the metal detector
and clams were excavated, with care taken to minimize distur-

bance to those clams not measured. To ensure that a random
sample was taken every time, a strip was randomly drawn through
the plot and clams were sampled in this area. Comparisons of
growth over the full experimental period could not be made be-
tween seasons. Interim calculations were made for growth after 5
mo for the summer transplant experiment.

RESULTS

Mortality

Apparent mortality of clams was estimated from the nimiber of
tagged cluckers retrieved after 5 mo for each season. A four-factor
ANOVA was performed using the factors "season,"" "site,"" ""size,"
and "density."" There was no significant effect of season or density
on apparent mortality (P > 0.05). nor any interaction involving
these factors. The pattern of mortality between sites was not the
same for large and small clams, giving a significant interaction {P
< 0.01) between site and size. Mortality for large clams was high-
est at Mid Point Wells, but for small clams it was highest at Low
Point Wells (Fig. 3). Survival was significantly different between
large and small clams (P < 0.01), with mortality higher for large
clams than for small ones (Fig. 3). In addition, a significant dif-
ference between sites (P < 0.01) was detected. Apparent mortality
was lowest at Lews Bay, the site of origin for all transplanted
clams (Fig. 3).

Survival was assessed from the number of clams retrieved at
the end of both summer (after 1 1 mo), and winter (after 5 mo)
transplants. The average survival was 75%-90% for large clams
transplanted in winter, and greater than 60'7f for large clams trans-

Is sp

Treatments
Figure 3, Mean percent apparent niortalilv, estimated from the re-
trieval of cluckers across treatments after 5 mo [from Marcli to July
for the summer transplant experiment (a) and September to January
for the winter transplant experiment lb(l. n = 3 replicated plots at each
site. Ip = large cockles (25-32 mm shell length! at packed density
(2()()/().25 m-|. Is = large cockles at spaced density (5(l/().25 nr). sp and
ss = small cockles (1(1-18 mm shell length) at packed and spaced den-
sities, respectively.

24

Stewart and Creese

planted in summer, with the exception of Lews Bay (Fig. 4). Even
for small clams, average survival was greater than 307f at all sites
except Low Point Wells in summer (Fig. 4|. Survival at Lews Bay
was confounded by storm events removing an entire shell bank
containing the large clams in the winter transplant experiment, as
well as many of the large clams in the summer transplant experi-
ment. Therefore, data from Lews Bay were excluded from the
analysis. Three-way ANOVAs were performed separately for each
of the summer and winter transplants for the Mid Point Wells and
Low Point Wells sites only, with the other factors being ""size" and
"density."" With Lews Bay excluded, the two Point Wells sites
effectively become "shore level"" treatments. For the summer trans-
plant experiment, both the "size x density"" interaction (P = 0.04)
and the "shore level x size"" interaction (P < 0.01 ) were significant.
Survival for large clams was similar between Mid Point Wells and
Low Point Wells, but survival of small clams was lower at Low
Point Wells (Fig. 4). Significantly more large clams were retrieved
than small ones (P < 0.01 ). and overall survival of large and small
clams was highest at Mid Point Wells {P < 0.01 ). The significant
"density x size"" interaction is due to fewer small clams in the
spaced density treatment surviving than in the packed density
treatment (Fig. 4). For large clams, survival was similar between
density treatments (Fig. 4). Results for winter transplants are simi-
lar, but the "size x density"" interaction was not significant (P =
0.08) (Fig. 4).

90
75
60
45
30
15
0

(a) Lews Bay
Summer

X

I ' ■ I II I

Ip Is sp ss control

9(1 -

(d) Lews Bay

Winter

75 -

60 -

pS

45 -

rn 1

30 -

15 -

N/A

1 1

1

1

1

Ip Is sp ss control

90
75
60
45
30
15
0

(b) Mid Point Wells
Summer

i

90 -

75 -

—

-1

(e) Mid Point Wells
Wmter

60 -

-^

45 -

30 -

15 -

N/A

1

1

i

Ip Is sp ss control

Ip Is sp ss control

90 -
75 -
60 -

T

(c

) Low Point Wells
Summer

45 -
30 -

1

15 -
0 -

1

1

■ 1

Ip

Because the storm events that affected experiments at Lews
Bay affected only large clams planted low on the shore and not
small clams planted higher on the shore, two-way ANOVAs on
survival were run for small clams only. That is. the factor "size""
was removed and only "site"" (Lews Bay included) and "density""
were analyzed. For both summer and winter transplants, there was
no significant "site x density"" interaction. However, both site and
density were significant factors. In the summer transplant experi-
ment, survival of small clams at Low Point Wells was significantly
lower than at Mid Point Wells or Lews Bay (Student-Newman
Kews test. P < O.O.'i). Survival was not significantly different be-
tween Mid Point Wells and Lews Bay iP > 0.05). For the winter
transplant experiment, survival of small clams was significantly
different among all three sites (P < 0.05). with lowest values at
Low Point Wells (Fig. 4).

The majority of small clams not retrieved in the final sample
(either as whole or damaged valves) could not be accounted for
(Fig. 5). At Lews Bay, the majority of large clams not retrieved
were also missing (Fig. 5a and b) presumably due to the storm
events. However, at the Point Wells sites, the majority of large
clams not retrieved in the final sample were accounted for by
either whole valves or damaged valves, retrieved during visual
searches carried out during the year. Most retrieved valves were
still intact, with no evidence of damage that might indicate preda-
tion (Fig. 5). Damaged shells were mainly crushed half valves or
had chipped ventral margins.

sp ss control Ip Is sp ss control

Treatment Treatment

Figure 4. Mean percent survival taken from the number oflive tagged
cockles retrieved at the end of the experiment ( 1 1 mo for the summer
transplant and 5 mo for the winter tran.splant). n = 3 replicate plots.
Size and density treatments as in Figure 3. Disturbance controls (Low
Point Wells site only! are shown in grey.

90 -

75
60

45 -

30

15

0

(a) Lews Bay
Summer

90 -

(h) Lews Bay

75 -

Winter

60 J

fi

45 -

fl

30 -

15 -

Ip Is

control

Ip Is sp ss control

i 90 -

(c) Mid Point Wells

ij

Summer

^ /s-

^ 60-

c 45 -

g_ 30 -

p

S 15-
2 0-

^t--^tL

90 -
75 -

(d) Mid Point Wells

Winter

60 -

45 -

}

i

30 -

15 -
0 -

_^yj^

h~-^

k — 1 —

Ip Is sp ss control

Ip Is sp ss control

90 -

75 -

(e) Low Point Wells -j
Summer

60 -

T

45 J

30 -
15 -
0 -

k

t

t"

X

90 -

75 -

(f) Low Point Wells
Winter

60 -
45-
30 -•

li' ■ 1 missine
1 1 whole
I^H damaged

15 -

Ip

Is sp
Treatment

Is sp ss control
Treatment

Figure 5. Mean percent of cockles missing, and mean percent of dam-
aged and whole valves retrieved by visual searches at the end of the
experiments ( 11 mo for summer experiment and 5 mo for w inter ex-
periment). II = 3 replicate plots at each site.

Transplants of the New Zealand Little Neck Clam

25

Growth

Mean shell length was initially taken tVom random samples of
20 individuals taken from each plot every 2 mo. These individuals
were considered representative of the experimental population.
Inter-replicate variation was found to be virtually zero, so replicate
treatments were pooled for graphical representation. Because there
was little change in mean si^e until toward the end of the experi-
ment (when water temperature increased), the difference in growth
between treatments was analyzed in terms of the change between
the initial mean shell length of transplants and the mean shell
length at the end of the experiment.

Growth was analyzed separately for each expermient using a
three-way ANOVA with the factors "site," "size class," and "den-
sity." For both seasons, there was a significant (P < 0.05) three-
way interaction ("site x size x density"), but some patterns can be
seen in the data. Large clams grew only 1-2 mm during the ex-
periment (Fig. 6). Small clams, however, showed pronounced

i 2

1 -

(b)

i

I I Lews Bay
^M Mid Point Wells
I ~1 Low Point Wells

N/A

sp ss control

Treatment
Figure. 6. Mean change in shell length (mm) after 11 months (March-
January) for the summer transplant experiment (a) and alter 5 months
(March-July and September-January) for the summer (b) and winter
(c) transplant experiments, respectively. // = 3 replicate plots at each
site. Control plots (Low Point Wells only) for disturbance are shown in
the gray bars.

growth in some treatments, particularly at the Low Point Wells site
(Fig. 6). Interestingly, growth in the small clams was readily iden-
tifiable by direct observation of the shell. A clear disturbance mark
could be seen in the shell, indicating when they were first placed
out in the field. No other clams in the area showed such as pro-
nounced growth check. Clams taken from the high shore level at
Lews Bay and transplanted to Point Wells grew more than those
leturned to the site of origin at Lews Bay (Fig. 6).

DISCUSSION

Although the initial collection and tagging of clams did appear
to cause a clear gi'owth check mark, the disturbance of clams for
ongoing sampling during the experiment appeared to have no ef-
fect on growth or survival. The absence of a digging effect is
consistent with results from previous manipulations of these clams
(Martin, unpubl. data).

Apparent mortality of large clams was lowest when they were
returned to their site of origin at Lews Bay. At the end of the
experiment, however, no large clams transplanted in winter and
few large clams transplanted in summer were retrieved from Lews
Bay, due to storm events. Until this point, mortality, estiirtated
both by the numbers of cluckers retrieved and by observations
made during ongoing sampling, was lower than for the Point Wells
sites. We are confident that this trend of lower moitality at the site
of origin would have continued in the absence of storm inteifer-
ence. Lower mortality for clams returned to the site of origin
indicates that survival of transplanted clams was affected by en-
vironmental characteristics of particular sites, because all clams
underwent the same transplant process.

The majority of small clams not retrieved at the end of the
experiment could not be accounted for. These clams may have
either died and their shells were lost, or they may have migrated
out of the area. Small clams were observed to move away from the
initial experimental plots more than large clams. If emigration is
the main source of "loss," and these clams remain within the
general vicinity of the transplant plot, then enhancement may still
be considered successful, because these clams will still contribute
to the population. If they have migrated away from the area en-
tirely, then the effect on enhancement success is the same as mor-
tality. Tethering of clams would allow better assessment of wheth-
er these clams are dying (with subsequent loss of their shells) or if
they are migrating. However, we suggest that a minimum survival
rate for transplanted clams of 30% (as achieved in our experiment),
although not great, may be a realistic management option and
preferable to doing nothing and accepting the piesent situation of
declining stocks and poor recruitment.

The majority of enhancement projects are conducted using ju-
venile shellfish, simply because large numbers can be produced in
a hatchery and they do not require costly on-growing to a larger
size before planting out. However, planting out of larger shellfish
is often more successful (Peterson et al. 1995; Marelli & Arnold
1996), a result attributed to high mortality of juvenile shellfish,
possibly from predation. This inverse relationship between size
and mortality rate is often explained in terms of prey size refuge
(Whetstone & Eversole 1977; MacKenzie 1979; Arnold 1984;
Kraeuter & Castanga 1989; Peterson et al. 1995; Marelli & Arnold
1996). For this reason, Marelli and Arnold (1996) consider that
broadcasting (i.e., the seeding out of very large numbers of un-
protected juveniles) is not an effective stock enhancement tech-
nique.

26

Stewart and Creese

Our experiments enabled a comparison between the survival ot
small and large transplanted clams. As predicted by the published
studies cited above. surN ival of large clams was much higher than
for small clams (generally >60'-f and up to 9()'/f ).

The survival rates obtained in this study compare favorably
with transplants of the hard clam Meaeiu'iUi iiwircuciia (mainly
juveniles) in the United Slates. Peterson et al. (IW5) achieved
35% survivorship over 14 mo. Marelli and Arnold (1996) were less
successful with 95% mortality after 80 d. hi earlier trials by Flagg
and Malouf (1983), greater than 10% survival was only achieved
for those clams larger than 20 mm. and even clams of this size
suffered 100% mortality in areas with large whelk populations. A
minimum survival for small clams of 30% could well be accept-
able for enhancement, but a greater initial number of transplants
would be required to compensate for subsequent losses. To achieve
the desired level of enhancement, it may also be necessary to
follow the initial transplant with a number of smaller secondary
transplants. It would be laborious to collect such large numbers of
A. snachhuni from the field, and enhancement may only be fea-
sible if viable hatchery production, using local spawning stock,
could be established.

Damaged shells recovered in this study had either crushed
valves, likely to be attributable to birds or crabs, or chipped ventral
margins, likely due to whelks. No large crabs were observed in the
area and the only birds observed were lone pairs of oystercatchers.
Haeiustriipus ostralegiis. Other potential predators may have been
stingrays or fish, because a number of feeding pits were observed
in the area. However, predation by stingrays would have left a
noticeable disturbance in the experimental grid, which v\as never
observed.

Peterson et al. (1995) found that survi\orship was greater for
Mercenaria mercenaria clams planted in late fall/winter, but av-
erage growth was greater for clams planted in late summer. How-
ever, clams transplanted in summer were in the field for at least 2
mo longer than winter-transplanted clams. This temporal differ-
ence is mentioned, but no adjustments were made for comparison
of the two seasons. Thus, the period of exposure was compared,
and not season.

In our study, growth of transplanted A. Muichhiini was more
dependent on the time of year than on the treatment. Large clams
showed minimal growth, probably due to having already attained
near maximum size of 35 mm (Larcombe 1971: Dobbinson et al.
1989). Small clams showed pronounced growth at some sites. This
growth did not begin until late winter (September) for clams
planted out in March, and was most noticeable in the spring (No-
vember). For those clams planted out in September, an increase in
size was already noticeable 2 mo later, in November. Small clams

showed the greatest increase in shell length when transplanted,
from high on the shore at Lews Bay (the original site) to low on the
shore at Point Wells. This same shore level effect was found b\
Dobbinson et al (1989) in Otago Harbour, southern New Zealand.
A. stutchburyi low on the shore are generally larger than those high
on the shore (Wood cited in Morton & Miller 1973; Larcombe
1971: Dobbinson et al. 1989). Larcombe (1971) hypothesizes that
this is due to poor grow th conditions, in particular food availability
(due to increased exposure time), restricting growth at higher shore
levels. Clams transplanted to low on the shore al Point Wells grew
considerably more than those returned to the site of origin at Lews
Bay, indicating that clams high on the shore at Lews Bay are
unlikely to realize their full growth potential. If this is the case,
then translocating these clams to areas lower on the shore at en-
hancement sites may be a way of increasing the biomass yield
(Dobbinson et al. 1989).

Dobbinson et al. (1989) found virtually no effect of density on
growth of A. sliilc-lihiiiyi. Density manipulations by Stephenson
(1981) and Blackwell (1984), however, resulted in apparent in-
traspecific competition, limiting growth and increasing mortality.
Martin (1984) found higher growth rates in treatments where den-
sity was experimentally reduced. The effect of density on growth
and survival in our study is difficult to interpret because of an
interaction with the effect of size. Higher retrieval of clams in
densely packed treatments may have been because clam movement
was restricted at the higher density, and therefore more clams were
retained. If so. retention of transplanted clams may be improved if
they are planted out in dense clumps rather than spaced over a
beach. This would also make post-transplant monitoring easier.

In conclusion, high survival for large clams and reasonable
survival for small clams indicates that the transplant of New
Zealand clams is a feasible technique for enhancement. It is rec-
ommended that transplant of clams for enhancement should be
undertaken with adult clams, because these show the highest sur-
\ i\ al. are easier to collect, and are more likely to remain within an
area. Recent studies have demonstrated that infaunal bivalve re-
cruitment can be dependent on the adult density (Peterson & Sum-
merson 1992; Peterson et al. 1996; Arnold et al. 1998). Large
clams may therefore also have the added benefit of adding to the
population sooner (through spawning and subsequent recruitment
of new clams), thereby speeding the recovei^ of an area.

ACKNOWLEDGMENTS

This work was funded in part by the Auckland Regional Coun-
cil. The transplant experiment was achieved with the help of many
students at the Leigh Marine Laboratory. The manuscript was
improved by the comments of an anon\ nious reviewer.

LITERATURE CITED

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composition on the rate of predation of the blue crab, CalUnectes
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Arnold, W. S., D. C. Marelli, C. P. Bray & M. M. Hamson ( 1998). Recruit-
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Zealand.

Blackwell. R. G. 1984. Aspects of the population dynamics of Chiom-

stutchburyi in Ohiwa Harbour, Bay of Plenty. New Zealand. Ph.D.

thesis. University of Auckland, New Zealand.
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surveys: 1993-1995. MAF Fisheries. Auckland. 79 pp.
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Pac. Comerw Biol. 2:55-63.
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Shellti.sh Res. 8:197-212.

Transplants of the New Zealand Little Neck Clam

27

Flagg. P. J. & R. E. Malouf. 1983. Experimental plantings of juveniles of
the hard clam Menenaria mercenaria (Linne) in the waters of Long
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402.

James, R. J. & P. G. Fairweather. 1995. Comparison of rapid methods for
sampling the pipi, Donax delloides (Bivalvia: Donacidae), on sandy
ocean beaches. Mar. Freshwater Res. 46:1093-1099.

Kassner, J. & R. E. Malouf. 1982. An evaluation of "spawner transplants"
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Larcombe. M. F. 1971. The ecology, population dynamics and energetics
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surveys of shellfish populations in the Auckland region, 1997-98, and
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257-272.

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Jimnml of Shellfish Research. Vol. 21. No. 1. 29-32, 2002.

MORTALITIES OF ENSIS ARCUATUS (JEFFREYS) (SOLENACEA) IN WESTERN IRELAND

EDWARD FAHY,' MARIA LYONS ALCANTARA,' MARK NORMAN," RONAN BROWNE,^
VINCENT ROANTREE,- AND NICK PFEIFFER'

'Marine Institute. Ahhatstown. Dublin 15. Ireland: 'Taighde Mara Ted. Carna. Co Galway. Ireland:
'Atlantic Diving. 25 William St W., Galway. Ireland

ABSTRACT Monalities of the razor clam Ensis arcuatus were widespread in western Ireland in the spring months of 2001 . Loss of
biomass from one razor clam bed was estimated at 74%. Larger razor clams were more susceptible. Histological and bacteriological
examinations and TEM were carried out on moribund and live individuals but no pathological cause was identified. Gonadal staging
revealed that large Ensis arcuatus were partially or completely spent. Mortality is explained as a post-spawning phenomenon, which
was unusually severe in 2001.

KEY WORDS: Ensis arcuatus. razor clams, shellfish mortalities, western Ireland

INTRODUCTION

In 1997. a hydraulic dredge fishery for razor clams commenced
in the Irish Sea where extensive beds of Ensis siliqiia had been
discovered. A market developed, largely in Spain, and. when the
Irish Sea fisheries became exhausted, additional beds were sought
on the western Irish seaboard. The inshore waters of Cos Galway
and Mayo contain thinly dispersed E. arcuatus. which form suf-
ficiently dense concentrations to reward commercial exploitation
only in special and limited circumstances. These patches are in-
variably in the lee of reefs and islands that provide shelter from the
Atlantic swell.

Investigations were undertaken on these razor clam beds to
provide biological data on the growth and reproduction of Ensis
arcuatus (Fahy et al. 2001b); in the course of these studies we
encountered the phenomenon described here, which is evaluated in
the context of information emerging from the wider biological
enquiry.

Investigations on the local ecology of E. arcuatus had been
carried out in Cill Chiarain Bay. Co Galway. in August 2000. In
March of the following year, a mass mortality of the species was
reported there by a local fisherman, prompting a second assess-
ment. Cill Chiarain Bay |9°45'W: 53"20'N) is occupied by a major
Irish shellfish cooperative. Comharchuman Sliogeisc Chonamara
Teo, managing oyster and scallop, and concern for the possible
involvement of a disease in the mortalities prompted further in-
vestigations. Other shellfish species were not. however, similarly
affected, and the fisherman who first reported the event also re-
ported mortalities of Ensis arcuatus over a wide area south of
Slyne Head (10°10'W:53°7'N), extending to Inishmann in the
Aran Islands (9°36'W;53°24'N). Survey work by one of the au-
thors (E. F.) in the vicinity since has confirmed that the phenom-
enon was widely distributed and that it has affected large numbers
of the species.

MATERIALS AND METHODS

The distribution of E. arcuatus in a part of Cill Chiarain Bay
was first investigated in August 2000. Divers sampled by pouring
1 L of granular salt over a quadrat 0.33 m" and collecting all razor
clams which emerged from the substratum within it. Initially, the
area was investigated along north-south and east-west transect
lines but. when heavier densities of the animals were located (the
razor clam "bed"), sampling was concentrated on those areas. The

length of every individual was measured on a fish measuring board
and the biomass in each quadrat was estimated from a weight at
length curve, the parameters of which were calculated in August
2000. Repeat sampling of the bed was conducted on April 23. May
29. and August 23, 2001.

Samples of razor clams. Ensis arcuatus. taken froin Cill Chi-
arain Bay in April (24 individuals) and May 2001 (29 individuals)
were processed for histology. Animals were dissected and imme-
diately fixed in Carson's fixative for 48 h. Cross sections were cut
so as to include as many organs as possible. Tissue samples were
processed in an automatic processor for 10 h. In the processor, the
tissues were passed through different grades of alcohol and xylene
and then impregnated and embedded in paraffin wax. Tissue sec-
tions were cut at 3 p.m and stained in an automatic stainer with
hematoxylin-eosin; they were then mounted on silinized slides and
air dried.

Some of the razor clams collected in April displayed postmor-
tem tissue changes, so the following month a distinction was made
between 13 moribund individuals that were lying on the surface of
the substratum and 29 live razor clams that emerged from the
substratum in response to granular salt having been spread within
a quadrat.

The stage of gonadal development was assigned to the samples
collected in May following microscopic examination using the
terminology of Caspar and Monteiro (1998): the six stages of
gametogenic development progress from stage 0 (inactive) through
stages 1 (early active gametogenesis). 11 (late active gatnetogen-
esis). Ill (ripe), IV (partially spent), to V (spent).

Tissues from two moribund individuals were fixed in Carson's
fixative for 48 h for electron microscopy. They were wa.shed sev-
eral times in 24 h in a bath of cold cacodylate buffer (at 4°C) and
then fixed in 3% glutaraldehyde. Samples were washed in caco-
dylate buffer then postfixed in 1% Os04 (for 1 h at 4°C) and
washed again in cacodylate buffer, dehydrated in ascending grades
of ethanol, and subsequently embedded in epoxy resin. Thin sec-
tions were cut with a diamond knife using an ultramicrotome.
stained with uranyl acetate and lead citrate, and examined under an
electron microscope.

Bacteriological culture plates were used for primary isolation
of pathogens. The media employed included blood and seawater
agars and thiosulfate citrate bile salt sucrose agar (TCBS) for
detection of Vibrio. A BioNor (Norway) monoclonal antibody
agglutination kit was used to delect the possible pre.sence of
V. aiiguillarum.

29

30

Fahy et al.

TABLE I.

Single-factor ANOVA comparing razor clam biomasses in (ill Chiariiin on four occasions: August 200(1. and April, May,

and August 2001.

Groups

36982
37012
37104

Source of Variation

Between Groups
Within Groups

Total

Count

21

24

SS

11019.4

41477.1

428496.5

DF

50
52

Summary

Sum

2502.274
606.9071

2657.337

ANOVA

MS

5509.698
8349.542

Average

119.1 559
75.86338
110.7224

P-value

0.65988

0.521356

Variance

8031.401
2704.084
10344.37

F crit

3.182606

RESULTS

When the razor clam bed was exainined in April 2001, dead
and dying razor clams were much in evidence on the substratum or
standing half-clear of it. together with freshly empty but undam-
aged shells. Similar conditions obtained when the site was visited
I mo later. Razor clams on the bed in Cill Chiarain Bay are not so
densely concentrated when compared with parts of the Ensis sili-
cjiia bed on the east coast, for example (Fahy et a!., 2001a) and
there was wide variance in estimates of biomass on each occasion
it was sampled. A single-factor analysis of variance (ANOVA)
suggests that mean biomass altered highly significantly (Table I )
and more detailed comparisons revealed the differences in biomass
to be highly significant between August 2000 and May 2001 (P <
0.001 ); biomass also differed highly significantly between August
2000 and August 2001. The reduction in biomass between August
2000 and May 2001 was 74% (Fig. 1 1.

Length frequency distributions of live razor clams recovered by
divers in August 2000, May 2001, and August 2001 are shown in
Figure 2. along with the length frequency of moribund animals in
April 2001. In May. there were signs that some of the larger
animals recorded the previous August (corresponding with the
moribunds recorded in April 2001), were absent from the popula-
tion, although smaller size categories were not recorded either in
May 2001. A notable difference between the years is the large
incidence of juveniles, corresponding to recently spatted 0-group
animals in August 2001. To compare the year-to-year changes in
length distributions, therefore, only animals greater than 7 cm
were considered. The two length frequencies differ significantly
(P < 0.05) (Table 2); contrary to expectation, however (the mori-
bunds had apparently been the largest size group), the greatest
discrepancy is in the abundance of small- to medium-sized animals
in the second year.

Gonadal staging of live and moribund razor clams in May
(Table 3) revealed that the majority were female, but all were
either stage IV or V (partially or completely spent).

Histological examination of razor clams sampled in April 2001
revealed that ciliates were occasional on the gills, and there was a
low incidence of Nematopsis oocytes (Gregarines) in the connec-
tive tissue of the mantle and gills of some animals. Low numbers
of Chlamydia-Wke organisms were found in some epithelial cells
of the digestive gland of some razor clams. The levels of infesta-

tion of any pathogen were so low that they could not be associated
with the mortalities. Some animals presented postmortem tissue
changes. Abnormal nuclei were found in the connective tissue cells
of some razor clams. Transmission electron microscopy (TEM)
analysis of these samples confirmed that the abnormalities were
related to necrosis and not to a pathogen.

Some razor clams collected in May 2001 had Clilamydia-Uke
organisms in the epithelial cells of the digestive gland and some
had Neimilopsis oocytes in the connective tissue. One individual in
May had a heavy infestation of metazoan sporocysts. But again,
mortalities could not be attributed to any of these. The presence of
an unexplained "deposit" in some digestive diverticular cell cyto-
plasm was noted.

Bacteriological analysis indicated the presence of Vibrio sp.
(possibly V. fluvialis). the natural flora of the environment. Results
of serological agglutination tests for V. anguilUiniiii were negative.

DISCUSSION

Occasional mass mortalities of razor clams. Ensis sp.. are at-
tributed to several causes, among them storms (Tebble 1966) and

500

400

E

CO

300

0}

a

200

CTI

100
0

-100

-200

9 9 9 9 9

9 9 9
Q. ro 3
< 5 ->

Figure 1. Mean values (±1 SD) of razor clam bioma.ss in Cill Chiarain
Bav at times between August 2000 and August 2001.

Razor Clam Mortalities

31

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

25
20
15
10
5
0

May-01

n

^x

R

n

p

'f-^'

i

rr77

m

i

■:».

\ — 1 — 1 — 1 — "^

_

^

J

,.,i:

8 10 12 14 16

18

>. 16
u

S 14
i-12
t 10

Ol 8

c 6

01

!£ 4

0)

Q. 2

0

15

/

aiug-01

rn

1

i

—

-

Tm

?/'

',<:

_P3

-

□EZ] -

m

^,

■

s^

0 12 3 4 5 6 7

9 10 11 12 13 14 15 16

40
30
20
10

Moribunds, April-01

w\

lai i£2 -.^j^ '<^

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Length, cm

Fijjure 2. Length frequency distributions of Ensis aniialiis in August
2UUU and in May and August 2001, and distributions of moribund
animals In April 2001.

"adverse environmental conLlitions" (Howard 1998). Mortalities of
the Pacific razor clam. Siliiiiici puliiUi. explained by infection with
Rick('ltsia-\ike organisms, referred to as nuclear inclusion X. have
been reported in several locations in the United States (Elston 1986).
Razor clam mortalities in western Ireland in the spring of 2001
were too widespread to be attributed to a local environmental
explanation. They occuired within (and outside) a large shellfish
cooperative and there was concern lest the oysters and scallops that
are managed within the cooperative might be infected with a patho-
gen, but simultaneous mortalities were not observed among these.

TABLE 2.

Length frequencies (above 7 cm) of razor clams in August 2000 and
August 2001, compared by chi-square.

Length

.August

August

August

August

(cm)

00

01

00

01

Chi-square

7

4

1

8

7

6

12

8

2.0

9

9

3

9

5

3.4

10

11

5

11

5

7.3

II

12

13

12

I-"?

0.1

12

14

24

14

24

4..S

1,^

21

2.^

21

23

0.2

14

1(1

19

21

")T

0.0

l.s

5

3

16

1

(1

100

IDU

100

100

17.6

N - \ = 6; at 6 degs of freedom, chi-square = 12.6. therefore. P < 0.05.

The razor clams that made up the mortalities were apparently
predominantly large E. arcuatus. the dominant bivalve species in
this vicinity (Fig. 2). Although it is accepted that larger individuals
would be more visible to a diver collecting specimens, the size
groups which appeared to have been removed from the population
are of small to medium size (Table 2). when August 2000 is
compared with August 2001. although sampling in April and May
2001 suggested the largest clams were most affected. The question
is why the small- to medium-sized razor clams were not better
represented among the moribunds. Razor clams are highly mobile,
probably the most mobile of the bivalves after scallops, and they
are known, for example, to rapidly recolonize favorable ground
after dredging operations, so some immigration to the depleted
clam bed from the sunounding lower densities is likely to have
taken place after the event. E. arcuatus is widely distributed in the
vicinity and its length frequency distribution in the areas of great-
est clam concentration (the "bed") was similar to that among the
more thinly dispersed population in the waters surrounding it in
August 2000 (Fahy et al.. 2001b). The razor clam bed in this part
of Cill Chiarain Bay is understood to occupy more sheltered and
hence more favorable conditions for the animals.

Recorded mortalities were confined to animals of mature size
(the smallest mature E. arcuatus recorded in the course of biologi-
cal investigations was 8.5 cm, corresponding with an age of 2-3 y).

TABLE 3.

Gonadal staging for apparently healthy (livel and moribund razor
clams from Cill Chiarain Bav in May 2001.

Gonadal

Developmental

Nu

liber

of

Status

Sex

Stages

Ind

ividuals

Percentage

Moribund

Female
Male

V
IV
V
IV

5
1
1
2

56
11
11
22

Live

Female

V
IV

13
4

65
20

Male

V
IV

1
2

5
10

32

Fahy et al.

Although it is possible that the smallest dead razor clams were
undersampled. the mortalities appeared to be heavily skewed to-
ward the largest, oldest individuals. There are signs that some spawn-
ing by E. anuatus takes place in every month of the year, but it is
more prevalent in the population from October to April; there were
signs of only one spatfall. on the other hand, in June or July. The
latter would coiTespond with spawning in March-April, which must
therefore be the most significant spawning time in this species.

If postspawning mortality is a normal occurrence in E. arciia-
riis. the incident in the spring of 2001 must have been exceptional.
A regular loss of 749?- biomass. although it was followed in this
case by a considerably larger spatfall than in the previous year,
would have .serious consequences for populations of a species with

a longevity of up to 18 y. of which 21 Vc of the population on the
bed and .^8.4% of the more w idely dispersed population in the Bay
in 2000 were more than 10 y old. These are the age groups that
contribute most to the biomass.

A natural postspawning mortality is the proffered explanation
for the razor clam deaths in Co Galway in 20(1 1 .

ACKNOWLEDGMENTS

The authors offer thanks to Frank Berthe and Bruno Chollet of
the IFREMER EU reference laboratory for shellfish diseases for
their continuous advice and for carrying out the electron micros-
copy studies.

Elston, R. A. 1986. An intranuclear pathogen (nuclear inclusion X (N1X|)
associated with massive mortalities of the Pacific razor clam. Silicjuu
patulci. J. Invert. Pathol. 47:93-104.

Fahy. E. & J. Gaffney. 2001a. Growth statistics of an exploited razor clam
(.Ensis siliijua) bed at Gomianstown. Co Meath. Ireland. In: G. Bumell
(ed.) Coastal Shellfish - a sustainable resource. Hydrohiologia.
465:139-151.

Fahy. E.. M. Nomian. R. Browne. V. Roantree. N. Pfeiffer, D. Stokes. J.
Carroll & O. Hannaffy. 200 lb. Distribution, population structure, growth

LITERATURE CITED

and reproduction of the razor clam Ensis anuatus (Jeffreys) (Solenaceae)
in coastal waters of western Ireland. Ir. Fish, hivest. 10:24 pp.

Caspar. M. B. & C. C. Monteiro. 199S. Reproductive cycles of the razor
clam Ensis siliquu and the clam Venus striatula from Barrinha. South-
ern Portugal. J. Mar. Biol. Assoc. U.K. 74:939-954.

Howard. A. 199S. Razor clam injuries at Paignton Beach. Shellfish News 6:6.

Tehble. N. 1966. British bivalve seashells: A handbook for identification.
London: Bntish Museum.

Journal iif Shellfish Research. Vol. 21, No. 1. ?.^-«), 2002.

ECOLOGICAL AND MORPHOLOGICAL FEATURES OF THE BIVALVE ASTARTE BOREALIS
(SCHUMACHER, 1817) IN THE BALTIC SEA NEAR ITS GEOGRAPHICAL RANGE

MICHAEL L. ZETTLER

Baltic Sea Research Institute. Seestr. 15. D-181 19 Rostock. Germany

ABSTRACT During 1444 and 2000 niacKvoobenthos surveys were made in the Mecklenburg Bight (western Baltic Sea). In total 1 16
stations were investigated between March and September. Astaite horealis showed a wide distribution at depths between 12 and 26.5
m. Mean abundance at these depths was 47 ind./m- with a biomass (AFDW) of 0.5 g/m'. Maximum densities observed at these depths
were 541 ind./m" and 16 glrcr. respectively. In comparison to a data set of the 1960s, a decreased A. horealis population was found.
A. horealis ranged from 1.2 to 28.7 mm in shell length. Most (78%) individuals in the population measured <IO-mm shell length,
indicating strong recruitment in the Bight during recent years. Larger size classes (>20 mm) were observed only sporadically and in
low numbers. Shell length to wet weight and ash free dry weight correlations are given. Mean wet meat yield was 14.5%. The individual
ash free dry weight decreased with increasing shell length from 9.1% (<5 mm) to 5.6% (>25 mm) with a mean value of 7.4%. All results
were compared with data from populations in Russian Arctic and adjacent waters.

KEY WORDS: Astarte horealis. distribution, abundance, size, meat yield, Baltic Sea. Mecklenburg Bight

INTRODUCTION

The bivalve, Astarte horealis. is an arctic-boreal species that
occurs in Arctic. North Atlantic and adjacent waters (Zettler 2001 ).
Some data are known from the North Pacific waters such as Japan,
Sea of Ochotsk. Behring Street, British Columbia and at the Aleu-
tian Islands (e.g., Coan et al. 2000; Higo et al. 1999; Skarlato
1981). A. horealis find its most extensive distribution in Arctic
waters of Russia (e,g,, Antipova 1978; Filatova 1957; Gagaev
1989; Matveeva 1977). It extends from the Barents Sea, via Kara
and Laptev Sea to Chukchi Sea and to areas of northern Alaska.
Greenland (Ockelmann 1938) and Spitzbergen (Hiigg 1904). At
the Grand Banks off Newfoundland, it reaches high abundance in
sandy bottoms around 130 m depth (Prena et al. 1999). In Europe
this bivalve extends from Iceland (Thorarinsdottir 1997) and off
Faeroes and Norway (Brattegart & Holthe 1997) to the northern
North Sea (Johansen 1916) via Kattegat (Rasmussen 1973) into the
western Baltic Sea and reaches there, its eastern limit of distribu-
tion in the Bornholm basin (see Fig. 1) (Demel & Mulicki 1954;
von Oertzen & Schulz 1973). Thus, the Baltic population repre-
sents the most southern occurrence, an outpost of the mainly arctic
area of distribution. The largest populations in the Balic are found
in the Kiel and Mecklenburg Bights in depths below 15 m (KCi-
hlmorgen-Hille 1963; Schulz 1969; Zettler et al. 2000). A. horealis
is among the longest living species in the Baltic and is an impor-
tant indicator of environmental conditions. Beside salinity and
sediment structure, oxygen concentration has a strong influence on
the composition of Baltic Sea fauna and flora. Although A. horea-
lis is highly resistant to oxygen depletion (von Oertzen 1973;
Oeschger 1990) frequent and long lasting periods of anoxic con-
ditions finally diminish or kill the species. This has resulted in a
severe decrease of the Baltic Sea population oi A. horealis during
recent decades in the deeper parts of the Mecklenburg Bight (Gos-
selck et al. 1987; Schulz 1968).

Information on the population biology and morphological fea-
tures (growth, age. size) of this species is limited. Some investi-
gations on production, growth, population size, and morphological
features were carried out in Russian Arctic Waters (Antipova

Phone: -i-OO 49-381-5197-236; Fax: -l-OO 49-381-5197-440; E-mail:
michael.zettler@io-wamemuende.de

1978; Gagaev 1989; Matveeva 1977). Within the framework of an
autecological analysis of glacial relict species in the Baltic Sea.
investigations on the reproduction of A. horealis were earned out
in addition to experiments concerning its resistance and metabolic
adaptations (von Oertzen 1972. 1973; von Oertzen & Schulz
1973). Schaefer et al. (1985) studied biometric features of A. ho-
realis in Kiel Bight, the westernmost part of the Baltic Sea. They
investigated several relationships between shell length and weight
and their applicability for taxonomical distinguishing of species of
the genus Astarte.

The purpose of this study was to investigate the distribution,
frequency and biomass of A. horealis in Mecklenburg Bight as the
first extensive study on the population characteristics of this im-
portant indicator species near its geographical range. A further aim
was to compare these results with existing data of the 1960s com-
piled by Schulz ( 1969). From one monitoring station (stn. 018). we
have a long time data set to show the development of A. horealis
within the last decades.

.Area of Invesligalioii

The Mecklenburg Bight is part of the Belt Sea and belongs to
the transition area between North Sea and Baltic Sea (Fig. 1 and
Fig. 2). It is connected with Kiel Bight via Fehmanibelt and with
Kattegat via the Belts. To the East, the Kadet Trench crossing the
Darsser Rise connects it with the Baltic proper.

During 1999 and 2000 macrozoobenthos surveys were made in
the Mecklenburg Bight. In total 116 stations were sampled be-
tween March and September (Fig. 2). Station depth ranged from 5
to 29.6 m. The sediment varied from fine sand at the shallowest
stations to sand mixed with silt and clay at the deepest stations.
Sediment characteristics and current data for the area have been
published by Lange et al. ( 1991 ).

MATERIALS AND METHODS

Profiles of salinity were recoi'ded throughout the water column
using a CTD (conductivity/temperature/depth probe) system.
Samples for bottom water oxygen were taken with a 5-1 water
sampler (mounted on the CTD) at 0.5 m above the bottom and
oxygen levels determined by Winkler titration. Benthic samples
were taken with a 0. 1 m~ Van Veen grab. Due to sediment con-
ditions, grabs of different weights were used. Three replicates of

33

34

Zettler

56,5

100 10.5 11,0 11 5 120 12.5 13.0 13.5 14 0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 190

Longitude (E)

Figure 1. Distribution nf Aslarle hiirealis within the Baltic Sea. The sources of the data are Deniel anti Mulicki (1454), Kuhlmorgen-Hille ( 1963),
Liiwe (1963), von Oertzen and Schul/, (1973). Petersen (1918) and own observations. Due to a strou}; decrease and wide disappearance of this
species mainly in the liornholni and Arkona ISasins not all dots represent recent locations. (KB-Kiel Bight, MB-Mecklenburg Bight, AB-Arkona
Basin, BB-Bornholni Basin, SF-Slupsk Furrow, circled area refer to the present study and Fig. 2).

grab samples were carried out at each station. The samples were
sieved thi'ough a 1-mm screen and animals preserved with 4%
formaldehyde in the field. For sorting in the laboratory a stereo-
microscope with 10— lOx magnification was used.

The shell length of all collected individuals was measured with
a vernier calipers to the nearest 0.1 mm for the length-frequency
distribution and the length-meat weight relationship. In total about
414 specimen were measured. The valves and the wet meat of the

54.5 -

<D

CO

54.0 -

11.0

12.5

13.0

11.5 12.0

Longitude (E)

Figure 2. A map showing the investigation area with 116 stations in the Mecklenburg Bight (circled stations refer to text and Figs. 5 and 8).

ASTARTE BOREAUS IN THE BALTIC SEA

35

11.0

11.5 12.0

Longitude (E)

12.5

13.0

b)

54.5

54.0

LObeck

11.0

12.5

13.0

11.5 12.0

Longitude (E)

Figure 3. Distribution o{ Astarte borealis {ind./m-|: (a) in 19y9/2(t()(t and (h) during tlie investigation period 1962-1965 (Schuiz 1969).

specimen were weiglited separately. Dry weight (DW) and asli free
dry weight (AFDW) were determined to the nearest 0.01 nig.
Length-frequency distribution for each station was calculated for 5
mm size classes. For the shell length to height relationship. 221
individuals from the 1999/2000 survey and 49 valves of A. borea-
lis from the Zoological Collection of the University of Rostock
(sampled in the 1980s from the Mecklenburg Bight) were mea-
sured.

The distribution map of .4. borealis in the Mecklenburg Bight
was made using Surfer 7.0 programme of Golden Software Inc.
The recent distribution was compared with the results from Schuiz
(1969), whose data were transformed into the Surfer program to
obtain a comparable map. For the long time series ( 1985 to 2000)
of the monitoring station 018. the data of the Baltic Sea Research

Institute Warnemiinde and data from literature were used (Al-
Hissni 1989; Voigt 1991).

RESULTS

Bottom Water Variables

Salinity throughout the water column ranged between 7.5 and
27.8 psu, while bottom water salinity of areas inhabited by .4.
borealis varied between 1 1.0 and 26.3 psu in 1999/2000. No oxy-
gen depression was observed during the surveys. Up to a depth of
1 8 m, more than 5.0 mg/1 oxygen was measured. In deeper parts of
the Bight the oxygen content decreased to a minimum of 1.0 mg/1.
A. borealis were found in an oxygen range of 1.5 to 8.1 mg/1. The

36

Zettler

54.5 -

S

54.0

Lubeck

11-0

11.5 12.0

Longitude (E)

12.5

13.0

Figure 4. Biuniass distribution o{ Aslarte borealis (AFDW g/nr) in 1999/2000.

mean oxygen content in area.s inhabited hy the bivalve was 6.27 ±
1.98 mg/1.

Distribution, Abundance and Bioniass

In 1999/2000. A. borealis was distributed between 12 and 26.5
111 depth in the Mecklenburg Bight. The species showed a very
patchy density distribution (Fig. 3a). In the shallow areas 10 ni
depth with fine sand sediments, no A. borealis were found. Fur-
thermore, the muddy zones of the outer Mecklenburg Bight and the
outer Kadet Trench with strong currents and stony substrates were
not inhabited. The highest abundance was found in the southeast-
em part of the Bight with a ma.ximum of 400 to 500 ind./m" and
an AFDW of 4 g/m~ (water depth around 16.5 m). Thirty-tlve
years ago (in the mid 1960s). A. borealis reached medium densities
between 10 and 50 ind./m"^ with a maximum of 100 ind./ni" in the
eastern central part of the Bight (Schulz, 1969) (Fig. 3b). At depths
below 20 m of the innermost area (Lubeck Bay) and western outer
Bight no A. borealis were found. The comparison of the main
distribution areas of the 1960s with the results of the present study
showed a clear change.

The highest biomass was observed at the coast off Holstein and
Mecklenburg (water depth about 16.0 m) with an ash free dry

S~ 500

G

5 400

~jr 300

u

a

S 200

5 100

nir^

i

Sin. 018

54" 1 1 0 N
ir46,0E
19 5ra

1 ' ' ' ' 1 ' ' ' '

1965-1985 1986 1987 1988 1989-1994 1995 1996 1997 1998 1999 2000

Figure 5. Changes in mean ah)undance (±S.I).) of Astarle borealis at
stn. (lis over the last 15 years in comparison with data from the mid
1960s (Schulz 1969).

weight (AFDW) of 5-16 g/nr (Fig. 4). Within water depths of 12
and 27 m.A. borealis reached a mean abundance of 47 ind./m" and
a biomass (AFDW) of 0.5 g/ni~.

The development of the abundance of A. borealis at stn. 018
(see Fig. 2 for location) during the last 15 years is shown in Figure
5. From 1985 to 1989 abundance increased from around 50 ind./m"
to about 400 ind./nr. By the mid 1990s and through to 2000
abundance at the same station declined dramatically to 10-20
ind./ni".

In the present study the highest abundance was observed in
depths between 12 and 22 m (Fig. 6). In this depth range. A.
borealis settled in an abundance of between 20 and 100 Ind./ni".

20

Abundance (ind./m^)

40 60 SO

too

120

a
■o

6
8

10
12
14
16
\i
20
22
24
26
28
30

1

n=2

n=7
n=16

1

1

, n=13

''J

an=7

n = >

■■ ■ ' ■ ' ' ' ' ■ ■ . :'.l 1

] n 11

fl7

^^

d n

] , ,ln=8

n=13
'JD n=13

n=6
n=2

■ abundance
□ biomass

—i

' 1

1

' hJ

0.5 1 1,5

Biomass (AFDW g/m^)

2,5

Figure 6. The vertical distribution of abundance (ind./nr) and bio-
mass (.AFDW g/nrl iti. Aslarle borealis in the Mecklenburg Bight in
1999/2000 (n = number of included stations of each depth interval!).

ASTARTE BOREALIS IN THE BALTIC SEA

37

28
26
24
22
I 20

■~ 16

M 14

1 12

5 10

»}

, . >ol

^<V^ 1

!^°

1

P

f»*

^ 1

oS^

1

:_._: . __^ .^^ . .__ 1

1 1 Qi^**^ i

L

'

n

j^^

1

1 _rf?r^

y = 0.8664X + 0.0533
R- = 0,9965
n=271

! Lg^^

^-j^i

^^ 1

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
Shell length in mm

Figure 7. Shell height-length relationship of Mtarlv boeralh from the
Mecklenburg Bight.

The biomass (AFDW) reached average amounts of 1.8 g/m" at
15-22 m with a maximum of 2.4 g in 15-16 m interval. In shal-
lower and deeper areas no or only single specimens were found.

Population Structure, Meat Yield and Growth

Measured shell lengths of ,4. borealis ranged from 1.2 to 31.2
mm (Note, this range included both the survey and the collection
material, see earlier). The shell length to shell height relationship

is linear as indicated in Figure 7. The mean ratio length to height
was 1.15 ± 0.04 and \aried from 1.05 in minimum up to 1.25 in
maximum, independent of shell length.

The population structures of selected stations are shown in
Figure 8. The size structure varied between the stations. The 0-10
mm size class composed about 78% of the population. Larger size
classes (>20 mm) were observed only sporadically and in low
numbers. The dominant 0-5 mm size class at most of the inves-
tigated stations represents the survivors of the settlement during
the last two years. Only at stn. 25 was the size structure dominated
by older specimens.

Relationships between shell length and individual weights
(whole wet weight, wet meat weight and ash free dry weight) are
shown in Figure 9. These graphs include all measured specimen
(1.2 mm to 28.7 mm) from the 1999/2000 survey. The smallest
meat wet weight was 0.09 mg at a shell length of 1.2 mm and the
largest one was 1.17 g at 28.3-mm shell length. The estimated
mean regression line indicates the average meat yield per unit shell
length of y4. borealis. Differences between the station means were
not statistically significant. The ash free dry weight \'aried between
0.08 mg (1.9 mm in length) and 0.29 mg (28.5 mm in length). The
estimated regression lines of weights for different stations did not
differ significantly. The results indicate that throughout the Mecic-
lenburg Bight A. Iwrealis contained approximately the same meat
per unit shell length for the range of length considered. The mean
wet meat yield (percentage of total wet weight) of different size

00 n

n%

— 1

Cf^ .

Stn. 25

27th Apr 1999

16.4 m
n=31

^

40 ■

■

1

20 -

■

■ ,

1

0 1

1 — ^^Ma

IJ

Stn. 45

28th Apr 1999
16.5 m
n=132

^

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^/^^■^Tjir-v.,^^,:^

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/

.f<Tr. - *r"'w'^^'A_y

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'

/

# ' • •

" '. ^ -yxyT

\

)''

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^L*. .*

^

?

. • .

^o' "■

PL.

540

( -J

^

• y^

^

<^

i4

K

Stn. 04

24th Aug 2000
18.5 m
n=36

I ongitude (E)

Stn. 32

27th Apr 1999
16.9 m
n=85

Stn. 72

01st May 1999
19.2 m
n=74

5-10 10-15 15-20 20-25 25-30

0-5 5-10 10-15 15-20 20-25 25-30

0-5 5-10 10-15 15-20 20-25 25-30

Figure 8. Shell length-frequency distribution for 5 mm size classes oX Astarte borealis at several stations in depths between 16.4 m and 20.6 ni
in Mecklenburg Bight in 1999/2000.

38

Zettler

10 15 20

Sbell length in mm

Figure 9. Estimated relationships between shell length and whole wet
weight ( + ), wet meat weight (xi and ash free dry weight lo) tor Astartc
horealis. The corresponding equations lor the calculated best fit re-
gression lines are given in the figure.

classes varied in a range between 12.21% and around \6A'7c with
a mininuini at SVc and a maximum at 23^7^ (Fig. 10). The mean
meat was 14.57c. With 9.1%, the highest amount of organic con-
tent (ash free dry weight) appeared in the 0-5 mm shell size class.
The lowest organic content was 5.67f in the 25-30 mm size class.
The difference was significant (Mest. p < 0.001). The mean or-
ganic content of dry weight ranged from 4.05% to 31.2% with a
mean value of 7.4%.

DISCUSSION

Distribiiliiin, Abundance and Hinmass

The occurrence of A. hnrcalis in the Mecklenburg Bight has
been known since the 19th century when the first investigations on
the henthic fauna of the Baltic Sea took place (von Martens 1871;
Wiechmann 1869/70). While the main Baltic populations live in
the Kiel Bight and in parts of Sound and Belt, the distribution in
the Mecklenburg Bight and the Arkona Basin represent the most
eastern recent occun'ence of this species in the Baltic Sea (see Fig.
1 and von Oertzen & Schulz 1973). Due to the decreasing salinity,
A. horealis has its natural limit of distribution in areas east of
Arkona Basin. Formerly, the distribution reached the deep parts of
the Bomholm Basin as far as the Slupsk Furrow (Demel & Mulicki
1954; Jaeckel 1952; von Oertzen & Schulz 1973). Due to long

Shell length in mm

Figure 10. Mean individual percentage of wet meat yield and ash free
dry weight in 5-mni size classes of Astarte horealis from the Mecklen-
burg Bight in 19W/2(H)0 (±S.D.).

lasting oxygen depletion in the last decades the bivalve had nearly
disappeared in this area (.Andersin et al. 1978). Today. A. horealis
occurs in this region onl\ in the Slupsk Furrow and only in depths
between 60 and 70 m (Warzocha 1995).

The investigation of Schulz ( 1969) from 1962 to 1965. giving
ihc distribution pattern of A. horealis in the Mecklenburg Bight,
was used for comparison with the present study. In the 1960s,
densities were between 10 and 50 ind./nr in mean (maximum 320
ind./nr). During the 1980s, the abundance of the bivalve increased
in waters near 20 m depth (e.g.. at the monitoring station 018)
(Kohn 1989; Voigt 1991). Due to a long period of oxygen deple-
tion In the deeper part of the LCibeck Bay (the inner part of the
Mecklenburg Bight), no A. horealis were found in the 1980s (Gos-
selck et al. 1987; Prena et al. 1997). In the 1990s, the density of A.
horealis decreased in shallower regions of the Mecklenburg Bight,
loo. Recently, a mean abundance of approximate 50 ind./m" In
depths between 1 1 and 26 m could be observed. The highest abun-
dance and biomass was observed in the region betw een 1 6 and 22
m. The mean biomass reached 1 g/nr AFDW (17 g/ni" wet
vselght). In comparison, at the beginning of the 1950s during the
Inxestlgations of Ktihlmorgen-Hllle (1963) In the Kiel Bight A.
horealis occurred In a mean abundance of between 4 and 52 Ind./
nr. At the beginning of the 1970s Arntz et al. (1976) observed in
the Kiel Bight the highest frequency and abundance in depths
between 10 and 20 m with 74-83 ind./m" (max. 570 ind./nr. 1240
g/m- wet weight). In adjacent waters of the Arkona Basin Liiwe
(1963) found A. horealis In quite high amounts of 13 g/nr wet
weight. The largest biomass with about 70 g/ni" he observed off
the Island of Falster and at the entrance of the Sound. Outside the
Baltic In Russian Arctic waters, the main distribution area of the
species, A. horealis colonises the littoral zone and reaches an abun-
dance of about 200 Ind./nr and a biomass of about 620 g/nr wet
weight (Antipova 1978; Gagaev 1989; Matveeva 1977). .4. horea-
lis belongs to the most productive bivalves in this region (Gagaev
1989).

According to von Oertzen ( 1972). A. horealis has an extremely
prolonged period of ripe eggs and sperms. The main spawning
season is presumably In spring with the possibility of "portion
spawning" the whole year (Kohn 1989; Matveeva 1977; von
Oertzen 1972). .4. horealis prefers sandy substrates and mixed
sediments avoiding muddy sediments (e.g.. Arntz et al. 1976;
Schulz 1969). In the deeper parts (>22 m) of the Mecklenburg
Bight, oxygen depletion In late summer prevents successful re-
cruitment and growth of the juveniles. However, A. horealis Is one
of the most tolerant species to oxygen deficiency and to hydrogen
sulphide (von Oertzen 1973; Oeschger 1990). Probably, the pos-
sibility of the high tolerance to oxygen deficiency Is restricted to
the adults, whereas the juveniles are more susceptible, causing
settlement only In favourable years and depths. The lack of re-
cruitment events Is described for populations of Kiel and Meck-
lenburg Bight by Werner et al. (1974) and Kohn (1989). In shal-
lower zones (between 15 and 20 m), no major hypoxic episodes
have been observed in the recent past (Matthiius et al. 1999).
Although recruitment can take place In the shallower areas, con-
ditions for growth are suboptimal. probably due to the lower nu-
trition supply, lower salinity and higher temperature (.Arntz et al.,
1976; von Oertzen. 1973). In depths shallower than 1 1 m the mean
salinity, 8-12 psu, is probably too low. The range of potency of A.
horealis is 8-35 psu with an optimum between 14 and 30 psu
(.laeckel 1952; von Oertzen 1973).

ASTARTE BOREALIS IN THE BALTIC SeA

39

Population Siriicliire

The largest living .4. borealis found in this survey was 28.3 mm
long and 24.7 mm high. Valve material of the Zoological Collec-
tion of the University of Rostock (sampled in the 1980s) had a
maximum length of 31.2 mm (26.4-mm height). Lenz (1882)
found specimen of 36 mm in length {31-mm height) in the Lijbeck
Bay. The shell is quadrate to subtrigonal and compressed v\ith a
total shell length of 38 mm at boreal outpo.sts (e.g., Baltic Sea.
Jaeckel, 19?2) and 55 mm in the Arctic Sea (Coan el al. 2000.
Filatova 1957) with a mean of 25-45 mm (Dance 1977). In gen-
eral, the shell length exceeds the height and the height/length
indices vary from 0.8 to 0.9 (e.g.. Ockelmann 1958). However. A.
borealis is a variable species with several forms and varieties (see
Zettler 2001). In the past, the great variability in morphological
features resulted in a number of new species and subspecies de-
scriptions (see Zettler 2001 ). Recent investigations of Hopner Pe-
tersen (2001) show a large variety of shell morphology within the
genus Astarte. The material discussed in the present study had a
high constancy in respect to the relation between length and height
(Fig. 7).

Statements on population dynamics or size structure of A. bo-
realis are very sparse in the literature. Some Russian studies
(Gagaev 1989; Matveeva 1977) and few investigations on Baltic
populations (Kohn 1989; Voigt 1991: Werner et al. 1974) have
been carried out. During the studies of Kohn (1989) and Voigt
(1991) in the Mecklenburg Bight the populations were dominated
(89%) by individuals <6 mm in length. Larger sizes were observed
only sporadically. In the present study, juvenile individuals were
dominant at most of the stations investigated. Adult specimens
(>20 mm) were only observed in high abundance in the inner part
of the Mecklenburg Bight (Fig. 8). Kohn (1989) pointed out that
only individuals >20 mm are reproductive. In the White Sea A.
borealis reaches sexual maturity in sizes >17 inm (Matveeva
1977). The maintenance of the stock in the Mecklenburg Bight
depends on few adults and/or probably on drifting of lecitothrophic
(non-pelagic) larvae from regions nearby. In areas with strong
currents, longer immigration distances are imaginable (Rasmussen
1973). In the Kiel Bight and in Russian Arctic waters however, the

population structures are more balanced (Werner et al. 1974;
Gagaev 1989).

Meal Yield

The mean wet meat yield of .4. borealis of 14.5% in the present
study is similar to reported values of Kohn (1989). He found
organic contents (wet) between 9% and 30% for a population in
the Mecklenburg Bight. Ansell ( 1975) observed a soft tissue con-
tent for/4, elliplica of 14.3-23.5% and dry tissue weights of 3-5%
in British waters depending on the reproductive cycle. In the
Mecklenburg Bight, in the present study, the mean individual ash
free dry weight of A. borealis decreased from 9.1% to 5.6% with
increasing size classes. The increasing mean wet yield and the
decreasing ash free dry weight with increasing shell length indi-
cates relatively higher water content of bigger bivalves. The length
specific wet weight (shell length - wet weight relationship) in this
study was similar to that reported for A. borealis of the Kiel Bight
and Russian Arctic waters. In the Mecklenburg Bight, the calcu-
lated wet weight for an individual of 25 mm shell length was 3.42
g. The wet weight of .4. borealis in the East Siberian Sea reported
by Gagaev ( 1989) was 3.49 g and in Kiel Bight 4.22 g (Schaefer
et al. 1985). According to Kohn (1989) the meat wet weight of a
25-mm specimen of A. borealis is 534 mg compared with 572 mg
in the present study.

In conclusion, the present study shows ecological and morpho-
logical features of Astarle borealis in the Mecklenburg Bight near
its eastern distributional boundary within the Baltic Sea. Further
investigations must deal with growth rates and morphology of A.
borealis within the Baltic and adjacent waters. The causes of the
decline of this cold-adapted arctic-boreal species in much of the
Baltic remain unclear. The comparative population dynamics at
different water depths and/or within different sediment structures
and the dispersion and settlement patterns of the larvae in this
"border" area are of special interest.

ACKNOWLEDGMENTS

The author thanks Christine Peters, Ines Glockzin for helpful
technical assistance, the Zoological collection of the University of
Rostock for supplying valve material, and the referees for their
helpful and interesting remarks.

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.lounial of Shellfish Rcscirch. Vol. 21, No. 1, 41-49, 2002.

IS SURVIVAL GENOTYPE-DEPENDENT IN NORTH AMERICAN POPULATIONS OF FARMED

BLUE MUSSELS, MYTILUS SPP?

R. W. PENNEY AND M. J. HART

Departinenl of Fisheries and Oceans. Science. Oceans, and Environment Brandt. P. O. Box 5667
St. Johns. Nfld. Canada. AlC 5X1

ABSTRACT We monitored survival and allele frequencies at five enzyme loci in single year-class cohorts of cultured Mytilus spp.
during a 14 month period from sleeving of seed to harvest at three commercial mussel farms in Notre Dame Bay, Newfoundland,
Canada. Amona-site genetic heterogeneity at all five individual loci and over all loci combined was evident among the three seed
sources. Significant temporal genetic heterogeneity occurred at all three sites. The observed intra-site temporal heterogeneity was not
related to genolypic state smce neither the relative proportions of homozygotes and heterozygotes at each of the five loci individually,
nor the mean heterozygosity over five loci, changed significantly within sites over time. Significant change in genotypic frequencies
at the Gpi locus was the primary contributing factor to the overall temporal genetic heterogeneity within each of the three sites.
Significant temporal changes in genotypic frequencies at each of the Mpi. Lap. Pgm. and Oilh enzyme loci inconsistently appeared
among the three sites. A significant directional shift in genotypic frequencies, consistent across all three sites, gave evidence of
genotype-dependent survival selection differentially favoring survival of Gpi genotypes with electrophoretically slower alleles in
comparison to genotypes with electrophoretically faster Gpi alleles during sub-tidal rope culture of Mytihis spp. populations in
Newfoundland. We also conclude that temporal genetic heterogeneity is a common occurrence in suspended rope culture of New-
foundland blue mussel populations and is likely a significant contributory factor in the extensive geographic genetic population
structuring previously reported among mixed M. ediilis - M. Irossiiliis populations.

KEY WORDS: Mytihis. genotype-dependent, survival, genetic heterogeneity

INTRODUCTION

In northeastern North America, two separate mytilid species.
Mytilus ediilis and M. trossiiliis are recognized (Koehn et al. 1984;
Varvioetal. 1988; McDonald et al. 1991). In Atlantic Canada, the
distribution of both species is widespread (McDonald et al. 1991;
Mallet & Carver 1992) and the two form hybridized mixed-species
assemblages (Saavedra et al. 1996). Newfoundland is. apparently,
a zone of natural distributional overlap between these two mytilid
species since most populations consist of mixtures of both species
varying widely in relative frequency (Bates & Innes 1993; Penney
& Hart 1999).

Within the overlap zone in Newfoundland, sites in close prox-
imity to each other display inter-site genetic heterogeneity on a
magnitude scale comparable to sites much larger distances apart
(Penney & Hart 1999). Such scales of genetic heterogeneity sug-
gest the potential for significant stock- or site-related variability in
growth, survival, or other characteristics among adjacent sites
throughout the mixed-species zone. For commercial industry, fac-
tors that significantly affect productivity within and among mussel
farms is of great importance. Genotype-dependent temporal sur-
vival selection processes have previously been noted in inter-tidal,
mixed-species communities involving M. galloprovincialis and M.
ediilis in England (Gardner & Skibinski 1991; Skibinski & Rod-
erick 1991; Gardner 1994) and in a mixed M. ediilis and M. tros-
siiliis intertidal assemblage in Nova Scotia (Pedersen et al. 2000).

In cultured stocks, inter-site variation in mortality rates related
to genetic (stock) differences are known to exist (Mallet et al.
1987, 1990) and may significantly affect production in suspended
culture (Mallet & Carver 1989). There is also evidence that be-
tween-site mortality differences are maintained when the stocks
are transferred to new sites (Mallet et al. 1990; Myrand & Gaud-

Corresponding author. R. W. Penney. Department of Fisheries and Oceans.
Science. Oceans, and Environment Branch. P. O. Box 5667 St. John's,
Nfld, Canada. AlC 5X1. E-mail: penneyrate'dfo-mpo.gc.ca

reault 1995) suggesting that among-site survival variation is re-
lated to stock genetics rather than environmental factors. Mortality
and growth variation related to genotype {M. ediilis vs. M. tros-
siiliis) was reported to significantly influence productivity at one
fami site in Nova Scotia (Mallet & Carver 199.5). Since the com-
bination of stock mortality and growth rale primarily define pro-
duction in suspended culture, among-site stock and/or genotype-
dependent variation in either parameter due to selective processes
may be an important determinant of inter-site variability in fartn
production indices.

In this study, we investigate survival patterns in three hybrid-
ized M. ediilis - M. trossidiis assemblages of cultured mussels in
Newfoundland. Canada, over the latter phase of the production
cycle, namely from sleeving of seed mussels to harvest. We use
empirical observations of temporal change in allele frequencies at
five enzyme loci to test the hypothesis that genotype-dependent
selection processes occur within rope-cultured mixed M. ediilis-
M. trossiiliis populations and. by inference, may be a significant
contributory factor influencing genetic heterogeneity, survival, and
production variability among mussel farms in Newfoundland.

METHODS

Seed mussels from collector ropes were sleeved in standard
commercial plastic mesh on each of three farms in Notre Dame
Bay. Newfoundland in August, 1998 (Fig. 1), In each case, the
.seed mussels were derived from collector ropes set out on each
farm in 1997. Seed mussels were mechanically de-clumped prior
to sleeving to ensure random distribution across all sleeves. Each
sleeve was 1 meter in length and was vertically suspended at
0.5-meter intervals from horizontal mainline ropes with flotation
sufficient to ensure stable suspension at depths of approximately
10-12 meters. One week after gear placeinent, three replicate
sleeves were retrieved from each farm site. From measured sec-
tions of each sleeve, all the mussels were removed and counted and
100 individuals were randomly selected for allozyme analysis (to-
tal of 300 from each site). The one-week delay in taking the first

41

42

Penny and Hart

Burnt Arm

\

Thwart Island

,r 55= 53", Notre Dame Bay

Canada -Atlantic Coast

Figure 1. Geographic location of the three farm sites in Notre Dame Bay. Newfoundland.

sample was necessary to allow tor mussel accUmation and attach-
ment to the sleeving material and thus eliminate drop-off of any
mussels that failed to properly attach themselves via their byssal
threads.

Each farm site was similarly sampled in May. 1999 and again
in October, 1999 at which time the majority of mussels had
reached or exceeded the minimum commercial harvest size of 55
mm. Survival over time was calculated as the difference in mean
number of mussels sleeve"' in May. 1999 and in October. 1999.
compared to the initial sample. In this context, changes in survival
do not distinguish between changes due to mortality versus drop-
off from the culture gear.

Hepatopancreas tissue was excised from each selected mussel,
lyophilized. and stored at 5°C for later allozyme analysis. Five
polymorphic loci were investigated: mannose phosphate isomerase
{Mpi, EC 5.3.1.8). aminopeptidase-I {Lap. EC 3.4.1 1.-), phospho-
glucomutase {Pgin. EC 2.7.5.1), glucose-6-phosphate isomerase
{Gpi. EC 5.3.1.9), and octopine dehydrogenase (Odh, EC
1.5.1.1 1). A small amount of freeze-dried material was ground to
a fine powder with 0.5M Tris HCL pH 8.0 buffer containing 20%
glycerol and 0.2% NAD. Subsequent electrophoresis and staining
on cellulose acetate plates followed the general procedures of He-
bert and Beaton (1989). although a constant current of 2 mA per
plate was used during the electrophoretic runs. The procedure for
Lap was modified to run with Tris glycine pH 8.6 buffer. For Odh,
we modified the stain for Cpi suggested by Hebert and Beaton
(1989) to use octopine as the substrate instead of fructose-6-
phosphate. Allele nomenclature is similar to that employed by
previous authors (Koehn et al. 1984; McDonald & Koehn 1988)
with the exception that alleles at the Odh locus were numbered
consecutively from 0-9 in order of increasing electrophoretic mo-
bility from the origin. The latter exception was necessary since we
were unable to match Odh electromorphs on our plates with pre-
viously published allele frequencies.

Analysis of allele frequencies for population differentiation and
probability estimation of log-likelihood G tests of genetic hetero-
geneity within and among genotype classes and within and among
sites utilized the software package F-Stat for WindowsT^. version
2.9.1 (Goudet et al. 1996). All other statistical analyses used the
SAS software system (SAS Institute Inc. 1988). Significance lev-
els of all test statistics involving multiple comparisons were Bon-
ferroni-adjusted. Non-parametric analysis of variance of ranks and
Rruskal-Wallis rank test statistics (SAS Institute Inc. 1988; Sokal
& Rohlf 1995) were calculated to test for directional, intra-site,
temporal trends in genotypic frequency distributions at the Gpi
locus. For these analyses, the twelve observed Gpi alleles were
assigned an ordinal rank score based on electrophoretic mobility,
with faster alleles scoring lower than slower alleles (e.g., C/w' '" =

rank 1 ; Gpi

rank 2, etc.). Thus for diploid loci, the genotypic

rank assigned each individual is the mean rank score of its two
constituent alleles (e.g.. for an individual of genotype Gpi""'" .
rank = 1 .5, etc.). For Kruskal-Wallis tests with n > 2 sample dates,
SAS calculates a x" approximation of the Kruskal-Wallis test sta-
tistic for significance testing (SAS Institute, Inc. 1988).

RESULTS

Seed mussels were collected from three farms located in Burnt
Arm, Charles Arm. and at Thwart Island in Notre Dame Bay on the
northeast coast of Newfoundland (Fig. 1 ) and transferred to com-
mercial plastic mesh sleeves in August, 1998, for grow-out. Elec-
trophoretic assays of the three seed mussel populations detected
four alleles at the Mpi locus, six at the Lap locus, eight at the Pt;iii
locus, eleven at the Odh locus, and twelve at the Gpi locus. Gene
diversity per locus at the three sites was very low (<0.20l at the
Odh locus, moderate (0.40-0.50) at the Mpi locus, relatively high
(0.60-0.75) at Lap and Pgm. and highest O0.80) at Gpi. Log-
likelihood G lest scores from inter-site comparison of the three

Genotype-Dependent Survival in Mussels

43

seed mussel popiikitidiis in August 1998, revealed significant (p <
0.001) among-site population genetic heterogeneity at all loci in-
dividually as well as o\er all loci combined (Table 1 1. At the Mpi
locus, Mpi^"" predominated at Burnt Arm and Charles Arm while
Mp/'"* was the allele at highest frequency at Thwart Island (Fig. 2).
Pgm'°" was the most common allele at the Pgm locus at both
Burnt Arm and Charles Arm while the frequency of Pgiii^ ' ' was
highest at Thwart Island. At the Lap locus, Lcip''^ was the most
common allele at Charles Arm but at Burnt Arm and Thwart Island
the frequency of Lap'"^ was greatest. Gpr"^ predominated at
Charles Arm while, at both Burnt Arm and Thwart Island, the
frequency of Gpt"" was highest. At Odh. the three sites differed
only in frequency of relatively rare alleles. The frequency of Odlr"
was greatest for all three sites.

In August, 1998, mean mussel density on the sleeves varied
among sites from 450 mussels m~' at Charles Arm to 790 mussels
m~' at Burnt Arm and 890 mussels m"' at Thwart Island. Survival
to May, 1999 (calculated as within-site change in mean mussel
density since August, 1998) was not significantly different among
sites (ANOVA. P > 0.05). Mean survival during the first 9 months
of grow-out (to May, 1999) was 81.2% at Charles Arm. 76.9% at
Thwart Island, and 78.0% at Burnt Arm (Fig. ?<). After 14 months
on the sleeves (October, 1999), mean survival at Charles Arm was
significantly less than the other two sites (ANOVA, P < 0.05).
Mean survival at Charles Arm was only 37.4% compared to 5 1 .6%
at Thwart Island and 51.7% at Burnt Arm. Log-likelihood G test
scores for among-site comparisons of population genetic structure
after 9 and 14 months of grow-out (May and October, 1999 re-
spectively) were also significant (Table I) indicating the initial
among-site genetic heterogeneity was maintained during the
course of the grow-out period.

During the same 14 month grow-out period, significant intra-
site temporal changes in allele frequencies at one or more loci were
found at all three sites (Table 2). After the first 9 months of
grow-out (to May, 1999), log-likelihood G test scores were sig-
nificant over all loci combined at all three sites indicating signifi-
cant temporal change in genetic structure had occurred over this
period. Significant intra-site temporal population differentiation at
the Gpi locus was evident at all three sites. Intra-site population
genetic differentiation was also evident at the Mpi locus at Burnt
Arm, the Pgm and Odh loci at Charles Arm, and at the Odh locus
at Thwart Island. This pattern continued through to October, 1999
after 14 months of grow-out by which time further genetic differ-
entiation at the Odh locus had become evident at Burnt Arm and
at the Lap locus at Thwart Island.

To determine whether the observed intra-site temporal genetic
differentiation revealed by the log-likelihood G tests gave evi-

TABLE 1.

Log-likelihood G test (dloudet el al. 1996) probability estimates for

inter-site population genetic differentiation al the time of socking

(August, 19981, in May, 1999, and at harvest in October, 1999.

Locus

Date

Mpi

Lap

Pgm

Gpi

Aug 98
May 99
Oct 99

^^^

***p<0.001

dence of selective survival patterns, we further examined the
dataset for (a) non-random mortality related to genotypic state
(e.g.. differential survival of homozygotes vs. heterozygotes) at
either of the five loci individually or over all loci combined; and
(b) non-random mortality favoring specific single locus genotypes.
With respect to (a), for all five loci individually, the relative pro-
portions of homo/ygotes vs. heterozygotes within all three sites
did not significantly change over the 14 month grow-out period
(Fisher's exact test; P > 0.05). Within-site variation in mean multi-
locus heterozygosity was also not significantly related to time
(sample date) and the interaction effect of time x site was not
significant (ANOVA; P > 0.05) at all three sites (Table 3). Thus,
we conclude the observed intra-site temporal variation in popula-
tion genetic structure revealed by the log-likelihood G tests is not
explained by selective survival related to genotypic state at either
of the five loci individually or on the basis of multi-locus het-
erozygosity (multi-locus homozygotes vs. heterozygotes). Multi-
locus heterozygosity did significantly vary however among sites
(ANOVA. P < 0.01; Table 3). Mean multi-locus heterozygosity
was lowest at Charles Arm. highest at Burnt Arm and intermediate
between these two at Thwart Island (Fig. 4). However, only the
May and October means at Burnt Arm and Charles Arm were
significantly different (Tukey. P < 0.05).

With respect to (b). we then tested the hypothesis that the
observed intra-site temporal genetic differentiation could be ex-
plained by non-random mortality favoring specific single locus
genotypes. To accomplish this, we tested for temporal patterns of
change in genotypic frequencies (x^) with specific individual al-
leles (homozygotes and heterozygotes combined) versus those
without at each site x locus combination wherein the log-
likelihood G test was significant. Homozygotes and heterozygotes
were combined for this analysis due to the previously noted non-
significant temporal change in frequencies of homozygotes and
heterozygotes at all three sites. With respect to the Lap, Pgm. and
Odh loci, the observed temporal changes in frequency of geno-
types with or without all individual alleles were not significant
after Bonferroni-adjustment of significance levels for multiple test
comparisons. For the Mpi locus, a significant G test score was
observed only at Burnt Arm (Table 2). Al this site, the relative
frequencies of genotypes which included the Mpi""' allele were
significantly reduced while the relative frequencies of genotypes
which incorporated the Mpi"''' allele were significantly increased
during the August. 1998, to October, 1999 period. These frequency
changes remained significant after BonfeiToni correction of prob-
abilities for multiple comparisons. Frequency changes at Burnt
Arm for genotypes with all other Mpi alleles were not significant
after Bonferroni correction of probabilities.

At the Gpi locus, x' tests of temporal patterns of change in
genotypic frequencies involving specific alleles varied across the
three sites (Fig. 5). At Burnt Arm. significant (x", P < 0.05) fre-
quency reductions occurred within genotypes which included the
Gpi"", Gpi"'-, and Gp/'"^ alleles, while genotypic frequencies with
— the Gp('\ Gp/^', Gpi^^, and Gpi" alleles all increased. At Charles
Arm. a significant reduction in individuals with genotypes that
qjIj ^11 included Gpi"" also occurred while genotypic frequencies with

Gpi"'", Gpi"", and Gpi*^" all increased. At Thwart Island, a trend
towards increasing frequency of individuals with Gpi''^ and G/x**'
occurred with reductions in the frequency of individuals with the
more electrophoretically mobile Gpi'"^ and Gpi'"\ Frequency
changes over time for all other Gpi alleles at each of the three sites

44

Penny and Hart

09

08

05

04

< 03

0 1

. 04

< 02

■ Burnt Arm
n Charles Arm
B Thwart Island

94 90

Mpi Allele

■ Burnt Arm
D Charles Arm
B Thwart Island

96
Lap

94
Allele

08

07

06

g 0.5
a>

3

S 04
«

M 03

<

0,2

0 1

0

0.4

035

03

^ 0.25

0}
3
IT

S 0.2

u.

a>

M 015
<

01
0 05

[J_

114' 111

■ Burnt Arm
DCharles Arm
H Thwart Island

Jk

106 100

Pgm Allele

■ Burnt Arm
DCharles Arm
H Thwart Island

107' 102 100

98 96 93 89 86 83'
Gpi Allele

*" ^ 1" —

■ Burnt Arm

DCharles Arm
1 BThwart Island

S »^ B-Ri -Tk,

09

08

07
u

£ 06

O"

S 05
u.

I 04

<

0.3

02
01

0

0' 1 2 3 4 5 6'

Odh Allele
Figure 2. Inttr-site comparison of allelt frequencit's at the Mpi. Pfiin. Uip. Gpi, and Odh enzyme loci in seed mussel populations at Burnt Arm,
Charles Arm, and Thwart Island at the start of the rearing trials in August, 1998. Allele frequency numhers with a ' (e.g., I'gm allelel 14' l include
frequencies of adjacent rare alleles.

were not significant (\^. P > 0.03) when significance levels were
Bonferroni-adjusted for nuiltiple comparison tests.

However, while the frequencies of the same individtial Gpi
alleles were not consistently significantly differentiated among
sites over time, the overall Gpi genotypic frequency distribution
significantly shifted in favor of electrophoretically slower alleles at
all three sites (Table 4). At the Gpi locus, mean genotypic rank
scores within all three sites significantly increased over time. Also.
Kruskal-Wallis H tests revealed the median genotypic rank score

also significantly increased within all three sites over time. These
results indicate a consistent directional change in the overall ge-
notypic frequency distribution had occurred at all three sites. Gpi
genotypes carrying alleles of higher electrophoretic mobility were
significantly reduced in favor of those with alleles of relatively
lower electrophoretic mobility at all three sites during the 14-
month grow-out period. We infer this directional shift in Gpi ge-
notypic frequencies which is consistent across all three sites is
evidence of genotype-dependent selective survival processes dif-

Genotype-Dependent Survival in Mussels

45

100

^--^

- Charles Arm

-Thwart Island
Burnt Arm

J

90

- - » -

— -A-

2 80

I

Survivorship

8 S

50

\ >■

40

\

August
1998

May
1999

October
1999

Figure, i. Comparative survival from sleeving in August, 1998, to harvest in October, 1999 at the three farm sites. Plotted points are means of
replicate socks with ±2 se.

ferentially favoring alleles of relatively lower electrophoretic mo-
bility at this locus during sub-tidal, rope culture of Mytihis spp. in
Newfoundland.

DISCUSSION

Two recent studies {Gilg & Hilbish 2000; Pedersen et al. 2000)
have provided evidence that both cohort niixuig as well as selec-
tive survival processes work to produce significant temporal ge-
netic heterogeneity in mixed-species, intertidal mytilid communi-
ties. However, ours is the first to examine the potential for similar
selection processes within suspended rope-cultured populations in
commercial culture. In the present work, we have found significant
within-site genetic heterogeneity among samples taken over a 14
month grow-out period for three mixed-species populations. The
probability that our results were affected by cohort mixing seems
remote. This would have required an undetected re-settlement on

TABLE 2.

l.og-likelihood G test ((loudet et al. 199A) probability estimates for

intra-site temporal genetic differentiation during the 14 month
grnwout period at all loci individually and over all loci combined.

the culture sleeves with a new larval cohort some time after the
original gear setup date in August. 1998. Such an occurrence
would have been easily detected due to the disparity in length
frequencies between the older mussels and the new cohort on the
sleeves. Our length frequency data show no evidence of such
cohort mixing. A more parsimonious explanation for the observed
intra-site differences among sample dates is that temporal genetic
heterogeneity occurred within the original populations from time
of sleeving in August. 1998. through to harvest size in October.
1999.

Both Gilg and Hilbish (2000) and Pedersen et al. (2000) re-
ported genotype-dependent mortality patterns in Mytilus spp.
populations that resulted in population structuring along species
lines. In the case of the former, an intertidal mixed population of
newly-settled M. edulis. M. galloprovincialis. and their hybrids in
SW England became heterogeneous on the basis of tidal height. In
the latter, a directional selective mortality pattern favoring M. tros-
siihis at the expense of M. edulis was demonstrated in a mixed
intertidal M. edulis and M. trossidiis assemblage in Nova Scotia. In
our study, the mussel populations at all three sites could be clas-
sified as hybridized, mixed-species (M. edulis, M. trossulus. and
their hybrids) stocks based on their Mpi genotypes (Varvio et al.

Interval
(months!

Loc

us

TABLE 3.

Analysis of variance (ANOVA) test results of
number of heterozygous loci (out of five) per ind

variation
ividual an

Site

Mpi

Lap

Pgm

Gpi

Odh

All

in the
nong the

Burnt Arm

three sites

and sample

dates. F ratios without su

perscripts

are not

9

**

n.s.

n.s.

*

n.s.

■*

significant tP >0().S|

14

9
14

*

n.s.
n.s.

n.s.

n.s.
n.s.

n.s.

*

***
***

*

***

***

***

Charles
Arm

Variable

d.f. MS

F

*

***

***

***

Date

2 0.62

0.56

Thwart

Site

2 6.31

5.72**

Island

9

n.s.

n.s.

n.s.

***

*

**

Date X Site

4 0.07

0.06

14

n.s.

*

n.s.

*+*

**

***

Residual

2652 1.05

* P < 0.05; *

*P<O.OI;

'** P <

0.001

** P< 0.01

46

Penny and Hart

Si

E

- - • -

-Thwart Island

--A-

■ Burnt Arm

August
1998

May
1999

October
1999

Figure 4. Mean multi-locus lieterozygositj (mean number of heterozygous loci out of fivel ±2 se in August 1998, May 1999. and October 1999
at each of the three farm sites.

1988; McDonald & Koehn 1991). All three populations showed
evidence of significant temporal genetic heterogeneity over the 14
months from sleeving to harvest. However, the relati\e proportions
of Mpi genotypes changed only at one site. Burnt Arm. This seems
to go against the general applicability of the hypothesis that ge-
notype-dependent mortality among Mpi genotypes (e.g.. M. ediilis,
M. troxsiilits. or their hybrids) might be the primary cause of the
temporal genetic heterogeneity we observed among cultured New-
foundland stocks as Pedersen et al. (2000) found u ithin one rocky
intertidal community in Nova Scotia.

Several factors may be contributory to the different conclusions
we have reached compared to Pedersen et al. (2000). Presumably,
selective processes favoring one genotype over another will only
manifest themselves under conditions that induce differential de-
grees of stress. Since both M. ecliilis and M. trossulus are endemic
throughout Atlantic Canada, we may infer that both species are
generallv adapted to the range of environmental conditions and
habitat types encountered throughout that range. However, it is
entirely likely that naturally occurring pelagic larval dispersal and
settlement will sometimes result in mussels being placed in habi-
tats where differential selection of genotypes can occur. The in-
tentional transfer of seed mussels to new areas, as in the case ot
commercial mussel farming, could likewise produce an environ-
ment where differential selection could occur. Thus the appearance
of differential selection processes would be site-specific, perhaps
even on micro-geographic scales, and may change with time on
any particular site as stressful conditions appear and disappear due
to natural environmental variability. The range of environmental
conditions experienced by rope-cultured, sub-tidal mussels is less
extreme and likely less stressful than those experienced by natural
inter-tidal assemblages and this may explain the different conclu-
sions we have reached compared to Pedersen (2000). Alterna-
tively, selection processes may also change with age (Gilg & Hil-
bish 2000). Our study was not directly comparable to that of
Pedersen et al. (2000) on the basis of age since we started our work

with seed mussels considerably older than the newly recruited
mussels they studied.

In our study, the temporal genetic patterns we observed may
have been produced not by mortality per se but rather by drop-off
from the culture gear. On vertically suspended sleeving substrates
used in commercial mussel culture, selective processes affecting
strength of attachment by byssal threads may result in drop-off.
While not sufficient to cause mortality per se. such episodes nev-
ertheless cause loss to the culture system and result in apparent if
not real mortality. Such selective processes may not be apparent in
intertidal habitat where mussels with similariy weakened byssal
attachment are physically better supported and thus better able to
retain their placement. We know that byssal thread formation var-
ies in mytilids in response to environmental variation (Young
1983). Strength of attachment also varies among species due to
interactions between hydrodynamic forces and interspecific varia-
tion in shell shape (Willis & Skibinski 1992: Bell & Gosline 1997).
Co-occurring cultured M. ediilis. M. trossulus and their hybrids
differ in shell shape within the populations we studied (Penney &
Hart 1999). Whether or not interactions among strength of attach-
ment, genotype-dependent physiological variability, and/or shell
shape contributes to the temporal genetic heterogeneity we ob-
served is the subject of continuing research efforts.

Previously, heterozygosity at individual loci or mean heterozy-
gosity over several loci have been implicated in selective survival
mechanisms among mytilid populations; (Koehn & Gaffney 1984,
Tremblay et al. 1998). A higher degree of mean multi-locus het-
erozygosity over several enzyme loci has been associated with
lower energy requirements for maintenance metabolism (Diehl et
al. 1986; Hawkins et al. 1986. 1989) thus imparting a differential
survival advantage to heterozygotes. In the present work, variable
survival in relation to heterozygosity, either at individual loci or as
a mean over all loci, was not a significant factor.

In our study, all enzyme loci did not contribute equally to the
observed temporal genetic heterogeneity. Only the Gpi locus pro-

Genotype-Dependent Survival in Mussels

47

s

g 30

i 20

■ August
a May
a October

H

uffl

h

iH

98 96 93

GPI Allele

■ August
DMay
D October

1

I

Mi

107 102 100 98 96 93 89 96 83

GPI Allele

■August

DMay
□ OctotDer

^iwm

Thwart Island

ly

m

Figure 5. Intra-site change in genotypic frequencies with various Gpi
alleles (homozjgotes and heterozygotes combined I from August, 1998
to October, 1999. Arrows indicate specific alleles for Hhich the x" test
for change in frequency o\ er time of those with the allele I homozygotes
and heterozygotes combined! vs. those svithout was significant after
Bonferroni adjustment of significance levels for multiple tests. Arrow
direction indicates an increase (upwards) or decrease (downwards) in
frequency over time. Alleles without arrows did not significantly
change in frequency over time. Allele frequency numbers with a " (eg.
Gpi allele 83') include frequencies of adjacent rare alleles.

duced significant temporal genetic heterogeneity witli any pattern
suggesting the presence of consistent selection processes. Tempo-
ral variation among eiectrophoretic variants at any locus implies
the existence of selective processes differentially imparting a sur-
vival advantage over time. This does not necessarily imply a direct
relationship between polymorphisms at a particular locus and sur-
vival. Variation at individual enzyme loci may simply serve as an
indirect marker of another linked genetic condition that is subject
to selective pressure (Beaumont et al. 1990). During our commer-
cial rearing trials at three sites in Newfoundland, the population

TABLE 4.

Mean genotypic rank scores, non-parametric analysis of variance of

ranked genotypic frequencies, and x' approximation of the

Kruskal-Wallis test for differences among median genotypic rank

scores within sites over time at the Gpi locus.

Kruskal-
Wallis
Mean Non-Parametric H (x'
Site Sample Date Rank F Score approx.)

Burnt .Arm

August. 1998
May. 1999
October. 1999

5,78
5.96
6,49

20.28***

37.04***

Charles Arm

August. 1998
May, 1999
October. 1999

5.4.^
5,72
5.73

3.30*

6.43*

Thwart Island

August. 1998
May, 1999
October, 1999

6.33
6.64
6.60

4.78**

12.09**

P < 0.0001 ; ** P <Q.0\: * P < 0.05

genotypic frequency distribution at the Gpi locus was shifted over
time in favor of electrophoretically slower alleles. We infer from
this frequency shift that selective survival processes favoring sur-
vival of Gpi genotypes incorporating relatively slower alleles exist
during sub-tidal rope culture of blue mussels in Newfoundland.
Although, this pattern of temporal change (high mobility alleles
reduced, low mobility alleles increased) was consistent among all
three sites, the same Gpi alleles were not always significantly
implicated across all three populations. We found no evidence that
any individual Gpi allele was consistently selected for or against at
all three sites during the grow-out period. With the current dataset.
we are unable to identify a causal mechanism that would ad-
equately explain these observed patterns. However, our failure to
detect a significant relationship between survival and the same
specific individual Gpi alleles which was consistent over all three
sites may be related to the widely different genetic population
structure at these three sites. All three sites had allele frequencies
significantly different from one another at all loci on all three
sample dates during the rearing trials. A different result may well
have occurred if the three populations had been genetically homo-
geneous initially. Until such time that a causal physiological
mechanism is found, our inference of a genotype-dependent selec-
tive relationship between Gpi polymorphisms and survival in sub-
tidal mussel culture based on directional shifts in genotypic fre-
quencies will remain contentious.

Se\eral previously published studies offer some insights which
may help identify a causal physiological mechanism for the ob-
served relationship between Gpi genotype and survival in sub-
tidal, rope culture. The Gpi locus exhibits a considerable degree of
polymorphism among most species of bivalve molluscs (Koehn et
al, 1976) including North American mussel populations (Mc-
Donald et al, 1991; Penney & Hart 1999). Latitudinal clines that
parallel thermal gradients have been observed at the Gpi locus in
a variety of marine finfish and invertebrate species (see Hall 19S.5)
suggesting a possible relationship between allelic variation at the
Gpi locus and temperature. Available evidence in support of this
possibility is inconclusive. In a series of laboratory experiments
with larval and juvenile mussels. Beaumont et al. (1988. 1990)
recorded significant genotype-dependent mortality at the Gpi lo-

48

Penny and Hart

cus. However, in neither case was this related to either temperature
or sahnity.

In contrast, the worl^ of Hall (1985) does provide some evi-
dence to support a potential relationship between Cpi and tem-
perature. Glucosephosphate isomerase (Cpi) acts in the glycolytic
pathway catalyzing the interconversion of fructose-6-phosphate
and glucose-6-phosphate (Hall 1985). In experiments involving
purified extracts of glucosephosphate isomerase derived from
Gpi'°" and Gpi'^'' genotypic individuals. Hall (1985) demonstrated
the two variants had similar catalytic activity at 5-1 0°C but the
electrophoretically faster G/)/'"" enzyme became more efficient at
higher temperatures between 15-25^C. In a similar experiment
involving the sea anemone. Merridiiim senile. Hoffmann (1984)
noted superior activity at higher temperatures for genotypes with
the electrophoretically faster of two Gpi alleles and that the popu-
lation variation in frequency of these two alleles exhibited a lati-
tudinal cline along the eastern North American coast with the
faster allele dominant in populations south of Cape Cod.

Both these studies suggest a possible selective advantage fa-
voring relatively faster Gpi alleles in high temperature environ-
ments and further suggest a possible explanation for the observed
decline in electrophoretically faster alleles in favor of slower ones
in our study. The seed mus.sels in our study are at least in part
derived from spawning of intertidal mussels which must regularly
encounter periods of high air temperatures in the 20-25"C range
during periods of emersion in summer. We may speculate that this
produces selective temporal adaptation among intertidal popula-
tions in favor of electrophoretically faster Cpi genotypes while
electrophoretically slower Gpi genotypes would be favored in sus-
pended subtidal culture where temperature maxima are much
lower (<20°C). Alternatively, the observed temporal variation at

the Gpi locus may be luiked to other factors unrelated to tempera-
ture such as selection for continuous immersion versus periodic
emersion, a factor which might provide a different genotype-
survival relationship in natural, intertidal populations versus those
in sub-tidal rope culture.

In summation, our work has established that temporal genetic
differentiation is apparently a common feature among rope-
cultured blue mussel populations in Newfoundland and a Gpi ge-
notype-dependent selective survival relationship significantly in-
fluences survival of Mytiliis spp. during sub-tidal rope culture.
This temporal genetic heterogeneity is likely a significant con-
tributory factor in the extensive geographic genetic population
structuring noted earlier among mixed M. echilis - M. trossidus
populations in Newfoundland (Penney & Hart 1999). Identifica-
tion of causal mechanisms controlling these temporal shifts in
genotypic frequencies may permit commercially motivated selec-
tion of seed stocks with enhanced survival probabilities for culture
operations. Commercial utilization of such seedstocks may help
boost farm production and profitability.

ACKNOWLEDGMENTS

We thank the owners and staff of Thimble Bay Farms Ltd.,
B&C Mussel Growers Ltd., and Noel Bros. Mussel Farms Ltd. for
their support and assistance maintaining experimental mussel gear
at their respective farm sites. Special thanks to Nadine Templeman
for her mifailing efforts in the laboratory conducting electropho-
retic assays. An earlier draft of this manuscript was much im-
proved by the helpful suggestions of Dale Parmiter and Geoff
Veinott, Fisheries and Oceans Canada, St, John's, Nfld.. and by
two anonymous reviewers.

LITERATURE CITED

Bates. J. A. & D. J. Innes. 1995. Genetic variation among populations of
Myliliis spp. in eastern Newfoundland. Men: Biol. 124:417— 124.

Bell. E. C. & J. M. Gosline. 1997. Strategies for life in tlow: tenacity,
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Jonmal of Shellfish Research. Vol. 21. No. 1, 51-57. 2002.

EFFECTS OF FOOD QUALITY AND QUANTITY ON FEEDING AND ABSORPTION IN

BLACK-RIBBED MUSSELS, SEPTIFER VIRGATUS (WIEGMANN) (BIVALVIA: MYTILIDAE)

DOMINATING WAVE-EXPOSED HABITATS IN HONG KONG

QIN-FENG GAO,' ZHENG-LIN WANG,' WAI-HING WONG,' AND SIU-GIN CHEUNG' *

^Department of Biology and Chemistry. Cit}- University of Hong Kong. Tat Chee Avenue, H(mg Kong
SAR, China; 'Department of Oceanography, The Ocean University of Qlngdao. Qlngdao, China;
^Department of Ecology and Evolution. State University of New York at Stony Brook. Stony Brook.
New York H 794-5245

ABSTRACT The black-ribbed mussel Septifer virgattis is an ecologically important species dominating exposed rocky shores in
tropical and subtropical areas of Asia. The present study investigates its feeding and digestive responses to vanations in food quantity
and composition under laboratory conditions. Individuals were exposed to either one of five rations composed of different percentages
of silt and the green microalga Dunaliella tertiolecta. Clearance rate (CR: 1 h"') was correlated weakly with both food quantity and
quality whereas rejection rate (RR: 1 h"') showed a strong positive correlation with total particulate matter (TPM: mg 1"'). with the
lowest threshold of TPM for pseudofaeces production estimated at 10 nig 1"'. Organic fraction in pseudofaeces was also significantly
lower than organic content in water (f). evidence of pre-ingestive selecfion. Absorption efficiency (AE) was a negative exponential
function of TPM and a negative quadratic function of organic ingestion rate (OIR). Gut passage time (GPT) showed a negative
relationship with food quantity and ingestion rate (IR: mg h"'). As food concentration increased (TPM). OPT of S. virgatiis remained
high when OIR was low (<0.4 mg h"'); further increases in OIR re.sulted in reductions of both GPT and AE. A prolonged GPT at low
OIR resulted in a rapid increase in AR from 0,1 to 0.3 mg h~'; further increases in OIR, however, did not cause a significant increase
in AR. The existence of the regulative mechanism facilitates maximum absorption in wave-exposed habitats characterized by low
seston concentrations.

KEY WORDS: Seplifer virgatiis. feeding, seston composition

INTRODUCTION

Both the concentration and composition of suspended particles
in intertidal seawater undergo large tluctuations as consequences
of the effects of tides, waves, currents, discharges of freshwater
from rivers, and temporal variations in the abundance of phy-
toplankton and zooplankton. The feeding behavior of suspension-
feeding organisms, therefore, is affected strongly by fluctuations in
the food environment. To compensate for fluctuations in food
availability, various mechanisms have been developed in bivalves
which include regulation of feeding rate, production of pseudo-
faeces, selection of nutritious particles, and alteration of digestive
efficiency (Hawkins et al. 1990; Bayne et al. 1993; Bacon et al.
1998). The relationships between suspension feeding and food
concentration and quality have been studied in temperate, but not,
generally, tropical bivalves. Such studies have provided insights
into the trophic roles of bivalves in temperate aquatic ecosystems
(Navarro et al. 1992; Hawkins et al. 1996) and identified optimum
food conditions for aquaculture purposes (Beiras et al. 1993; Na-
varro et al. 1996).

In contrast to the Green mussel Penui viridis which is exten-
sively cultured in southeast Asia and dominates sheltered and pol-
luted harbours in Hong Kong characterized by turbid waters, the
Black-ribbed mussel Septifer virgatus (Wiegmann) dominates
wave-exposed rocky shores in Hong Kong characterized by clean
water and low seston concentrations (Morton & Morton 1983;
Seed & Richardson 1999). It forms a conspicuous band appro.xi-
mately 1 .0 m wide in the eulittoral and the structural complexity of
the mussel bed provides refuge and suitable habitat for a wide
diversity of associated organisms (Ong Che & Morton 1992;
Seed & Brotohadikusumo 1994). The species matures at a shell

*Corresponding author.

length of approximately 15 mm, about I year after recruitment
and, like other local mytilids, 5. virgatus is dioecious with a bi-
modal pattern of spawning and recruitment in any one year (Mor-
ton 1995). Although it is an ecologically important species on
wave-exposed shores, far less is known about its ecology (Seed
& Richardson 1999) and feeding behaviour as compared with
that of temperate species such as Mylilus edulis and the tropical
and subtropical mussel Perna viridis (Hawkins et al. 1998; Wong
& Cheung 1999, 2001a,b). The present study is the first to in-
vestigate the feeding and digestive responses of S. virgatus to
variable food quantity and quality under laboratory conditions.

MATERIALS AND METHODS

Collection and Maintenance of Experimental Animals

In December 1999, individuals with shell lengths of between 38
and 42 mm were collected from the eulittoral population of 5f/;-
tifer virgatus at Cape D'Aguilar on the southeastern extremity of
Hong Kong Island. Hong Kong, and transported to the laboratory.
Individuals of this size range were used because this was the
dominant size group in the habitat. Epibionts on the shell of each
individual were removed, and the mussels acclimated to laboratory
conditions for 25 days before experimentation. During acclima-
tion, individuals were fed daily with the green microalga. Du-
naliella tertiolecta.

Preparation of Diet

Five diets with different quantities of sediments and the mi-
croalga Dunaliella tertiolecta were prepared. The sediments were
collected adjacent to the population of Septifer virgatus at the
study site and dried and ashed at 600^C to burn off organic matter
in the sediments. The residual matter was grounded up in a pestle,
sieved through a 37 p.ni sieve and mixed with D. tertiolecta, for

51

52

Gao et al.

which the organic matter content had been pre-deterniined. Diet
characteristics, including total particulate matter (TPM: mg I ' ),
particulate organic matter (POM: mg T'l. particulate inorganic
matter (PIM: mg I"') and organic content (f = POM/TPM), are
identified in Table 1 . The ranges of TPM and f used in the present
study were comparable to those reported for the study site where
TPM ranged from 4.5 to 36.0 mg P' with an annual mean of 4.5
mg r ' and f ranged from 0. 1 5 to 0.57 with an annual mean of 0.33
(unpublished data).

Experiimnlal Procedures

Each individual of Seplifer vir_i;ari(.\ was kept in a separate
beaker (250 ml) and supplied with one of the diets via continu-
ously-flowing seawater pumped from a reservoir by an 8-channel
peristaltic pump. A beaker without animals was considered as the
control. The experiment was replicated so that results of fourteen
mussels were obtained for each diet. The particles in the reservoir
were kept in suspension by aeration and stirring. A preliminary
experiment was done to detemiine the appropriate flow rate to be
used in this study. We have tested four flow rates (20, 40, 60, and
80 ml min"') with seven individuals each. Results showed that
clearance rate was significantly lower al 20 ml min"' whereas no
significantly difference was obtained among the other three flow
rates. A flow rate of 40 ml min"'. therefore, was used in this study
The reduction in particle concentration at this flow rate was found
to be less than 40%. This helped to ensure significant particle
reduction between inflow and outflow for accurate determination
of CR at the same time avoiding recirculation of water in the
beakers which would otherwise have resulted in underestimation
of CR (Hildreth & Crisp 1976; Hawkins et al. 2001). Seawater
samples were collected from a beaker without an experimental
mussel (i.e.. the control) at fixed time intervals of about 30 min.
The collected samples were filtered through ashed and pre-
weighed 25 mm GF/C filters and rinsed with isotonic ammonium
acetate solution. The filter papers were dried in an oven at 90°C.
weighed, ashed in a muffle furnace at 450'C and reweighed to
determine TPM (mg 1"'), POM (mg 1"' ). PIM (mg I"') and f values
for the filtered particles.

Before the start of the experiment, individuals of Seplifer vir-
gcitii.s were kept in filtered seawater to empty their guts for about
50 min which was the maximum evacuating time being obtained in
a preliminary experiment for a 40 mm individual. The maximum
evacuating time was determined by feeding the mussels with the
microalga Dunaliella tertiolecta for 24 h. They were then starved
in beakers containing flowing filtered seawater and pseudofaeces

TABLE 1.

Characteristics of experimental diets.

TPM

PIM

POM

Diet

(mg 1"')

(mg !"')

(mg 1"')

f (POM/TPM)

A

6.13 ±0.21

3.72±O.I2

2.41 ±0.11

0.39 ± 0.(J07

B

X.l()±().22

5.69 + 0.18

2.41 ±0.1!

0.30 ± 0.01 1

C

10.08 ±0.72

8.01 ±0.74

2.07 ±0.1 2

0.22 ±0.02

D

1().3.S± !.(.).>

9.28 ± 1 .00

1 .07 ± 0. 1 1

0.11 ±0.01

E

20.33 ± 1.28

14.34 ±0.06

5. 98 ± 0.46

0.30 ± 0.02

TPM: total paniculate matter: PIM: particulate inorganic matter: POM:
particulate organic matter; f: organic content of suspended matter. Values
are mean ± S.D.

and faeces produced were collected continuously. The time when
no more faeces and pseudofaeces produced was considered as the
maximum evacuating time which was estimated al 50 min. After
emptying the gut. all pseudofaeces and faeces produced in the first
hour were removed. Each individual was then exposed to an ex-
perimental diet for 150 to 180 min and faeces and pseudofaeces
were collected with caution during and at the end of the experi-
ment to prevent resuspension of faeces and pseudofaeces. After
food supply was stopped, the mussels were maintained in the
beakers for another 40 to 50 min until no more faeces and pseudo-
faeces were produced. The minimum gut passage tiine rather than
the mean gut passage time (Decho & Luoma 1991) was mea-
sured and defined as the difference in time between initial filter-
feeding by each individual and the first appearance of faeces dis-
regarding extracellular and intracellular digestion. The total, inor-
ganic and organic weight of pseudofaeces and faeces were
determined by the same methods as those described for seawater
samples. The following rates were then computed: total matter
rejection (RR; mg h"'l, organic matter rejection (ORR: mg h"'),
inorganic matter rejection (IRR: mg h~'), total egestion (defeca-
tion) (ER: ing h~'). organic matter egestion (OER: mg h"') and
inorganic matter egestion (lER: mg h"') (Hawkins et al. 1996).

Calculation of the Feeding Parameters

Food processing rates (feeding and absorption) were calculated
following Iglesias et al. (1992). Assuming that absorption of in-
organic matter through the digestive system was negligible (Cran-
ford & Grant 1990). the sum of IRR and lER was considered to
represent the rate of inorganic matter filtration (IFR; mg h~').
Clearance rate (CR; I h~'l was then estimated as CR = IFR/PIM.
Filtration rate of total particulate (FR: mg h"') was computed
as FR = CRxTPM and filtration rate of particulate organic matter
(OER: mg h"') as OER = CRxPOM. Ingestion rates of total
particulate matter (IR; mg h"') and of particulate organic matter
(OIR: mg h"') were estimated as IR = FR-RR and OIR = OFR-
ORR and hence, the organic content of absorbed food could be
estimated as AR (mg h~') = OIR-OER. and absorption efficiencv
(AE) = AR/OIR.

Preingestive selection efficiencies for total filtered organic mat-
ter (SEo) were estimated as: SEo = I - (p/f) (Navano et al. 1992).
where p is the organic content of the pseudofaeces and f is the
organic content of the suspended matter. In view of a very narrow
size range of animals we used, no attempt has been made to
weight-standardized the measured feeding rates.

Statistical Procedures

To obtain functional relationships between feeding responses
and food parameters, a set of regression equations was fitted to
experimental data, following standard least-squares procedures.
Regression analyses were performed by simple linear and non-
linear procedures, depending on the most appropriate function to
be fitted in each case (Zar 1999). Multiple regression analysis was
conducted when feeding behavior was correlated with more than
one diet parameters, and the coUinearity between the independents
was tested with coUinearity statistics of SPSS (Belsley et al. 1980;
SPSS Inc. 1999a). When the independent(s) were highly correlated
with others, they would be eliminated from the independent list
and the model was reconstructed until all the intercorrelations
between the independents were removed from the regressive
model. Residuals were also analyzed to check the normality, con-

Feeding Responses of Sept/fer virgatus

53

slant variance of predicted dependents and other necessary as-
sumptions of the regression model. Data were transformed if nec-
essary to meet the regressive requisites, i.e.. nomiajity of datum
distribution and homogeneity of variances.

Analysis of variance (ANOVA) was used for comparisons
among different treatment groups. Prior to analysis, raw data were
diagnosed for normality of distribution and homogeneity of vari-
ance with Kolmogorov-Smirnov test and Levene test, respectively.
The specific function and procedures followed for each feeding
response will be presented with the results. All the statistical pro-
cedures were performed with software SPSS for windows, release
9.0 (SPSS Inc. 1999a.b).

RESULTS

Figure 1. The relalicinship between filtration rates (FR: mg li 'l and
rejection rates (RR: nig h~'l in S. virgatus.

Clearance Rales and Filtration Rates

The relationship between CR and food availability was weak
but significant and can be described by the multiple regression
equation as:

CR = 0.18-1- 0.09/TPM -i- 0.13 ftr"
P < 0.001)

0.19. F,,,, = 6.56.

There was no significant relationship between FR and f. FR. how-
ever, was positively related to POM and the equation that describes
the relationship is:

FR = 0.79 -I- 0.57 POM (r- = 0.72, F, ^.^ = 185.57.
P< 0.001)

Pseudofaeces Rejection, Selection Efficiency and Ingestion of
Filtered Food

The organic content of particulate matter (f) was significantly
higher than that of the pseudofaeces for Diet C. D and E of which
pseudofaeces were produced (Table 2). As TPM increased, pseud-
ofaeces production increased with the increase in FR (Fig. 1 ) and
can be described by the equation:

RR = -0.439 -f 1.014 hi(FR) (r" = 0.84. F.^^ = 365.04.
P< 0.001).

There was a significant positive relationship between TPM and RR
(RR = 0.54 + 0.08 TPM (r- = 0.786. P < 0.001 )). No significant
relationship, however, could be established between RR and f.
showing that the rejection rate was related to the quantity (TPM)
but not the quality (f) of suspended matter. As pseudofaeces were
only produced for Diets C. D. and E. the lowest threshold of TPM
for pseudofaeces production was about 10 mg P'.

Selection efficiency (SEo) is a measure of the efficiency in
selecting the organic fraction of food when pseudofaces are being
produced. There was an inverse relationship between SEo and

TABLE 2.

Comparisons between the organic content of the diet (f) and that of

the pseudofaeces (e) for diet C, D and E where pseudofaeces

were produced.

f e

Diet (mean ± SD) (mean ± SD)

value

Degree of
Freedom

c

(J.22 ± 0.02

0.12 ±0.058

5.10

D

0.11 ±0.01

0.071 ±0.016

6.91

E

0.30 ± 0.02

0.199 ±0.068

5.56

8 <0.001

7 <0.001

13 <0.001

TPM. No significant relationship between SEo and f. however,
could be established. The corresponding equation is:

SEo = 0.77 -t- 1.51/TPM (r" = 0.49. F,

66.34. P< 0.001)

Ingestion rate (IR) is estimated as the difference between filtered
food and rejected pseudofaeces. Both IR and OIR were not sig-
nificantly correlated with f but IR was a positive power function of
TPM and OIR a linear function of POM which can be described by
the following equations:

IR = 1.02 TPM""* (r- = 0.82. F, „« = 328.1 1, P < 0.001)

OIR = 0.048 + 0.186 POM (r' = 0,91. F, ^g = 672.81.
P< 0.001)

The more the particles were filtered, the more they were in-
gested, resulting in a positive linear relationship between IR
and FR:

IR = 0.50 + 0.64 FR (r- = 0.94, F.^g = 1013.45, P < 0.001)
Absorption Rate and Absorption Efficiency

AR was a positive linear function of f (Fig. 2). As OIR in-
creased. AR increased rapidly until OIR reached about 0.4 mg h"'
(Fig. 3). The rate of increase of AR. however, was slower when
OIR increased further. The corresponding equations are:

AR = 0.09 + 0.85 f(r- = 0.63, F, ^^ = 126.27. P < 0.001)

AR = 0.45 - 0.05/OIR (r' = 0.74. F,

192.70. P< 0.001)

0.6
0.5

-~ 0.4
g 0.3

oi

0.2

0.1

0

0.1

0.2

0.3

0.4

0.S

Figure 2. Absorption rates (AR: mg h ') as a function of organic
content (f) of seston in S. virgatus.

54

Gao et al.

0.6

♦

0.5'

^ ♦♦

♦

^ 0-4'

^¥H 9f~

♦

♦♦ ♦

■= 0.3

/* ♦

♦

♦

OX)

E

/<•♦

♦

-S 0.2-

k

ck:

^

< 0.1-

f

0

0

0.4

0.8

1.2 1

OIR(mg

h^)

Figure 3. The relationship between absorption rates ( AR: ni;; h ' ) and
organic ingestion rales (OIR: nig h ') in S. rirgatiis.

AE decreased exponentially with an increase in TPM in the water
and was a negative quadratic function of both POM (Fig. 4) and
OIR (Fig. 5). The corresponding equations are:

AE = 0.962e-""-*''^™ (r- = 0.42. F, „, = 61.28. P < 0.01 1

AE = 0.726 + 0.037 POM - 0.017 POM' (r" = 0.73.

F,.

!.03. P < 0.001)

AE = 0.77 + 0.07 OIR - 0.38 OIR- (r" = 0.74.
F2.67 = '^6.36. P< 0.001)

Gut-Passage Time

Gut-passage time (GPT) for particles was a negative exponen-
tial function of TPM (Fig. 6) and a negative quadratic function of
OIR (Fig. 7). showing that the time the food particles stayed in the
gut was prolonged when OIR was low. GPT. however, was much
reduced when OIR increased. The respective equations are:

213.34. P< 0.001
= 0.42.

GPT = jv._i^ e u - vj. /u, 1 , ,,x

GPT = 21.28 + 49.07 OIR - 43 04 OIR' (r
F,„7 = 24.16. P< 0.001)

DISCUSSION

Rates of Clearance, Filtration and Pseudofaeces Production

Food availability is regarded as one of the most important
factors affecting the feeding behaviour of bivalves. To compensate

1 1
0.8
0.6

<

0.4

0.2

0

3 4

POM(mgr')

Figure 4. Absorption efficiency (AE) of .S. virgalus as a function of
particulate organic matter (POM: mg I"') in the water.

0.4

0.8
OIR(mgh"')

1.2

1.6

Figure 5. The relationship between absorption efficiency (.\E| and
organic ingestion rates (OIR: mg h') in .S. virgalus.

for fluctuations in lV)od availability, various mechanisms have been
de\ eloped which include regulation of feeding rate, pseudofaeces
production, selection of nutritious particles and alterations to di-
gestive efficiency (Hawkins et al. 1990: Bayne et al. 1993: Bacon
et al. 1998). As particle concentration increases, ingestion is com-
monly regulated by a reduction in clearance rate and the produc-
tion of pseudofaeces in mussels (Widdows et al. 1979). scallops
and clams (Navarro et al. 1992: Bacon et al. 1998). A similar
reduction in clearance rate with food availability was also recorded
for another locally dominant mussel, i.e.. Perna viridis. under both
laboratory (Wong & Cheung 1999) and field conditions (Hawk-
ins et al. 1998: Wong & Cheung 2001a.b). In contrast, the
correlation between clearance rate and food availability (TPM and
f) in Scptifer virgatits was weak, showing that ingestion of par-
ticulate organic matter was not regulated through clearance rate. In
M\tihis irussiiliis. CR was independent of increasing seston quality
at relatively high seston loads (20 and 50 mg T') (Arifin &
Bendell-Young 1997). Positive coiTelations between clearance
rates and the TPM of natural seston have been reported upon for
Mytilus edidis (Newell & Shumway 1993: Hawkins et al. 1996)
and Cerastodenna edide (Iglesias et al. 1992). CR of Crassostrea
gigas was maintained high and constant when TPM was 50 mg 1"'
but decreased with further increases (Barille & Prou 1994). Nev-
ertheless, all species should have an upper limit 10 the rate of
panicle processing (Bayne & Newell 1983). It was expected,
therefore, that the CR of 5. virgatiis would ultimately decrease
when TPM increased to a level higher than that experienced in the
present study.

With clearance rate relatively independent of food quantity,
filtration rate increases with POM, as shown here for Septifer

60
50

p 40
1 30

«5 20 ^
10

10

15

20

25

TPM (mg r')

Figure 6. Gut passage time (GPT: min) of S. virgatus as a function of
total particulate matter (TPM: mg I"') in the water.

Feeding Responses of Septifer v/rgatus

55

0 0.4 0.8 1.2 1.6

OIR(mgh"')

Figure 7. The relationship between gut passage time K;PT: min) and
organic ingestion rates (OIR: mg h"') in S. virgatiis.

virgaliis. Reports on the relationship between fihration rate and
food concentration, however, were variable. Winter (1973) showed
that the filtration rates of M. ediilis decreased with increasing food
concentration such that the bivalves could keep the number of
filtered algae in unit time relatively constant within the range of 10
X 10'' to 40 X 10'' cells I"'. Contrary results, however, were re-
ported by Winter (1978) and Riisgard and Randlov (1981), that is,
that filtration rates increased quickly with increasing particle con-
centration. Griffiths and Griffiths ( 1987) reviewed the relationship
between food quality and quantity and filtration rate of various
species of suspension-feeding bivalves and concluded that con-
flicting experimental results were largely attributed to differences
in particle concentration. At low concentrations, a positive rela-
tionship between filtration rate and food quantity was established.
When similar experiments were conducted at low to intermediate
particle concentrations, and the concentration ranges were rela-
tively narrow, no obvious relationship between filtration rate and
particle concentration was observed. In the present study, four out
of the five treatment groups had particle concentrations either
equal to or lower than 10 mg I"'. A reduction in filtration rate,
therefore, is expected at higher particle concentrations when CR
decreases.

Pseudofaeces production is regarded as one of the most Impor-
tant regulative processes in bivalves to organically enrich ingested
matter by the selective rejection of inorganic matter versus organic
matter and thus, as a consequence, to optimize energy uptake. The
production of pseudofaeces is strongly related to both the density
and organic content of suspended seston In the water (e.g., Foster-
Smith 1975; Bacon et al. 1998: Baker et al. 1998; Benlnger et al.
1999). Septifer virgatus started to produce pseudofaeces when
TPM was -10 mg P'; its production rate was also highly corre-
lated with TPM and FR. Ingestion rate, therefore, increased across
the full range of food availability. This agrees with the observa-
tions of Ki0rboe at al. (1980) and Hawkins et al. (1996) on Mytihis
edulis but is not consistent with those of Widdows et al. (1979)
who demonstrated that maximum IR coincided with the threshold
TPM above which pseudofaeces were produced. Hawkins et al.
(1996) anticipated that IR would reach maximal values in asso-
ciation with reduced CR and/or an Increase In the ratio RR/FR, as
observed for M. edulis feeding upon pure strains of cultured algae
(Foster-Smith 1975).

The SEo index represents the efficiency with which bivalves
can organically enrich ingested matter by selectively rejecting In-
organic matter via the palps. For both C. edide (Iglesias et al. 1992,

1996) and P. viridis (Wong & Cheung 1999), SEo was a curvi-
linear function of f whereas in another study on C. ediile, SEo was
positively related to f and negatively related to seston concentra-
tion (Navarro & Widdows 1997). In S. virgatus. SEo varied from
0.74 to 1.0 and was negatively correlated with TPM but not sig-
nificantly correlated with f. Such high values of SEo in S. virgatus
can probably be attributed to low seston concentrations and it Is
expected that SEo would decrease when seston concentration In-
creased beyond that experienced by the individuals used in this
study. A maximum selection efficiency of 0.60 was obtained for C.
edule when fed at low seston concentrations, and this decreased to
the lowest value of 0. 1 0 when seston concentration increased to
600 mg r' (Navarro & Widdows 1997). The SEo of Permi
viridis was comparable to S. virgatus, with a maximum efficiency
of 0.75 (Wong & Cheung 1999).

The present study demonstrates inter-specific differences In
pre-lngestive feeding behaviour between two mussels in Hong
Kong. S. virgatus Is a dominant species on exposed rocky shores
in the eastern waters of Hong Kong characterized by low TPM
values (Morton & Morton 1983). Seasonal variations in TPM at
Cape d'Aguilar, Hong Kong, where the animals used in the present
study were collected from, have been studied for one year (unpub-
lished data). TPM varied from 4.5 to 36 mg T' with an annual
mean of 9.5 mg 1"'. With the relative independence of clearance
rate from particle concentration, and positive relationships be-
tween RR and FR and between IR and FR, the ingestion rate of
organic particulate matter, therefore, could be enhanced under the
low food concentrations which prevail In this environment. In
contrast to S. virgatus. P. viridis occurs predominantly in sheltered
environments in which the water Is often turbid and sometimes
heavily polluted (Lee 1985; Cheung 1993). This species possesses
large labial palps with strong ciliary rejection tracts (Morton 1987;
Seed & Richardson 1999) to cope with the high sediment load-
ings often associated with sheltered, low energy habitats. Ingestion
of particulate organic matter by P. viridis was regulated through
pseudofaeces production, as herein demonstrated for S. virgatus.
although the rate was much higher at -9 mg h"' (Wong & Che-
ung 1999) as compared with S. virgatus of a similar size (-1.6 mg
h"'). Regulation was further enhanced by controlling clearance
rate which has not been observed in S. virgatus.

Absorption and Gut Passage Time

The absorption efficiency of suspension-feeding bivalves has
been shown to change over time scales according to variability In
seston characteristics (Bayne & Newell 1983) and is mainly con-
trolled by organic food quantity and/or quality when feeding on
either natural seston or on suspensions resembling natural condi-
tions (Bricelj & Malouf 1984). In the present study, the AE of S.
virgatus was negative quadratic functions of both POM and OIR.
Digestion in bivalves is biphasic involving the stomach and diges-
tive diverticula as compartments for extra- and intra-cellular di-
gestion, respectively (Purchon 1968). Food may undergo extra-
cellular digestion in the stomach followed by intestinal absorption
and is voided as "Intestinal faeces"". Some of the food may then
also undergo intra-cellular digestion and absorption within the di-
gesfive diverticula and eliminated as "glandular faeces'" (Bricelj
& Malouf 1984). With higher rates of food processing, the pro-
portion of particles by-passing the digestive diverticula would in-
crease and result In reductions in both GPT and AE (Navarro &
Iglesias 1993). In models combining gut passage time and feeding

56

Gao et al.

behaviour. Willows (1992) also showed that GPT generally de-
creased with an increase in the quality of food at high levels of
food availability. These observations agree with the present study,
i.e.. as food concentration increased (TPM). the GPT of S. virgatus
remained at high levels and was relatively constant when OIR was
low {<0.4 nig h"' ). Further increases in OIR resulted in a reduction
in GET. The high and constant values of AE under relatively low
OIR (up to 0.4 mg h"'), therefore, might be attributed to the
prolonged GPT and resulted in a rapid increase in AR from 0. i to
0.3. Further increases in OIR. however, did not cause significant
increases in AR. Similar observations were also reported upon for
Placopecten magellanicus (Brillant & MacDonald 2000) and
Aulacomya ater (Griffiths & King 1979) that AE of the latter
species decreased rapidly with increasing algal concentration and
AE approached zero when algal concentrations were >32 x 10''
cells r'. Using algal concentrations ranging from 50 to 100 x 10^
cells r' (equivalent to 1.25 - 2.5 mg 1"' POM). Gerdes (198.3).
however, found that AE of the Pacific Oyster. Crassostrea gigcis.
was independent of ration with AE of -75% being obtained for all
three kinds of diet with different TPM and POM. As the range of
POM used in our study on S. virgaius varied from 1 to 6 mg 1"' as
compared to a narrower range ( 1.25 - 2.5 mg 1"' ) used by Gerdes
( 1983). AE of C. gigas is expected to decrease, as what has been
observed in 5. virgatiis and other bivalves, when POM increases
further.

The ranges of seston quantity (TPM) and quality (f) used in this
study were 20 mg 1"' and 0.4, respectively, and within the ranges

recorded for the habitat where 5. virgciiiis was collected for this
study. AR reached asymptote when OIR was only one third that of
the highest value. This suggests that S. virgarns is particularly
well-adapted to a low seston concentration environinent. Food
quality was also an important factor controlling AR. For example,
AR is a positive quadratic function of POM in P. viriiiis (Wong
& Cheung 1999) and is positively linearly related to the different
measures of diet quality in Placopecten inagellanicus. even when
the organic content of seston (f) was as high as 80% (Cranford
1995). This agrees with the results obtained for 5. rirgatits in this
study.

The present study demonstrated regulative mechanisms in the
subtropical mussel S. virgatiis which is dominant on locally clean
and wave-exposed habitats characterized by low seston concentra-
tions. As food concentration increases, particulate organic matter
ingestion is enhanced through pseudofaeces production with pref-
erential ingestion of particulate organic matter, although clearance
rate is relatively independent of food quantity. GPT was a negative
function of TPM and OIR such that AE is relatively constant and
absorption rate reaches asymptote at low seston concentrations.

ACKNOWLEDGMENTS

The work described in this paper was substantially supported
by a grant from the Research Grants Council of the Hong Kong
Special Administrative Region. China (CityU Project No.
9040279). We thank Prof Brian Morton and two anonymous re-
viewers for constructive comments on this manuscript.

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COMPARATIVE SETTLEMENT DEPTHS OF MYTILUS EDULIS C. LINNAEUS, 1758 AND
M. TROSSULUS GOULD. 1850: I. A MESOCOSM STUDY

K. R. FREEMAN,' * E. KENCHINGTON,' AND S. P. MACQUARRIE"

^Department of Fisheries & Oceans, Bedford Institute of Oceanograpliy. P.O. Box 1006. Dartmouth,
Nova Scotia B2Y 4A2. Canada: 'Department of Biology. Dalhousie University, 1355 Oxford Street,
Halifax, Nova Scotia B3H 4J I, Canada

ABSTRACT Production on many commercial mussel farms in Eastern Canada is hampered hy the presence of Mytilus trossulus. a
comparatively thm-shelled but close relative of M. ediili.s. the principal species collected and grown. Exploitation of a species-based
difference in depth preference could potentially increase the collection of M. edulis at mixed species sites. As a first step in the
investigation, larval cultures of each species were reared to settlement in replicated mesocosms in a study conducted at Dalhousie
University's Aquatron facility in Halifax. Nova Scotia. Depth preference for settlement was examined with and without a thermocline.
Both M. edulis and M. trossulus had the largest percentage of larvae settling at the surface. Settlement occurred below the thermocline
at 6 m in both species, although in significantly lower proportions. In mixed water, a significant species-depth interaction was detected.
This was driven by a significantly large number of M. edulis spat settling at 8 m. a pattern not found in M. imssuliis-

KEY WORDS: Myiilus edulis. M. trossulus, settlement, depth, mesocosms

INTRODUCTION

Farm recruitment 'of mussels in Eastern Canada depends on the
timing and inter-relationship of a number of natural events coin-
bined with judiciously applied husbandry practices. The deploy-
ment of a suitable settlement medium, at an appropriate time, is
key to maintaining an uninterrupted cycle of spat collection, grow-
out, and harvest. Traditionally, spat collecting is performed on the
farm site, usually by deploying plastic mesh (Vexar^^") on head-
ropes near the surface several days before the anticipated onset of
settlement. This technique has proven successful over the rela-
tively short history of Eastern Canadian mussel farming. Never-
theless, spat collections at some farms comprise two species {Myti-
lus trossulus Gould, 1850 and M. edulis C. Linneaus 1758) and the
presence of the former is cause for concern to many growers
(Freeman 1996).

Although it was realized early in the industry that certain farms
often produced mussels of varying quality, it was not initially
appreciated, at least in certain cases, that the problem was species-
based. After this had been demonstrated (Koehn et al. 1984). in-
vestigations into distributions of the two species showed that East-
em Canadian Mytilus populations vary from purely one or the
other species to varying proportions of each (Penney & Hart 1999;
Mallet & Carver 1999). Although the two are often superficially
similar, M. trossulus is characterized by comparatively fragile
shells and generally lower ineat weight per shell length. For ex-
ample, it has been found that the initial number of M. trossulus
farmed would have to be multiplied 1 .7 times to achieve the same
economic return as with M. edulis alone (Mallet & Carver 1995).
Furthermore, anecdotal reports suggest that on some farms the
proportion of M. trossulus to M. edulis is gradually increasing
(Freeman 1996). Importation of pure M. edulis seed has been the
sole means producers have to overcome production shortcomings
associated with farm-collected M. trossulus. however, researchers
have been looking for other options. It had been believed, for
example, that comparison of early life events of both species might
suggest alternate husbandry strategies at sites with both species.

where growers inight wish to harvest only the more commercially
desirable M. edulis (Freeman 1996)

Examination of spawning times and duration of larval phases
revealed close synchrony between M. edulis and M. trossulus
(Freeman et al. 1994). indicating that any separation technique
employed as part of routine husbandry would have to be based on
some other criterion. Anecdotal references to different settlement
depths for each suggested that looking for larval depth variations
between the two would be an appropriate first step. It is presumed
that initial settlement would "preferentially" occur at a depth cho-
sen by the lar\ ae upon their attainment of competence, assuming
the larvae had immediate accessibility to a suitable substrate at that
depth. Following recent success of mesocosm studies of Pla-
copecten magellanicus larvae at the Dalhousie University Aqua-
tron in Halifax, Nova Scotia (e.g., Manuel et al. 1996; Gallagher et
al. 1996). additional experiments were perfonned at the same fa-
cility using laboratory-reared larvae of M. edulis and M. trossulus
(MacQuarrie 1995; Freeman & MacQuarrie 1999).

Here we present data on depth settlement preferences of both
A/, edulis and M. trossulus as observed in the laboratory. Settle-
ment behavior was examined in columns of mixed water, and
when a thermocline was present at 6 m.

METHODS

Broodstock Selection

*Corresponding author.

Naturally conditioned broodstock animals were obtained from
a commercial mussel farm near Lunenburg. Nova Scotia. The col-
lection was made in early June 1995. a time corresponding to peak
natural spawning at this site. Animals were selected on the basis of
apparent shell weight (a completely subjective judgment) com-
bined with shell length to shell height ratio derived from direct
measurements. Those animals with a shell length to shell height
ratio >2.2 and deemed 'light weight" for their length, were con-
sidered M. trossulus. whereas those animals with a ratio of <2.0
and deemed 'heavy" for their length were regarded as M. edulis.
Subsequent isoenzyme analysis in earlier work confirmed the ac-
curacy of this visual identification method at this site (Freeman et
al. 1994). Furthermore, a more recent shell analysis of mature.
DNA-identified mussels has indicated that the length-to-height

59

60

Freeman et al.

TABLE 1.
Chronology of mesocosm experiment. 1995. M. editlis lar\ae produced on June 13, and M. trossiiliis larvae produced on June 20.

Date & Age (days

1 of Mussels at

Interval Collector

Larval Age
at Placement

Larvae per
Mesocosm (xlO")

Species

Replicate

Placed

Deployment

Removal

M. edulis

El

June 18

.i days

1.43

July 7 (24)

July 31 (48)

M. edulis

E2

June 18

5 days

1.43

July 7 (24)

July 31 (48)

M. edulis

E3

June 18

5 days

1.43

July 7 (24)

July 31 (48)

M. edulis

NtE4

June 18

5 days

1.43

July 7 (24)

July 31 (48)

M. trossulus

Tl

June 22

2 days

1.60

Juh 11 (21)

July 31 (41)

M. trossulus

T2

June 22

2 days

1.60

JuK 11 (21)

Julv 31 (41)

M. trossulus

T3

June 22

2 days

1.70

Julv 11 (211

July 31 (41)

M. trossulus

NtT4

June 22

2 days

1.70

Jul) 11 (211

July 31 (41)

Nt

no thermocline mesocosm.

proportions, as a guide to species identification at Corl<ums Island,
remain valid. Greater assurance of species identification was at-
tained by selecting animals toward the extreme ends of the shell
length/shell height scale, which at this site, ranged from approxi-
mately 1.75 to 2.50. One hundred putative M. editlis and 92 M.
trossulus were chosen from several dozens of specimens exam-
ined. These animals were taken to the former Halifax Fisheries
Research Laboratory. Halifax. Nova Scotia.

Spawning and Fertilization

Animals were held in ultraviolet-irradiated, l-fjim filtered am-
bient teiT)perature (<10'C) seawater to encourage gut content re-
lease prior to spawning attempts. On spawning occasions, mussels
were brushed clean of epiphytic growth, wiped with dilute bleach
solution, rinsed in 1-jji.m filtered seawater. and then placed in 500-
mL containers, one animal per container. Seawater (20°C) was
added to each container and changed after the first 30 min if
spawning had not commenced; any mussel not spawning after this
first rewarming was discarded. Sperm-seawater mixtures from
several M. edulis males were combined in one labeled container,
and egg-seawater mixtures from several females of the same spe-
cies were combined in a second labeled container. To remove fecal
matter and other detritus, all the eggs were passed through a 100-
(jim filter, and the sperm mixtures were passed through a 40-|xm
filter. Gametes from M. trossulus were treated similarly. M. edulis
broodstock was spawned on June 13, and M. Irossulus on June 20,
1995.

Within species, pooled sperm was well mixed and 25 mL of the
mixture were added to the pooled eggs in a 20-L bucket and gently
mixed again. Egg-sperm mixtures were examined microscopically

to ensure that the sperm concentration was not excessive; further
sperm additions were made if concentrations appeared low. Two
hours following fertilization, a well-stirred sample of each batch of
eggs was examined micro.scopically and the percentage of fertil-
ization was estimated by noting the proportion of intact eggs to
embryos having attained at least the first division. Within 3 h of
fertilization, each batch of fertilized eggs was separated into seven
roughly equal lots, placed into seven 135-L plastic containers,
filled with treated (20°C, 1 |jim) seawater, and left standing. One
day later, the upper 80% of each container was carefully siphoned
off and retained. The final 207f containing detritus, unfertilized
eggs, and many substandard trochophores, was discarded. The
135-L containers were sanitized, the retained larvae replaced in
them, and refilled with treated seawater. At 48 h, the entire con-
tents of the 135-L tubs, now containing straight-hinge (veliger)
larvae, were siphoned through 40-|xm filters, and rinsed with, then
resuspended in treated seawater. At this stage, the M. trossulus
larvae were enumerated using a Coulter Counter, then equally
divided among four sanitized, covered, 20-L buckets and trans-
ported to the 10-m-deep tower tank at Dalhousie University (for a
complete description of the facility, see Gallagher et al. 1996). For
logistic reasons, M. edulis larvae were held for 5 days at the
Department of Fisheries Laboratory before final counting, separa-
tion into four lots, and being transported to the tower tank. While
being held at the Fisheries laboratory. M. edulis larvae were fed
Tahitian Isnclirysis daily, and the water was changed at 2 and 4 days.

Tower Tank Procedure

Each mesocosm was a 8.5-ni-deep by 0.6-m-diameter polyeth-
ylene cylinder sealed at the bottom, supported at the surface by a

TABLE 2.
Nested ANOVA of the transformed percentage of mussel spat settled at depth by species.

Source

DF

Sum of Squares

F Ratio

Probability
> F

Power

Sp. code

1

Depth

8

Sp. code X depth

8

Mesocosm |Sp, code]

4

Depth X mesocosm [Sp. code]

32

O.()0y075
I2.4378.'i()
0.364035
0.273,567
2.099770

0.2934

0.5903

0.083

50.2592

<0.0001

1.000

1.4710

0.1896

0.600

2.2109

0.0799

0.610

2.1212

0.0072

0.990

This analysis was based on dala from mcsocosms with no Ihermoclinc.

Mytilus Settlement in a Mesocosm

61

TABLE 3.
M. edulis: Comparisons for all pairs of depths (m) using Tukey-Kramer honestly significant difference (HSD) test.

Depth

0

2

3

1

4

5

6

8

7

II

-0.35

-0.02

0.03

0.04

0.08

0.16

0.57

0.59

0.83

2

-0.02

-0..^5

-0.30

-0.29

-0.24

-0.16

0.24

0.26

0.50

3

0.30

-0.30

-0.3.S

-0..34

-0.29

-0.21

0.19

0.21

0.45

1

0.04

-0.29

-0.34

-035

-0.31

-0.23

0.18

0.20

0.44

4

0.08

-0.24

-0.29

-0.31

-0.35

-0.27

0.13

0.16

0.39

5

0.16

-0.16

-0.21

-0.22

-0.27

-0.35

0.05

0.08

0.32

6

0.57

0.24

0.19

0.18

0.13

0.05

-0.35

-0.33

-O.OS

8

0.S9

0.26

0.21

0.20

0.16

O.OS

-0.33

-0.35

-0.11

7

0.83

0.50

0.45

0.44

0.40

0.32

-0.09

-0. 1 1

-0.35

Positive values in bold show pairs of means that are significantly different at a = 0.05.

A thermocline was present at 6 m.

Depths are ordered to position significantly different pairs together.

styrofoam floatation collar, and filled with 2.40 m' of 1.0-(jini
filtered water. Hot and cold water circulating in vertically adjust-
able piping circling the inside walls of the tank maintained a ther-
mocline, in this instance at 6 m. The temperature above the ther-
mocline was 19°C: below it 9°C. Of the M. edidis larvae. 1.43 x
1 0" were placed in each of four mesocosms suspended in the tower
tank. For M. trossuhis, two mesocosms were charged with 1 .65 x
10'' larvae and two others with 1.70 x 10*" larvae. One of the latter
M. twssulus and one M. edulis mesocosm was fitted with an outer
polyethylene bag containing water and an airlift system, which
kept the water stirred. This system eliminated the thermocline in
these two mesocosms, and held their internal temperatures at 12°C,
top to bottom. Logistic constraints eliminated the possibility of
replicating the nonthermocline mesocosms. A 12-h day/night pho-
toperiod established during the larval stages was continued to the
end of the settlement experiment. The feeding regime established
by the mid-larval period was also continued. Mixtures of Tahitian
hochysis and Chaetoceros gracilis were distributed throughout the
depths of each mesocosm by use of perforated hoses. Final con-
centrations of 5.0 X lO' cells/mL of each algal species were main-
tained to the end of the experiment. Algal counts were assessed
and adjusted every 2 days.

Collectors

Duplicate, preconditioned. 13-inm diameter. 25-cm lengths of
polypropylene rope were suspended by weighted monofilament
lines in all eight mesocosms. The portions of rope were positioned
more or less horizontally, at l-m depth intervals, from the surface
to 8.0 m. The use of fine-diameter, smooth-surfaced monofilament
line was intended to discourage newly settled spat from migrating
vertically. The collectors were installed in the M. edidis meso-
cosms when these larvae were 24 days old, and in the M. trossiilus
mesocosms when these larvae were 21 days old. In each case,
settlement began about 5 days after collector installation.

On July 31, the 25-cm segments of polypropylene rope from
these collectors with their attached spat were retrieved, placed in a
container of 80% ethyl alcohol, and labeled by species, replicate,
and depth. M. edidis collectors were removed at 48 days of age;
those of A/, trossiilus were removed at 41 days. Spat were subse-
quently separated from the rope segments with a light brushing,
then counts of the total number of spat per seginent were done
microscopically. The chronology of events is shown in Table I.

Data Treatment

Thermocline at 6 m

Spat counts from the collectors (two replicates for each meso-
cosm with a thermocline) were converted to percentages settled at
each depth per collector, log,,, transformed, then tested for nor-
mality using the Shapiro-Wilk W test. Normal Q-Q plots of the
residuals were visually inspected for deviations. For each factor,
homogeneity of variance was assessed with Levene's test. Data
were then subjected to analysis of variance (ANOVA). All analy-
ses were computed with JMP® version 3.1 software. The initial
analysis was designed to test the hypothesis that there was no
significant difference between species in settlement depth distri-
bution in the presence of a thermocline at 6 m. The mesocosms
were nested within each species. The model is expressed as

(0

a

(A

Si

E

3

z

■o
«

E

o

—

therrr

ocline

1.5-

i

-

\

-*-

T

1.0-

I"

S--

■*~^

0.5-

- A

^

i

-

-Y

—

0.0-

I

I

1

012345678
Depth (m)

Figure 1. Log||,-transformed percent number of M. edidis spat. Mean,
standard error, and standard deviation are indicated for each depth
(0-8 m). The mean across all depths Is also indicated (solid line). A
thermocline Has present at 6 m in this experiment.

62

Freeman et al.

TABLE 4.
M. trossitliis: Comparisons for all pairs of depths (m) using Tukey-Kramer honestly significant difference (HSD) test.

Depth

0

4

1

2

5

3

6

7

8

0

-0.46

0.00

(l.lll

0.02

0.07

0.21

0.53

0.59

0.67

4

0.00

-0.46

-0.45

-0.44

-0.39

-0.25

0.06

0.12

0.20

1

0.01

-0.45

-0.46

-0.45

-0.40

-0.26

0.06

0.12

0.20

2

am

-0.44

-0.45

-0.46

-0.41

-0.27

0.05

0.11

0.19

S

0.07

-0.39

-0.40

-0.41

-0.46

-0.32

-0.01

0.05

0.13

3

0.21

-0.25

-0.26

-0.27

-0.32

-0.46

-0.15

-0.09

-0.01

6

0.53

0.06

0.06

0.(15

-0.01

-0.15

-0.46

-0.40

-0.32

7

0.59

0.12

0.12

tl.ll

0.05

-0.09

-0.40

-0.46

-0.38

8

0.67

0.20

0.20

0.19

(Ui

-0.01

-0.32

-0.38

-0.46

Positive values show pairs ot means that are significantly different at a = 0.05.

A thermocline was present at 6 m.

Depths are ordered to position significantly different pairs together.

X,ju = (X + A, + B, + AB, + C(B)„„ + AC(B),„„ + e,„j,)

where A is depth with nine levels {0. 1. 2, 3. 4. 5. 6, 7. and iS in)
treated as an ordinal value, B is species with two levels (M. ediilis
and M. tros.siiliis). and C is mesocosm with three levels nested
within species. Depth was orthogonal to both factors.

Tukey-Kramer honestly significant difference (HSD) tests were
perfomied in post-hoc analyses of all ANOVA results. Multiple
comparisons of all pairs of means were performed. The signifi-
cance level (alpha) in the one-way ANOVA for all comparisons
was set at 0.05. The mean values of the log,o-transformed per-
centages were plotted for each depth and species along with de-
scriptive statistics.

No Thermocline

A second model was used to test the hypothesis of no differ-
ence in the percent of spat of each species settling al depth, in the
absence of a thermocline. Because there was only one mesocosm
per species, a two-way ANOVA model was used;

X„, = ix + A, + B, + AB„ + e,„

where A is depth with nine levels (0, 1, 2, 3. 4. 5, 6, 7, and 8 m),
B is species code with two levels (M. cthilis and M. irnssiilus). and
AB is the interaction term. The data were converted to percentage
settled at each depth per collector rope and log,,, transformed.
Normality, homogeneity of variance, and post-hoc tests were per-
formed as above.

RESULTS

Spawning and Fertilization

Of the putative M. edulis subjected to spawning inducement, 28
females and 27 males (55*"/^) spawned. Of the putative M. irossuhis
subjected to spawning inducement, 18 females plus 22 males
(43%) spawned. Two hours after batch fertilization, 84% of the M.
edulis eggs had progressed to at least first divisions and for A/.
trossiiliis. 86%. From past experience with broodstock frotii the
same farm, the percentage spawning and the subsequent progress
of fertilization and embryo development fell well within expecta-
tions. Approximately 5.72 x 10'" M. edulis larvae and 6.60 x lO''
M. trossulus 2-day-old veliger larvae were taken to the Dalhousie
University tower tank.

Analyses (if Spat Settlement, Thermoeline Present at 6 m

The transformed data produced a distribution that was not sig-
nificantly different from normal at P = 0.01 (Shapiro-Wilk W
test). Untransformed percentages did not meet the assumptions of
the tests. The variances were homogeneous within depth and spe-
cies according to Levene's test (P = 0.45 and 0.74, respectively).
Normal Q-Q plots of the residuals indicated that the residuals were
normally distributed.

The ANOVA model explained approximately 80% of the vari-
ance in the data set (adjusted R- = 0.81 ). Table 2 provides the test
results. The.se indicate that both species had significantly different
numbers of spat settling at different depths and that those distri-
butions differed according to mesocosm. The spat were not uni-
formly distributed by depth, however, no significant differences
between the species were observed.

(S
Q.
CO
(0

3

"3

(0

It)
o

^

.Q

E

■a

4)

E

(0

c

O)

o

thermocline

Depth (m)

Figure 2. Log|„-transformed percent number of M. trossulus spat.
Mean, standard error, and standard deviation are indicated for each
depth (0-8 m). The mean across all depths is also indicated (solid line).
A thermocline was present at 6 m in this experiment.

Mytilus Settlement in a Mesocosm

63

TABLE S.
Two-way ANOVA of the transformed percentage of spat settled at depth by species.

Source

DF

Sum of Squares

F Ratio

Probability > F

Power

Sp. Code

Depth

Sp. Code * Depth

1
8
8

0.007876
0.947826
0.609815

0.3184
4.7893
3.0814

0.5795
0.0028
0.0225

0.083
0.877
0.559

No thermocline was present.

Post-hoc analyses of the depth distributions of spat of each
species in the presence of a thermocline at 6 m depth were per-
formed. For M. ediilis, the number of spat settling at the surface
(depth 0 m) was significantly greater than those settling at any
other depth except 2 m. All collections taken above 6 m were
significantly greater than those settling below 6 m. where the
thermocline was positioned (Table 3). The mean values of the
transformed data are illustrated in Figure 1 for each depth. The
general pattern is seen quite clearly. These results are mirrored in
the depth settlement distribution of M. rmssuliis. however, the
significance of the pattern is not as strong (Table 4. Fig. 2). In this
species the surface samples (0 m) had significantly more spat than
any other depth, whereas the shallower depths generally had sig-
nificantly more spat than those below the 6 m thermocline.

Analyses of Spat Settlement, No Thermocline

The transformed data produced a distribution that was not sig-
nificantly different from normal (Shapiro-Wilk W test, P =
0.873). The variances were homogeneous within depth and species
according to Levene's test (P = 0.06 and 0.098, respectively).
Normal Q-Q plots of the residuals indicated that the residuals were
normally distributed. The results of this analysis are summarized in
Table 5. Once again, there is a significant effect of depth, but no
difference between the species. The interaction term is also sig-

nificant and appears to be influenced by the differences in the
means of each species below 6 m (see Figs. 3 and 4).

Post-hoc analyses were performed on the data. M. ediiUs had
large percentages of spat setting at the surface and also on the
deepest collectors (8 m). This suggests that the thermocline was a
barrier to settlement (Table 6, Fig. 3). because there was low
settlement below 6 in when the thermocline was present, but high
numbers when the water was mi.xed. However, the only signifi-
cantly different pair of depths was between 0 and 6 m (Table 6).
with a greater percentage of spat at the surface. In contrast. M.
irossidits did not show an increase in settlement with depth when
the thermocline was removed. The only significantly different
pairs of depths were between 0 and 8 m (Table 7. Fig. 4l. This
difference between the species at the 8-m depth accounts for the
significant interaction term in the ANOVA (Table 5).

CONCLUSIONS

Larval depth distributions of M. edults and M. trossidus have
been examined with a similar experimental design to that used in
our study (MacQuarrie 1995; Freeman & MacQuarrie 1999). Half-
way through the larval stage, vertical distributions over time in
larvae of both species were similar to each other, with major
concentrations found just above the 6 m thermocline. with slightly
lesser amounts at the surface, and lower numbers elsewhere. This

3

■o

"5

I

E

3

■o

«

E

c
m

?

Depth (m)

Figure 3. Log,,|-transformed percent number of A/, ediilis spat. Mean,
standard error, and standard deviation are plotted. The .sample mean
across all samples is illustrated (solid linel. No thermocline was present
in this experiment.

(0

I

a

o

n
E

3
Z

■o

4)

E

(0

c

o

Depth (m)

Figure 4. Log,„-transformed percent number of M. trossulus spat.
Mean, standard error, and standard deviation are plotted. The mean
across all samples is illustrated (solid line). No thermocline was present
in this experiment.

64

Freeman et al.

TABLE 6.
M. cdulis: Comparisons for all pairs of depths (m) using Tukcy-Kramcr honestly significant difference (HSD) test.

Depth

0

8

2

5

3

4

1

7

6

0

-0.56

-0,46

-0.36

-0.28

-0.26

-0.24

-0.20

-0.13

0.02

8

-0.46

-0.56

-0.46

-0.37

-0.35

-0.34

-0.30

-0.23

-0.08

2

-0.36

-0.46

-0.56

-0.47

-0.45

-0.43

-0.39

-0.32

-0.18

5

-0.28

-0.37

-0.47

-0.56

-0.53

-0.52

-0.48

-0.41

-0.26

3

-0.26

-0.35

-0.45

-0.53

-0.56

-0.54

-0.50

-0.43

-0.29

4

-0.24

-0.34

-0.43

-0.52

-0.54

-0.56

-0.52

-0.45

-0.30

1

-0.20

-0.30

-0.39

-0.48

-0.50

-0.52

-0.56

-0.49

-0.34

7

-0.13

-0.23

-0.32

-0.41

-0,43

-0.45

-0.49

-0.56

-0.41

6

0.02

-0.08

-0.18

-0.26

-0.29

-0.30

-0.34

-0.41

-0.56

Positive values in bold show pairs of means that are significantly different at a

No thermocline was present.

Depths are ordered to position significantly different pairs apart.

0.05.

clustering of liirvae at the thennocliiie was not surprising, because
the behavior has been noted in field studies of lamellibranch larvae
(e.g.. Southward & Barrett 1983; Scrope-Howe & Jones 1986). In
addition, M. rrossulus larvae lack a diel migration (MacQuarrie
1995), although Freeman and MacQuarrie (1999) report a modest
( 1.6 m) mean diel migration in 1 1 -day-old M. edulis larvae with a
thermocline present. However, these diel migrations are unlikely
to be statistically significant and are in contrast to observations
made on scallop larvae (Manuel et al. 1996).

Although thermocline accumulations of larvae decreased as the
veligers matui-ed, the settlement patterns, as reported here (Figs.
1^), are incongruous, becau,se it was expected that a reflection of
the earlier attraction to the thermocline would be clearly evident in
the distribution of spat which were harvested immediately upon
settling. M. eiliiUs and M. tiossuhis showed similar patterns of spat
distribution with depth in the presence of a thermocline. Spat of
both species settled below the thermocline. but at lower numbers
than above and the highest numbers were recorded at the surface.
In mixed water, M. edulis showed a strong preference for settle-
ment at depth (8 m). a feature not seen in M. trossidus. although
both had high numbers settling at the surface. Reasons for these
different patterns are currently speculative, but a recognized weak-
ness in the experiment was that we did not obtain genetic data to
support the status of broodstock used. This leaves open to question
whether these laboratory observations might have been affected by

species contamination. Nevertheless, in subsequent field experi-
mentation (see Kenchington et al. in this issue), DNA markers on
field-collected mussels support the observation of behavioral dif-
ferences by species seen in the mesocosm work.

The industry custom of deploying spat collectors at or near the
surface suggests that yields are better there, as was observed in this
study. The high degree of similarity in the distribution of the spat
of both species augurs little hope for growers wishing to prefer-
entially select M. edidis over M. trossidus at mixed species sites.
However, the significant species x depth interaction in the mixed
water offers some basis for further hypothesis testing. Further-
more, the coiTiplexity of natural conditions may provoke disparities
between the species that are not seen under the controlled condi-
tions of the tower tank.

ACKNOWLEDGMENTS

The authors are grateful for technical assistance provided by
Mr. Ron Duggan and Ms. Natalie Randall, and for the broodstock
animals supplied by Mr. Dale Cook, Corkums Island Mussel Farm,
Lunenbuig. Nova Scotia. Prof. Ron O'Dor, Dalhousie University,
provided funding and suppoil for this project through the Interim
Funding Research Program, an Atlantic Canada Opportunities
Agency-funded program in collaboration with Dr. E. Kenchington.
The authors thank Mr. Dale Roddick for his critical review of the
manuscript.

TABLE 7.
M. trnssulus: Comparisons for all pairs of depths (m) using Tukey-Kramer honestly significant difference (HSD) test.

Depth

0

1

3

4

2

6

7

5

8

0

-0.68

-0.55

-0.47

-0.33

-0.25

-0.11

-0.09

-0.06

0.12

1

-0,55

-0.68

-0.60

-0.46

-0.38

-0.24

-0.22

-0.19

-0.01

3

-0.47

-0.60

-0.68

-0.54

-0.46

-0.32

-0.30

-0.27

-0.09

4

-0.33

-0.46

-0.54

-0.68

-0.60

-0.46

-0.44

-0.41

-0.23

2

-0.25

-0.38

-0.46

-0.60

-0.68

-0.55

-0.52

-0.50

-0.31

6

-0.11

-0.24

-0.32

-0.46

-0.55

-0.68

-0.66

-0.63

-0.45

7

-0.09

-0.22

-0.30

-0.44

-0.52

-0.66

-0.68

-0.66

-0.47

5

-0.06

-0.19

-0.27

-0.41

-0.50

-0.63

-0.66

-0.68

-0.50

8

0.12

-0.01

-0.09

-0.23

-0.31

-0.45

-0.47

-0.50

-0.68

Positive values in bold show pairs of means that are significantly different at a = 0.05.

No thermocline was present in this experiment.

Depths are ordered to position significantly different pairs apart.

Mytilus Settlement in a Mesocosm

65

LITERATURE CITED

Freeman. K. R., K. L. Perry. T. G. DiBacco & D. J. Scarratt. 1994. Ob-
servations on two niytilid species from a Nova Scotian mussel farm.
Can. Tech. Rep. Fish. Aqua!. Sci. 1969:47 p.

Freeman. K. R. 1996. An examination of biological and other factors
affecting mussel aquaculture development in the Scotia-Fundy Region
of Nova Scotia. Can. Tech. Rep. Fish. Acjuat. Sci. 2125:32 p.

Freeman, K. R. & S. P. MacQuarrie. 1999. Reproduction and prc-
settlement behavior of Mylihi.t edulis and Mylihis trossiilus in con-
trolled environments: Implications for mussel culture in mi.xed-species
assemblages. Bull. Aquacult. Assoc. Can. 99: 1 7-2 1 .

Gallagher. S. M.. J. L. Manuel, D. A. Manning & R. O'Dor. 1996. Onto-
genic changes in the vertical distribution of giant scallop larvae. Plu-
cvpecten magellanicus. in 9-m deep mesocosms as a function of light,
food, and temperature stratification. Mar. Biol. 124:679-692.

Koehn, R. K.. J. G. Hall, D. J. Innes & A. J. Zera. 1984. Genetic differ-
entiation of Myiilus edulis in eastern North America. Mar. Biol. 79:
117-126.

MacQuarrie. S. P. 199.5. The vertical distribution and depth of settlement
of mussel larvae. Mytilus irossulus. in 8. .5 m deep mesocosms under
controlled conditions. Honors Thesis, Department of Biology, Dalhou-
sie University. Halifax. Nova Scotia. 55 p.

Mallet. A. L. & C. E. Carver. 1995. Comparative growth and survival

patterns of Mylihis Irossulus and Mytilus edulis in Atlantic Canada.

Can. J. Fish. Aquat. Sci. 52:1873-1880.
Mallet, A. L. & C. E. Carver. 1999. Maritime distribution and commercial

production performance of Mytilus edulis and Mytilus Irossulus. Bull.

Aiiuucul. Assoc. Can. 993:7-13.
Manuel, J. L.. S. M. Gallagher, C. M. Peaice, D. A. Manning & R. K.

O'Dor. 1996. Veligers from different populations of sea scallop Pla-

copecten magellanicus have different vertical migration patterns. Mar.

Ecol. Progr Ser. 142:147-163.
Penney. R. W. & M. J. Hart. 1999. Distribution, genetic structure, and

morphometry of Mylihis edulis and M. Irossulus within a mixed species

zone. / Shellfish Res. 1 8:367-374.

Scrope-Howe. S. & D. A. Jones. 1986. The vertical distribution of zoo-
plankton in the Western Irish Sea. Estuar. Coast. Shelf Sci. 22:785-
802.

Southward. A. J. & R. L. Barrett. 1983. Observations of the vertical dis-
tribution of zooplankton. including post-larval teleosts. off Plymouth in
the presence of a thermocline and a chlorophyll-dense layer. J. Plank-
ton Res. 5:599-618.

Journal of Slwllthh Racairh. Vol. 21, No. I, 67-73. 2002.

COMPARATIVE SETTLEMENT DEPTHS OF MYTILUS EDULIS C. LINNAEUS, 1758 AND M.
TROSSULUS GOULD, 1850: II. FIELD OBSERVATIONS

E. KENCHINGTON, K. R. FREEMAN, B. VERCAEMER, AND B. MACDONALD

Department of Fisheries & Oceans. Bedford Institute of Oceanoi;raphy. P.O. Bo.x 1006.
Dartmouth. Nova Scotia B2Y4A2. Canada

ABSTRACT Controlled niesocosm experiment', u^ing larvae of Mytilus ediilis and M. trossiilus have shown that the two species have
different settlement patterns with respect to depth, and that the presence of a thermocline can modify the depth distribution. A field
program was conducted over 3 y at one site, and at two sites during the final year to determine the settlement patterns of these species
in the wild. In all analyses, there was a greater percentage of M. edulis postlarvae settling at ? m than of M. irossulus. In addition to
opening up cenain ecological questions, these results show promise for direct application to husbandry practices at mussel farms having
both species, where preferential collection of the M. edtili.<: is desirable.

KEY WORDS: Mytilus ethilh. M. irossulus. settlement, depth, annual variability, spatial variation

INTRODUCTION

The relative depth at which Mytilus edulis C. Linnaeus, 1 758
and M. trossulus Gould, 1 850 settle is of direct concern to aqua-
culturists in Atlantic Canada, and elsewhere, most of whom de-
pend upon the collection of spat from the wild. Although similar in
morphology, the lower yield and propensity for shell breakage
observed in M. trossulus can cause significant production losses on
mixed species farms (Mallet & Carver 1995).

The possibility of using ecological infortnation (o preferentially
collect M. edulis postlarvae over M. trossulus has been explored
(e.g.. Freeman et al. 1994). Freeman et al. (2002) have shown that
in experiments conducted an indoor tank at Dalhousie University
(Halifax, Nova Scotia, Canada), the two species have different
depth preferences for settlement. Both species had high percent-
ages of postlarvae settling at the surface, however, in the absence
of a thermocline, M. edulis showed a strong preference for settling
at 8 m depth, which was not seen in M. trossulus. Should these
results be confirmed in the field, they offer a practical solution to
growers who have traditionally deployed surface collectors.

In designing our experiment, we were particularly concerned
with identifying patterns that were stable in time and space in order
to generate advice to the mussel industry. Although our field ex-
periments were limited in many ways, we were able to examine
postlarval settlement patterns in M. edulis and M. trossulus with
depth through the spawning season, across 3 y at one site, and
between sites in 1 y.

MATERIALS AND METHODS

Stud\ Sites

Two study sites were chosen to examine temporal and spatial
variability in the depth preferences of the mussel spat. One site was
located in St. Margarets Bay and the other in Ship Harbour, Nova
Scotia (Fig. 1 ). Both sites were associated with mussel farms that
collect, as pan of their operation, wild spat for culture, and were
known to have both M. edulis and M. trossulus. Although both
sites were located in coastal inlets, the oceanography of the two
sites is quite different.

Parrang Cove, St. Margarets Bay

St. Margarets Bay is situated approximately 40 km west of
Halifax, Nova Scotia. Our studv site was located near the head of

Figure 1. Location of sampling sites at Parrang Cove in St. Margarets
Bay and at Ship Harbour. Nova Scotia.

the bay at Parrang Cove (44'-37'N, 65°56'W) on the eastern shore-
line (Fig. 1 ). St. Margarets Bay is one of the larger bays along the
Nova Scotian Atlantic coast and has an axis length of 23.7 km, a
surface area of 138.0 knr, a total volume of 5191 x 10*' m\ and
an opening of the sea that is -5 km wide. The bay is subjected to
freshwater input from a watershed of 819.1 km"^ (Gregory et al.
1993). In addition to direct runoff, freshwater inputs come from
five rivers and the tidal/freshwater volume ratio is 416.32 (Gregory
et al. 1993). There is a large, deepwater basin of 80 m maximum
depth, and there is a sill at a depth of 35 m (Piatt et al. 1972).

As with all coastal inlets in Nova Scotia, the water in winter is
well mixed. A thermocline establishes in late spring or early sum-
mer between 4 m and 10 m, but in summer the warm surface layer
may extend to 30-36 m, depending an direction and strength of
winds (Piatt et al. 1972). The thermocline can be destroyed
through sustained SE or NW winds and can also be pushed off-
shore by the summer southwesterlies resulting in a cold upwelled
nearshore area.

67

68

Kenchington et al.

Ship Harbour

Located 90 km east of Halifax, the much smaller Ship Harbour
has an axis length of 8.3 km, a surface area of 6.6 knr. a total
volume of 47 X 1 0'' m\ and an opening to the sea at the mouth of
0.6 km. It is subjected to freshwater input from a watershed of
444.7 km" (Gregory et al. 1993), including two rivers and two
brooks. The tidal/freshwater volume ratio is 23.87 (Gregory et al.
1993). There is a small, deepwater basin of 27 m and an extensise
sill at 7 m.

As with other inlets along the coast, in Ship Harbour the water
column turns over in the fall, with a theriin)cline reestablishing
according to wind direction and velocity, the next spring. Ther-
moclines are strongest in the summer months, establishing around
5 m (Strain 2002). Within Ship Harbour, the study site was located
near the head of the harbour at 44 48.65'N, 62"30.53'W, along the
northeastern shoreline (Fig. 1).

Sampling Design

Mussel growers at each site reported selllemenl beginning in
mid- to late July and continuing through August and into Septem-
ber. Settlement very late in the year occasionally occurs (Decem-
ber), and may be attributed to either a second fall spawning or to
a protracted spawning period. In the first year of sampling ( 1998),
replicate collector ropes were deployed at Parrang Cove on July
24. These were removed on July 31 and replaced with a new pair
of collectors. Spat collection continued through the spawning sea-
son (Table 1). The schedule for the deployment and retrieval of
collectors is given in Table 1 . Sampling continued at Parrang Cove
at a reduced frequency in 1999 and 2000, according to the schedule
in Table 2. In 2000, replicate collectors were also placed at Ship
Harbour in order to examine geographic variation in the observed
setting pattern. These were deployed and retrieved on the same
dates as the Parrang Cove collectors (Table 2).

During 1998, a small experiment was run to determine whether
the active postlarval mussels would move along the collector rope
after settlement. Two collectors were left in the water from July 24
until October 16 (85 days) at Panang Cove, in order to compare
the depth distribution of older mussels with that of newly settled
spat.

Spat Collection

Spat were collected using 5-m lengths of 13-mm-diameter yel-
low polypropylene rope, weighted at the lower end and suspended
at each site from buoyed, surface headropes. Prior to deployment,
all collectors were soaked for 2 wk in running seawater to remove
any chemicals that might inhibit settlement.

As collectors were retrieved, they were cut into l-m lengths and

TABLE L

Deployment and retrieval dates of mussel spat collectors at Parrang
Cove, Nova Scotia in 1998.

TABLE 2.

Deployment and retrieval dates of mussel spat collectors at the two

experimental sites grouped according to three time periods used in

the ANOVAs.

Time

Deployment Dale

Retrieval Date

.Inly 24
July 31
August 7
September 4
September 1 8
October 2

July 31
August 7
August 21
September 18
October 2
October 16

Deployment

Retrieval

Time

Location

Year

Dale

Date

1

Parranc Cove

199S

July 24

July 31

1

1999

July 14

August 1 1

1

20()(J

July 18

August 15

1

Ship Harbour

2000

July 19

August 15

T

Parrang Cove

1998

August 7

August 21

">

1999

August 1 1

September 8

T

2000

August 15

September 12

T

Ship Harbour

2000

Au2ust 15

September 12

3

Parrang Cove

1998

September 4

September 18

3

1999

September 8

October 6

3

2000

September 12

October 10

3

Ship Harbour

2000

September 12

October 10

placed in polyethylene bags and sealed. Care was taken to avoid
cross-contamination of the sample segments. In the laboratory,
spat were preserved by adding SSCf ethanol to each bag. Subse-
quently, each l-m rope portion was removed and the attached spat
were separated from the rope by a gentle brushing, and then placed
in vials with 80% ethanol. Although mussels from all five l-m
lengths were saved, only those from 0-1, 2-3, and 4-5 m were
analyzed (hereafter referred to as 1, 3, and 5 m), with one excep-
tion. All five l-m rope segments were analyzed from the two
collectors which were deployed at Parrang Cove on July 24, 1998,
and left in the water until the fall. The number of spat at 1, 3, and
5 m were recorded for two time periods at each of two sites in
2000.

Species Determination

Approximately 30 individual spat from each sample depth rep-
licate/time/site were subsequently decanted and examined under
the microscope to ensure that the shell contained tissue. In some
cases, there were too few animals to meet the sample size objec-
tive, in which case all available animals were sampled. For the two
collectors left in the water for 85 days, only animals greater than
9 mm were analyzed in order to ensure sampling of animals from
the earliest settlement period.

Individuals were then separately picked, crushed, and rinsed
with high TE ( 1 M Tris 0.5 M ethylenediaminetetraacetic acid
[EDTA], pH 8) and ddH,0 three subsequent times (Heath et al.
1995). Tissue was resuspended directly in a 20.4-|j.L extraction
solution ( 19 |jiL H,0. 1 fjiL 10 x SFG buffer, 0.4 fjiL proteinase K)
and digested overnight at 37"C. The reaction was stopped by boil-
ing samples for 10 min before performing polymerase chain reac-
tion (PCR) amplification. The internal transcribed spacer (ITS)
region of the nuclear DNA was used as a species-specific marker
(Heath et al. 1995). ITS primers were purchased from Operon
Technologies, Inc. Twenty-two microliters of reaction mixture
containing I x PCR buffer, 2 niM MgCK, 0.5 [xM of each primer,
0.2 niM dNTP, and 1 .0 U Taq polymerase were added to 2 p.L of
DNA solution. The thermal cycling protocol was used as described
in Heath et al. (1995). PCR-amplified ITS fragments (approxi-
mately 1250 bp) were verified by running 8 |j.L of the amplifica-
tion product on XJr agarose gels. Successfully amplified ITS frag-

Mytilus Settlement in the Field

69

ments were digested with the endoiuieleiise Hhul (MBI Fernien-
tas). Ten mieroliters of digestion mixture containing l(!)x en/ynie
buffer and 5.0 U of Hhal was added to 13 (xl of amplification
product. The Myuhis species-specific restriction fragment length
polymorphisms (RFLPs) were then visualized on 2% agarose gels
and samples were identified as M. edidis. M. trossiilus. or hybrid.

Statistical Analyses

The percentage of M. echilis spat was determined for each of the
rope segments. Hybrids were rare and were not included in the
percentage calculations, Arcsin-transformed data were tested for
normality with the Kolmogorov-Smirnov (K-S) test. Levene's test
was used to test the homogeneity of the error variance. Normal
Q-Q plots of the residuals were visually inspected for deviations.
Transformed data were subject to analysis of variance (ANOVA).

A full factorial ANOVA was calculated froin the 1998 data
series collected at Parrang Cove (Table 1 ) with two factors. Time
(six consecutive sampling periods) and Depth (1. 3. and 5 m).
using a type III sums of squares. For the two collectors which were
left in the water froin July 24 through to October 16, a one-way
ANOVA with Depth as a factor (1, 2. 3. 4. and 5 m) was per-
formed. The results of these analyses inspired the subsequent field
prograin to determine the temporal and spatial stability of our
observations.

To determine whether there were annual variations in the spe-
cies-specific settlement patterns of Mytilns. a three-factor, full-
factorial ANOVA was calculated using type III sums of squares.
Specifically, the data collected from Parrang Cove (Table 2) were
analyzed to examine whether the percentage of M, edulis spat
varied in response to Year of collection (1998. 1999, 2000). Time
of collection during the spawning season (three periods of collec-
tion) and/or depth (1.3. and 3 m). Including the 1998 data created
a lack of independence between this analysis and the previous
ANOVA; however, excluding the 1998 results did not change the
significance of the terms and so the full analysis is presented here
for its greater interpretive value.

A similar three-way ANOVA was calculated to examine
whether the percentage of M. edulis spat varied in response to the
Site of collection (i.e., Parrang Cove or Ship Harbour). Time dur-
ing the season, and Depth (Table 2). Data for this analysis were
collected in only 1 y. 2000.

All interaction terms and main effects were tested, however, the
three-way interaction term in the last two analyses was nonsignif-
icant and so this term was removed from the equation and the
analyses were rerun, testing only main and two-way effects.
Tukey-Kramer honestly significant difference (HSD) pairwise
multiple comparison tests between each pair of means were per-
formed in post-hoc analyses of all ANOVA results with a signifi-
cance level of 0.05.

RESULTS

The 1 998 data from Parrang Cove were analyzed prior to the
1999 field season in order to determine the design of subsequent
fieldwork. The 1998 field season was designed to test the hypoth-
esis, with higher power, that there was no significant difference in
the percentage of M. edulis spat settling at different depths.

The arcsin-transformed percentage of M. edulis spat data dis-
tribution was not significantly different from a normal distribution
(K-S Z = 0.628; P = 0.825). The variances were homogeneous
and the residual plots supported a random distribution of the error

term. The two-way ANOVA model explained 71% of the variance
in the data set (adjusted R' = 0.71 1; F = 5.92; P = 0.00). There
was no significant interaction between Depth and Time, however,
the factors Depth and Time of collection were both highly signifi-
cant (Table 3).

Post-hoc analyses of the data using Tukey's HSD multiple
comparisons test revealed highly significant differences between
the transformed percentage of M. edulis spat settling at 1 m depth
and the percentage settling at 3 and 5 m {P = 0.004 and 0.000.
respectively). There was no significant difference between the per-
centage of M. edulis spat settling at 3 and 5 m (P = 0.102).
Examination of the mean values for each depth shows that there
are significantly fewer M. edulis at 1 m, or conversely, that there
are more M. irossulus. The nonsignificant interaction term indi-
cates that this pattern did not change during the course of the
setting season.

The percentage of M. edulis spat settling also showed a sig-
nificant difference with Time (Table 3). The post-hoc analyses
(Table 4) did not have any clear pattern in these differences other
than period 4 (deployed September 4th and retrieved on the 18th)
which had a significantly lower percentage of M. edulis across all
depths than most other sampling periods (except period 2). Al-
though no pattern of species-specific spat settlement was known to
exist during the setting season, a significant Time factor causing
higher percentages of one species over the other through the sea-
son can be explained by events such as differential spawning time,
larval periods, and/or current changes.

Did mussel spat change their position along the ropes when
allowed to stay in the water for longer periods'? As in the above
analysis, depth was a significant factor in the one-way ANOVA {F
= 11.95; P = 0.009; power 0.93). Post-hoc analyses using
Tukey's HSD test indicated that there was no significant difference
(a = 0.05) between the percentage of W. edulis at 1.2. 3. or 4 m.
but that significantly more M. edulis were found at 5 m than at
these depths (P = 0.032. 0.008. 0.14. and 0.47. respectively).
These animals were all over 9 mm in length, indicating that they
had settled on the collectors early in the season. Therefore, the
preference of M. edulis to settle at 5 m depth as observed in the
newly settled spat, persists through 83 days to at least 9 mm shell
length.

Annual Variation in Setting Pattern

The results from the 1998 program indicated that in the field.
M. edulis prefers to settle at 3 and 5 m depth, while M. trossulus
is dominant near the surface to 1 m. However, we felt that such
results could not be generalized until the annual stabilitv of the

TABLE 3.

Two-way ANOVA of the arcsin-transformed percentage of M. edulis

spat settling at Parrang Cove, Nova Scotia, by time (six periods

during set) and depth (I, 3, 5 m) with two-way interactions.

Observed

Factor

df

SS

F

P

Power"

Time

.s

1 .93

8.45

0.00

1. 00

Depth

T

1.64

18.02

0.(»

1.00

Time X Deptli

Id

I.OI

2.:i

0.72

(1.72

Residual

17

0.77.'i

■' Computed at a = 0.05.

70

Kenchington et al.

TABLE 4.

Signincant (a = (t.05) Tukev's HSD nuiltipir comparisons test in a

post-hoc analysis of the translbrnicd mean percentage of .1/. ediilis

spat settinj; at six different periods at Tarran;; Cove, No>a Scotia,

during the summer of 1998 (see Table 1 for dates).

(ll Set
Time

(J) Set
Time

Probabiiil\

0.034
0.003
0.008
0.045
0.034
0.045
0.002
0.004
0.003
0.002
0.008
0.004

pattern could be determined. Annual ditTerences in the percentage
of M. edidis spat setting at depth during three time periods in each
of 3 y were examined.

The three-factor ANOVA model explained TO'^i- of the \ ariance
in the data set (adjusted R- = 0.702: F = 7.66; P = 0.00) and
identified a significant intei'action between Year of collection and
Time of collection during the spawning season (Table 5). When
the adjusted mean values of the transformed data are plotted (Fig.
2), it can be seen that this interaction effect is due to a much higher
percentage of M. edulh sampled in 199S during the first collection
period (July 14— August 15) than in a similar time period in 1999
and 2000. The second time period (sampled between August 7 and
September 12) showed relatively high percentages of M. eduUs
each year, whereas in the third time period (September 4-October
10) the percentage of A/, edidh was very low in 199S but lemained
high in 1999 and 2000. These results indicate that the percentage
of M. edulis vs. M. rmssidiis spat varies through the season and is
not piedicted front year to year.

Depth was the only significant main effect in this analysis
(Table .'^). but was never significant in an interaction. Figuie 3

TABLE 5.

Three-Hay ANOVA of the arcsin-transformed percentage of M.

edulis spat settling al Parrang Co\e, Nova Scotia, by year (1998.

1999, 2U00), time (three periods during set), and depth (L 3, 5 m)

with tHo-«av interactions.

Observed

Factor

df

SS

F

P

Power"

Year

2

0.044

0.69

0.51

0. 1 6

Time

~i

0.181

2.S5

0.07

0.52

Depth

t

2.733

42.94

0.00

1.00

Year x Time

4

1.031

8. 10

0.00

0.99

Year x Depth

4

0.145

1,14

0.35

0.32

Time x Depth

4

0.254

1 .99

0.12

0.54

Residual

33

1.050

■' Conipuled at a

= 0.05.

3

0.9

0.8

0)
O)
CO

c

V

S 0.7

V

c

(/)
u

< 0.6

c

(C

u

0.5

•

» •

/

* \
/ \

-

1998

- - - - 1999

— - -2000

1

0.4

1 2 3

Time of Set (Consecutive Periods)

Figure 2. The interaction between year (1998, 1999, and 2(I(M)) with
sampling period in the transformed adjusted mean percentage of M.
edulis spat collected at Parrang Cove, St. Margarets Bay, Nova Scotia,

shows the strong change in the median value of the transformed
data with depth. Post-hoc analysis using Tukey's HSD identified
significant differences between all paiis of means of each depth,
with the greatest percentage of M. edulis spat settling at the 5 m
depth and the least at 1 m.

Geographic Differences

The above-described anaKses confirmed that the ohser\ations
first documented at Parrang Cove in 1998 were repeated in 1999
and 2000, with a greater percentage of M. edulis occurring at depth
(5 ml and a greater percentage of M. trossuhis occurring at the
surface ( 1 m). The final analysis w as designed to determine wheth-
er this pattern was particular to Parrang Cove, or whether it also
occuiTcd at other locations.

The three-factor .ANOVA model explained 66% of the variance
in the data set (adjusted R- = 0.660; F = 6.23; P = 0.00) and
identified a significant interaction between the Site of collection
and Time of collection during the season (Table 6). In all cases,
there is a greater percentage of M. edulis spat at Parrang Cove than
at Ship Harbour. However, in the first sampling period and in the
last (third) sampling period, the relative difference between the
sites is the same, whereas in the second period there are fewer M.
edulis postlarvae (and therefore more M. trossulus) at Ship Har-
bour relative to Parrang Cove (Fig. 4).

LInlike the previous ANOVA, all three main effects were sig-
nificant. The boxplots of the median values by depth and site are
illustrated in Figure 5. As in the previous analysis, there is sig-

Mytilus Settlement in the Field

71

a

a.
m

(0

I

0)

u

91
Q.

(0

o

Depth (m)

Figure 3. Boxplots (median bar, upper and lower quartiles. and outer
fences marking extent of data beyond the quartiles) of the arcsin-
transformed percentage of A/, edulis spat setthng at Parrang Cove, St.
Margarets Bay, Nova Scotia, at three depth intervals.

1 -
0.9 -

s

0.8 -

s
\ s
\ s

edulis spat)

o o

CD ~J

\ s

* \

V \

(Percentage M.

o o
*> en

• \
*\

\ *

\ ft

\ V

\ ft
\ ft

\ V

c

\

8 0.3-

- - - - Time Penod 1

\

\

<

— . —Time Period 3

\

0.2 -

\

0.1 -

\

0 -

■ 1 1

Parrang Cove Ship Harbour

Figure 4. The interaction between site with sampling period in the
transformed adjusted mean percentage of M. edulis spat collected at
the two locations in Nova Scotia in 2000.

nificantly more M. edulis spat settling at 5 m depth than at the
surface meter (Fig. 5). The third time period had the largest per-
centage of M. edulis spat across all sites and depths. Therefore, as
in the previous analysis, depth is an important factor in determin-
ing the percentage of M. edulis spat, regardless of site or time
period, or year of collection.

The total numbers of postlarvae at the 1, 3 and 5 m depth
intervals from a single collector rope from each site on two dates
are provided in Table 7 in order to place the percentage data in

perspective. As expected the total number of mussels collected was
highly variable and ranged from 134 to over 10,000 per metre. The
experimental design was not adequate to statistically analyze these
data and so additional counts were not completed. However, it can
be seen that large numbers of postlarvae did settle below the sur-
face and that the total numbers of M edulis collected at depth was
consistently higher than the number setting at the surface (Table
7). M. tnissulus was more variable being present in the highest
numbers at the surface in two collections, at 5 m in one and

TABLE 6.

Three-way ANOVA of the arcsin-transformed percentage of M.

edulis spat settling in 2000 by site (Parrang Cove, Ship Harbour),

time (three periods during set), and depth (1, 3. 5 ni) with

Iwo-wav interactions.

Observed

Factor

df

SS

F

P

Power"

Site

1

L55y

28.56

0.00

0.99

Time

2

0.747

6.84

0.01

0.88

Depth

2

1.199

10.98

0.00

0.98

Site X Time

2

0.434

3.98

0.03

0.65

Site X Depth

->

0.225

2.06

0.15

0.38

Time x Depth

4

0.256

1.17

0.35

0.31

Residual

.^6

17.853

° Computed at a

= 0.05.

ll

i

Depth (m) Parang Cove Ship Harbour

Figure 5. Boxplots (median bar, upper and lower quartiles. and outer
fences marking extent of data beyond the quartiles) of the arcsin-
transformed percentage of M. edulis spat sampled in 2000 at three
depth intervals (A), and at two sites in Nova Scotia, Canada (B).

72

Kenchington et al.

TABLE 7.

The absolute number of mussel postlanae per 1 m rope at I. 3 and 5 m depths collected at Parranye Cove and Sheet Harbour. Nova Scotia
at two sampling dates in 2tM)(l. The percentage of mussels Identified as M. ediilis usinj; DNA markers is indicated, as well as the estimated

numbers of M. edulis ( N ,

jnd I/. Irosiiiliis (N„ ,

postlarvae.

Date

Location

Depth

(m)

% M. edulis

^IM. cdiilh

Aug. 1?. 2(100

Parrang Cove

1

476

3 1 .3

3

IS4

61.9

5

367

60.7

Ship Harbour

1

569

6.7

3

831

10.7

5

6560

65.5

Ocl. 10. 2000

Parrang Cove

1

6030

71.9

3

10750

68.8

5

7470

80.0

Ship Harhiiur

1

741

15.2

3

134

64.7

5

140

100,0

149

114

223

38

89

4297

4336

7396

5976

113

87

140

327

70

144

531

742

2263

1694

3354

1494

628

47

0

homogenously distributed through the depth range in the fourth
sample (Table 7).

Hybrids

Of the 24X5 animals scored using the DNA technique. 8.4%
(208) were hybrids. The hybrids were not present in sufficiently
high numbers to permit a robust analysis of their distribution.
However, hybrids were relatively abundant in the October 2 rep-
licates sampled at Parrang Cove in 1 998 (Table 1 ). with the per-
centages ranging from 5% to 58%. In these two samples, the
greatest percentage of hybrids was at the surface.

DISCUSSION

The mixing of seawater due to tides, winds, currents, and up-
welling, and the seasonal establishment of thermoclines. charac-
terize inshore marine waters around Eastern Canada and in similar
temperate waters elsewhere in the world where Myiilus is found.
The larval period is therefore one of great uncertainty, where the
dynamic environment must be contended with while simulta-
neously searching for food and avoiding predators. Under these
conditions, it is remarkable that we were able to observe a con-
sistently and significantly higher percentage of M. edulis postlar-
vae at 5 m depth. This depth preference observed in the newly
settled spat persists through 85 days to at least 9 mm shell length,
despite the fact that postlarval mussels are very active and have an
ability to repeatedly attach themselves to various substrata prior to
choosing a final settlement location (e.g.. Bayne 1964; Dare &
Davies 1975). Further, the total number of M. edulis collected at
depth was greater than at the surface, to the extent that this was
examined.

Pineda (2(J0()) identified three processes affecting larval settle-
ment in invertebrates; ( 1 ) processes influencing the larval pool; (2)
physical transport; and (3) microhydrodynamics. substrate avail-
ability, and behavior. Larger-.scale processes governing physical
transport, and coupled to the onset and duration of spawning,
likely explain the variation in the relative percentage of each spe-
cies through the spawning season, across years, and between sites,
observed here and elsewhere (e.g.. Seed & Suchanek 1992). How-
ever, the significant difference in postlarval depth distribution be-
tween the species is consistent with a behavioral differences.

Freeman et al. (2002) observed the settlement of M edulis and
M. trossulus separately in the laboratory, under mixed water con-
ditions and with a thermocline. In all cases the greatest percentage
of postlarvae settled at the surface, however, under mixed water
conditions the postlarvae of M. edulis also had a large settlement
at 8 m. a pattern not seen in M. trossulus. These data support an
innate behavior as opposed to a response created through inter-
specific competition (e.g.. Petersen 1984). Furthermore, the lim-
ited data that we obtained on hybrids identified a settlement pattern
similar to M. trossulus. We were unable to determine which spe-
cies dammed these hybrids, however, the possibility of maternal
inheritance of setting depth is intriguing.

Mussel larvae are known to show behavioral responses to light,
gravity, pressure, and substratum (e.g.. Bayne 1976; Seed &
Suchanek 1992; Young 1995). To cite only a few examples: com-
petent larvae of M. edulis migrate to deeper water in the North Sea
(Bayne 1976) and prefer to settle subtidally below 3 m in the
Wadden Sea (Pulfrich & Ruth 1993), although Dobretsov and
Miron (2001 ) observed the opposite in the White Sea. with pedi-
veligers migrating toward the surface to set primarily at 1 .5 m. M.
galloprovincialis Lamark, 1819 is more homogeneously distrib-
uted with depth than M. edulis and M. trossulus (as observed in
this study). Hov\ever M. galloprovincialis shows greater settle-
ment at 5 and 10 m depth in northwest Spain (Caceres-Martinez &
Figueras 1998). reflecting the pattern seen in M. edulis. In addi-
tion, variation in settlement depth preference of mytilid species can
be influenced by the presence of phytoplankton and predators.
With the literature confounded by difficulties with species identi-
fication (cf. Johannesson et al. 1990). it is premature to determine
whether the observations reported here are representative of the
two species.

Miron et al. (1999) have suggested that competent barnacle
larvae position themselves at a depth in the water column corre-
sponding to the position where they might settle on shore. If this
were true for mussels, then photonegative and geopositive behav-
ior would ensure a greater depth with less exposure when setting
on a fixed substrate (e.g.. shore, pilings). Conversely, the photo-
positive and geonegative behavior hypothesized for M. trossulus
would ensure a distribution above mean low water spring tide
(MLWS). with greater exposure to desiccation, freshwater, and

Mytilus Settlement in the Field

73

temperature fluctuations associated with life in the upper intertidai
environment. This tolerance is consistent with the more euryhahne
nature of M. trossulus.

For aquaculturists, positioning mussel collectors on the bottom
or at 5 m depth would ensure the preferential collection of M.
edulis over M. trossulus. at least in Nova Scotian waters. This is in
contrast to the current practice of deploying plastic mesh on head-
ropes near the surface for several days before the anticipated onset
of settlement, a practice which may favor the collection of M.
trossulus and increase the relative proportion of this species on the
lease sites over time. The absolute numbers of spat were not re-
corded for all collectors and density is expected to be much more
variable because of large-scale processes and microhydrodynamic
conditions. However, in the collectors examined, placing the col-
lectors below the surface rarely resulted in reduced settlement,
although the total number of post larvae was highly variable with

respect to depth of collection. Nevertheless, we encourage mussel
growers to experiment with subsurface and bottom deployment of
collectors at sites where both M. edulis and M. trossulus occur.

ACKNOWLEDGMENTS

We thank Yonghong Shi, Melissa Cox, Angela Glass, Amy
Chisholm. Shawn Roach, Amanda Ginnish. and Liqin Cao for their
assistance in the laboratory; Amy Thompson for her help in the
field. We thank Dr. Gareth Harding (Bedford Institute of Ocean-
ography. Dartmouth, N.S.) for reviewing this manuscript. This
work could not have been done without the co-operation and as-
sistance of the mussel growers. Mr. Joe Levy {Parrang Cove), and
Mr. John Stairs (Ship Harbour). This project was supported by the
Department of Fisheries and Oceans, Canada, the Human Re-
sources Canada Science and Technology Youth Internship pro-
gram, and an NSERC research grant to EK.

LITERATURE CITED

Bayne. B. L. 1964. Primary and secondary settlement in Mytilus edulis L.

(Mollusca). J. Aiiim. Ecol. 33:513-523.
Bayne, B. L. 1976. The biology of mussel larvae. In: B. L. Bayne, editor.

Marine mussels: Their ecology and physiology. Cambridge University

Press: London, pp. 81-120.
Caceres-Martinez, J. & A. Figueras. 1998. Mussel (Mytilus galloprovin-

cialis Lamarck) colonization on artificial substrates in the Ria de Vigo

of NW Spain. J. Shellfish Res. 17:153-157.
Dare. P. J. & G. Davies. 1975. Experimental suspended culture of mussels

(Mytilus edulis L.) in Wales using spat transplanted from a distant

settlement ground. Aquaculture 6:257-274.
Dobretsov. S. V. & G. Miron. 2001. Larval and post-larval vertical distri-
bution of the mussel Mytilus edulis in the White Sea. Mar. Ecol. Prog.

Sen 218:179-187.
Freeman, K. R.. K. L. Perry. T. G. DiBacco & D. J. Scariatt. 1994. Ob.ser-

vations on two mytilid species from a Nova Scotian mussel farm.

Canadian Technical Repeport in Fisheries and Aquatic Sciences

1969:47 pp.
Freeman. K. R., E. Kenchington & S. Macquarrie. 2002. Comparative

settlement depths of Mytilus edulis C. Linnaeus, 1758 and M. trossulus

Gould. 1850: I. Laboratory Observations. J. Shellfish Res. 21:000-000.
Gregory. D.. B. Petrie. F. Jordan & P. Langille. 1993. Oceanographic.

geographic, and hydrological parameters of Scotia-Fundy and southern

Gulf of St. Lawerence inlets. Canadian Technical Report in Fisheries

and Aquatic Sciences 143:248 pp.
Heath, D. D., D. R. Hatcher & T J. Hilbish. 1996. Ecological interaction

between sympatric Mytilus species on the west coast of Canada inves-
tigated using PCR markers. Moi Ecol. 5:443-447.
Johannesson, K., N. Kautsky & M. Tedengren. 1990. Genotypic and phe-

notypic differences between Baltic and North Sea populations of Myti-
lus edulis evaluated through reciprocal transplantations. II. Genetic
variation. Mar. Ecol. Prog. Sen 59:211-219.

Mallet, A. L. & C. E. Carver. 1995. Comparative growth and survival
patterns of Mytilus trossulus and Mytilus edulis in Atlantic Canada.
Can. J. Fish. Aquat. Sci. 52:1873-1880.

Miron. G., L. J. Walters, R. Tremblay & E. Bourget. 1999. Intertidai bar-
nacle distribution: A case study using multiple working hypotheses.
Mar Ecol. Prog. Ser 197:205-219.

Petersen, J. H. 1984. Larval settlement behaviour in competing species:
Mytilus califimuanus Conrad and M. edulis L. J. Exp. Mar. Biol. Ecol.
82:147-159.

Pineda. J. 2000. Linking larval settlement to larval transport: Assumptions,
potentials, and pitfalls. Oceanogr. Eastern Pacific 1:84—105.

Piatt. T.. A. Prakash & B. Irwin. 1972. Phytoplankton nutrients and flush-
ing of inlets on the coast of Nova Scotia. Naturaliste Can. 99:253-261.

Pulfrich. A. & M. Ruth. 1993. Methods for monitoring the spatfall of
mussels [Mytilus edulis L.) in the Schleswig-Holstein Wadden Sea.
ICES 199SCM 1993/K:42.

Seed, R. & T. H. Suchanek. 1992. Population and community ecology of
Mytilus. In: E. Gosling, editor. The mussel mytilus: Ecology, physiol-
ogy, genetics and culture. Developments in aquaculture and fisheries
science, vol. 25. New York: Elsevier, pp. 87-169.

Strain. P. M. 2002. Nutrient dynamics in Ship Harbour. Nova Scotia. Al-
njosphere-Ocean 40:in press.

Young, C. M. 1995. Behavior and locomotion during the dispersal phase of
larval life. In: L. McEdward, editor. Ecology of marine invertebrate
larvae. New York: CRC Press, pp. 249-278.

Joiirihil ,:t Shellfish R<'sc,inh. Vol. 21. No. 1. 75-79. 2(102.

ROUTES OF INTRODUCTION OF THE MEDITERRANEAN MUSSEL (MYTILUS
GALLOPROVINCIALIS) TO PUGET SOUND AND HOOD CANAL

ANTHONY S. ANDERSON, A. LELANIA BILODEAU,* MATTHEW R. GILG, AND
THOMAS J. HILBISH**

Dcpartmcnl oj Biological Sciences, University of South Carolina. Cohunhia. South Carolina 29208

ABSTRACT To test alternative routes of introduction we describe the distribution o( Myliliis galloiymvincialis. M. Irossidus, and their
hybrids in Puget Sound and Hood Canal. Native mussels. Myiihis imssniiis. dominate the blue mussel communities of Puget Sound
and Hood Canal; at most sites M. i^idloproyincialis alleles were rare or absent. M. galloproYuicialis alleles were present but uncommon
(-.')%) in mussel populations in the southern portion of Puget Sound, and were nearly absent in populations in the northern Puget Sound
and the Hood Canal. The only locations where M. gallopiovincicilis alleles are locally common are sites where they are likely to have
been repeatedly introduced. These include sites near mussel farming operations and near the Port of Seattle and Bremerton Naval
Shipyards. The results indicate that both aquaculture and shipping activities have been responsible for the repeated introduction of M.
aulloprovincialis to Puget Sound. Although hybridization between M. galloprovincialis and M. trossulus occurs, there was little
evidence for advanced introgression between these two species.

KEY WORDS: introductions, mussels. Myiiliis gtilloprovincialis

INTRODUCTION

The invasion of nonindigenous species has become of ecologi-
cal and economic concern in the last few decades (Schiiiit/. &
Simberloff 1997). The effects of species introduction range from
localized and limited to those that severely degrade native ecosys-
tems and have immense economic consequences (Geller et al.
1994; Ruiz et al. 1997; Ruiz, et al. 20001. The unpredictable eco-
logical consequence of introducing nonnative species makes such
introductions generally undesirable.

Global commerce is responsible for the majority of both inten-
tional and unintentional species introductions. In marine systems a
major route of species introduction has been through the transport
and discharge of ballast water from ships (Geller et al. 1994; Ruiz
et al, 1997; Ruiz et al, 2000), For example, Carlton and Geller
(1993) found that the ballast water from 159 cargo ships in Coos
Bay, Oregon, contained a minimum of 367 marine species, A
second, potentially major, source of species introductions is aqua-
culture endeavors, particularly fartiis and fisheries. The decline of
traditional fisheries has lead to a rapid increase in aquaculture as a
source of aquatic products (Johnson 1998); presently 20% of the
world's seafood conies from aquaculture. The cotnmercial culture
of aquatic organisms has led to the intentional and accidental in-
troduction of nonnative species in many parts of the world (Bartley
& Subasinghe 1996).

One marine species that has been both intentionally and acci-
dentally introduced by these activities is the tiiussel Mytihis fial-
loprovincialis. This mussel has been unintentionally introduced to
South Africa (Grant & Cherry 1985), the Sea of Japan (Wilkins et
al, 1983), and California (McDonald & Koehn 1988), Larvae of M,
galloprovincialis are commonly found in ballast water of transport
ships (Geller et al, 1994), making unintentional introductions
likely, M. galloprovincialis is also the subject of large aquaculture
farms in Europe and has been intentionally introduced to Puget

♦Present address: USDA-ARS. Catfish Genetics Research Unit, 127 Ex-
periment Station Rd., Stoneville, MS .^8776.
**CorTesponding author. Fax: (803) 777-4002. E-mail: hilbish@biol.sc,edu

Sound and the Georgia Straits as pail of culture efforts in the
northeastern Pacific (Heath et al. 1995).

Mytihis galloprovincialis is a member of a complex containing
two other species of blue mussel, M. ediilis and M. trossulus. M.
galloprovincialis is native to the Mediterranean and the Atlantic
coast of southern Europe, M. eduhs is native to temperate Atlantic
waters of Europe and North America, and M. trossulus is native to
cold-temperate waters of the northern Pacific and Atlantic (Gos-
ling 1992). M. galloprovincialis is the most warm-adapted of the
three species (Hilbish et al, 1994), which perhaps contributes to its
propensity for introduction. All members of the blue mussel spe-
cies complex will hybridize in nature. In particular, extensive hy-
bridization between M. galloprovincialis and M. trossulus occurs
in California (Rawson et al. 1999) and between M. galloprovin-
cialis and M. edulis in Europe (Skibinski et al. 1983). The capacity
to interbreed with congeneric species expands the possible conse-
quences of introducing non-native species; they may genetically
contaminate native species as well as ecologically displace them.
The fate of hybrids, however, is uncertain. In Europe hybridization
between M. galloprovincialis and M. edulis is extensive resulting
in high levels of introgression between the species (Quesada et al.
1998; Rawson & Hilbish 1998). In contrast, hybridization between
M. galloprovincialis and M. trossulus is prevalent, but results in
very limited introgression (Rawson et al. 1999).

This study describes the distribution of Mytihis galloprovincia-
lis. M. trossulus. and their hybrids in Puget Sound. M. gallopro-
vincialis has been intentionally introduced to Puget Sound through
aquaculture efforts, but also may have been repeatedly introduced
via shipping activities. If M. galloprovincialis is present in Puget
Sound as a result of shipping activities, we expect high concen-
trations of M. galloprovincialis alleles to be present around inajor
shipping ports, particularly in the vicinity of Seattle, Alternatively,
if aquaculture activities are responsible for the presence of M.
galloprovincialis. we expect to find alleles indicative of this spe-
cies in the vicinity of culture operations. In this study we also have
used molecular markers that distinguish M. galloprovincialis and
M. trossulus to determine whether genetic contamination of native
mussels is occurring in Puget Sound and to infer the possible fate
of hybrid mussels.

75

76

Anderson et al.

METHODS AND MATERIALS

Mussels were collected in the summer of 1997 and 1998 from
28 locations in Puget Sound and Juan de Fucu Strait and five
locations in the Hood Canal (Fig. 1 ).

Shell length of each mussel was measured from the anterior to
posterior margin to the nearest 0.1 mm. We attempted to analyze
both small and large mussels from each location, but this was not
always possible because the size structure of the mussels varied
widely among sites. We analyzed between 16 and 64 mussels from
each location. Each mussel was dissected and a section from the
margin of the mantle was removed and preserved in 95'7f ethanol.
Total cellular DNA was extracted from each tissue sample as
described by Rawson et al. ( 1999). The Glu-5' gene was amplified
using the polymerase chain reaction (PCR) as described by Raw-
son et al. (1996) using the primers of Inoue et al. (1995). PCR
amplification of the Glu-5' marker produces species-specific prod-

iM^?M^

Figure 1. Location ol'saniplin)> sites in Puget Sound and Strait of Juan
de Fuca: Beliingham Bay = BH, Anacortes = AC, Deception Pass
Marina= DP, Oak Harbor = OH. Keystone Ferry = KF, Freeland = FL,
Mutiny Bay=MB, Possession Point = PO, Edmonds = ED, Poulsljo =
PB, Silverdale = SD, Shilshole Bay = SB, Elliot Bay Marina = EB,
Seattle Pier 91 = SE, West Seattle = VVS, Manchester Stale Parli = MC,
Seahurst County Park = SH, Purdy = PV, Saltwater State Park = SV\ ,
Point Defiance = PD, Grape>ie« Marina = CV, Penrose Point Stale
Park = PP. .Joemma Beach Slate F'ark = JB, Carlyon = CL, Tollen
Inlet = TI, Taylor United = TU, Sleilacoom Marina = ST, Tolmic Slate
Park = TL, and Priest Point = PR. Location of .sampling sites in Hood
Canal: Seal Rock = SR, Mike's Marina = MM. Potlalch State Park =
PL. Twanoh State Park = TVV. and Belfair State Park = BL.

ucts which differ in size for Mylilus ediilis. M. trossuhis. and M.
gcilloprovinicialis (Rawson et al. 1999). A second locus, internal
transcribed spacer (ITS), was amplified using the procedures de-
scribed by Rawson et al. (1999), cut with the restriction enzyme
Sau96. and run on a 1.5% agarose gel. This produces a species-
specific restriction pattern that distinguishes M. trossulus from M.
gallopmv/iiiiali.s (Rawson et al. 1996). Mussels from three loca-
tions (Silverdale. Totten Inlet, and Edmonds) were also assayed at
a third nuclear locus. Mal-I. according to the protocol given by
Rawson et al. (1999).

RESULTS

The M. galloprovincialis allele frequency for the Glu-5' and
ITS genes at each site is presented in Table 1 . Alleles specific to
M. galloprovincialis are not generally very abundant in Ptiget
Sound. Outside of the Totten Inlet (where Taylor United. Inc.,
cultivates M. galloproviiulalis) the average frequency of the M.
galloprovincialis allele was 0.032 for Glu-5' and 0.019 for ITS
(counting only sites for which both genes were scored). The fre-
quency of M. galloprovincialis alleles at Glu-5' and ITS was
highly correlated (r = 0.775. P < 0.05. sites without M. gallopro-
vincialis alleles at both loci and the Taylor United site were ex-
cluded). In the central and southern Puget Sound. M. galloprovin-
cialis alleles are relatively common in three locations. The Totten
Inlet contained relatively high frequencies of M. galloprovincialis
alleles. Mussels farmed by Taylor United in the Totten Inlet con-
tained only M. galloprovincialis alleles at both loci. Wild mussels
from a floating dock near the middle of the inlet (Totten Inlet) also
had relatively high frequencies of M. galloprovincialis alleles.
Mussels from the Carlyon Marina near the mouth of Totten Inlet.
however, had low frequencies on M. galloprovincialis alleles,
similar to that observed in other mussel populations in the southern
Puget Sound (Table 1). Alleles specific to M. galloprovincialis
were also common at Silverdale and at sites north and south ot
Seattle (Edmonds. Shilshole Bay. and Seahurst) (Table 1. Fig. I).
Silverdale is near the Bremerton Naval Shipyards, whereas the
other three sites are near the Port of Seattle at Elliot Bay. Surpris-
ingly. M. galloprovincialis alleles were absent from samples col-
lected within Elliot Bay (Elliot Bay Marina. Seattle Pier 91. and
West Seattle) (Table I ). Mussels with M. galloprovincialis alleles
were absent from samples collected in the Hood Canal and from
the northern portion of Puget Sound and Juan de Fuca Strait (Table 1 ).

To evaluate the extent of introgression between the two species
mussels from the three sites with the highest frequency of Myiihis
galloprovincialis alleles (Silverdale. Totten Inlet, and Edmonds)
were assayed at all three genetic markers and pooled into multilo-
cus genotypic classes. Mussels homozygous for M. trossulus al-
leles at all three loci were designated M. rrossulus. Those homozy-
gous for M. galloprovincialis alleles at all three loci were desig-
nated M. galloprovincialis. Mussels heterozygous for M. trossuhis
and M. galloprovincialis alleles at all three loci were designated Fl
hybrids. Those homozygous for M. trossulus alleles at one locus
and M. galloprovincialis alleles at another locus were designated
F2 hybrids. Of the remaining possible genotypes, those that con-
tained four or five M. trossulus alleles were designated as M.
trossulus back-crosses and those that contained one or two M.
trossulus alleles were designated M. galloprovincialis back-
crosses. Sorting individuals into the categories listed above does
not necessarily indicate their genealogical origin; genuine F2 and
back-cross matings should generate a wide variety of multilocus

MyTILUS GALLOPROVINCIAUS IN PUGET SOUND

77

TABLE 1.

Allele frequency of Clii-S' and ITS at each sample site in the Pujjel Sound and Hixid Canal: Sample size (;/) for each locus and the size

ranye and habitat sampled are also indicated.

Habitat

Length (mm)

Glu-5'

ITS

Site

Frequency

II

Frequency

II

Puget Sound

Bellinghani Buy

Intertidal

25-40

0.00

22

0.00

18

Anacorles

Suhtidal

>40

0.00

32

0.00

28

Deception Pass Marina

Suhlidal

>40

O.fK)

30

0.00

28

Oak Harbor

Intertidal

35-40

0.00

32

0.00

32

Keystone Ferry

Intertidal

>35

0.00

28

0.00

32

Freeland

Intertidal

>40

0.00

26

0.00

30

Mutiny Bay

Intertidal

15-20

0.00

28

0.00

30

Possession Point

Subtidal

20-25

0.00

28

Edmonds

Subtidal

>35

0.19

26

0.16

32

Poulsbo

Subtidal

>45

0.00

32

0.00

26

Shilshole Bay

Subtidal

<10and>35

0.12

68

Silverdale

Suhtidal

5 to >70

0.35

126

Elliot Bay Marina

Suhlidal

<10 and >35

0.00

66

Seattle Pier 91

Intertidal

25-35

0.00

28

0.00

28

West Seattle

Suhtidal

<10

0.00

24

Manchester State Park

Intertidal

3-35

0.00

48

0.00

20

Seahurst County Park

Intertidal

<5 and >40

0.1')

32

0.04

22

Purdy

Intertidal

<10and>40

0.04

52

Saltwater State Park

Intertidal

<15 and 25-35

0.04

28

0.04

26

Point Defiance

Intertidal

20-40

0.07

28

0.04

24

Grapeview Marina

Subtidal

< 10 and >35

0.09

32

Penrose Point State Park

Subtidal

5-20 and >40

0.02

60

0.00

22

Joemnia Beach State Park

Suhlidal

10-40

0.02

58

0.00

32

Carlyon

Suhlidal

35-40

0.08

26

0.07

28

Totten Inlet

Suhlidal

35-50

0.21

24

0.33

26

Taylor United

Subtidal

>60

1 .00

58

1. 00

30

Steilacooni Marina

Both

<I5 and>35

0.00

62

Tolmie State Park

Intertidal

5-25

0.00

44

Hood Canal

Seal Rock

Intertidal

5-35

0.00

54

0.00

24

Mike's Marina

Suhtidal

5-20 and 35-40

0.00

30

Potlatch State Park

Intertidal

5-15 and 35-45

0.00

30

0.00

30

Twanoh State Park

Intertidal

5-15 and 40-45

0.00

32

0.00

22

Belfair State Park

Intertidal

5-10 and 35^0

0.00

26

0.00

20

genotypes, including individuals that are homozygous for alleles
from one species at all three loci and mussels that are heterozygous
at all three loci. It is important to note, however, that mussels
homozygous for M. irossidus alleles at one gene and homozygous
for M. galloprovwcUdis alleles at another can only be the product
of F2 or other advanced introgressive crosses; they can not be the
progeny of either Fl hybridization or first-generation back-
crossing.

Of the 55 mussels from Silverdale. Edmonds, and Totten Inlet
assayed for all three genetic markers. 5 1 % had genotypes consis-
tent with pure Mytihis trossulus and 22% appeared to be pure M.
galloprovincialis (Table 2). Putative Fl hybrids were rare, com-
prising only 9% of these samples. Putative back-crosses to M.
trossulus appear to be about four times more common than back-
crosses to M. galldpnivincUilis (15% versus 4%. Table 2). No
individual was observed, in either the overall sample (Table I ) or
this subsample (Table 2). that had a genotype exclusively compat-
ible with F2 or other advanced hybridization.

Silverdale was the only site with a high frequency of M. gal-
loproriiwialis alleles where a wide range of size clas.ses was avail-

able for analysis (Table 2). Mussels between 5 and 60 mm shell
length were similar in genetic composition; the frequency of M.
trossulus alleles was between 0.73 and I. depending on locus, and
M. trossulus genotypes predominated. Mussels >60 mm shell
length were dominated by M. galloproviiiciiilis genotypes and al-
lele frequencies (0.75-0.77. depending on locus) (Table 2). M.
galloproviiicialis may either grow faster or attain a larger size than
M. trossulus. be selectively favored, or historical variation in re-
cruitment success may have favored M. galloproviiuidlis some-
time in the past, but not more recently. The presence of putative Fl
and M. galloprovincialis back-cross genotypes among the largest
mussels at Silverdale suggests the observed relationship between
size and allele frequency is a long-term and recuning feature at
this location and not the result of historical variation in recruit-
ment.

DISCUSSION

Native mussels. Mytihis trossulus. dominate the blue mussel
community in Puget Sound; at most sites M. galloprovincialis

78

Anderson et al.

TABLE 2.

Multilocus genotypes of mussels from Silverdale. Edmonds, and
Totten Inlet: The number of mussels of each yenolype Is indicated.

(Jenotype

Trossulus

Back-tross

Fl

Back-gallo

Gallo

Silverdale, length (mm)

0-20 3

->

0

0

0

20-40 4

3

0

0

0

40-60 7

1

1

0

1

>60 1

0

3

T

6

Edmonds 8

1

0

0

-)

Totten Inlet 5

0

1

0

3

Trossulus = mussels homozygous for M. irossuliis alleles at Glu-5'. ITS.
and MalT, Gallo = mussels homozygous for M. f>aHoprn\incialis alleles
at all three loci; and Fl = mussels heterozygous at all three loci. Back-
tross and Back-gallo indicate mussels with genotypes consistent with M.
trossulus back-crosses and M. galloprovincialis back-crosses, respectively,
as specified in the text. No mussels were observed that were homozygous
for M. Inissulus alleles at one locus and homozygous for M. galloprdvin-
cialis alleles at another (i.e., F2 genotypes).

alleles were rare or absent. The dislributioti of M. gaUiipiinincudis
alleles varies regionally within Puget Sound. M. gaUoprovimudis
alleles are present but unccMiimon (-59?-) in mussel populations in
the southern portion of Puget Sound, and appear to be nearly
absent in populations in the northern Puget Sound and the Hood
Canal. The only locations where M. galloprovincialis alleles are
locally common appear to be sites where they are likely to have
been repeatedly introduced. These include the Totten Inlet where
Taylor United. Inc.. maintains a mussel farm, and sites neai the
Port of Seattle and Bremerton Naval Shipyards.

Aquaculture operations appear to be the source of the high
frequency of M. galloprovincialis alleles in mussel populations
from the Totten Inlet. Taylor United, Inc., maintains a grow-out
facility in the inlet that produces 1-1.5 millioti pounds (live
weight) of M. galloprovincialis per year (Gordon King. pers.
comm.). The frequency of alleles specific to M. galloprovincialis
among "wild" mussels within the inlet ranged between 21% and
.3.3%, depending on the locus assayed. However, the impact of the
aquaculture operation is highly restricted; mussels sampled at the
mouth of the inlet (Carlyon Beach) had a low frequency of M.
galloprovincialis alleles that was not readily distinguished frotii
the background frequency of these alleles found in other mussel
populations in the southern Puget Sound, This result is surprising
because the Totten Inlet is about 1 2 km long and mussel larvae
could potentially disperse over much greater distances during their
development in the plankton of several weeks. These results sug-
gest that the circulation patterns in Totten Inlet retain larvae w ithin
the inlet.

Two additional lines of evidence suggest that the genetic im-
pact of aquaculture operations on the genetics of indigenous mus-
sel populations tnay be tniniinal. First, Taylor United has previ-
ously cultured M. galloprovincialis at Freeland, on Whidby Island
in the northern Puget Sound from 1992 to 1998, No M, gallopro-
vincialis alleles were found among wild mussels at Freeland or at
any other location on Whidby Island in 1997. Second. Taylor
United tnainlains hatchery and nursery operations for the culture of
M, galloprovincialis in Dabob Bay at the northern end of the Hood
Canal. M. galloprovincialis alleles were viitually absent from all

sites sampled within the Hood Canal, These lesults indicate that
either cultured mussels are unlikely to escape aquaculture opera-
tions or. if they do. they have relatively little impact on resident
populations o( M. rrossnhis. Surveys conducted by Taylor United
indicate that in 1996 and 1997, the frequency of M, galloprovin-
cialis alleles among mussels on the shore near their mussel farm on
Whidby Island was 2,8% and \3'7f. respectively (Gordon King,
pers, comm,), Taylor United's mussel farming operations on
Whidby Island were discontinued due to high mortality rates
among M. galloprovincialis. These results all suggest that M. gal-
loprovincialis is only marginally successful in northern Puget
Sound.

It is worth noting that 100% of the mussels obtained from
Taylor United's grow-out operation in the Totten Inlet were ho-
mozygotes for M, galloprovincialis alleles at both Glu-5' and ITS,
This result indicates that Taylor United has been highly successful
at maintaining pure cultures of M. galloprovincialis for several
generations, despite the presence of resident populations of M.
irossidas as potential sources of contamination at both the grow-
out and the hatchery/nursery locations.

Shipping also appears to be an important vector for the release
of M. galloprovincialis into Puget Sound. The two greatest con-
centrations of M, galloprovincialis alleles outside of the Totten
Inlet are near major shipping areas. The highest frequencies of M.
gall(}provincialis alleles outside of the Totten Inlet were observed
at the Edmonds and Shilshole Bay Marinas north of Seattle.
Seahurst County Park south of Seattle, and Silverdale. near the
Bremerton Naval Base. Shipping appears to be the primary inecha-
nism responsible for the introduction of nonnative marine species
throughout the world (Carlton & Geller 1993; Ruiz et al. 1997:
Ruiz et al. 2000). Geller et al. (1994) documented that larvae of M.
galloprovincialis may be transported in ballast water of ships. The
occurrence of M, galloprovincialis alleles near major shipping
terminals in Puget Sound suggests that shipping may be respon-
sible for the repeated introduction of this species to Puget Sound,
It is also suggestive that M. galloprovincialis alleles are absent
from the Hood Canal, which does not have any major shipping
activity. Notably. M. galloprovincialis alleles were absent from
within Elliot Bay near the Port of Seattle (Elliot Bay Marina. Pier
91 and West Seattle), which may indicate that ships release their
ballast water before entering Elliot Bay.

Although M. galloprovincialis appears to have been repeatedly
introduced into Puget Sound by both aquaculture and shipping
activities, the fate of these introductions is tenuous. Mussels from
the southern Puget Sound with M. galloprovincialis alleles appear
to mostly be the result of back-crosses with M. irossnlus. There
was little evidence for pure M. galloprovincialis mussels outside of
Totten Inlet and Dyes Inlet (Silverdale), Even in the three areas
with the highest frequency of M. galloprovincialis alleles there
was no evidence for advanced introgression. as indicated by the
presence of genotypes that are exclusively compatible with being
the progeny of F2 hybridization. These results indicate that when
pure M. galloprovincialis are introduced, they occasionally hybrid-
ize with M. trossulus and then Fl hybrids may subsequently back-
cross to M. trossulus. Continued hybridization is not apparent
within Puget Sound. Rawson et al. ( 1999) examined hybridization
between M. galloprovincialis and M. trossulus in California, They
also found that even though mussel assemblages often contained
high frequencies of both species, hybrids were relatively uncom-
mon and there was little evidence for advanced levels of introgres-
sion. Although the mechanism is presently unclear, the results of

Mytilus galloprovinc/alis in Puget Sound

79

Rawson et al. (1999) and of this study indicate that the "genetic
pollution"" of native populations of M. irossiiliis by introduced M.
guUopwvincialis is limited. Fuilher work is necessary to examine
the mechanism(s) that determine the genetic fate of M. gallopro-
vincialis alleles introduced by hybridization to resident popula-
tions of M. trossidiis. It should also be noted that although intro-
gression between these two species appears to be limited. M. gal-
Inprovincialis has been successfully introduced in California and

in the Sea of Japan, and the ecological impact of these introduc-
tions on native mussel populations is presently unclear.

ACKNOWLEDGMENTS

We thank Gordon King and Taylor United. Inc.. for providing
samples and information on local mussel farming operation. We
acknowledge the support of NSF grants DEB-9807055 and OCE-
9731277 toT.J.H.

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Rawson P. D. & T. J. Hilbish. 1998. Asymmetric introgression of mito-
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Rawson P. D.. C. L. Secor & T. J. Hilbish. 1996. The effects of natural
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Rawson P. D.. V. Agrawal & T. J. Hilbish. 1999. Hybridization between
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Ruiz G. M., P. W Fofonoff, J. T. Carlton, M. J. Wonham & A. H. Hines.
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Joiinml of Shellfish Research. Vol. 21. No. 1. 81-86, 2002.

TEMPORAL AND SPATIAL VARIABILITY IN DREDGING INDUCED STRESS IN THE GREAT

SCALLOP PECTEN MAXIMUS (L.)

JULIE A. MAGUIRE,' MAURICE O'DONOGHUE,^ STUART JENKINS,^ ANDREW BRAND,'
AND GAVIN M. BURNELL'

'Aqiiaciilturc Development Centre. Dept.. of Zoolofiy and Aninuil Ecology. University College Cork. Lee
Maltings. Prospect Row. Cork. Ireland: -Department of Microbiology, University College Cork. Cork,
Ireland; ''Port Erin Marine Laboratory, University of Liverpool, Port Erin, Isle of Man, United Kingdom

.ABSTRACT Temporal and spatial vanability in the stress caused to the great scallop Pecten maximus by dredge capture was
determined using the adenylic energetic charge (AEC) of the striated muscle as an index. Samples were taken on a seasonal basis from
dredge captured scallops of a single fishing ground in the north Irish Sea. All samples were taken from individuals under the minimum
legal landing size. On one date samples were taken from scallops from three separate grounds with contrasting bottom characteristics.
At all dates dredge captured scallops had a significantly reduced level of AEC compared to control scallops. This reduction was greatest
in February when glycogen levels in the muscle tissue were shown to be at a minimum. There was little or no variability among fishing
grounds in the effect of dredge capture on AEC levels. An additional aim of this study was to determine if scallops that encounter
dredges but are not captured suffer a reduction in AEC levels. Scallops collected from dredge tracks immediately following the passage
of dredges showed a significant reduction in AEC compared to control, scallops collected from outside the tracks. However, the
reduction in AEC was not as great as in captured scallops.

KEY WORDS: scallop. Pecten iihi.\iiims. dredging effects, adenylic energetic charge, seasonal effects, glycogen

INTRODUCTION

Most fishing techniques aim to capture a single or limited num-
ber of target species over a minimum size. This size may be
dictated by markets or by government legislation. In scallop dredg-
ing, as in many other fisheries, a proportion of the captured target
species is discarded owing to their small size. These undersized
discards may be damaged during capture, for example suffering
chipped valve margins or separation of the hinges, or may suffer
stress from fatigue and desiccation. The level of mortality of these
discarded scallops is unknown but may be considerable. Several
studies have speculated on the levels of dredging induced mortality
in relation to scallop size, sorting time and conditions on deck
(Medcof & Bourne 1964), substrate type (Naidu 1988), catch
weight (Chapman et al. 1977) and type and performance of gear
(Gruffydd 1972).

Damaged scallops that are left on the seabed, or are discarded,
are likely to show high mortality owing to the aggregation of
predators to dredge tracks (e.g„ Caddy 1973: Kaiser 1994: Medcof
1964) and to damaged bycatch (Veale et al. 2000). However, the
majority of undersized discarded scallops show no signs of exter-
nal physical damage. In the Irish Sea Jenkins et al. (2001 ) showed
that less than I09c of scallops {Pecten moAiniiis} encountering
dredges showed any signs of damage while Shepard and Auster
(1991) estimated that damage to the scallop PUwopecten inagel-
lanicus, that encounter dredges but are not captured, can vary
between T7ir and 25"/^ depending on substrate type. Although dis-
carded scallops may have low levels of datnage, they are likely to
be highly stressed from the process of capture. Little is known
regarding the effects of fishing induced stress on subsequent sur-
vival of scallops.

Dredging induced stress can be assessed in scallops using a

Correspondmg author. Julie Maguire, Aquaculture Development Centre,
Dept., of Zoology and Animal Ecology, University College Cork, Lee
Maltings. Prospect Row. Cork, Ireland. Phone: +35.^-21-490-4053; Fax:
^-353-2 1-427-7922. E-mail; niaguirejuliels'hotmail.co

variety of biochemical indicators. Maguire et al. (in press, a) dis-
cussed the usefulness of various techniques for stress assessment
in scallops and found Adenylic Energetic Charge (AEC) to be the
most effective in the measurement of acute dredging stress. AEC
was first proposed as a stress index by Atkinson (1968) and is
defined by the ratio:

AEC = (ATP + O..SADP) -H (ATP + ADP -i- AMP)

where: ATP = adenosine tri-phosphate, ADP = adenosine di-
phosphate and AMP = adenosine mono-phosphate.

The AEC ratio varied from 0 to I i.e., 0 (all nucleotides were
energy deficient AMP molecules) or 1 (all nucleotides were energy
rich ATP molecules). In optimal conditions animals typically dis-
played AEC levels >0.8 while under stress conditions values
ranged from 0,5 to 0,75, Such animals had a reduced growth rate
and did not reproduce, but recovered when returned to optimal
conditions. Severely stressed animals yielded values of <0,5 and
these animals had a negative scope for growth and did not recover
(Duncan 1993: Ivanovici 1980), Subsequently many studies have
been carried out using AEC as a stress index in scallops, for
example P. imigellaiuciis (de Zwaan et al, 1980; Livingstone et al,
1981 ) and P. nui.\iiiu(\ (Fleury et al. 1997; Maguire et al. 1999a:
Maguire et al. 1999b). Maguire et al. (in press a) used AEC as a
stress index to investigate the effect of dredge capture on under-
sized scallops. They found no difference in AEC levels between
different lengths of tow (15, 30 or 45 minutes) but found that
emersion following dredging had an added stress effect. In this
study we aimed to investigate different aspects of dredging on
scallops i.e., effects of different seasons, ground types and to as-
sess the stress caused to scallops that come in contact with dredges
but remain uncaught on the seabed.

We aimed to exatiiine dredging induced stress in the great
scallop Pecten maximus on a seasonal basis to determine if sea-
sonal reproductive cycle in this species affected the extent to
which it is negatively affected by dredge captui-e. Seasonal vari-
ability in AEC levels has been examined in a number of inverte-
brate species including the crayfish, Procamhanis acinus aculus

81

82

Maguire et al.

(Dickson & Giesy 1982). the oysters Ostica ediilis and Crassos-
trea gi,i;a.s (Moal et al. 1989b; Moal et al. 1991 ). the nuissel M\ri-
his ediilis (Zaroogian et al. 1982) and the clam CanUiim sp.
(Picado et al. 1988). Such variability has generally been associated
with the reproductive cycle. Seasonal changes in biochemical com-
position of the scallop P. maxhmis have been well documented
(Ansell 1978; Maguire & Burnell 2001 ). Carbohydrate reserves are
built up during periods of greater food availability dtiring the sum-
mer and used up in the winter. In P. iiui.xiiiuis the main period tor
gonadal proliferation takes place between November and February
by the mobilisation of glycogen and protein reserves from the
adductor muscle and lipid from the digestive gland. P. inaximiis
may spawn over a long period from spring to autumn (Ansell
1978). Gametogenesis represents a period of high-energy demand
in scallops and when external food supplies are limited, gamete
production occurs at the expense of biochemical components in
somatic tissues (Calow 1985). In this study on each sampling day
we measured the glycogen content of both the striated adductor
muscle and the gonad in order to assess the reproductive state of
the scallops. Brokordt et al. (2000) showed that the reproductive
state of the scallop Clilaiuys isUindka affected its ability to escape,
presumably as a result of changes in the energetic state of the
individual. Therefore, it would be reasonable to hypothesize that
seasonal variability in the energetic state of scallops would affect
the degree to which they are negatively affected by dredging.

In addition to the major aim of this study, the determination of
temporal variability in dredging induced stress, two further objec-
tives were pursued. Initially, field trials were conducted at three
different fishing grounds to assess the degree to which dredging
induced stress varies spatially. Fishing grounds were specifically
chosen with contrasting bottom characteristics. Diving was under-
taken to collect scallops that encountered dredges but were not
captured. AEC was measured to determine the extent to which
these scallops were negatively affected by this experience.

MATERIALS AND METHODS

Seasonal Field Trial

The field trial was carried out on the commercial scallop
ground known as the Chickens, off the south west of the Isle of
Man in the North Irish Sea (53°58.75'N. 04°52.7rW). The scal-
lops were caught using a Newhaven type spring-loaded dredge
(width - 75 cm) with a toothed cross bar (tooth spacing - 66 mm;
tooth length - 100 mm), a collecting bag made from case hardened
steel rings (diameter - 70 mm) and a net mesh cover (mesh size -
100mm). Eight dredges were towed at a speed of 5 kph for forty-
five minutes on 4 dates, March 15th, June 5th, October 6th (2000),
and February 14th 2001 . On each date two tows were made. At the
end of each tow at least 15 scallops below the minimum legal
landing size (<l 10 mm shell height) were taken from two random
dredges and samples of muscle and gonad taken for AEC and
glycogen analysis. Each scallop was opened and two small por-
tions of the striated adductor muscle and one portion of gonad
weighing approximately 0.2 g were removed using a scalpel.
Samples were wrapped separately in foil and immediately placed
in liquid nitrogen.

On each date 10 undersized scallops were taken from the
dredges and transferred alive to Port Erin Marine Laboratory and
placed in tanks containing running seawater. Samples of muscle
were taken one week later as described above. These samples acted
as controls for AEC analysis. A preliminary experiment showed

that the AEC levels of post-dredged scallops returned to normal
after 24 hours (at most) of recovery in seawater (Maguire et al.. in
press b). Scallops placed in tanks for a full week had made a
complete recovery and thus this protocol was adopted for the con-
trol animals in this study.

Cumparisdii of Scallops from Different Grounds

This field trial was cauied out in June 2000 on three commer-
cial scallop fishing grounds off the Isle of Man (Fig. I ); Chickens
(53°58.75'N, 04°52.7rW). Laxey (54°I3.50'N. 04°21.38'W) and
Bradda Inshore (54°06.5'N, 04°47.76'W). The substratum on all
grounds was generally coarse sand or gravel, often overlain with
pebbles, cobbles and dead shell. The abundance of pebbles and
cobbles, as indicated by the number caught during dredging varied
greatly among the three grounds sampled. At Laxey there were
relatively few and the substratum was predominantly sandy with
areas of dead maerl. The Chickens and Bradda Inshore grounds
were both made up of variable gravel sediment w ith large numbers
of pebbles and cobbles at Bradda Inshore and intermediate
amounts at Chickens.

Scallops were caught using the same method as above. Samples
of muscle for AEC analysis were taken from 15 undersized scal-
kips from random dredges from two tows.

Stress in Son-Captured Scallops

In June 2000 dredging was undertaken on the Bradda Inshore
scallop ground as part of a study into the impacts of dredging on
benthic megafauna (see Jenkins et al. 2001 for details). Divers
collected scallops that had encountered dredges but had not been
captured by following the tracks on the seabed formed from the
passage of the dredge. Forty-two scallops from within the dredge
tracks (impacted scallops) and 14 scallops from outside the tracks
(control scallops) were collected between 15 and 45 minutes after
the passage of the dredges. Dredging also took place and two tows
were cairied out. For each tow. 15 undersized animals were re-
moved from the dredge bags. On surfacing all scallops were im-

Figurc 1. Map of dredged sites.

Seasonal Effects of Dredging on Scallops

83

mediately opened and samples of striated adductor muscle taken
tor AEC analysis.

Biochemical Analyses

All samples were stored in liquid nitrogen until analysis. Ade-
nylic Energetic Charge (AEC) of the striated adductor muscle was
determined using the technique of Moal et al. ( 1989a). Maguire et
al. ( 1999b) conducted an experiment to assess the effect of stress
on the AEC level of both the smooth and striated muscle of the
king scallop. A greater significant AEC decline was shown be-
tween treatments in the striated muscle. For glycogen analysis
samples were withdrawn from the liquid nitrogen and freeze dried
using a HETOSICC CD 53-1 freeze dryer. The glycogen content
was analyzed using a miniaturization of the Dubois et al. (1956)
method.

Statistical Analyses

After testing for normality, non-parametric data were normal-
ized by log transformation or arcsine square root transformation.
One-way analyses of variance (ANOVAs) were used to test sig-
nificant differences between treatments and (/ posteriori Tukey test
was used to contrast treatments. The level of significance was set
at 0.05.

RESULTS

Seasonal Field Trial

Figure 2 shows the effect of dredging on the AEC level of
scallops from the Chickens scallop fishing ground off the coast of
the Isle of Man. The AEC level decreased significantly in the
dredged scallops from the control samples at each sampling period
(P < 0.001 ). However, the AEC levels of the control samples
varied throughout the year but not significantly. The highest AEC
levels in the control animals were found in October (0.90 ± 0.01)
and the lowest levels were found in February (0.77 ± 0.05). Be-
cause of this seasonal AEC variation in the control animals the %
AEC decrease from the control to dredged scallops was calculated
for each season. The AEC reduction was highest in February
(44.8% ± 3.3) and June (43.4% ± 2.9) and significantly less (F, ,^3

I tlritli;!- Il,.(i

(I3,8°C)

(8.0°CI

a

ab

I

IlL

June Oclober

February

Figure 2. The seasonal effects of dredging on the AEC level (mean ±
s.e) of scallops from Chiciiens (March 2(10(1 to February 2()(M I. Sea
water temperatures shown in parenthesis. .\n\ two means sharing a
common letter between each treatment are not significantly different
aiP < 0.05 (Tukev test).

= 3.476. P < 0.(15) in March (33.6% ± 2.8). The lowest absolute
value of AEC in the striated muscle of scallops following dredging
was also in February (0.43 ± 0.03) and the highest was in October
(0.53 ± 0.01). The AEC level reached in scallops sampled in
February was significantly different (F|3f,3 = 4.942, P < 0.05)
from all the other dates sampled except for June. No difference in
the AEC levels of dredged scallops was found between any of the
other sampling dates.

The percentage of glycogen content in the striated muscle of
the animals showed a similar pattern to the AEC results found in
the control animals (Fig. 3). The lowest glycogen levels were
found in the scallops sampled in February (4.38% ± 0.27). with

levels over three times higher in October (F,

83.32, P <

0.001 ). The % glycogen content in the gonad also varied signifi-
cantly throughout the year (F.^^,, = 331.42, P < 0.001). The
glycogen levels in the gonad ranged from a minimum in October
(0.30% ± 0.05) to a maximum in June (10.33% ± 0.43).

Sea water temperatures decreased from a maximum on August
29th 2000 (I5.7°C) to a minimum value on the 17th of January
2001 (7.3°C). On the specific sampling days the temperatures are
shown on Figure 2.

Comparison of Scallops from Different Grounds

There was no difference in the AEC levels of scallops dredged
at different sites in June (Fig. 4). The AEC level varied in scallops
from 0.46 to 0.48 on all three grounds.

Stress in Non-captured Scallops

.Scallops that came in contact with the dredge but remained
untaught had a significantly lower AEC level than the control
scallops (tj,,, = 7.401, P < 0.001 ). However, the reduction in AEC
was not as great as in captured scallops. Figure 5 showed that the
AEC level in captured scallops was significantly lower (tjj =
6.869. P < 0.001 ) than in the non- captured scallops.

DISCUSSION

The results from this study suggest that the AEC response to
dredging of undersized scallops is different according to the season
of the year. Lowest AEC levels in dredged and control scallops

s. 10

a

In

a

February

Figure 3. The seasonal % glycogen content (mean ± s.e) of scallops
from Chickens (March 2000 to February 2001). Any two means shar-
ing a common letter between each treatment are not significantly dif-
ferent at P < 0.05 (Tukev test).

84

Maguire et al.

I

0,9
0.8

B 0.7

a

o
'^ 0.6

0.5

0.4

0.3

■ Dredge Bag
D Control

J

I

■1

Laxey

Figure 4. The effects of dredjjinK on the AEC ratio (mean ± s.e) of
scallop.s from three sites off the Isle of Man (June 20(11).

were found in February and highest levels in October. This ciiin-
cided with the troughs and peaks of the glycogen content in the
adductor muscle of the test animals. Reserves are built up during
periods of greater food availability in the summer. However, en-
ergy requirements for maintenance are high therefore rapid utili-
zation of reserves takes place during the winter. Many demands are
placed on the metabolism of scallops during the winter from the
stresses imposed from internal physiological drives such as go-
nadal proliferation. However, during unfavorable winter condi-
tions these energy demands may be insufficient to support gonadal
development (Ansell 1978). The greatest percentage of AEC de-
crease from control to dredged scallops was also in February
(44.8%). Therefore, the additional stress effect of dredging at the
end of winter had a gieater negative effect on the AEC level of
scallops whose energy reserves were already low. Similarly,
Brokordt et al. (2000) showed that the mobilization of reserves had
a detrimental impact on the escape response of the Iceland scallop,
Chlainys islandica.

Moal et al. ( 1989b), also found that the AEC response to the
stressor aerial exposure was different according to the time of year
in the oyster Crassostrea gigas. The animals were subjected to a
three hour emersion period in January, May, and July. Lowest
AEC values were found in July and these levels were invei'sely
correlated with temperature. Similarly, in our study a large % AEC

Dredge Track

Control (Dived)

Figure 5. Comparfson of the AEC levels (mean ± s.e) of dredged scal-
lops and scallops left behind on the dredge track from Bradda in .June,

decrease from control to dredged animals was recorded in June
(43.4%) and October (41.1%) when temperatures were highest.
The June sample coincided with the spawning season of the scal-
lop population in the Isle of Man. Moal et al. (1991), also found
low AEC levels during the summer in the farmed oysters C. gigas
and correlated this decrease with the reproductive state of the
oysters.

Many authtirs have suggested that AEC levels in molluscs be-
low a value of 0..^ result in a negative scope for growth, which
would eventually lead to mortality even if the animals were trans-
ferred to optimal conditions (Ivanovici 1980; Duncan 1993). How-
ever, in this study scallops dredged in February and June had AEC
levels <0.5. However, no mortality was recorded in the control
scallops. Similarly in a previous study by Maguire et al. ( 1999a).
juvenile scallops were transported in polystyrene boxes for 12
hours and the AEC level of the spat decreased form 0.88 to 0.42.
Recovery of these animals was initially rapid and AEC levels had
increased to 0.68 after only two hours in optimal conditions and
had returned to normal after 24 hours. In addition dredge captured
scallops that are returned to the laboratory and maintained in run-
ning seawater showed consistently low levels of mortality (Jen-
kins, unpublished data). Although the stress and subsequent reduc-
tion in AEC levels in dredge captured scallops may not lead di-
rectly to mortality, such scallops may be more susceptible to
predation (Thompson et al. 1980) through a reduction in their
escape response or inability to recess (Minchin et al. 2000).
Maguire et al. (in press a) found that the AEC levels of dredged
scallops (30 minute tow length) had returned to normal after 3 days of
recovei7 but had not recessed into the sediment in that time period.
Jenkins and Brand (in press) have shown that the escape response in
P. iiuLxinius was significantly reduced following simulation of dredg-
ing for up to 24 hours. Such observations suggest thai predator in-
duced mortality may be significant for stressed discards.

In general, the percentage of glycogen content of these Isle of
Man scallops was quite low in the striated adductor muscle ranging
from 4.4% to 16.4%. In a study of a population of scallops in
Bantry Bay, Ireland the glycogen content ranged from 16.5% to
45.9% (Maguire & Bumell 2001). Ansell (1978) studied the stor-
age of reserves in the adductor muscle of some scallop populations
in the U.K. and found that the % glycogen content generally varied
from 2.2% to 24.0%. but in one population the glycogen content
was as high as 39%. He also suggested that glycogen content could
vary between sites and between different years.

There was no difference in the AEC values between the three
different sites studied. The effect of the dredging process was the
dominant stressor on the animals so much so that the subtle dif-
ferences between the sites had little added impact on the AEC
decrease. Similarly, Verschraegen et al. ( 1985) found no difference
in the AEC levels of the polychaete species Nereis diversicolor
and Nephtys sp. between sites in the Western Scheldt estuary,
Belgium. Also, Zaroogian et al. (1982) found no difference in AEC
levels of the mussel Mytilits ciiiilis in two sites.

Scallops that came in contact with the dredge but remained
uncaught and were found on the dredge track were stressed (AEC
level = 0.75). Although the AEC level was not as low as in
captured scallops it is important to demonstrate that the impact of
dredging is not confined solely to those scallops landed on deck.
Owing to the relatively low efficiency of scallop dredges (Dare et
al. 1993) far more scallops will encounter dredges and remain on
the seabed than are captured. These non captured animals which

Seasonal Effects of Dredging on Scallops

85

Include all sizes, may suffer enhanced mortality due to dredge
induced stress in the same way as undersized discards. Also, it was
interesting to note that the collection and transportation of scallops
from the sea-bottom to the surface did not induce stress in the
control animals.

CONCLUSIONS

1 . AEC levels in dredged and control animals, and % glycogen
in the striated muscle of dredged scallops, were lowest in
February and highest in October.

2. The largest % AEC decrease from control to dredged scal-
lops was also in February and the smallest decrease was in
March.

3. There was no difference in the AEC level of dredged scal-

lops from three sites with different ground types around the
Isle of Man.
4. Scallops that came in contact with the dredge but remained
uncaptured on the dredge track had a reduced AEC level but
this level was significantly higher than the AEC level of
captured animals.

ACKNOWLEDGMENTS

This study was carried out with financial support from the
Commission of the European Communities, Agriculture and
Fisheries (FAIR) specifically RTD programme PL-4465,
ECODREDGE. It does not necessarily reflect its views and in no
way anticipates the Commission's future policy in this area.

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Fleury. P. G., C. Mingant & A. Castillo, 1997. A preliminary study of the
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Gruffydd. L. I. D. 1972. Mortality of scallops on a Manx scallop bed due
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Ivanovici, A. M. 1980. The adenylate energy charge in the estuarine mol-
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Jenkins, S.. B. Beukers-Stewart & A. Brand. 2001. Impact of scallop
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301.

Jenkins. S. R. & Brand A. R. The effect of dredge capture on the escape
response of the great scallop. Pecten ma.ximus (L.): implications for the
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Maguire. J. A.. D. Cashmore & G. M. Bumell. 1999a. The effect of
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Shepard. A. N. & P. J. Auster. 1991. Incidental (non-capture) damage to
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Thompson. R. J.. D. R, Livingstone & A. de Zwaan. 1980. Physiological

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Zaroogian. G. E.. J. H. Gentille. J. F. Heltshe, M. Johnson & A. M.

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Joiimal of Shellfish Research. Vol. 21. No. 1. 87-91. 2002.

REDUCED GROWTH OF ICELAND SCALLOPS CHLAMYS ISLANDICA (O.F. MULLER)
CULTURED NEAR THE BOTTOM: A MODELLING STUDY OF ALTERNATIVE HYPOTHESES

MARCEL FRECHETTE' AND GAETAN DAIGLE^

'histinn Mauricc-Lanumtagne. Miuistere des Peches et Oceans. C.P. WOO, Moni-Joli. QC. G5H 3Z4,
Canada: 'Departcnient de Mathemaliqiies et de Statisticjiie. Facidte des sciences et de genie. Pavilion
Ak'xandre-Vachon. Universite Laval. Quebec. QC. GIK 7P4. Canada

ABSTRACT Slow growth ol CliUiiiiys islamlica liL-ld in pearl nets near the bottom is usnallv attributed to poor seston quality owing
to higher eoneentration of resuspended inorganie particles near the bottom. In a recent contribution, we hypothesized that current speed
variations in boundary layers and feeding behavior may also result in slower growth of Iceland scallops kept in pearl nets near the
bottom, independently of resuspension effects. Here we present computer simulations of growth of .scallops kept in pearl nets at two
population densities and various heights above the bottom. Our simulations suggest that resuspension may account for experimental
results hut that food depletion within the pearl nets i.s required. Our simulations also suggest that a direct positive effect of current speed
on clearance rate or physiological regulation of clearance rate, both coupled with seston depletion within pearl nets, may both e.xplain
experimental patterns. Based on the profile of growth across height, our results also provide a criterion for interpreting results of
experiments designed to identify the actual control mechanism.

KEY WORDS:

Chlmnw isluudica. density dependence, growth, scallop, seston quality

INTRODUCTION

Iceland scallops iChkunys iskmdica O.F. Miiller) cultured in
pearl nets typically exhibit reduced growth rate near the botloni
(Wallace & Reinsnes 1984; Wallace & Reinsnes 1985; Thorarins-
dottir 1994). Because reduced se.ston quality dramatically reduces
assimilation efficiency of Iceland scallops (Vahl 1980), near-
bottom growth reduction is usually attributed to low .seston quality
due to local resuspension. Resuspended particles indeed tend to be
of poor trophic quality as compared to planktonic food, the stron-
ger the resuspension forces the larger the difference (Demers et al.
1987; Thomsen & Gust 2000).

Boundary layers are zones of strong change in cutxent speed
with respect to distance frotn the interface between fluids and solid
surfaces. They are universal features of flow near such disconti-
nuities. Bottom resuspension results from shear in the benthic
boundary layer (BEL: e.g., Muschenheim 1987; Thomsen & Gust
2000). Therefore if organisms are held across a resuspension gra-
dient near the bottom, they will also experience a current speed
gradient. It follows that the seston quality hypothesis cannot be
invoked as the sole explanation to near-bottom vertical differences
in scallop growth without implying that the scallops were also held
across a current speed gradient and that cuirent speed effects were
negligible.

Fluid movement and associated physical processes are funda-
mental to the biology and ecology of aquatic organisms (Vogel
1981; Wildish & Kristmanson 1997). Trophic dynamics of scal-
lops are no exception. For instance, Wildish et al. ( 1992) reported
that current speed affects growth in a non-monotonous way
through its effect on clearance rate. At low current speed, clear-
ance rate increases with current speed until a maximum is reached
at about 10 cm s~'. Higher current speed results in clearance rate
inhibition. These patterns are reflected in growth (e.g., Wildish et
al. 1987; Cahalan et al. 1989; Eckman et al. 1989; Wildish et al.
1992; Wildish & Saulnier 1992) but in pearl net culture, they may
interact with seston depletion within the pearl nets (Claereboudt et

Corresponding author. Marcel Frechette. Institut Maurice-Lamontagne.
Ministere des Peches et Oceans, C.P. 1000, Mont-Joli, QC, G5H 3Z4,
Canada.

al. 1994b). Furthermore clearance rate may respond to temporal
variability in phytoplankton concentration and cuirent speed, in-
dicating an effect of feeding history (Pilditch & Grant 1999).
Therefore near-bottom effects may be ascribed to direct and indi-
rect effects of current speed as much as to sediment resuspension.

In a recent study of growth and survival of Iceland scallops
kept in pearl nets, we tested the effect of site, of height above the
bottom (0.1 m versus 2.0 m) and of group size within the pearl nets
(Frechette & Daigle, in press). Our analysis also included fluctu-
ating asymmetry as a proxy of individual effects. The group size
treatment allowed testing for containment effects within the pearl
nets. Available data showed no evidence of phytoplankton vertical
depletion in the water column. However, we found slower growth
near the bottom. This is similar to previous reports (Wallace &
Reinsnes 1984; Wallace & Reinsnes 1985; Thorarin.sd6ttir 1994).
We also found that at 2.0 m above the bottom there was a group
size effect, but not at 0.1 m. High density groups grew slower than
low density groups. This is consistent with food depletion within
the pearl nets and has been observed repeatedly in growth experi-
ments (Parsons & Dadswell 1992; Claereboudt et al. 1994a;
Claereboudt et al. 1994b). The reasons for the absence of a group
size effect at 0.1 m. however, are not clear. Food within the pearl
nets was presumably more depleted at 0.1 m height, down in the
BBL. where current speed is slower than at 2.0 m height. This should
have generated a group size effect at 0.1 m as found at 2.0 m.

The goal of the present study was to explore three hypotheses
for the group si/.e*height interaction found in the growth experi-
ment. We modeled growth according to various control factors: ( 1 )
with increasing inorganic particle concentration near the bed, due
to resuspension; (2) with a direct positive effect of current speed
on clearance rate; (3 1 with an indirect effect of current speed on
clearance rate because of physiological regulation of clearance rate
in response to more intense phytoplankton depletion in the pearl
nets held near the bottom. To simplify matters, we modeled the
effect of each factor separately.

METHODS

We modeled soft tissue growth of individual scallops contained
in hypothetical pearl nets, as a function of non-dimensional current
speed, f/Vf/o, with U. and (/^ being respectively current speed at

87

Frechette and Daigle

heights c and 2.0 m above the bottom. We assumed that the benthic
boundary layer extended to 2.0 m above the bottom and that U-
increased logarithmically with height (Grant et al. 1984). A first
set of simulations was made assuming that no seston depletion
occurred in the pearl nets. In the second set of simulations, we
assumed that seston depletion occurred in the pearl nets. Outside
the pearl nets, phytoplankton concentration was assumed to be
constant across heights (see Frechette & Daigle. in press). We
assumed that there were no interactions between individual scal-
lops otherwise than through their effect on bulk food concentration
in the pearl nets. We modeled flesh growth as the balance between
energy intake and energy losses, based on the Scope for Growth
approach (Bayne & Widdows 1978). although more mechanistic
approaches to this problem are possible (e.g.. van Haren &
Kooijman 1993).

The numerical model has two state variables, scallop tlesh mass
{in, g) and phytoplankton concentration in the hypothetical pearl
nets (P. J L~'). We assumed that survivorship was 60% and 80%
in pearl nets held at 0.1 m and 2.0 m. respectively (Frechette &
Daigle. in press). Mortality was spread evenly through time, one
individual at a time. Flesh mass growth (d;»/d/) is driven by net
energy balance, with

dm/dt = C| -[(c, -CR-P- AE)-R] (1)

where r, is a conversion factor from J to mass (c, = O.OOO.'il;
Dauvin &. Joncourt 1989), c, is a parameter accounting for flow
effects on clearance rate (see later). CR is clearance rate (L day"' ).
AE is assimilation efficiency (dimensionless) and R is respiratory
losses (J day"'). To model phytoplankton in the pearl nets, we
assumed that pearl nets were analogous to tlow-through chambers,
with flow rate being directly proportional to current speed. It is
assumed finally that mixing is complete and that water displace-
ment by scallops has negligible effect on residence time. Phy-
toplankton conservation in the pearl nets is given by

dP/dt = (V ■ e,)"' • [c, ■ C4 ■ V ■ (P, - PJ - N • (P, ■

CR)]

(2)

where dP/dr is the rate of change of phytoplankton concentration
in the pearl nets. V is the volume of the hypothetical pearl nets, f,
and fj are parameters (see later). P, and P, are phytoplankton
concentration (J L"') at the inflow and the outflow of the pearl
nets, respectively, N is the number of scallops in the pearl nets. In
all simulations. P, = 15.6 J L"'. Assuming that the energy content
of phytoplankton is 10.4 J mg"' (Bayne & Widdows 1978.
Thompson 1984), this specifies a particulate organic matter (POM)
concentration equal to 1.5 mg P'. constant along the vertical.
Under the assumption of complete mixing within the pearl nets. P
is estimated by P, (Northby 1976).

Growth was simulated for 360 days with no seasonal signal in
P,. Temperature was not taken into account. The values of V (40
L), (S. f,. C4 and basic flow rate v (1000 L day"') were set arbi-
trarily to provide the desired conditions in the pearl nets-either
seston depletion or no depletion, and flow rate variations as a
function of height. The parameters cs and tj are the ratio of cunent
speed at height ; to current speed at 2 m above the bottom, c s and

Cj e {0.1, 0.2, 0.3 1 ). depending on the situation modeled.

They are used to mimic the effect on flow rate of change in cuirent
speed with height in the benthic boundary layer.

In the first set of runs where phytoplankton depletion in the
pearls nets is negligible, c, = 1000. In the second set of runs,
where depletion occurs, c, = 1. To model the impact of a positive

effect of current speed on CR, we assumed that c, and q increase
proportionally to current speed (Wildish et al. 1992) and ran the
model with is and Cj equal to 0.1, 0.2 1 successively. Clear-
ance rate (1 day'' ind.^'l was given by CR = 2A ■ 3.09 ■ m""'
after Vahl (1980).

To model the expected results of an indirect effect of cuiTent

speed, we set c, = 1 and c^ equal to 0.1. 0.2 1 as abme.

Clearance rate was given by CR = 24 ■ (5 • exp(-().4 • P ) -1-
3.09 • /;;'"'"). The exponential part of the equation allows realistic
mimicry of physiological regulation of clearance rate in response
to phytoplankton concentration, at least as observed in Plu-
copecten maf^cllaniciis (Bacon et al. 1998) and Argopecten irra-
dians (Cahalan et al. 1989). In the cases of direct and indirect
effects of current speed, AE = 0.5.

To model the effect of resuspension. we first constructed a
suspended inorganic sediment (PIM) concentration profde above
the bed. based on Rouse's theory, according to which we have C
= C„ • (fl/c)''. where C is sediment concentration ( mg T' ) at height
z above the bed. C_, is a reference concentration at a reference
height a (C„ = I mg l"'; a = 200 cm), and q = wJkU-. where
w^ is sediment mean fall velocity (n\ = 0.05 cm s"'). k is von
Karman's constant (0.41 ) and U. is friction velocity (U. = 1 cm
s"') (see e.g., Muschenheim 1987). Assuming that velocity above
the scallop bed followed the law of the wall, with U, = 1/ K"'ln(ry
z,,) (e.g.. Grant et al. 1984). and estimating z,, = 0.2 cm by means
of Nikuradse's parameterization of bed roughness iz„ = A:,, • 30"'.
where A,, = 7 cm. the approximate size of scallops and pebbles
recovered from scallop dredge tows on the donor site of our
growth experiment), we constructed an hypothetical tlow profile
above the bed. We then found the heights at which we had UJU2
e |0. 1. 0.2 1 I and deduced the conesponding PIM concen-
tration profile. Knowing PIM as a function of UJU^ (POM is
assumed to be constant across heights), we determined EA at
height as EA = sin-( 139.6-2. 14 • arcsin( 1 - (PIMKPIM
+POM))f^), as modified from Vahl (1980). In all cases, respira-
tion is given by P = (155.2 fxl O, ■ m'"*")-19.9 • IQ-' J jjilO."'
(Vahl 1978).

RESULTS

Results ol the growth simulations with and without depletion in
the hypothetical pearl nets are shown in Figure 1 and Figure 2,
respectively. All results are reported as a function of UJU2, which
scales as In ; under the assumption of a logarithmic current profile
above the bed. In all cases, growth at 2.0 m above bottom did not
vary because this was the reference level, and was assumed to have
constant current speed and suspended sediment concentration.
Growth at height c. however, decreased with decreasing C/if/,.
Without food depletion in the hypothetical pearl nets, growth de-
creased in a curvi-linear fashion with UJU2. with upward concav-
ity for both resuspension and a direct effect of current speed on
clearance rate as control mechanisms. Because there was no food
depletion to generate group size effects, data for different group
sizes overlap in Figure 1. Therefore there was no interaction be-
tween height above bottom and group size. With food depletion in
pearl nets, however, growth patterns were richer (Fig. 2). For
UJU. = 1. growth at height ; was higher than at 2.0 m because
mortality at the lower level was more severe than at 2.0 m and
therefore per capita food availability was higher. Resuspension and
a direct effect of flow on clearance rate resulted in a curvilinear
decrease of growth as UJU2 decreased, with upward concavity

Near-bottom Effects on Iceland Scallop Growth

89

50

A

-,

O)

/

c/, 40

-

/

(/>

/

m

4

E 30

-

/

CO

/

13

J

-D 20

-

/

>

•o

E 10
n

0.0

0.5

1.0

vertical and with phytoplankton depletion within the pearl nets
{Claereboudt et al. 1994a; Claereboudt et al. 1994b). may have
generated the height*group size interaction (Fig. 2A|. Because
within pearl net phytoplankton depletion is required, this mecha-
nism is different from the hypothesis of a straightforward negative
effect of poor seston quality on assimilation efficiency which is
usually invoked to explain near-bottom effects on growth of Ice-
land scallops held in pearl nets (Wallace & Reinsnes 1984. 1985;
Thorarinsdottir 1994). Clearly different group sizes should be used
routinely in growth experiments to test for containment effects.

The second mechanism tested, that is. a direct positive effect of
higher current speed on clearance rate (Wildish et al. 1992).
coupled with seston depletion within the pearl nets, also resulted in

O)

O

O

"D
>

C

20

10

0.0 0.5 1.0

Current speed ratio ^U ,/U 2)

Figure 1. Simulated soft tissue growth «i(h(iul pliytoplanl^ton deple-
tion in the hypothetical pearl nets. Diamonds: height : above bottom;
squares: 2 m above bottom: solid symbols: 5 individuals per hypo-
thetical pearl net: empty symbols: 1(1 individuals per hypothetical
pearl net. Here empty symbols are hidden by solid symbols. \: effect
of particle resuspension at the bottom. B: effect of positive relation
between current speed and clearance rate.

(Fig. 2A. B). An indirect effect of current speed, however, gener-
ated a response with downward concavity (Fig. 2C). In all three
cases, the difference between group sizes decreased with decreas-
ing UJUy

DISCUSSION

Our simulations indicate that decreasing UJU2 ratio resulted in
a height effect on simulated growth. However, phytoplankton
depletion within the hypothetical pearl nets was required to gen-
erate a height*group size interaction similar to that found in the
actual growth experiment (Frechette & Daigle. in press). The
chances of occuirence of a significant height*group size interac-
tion increased with decreasing height c because the effect of group
size on scallop growth decreased with decreasing current speed.
The lower the UJU2 ratio, therefore, the better the simulations
mimicked the experiment. The key factors implied here are seston
depletion within the pearl nets and vertical differences in cunent
speed owing to the BBL.

Our simulations show that the three mechanisms tested might
have generated the height*group size interaction. Vahl (1980) re-
ported that seston quality had a strong negative effect on growth of
Iceland scallops. According to our simulations, higher PIM near
the bottom, coupled with homogeneous POM concentration in the

12

S 10
(/>

CO

E

^ 6 -

to

"D
>

c

0.0

0.5

1.0

0.0

0.5

1.0

O)

o
E

o

"O
>

C

0.0 0.5

Current speed ratio {U 2IU2)

.0

Figure 2. Simulated soft tissue growth with phytoplankton depletion
in the hypothetical pearl nets. Symbols as in Figure 1. A: effect of
particle resuspension al the bottom. B: effect of positive relation be-
tween current speed and clearance rate. C: effect of clearance rate
regulation in response to seston concentration within the hypothetical
pearl nets.

90

Frechettb and Daigle

a heighI*group size interaction (Fig. 2B). It lias been argued, him-
ever, that the positive relationship between clearance rate and cur-
rent speed may be an artifact of flow removing seston depletion
envelopes around individuals (Lenihan et al. 1996). There is in-
deed evidence that phytoplankton concentration may be structured
in space (cm scale) by exhalent jets of suspension feeders (Moni-
smith et al. 1990; ORiordan et al. 1993; O'Riordan et al. 1993). In
addition. Claereboudt et al. (1994b) found no evidence of positive
effect of current speed on sea scallop growth. Although the ongo-
ing considerations provide little support for a positive response ot
clearance rate to cuirenl speed, it should be noted that Butman et
al. (1994) found such a positive response in a mussel population
contained in a flume.

The third mechanism tested, that is. an indirect effect of flow
mediated through changes in phytoplankton concentration within
pearl nets coupled with clearance rate regulation (clearance rate is
assumed to increase with decreasing phytoplankton concentration;
e.g.. Bacon et al. 1998). also resulted in a height*group size in-
teraction. The relation between growth and UJLK was curvilinear,
with downward curvature. In the case of resuspension and ot a
direct effect of flow, the relation was also curvilinear, but with
upward curvature. Therefore, assuming that regulation of clear-

ance rale in Iceland scallops resembles that of sea scallops and bay
scallops, results of a growth experiment with different heights
tested may provide information as to whether the height*group
si/e interaction was attributable mainly to physiological regulation
or to environmental forcing. We did not study the effect of inhibi-
tory high flows on clearance rate (Eckman et al. 1989; Wildish el
al. 1992) because it was inconsistent with lower growth near the
bottom.

In summary, our simulations suggest that the original seston
quality hypothesis alone is insufficient to account for the decrease
in growth of scallops near the bottom. An additional factor is
required, that is. seston depletion within the pearl nets and its
interaction with the BBL. Furthermore, two other mechanisms,
independent of resuspension. may explain the growth pattern in the
BBL. Both require food depletion within the pearl nets and inter-
action with the BBL. The first mechanism is a direct positive effect
of higher current speed on clearance rate and the second mecha-
nism involves clearance regulation In response to food depletion.
Because our simulations allow predictions about the profile of
growth across height, growth experiments may provide evidence
on the factor actually causing near bottom reduction in growth.
pro\ ided that the processes simulated above act independently.

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Claereboudt. M. R.. J. H. Himmelman & J. Cote. |y94h. Field evaluation
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Eckman. J. E.. C. H. Peterson & J. A. Cahalan. 1989. Effects of tlow speed,
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Frechette, M. & G. Daigle. In press. Growth, survival and tluctuating
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Grant, W. D., A. J. I. Williams & S. M. Glenn. 1984. Bottom stress
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Monismith. S. G.. J. R. Koseff, J. K. Thompson, C. A. O'Riordan & H. M.
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Muschenheim. D. K. 1987. The dynamics of near-hed seston dux and
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O'Riordan. C. A.. S. G. Monismith & J. R. Koseff. 1993. A study of
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O'Riordan. C. A., S. G. Monismith & J. R. Koseff. 1995. The effect of
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Parsons. J. G. & M. J. Dadswell. 1992. Effect of stocking density on
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Brunswick. Acpiaculrnre. 103:291-309.

Pilditch. C. A. & J. Grant. 1999. Effect of variations in How \elocity and
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Thompson. R. J. 1984. The reproductive cycle and physiological ecology
of the mussel Myiilus eihilis in a subarctic, non-estuarine environment.
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Thorarinsd(ittir. G. G. 1994. The Iceland scallop. CliUimys isUunlica (O. F.
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Vahl. O. 1978. Seasonal changes in oxygen consumption of the Iceland
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143-1.54.

Vahl. O. 1980. Seasonal variations in seston and in the growth rate of the
Iceland scallop. Clilamys islandica (O.F. Miiller) from Balstjord. 70 N.
./, £a/;. Mar Biol. Ecol. 48:195-204.

van Haren, R. J. F. & S. A. L. M. Kooijman. 1993. Application of a

Near-bottom Effects on Iceland Scallop Growth

91

dynamic energy budget model to \'Iynhi\ Cihilis (L.). Neth. J. Sea Res.
31:119-133.
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Wallace, J. C. & T. G. Reinsnes. 1984. Growth variation with age and
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Journal of Shellfish Rexfunh. Vol. 21, No. 1, 93-101. 2002.

ULTRASTRUCTUR-\L CHARACTERISTICS OF SPERMATOGENESIS IN DIPLOID AND
TRIPLOID CATARINA SCALLOP (ARGOPECTEN VENTRICOSUS SOWERBY II. 1842)

ROSALIO MALDONADO-AMPARO AND ANA M. IBARRA

Aqiuicidturul Genetics Laboratory. Centra de Investigaciones Biologicas del Noroeste. La Paz

Mexico

ABSTRACT Spermatogenic stages in diploid and triploid catarina scallop (Argopecten ventricosus) were studied by light and
electron microscopy at two different ages. In nine month old triploid scallops, a reduced number of spermatogonia and pnniary
spermatocytes were seen in the male acini, and although no spermatids were found, a few spermatozoa were observed. All triploid
spermatogenic stages and their nuclei were larger in diameter than those in diploids. Ultrastructure analysis reveled that spermato-
genesis in tnploids was almost completely arrested early, at the primary spermatocyte stage, indicating the halt occurs during the
prophase stage of meiosis 1. before the first reductional division. At age 12 months a large number of hemocytes invade some of the
remaining male acini of triploids. which possibly have a trophic role after development is halted. At this age. the male part of the gonad
in most analyzed scallops is completely replaced by female acini, with some developing oocytes. Checkpoint mechanisms recently
described as "meiotic checkpoints" are proposed as possibly being involved in the partial or total sterility resulting from the triploid
condition.

KEY WORDS: Argopecten renlricosiis. sterility. EM. triploid. pectinid. spermatogenesis, meiotic-checkpoint

INTRODUCTION

Triploid sterility has been reported for a number of mollusk
species, but in most cases the sterility is not reflected in a complete
lack of gametogenic stages, but rather on a delayed process and a
reduced number of fully developed gametes (Allen et al. 1986;
Komaru & Wada 1989, 1990; Allen 1987; Allen & Dov.'ning 1990:
Guo & Allen 1994b; Co.x et al. 1996; Eversole et al. 1996; Kiyo-
moto et al. 1996: Ruiz-Verdugo et al. 2000). Whereas the most
common characteristic in triploid mollusks has been reported to be
the reduced development of the gonad and gametes, the reversal of
gonad sex in a known functional hermaphrodite has been only
reported to occur in catarina scallop (Ruiz-Verdugo et al. 2000).

In as much as several studies comparing diploids and triploids
have been done on gametogenic stages by light microscopy, partial
or total ultrastructure analysis of abnoimal gametogenesis in poly-
ploid mollusks has been done only for triploid Pacific oyster Cras-
sostrea gigas (Komaru el al. 1994), triploid Sydney rock oyster
Saccostrea commercialis (Cox et al. 1996), and triploid and tetra-
ploid Mediterranean blue mussel Mytihis giilloproviiicialis (Ko-
maru et al. 1995: Kiyomoto et al. 1996). Spermatogenesis in dip-
loid catarina scallop Argopecten ventricosus at the ultrastructure
level has not been studied before, although it has been done for
other pectinid species, Pecten mcLximits (Dorange & Le Pennec
1989a: Beninger & Le Pennec 1991).

The catarina scallop, Argopecten ventricosus. is a functional
hermaphrodite pectinid in which the gonad is divided into two
parts, one female and one male. When triploidy is induced in this
species, a gradual suppression of functional hermaphroditism has
been noted to occur, with only female gametogenic stages devel-
oping in the male part of the gonad in one-year-old scallops (Ruiz-
Verdugo et al. 2000). As reported for oysters (Guo& Allen 1994a)
and Manila clams (Utting et al. 1996), as well as for this panicular
species (Ruiz-Verdugo et al. 2001 ), the number of oocytes formed
was reduced when compared to diploids. In a continuance of those
studies with triploid catarina scallop, we report here the effects of

triploidy on spermatogenesis, studied as the comparative structure
and ultrastructure of 9-nionth-old and 1 2-month-old diploid and
triploid catarina scallops.

Corresponding author. Ana M. Ibarra Aquaciiluiral Genetics Laboralon:
Centra de Investigaciones. Biologicas del Noroeste. S.C. A.P. 128. Mar
Bermejo # 195. Col. Playa Palo Santa Rita.La Paz B.C.S. 23000. /Mexico.
FAX: -I- 52-612-125-3625: E-mail: aibarra@cibnor.mx

MATERIALS AND METHODS

Scallops

Diploid and triploid catarina scallops about nine months age (6
cm in shell length), kept in a growout urea at Bahi'a Magdalena in
Baja California Mexico, were transported to the Genetic Labora-
tory of CIBNOR (Centro de Investigaciones Biologicas del No-
roeste). and placed under inaturation conditioning for 20 days. A
second group of scallops that were not conditioned for maturation
in the laboratory was sampled from the field area at age 12 months.
The triploid scallops were produced using cytochalasin-B (0.5 mg/
L) as described by Ruiz-Verdugo et al. (2001). and the diploids
were from a control group the same age.

Maturation Conditioning

Each ploidy group was stocked (five scallops per 40-L tank)
and kept under standardized controlled maturation conditions dur-
ing 20 days (Ramirez et al. 1999). Feeding consisted of adding a
mixture of Isochrysis gulhana. Monocluysis lutheri, and Chaeto-
ccros muelleri at a total concentration of 4 x 10** cells / scallop /
day. Water temperature was kept at 19-20°C. salinity at 36%f, and
dissolved oxygen from 7-9 mg / L. The maturation room was
provided with a 12-hour light and 12-hour dark photoperiod.

Sampling and Histology for Light Microscopy

After the 20-day maturation-conditioning period, the gonads
from 20 scallops per ploidy group were fixed in buffered formal-
dehyde ( 109r), dehydrated in a graded ethanol series, and embed-
ded in paraffin. Duplicated sections, 5 (xin thick, were obtained
from each gonad, and stained with hematoxilin-eosin (H&E) and
Feulgen.

Spermatogenic stages in diploid and triploid scallops were
measured using image analyses. Images were taken with an Olym-
pus BX-41 microscope, with an integrated camera (CoolSNAP-
Pro). The images were analyzed with SigmaScan Pro 5, obtaining
an area by digitalizing contours and estimating diameters from the

93

94

Maldonado-Amparo and Ibarra

area for cytoplasm and nucleus of each spermatogenic stage. To
correct for scale differences, for each microphotograph evaluated
the image analysis program was calibrated accordingly to the mi-
croscope objective used for those images. For each ploidy group
and stage (spermatogonium, spermatocyte, spermatid, and sperma-
tozoa), 30 cells were measured.

Diameters of each spermatogenic stage for the two ploidy
groups were analyzed with a single factor (ploidy) ANOVA, and
mean differences tested with the F-test (Neter et al. 1985). Sig-
nificance was set at P < 0.05.

To establish the impact of triploidy on both nuclei and cell size,
a ratio between nucleus diameter and total cell diameter was esti-
mated by dividing the nucleus diameter by the total cell diameter.
Additionally, the increase in area (Area = 3.1416 x radius") in
triploids was estimated for each cell and nuclei from the following
relationship; |(Area in 3n x 100 / Area in 2n ) - 100].

Electron Microscopy

Five gonad samples from each ploidy group were taken 20 days
after laboratory conditioning (9-month old) as well as five from
each ploidy group from 12-month-old field scallops.

The methodology described by Komaru et al. (1994) was fol-
lowed for transmission electron microscopy (TEM). Gonad
samples (2 mm") were fixed with a 4% gluteraldehyde solution in
0.1 M Sorensen's phosphate buffer, pH 7.5. for two hours at 4 'C.
The pieces were then washed in Sorensen's buffer solution three
times for 30 minutes, and post-fixed in \% osmium tetraoxide
(OsOj) in the same buffer for one hour, at 4°C. Samples were then
dehydrated in ethanol (25, 50, 70, 95, and 100%) for 20 minutes in
each concentration, and twice in 1009f propylene oxide for 20
minutes. They were embedded according to the protocol of the
commercial Kit Embed 812 Electron Microscopy Sciences (Luft
1961). Sections of 900A were obtained with an ultramicrolome
(Reicher, Model 0MU3), mounted on a grid and stained with 29c
uranyl acetate for 20 minutes and lead citrate for five minutes
(Komaru et al. 1994). Photo microscopy was done using a JEOL
JEM-1200EX1ITEM.

Diameters of cell and nuclei of spermatogenic stages captured
by photographic prints of TEM were measured after scanning the
photomicrographs. To correct for amplifications, the size bar of
each EM photomicrographs was used as a reference. All measure-
ments were done using the image analyzer SigmaScan Pro 5. The
number of stages measured varied, and depended on the number
found in photomicrographs (6 spermatogonia, 15 spermatocytes
and spermatids, 30 spermatozoa nucleus, mitochondria, and tla-
gella, and 21 for acrosome length and invaginations in spermato-
zoa). The low number of captured spermatogenic stages in the
triploids did not allow for measurement.

Scanning electron microscopy (SEM) gonad samples (4 mm")
were fixed, post-fixed, and dehydrated in the same manner.
Samples were dried in a SAMDR1-PVT-3P critical point dryer and
coated with gold in an EDWARDS S150B sputter coating system
(Komaru et al. 1994). Photo-microscopy was done using a JEOL
JSM-5410LV SEM. All electron micro.scopy work was done at the
Institute of Cellular Physiology in the National Autonomous Uni-
versity of Mexico (UNAM).

RESULTS

Gonad stmcture of nine-month diploid and triploid scallops are
presented in Figure 1. In diploid scallops the characteristic syn-

chronized development of sperm and oocytes in the gonad was
seen (Fig. I A). Compared to diploids (Fig. IC). gametogenesis in
triploids was retarded (Fig. IB), and spermatogenesis was largely
abnormal with only a reduced number of acini showing develop-
ment of spermatogenic stages (Fig. ID).

A summary of differences in mean size of spermatogenic stages
between the two ploidy groups is in Table 1. The acini of triploid
scallops were significantly larger (55%. P = 0.01) than those in
diploids. There were also significant differences between ploidy
groups in the diameter of all spermatogenic stages, as well as in
their nucleus. Triploid scallops not only had larger nuclei area in
spermatogonia and spermatocyte than diploid scallops (37% and
1 12% larger area in 3Ns. respectively), but also more cytoplasm
area (78% and 1 19% more in 3Ns). Spermatids were not found in
triploids, and comparative measurements between ploidy groups
were not possible, but the few spermatozoa found indicated that
triploids had a 50% larger area than diploids (Table 1 ). The
nucleus/total cell ratio of spermatogonia was smaller for triploids
(0.67) than diploids (0.76), whereas for spermatocytes it was ap-
proximately the same ratio for both ploidy groups (0.88 for 2Ns,
0.86 for 3Ns).

Diploid Male Gonad Structure and Vltrastructure

The details of the male part of the gonad of a diploid scallop are
shown in Figure 2 and Figure 3. Male germinal cells were grouped
in acini ranging from 120 to 200 ixm in diameter. Each acinus
contained a variety of developing stages distributed in a centripetal
pattern from the inner acinus wall to the lumen (Fig. 2A). The
spermatogonia (Spg) are oval shaped, and located nearest to the
inner wall, whereas the spermatocytes (Spc) are smaller and loose
from the acinus wall, positioned between the Spg and the lumen of
the acinus. Mature spermatozoa (Spz) are almost exclusively con-
fined to the central lumen of the acinus (Fig. 2A). The Spg had a
diameter in ultrastructure measurements of 6.95 [jim. and a nucleus
4.39 p-iii. The Spc appeared to be more numerous than the Spg
(Fig. 28). with a diameter approximately 3.92 |xm. and a nucleus
2.93 p.m. The Spc cytoplasm contained a complement of or-
ganelles, Golgi bodies (Gb) and mitochondria (Mi), very similar to
those in the spermatogonia. The nucleus of primary spermatocytes
(Spcl ) exhibits scattered electron-dense chromatin (Fig. 2C), and
its cytoplasm contains some Mi and Gb, Spermatids (Spm) in their
early stages are spherical cells about 2.63 (jtm in diameter by
ultrastructure measurements, with a dense nucleus, also spherical,
about 1.95 |jLm in diameter (Fig. 2D). In the cytoplasm of the
maturing Spm. Mi can be seen in what will be the basal pole of the
future Spz.

The Spz have a proximal acrosome (Ac), nucleus (Nu). and a
maximum of five cross-sectioned Mi in its distal part (Fig. 3A).
where the tlagellum (Fl) emerges. The anterior part of the sper-
matozoa, where the acrosome is. appears slightly denser than the
nucleus. The ultrastructure of the spermatozoa in diploid Ar-
gopecten ventricosus can be divided into three main components:
a sperm head consisting of the Ac and Nu, a middle piece con-
sisting of two centrioles and the mitochondria, and a tail or fla-
gella. The acrosome is invaginated toward the nuclear surface
forming a conical structure about 0.51 [xni in height, and it is
composed of an outer layer with electron dense material and an
inner layer of electron lucent material (Fig. 3A). The spherical
electron-dense Nu is 1 .82 |xm in diameter and presents an anterior

Spermatogenesis in Diploid and Triploid Scallop

95

-^-J^f

Figure I. Diploid and li iplcjid liLiht photomicrographs. A. Diploid female and male gonads in advanced stages of gametogenesis. B. Triploid
female and male parts of the jjonad at 9 months old. C". Diploid male portion of the gonad in advanced stage of spermatogenesis. D. Triploid male
part of the gonad with few spermatogenic stages developing.

invagination (Ai) 0.23 [xm in depth (Fig. 3A). and a posterior
invagination (Pi) 0.29 ^jim in depth (Fig. 3B). The distal mid-part
of the spermatozoa head contains a ring of three to five cross-
sections of mitochondria, each about 0.76 jjim in diameter around
two centrioles (Fig. 3B. 3C). The proximal centriole (Pc) is joined
to the nuclear envelope by a satellite body (not shown) found in the
post-nuclear fosse. The distal centriole (Dc) is joined and forms the

TABLE 1.

Mean diameter (SD) of spermatogenic stages (n = 30) in diploid and

triploid catarina scallop Argopecten veiitricosus measured bv image

analyses — light microscop), and percent increase area in triploids

with regard to diploids.

Diameter (jini)

Spermatogenic stages

Diploid

Triploid

increase in 3N

Acinus'

167(36)°

259(47)*"

141

Spermatogonia'

6.30(0.43)°

8.41 (1.64)''

78

Spermatogonia nucleus"

4.81 (0.36)"

5.62(1.11)"

37

Spermatocyte"

3.81 (0.31)-'

5.64(0.44)''

119

Spermatocyte nucleus"

3.34(0.32)-

4.86(0.36)"

112

Spermatid'

2.62(0.25)

n.a.

Spermatozoa' head

1.69 (0.08 )■■

2.07(0.15)"

50

Number of sperm/

1000 (im-

250.000

2-3

Superscript numbers in spermatogenic structures indicate magnification
used for measurements: 1 = 4x; 2 = I Ox; 3 = lOOx. n.a. = not available
for measurements. Different letters between pioidy groups indicate signifi-
cant differences (P < 0.05).

basal body of the Fl, which has a diameter of 0.31 |j.ni. Granules
of glycogen (Gi) are delected between the mitochondria cross-
section (Fig. 3B). Transversal cuts of the tlagellum show the clas-
sical structure of nine external microtubule doublets (Md) and two
internal single microtubules (Fig. 3D).

Triploid Male Gonad Strucliire and Ultraslriiclure of Nine-Montli-Old
Scallops Conditioned in the Laboratory

Male acini of triploids were larger than in diploids, with a
diameter varying from 197 to 387 p.m. On the acinus wall dark
colored cells representing Spg can be seen, and detached from the
acinus wall some Spc were present. In some of the triploid scal-
lops. Spz were seen sparsely distributed in the lumen of the acinus
when using the Feulgen stain (Fig. 4A), and also when using
scanning electron microscopy (Fig. 4B). The overall structure of
the spermatozoa produced by a triploid catarina scallop appears to
be similar to those produced by a diploid.

The spermatogonia were oval, and had the chromatin uniformly
dispersed in small aggregations, with numerous mitochondria in
the cytoplasm (Fig. 4C). As in diploids, the primaiy spermatocyte
had a smaller size than the spermatogonia, and their cytoplasm
contains a complement of oiganelles very similar to the spermato-
gonia (Fig. 4D). In the nucleus, the chromatin, set out in clusters,
indicates the spermatocytes were in zygotene-pachytene stages of
prophase I. There was no evidence in transmission electron mi-
croscopy of secondary spermatocytes and spermatids, although as
already stated, some spei'inatozoa were observed by scanning elec-
tron microscopy (Fig. 4B).

96

Maldonado-Amparo and Ibarra

Figure 2. Diploid gonad structure and ultrastructure. A. Light microscopy (LM) of male acinus with spermatogonia (Spg) attached to the wall
(W), spermatocytes (Spc), and spermatozoa (Spz). B. TEM of sperniatogenic stages: Spg. Spc. spermatid (Spni), and Spz. C. TEM of primary
spermatocyte with multiple mitochondria (Mi) and Golgi bodies (Gb). D. TEM of Spm with mitochondria at posterior pole, transversal cuts of
flagella (Fl).

Triploid Male Gonad Strucltire and Vllrastructure of Field Scallops
Sampled at 12 Months Old

The male part of the gonad in some triploid catarina scallops at
age 12 months had a mixture of male and female acini, with both
male and female gametogenic stages occurring in different acinus.
However, most of the male germinal cells were in the process of
degeneration. Large numbers of hemocytes were invading the male
acini (Fig. 5A, 5B). which were characterized by the presence of
degenerating spermatogonia and primary spermatocytes (Fig. .SC).
The hemocytes were of the basophilic granulocyte type, with a not
centrally positioned oval-rounded nucleus 4.5 \x.m in diameter,
condensed chromatin, and an entire cell approximately 8 p,m in
diameter (Fig. 5D).

In most triploid scallops a gonad sex change occurred, as the
male part of the gonad (the most distal part) was completely sup-
pressed and female germinal structures were replacing the male
germinal structures (Fig. 6A). Oocytes developing in the male part
of the gonad of triploids (Fig. 6B) were similar to those found in
the female part of the gonad of a diploid (Fig. 6C). They contained
numerous vitelline inclusions, cortical granules and a vitelline
coat.

DISCUSSION

The ultrastructure of diploid spermatogenesis was similar to
that described by Dorange and Le Pennec ( 19S9a) for Pecten ma.xi-
mus. with some size and morphology differences which is not
surprising, as closely related species are known to have similar but

not identical ultrastructure of gametic stages (Le Pennec & Be-
ninger 1997).

Differences in cell size between triploid and diploid moUusks
have been reported only for egg size (Guo & Allen 1994a, 1994c:
Eversole et al. 1996; Utting et al. 1996; Ruiz-Verdugo et al. 2001 )
or sperm size (Komaru et al. 1994), but in triploid fish species an
increase in size of different cell types has been reported (Valenti
1975: Ueno 1984: Small & Benfey 1987: Aliah et al. 1990). In the
present study not only spermatogonia, but also all other sperniato-
genic stages measured indicated a larger cell size in triploids than
diploids. Fuilherniore. an increase in spermatogenic stage size was
not necessarily a correlate of increase in nucleus size. For example,
there is an expected increase in the nucleus/total cell ratio of sper-
matocytes when compared to that same ratio in spermatogonia
occurring as a consequence of nuclear distention (Dorange & Le
Pennec 1989a). However, whereas the increased ratio in sperma-
tocytes was observed in this study for both ploidy groups, the
nucleus-total cell ratio in spermatogonia was smaller for triploids
than diploids, and this occuned despite the larger nucleus in trip-
loid spermatogonia than in diploids. This, as well as the increase in
area estimated for triploid spermatogonia cell and nuclei indicates
that a larger increase in cell size than nucleus size occurs in triploid
spermatogonia of catarina scallop, and might be caused by a larger
number of organelles (mitochondria, endoplasmic reticulum, ribo-
somes) than tho.se found in diploids, as previously proposed by
Guo and Allen ( 1994a) to explain the increased egg size of triploid
oysters. These authors proposed that the increase egg size could be
a consequence of a larger nucleus requiring a larger cytoplasm.

Spermatogenesis in Diploid and Triploid Scallop

97

500 nm

^i^*^*^ 500 nm

Figure 3. Diploid spermatozoa ultrastructure. A. TEM of section througli a spermatozoon head, sliowinj; airosome (Ac), anterior invagination
(Ai), nucleus (Nu), and two cross-sections of mitochondria (Mi). B. TEM section of the spermatozoa mid-part; posterior invagination (Pi),
proximal centriole (Pc), distal centriole (l)c), glycogen granules (Gi), mitochondria (Mi) and llagella (Fl). C. TEM of transverse section through
the mid-part of spermatozoa showing five cross-sections of mitochondria at most. D. TEM of transverse section through flagella showing the
classical structure of 9 external microtuhule doublets (Md). and 2 internal.

such that allocation of nutrients and organelles is in adequate pro-
portions during cell divisions. Unfortunately, because of the few
spermatogenic stages found in triploids when electron micixiscopy
was done, quantification of the number of organelles could not be
accomplished.

The spermatozoa of triploid catarina scallop was morphologi-
cally similar to that in diploids, although as shown by Komaru et
al. (1994) for the Pacific oyster, it showed a larger head than that
in diploids. The diameter differences observed between diploid
and triploid spermatozoa of catarina scallop correspond to an area
for triploid sperm of 1.5 times that of a diploid sperm, and is most
probably a consequence of an increased amount of DNA in the
sperm produced by triploids. Komaru et al. (1994) found an in-
crease area of 1.4 times in sperm from triploids when compared
with diploids, and the increase in area in triploid spermatozoa of
the Pacific oyster has been shown to agree with an increased
amount of DNA (1.5 of that in sperm from diploids) by flow
cytometry (Allen 1987; Guo & Allen 1994a). In spite of the size
increase, the spermatozoa of triploid catarina scallop had the same
number of cross-sectioned mitochondria than diploids. A larger
number of organelles in gametic cells have been demonstrated
only for tetraploid Mediterranean blue mussel, but not for sperm
produced by triploid Pacific oyster. That is. Komaru et al. (1995)
found that spermatozoa from tetraploid Mediterranean blue mus-
sels had from 5 to 7 mitochondria with a mode of 6, whereas that
from diploids had 5. Contrary to that, sperniatozoa from triploid
Pacific oyster, although larger than that in diploids, had the same
number of mitochondria as diploids (Komaru el al. 1994).

The observation of spermatozoa in triploid catarina scallop,
even if in low numbers was important because previously. Ruiz-
Verdugo et al. (2000) were unable to observe sperm in triploids
from this same species, but their histology techniques were limited
to H&E staining and light microscopy. In the present study, the use
of the Feulgen stain for light microscopy allowed for the finding of
some spermatozoa in isolated acinus, an obser\ ation that was cor-
roborated when spermatozoa were also found by scanning electron
microscopy. However, even if present, the rare occasions in which
spermatozoa were found indicates that the triploid condition in
catarina scallop results for the most part in an anest of spermato-
genesis early during the process of gamete formation, at the pri-
mary spermatocyte stage. That is, the arrest occurs at meiosis I,
when the secondary spermatocyte would be produced by the first
meiotic reduction (Beninger & Le Pennec 1991). The nearly com-
plete arrest of meiosis at the primary spermatocyte stage has been
previously noted by ultrastructure analysis in other bivalves such
as Saccostrea commercialis (Cox et al. 1996) and Mytilus gallo-
provincialis (Kiyomoto et al. 1996). Contrary to that, in triploid
Crassostrea gigas spermatogenesis has been reported to be re-
duced, but not arrested al the primary spermatocyte as spermatid
and spermatozoa production occurs (Allen & Downing 1990), the
last one being capable of fertilizing eggs (Guo & Allen 1994a).

The finding that in older triploid catarina scallop the male
portion of the gonad was replaced with female acini is similar to
that reported by Ruiz-Verdugo et al. (2000) after studying the
annual gainetogenic cycle of diploids and triploids for this scallop.
The few gametic stages still present in the male acini of scallops

98

Maldonado-Amparo and Ibarra

Figure 4. Triploid structure and ultrastriiilure of male part of the gonad. A. \M of section througli a male acinus witli few spermatogonia (Spg)
hv the Hall (\V(. spermatocytes (Spc). and spermatozoa (Spz). B. Scanning electron microscopy (SEM) of spermatozoa found in a male acinus,
with electron dense nucleus (Nu), acrosome (Ac), mitochondria (Mi) at the base, and flagella (Kl). C-D. TEM of section through a male acinus
with spermatocytes (Spcl), spermatogonia (Spg), and haemocyte (He).

12 momhs old were being lysed and possibly recycled through
phagocytic activity, as demonstrated by the presence of laige num-
bers of hemocytes. which are known to play a role in phagocytosis
of degenerating pectinids gametogenic structures (Dorange & Le
Pennec 1989a. 1989b; Beninger & Le Pennec 1991). The
hemocytes were the typical ones described for other bivalves such
as Mya areiiaria and Mytihis edulis (Cheng 1981 ).

With this study we have demonstrated that the male gonad of
triploid catarina scallop is almost but not completely sterile, as the
lack of male gametes occurs until after spermatogenesis begins and
is arrested, observing later oogenesis in male acini. The cause of
the reversal of sex in male acini of catarina scallop is not known,
but might be a consequence of the arrest of meiosis signaling,
possibly through a neuroendocrine mechanism, an arrest of syn-
thesis or release of some type of maleness factor required for
spermatogenesis. Neuroendocrine factors are known to be neces-
sary for development of male and female gonad in some mollusks
(Feral et al. 1987), or only needed for development of the male
gonad in functional hermaphrodites for which female gonad results
from auto-differentiation (Gomot & Griffond 199.'?). The existence
of those neuroendocrine factors has been demonstrated bv isola-
tion in other mollusks (\an Minnen et al. 1989). but not in pec-
tinids.

With regard to the arrest of meiosis. it has generally been
presumed that the primary cause for triploid sterility, measured as
inability to fonn viable and large number of gametes as in diploids,
is the inability of chromosomes to pair and complete synapses
because of multivalent formation. However, it has been demon-
strated at least for one species that this is not the cause of sterility.
Guo and Allen (1994a) established that for the Pacific oyster.

Crassostrea gigas. the normal synapse of chromosomes and nor-
mal segregation was not a necessary event to obtain mature ga-
metes in triploids, as they observed that the extra set of chromo-
somes segregated randomly in spawned eggs from triploids. To
explain the sterility induced in triploids it is interesting to point to
new research being done in the molecular genetic field. During the
last decade a series of "housekeeping" mechanisms or "check-
points" operating in the cell cycle during meiosis have been de-
scribed for a number of experimental organisms. Their function is
to detain further de\elopment when problems during replication,
recombination, or segregation are present in a cell, and it is pos-
sible to explain some of the abnormal events occurring in triploids
gametogenesis by considering them. These checkpoints act by
means of protein complexes signaling abnormalities in chromo-
some behavior, and effector proteins acting on those signals to
delay or arrest the meiolic process (Roeder & Bailis 2000). For
example, among the described checkpoint mechanisms for meiosis
is a "S-phase or premeiotic replication checkpoint." which func-
tions on detecting chromosomal lesions after replication, but be-
fore recombination (Murakami & Nurse 1999). A second check-
point mechanism, the "recombination or pachytene checkpoint." is
known to act on gametogenic cells that have not completed cor-
rectly the recombination between sister chromatids during meiosis
I. or in which the synaptotemal complex is defective, and it will
arrest meiosis at pachytene (Murakami & Nurse 1999. 20()0;
Roeder & Bailis 2000; Tarsounas & Moens 2001). A third check-
point mechanism, the "metaphase checkpoint," has been described
for arresting meiosis in metaphase I. by blocking the metaphase to
anaphase transition when chromosomes are misaligned on the
spindle apparatus. An interesting aspect of this checkpoint mccha-

Spermatogenesis in Diploid and Triploid Scallop

99

►»f^i

"^-^i^

%.

T, .-^_

fi

B

He

Dsc-^ «^ 45^ "•

ftlf^

A

;« ^^

Mi

Figure?. Triploid structure and ultrastructureshowingdegtniratioii ol male spermaliimiiii stages. \. I,M (it section through a male acinus with
spermatogonia (Spgl and spermatocytes (.Spc) closest to the Hall, and acinus tilled with haeniocytes (He). B. LM of connective tissue (Ct) at acinus
wall with He. C. TEM of Spc in degeneration process (Dsc), surrounded by multiple He. I). TEM at larger amplillcation of He and Dsc.

*•'>

Nu

'••' Vv^. :?**•: ,-..^ ,vi

^

:i:.vv.-Ve V,

'i^h r

■*-. <

• 5 pm ,

•'•

s . *

i.v :

■-*: •■.•.■; ••..;•.

:#si."

• • . "S' , -

^u'.v;.- \ .;:

•

!?*■.;;

. ;i' :J^ \ J.

;-.^

'Nu ;

. %^?vM..-'

''\<^r\^

•^j'S. •

' S ''

J-A

. .*.-

' X ■ 4 *■ '

..:,\-.^^5

$?*■

■<^^-;

,' lOjum J

Figure 6. Gonad structure and ultrastructure ol 12-ni(> old scallops. A. LM of male part of gonad in triploid occupied with female acini with
oocyte development. B. TEM of oocytes in male part of the gonad of a triploid, showing nucleus (Nu), vitelline inclusions (Vi). cortical granules
(Cg). and vitelline coat (Vc). C. TEM of oocytes from a diploid scallop with the same structures than that in triploids.

100

Maldonado-Amparo and Ibarra

iiisni is that it is l<nown to operate in inammalian males, halting
spermatocytes at metaphase I, but oocyte formation is not affected
or detained even when missegragation will result (for review see:
Roeder & Bailis 2000). By considering the possible phenotypic
effects of these three checkpoints on gametogenesis, we can par-
allel some of the known events in triploid gametogenesis. For
example, the delay in the onset of gametogenesis known to occur
in both sexes of triploids (Allen et al. 1986; Allen 1987; Allen &
Downing 1990; Guo & Allen 1994b; Cox et al. 1996; Eversole et
al. 1996; Kiyomoto et al. 1996; Komaru & Wada 1989. 1990;
Ruiz-Verdugo et al. 2000) can be explained by the first checkpoint
mechanism, the "replication checkpoint." If we can presume that
DNA replication in triploids will have a larger probability of errors
needing correction because more DNA is present, that could result
in a delayed onset of gametogenesis. The delay reported to occur
in triploid gametogenesis and not necessarily on their growth can
be explained by a difference between meiosis and mitosis: the
S-phase in meiosis usually takes from two to five times longer than
the S-phase in mitosis (Murakami & Nurse 2000). The second
checkpoint mechanism, the "pachytene checkpoint", agrees well
with the observation that spermatogenesis is arrested at the
pachytene-zygotene stage of prophase in meiosis I, at the primary
spermatocyte, and also agrees with the fact that a lower number of
vitellogenic and mature oocytes are formed in triploid catarina
scallop than diploids (Ruiz-Verdugo et al. 2000, 2001), as it is
known that in diploid scallops only oocytes that complete the
pachytene stage and enter the diplotene stage will begin the pro-

cess of vitellogenesis and become mature (Dorange & Le Pennec
1989b; Beninger & Le Pennec 1991 ). Further research on oocyte
type frequencies in diploid and triploid mollusks are necessary to
understand precisely at which stage oogenesis is halted.

In conclusion, this study has established spermatogenetic dif-
ferences between diploid and triploid catarina scallop Aii^opeclen
ri'iinicdsiis. pointing toward the halting of spermatogenesis occur-
ring during prophase of the first meiotic division. Future studies
are necessary to establish the level of ploidy and functionality of
the few produced male gametes in triploids.

ACKNOWLEDGMENTS

This research was supported by CONACYT grant 28256-B
to A. M. Ibarra. We thank Carmen Rodriguez-Jaramillo from
CIBNOR for histology technical support, and Dr. Mark West,
Rodolfo Paredes and Jorge Sepulveda from the Institute of Cellular
Physiology of the UNAM for their support to carry out the ultra-
structure analyses. We thank Dr. Elena Palacios for valuable sug-
gestions on the manuscript. We also thank Dr. Alejandro Lopez-
Cortes from CIBNOR for his advice on preserving tissues for
ultrastructure analysis, and Sergio Rosas and Gerardo Hemandez-
Garci'a for the photography prints. The Scan Pro program used for
the image analyses was that from Dr. Mario Monteforte at
CIBNOR. The senior author is a CONACYT and SEP (DECYTM)
Ph.D. fellow, and the results presented here are part of his thesis.
Dr. Ellis Glazier edited the Eniilish lannua.ae.

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241:339-342.

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Utting. S. D.. P. F. Millican & 1. Laing. 1996. The breeding potential and
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Valenti. R. J. 1975. Induced polyploidy in Tilapia aurea (Steindachner) by
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van Minnen, J.. R. W. Dirks. E. Vreugdenhil & J. van Diepen. 1989.
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Joiirihil of Shellfish Reseiiivh. Vol. 21. No. 1. 103-108. 2002.

REPRODUCTIVE CYCLE OF SPONDYLUS CALCIFER CARPENTER, 1857 (BIVALVIA:
SPONDYLIDAE) IN THE "BAHIA DE LORETO" NATIONAL PARK, GULF OF

CALIFORNIA, MEXICO

MARCIAL VILLALFJO-FUERTE, MARCIAL ARELLANO-MARTINEZ.
BERTHA P. CEBALLOS-VAZQUEZ, AND FEDERICO GARCIA-DOMINGUEZ

Centni liuevdisiiplimirio de Ciencias Marinas, histihilo Folireciiico Nacional. Apartado Postal 592. La
Paz. B.C.S. 23000. Mexico

ABSTRACT The reproductive cycle of SpoinlYlux calcifcr lYom Bahia dc Loreto. Gulf ofCalitornia, was studied from January 1998
to March 1999. Microscopic analyses established that this species is gonochoric. The size at spawning occurred at 1 13-mm in shell
height. S. calcifer e.xhibits a short spawning period from August to October followed by a long inactive period from November to
February. Increase in water temperature initiates the gonadal maturation process, while decrease in water temperature inhibits
gametogenesis. The threshold water temperature for spawning in S. calcifer. is 29°C. Nutrient reserves from the digestive gland are
mobilized and used by the gonad during the developing stage. Meanwhile, reserves stored in the muscle are used only after gamete
production has started, helping to support the energetic output during the ripe and spawning stages. In Bahfa de Loreto, the period of
highest phytoplankton abundance in bottom waters coincides with the onset of gametogenesis in .S. calcifer. This suggests that S.
calcifer also depends on food intake for oocyte growth.

KEY WORDS: spawning season, histology, energy transfer. Spoiulyliis calcifer. Gulf of California

FNTRODUCTION

Spaiidylns calcifer Cuipenter. 1857 cotntnonly natned "Buitu
clam." belongs to the Spondylidae Family. It is geographically
distributed from the Gulf of California to Peiu. This clam can be
found from intertidal to subtidal zones, attached by the right valve
to exposed boulders or under rock ledges (Skoglund & Mulliner
1996). At present in Loreto, Baja California Sur. Mexico, this
species is not commercially exploited; however divers capture it
(only in small c|uantities) for human consutiiplion year around.

This clam is the largest species of the genus Spondxlus. S.
calcifer is considered in danger of extinction (Baqueiro et al.
1982). and is currently protected by Mexican laws (Norma Oficial
Mexicana 1994). Despite the above, there are no published records
on its biology. Few articles have been published about the genus
Spondyhts. most focusing on the taxonomy, anatotny. tnorphology.
habitat, and other topics ( Yonge 1973: Dakin 1928 a.b: Mata et al.
1990; Parth 1990; Okutani 1991; Skoglund & Mulliner 1996).
There is only one study about the reproductive cycle and spawning
season of Spondyhts leucacanthiis (Villalejo-Fuerte & Garci'a-
Domt'nguez 1998).

The present study describes the reproductive cycle throughout
15 months, and the spawning season of 5. calcifer. from histologi-
cal analysis and measurements of the volumetric fraction of oo-
cyte. Additionally, the relation between the reproductive cycle and
gonadic. digestive gland and muscle yield indexes is analyzed.

MATERIALS AND METHODS

Sampling was conducted in Bahia de Loreto. Gulf of Califor-
nia. Mexico (25°48'54". 1 1 1°15'45") (Fig. 1 ). Fifteen to .30 speci-
mens of S. calcifer were collected monthly, from January 1998 to
March 1999. by scuba diving at lO-m depth. Shell height and wet
weight of the gonad, digestive gland, muscle and total soft body
were recorded for each clam after fixation in a neutral 10% for-
malin solution prepared with sea water. Water temperature was
recorded at each sampling time. The photosynthetic pigment con-

centration (mg chlorophyll/m') in Bahia de Loreto frotn January
1998 to March 1999 was obtained from Seawifs Project. NASA/
Goddard Space Flight Center, and this was considered as an esti-
mation of the food availability for the clams.

Becau.se 5. calcifer does not exhibit sexual ditiiorphism, indi-
vidual sex was determined through histological analysis. The sex
ratio for the total sample was obtained. The null hypothesis of a 1:1
sex ratio was established and its significance was tested using a
chi-sqtiared analysis (Sokal & Rohlf 1979).

For histological studies, gonads were dehydrated in an alcohol
series and embedded in paraplast. Sections (7 |j.m) were placed on
slides and stained with hematoxylin-eosin (Humason 1979).

The classification of gonadal development was similar to that
oi S. k'Hcacantlms (Villalejo-Fuerte & Garci'a-Domi'nguez 1998).
which includes five development stages: undifferentiated, devel-
oping, ripe, spawning, and spent. To facilitate the description of
the reproductive cycle, the tnonthly relative frequencies for all
gonadal development stages were calculated.

The size at spawning in the population is defined as the small-
est length at which 50% of females and males are spawning (Som-
erton 1980). The size at spawning was estimated as the shell height
at which the 50% of cumulative frequency of clams in the spawn-
ing stage was attained.

Three indexes were calculated: gonad index (GI). digestive
gland index (DGI) and muscle yield index (MYI). Each index was
calculated by dividing the gonad, digestive gland or tnuscle wet
weight (respectively) by the total soft body wet weight, and ex-
pressing the results as a percentage (Sastry 1970).

Futtherniore. the monthly proponion occupied by developing
and mature oocytes combined (volume fractions) were estimated
by stereology (Lowe et al. 1982; MacDonald & Thompson
1988). The gamete volume fraction (GVF) was obtaitied frotn
point counts in a Weibel eyepiece graticule mounted in an ocular
microscope and applied to a gonadal section ( lOOX). Three repli-
cates were done for each gonad. The algorithm of Lowe et al.
(1982) was applied: GVF = No. of positive counts/total points

103

104

ViLLALEJO-FUERTE ET AL.

100

111"4'2" 26'6-23"

Figure 1. Location of sampling station in Bahia dn Liireto, Gulf of
California. Mexico.

counted X 100. GVF was calculated as the sum nf the \alues for
developing and mature oocytes in all females. The monthlv mean
GVF values were plotted.

A Spearman-rank correlation analysis was used to investigate
the relationship between monthly mean values of GI. DGI. MYI.
GVF. water temperature, and photosynthetic pigment concentra-
tion. As GI. DGI. and MYI are percentage values, the arcsine
transformation (Sokal & Rohlf 1979) was used to attain data nor-
mality and homoscedasticity for statistical analysis.

RESULTS

A total of 22.S clams were captured, of v\hich 279c were fe-
males and 32% were males. The remaining (41%) were undiffer-
entiated. The sex ratio for the total sample was 0.8 F:! M and did
not significantly differ from the expected ratio of 1:1 (P > O.O.'S).
Shell height ranged from 70.5 mm to I6.'i.2 mm. with the mode at
1 15 mm.

The reproductive cycle of S. cakifer was remarkably seasonal
(Fig. 2). In January and February of both years, the majority of
clams was inactive. Gametogenesis started in February in a small
proportion (5.3%) of the clams. By March 52.9% of the clams
were in the development stage. Development continued until Au-
gust with the highest proportion occurring in April (93.7%). Ripe
clams were found from April (6.2%) to July when the highest
proportion was observed (80.0%). A small proportion of ripe
clams was found in September (11.1%). Spawning was found in a
higher proportion during August and September (40.0% and
33.3%, respectively) and dropped drastically in October (7.7%).
From November to February, onlv undifferentiated and spent
stages occurred.

Figure 3 shows that the size at spawning in the population of S.
calcifer is 1 13-mm in shell height: however, individual organisms
may begin spawning at 86 mm in shell height.

The GI shows its peak values during May. June, and July,

J F
1998

Q] Undifferentiated
■ Spawning

m Developing
^^ Spent

jRipe

Figure 2. Kepn)ducti>e cjcle u\ Spondylus cakifer in Bahia de Loreto,
Gulf of California. Relative frequency of gonadal stages between Janu-
ary IWS and March 1999. Observations of males and females are
combined.

coinciding with the occurrence of the highest frequencies of clams
in the ripe stage. The GI declined from August to October, coin-
ciding with the spawning season. A period of low values occurred
from November to March, coinciding with the highest frequencies
of spent and inactive stages (Fig. 4a).

The DGI shows high values in February, declining from March
to June lea\e as is coinciding with the developing and ripe stages.
In August and September (months in which the highest proportions
of clams in spawning stage occur), the DGI values decrease. Af-
terwards, from October to March, DGI values start to rise again,
coinciding with a reproductive inactivity of the specimens (Fig.
4b).

The MYI values decreased according to the maturation process,
beginning the decrease in April, two months after the onset of
gonadal development (February). The lowest MYI value was
found in January 1999 and the higher \alue was found in March
1999 (Fig. 4c).

The volumetric fraction of gametes reflected the conad devel-

100

80

90 100

160

110 120 130
Height (mm)

Figure 3. .Size at spawning of 50% of the population of Spondylus
cakifer in Bahia de Loreto, (Julf of California, Mexico.

Reproductive Cycle of S. calcifer

105

c
o

o

CD

(U

E
o

JFMAMJJASONDJFM

1998 1999

Figure 4. Variation in tlie mean values of a) gonad index, b) digestive
gland index, c) muscle yield index, and d) oocyte volumetric fraction.

opment stages. Values increased when development and ripe
stages were present, and they decreased concurrently with the
spawning stage. Low values occurred when the inactivity and
spent stages prevailed (Fig. 4d).

Seawater temperature varied seasonally fruni 19'C to 29°C
during the study period. Temperature increased from March to
August, reaching its peak (29°C) from August to October, then
decreasing from November to February. The lowest temperature
was recorded in February, both years (19.5°C and 19°C) (Fig. 5).

Photosynlhetic pigment concentration (mg chlorophyll/m'') in
Bahia de Loreto was greater in the colder months than in the
warmer ones. The ma.ximum \alue was in March 99 (2.1 mg
chlorophyll/m') and the minimum was in September (0.2 mg chlo-
rophyll/m'') (Fig. 6).

GI and GDI values were negatively correlated, with statistical
significance (P < 0.05). GVF had a significant positive correlation
{P < 0.01) with GI and a significant negative correlation with
photosynlhetic pigment concentration (P < 0.05). GDI showed a
significant negative correlation with temperature (P < 0.05) and a
significant positive correlation with photosynthetic pigment con-
centration (P < 0.01). The photosynthetic pigment concentration
showed a significant negative correlation with temperature {P <
0.01). No significant correlation was found between MYI and any
other variable (P > 0.05).

DISCUSSION

In the Gulf of California low densities of S. calcifer have been
reported, ranging from 1 clam per 100 m"^ (from 1 to 25 ni deep)
to 1-5 clams per 25 m"(>25 ni depth! (Baqueiro et al. 1982). In
this study, the number of organisms captured per month corre-
sponds to these low population densities.

The histological analysis revealed that S. calcifer is a gono-
choric species as no hermaphrodite or sex-reversal specimens were
collected. However hermaphroditism has been reported in 3.S'* of
clams studied for S. leiicacanllms from the same locality (Villa-
lejo-Fuerte & Garci'a-Domi'nguez 1998). The sex ratio of 5. calci-
fer was not different from 1 : 1 , as also found for other bivalves like
Mercenaria mercenaria (Heffernan et al. 1989), Venus siriatiila
(Gaspar & Monteiro 1998). and Megapitaria squalida (Villalejo-
Fuerte et al. 2000). No references regarding the sex ratio for this
species were found in the literature. However. S. leucacanthus. a
related and sympatric species, presents a sex ratio different from
parity (Villalejo-Fuerte & Garcia-Dominguez 1998).

The size at spawning in S. calcifer occurs at 1 13-mm in shell
height, although specimens may start to spawn at 86-mrn in shell
height. In contrast, in the sympatric species S. leucacanthus, the
size at spawning is 75-mm in shell height with some spawning
specimens being as small as 4()-mm in shell height. This difference
in size at spawning may result from 5. calcifer being a the larger
species within the genus Spondylus.

The reproductive cycle of S. calcifer was remarkably seasonal.
S. calcifer exhibits an annual reproductive cycle with a short
spawning period from August to October followed by a long
inactive period largely during the winter (November-February).
The reproductive cycle of 5. calcifer was similar to the one de-
scribed for 5. leucacanthus (Villalejo-Fuerte & Garcia-Dominguez
1998). In both species, recruitment occurs once per year with a
similar short spawning period. These characteristics coiTespond to
a conservative reproductive strategy, similar to the one reported for
5. leucacanthus (Villalejo-Fuerte & Garcfa-Dominguez 1998).

Gametogenic cycles are generally ruled by external environ-
inental factors that may trigger and synchronize the "timing" oi
the different stages (Lubet 1983). The synchronization o'i the

106

VlLLALEJO-FUERTE ET AL.

J FMAMJ JASONDJ FM

1998 1999

Fijjure 5. Variation of surface teniperatiiri' from January 1998 to
March 1999 in Bahia de Loreto, Clulf of California, Mexico.

gonadal cycles in ;i population is probably also the result of some
kind of exogenous regulation (Gallardo 1989). Most studies
consider that temperature is the important environmental factor in
the regulation of bivalve reproduction (gametogenesis and spawn-
ing) (Giese and Pearse 1974; Sastry 1979; Barber & Blake 19S1 ).
However, in this study temperature was not correlated with Gl.
Nevertheless, the increase in water temperature coincided with
the beginning of gametogenesis. The above suggests that the
rise of water temperature may be responsible for triggering the
gonad maturation process, however, laboratory studies are need to
confirm it. In this re.spect. it had been previously observed that
sudden increases in water temperature appear to be the final cue
for stimulating maturation and ovulation in some fish species (Bye
1990).

In S. calcifer the inactive period (undifferentiated and spent
stages) occurs from November to February, when water tempera-
ture drops (from 29"C in October to 19°C in February). Thus, it is
reasonable to state that the decrease in water temperature inhibits

J F M A M
1998

J J A

S O N D J F
1999

Figure 6. Variation of photosynthetic pigments concentration from
January 1998 to March 1999 in Bahia de Loreto, Gulf of CaHfornia,
Mexico.

gametogenesis. but newly laboratory studies are necessary to con-
firm this conclusion. Similarly, a cooler temperature inhibits ga-
metogenesis in SpisuUi solidissiiiiii (Kanti et al. 1993). On the
other hand, spawning in .S'. calcifer. only occurs when water tem-
perature is at least 29'C (August to October). This fact suggests
that 29 C is the threshold water teinperature at which spawning
occurs in S. calcifer. A threshold temperature for spawning has
also been reported for some oyster species (Burrell 1985). In con-
trast, in other clam species, like Paphies donacina and Cerusto-
dcvuia cdide spawning occurs within a relatively wide temperature
range (Navarro el al. 1989; Marsden 1999).

The bivalve gamete production is strongly infiuenced (set in
a seasonal context) by environmental factors such as tempera-
ture but also by food availability (MacDonald & Thompson
1985). However, both factors may be strongly related. In this
work, the photosynthetic pigments concentration was negatively
correlated with temperature. Then the higher food availability for
clams (expressed as photosynthetic pigment concentration) were
during the cold months coinciding with the resting period. On the
other hand, the spawning season of S. calcifer coincide with the
lowest food availability (August to October). A relation of spawn-
ing time and food availability was found in Cldamys amandi
(Jaramillo et al. 1993). whereas Hinnites gigaiueti.s showed no
correlation between food availability and spawning (Malachowski
1988).

The DGI had a significant positive correlation with photosyn-
thetic pigment concentration, then the higher food availability
and the higher DGI values were during the inactive period of
reproduction. The trend observed for DGI suggests that a period
of accumulation of nutrient reserve in the digestive gland takes
place during the inactive period. These nutrient reserves are
mobilized and utilized by the gonad during the developing
stage. The significant negative correlation between DGI and
Gl confirms the above for 5. calcifer. In mollusks. it has been
observed that the onset of the oocyte growth phase is dependent
upon the accumulation and transfer of nutrient reserves from the
digestive gland to the gonad (Sastry 1968; Gabbott & Bayne
1973).

Despite MYl and Gl not being coirelated, MYI values start to
decrease in April, just two months after gametogenesis had started.
This suggests that there is a delayed in energy transfer from muscle
to gonad. The reserves accumulated in the muscle are used up after
gamete production had started, helping to support the energetic
output during the ripe and spawning stages. Similarly, in Ar-
gopecten irradian.s it appeared that lipids from the digestive gland
were used to fuel the beginning of gametogenesis whilst adductor
muscle reserves becoine important later on to complete gonadal
development (Barber & Blake 1981 ). A relationship between MYl
and Gl has been observed in Pectea ma.ximus (Comely 1974; Fav-
eris & Lubet 1991). Argopecleii irradian.s (Sastry 1966). Pari-
iiiipecren ycs.soensis (Mori 1975). Plucopecten magellanicus (Rob-
inson et al. 1981). and Argnpeclca circularis (Villalejo-Fuerte &
Ceballos-Vazquez 1996).

However, in pectinids and other bivalves, it has been proposed
that oocyte growth is dependent on two factors; food intake and
energetic storage in specialized organs (Sastry 1963. 1966. 1968;
Barber & Blake 1983). In P. magellaidcit.'i and A. circidari.s. the
energy for gamete maturation comes from both stored reserves and
ingested food (Thompson 1977; Robinson et al. 1981; Luna-
Gonzalez et al. 2000).

Reproductive Cycle of S. calcifer

107

ACKNOWLEDGMENTS

We are izratetui to The Iiistituto Pcilitecnicn Nacional (IPN) tor
fimding this work, and tor the grants (EDI and COFFA) to

the authors. We also wish to thank to Sofia Ortega for the data
of photosynthetic pigments concentration and to Mari'a Elena
Sanchez-Salazar for his editorial help on the English manu-
script.

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Sastry. A. N. 1979. Pelecypoda (excluding Ostreidae). pp. 131-192. In A.
C. Giese & J. S. Pearse (eds.). Reproduction of Marine Invertebrates.
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Skoglund, C. & D. K. Mulliner. 1996. The Genus Spondylus (Bivalvia:
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Sokal, R, R. & F. J. Rohlf 1979. Biometna. Principios y metodos esiadis-

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pana. 832 pp.
Somerton D. A. 1980. A computer technique for estniiatnijj the si/e of

sexual maturity in crabs. Can. J. Fish. Aqiuit. Set. 47:1488-1494.
Thompson. R. J. 1977. Blood chemistry, biochemical composition, and the

annual reproductive cycle ot the giant scallop. Placopecten magethiiu-

cus, from southeast Newfoundland. J. Fisheries Res. Board of Canada

34:2104-2116.
Villalejo-Fuerte. M. & B. P. Ceballos- Vazquez. 1996. Variacion de los

I'ndices de condicion general, gonadico y de rendimiento muscular en

Argopeeren eircularis (Bivalvia: Pectinidae). Rev. Biol. Trop. 44:591-594.

Villale|o-Fuene. M. & F. Garcia-Domfnguez. 1998. Reproductive cycle of
Spondyhis leiieaeanthiis Broderip. 1833 (Bivalvia: Spondylidae) at Isia
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Villalejo-Fuerte. M.. M. Arellano-Martinez. B. P. Ceballos-Vazquez & F.
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Yonge. C. M. 1973. Functional morphology with particular reference to
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Jnurmil of Shellfish Research. Vol. 21. No. 1, 109-112. 2002.

BACULOVIRUS-LIKE PARTICLES IN EPITHELIAL CELL OF DIGESTIVE DIVERTICULA OF

THE SCALLOP, PATINOPECTEN YESSOENSIS

YOUNG JIN CHANG,'* MIN-DO HUH," MYUNG-JOO OH,' AND YOSHIO SUGAWARA^

^Department of A</iiaciilliire, Piiky()ii,i> National University. Busan 60H-737, Korea; -Department of
Aquatic Life Medicine. Piikyong National University. Busan 608-737, Korea; ^Department of Aqualife
Medicine. Yosu National University, Yosu 550-749. Korea; * Department of Biotechnology, Senslm
University of Ishinomaki. Ishinomaki 986. Japan

ABSTRACT Virus-like particles were encountered in one epithelial cell of digestive diverticulum in a chnically healthy scallop.
Patinopecten yessoensis. Virions, showing typical rods, were found in the cytoplasm without marked cellular degenerative change and
were recognized as ovoid or spherical bodies, depending on sectional orientations. The envelope was appro.ximately 1 2 nrn in thickness
and the space between the envelope and nucleocapsid was about 10 nm. Average length and diameter of the complete virions were 520
and 130 nm. respectively. No occlusion bodies were observed. From this morphological evidence, it was considered that the virus
particles observed in one epithelial cell of digestive diverticulum in the scallop are likely to be a species of nonoccluded, baculovirus
type C, which are morphologically very similar to the baculovirus associated with white spot syndrome (WSSJ and baculoviral mid-gut
gland necrosis (BMN) in shrimp.

KEY WORDS: bivalve. Paimopeclen yessoensis, baculovirus. white spot syndrome (WSSl. baculoviral mid-gut "land necrosis
(BMN)

INTRODUCTION

A number of infectious diseases have been observed in bivalves
such as oysters, clams, mussels, and scallops. Because bivalve
production represents a considerable proportion of fisheries indus-
try in South Korea, much attention is now being paid to the cause
of the massive deaths occurring in bivalve farms along the south-
em coast. To date, diseases have been reported from 63 species of
parasites, 7 strains of bacteria, 2 strains of fungi, and 3 strains of
rickettsia or chlamydia. More recently, five kinds of viral diseases
were reported from oysters (Fisher 1988; Sindermann 1990; Couch
& Fournie 1993; Faisal & Hetrick 1994; Woo 1995). There are two
other kinds of diseases with unknown cause. For most of the
diseases cited, little is known regarding pathologic effects on the
host and methods of prevention and control.

Virus particles were, by chance, encountered in one epithelial
cell of digestive diverticulum during a study on the relationship tif
histological structures in digestive diverticula to nutrient accumu-
lation. The morphological characteristics of the particles are de-
scribed in comparison with other virus particles and discussed with
respect to pathogenic potential.

MATERIALS AND METHODS

Thirty scallops were sampled every month from March 1982 to
December 1983 from a bottom culture farm in Abashiri waters of
Hokkaido, Japan. Each individual, ranging from 8.5 to 1 1.2 cm in
shell height and from 62.9 to 197.4 g in total weight, was clinically
healthy. For transmission electron microscopy, all scallops were
necropsied and their digestive diverticula were carefully removed,
then diced into 1-mm cubes. All tissue cubes were prefixed with a
5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, 4°C). After
postfixation with 0.2 M cacodylate buffered 2% OSO4 for about an
hour, tissue blocks were dehydrated in a graded series of ethanol
and embedded with Epon 812. Ultrathin sections were obtained
using an ultramicrotome (Porter Blum II, Agwani, Massachusetts,

*Corresponding author: Fax: +82-51-628-7430; E-mail: yjchang@
pknu.ac.kr

USA) and stained with uranyl acetate and lead citrate. The grids
were examined with a transmission electron microscope (JEOL
lOOB, Tokyo, Japan) under an accelerating voltage of 80 kV.

RESULTS

As shown in Figure 1, one epithelial cell containing vu'al par-
ticles con-esponded to type I of the 3 types of cells that have been
classified by Chang et al. (1989). Because of the shortness of
apico-basal length, it appears to be embedded between the other
types of cells. Cytoplasm of the virus-containing cell was com-
pletely occupied basally by a densely packed mass of rough en-
doplasmic reticulum (RER) and apically by a round area contain-
ing a number of viral particles.

A great number of virions had a fence-like array, which again
formed several layers, roughly in parallel. Virions showed typical
rods in shape but, depending on the sectional orientation, they
were ovoid or spherical (Fig. 2). Virions were present among a
great number of small vacuoles that appeared to be a network of
proliferated, tubular smooth endoplasmic reticulum (TER). A layer
of variably sized, unidentified vacuoles surrounded the virus-
containing area (Fig. 3). The vacuoles, ranging from 520 to 1050
nm in diameter, were spherical in shape and clearly limited by
double membranes. They also had thread-like or fuzzy materials
with high electron density. No other morphological abnormalities
were found in this virus-infected cell.

Each virion had a nucleocapsid and an envelope of unit mem-
brane that was quite similar morpholtigically to baculovirus (Fig.
4). The average thickness of the envelope was 12 nm and the
average distance from the nucleocapsid was 10 nm. The average
length and diameter of virions were 520 and 130 nm, respectively.
No occlusion bodies were observed.

DISCUSSION

There have been few reports on viral diseases in bivalves, and
most of these were reported for oysters. Since the first identifica-
tion of birnavirus from clam, Telliiui tenuis (Hill 1976), retrovirus-
like particles from clam Mya arenaria. iridovirus-like particles
from oyster Crasso.strea aiif-iilula. and herpes-like particles from

109

110

Chang et al.

Figure I. \ iius-inlfcttd tvpf I till in dijiestive diverticula epithelium.
This cell was very similar to pancreatic exocrine cells of manunalian
pancreas. Cytoplasm near the basement membrane (BM) of the cell
contained many large flattened cisternae of the rough-endoplasniic
reticula (RER) and relatively few mitochondria (M). The apical part of
the cytoplasm was without marked degenerative change, but the cell
was filled with numerous viral particles (VP).

C. viifiinicd have been repurted (Perkins 1993). Snice oyster velar
virus disease (OVVD) caused by iridovirus was reported from
farmed C. i;igcis (Elston & Wilkinson 1985: Sindermann & Light-
ner 1988: Bower et al. 1994). descriptions followed on small and
nonenveloped virus particles from digestive diverticukmi of pearl
oyster Piiicicuhi inaxiiiia (Pass et al. 1987), green lip mussel Perini
canaliculus (Jones et al. 1966), herpes-like particles from
hemocytes of flat oyster Oslrea angasi (Hine & Thome 1977) and
enterovirus or calicivirus-like particles from digestive diverticula
of scallop Pectc'ii ninxiezelandiac and loheroa Puphlcs vcntricn-
sum (Hine & Wesney 1977).

Virus particles in this study were moiphologically different
from those particles previously reported, but were quite similar to
baculovirus that has not been described from bivalves. In general,
baculoviruses are rod-shaped DNA viruses, which are known to be

500 nm

Figure 2. Fence-like array of viral particles. Numerous viral particles
surrounded by tubular smooth endoplasmic reticula (TKRi were rod-
shaped, spherical, or ovoid in morphology, because of the difference in
viewing orientation.

infective only in invertebrates. Baculoviruses are divided into the
three subgroups of nuclear polyhedrosis \irus (NPV, type A),
granulosis virus (GV. type B). and nonoccluded virus (NOV, type
C) (Suidermann & Lightner 1988; Faisal & Hetrick 1994; Levy et
al. 1988).

From shrimps, six kinds of baculoviruses, including baculovi-
rus penaei disease (BP virus disease), monodon baculovirus dis-
ease (MBVD), baculoviral mid-gut gland necrosis (BMN), ple-
bejus baculovirus disease, and yellow-head disease (YHD), have
been described, although their pathogenesis is still poorly under-
stood (Faisal & Hetrick 1994). The causative agent responsible for
white spot syndrome (WSS). which was responsible for mass mor-
tality of shrimps in Taiwan during the last 7 years, was identified
to be a species of baculovirus (Wang et al. 1997).

All baculoviruses reported in shrimps range from 1.^0 to .MO
11111 in length and 3.3 to 74 nm in diameter. Virions in the scallop
were very similar in size to causative viruses responsible for BMN
and MBVD in shrimps. The finding that they did not form occlu-
sion bodies is consistent with the viruses responsible for BMN and
WSS in shrimps. The causative virus of BMN belongs to NOV
type C that is found only within the hypertrophied nucleus. BMN
frequently brings about mass mortality in an early larval stage of
shrimp. Histologically, BMN is characterized by necrosis of epi-
thelial cells in the hepatopancreas and mucosal membranes with a
variety of nuclear changes, including severe hypertrophy, margin-
ation and loss of chromatin, and loss of nucleoli (Momoyama &
Sano 1988: Momoyama & Sano 1989). In contrast, WSS is char-
acterized by homogeneous nuclei with a variable degree of nuclear
hypertrophy in nearly all kinds of tissue except for hepatopancre-
atic epithelium (Wang et al. 1997).

Even though grossly and micro.scopically no marked lesions
were found in association with the presence of viral particles in the
scallop, it was considered that the viral particles in this animal
should be carefully monitored for the pathogenic potential.

BaCULOVIRUS in DlGHSTIVE DIVERTICULA OF SCAI.LOP

III

lOOOnm

Hfiuri' 3. Ihf cluster ot lirai piiiiicks ";is siirioundud by unidentified \aiii()lcs (\ al.

200nm

JUKI.

Figure 4. Higlier magnification of Figure 2. Single envelope (arrow! and a central nuclcocapsid (NC| were recognized in each viral particle. Tlie
clear spaces between envelopes and nucleocapsids were recognizable from all orientations of viral particles.

LITERATURE CITED

Chang, Y. J.. Y. Sugawara & T. Nomura. Iy89. Structure and function of
digestive diverticula in the scallop, Palinopecten ye.ssoensis (Jay). Tn-
hoku J. Aj-hc. Res. 39:81-94.

Couch, J. A. & J. W. Fournie. 1993. Pathobiology of marine and esluarine
organisms. Boca Raton, FL: CRC Press. 552 pp.

Elston, R. A. & M. T. Wilkinson. 1985. Pathology, management and di-
agnosis of oyster velar virus disease (OVVD). Ac/iuiciilttire 48:189-
210.

Faisal. M. & F. M. Hetrick. 1994. Annual review of fish diseases. Vol. 4.
New York: Perganion Press, 433 pp.

Fisher, W. S. 1988. Disease processes in marine bivalve molluscs. Am.
Fish. Soc. Spec. Piihl. 18:315.

Mine. P. M. & T. Thorne. 1997. Replication of hcipes-like viruses in
haemocytes of adult flat oysters Osirea ani^iisi: An ultraslructural
study. Dis. Aquat. Oiff. 29:189-196.

Mine. P. M. & B. Wesney. 1997. Virus-like particles associated with cy-
topathology in the digestive gland epithelium of scallops Pccleii im-

niczelandiuc and toheroa Pupliies veiiliicusiim. Dis.
197-204.

Ac/iiiil. Oifi. 29:

Jones, J. B., P. D. Scotti. S. C. Deanng & B. Wesney. I96(i. Virus-like
particles associated with marine mussel mortalities in New Zealand.
Di.s. Aqiiut. Org. 25:143-149.

Levy, J, A„ F. C. Heinz & R. A. Owens. 1988. Virology. 2nd ed., Engle-
wood Cliffs, New York: Prentice Hall. 440 pp.

Momoyama. K. & T. Sano. 1988. A method of experimental infection of
kuruma shrimp larvae, Peiuieiis japonicus Bate, with baculoviral mid-
gut gland necrosis (BMN) virus. J. Fisli Dis. I 1:105-1 I I.

Momoyama, K. & T. Sano. 1989. Developmental stages of kuruma shrimp.
Peiuieiis japonicus Bate, susceptible to baculoviral mid-gut gland ne-
crosis (BMN) virus. J. Fisit Dis. 12:585-589.

Pass, D. A., F. O. Perkins & R. Dybdahl. 1987. Virus-like particles in the
digestive gland of the pearl oyster [Pinctada maxima). J. Iiwerl. Palhol.
51:166-167.

Perkins, F. O. 1993. Infectious diseases of molluscs. In: J. A. Couch & J.

j^2 Chang et al.

W Fourn,e editors Pathohiology of maruie and estuarme organisms. Wang. C. S., K. F. J. Tang, G. H. Kou & S. N. Chen. 1997. L.ght and

Boca Raton FL' CRC Press, pp. 255-287. electron microscopic evidence of white spot disease ,n the giant tiger

Smdermann. C.' J. & D. V. Lightner. 1988. Disease diagnosis and control shnmp. Penaeus monodon (Fabricins,. and the k-ruma ^hnmp. Pe-
rn North American marine aquaculture. New York: Elsevier. 431 pp. naeus japon,a.s (Bate), cultured in Taiwan. J. F,sh D,5 0.3-3-33L

Smdermann, C.J. 1990. Pnncipal diseases of marine fish and shellf-ish, vol. Woo. P. T. K. 1995. Fish diseases and disorders. Vol. K Protozoan and

^ San Diego: Academic Press. 516 pp. nietazoan infections. New York: Cab International. 808 pp.

Joiinuil ofShellfLsh Ri'.seanh. Vol. 21, No. 1. 113-118, 2002.

HAPLOSPORIDWM COSTALE (SEASIDE ORGANISM), A PARASITE OF THE EASTERN
OYSTER, IS PRESENT IN LONG ISLAND SOUND

I. SUNILA,' N. A. STOKES,- R. SMOLOWITZ,' R. C. KARNEY,^ AND E. M. BURRESON"

^ State of Connecticut. Depurtincnl of Agriculture, Bureau of Aquaculture. P.O. Box 97. Milford.
Connecticut 06460: 'Virginia Institute of Marine Science, College of William and Mary, P.O. Bo.\ 1346,
Gloucester Point, Virginia 23062; Marine Biological Laboratory, 7 MBL St., Woods Hole,
Massachusetts 02543: '^Manila's Vineyard Shellfish Group. P.O. Bo.x 1552, Oak Bluffs. Massachusetts 02557

ABSTRACT A haplosporidian parasite, Haplosporidium costale (seaside organism or SSO), is associated with high mortalities of
eastern oysters (Crassostrea virginica) in seaside bays of Virginia and Maryland. Its presence in Long Island Sound has been
tentatively suggested in several publications for the last 50 y. Positive identification of H. costale and differentiation from another
haplosporidian parasite, Haplosporidium nelsoni (MSX), from histological sections is difficult and requires the presence of spores. We
detected H. costale spores in 4 out of 5010 [Q.0V7c ) oysters collected from Long Island Sound in 1997-1999. In situ hybridization using
an oligonucleotide DNA probe designed to detect small subunit ribosomal DNA from Virginia's H. costale reacted positively with
tentative H. cosiale plasmodia in 5 oysters from Long Island Sound. In each case there was a coinfection of H. nelsoni. In Virgmia
and Maryland. H. cosiale has historically sporulated in all infected animals in May-June. In Long Island Sound, the rare sporulating
cases were detected in October-December, suggesting a different infection cycle.

KEY WORDS: Haplosporidium costale. eastern oyster, Crassostrea virginica. in silu hybridization. Long Island Sound

INTRODUCTION

A haplosporidian parasite. Haplosporidium costale. was iden-
tified as the causative agent of seaside organism (SSO) disease,
resulting in high mortalities of eastern oysters (Crassostrea vir-
ginica) on the Atlantic coast of Virginia (Wood & Andrews 1962).
Prevalence, mortality and infection cycle of H. costale have been
thoroughly studied in Virginia and Maryland, but information from
other geographical locations is scarce. In seaside bays of the Del-
marva Peninsula, the first plasmodia can be detected in the epi-
thelia of digestive tubules in April-May. Plasmodia rapidly pro-
liferate in May and .sporulate synchronously. Peak mortality of the
oysters is in May-June, when dying oysters release spores in sea-
water to initiate a new infection cycle. New infections remain
subpotent until spring of the following year. Mortality of oysters
has been 20% to 50% annually in the seaside bays of Virginia
(Andrews 1988: Andrews & Castagna 1978).

Reports of W. cosiale distribution north of Virginia's Atlantic
coast are inconsistent. Several reports suggest the presence of plas-
modia resembling H. costale in eastern oysters in Long Island
Sound. According to Andrews (1984, 1988), SSO disease ranges
from Cape Charles, Virginia, to Maine, but is important only in
high-salinity bays (>25%t) from Cape Henlopen, Delaware, to the
Virginia capes. He stated that the pathogen is regularly present
from New York to Massachusetts, but mortality has not been a
serious problem. However, a mortality event in Long Island Sound
in 1953 may have been caused by H. costale (Andrews 1988).
Sampling locations, dates, prevalences, or possible presence of
spores were not mentioned in these reports.

Newman (1971) studied 1,337 oysters from New Haven Har-
bor. Connecticut, from 1966 to 1967. He found five specimens
(0.4%) infected with plasmodia morphologically similar to H. cos-
tale. with one of the oysters moribund with heavy infection. No
sporulation was detected. Meyers (1981) described haplosporidia-
like Plasmodia (4%) in juvenile oysters (n = 68) collected from
Oyster Bay, north shore of Long Island. New York, from 1975 to
1976. He did not find similar organisms in adult oysters (/I = 145).
No effort was made to distinguish between different haplosporid-
ian species because sporulating stages were not present. In addi-

tion. Plasmodia morphologically identical to H. costale were ob-
served in oysters transplanted from the vicinity of New Haven,
Connecticut, to Tomales Bay, California, in 1967-1968 (Katkan-
sky & Warner 1970). Six specimens with tentative H. costale
infection (four moribund and two living) were reported, one with
spores. Total number of oysters studied was not mentioned.

Reports of tentative H. costale infections in Long Island Sound
reviewed above are based on histological examinations. Reliable
diagnosis of H. costale on histological sections is nearly impos-
sible when sporulating forms are not present. H. costale plasmodia
can be easily misdiagnosed as Haplosporidium nelsoni (MSX).
another haplosporidian oyster parasite enzootic to the area (for
review, see Ford & Tripp 1996). During routine monitoring for
oyster diseases in Connecticut, we found several oysters with
spores and plasmodia consistent with descriptions of H. costale.
We used a DNA probe, designed to detect H. costale from Virginia
as the in situ hybridization (ISH) probe for these specimens to
verify the presence of H. costale in Long Island Sound.

MATERIAL AND METHODS

The State of Connecticut. Department of Agriculture. Bureau
of Aquaculture routinely receives oyster samples for histological
diagnosis from Connecticut's commercial oyster companies. Most
of the seed originates from natural seed beds, though some hatch-
ery-raised seed is also used. Every oyster is transplanted an aver-
age of four times before it is marketed, which exposes it to possible
parasitic infections in several different sites. Seventeen oyster
samples were studied in 1997. 63 in 1998. and 87 in 1999. Each
sample consisted of 30 oysters, for a total of 5,010 oysters.
Samples represented the entire Connecticut shoreline and the north
shore of Long Island. New York. Of the 167 samples, 20 origi-
nated from New York. Fifty-six of the samples originated from
oyster nursery systems (5 from upwellers, 51 from suspended cul-
tures). 30 from off bottom cultures, and 81 from natural oyster
beds. Tissues were fixed in Davidson's fixative in 20%f artificial
seawater. Six-micrometer-thick paraffin sections were stained with
hematoxylin-eosin. Samples with H. costale or H. nelsoni pre-

113

114

SUNILA ET AL.

spores and spores were stained also with Ziehl and Hanis' hema-
toxylin according to Farley ( 1965).

The ISH procedure was conducted with 27 oysters, two with
presumed H. costale spores and 25 oysters with haplosporidian
Plasmodia. Specimens were selected for ISH as follows: (1 ) speci-
mens with haplosporidian plasmodia from samples in which H.
costale spores were detected; (2) specimens with small plasmodia
with central nucleoli, morphology that is considered to be charac-
teristic for H. costale; and (3) specimens with plasmodia in the
stomach, intestine, or digestive tubule epithelia, locations that are
considered to be characteristic for H. costale. For the ISH proce-
dure, 6-p.m-thick sections from these oysters were deparaffini/ed
and ISH was performed on consecutive .sections as previously
described (Stokes & Burreson 1995: Stokes & Burreson 2001).
Two commercially synthesized digoxigenin-labeled oligonucle-
otide DNA probes were used: a 22-base oligonucleotide
(SS01318) specific for H. costale (Stokes & Burreson 2001)
and a 2 1 -base oligonucleotide (MSXI347) specific for H. nelsoni
(Stokes & Burreson 1995). A negative control was performed
by substituting DNA probes with distilled water during hybridiza-
tion.

RESULTS

HaplosporicUuiii costale was detected in three locations on
Connecticut's shoreline: Norwalk. Branford. and Clinton (Fit!. I).

H. costale was diagnosed in seven different specimens either by
the presence of spores or by a positive ISH result. There were 17
oysters with haplosporidian spores among the 5,010 oysters stud-
ied (0.3%). Four oysters had a mixture of H. costale and H. nelsoni
spores (0.08%). The remaining 13 contained only H. nelsoni
spores. Locations, dates, and seed origin of oysters with spores are
listed in Table I.

H. costale and H. nelsoni spores ditfered m size, form, and
location in oyster tissues. H. costale spores (3 x 4 |j.m). the sporo-
plasm of which stained bright red with acid-fast stain, were de-
tected throughout the connective tissue. Prespores. which did not
retain acid-fast stain, occurred inside sporocysts throughout the
tissues. H. costale spores were found between vesicular connective
tissue cells surrounding the digestive diverticula (Fig. 2A), in con-
nective tissue of the gills, in the adductor muscle, in the heart,
between neurosecretory cells in the ganglia, and between kidney
tubules. On rare occasions, H. costale spores were detected inside
digestive tubule or digestive duct cells, in the lumens of the di-
gestive tubules and intestine, in epithelial cells of the intestine, or
in the follicles.

H. nelsoni spores (5x7 (j.m), which also stained bright red with
acid-fast stain, were in most cases restricted to digestive epithelial
cells (Fig. 2B). However, during intense sporulation in four speci-
mens there was an overspill to digestive duct cells and the con-
nective tissue surrouiidinsi the dicestive tubules. In cases with

73 eft'

CONNECTICUT

NEW YORK

ATLANTIC OCEAN

20 Miles

Figure. 1. A map of sampling stations with a positive diagnosis for H. costale in Long Island Sound.

Haplosporidium costale in Oysters

115

TABLE 1.

Haplosporidium costale (SSO) and Haplosporidium nelsoni (MSX) spores in eastern oysters in Long Island Sound.

Sampling

Sampling

Sampling

Shell Length

Spore

Seed

Date

Site

Location

(mm)

Type

Origin

09.28.97

Guilford, East River

41°16.05'N:72°39.62'W

104

MSX

Natural

10.14.97

Clinton. Cedar Island mud Hat

4ri5.97'N:72°32.00'W

7S

MSX

Natural

10.14.97

Clinton. Cedar Island mud tlat

41°15.97'N:72°32.00'W

101

MSX

Natural

12.17.97

Norwalk 1131

4r02.42'N:73''25.25'W

75

MSX

Natural

12.18.97

Norwalk 162

41''03.36'N:73°25.I2'W

69

MSX

Natural

01.08.98

West Haven Lot IB

41°15.47'N:72°55.26'W

42

MSX

Hatchery raised

09.18.98

Stratford 709

41°07.4rN:73°09.13'W

70

MSX

Natural

09.22.98

Milford 612

4I°1I.10'N:73''00.05'W

73

MSX

Natural

10.17.98

Branford 3 1 6

4ri5.42'N:72°44.40'W

7.'i

SSO, MSX

Natural

10.17.98

Brantord 168

41°15.77'N:72°45.9.VW

42

MSX

Natural

10.17.98

Branlord 179

41°15.73'N:72"45.63'W

64

SSO, MSX

Natural

11.21.98

West Haven Lot IB

4ri5.47'N:72°55.26'W

48

MSX

Hatchery raised

12.03.98

Clinton. Cedar Island Maruia

41''16.05'N:72°32.I0'W

24

SSO, MSX

Hatchery raised

09.22.99

Clinton. Cedar Island Marina

4ri6.05'N:72°32.10'W

76

MSX

Hatchery raised

09.22.99

Clinton. Cedar Island Marina

41°16.05'N:72°32.10'W

33

MSX

Hatchery raised

09.22.99

Clinton. Cedar Island Marina

41°16.05'N:72°32.10'W

26

MSX

Hatchery raised

11.16.99

Clinton. Cedar Island Marina

4r'16.05'N:72''32.10'W

29

SSO, MSX

Hatchery raised

extremely heavy sporulation. phagocytosed H. nelsoni spores were
observed in the vascular system hetnolymph sitiuses and veins, in
the mantle lobes and the gills, and being carried through stomach
or mantle epithelia via diapedesis. Spores that occurred in the
connective tissue were usually surrounded by aggregates of granu-
lar hemocytes. H. nelsoni spores were detected inside the lumens
of digestive tubules, digestive ducts, and the intestine. H. nelsoni
prespores, which did not retain stain in acid-fast reaction, were
detected exclusively inside digestive cells.

It was as probable that spores would be found in oysters origi-
nating from natural set as in oysters originating from hatchery-
raised seed (x" = 0.68 [not significant] [NS]). Oysters with spores
were detected in tnost areas of Connecticut's shoreline. No sporu-
lating specimens were detected in the north shore of Long Island,
New York. The size of an oyster with spores did not differ sig-
nificantly from the average size of the sample from which it was
taken {t = 0.08 [N.S]). The size of an oyster with spores also did
not differ significantly from the average sizes of all oysters
sampled for this study (/ = 0.97 [NS]).

ISH results with H. costale and H. nelsoni DNA probes are
summarized in Table 2. Five specimens had mixed infections, the
rest were infected only with H. nelsoni. Two specimens with
mixed infections had both types of spores present; three had only
Plasmodia. (An additional two specimens were diagnosed positive
for both H. costale and H. nelsoni on the basis of the presence of
spores [Table 1]. with seven positive specimens altogether.) The
two specimens with both spore types (Branford lots 179 and 316)
that were subjected to ISH had prominent H. costale infections.
Approximately 90% of the plasmodia hybridized with the H. cos-
tale probe and 10% hybridized with the H. nelsoni probe in these
samples (Fig. 3). Two other specimens with mixed infections
(Clinton and Norwalk 1131) had very light H. costale infections
(Fig. 4). More than 99% of the plasmodia hybridized with the H.
nelsoni probe and the very rare H. costale plasmodia would not
have been detected without the probe. H. costale plasmodia in the

Figure 2. Sporulation of H. costale and H. nelsoni in Long Island
Sound. (A) Digestive diverticulum of an oyster filled viith acid-fast H.
costale spores (Ziehl and Harris' hematoxylin). .Scale bar 100 pm. (B)
Cosporulation of H. costale and H. nelsoni. Small H. costale spores
occur in the connective tissue surrounding digestive tubules; larger H.
nelsoni spores occur inside digestive tubule. This is the same specimen
as in item (A) (Ziehl and Harris" hematoxylin). Scale bar 20 (im.

SUNILA ET AL.

tablp: 2.

ISH of Haplosporidmm coslale (SSO) and Haplosporidium nelsoni (MSX) in eastern oysters in Long Island Sound.

Sampling
Date

Sampling
Site

ISH with SSO
Sampling Slull Length and MSX Prohes

Location (mm) (Positive Result I

Location of Plasmodia

09.16.97 Norwalk Natural Bed
10.14.97 Clinton, Cedar island
mud flat

12.17.97 Norwalk 11.11

12.17.97 Norwalk 162

4I'04.85'N:73°23.55'W
4I°15.97'N:72'32.00'W

41°02.42'N:73°25.25'W

41 03.36'N;73 = 25.I2'W

10.17.98 Branford316

10.17.98 Branford 316

10.17.98 Branford 168

10.17.98 Branford 168

10.17.98 Branford 168

10.17.98 Branford 179

10.17.98 Branford 179
10.17.98 Branford 179

11.10.98 Stony Brook Harbor, NY

11.10.98 Stony Brook Harbor, NY

11.10.98 Stony Brook Harbor. NY

11.12.98 Oyster Bay. H. NY

11.12.98 Oyster Bay. H, NY

11.12.98 Oyster Bay, W. NY

11.12.98 Oyster Bay, W, NY

12.01.98 Milford 30.«i

12.01.98 Milford 305

12.15.98 Northport Bay. NY

1 1.17.99 Clinton, Cedar Island Marina
11.17.99 Clinton, Cedar Island Marina
12.07.99 Oyster Bay, J. NY

12.07.99 Oyster Bay, J, NY

80

40°54.3O'N;73 10.70'W
40'54..30'N:73' 10.70"W

40°54.30'N:73° 10.70' W
40°54.33'N:73°30.22"W

40"54.33'N:73''30.22'W

40°52..59'N:73°32.irW

40°52..59'N:73"32.irW

4PI1.I5'N:7,3°04.80"W
4ril.l5'N:73°04.80'W

40°55.87'N;73°22.87'W

4r 16.05'N;72 32.10'W
4ri6.05'N:72°32.10'W
4O°53..5O'N:73°30.23'W

40"53.50'N:73"30.23'W 68

110 MSX

113 SSO. MSX

SSO, MSX

66 SSO, MSX

12.17.97 Norwalk Manresa Island 4r04.42'N:73"24.55'W 70 MSX

41°I5.42'N:72'M4.4(1'W 68 MSX

41°15.42'N:72=44.40'W 75 SSO. MSX

4ri5.77'N:72'=45.95'W

41

MSX

41°15.77'N:72°45.95'W

41

MSX

41°15.77'N;72°45.95'W

6(1

MSX

41°15.73'N:72"45.63'W 62 MSX

41°15.73'N:72°45.63'W 70 MSX

41 = I5.73'N:72°45.63'W 64 SSO. MSX

123

MSX

127

MSX

107

MSX

76

MSX

87

MSX

92

MSX

84

MSX

78

MSX

24

MSX

92

MSX

67

MSX

65

MSX

9(1

MSX

MSX

Plasmodia throughout the tissues
MSX plasinodia (>99'7f ) throughout the
tissues, rare SSO plasmodia KlVr ) in the
gills
MSX Plasmodia (>99'/rl throughout the
tissues, rare SSO plasmodia (<l'7rl in the
gills and mantle
MSX Plasmodia (40%) throughout the
tissues; prespores in digestive tubules;
SSO Plasmodia (609f I throughout the
tissues
Plasmodia in the intestine, digestive duct
and epibranchial chamber epithelia; rare
Plasmodia throughout the tissues
Plasmodia in the intestine and stomach
epithelia; some plasmodia throughout the
tissues
MSX spores and plasmodia (10%) in the
digestive tubules; SSO spores and
Plasmodia (909i-') everywhere in the
connective tissue
A few Plasmodia in stomach epithelium;

several plasmodia throughout the tissues
A few Plasmodia in stomach epithelium;

several plasmodia throughout the tissues
A few Plasmodia in the stomach, intestine,
digestive tubule, and duct epithelia;
several plasmodia throughout the tissues
Plasmodia exclusively in the stomach,
intestine, digestive duct, and digestive
tubule epithelia
Plasmodia throughout the tissues
MSX spores and plasmodia ( 10%) in the
digestive tubules; SSO spores and
Plasmodia (90%) everywhere in the
connective tissue
Plasmodia throughout the tissues
A few Plasmodia in gill epithelia, several

Plasmodia throughout the tissues
Gaper; plasmodia throughout the tissues
Rare plasmodia exclusively in the stomach.

digestive tubule, and duct epithelia
A few Plasmodia in gill epithelia; several

Plasmodia throughout the tissues
Rare plasmodia exclusively in stomach

epithelium
Rare plasmodia exclusively in digestive

duct epithelia
Plasmodia throughout the tissues
Plasmodia throughout the tissues
Rare plasmodia exclusively in the stomach

and gill epithelia
Plasmodia throughout the tissues
Plasmodia throughout the tissues
Very rare plasmodia exclusively in

intestine and gill epithelia
Plasmodia in the stomach, digestive tubule,
and gill epithelia; some plasmodia
throuiihout the tissues

Haplosporidium costale in Oysters

117

• ■ - .. ■ •

Figure 3. ISH oCH. costale- and W. Hf/,v«;i(-inftcte(i oyster from Long
Island Sound. This is the same specimen as in Figure 2. (Al H. cosialv
DNA probe hybridizing with H. costale Plasmodia and prespores in the
connective tissue surrounding digestive tubules. iBl //. iielsoni DNA
probe hybridizing with H. nelsoiti Plasmodia and spores in digestive
tubules. (C) Higher magnification of bracketed area in item (A). (D)
Higher magnification of bracketed area in item (B). Scale bars 100 ^m.

light infections were detected in the gills and the mantle, and in the
heavy infections, were detected throughout the tissues.

ISH of an oyster with both H. costale and H. nelsoni spores is
illustrated in Figure 3. H. costale probe hybridized to Plasmodia
and prespores scattered in the connective tissue surrounding the
digestive diverticula (Fig, 3A and C), in the gills, and the mantle,
whereas H. nelsoni probe hybridized to plasmodia and prespores
mainly in the digestive tubules (Fig. 3B and D). This specimen
represented a terminal infection of H. nelsoni. when migration of
Plasmodia to digestive tubules had already occurred. Both probes
outlined mature spores but did not completely penetrate them.
Serial sections of an oyster with mixed infection of H. costale and
H. nelsoni plasmodia stained with hematoxylin-eosin and ISH with
the DNA probes are illustrated in Figure 4A, B, and C.

DISCUSSION

The presence of H. costale in Long Island Sound has been
suggested in several earlier reports (Andrews 1984; Andrews
1988; Katkansky & Warner 1970; Newman 1971 ); however, in the
absence of sporulating stages, positive identification has been im-
possible. Failure lo detect sporulating stages is easily explained on
the basis of the results of the present report; spores were detected
in only 0.08% of the oysters studied. We can assume that H.
costale has been enzootic to the area, but its presence was finally
verified, not because of increased prevalence, but because of in-
creased sampling effort and the use of species-specific diagnostic
tools. Furthermore, absence of spores in 1997 and in any of the
samples collected from the north shore of Long Island, New York,
is most likely due to small sample sizes (310 and 600 oysters,
respectively), which would give <0.5'7f probability to encounter a
sporulating specimen. High occurrence of positive specimens de-
tected in Branford-Clinton area (Table 1) was due to intensive
sampling in that area.

Detailed descriptions of sporulation and spore structures of H.
costale or H. nelsoni are presented by Couch et al. ( 1966), Rosen-
field et al. (1969), and Perkins (1969). Although morphological
characteristics of H. costale spores or plasmodia in our material

B

t

«

c

^^

4

*

*>'

• •.•-'

• » »

•

%..

•
••

».^'

^.

•

*

•p

•

»

•

•

^

V|-

•

•

•

c

•

':

.♦ •

► t

*

.. ; ••

r

•

. - V

«

Figure 4. Serial sections of an oyster with mixed haplosporidian plas-
modial infection. (.A) Hematoxylin-eosin stain. (B) ISH with H. costale
DNA probe detecting rare plasmodia in the gill. (Cl ISH with H. nelsoni
DNA probe of the same area shown in item (B). Scale bars 100 (im.

did not differ from those described in previous publications, H.
costale in Long Island Sound appears to have a different patho-
genesis than in the south. Gross signs as defined by Andrews
(1988) (emaciation, failure of new shell growth in spring, high

118

SUNILA ET AL.

epizootic mortality mid-May-iiiid-June. and discoloration of gap-
ers by spores) did not apply to our samples. In Virginia, infected
oysters sporulate synchronously in May-July, presenting an obvi-
ous, easily diagnosed stage. All plasmodia develop into sporonts.
and oysters die promptly after sporulation (Andrews 1984). H.
costale is considered to be a well-adapted parasite that infects new
oysters via spores that are released during mortality season (An-
drews 1982). Spores are usually detected in moribund oysters and
infection causes 20%-50% yearly mortalities (Andrews 1988).
Cosporuiation with H. iielsoni was reported previously by Couch
( 1967) in oysters collected from Chincoteague Bay, Virginia. The
six cases with both spore types were dead and dying oysters col-
lected in May and June during the characteristic H. costale sporu-
lation time for oysters in Virginia (Couch 1967).

SSO disease in Long Island Sound differed from the above
description in several ways. First, plasmodia and sporulation in the
present material were found in October to December. During this
time period, SSO disease in Virginia is subpatent and no plasmodia
are detected before spring (Andrews 1988). However, in a recent
paper describing H. costale probes also used in the present publi-
cation, Stokes and Bun-eson (2001) reported the presence of a
positive ISH to H. costale plasmodia in an oyster sampled in
October 1994 in Virginia. In the pre.sent paper, spores were de-
tected in live specimens with no evidence of a synchronous mor-
tality event. Spores were very rare, and infection always occurred
as a coinfection with H. nelsoiii. Because H. costale plasmodia
were also detected in the samples, sporulation may occur infre-
quently. Rare sporulation such as presented in this report cannot
sustain a widespread infection. Actual prevalence of H. costale in
the area is not known based on the results of the present study, but
the rare sporulation and the difficulty of finding positive speci-
mens with ISH suggest a low prevalence.

Possible mortality associated with H. costale is impossible to
estimate because of an H. iielsoiii epizootic that occurred in Long
Island Sound in 1997-1998 (Sunila et al. 1999). Oyster production
in Connecticut decreased from more than 300,000 bushels in 1996
to 170,000 in 1999, reflecting high MSX-associated mortalities.
According to Andrews (1984), H. iielsoni kills oysters much
quicker than H. costale, and depresses manifestation of SSO dis-
ease during MSX epizootics.

H. costale is usually restricted to high-.salinity bays with sa-
linities >30%c. Its lower limit is 25%f and salinities <20%c appear
to cause disease regression (Andrews 1979). It is possible that
salinity in Long Island Sound's oyster beds is not high enough to
sustain full epizootic H. costale activity. In Branford lots (Table 1 ).
salinity varies between 26%r and 21%o: in the Clinton Cedar Island
Marina (in the mouth of Hammonassett River), salinity varies from
139ff to 28'/ff. Oysters in the area are transplanted four times before
they are marketed. This exposes them to even lower salinities,
which may have provided a control for the disease.

Classically, the diagnosis of H costale relies on the presence of
sporulating stages and the site of initial infection, which for H.
costale is the epithelium of the digestive system and for H. nelsoni
is the gill epithelium. In addition, history of the sainpling area
relating to past H. costale or H. nelsoni infections directs the
diagnosis. In the present report, we were able to diagnose H.
costale in a new geographic area with a deviating sporulation time
by using DNA probes (Stokes & Burreson 1995; Stokes & Bur-
reson 2001). Further research to study the infection cycle, preva-
lences, and possible association with mortalities is under way.

ACKNOWLEDGMENTS

This study was funded in part by Sea Grant No. NA86RG0075;
VIMS contribution number 2461.

LITERATURE CITED

Andrews, J. D. 1979. Oyster diseases in Chesapeake Bay. Mar. Fish. Rev.

41:45-53.
Andrews, J. D. 1982. Epizootiology of late summer and fall infections of

oysters by Haplosporidium nelsoni. and comparison to annual life cycle

of Haplosporiditini costalis. a typical haplospiiridan. / Shellfisli Res.

2:15-23.
Andrews, J. D. 1984. Epizootiology of diseases of oysters (Crussostrea

virginica). and parasites of associated organisms in eastern North

America. Helgoliinder Meeresunters. 37:149-166.
Andrews, J. D. 1988. Haplosporidium costale disease of oysters. In: C. J.

Sindemiann & D. V. Lightner, editors. Disease diagnosis and control in

North American marine aquaculture. Amsterdam: Elsevier, pp. 296-

299.
Andrews, J. D. & M. Castagna. 1978. Epizootiology of Minchinia ciisltdis

in susceptible oysters in seaside bays of Virginia's eastern shore. 1959-

1976. J. Invert. Pathol. 32:124-138.
Couch, J. 1967. Concurrent haplosporidian infections of the oyster. Cras-

sostrea virginica (Gmelin). J. Parasitol. 53:248-253.
Couch, J., C. A. Farley & A. Rosenfield. 1966. Sporulation of Minchinia

nelsoni (Haplosporida, Haplosporidiidae) in Crassostrcci virginira

(Gmelin). Science 23:1529-153L
Farley, C. A. 1965. Acid-fast staining of haplosporidian spores in relation

to oyster pathology. J. Invert. Pathol. 7:144—147.
Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms. In:

V. S. Kennedy, R. 1. E. Newell & A. F. Eble, editors. The eastern

oyster. Crassostrea virginica. College Park, MD: Maryland Sea Grant

Book. pp. 581-660.

Katkunsky. S. C. & R. W. Warner. 197(1. The occurrence of a haplospo-
ridian in Tomales Bay, California. J. Invert Pathol. 16:144.

Meyers, T. R. 1981. Endemic diseases of cultured shellfish of Long Island.
New York: Adult and juvenile American oysters (Crassostrea vir-
ginica) and hard clams {Mercenaria mercenaria). Aqndcidtnre 22:305-
330.

Newman. M. W. 1971. A parasite and disease survey of Connecticut oys-
ters. Proc. Natl. Shellfish. Assoc. 61:59-63.

Perkms, F. O. 1969. Electron microscope studies of sporulation in the
oyster pathogen, Minchinia costalis (Sporozoa: Haplosporida). J. Para-
sitol. 55:897-920.

Rosenfield. A.. L. Buchanan & G. B. Chapman. 1969. Comparison of the
fine structure of spores of three species of Minchinia (Haplosporida,
Haplosporidiidae). / Parasitol. 55:921-941.

Stokes. N. A. & E. M. Burreson. 1995. A sensitive and specific DNA probe
for the oyster pathogen Haplosporidium nelsoni. J. Eukaryot. Micro-
biol. 42:350-357.

Stokes, N. A. & E. M. Burreson. 2001. Differential diagnosis of mixed
Haplosporidium costale and Haplospinidium nelsoni infections in the
eastern oyster, Crassostrea virginica. using DNA probes. / Shellfish.
Res. 20:207-213.

Sunila. I.. J. Karolus & J. Volk. 1999. A new epizootic of Haplosporidium
nelsoni (MS.X). a haplosporidian oyster parasite, in Long Island Sound,
Connecticut. / Shellfish Res. 18:169-174.

Wood, J. L. & J. D. Andrews. 1962. Haplosporidium costale (Sporozoa)
associated with a disease of Virginia oysters. Science 136:710-71 1.

Journal of Slwlirtsh Research. Vol. 21, No. 1. 119-125. 2002.

INFECTION INTENSITY, PREVALENCE, AND HISTOPATHOLOGY OF PERKINSUS SP. IN
THE MANILA CLAM, RUDITAPES PHILIPPINARUM, IN ISAHAYA BAY, JAPAN

KWANG-SIK CHOI,'* KYUNG-IL PARK.' KI-WAN LEE,' AND KAZUMI MATSUOKA^

' File ill ty of Applied Marine Science. Cliejii National University, 1 Ara 1-Dong, Jejii City Jeju-Do
690-756. Korea: 'Laboratory of Coastal Environmental Sciences, Faculty of Fisheries, Nagasaki
Universit}-, 1-14, Biinkyo-machi. Nagasaki 852-8521 . Japan

ABSTRACT Infection intensity, prevalence of infection, and pathologic features of Perkinsus parasitism among Manila clams
inhabiting in Isahaya Bay. Japan, were investigated. Ray's fluid thioglycollate medium technique followed by Choi's 2 M NaOH
digestion assay was applied to determine the infection intensity and prevalence. The infected tissues were also microscopically
examined from histological preparations. The prevalence of infection was STVr in clams sampled in February 2001. and the mean
infection intensity was .^."i 1,603 Perkinsus cells per clam or 225.701 Perkinsus cells/g of tissue. Perkinsus sp. was abundantly
distributed in the gill and vi.sceral mass, whereas it was rare in the adductor muscles and siphons. The total number of Perkinsus in
the clams was linearly correlated with the number of Perkinsus cells in the gill tissues (r = 0.908), suggesting that gill could be a
target tissue for efficient diagnosis of Perkinsus infection. Heavily infected clams exhibited white nodules on the surface of the mantle
as a consequence of inflammatory response to Perkinsus infection. Numerous trophozoites were observed in the connective tissue
around the gonads and gill filaments, suggesting that heavy infection with Perkinsus may exert potential deleterious effects on growth
and reproduction by interfering with the reproductive maturation and filtration activities of the clams.

KEY WORDS: Perkinsus. Ruiiitapes philippinarunt. infection intensity, histopathology, Isahaya Bay, Japan

INTRODUCTION

The protozoan parasite Perkin.siis sp. (Apicomplexa, Perkinsea)
has been known to cause mass mortalities worldwide in commer-
cially important shellfish, including oysters, scallops, clams, and
abalones (Andrews & Hewatt 1957; Mackin 1962; Lester & Davis
1981; Navas et ai. 1992; Blackbourn et al. 1998; Canestri-Trotti et
al. 2000; Park & Choi 2001; Liang et al. 20011. In particular. P.
atlaiuicus has been associated with mass mortalities of the venerid
clams of the genus Ruditapes (i.e.. Tapes or Venenipis) inhabiting
the Mediterranean and Atlantic coasts (Da Ros & Canzonier 1985.
Chagot et al. 1987, Sagrista et al. 1996). Perkinsus parasitism in
the Manila clam ("short-necked clam"), R. philippinanim. was also
recently confirmed in Japanese (Hamaguchi et al. 1998, Maeno et
al. 1999) and Korean waters (Choi & Park 1997; Park & Choi
2001; Lee at al. 2001). Perkinsus sp. distributed in Korea is be-
lieved to be responsible for the mass mortality of Manila clams.
This has occurred yearly, in late summer, since the early 1990s.
Park and Choi (20011 postulated that the decline in Manila clam
landings for the previous decade in Korea could be caused by
Perkinsus-associated mortalities occurring in every late summer.
As many other studies have reported. Perkinsus infection is asso-
ciated with high salinity, temperature, and density of clam popu-
lations (Andrews & Hewatt 1957; Soniat 1996; Burreson &
Ragone Calvo 1996; Cigarria et al, 1997).

Perkinsus-hke microorganisms have been discovered in Manila
clams distributed in Japan (Hamaguchi et al. 1998; Maeno et al.
1999) and China (Liang et al. 2001). Hamaguchi et al. (1998)
reported on the occurrence of Perkinsus in the clams collected
from Kumamoto and Hiroshima, in southern Japan. Comparison of
the DNA sequence of Perkinsus with P. atlanticus and P. olseni
indicated that the species of Perkinsus found in Kumamoto and
Hiroshima. Japan, is taxonomically very close to P. atlanticus and
P. olseni reported from Portugal and Australia (Hamaguchi el al.
1998). Manila clams are abundant in Isahaya Bay, southern Ky-

*CorTesponding author. Fax: 82-64-756-3493; E-mail: skchoi@cheju.cheju.ac.kr

ushu, Japan, where tidal flats are well developed and the clams are
commercially cultured. Ishii et al. (2001) reported that the clam
population in Ariake Sound has been declining significantly since
1987 due to overfishing, pollution, and predators. Because the
presence of Perkinsus in clam populations inhabiting Ariake
Sound was confirmed by Hamaguchi et al. (1998), detrimental
effects of Perkinsus parasitism on clam growth, as well as on
annual landings, cannot be Riled out in Isahaya Bay located on the
west of Ariake Sound. However, infection intensity and prevalence
of Perkinsus in the bay has not been reported previously.

Infection intensity and prevalence of Perkinsus parasitism on
Manila clams was investigated from a clam population distributed
in Isahaya Bay in the present study. This paper reports diagnosis,
histopathologic features of the infected clams, infection intensity,
and prevalence of Perkinsus parasitism in the clams collected in
February 2001.

MATERIALS AND METHODS

A total of 191 clams were collected from Isahaya Bay, Kyushu,
Japan, in February 2001 (Fig. 1). In the laboratory, shell length
(SL), width (SW), thickness (ST), and tissue wet weight (TWT) of
individual clams were recorded. Condition index (CI) was then
calculated for evaluating fatness of the clams as

CI = [TWT/(ST X SW X SD] x 1,000

For evaluating Perkinsus infection, the clams were placed in
two groups: one for histopathological examination and the other
for measuring total body burden, which is the infection intensity of
each clam as a total number of Perkinsus in a clam or number of
Perkinsus/g tissue. For histopathology. a longitudinal section was
made in the middle of the body, which included gills, digestive
glands, gonads, mantle, and foot. A 5-|xm thin section was cut for
each clam after dehydration. The sections were then stained with
Harris' hematoxylin and eosin Y. For measuring total body bur-
den, the whole fiesh of individual clams was immersed in fluid
thioglycollate medium (FTMl. fortified with nystatin and chlor-
amphenicol to prevent bacterial activity (Ray 1966). and placed in

119

120

Choi et al.

Np^'^Fukuoka |

• VD DsahayaBayX

130" 131°

Figure 1. Location of the sampling site, Isahaya Bay, Kyushu, Japan.

a dark area for a week. After immersion, the tissues were digested
in 2 M NaOH, and the number of Perkinsiis cells was counted
using a hemocytometer according to Choi et al. (1989). Total body
burden was then standardized as the number of Perkinsiis/g tissue.
To determine the distribution pattern of Perkinsiis per clam, the
gills, mantle, adductor muscle, and body containing the visceral
mass were excised separately from each clain and immersed in-
dependently in FTM. After 1 wk of immersion, the number of
Perkinsus cells in the tissue was measured as described above
(Choi et al. 1989). The infection intensity was standardized and
expressed as the number of Perkinsiis cells/g tissue.

RESULTS

Histopathological Observation of Perkinsus sp.

Numerous trophozoites were observed in the clams collected
from Isahaya Bay. Eccentric vacuoles, nuclei, and nucleoli were
observed from histological sections of the infected tissues (Fig.
2A). The diameter of the trophozoites, estimated microscopically,
ranged from 7.73 to 15.80 (j.m. with a mean of 10.98 p.m. Diam-
eters of the nuclei varied from 4.20 to 6.59 )xin, with a mean of
5.44 jjim, whereas the diameters of the nucleoli varied from 1.90 to
2.67 ixm, with a mean of 2.27 |xm. Most trophozoites in the gills
and mantle formed different sizes of clusters (Fig. 2A and B).

Perkinsus was predominantly found in gill filaments, mantles,
and digestive tubules, although a few of the trophozoites were
observed in the foot (Fig. 2B, C, and D). Heavy infection with
Perkinsus in the gill lamellae resulted in swollen connective tissue
with severe hemocyte infiltration (Fig. 2B). An inflammatory re-
sponse to the parasite was also observed in heavily infected clams
in the form of nodules on the mantle surface (Fig. 2C). Those
nodules appeared as white spots on the mantle surface and could
be observed even with the naked eye. Numerous Perkinsus tro-
phozoites were also observed around the digestive glands, indicat-
ing that Perkinsus could inhibit the digestive activity of the clams
in the visceral mass (Fig. 2D). Some clams exhibited mature eggs
or sperm even in February because of the influence of the warm
Kuroshio current in the bay. Perkinsus was also observed in the
connective tissues of female as well as male gonads (Fig. 2E and
F), indicating that Perkinsus infection in the Manila clam might
disturb the reproductive processes. Sporocysts of a cercaria-like
organism were also observed in the female gonad, although the
prevalence was much lower than that of Perkinsus (Fig. 2H); only
2% of total clams investigated were infected.

Prevalence and Infection Intensity of Perkinsus per Clam and in
Different Types of Tissues

The results of this Perkinsus infection survey conducted from a
clam population in Isahaya Bay are summarized in Table I. A total
of 191 clams with a mean SL of 31.0 mm and a mean TWT of
1.566 g were analyzed in the study. Prevalence, the percentage of
infection in the clams investigated, was 57.4%. Total body burden,
in terms of the total number of Perkinsus cells in individual clams,
varied from 0 to 2,609,375, with a mean of 351,603. Infection
intensity, as number of Perkinsus cells/g tissue, varied from 0 to
1,817,196, with a mean of 225,701 (Table I). No obvious corre-
lation was observed between the infection intensity and size of the
clams, as well as the CI.

Table 2 shows prevalence and infection intensity in various
types of clam tissues. Among the four types of tissues examined,
the gills showed the highest prevalence and infection intensity,
with 85.7I'7<- and 1.019.817 cells/g gill tissue, respectively. Preva-
lence of infection in the visceral mass was as high as that observed
in the gills, whereas the infection intensity as the number of Per-
kinsiis/g tissue was much lower than the value observed in the
gills. Infection intensity and prevalence was much lower in the
adductor muscle and siphons compared to that in the gills and
visceral mass. A positive correlation was observed between num-
ber of Perkinsus cells/g gill tissue and the total body burden (Fig.
3, /- = 0.908). The number of Perkinsus cells in the visceral mass
was also highly coirelated with the total body burden (;-^ = 0.893),
suggesting that the gill and visceral mass are the main target tis-
sues for Perkinsus infection in this species. Quantitative evaluation
of Perkinsus infection among the various tissue types indicated
that Perkinsus sp. is not evenly distributed in the clams; rather, it
is concentrated in the gills and visceral mass.

DISCUSSION

Histopathological Features of Perkinsus

Although histology is not widely used in the diagnosis of Per-
kinsus infection, the technique provides valuable information on
host-parasite cellular interactions (Hine & Thorne 2000; Diggles &
Hine 2001; Lee et al. 2001). In the present study, pathogenicity of
Perkinsus sp. was visually examined from histological prepara-
tions of the infected clams. Figure 2A shows typical Perkinsus
trophozoites displaying a Perkinsns-fipecifk "ring" structure (i.e.,
vacuole and nucleolus in a nucleus, Azevedo 1989; Azevedo et al.
1990; Auzoux-Bordenave et al. 1995: Perkins 1996; Park & Choi
2001). Trophozoite diameter measured in the present study was
somewhat comparable to the size reported by Hamaguchi et al.
(1998) and Maeno el al. (1999) in Japan. Trophozoite diameter
measured in our study varied from 7.73 to 15.80 p.m, with a mean
of 10.98 |xm. Hamaguchi et al. (1998) reported 5.3-32.5 jjim, with
a mean of 14.8 |j.m, as diameter of trophozoites in Manila clams,
and Maeno et al. (1999) reported 5.7-11.4 |a.m. In contrast, tro-
phozoites of Perkinsus sp. found in R. plulippinanini on the north-
em coast of China varied from 2 to 10 \i.m (Liang et al. 2001 1. The
size of trophozoites estimated in our study is also similar to the
size of P. olseni [which is taxonomically very close to Perkinsus
sp. found in Japan (Hamaguchi et al, I998)|, discovered in the
Australian black-ribbed abalone, Halioiis riilvci (Lester & Davis
1981).

Some heavily infected clams exhibited numerous clusters of
trophozoites on their gill plicae and digestive tubules with severe
hemocytic infiltration (Fig. 2B and D). Such a heavy infection in

Perkinsus Infection in Isahaya Bay, Japan

121

i^ai^:V/^:-'-

'^^

«

" !((■

Figure 2. Histopathological features of Perkinsus infection. (A) Mature trophozoites containing vacuole (V). Nucleolus (NL) occurs in nucleus
(NS). l.OOOx, scale bar = 5 nm. (B) Severe inflammatory reaction (asterisk) occurs around trophozoites in gill tissues. lOOx, scale bar = 100 jim.
(C) Early stage of nodule formation. Infiltration of hemocytes (asterisks) around trophozoites (arrows) results in the swollen connective tissue
of the foot. 20(»x. scale bar = 20 pm. (I)) Trophozoites in connective tissues of digestive glands. 400x. scale bar = 40 pm. (E) Trophozoites in
connective tissues of female gonad. \ oung oocytes (Ol are seen around the capsulated trophozoites. 400x, scale bar = 500 urn. (F) Grouped
trophozoites in connective tissues of male gonad. Concentrated hemocytes of the host enclose the trophozoites forming a capsule around
spermatogonia (SP). 400x. scale bar = 20 nm. (G) Inllanimation of host hemocytes (asterisk) in the mantle tissues. lOOx, bar = 100 fim. (H)
Sporocysts containing germ balls (GB). No inflammation observed. 200x. bar = 40 pm.

122

Choi et al.

TABLE 1.

Survey results of Perkiiisus infection in the clams distributed on
Isahaya Bay.

.V

Average

SD

Min

Max

SH (mml

191

21.4

2.1

15.6

25.9

SL (mml

191

31.0

3.1

22.3

39.9

TWT (g)

191

1.566

0.521

0.537

2.769

CI

89

0.167

0.028

0.059

0.2-32

Total PerlcinsLis

(cells/clam)

89

351.603

549.046

0

2.609.375

Unit Perkinsus

(cells/g twt)

89

225.701

365.002

0

1.817,196

Prevalence

150

57'7r

SH = shell height; SL = shell length; TWT = total tissue wet weight; CI
= condition index; SD = standard deviation; Min = minimum; Max =
maximum.

gill tissues may lower filtration efficiency and, in turn, cause re-
tarded growth, although the effects of Perkinsiis infection on fil-
tration activity of the clams has not been experimentally proven.
Infestation of Perkinsus in digestive tubules would cause digestive
tubule atrophy and exert deleterious effects on the food digestion
of the clams, as reported by Lee et al. (2001 ). Clams more heavily
infected with Perkinsus exhibited white nodules on their mantle
surfaces as well as gills, as was reported in other studies (Azevedo
1989; Navas et al. 1992; Montes et al. 1996; Almeida et al. 1999;
Lee et al. 2001; Park & Choi 2001). A cross-section of the nodules
revealed that they are the result of hemocytic encapsulations of
trophozoites and massive hemocytic infiltration around the mantle
tissues, evoking tissue inflammation (Fig. 2C). Several studies
also have reported that Perkinsus secretes extracellular enzymes
that perturb the host's immune system, which, in turn, allows the
host animal to become more susceptible to other pathogens (Gar-
reis et al. 1996; La Peyre et al. 1996; Faisal et al. 1999; Ordas et
al. 2000).

Perkinsus was also observed among the connective tissues of
female as well as male gonads (Fig. 2E and F), suggesting that
Perkinsus infection also affects the reproduction of the clams in
some way. Several studies have suggested that Perkinsus could ( I )
slow the gonad development process, or (2) reduce the reproduc-
tive output of the host animals by consuming net energy produc-
tion needed for gamete production (White et al. 1988; Wilson et al.
1988; Choi et al. 1993; Choi et al. 1994). In C. virginica. no
obvious correlation was observed between estimated fecundity and
P. mariniis infection (Choi et al. 1993). In contrast, the rate of egg
protein synthesis was found to be slower in C. virginica heavily
infected with P. marinus, whereas the rate was faster in oysters
with relatively low infection (Choi et al. 1994). This suggests that
the main effect of Perkinsus on host animal reproduction could be
retardation of gonadal maturation rather than reduced gamete pro-
duction. Reduced fecundity and impeded gonadal maturation were
observed in Manila clams heavily infected with Perkinsus sp. in
Korea (Park & Choi, in preparation). *"

Infection Intensity and Prevalence per Clam and in Various Types
of Tissues

Numerous methods have been applied in the examination of
Perkinsus parasitism since the first report on the occurrence of P.

marinus in the American oyster, Crassoslrea virginica (Mackin et
al. 1950). Perkinsus infection has been diagnosed using histology
(Perkins & Menzel 1966; Azevedo el al. 1990; Navas et al. 1992;
Sagrista et al. 1995; Sagristaet al. 1996; Montes et al. 1996; Bower
et al. 1998; Mine & Thome 2000), FTM assay (Ray 1952; Ray
1966; Choi et al. 1989; Bushek et al. 1994; Rodriguez & Navas
1995; Fisher & Oliver 1996; Ford 1996; Almeida et al. 1999),
immunological probes using Perkinsus-Hpecif\c antibodies (Choi
et al. 1991; Dungan & Roberson 1993; Maeno et al. 1999), and
polymerase chain reaction (PCR) techniques (Marsh et al. 1995;
Hamaguchi et al. 1998; Robledo et al. 1998; Figueras et al. 2000;
Coss et al. 2001 ). Among these methods. FTM assay has been the
most widely and frequently used in all types of Perkinsus diagno-
sis (Lester & Davis 1981; Azevedo 1989; Rodriguez & Navas
1995; Choi & Park 1997; Cigarria et al. 1997; Almeida et al. 1999;
Liang et al. 2001 ), although FTM assay was initially designed for
the detection of P. marinus (Ray 1953; Ray 1966). In the FTM
assay, suspected tissues are immersed in 10 to 15 mL of FTM
fortified with antibiotics for 1 or 2 wk. After immersion, hypno-
spores of Perkinsus developed in FTM and stained dark blue or
brown with LugoFs iodine and were readily identifiable under a
light microscope. The number of Perkinsus spores in FTM-assayed
tissues can be accessed after digesting the tissues with 2 M NaOH.
according to Choi el al. (1989). FTM assay combined with 2 M
NaOH digestion has been successfully used in the quantification of
P. marinus (Choi et al. 1989; Bushek et al. 1994; Fisher & Oliver
1996), as well as other Perkinsus species (Rodriguez & Navas
1995; Choi & Park 1997; Park 1999; Liang et al. 2001; Park &
Choi 2001).

Infection intensity and prevalence of Perkinsus sp. in Manila
clams in Isahaya Bay. Japan, is first reported in this study. Ray"s
FTM technique combined with Choi's NaOH digestion technique,
which has been used in many other studies, was successfully ap-
plied in the quantification of Perkinsus in this study. Prevalence of
infection and mean infection intensity of Perkinsus in the clams
collected in February 2001 was 579c and 225,701 spores per gram
tissue, respectively (Table I). Hamaguchi et al. (1998) also re-
ported the prevalence of Perkinsus in R. philippinarum collected
from Kumamoto. east coast of Ariake Sound, and Hiroshima. The
prevalence measured from Kumamoto was 87.5% in commercial
clam beds and 56.3% in natural clam beds. The prevalence values
reported by Hamaguchi et al. (1998) are similar to those estimated
from Isahaya Bay. The prevalence observed in Hiroshima. 93.8%
in commercial beds and 84.4% in natural habitats, was somewhat
higher than the prevalence measured in Kumamoto and Isahaya
Bay. No data on the infection intensity of Perkinsus in the Manila

TABLE 2.

Prevalence and average infection intensity of Perkinsus sp. among
various tissues of R. philippinarum in = 35).

Average Infection Intensity

Tissue

Prevalence

{Perkinsus Cells/g Tissue

Types

C/f)

\\ et Weight p SD)

Gills

85.71

1.019,817 ± 1,393,736

Siphons

45.71

66,579 ±152.537

Adductor muscle

42.86

56.331 ± 120.086

Visceral mass

82.86

120.918+ 164.124

SD = standard deviation.

Perkinsus Infection in Isahaya Bay. Japan

123

7 x105-

6 xl05-

5x105-

4 xlQs-

fl)

-)

3x10!-

rn

CO

■*—

2 xl05-

<l)

$

1 x105-

CO

C

c-

-!r

<li

U.

o

8 x105

a)

^

7 xlQs

t-

6 xlO*

15

5 x10i

o

1—

4 x105

3x105

2 x105

1 x105

0

y = 0.8822X + 111,703
R2 = 0.2763

Adductor muscle

1 x105 2 x105 3 xlO- 4 x105 5 x105 6 xlQs 7 xlQs

Number of Perkinsus / g siphons

0 1 <105 2x105 3x105 4x105 5x105 6x105

Number of Perkinsus I g adductor muscle

y = 0.1209X + 23.131
R2 = 0.908

1 x105 2 x10s 3 x105 4 x105

Number of Perkinsus I g gills

5 x105

8 x105t
7 xl05
6 x105
5 xl05
4 x105
3 x105
2 x105

I Xl05

c

0

y =

1.1653X

+ 21

.075

^

R2=

0.8933

♦/•

y^ ♦

♦ ♦

♦/^

r *

Visceral

mass

<^

«

1 xlO= 2 xlOS 3 x105 4 x105 5 xlO^ 6 x105 7 x105

Number of Perkinsus I g visceral mass

Figure 3. Correlation between the total number of Perkinsiislg tissue wet weight and the number Perkinsus/g of siphons, adductor muscle, gills,
and visceral mass.

clam are available so far in Japan with which to compare the
infection intensity measured in this study. The infection intensity
and prevalence observed in the present study is somewhat lower
than the values reported from neighboring countries. Prevalence of
Perkinsus infection in a survey of R. philippinariiin from Komsoe
Bay. on the west coast of Korea, where tidal flats are well devel-
oped and used as a clam culture ground, was almost 100%, with a
mean infection intensity of 709.028 spore/g tissue (Park & Choi
2001). Perkinsus infection reported from populations of R. philip-
pinarum in the northern Yellow Sea (38°50'26":39°27'09") was
also comparable to the values reported in the present study. The
prevalence varied from 209f to 1009f. with mean infection inten-
sity of 2 to 1.670.615 spores/g tissue in the northern Yellow Sea
(Liang et al. 2001).

No obvious correlation was observed between the infection
intensity and size of the clams, as well as the condition index, in
this study, although several studies have reported that Perkinsus
infection is often positively correlated with oyster or clam size.
Mackin (1951) and Ray (1953) found that C. rirginica juveniles
less than 1 y old have lower levels of infection compared to mar-
ket-sized oysters. Perkinsus sp. found in R. pliilippinaruni in Ko-
rea, as well as in China, also showed similar size-dependent in-
fection. Clams smaller than 13 mm in shell length normally exhibit
no infection while the larger clams appeared to be susceptible to
Perkinsus (Choi & Park 1997; Liang et al. 2001; Park & Choi
2001 ). Absence of any correlation between the clam size and Per-
kinsus infection intensity in this study could be explained by the
low prevalence of infection in the clams. In this study, 43% of the
clams examined showed zero infection, which, in turn, resulted in
a poor correlation coefficient. Several studies have indicated that
low Perkinsus infection prevalence matches well with low infec-
tion intensity in terms of number of Perkinsus cells/g tissue (Choi

& Park 1997l Liang et al. 2001; Park & Choi 2001). However, it
is unlikely that the observed low prevalence is related to the size
of clams, because no juvenile clams (i.e.. less than 15 mm SL)
were included in the analysis. The clams used in this study were
22.3-39.9 mm in shell length, with a mean of 31.0 mm. and all of
them are considered to be over 2 y old. Relatively low infection
prevalence in Isahaya Bay could be attributed to the density of
clams in their habitat. The clam density in the bay was observed to
be lower than the clam density reported from other commercial
clam beds, although the clam density in the sampling location was
not estimated. High infection intensity and prevalence of Perkinsus
is common among clam beds where the clams are intensively
cultured, resulting in high density (Da Ros & Canzonier 1985;
Choi & Park 1997: Liang et al. 2001; Park & Choi 2001). In a
high-density bed of clams, Perkinsus disease can be transmitted
quickly because any life stage of Perkinsus would be infectious
(Ray & Mackin 1954; Andrews & Ray 1988; Auzoux-Bordenave
et al. 1995; Perkins 1996).

Infection intensity of Perkinsus among different types of tis-
sues in R. pliilippinaruni was compared in this study using Ray's
FTM following the 2 M NaOH digestion (Ray 1966; Choi et al.
1989). Infection intensity of mantle, gills, siphon, and body con-
taining visceral mass was separately determined in this study for
diagnostic purposes. As shown in Table 2, the prevalence of in-
fection was found to be highest in the gills, followed by the vis-
ceral mass. Perkinsus density in the gills in terms of the number of
Perkinsus cells/g tissue was also the highest among various types
of clam tissues analyzed (Table 2). A strong positive correlation
was also observed between the numbers of Perkinsus spores/g
gill tissue and the total number of Perkinsiislg in whole clams (Fig.
3, r = 0.908 ). The number of Perkinsus spores in the vLsceral
mass was also strongly correlated with the number of Perkinsus

124

Choi et al.

spores in whole tissues (Fig. 3. r = 0.893). Parl< (1999) and
Rodriguez and Navas ( 1995) also observed strong positive coire-
lations between infection intensity in gill tissues and infection
intensity of whole clams in Korea and Spain. The high density of
PerkinsK.s in the gill and visceral mass indicates that a favor-
able condition for Perkinsus growth and reproduction is provided
in these tissues, where more energy is believed to be available
for Perkinsus (Choi et al. 1989). Our data suggest that gill assay is
an excellent alternative for whole clam assay (i.e., body burden
assay, Rodriguez & Navas 1995; Park 1999).

ACKNOWLEDGMENTS

We are grateful to the staff of the Shellfish Aquaculturc and
Research Laboratory of Cheju National University for their help
with FTM and histopathology. We also thank Mr. Ronald Nose-
worthy for his proofreading and improvement of the manuscript.
This study was supported from Cheju National University Grant
and Grant-in- Aid for Scientific Research of Japan (No. 1 1695080;
Study on Environment and Biology in the East China Sea and
Yellow Sea). We appreciate this support.

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Montes J, M. Durfort & J. Garcia-Valero. 1996. When the venerid clam,
Tapes decussatus. is parasitized by the protozoan Perkinsus sp. it syn-
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Navas. J. I., M. C. Castillo, P. Vera & M. Ruiz-Rico. 1992. Principal
parasites observed in the clams, Ruditapes decusastus (L). Ruditapes
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Venerupis aureus (Gmelin), from Huelva coast (S,W. Spain). Aqua-
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Ordas, M. C. A. Ordas. C. Beloso & A. Figueras. 2000. Immune param-
eters in carpet shell clams namrally infected with Perkinsus atlanticus.
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Park, K.-I. 1999. Occurrence of Perkinsus sp. in the Manila clam, Rudi-
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Park, K.-I. & K.-S. Choi. 2001. Spatial distribution of the protozoan para-

site. Perkinsus sp., found in the nianiia clam. Rudiui/ics philippinaruin
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Perkins, F. O. & R. W. Menzel. 1966. Morphological and cultural studies
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Ray. S. M. 1952. A culture technique for the diagnosis of infection with
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116:360-.361.

Ray, S. M. 1953. Studies on the occurrence of Dermocystidium marinum in
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Ray, S. M. 1966. A review of the culture method for detecting Dennocvs-
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Ray. S. M. & J. D. Mackin. 1954. Studies of pathogenesis of Dcrmocxs-
tidiuni marinum. Proc. Natl. Shellfish Assoc. 45:164-167.

Robledo. J. A. F., J. D. Gauthier, C. A. Coss, A. C. Wright & G. R. Vasta.
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Journal of Shellfish Research, Vol. 21, No. 1, 127-135. 2002.

SEASONAL CHANGES IN THE HISTOLOGICAL AND BIOCHEMICAL PROFILE OF THE

GONAD, DIGESTIVE GLAND, AND MUSCLE OF THE CALAFIA MOTHER-OF-PEARL

OYSTER, PINCTADA MAZATLANICA (HANLEY, 1856) ASSOCIATED WITH GAMETOGENESIS

PEDRO SAUCEDO, ILIE RACOTTA, HUMBERTO VILLARREAL, AND
MARIO MONTEFORTE

Ceutro lie Investigaciones Biologicas del Noroeste. S.C. Mar Bermejo 195, Col. Playa Palo de Santa
Rita, La Paz, Baja California Sitr, Mexico

ABSTRACT The relationship between the energy storage cycle and gametogenesis of the pearl oyster. Pmaada mazarlamca was
studied over an annual cycle (January to December 1999). Histological analysis, combined with oocyte examination and measurements
of carbohydrate, protein, total lipid, and triacylglycerides levels from gonadal tissue, digestive gland, and adductor muscle were
performed. One-way ANOVA was u,sed for assessing differences in the area of oocytes over time. Similarly, a two-way ANOVA was
applied tor differences in the biochemical composition of specimens over time and sex. The gametogenic cycle was affected by the
presence ot "La Nina" cold event during the first half of 1999. Gametogenesis commenced early in February and occurred synchro-
nously throughout the annual cycle. There were two reproductive peaks, one in spring (March to May) and other in summer (July to
September). A massive spawning was observed in September-October when water temperature was 29-29.5°C Carbohydrates either
stored or obtained from ingested food, were used as an immediate fuel for the production of oocytes, which grew and increased their
protein content dunng the first half of the year. Lipids and triacylglycerides also showed two important peaks in the female conadal
tissue and digestive gland, corresponding to the same peaks described histologically. Reserves stored in the muscle and digestive gland
were actively used for gametogenesis. Muscle proteins were mobilized to the gonad during the first half of the year while carbohy-
drates were used dunng the second half The digestive gland acted as a short-term storage site of carbohydrates and lipids during gonad
development. More studies on seasonal changes and energy storage and mobilization in pearl oysters are required, especially involving
the panicipation ot the mantle tissue.

KEY WORDS:

Bahia de La Paz, gametogenesis, metabolism, pearl oysters, temperature. Pmaada mazatlanica

INTRODUCTION

Marine bivalves show cycles of energy storage and utilization
that are closely related to gametogenic cycles (Gabbott 1975;
Bayne 1976; Barber & Blake 1981). Most species are capable of
storing nutrient reserves in their body tissues during periods of
high food supply, which are subsequently mobilized during times
of food shortage, decreased rates of feeding, and/or high energy
demand (Ansell 1974; Gabbott 1975: Bayne 1976; Sastry 1979;
Barber & Blake 1981, 1991; Epp et al. 1988). Gametogenesis
represents a period of particulariy high-energy demand, when both
maintenance costs and the cost of gamete synthesis must be met by
the food supply, stored reserves or a combination of both. The
reproductive and biochemical cycles are thus closely coupled in
most species of bivalves but vary in relation to environmental
factors, between species, and between populations of the same
species (Epp et al. 1988).

In addition, many marine bivalves inhabiting subtropical and
temperate areas follow an annual reproductive cycle with precise
periods of gonad maturation and spawning. Reproduction usually
demands considerable consumption of energy and the weight loss
during spawning can reach between 30 to 60% (Mathieu & Lubet
1993). In general, energy is stored prior to gametogenesis when
food is abundant in the form of lipid, glycogen, and protein sub-
strates, and subsequently is mobilized and utilized in the produc-
tion of gametes when metabolic demand is high (Bayne 1976;
Barber* Blake 1981. 1983, 1991).

Peari oysters are marine bivalves having a considerable eco-

Corresponding author. Pedro Saucedo. Centro de Investigaciones Biologi-
cas del Noroeste. S.C, Mar Bermejo 195, Col. Playa Palo de Santa Rita. La
Paz, Baja California Sur, Mexico, E-mail: psaucedo@cibnor.mx

nomic and commercial value because of their natural capacity to
produce pearis of high quality (Monteforte 1990, 1996). In spite of
this, the factors which affect reproductive success in these species
are pooriy studied and understood, although their basic reproduc-
tive biology has been well documented histologically (Tranter
1958a; Tranter 1958b; Wada 1959; Sevilla 1969; Chellam 1987;
Rose et al. 1991; Hernandez-Diaz 1993; Arizmendi-Castillo 1996:
Garci'a-Dominguez et al. 1996; Saucedo & Monteforte 1997;
Behzadi 1997).

Presently, the growing importance and value of peari oysters
has promoted the use of hatchery-produced stock, and as such, the
need for knowledge relating to broodstock conditioning and larval
rearing. However, in order to produce oocytes and spermatozoa of
the best quality, which will develop into strong and viable larvae,
it has been essential to understand the seasonal behavior of oysters
from wild population. This information will be used further to
determine the basic needs of broodstock in the laboratory. As an
initial step toward a better understanding of reproductive success
in the Calafia mother-of-pearl oyster Pinctada mazatlanica (Han-
ley), the relationship between the build-up and utilization of en-
ergy reserves and the annual cycle of gametogenesis and spawning
has to be investigated. The objective of this work was to study the
seasonal cycles of energy storage and depletion in the gonadal
tissue, digestive gland, and adductor muscle of this species.

MATERIALS AND METHODS

Specimen Collection and Dissection

From January to December 1999. twenty oysters were collected
monthly from the Submarine Experimental Farm of CIBNOR.
located in Caleta El Merito, in Bahi'a de La Paz, Mexico (24° 16' ;■;
and 110°19'W). Only adult specimens ranging 140 (±8 mm \-AJ)

111

128

Saucedo et al.

shell height were collected to assure the presence of both sexes in
the sample (Saucedo & Monteforte 1997). Water temperature was
recorded at the collecting site. Specimens were taken to the labo-
ratory to be dissected. The gonadal tissue (which develops gradu-
ally within the visceral mass at the expense of the interconnective
tissue matrix and the digestive gland, as gametogenesis proceeds)
and adductor muscle were excised from each specimen. The
former tissue component was preserved in Davidson's solution for
48 h and used for histological analyses. The muscle was preserved
at -80°C for biochemical analyses.

Histological Analysis

Preserved samples of gonadal tissues were dehydrated, embed-
ded in paraplast. sectioned at 5 |j.m along an anteroposterior plane,
and stained with hematoxyiin-eosin (Howard & Smith 1983). Ga-
metogenic state was characterized as either early, mid. or late
developing, ripe, partially spawned, and spent, based on a larger
version of Tranter's original scheme for pearl oysters (Tranter,
1958a, b). In this larger version, early development coiresponds to
small acini showing only few layers of spermatogonia and sper-
matocytes (in the testis) or oogonia connected to the follicular wall
(in the ovary). In mid development, acini tend to shrink and the
lumen becomes restricted, while oocytes and/or spermatids and
spermatozoa become increasingly common. Late development is
characterized by mature or maturing spermatozoa and oocytes
strongly packed within the follicular lumen, although immature
stages are still present.

The advancement of the oogenesis process was also evaluated
by counting the frequency of oocytes and measuring their size
variation over time (Grant & Tyler 1983). As a previous step,
oocytes were classified accordingly to their vitellogenic stage as
previtellogenic, vitellogenic, and postvitellogenic, following the
criterion of Gaulejac (1995) for the marine bivalve Pinna nobilis.
Aside from Gaulejac' s work, additional descriptions about the cy-
tological characteristics of each type of oocyte may be found in
Saucedo et al. (2001a).

The frequency was calculated by counting the total number of
each type of oocyte appearing in three randomly .selected areas of
the ovary. The size of oocytes was also assessed measuring thirty
oocytes of any type appearing in three random areas of the ovary.
The diameter was not used for the analysis because this dimension
usually provides data that vary considerably as a consequence of
the plane on which thin sectioning is made with the microtome.
Instead, the area is not affected that much by the sectioning process
and thus provides more homogeneous and reliable data about the
size of oocytes. Images were captured from the computer with a
digital Cole-Snap camera (I-niedia Cybernetics) and processed
with Sigma Scan Pro (vers. 5.0) designed for digital image analy-
sis.

Biochemical Analysis

For the biochemical analyses, preserved samples of gonadal
tissue and digestive gland were weighed and homogenized in 3 mL
of cold saline solution (NaCl 35% ) to obtain a crude extract. Simi-
larly, muscle samples were weighed and homogenized in 5 mL of
\0% trichloracetic acid (TCA) for carbohydrate analysis. Homog-
enized samples were then centrifuged at 3600 rpm at -5°C for 15
min, and the supernatant stored at -20°C for 24 h for further
analyses.

The TCA supernatant was directly used for carbohydrate analy-

sis in muscle. For other tissues, the saline crude extract was diluted
with 209f TCA. After centrifugation, 0.1 mL of the supernatant
was mixed with 1 niL of anthrone reagent (0.1 '/r dissolved in 767c
sulfuric acid), incubated for about 2 min at 90°C and immediately
cooled to 4'"C to stop further reaction (Van Handel 1965). Absor-
bance was read at 620 nm against a reagent blank and carbohydrate
was quantified using dextrose solution as standard.

For protein determination, the saline crude extract was diluted
1:5 in 0.5 N NaOH for soluble protein determination (Bradford
1976), using commercial chromogen reagent (Sigma) and bovine
serum albumin (Sigma) as standard solution. Absorbance was read
at 595 nm. For muscle protein determination, 0.01 g of tissue was
digested in 3 mL of 0.5 N NaOH for 24 h before following the
above methodology.

Commercial kits from Merck were used to determine the com-
position of total lipids (Merck num. 3321) and triacylglycerides
(GPO-PAP. Merck num. 1.1434.001). These methods were
adapted to a microplate using 20 jxL of supernatant and 200 |jlL of
enzyme chromogen reagent (Racotta et al. 1998; Palacios et al.
2000). Absorbance was recorded on a microplate reader (Lab-
systems, Uniskan II) at 560 nm for total lipids and 495 nm for
triacylglycerides.

Statistical Treatment

For studying the histological and biochemical variations related
to the ongoing gametogenic cycle, one-way ANOVA was used to
determine significant differences in the area of oocytes (factor T
with 12 levels or months). A two-way ANOVA was applied for
differences in the biochemical composition of gonadal tissue, di-
gestive gland, and adductor muscle over time and sex of specimens
(factor S with two levels). In addition, correlation analyses were
performed to assess the grade of relation between some histologi-
cal and biochemical parameters of the above tissue components.
For all analyses, the significance level was set at P < 0.05 (Sokal
& Rohlf 1981).

RESULTS

Variation of Temperature

Part of 1 997 and 1 998 were warm years because of the pres-
ence of "El Nino" phenomenon in waters of Bahia de La Paz. As
a consequence, the second half of 1998 and first half of 1999 were
"Niiia" years characterized by abnormal cold-water temperatures
(LIuch-Belda et al. 1999). The chronology and duration of the
reproductive stages defined in this study over the annual cycle
January to December 1999 are based in this abnormal cold event.
Figure 1 presents the historical register of mean temperature varia-
tion recorded at Bahi'a de La Paz from 1990 to 1999. Values of
water temperature in the present study showed a relatively high
variation of ±1-2.5°C from the summer and winter average values
registered during such period of time.

Histological Analysis

Reproductive Cycle

The annual gametogenic cycle is depicted in Figure 2. Inactive
gonads (unable to be sexed) were found only in January and Feb-
ruary. Gonadal de\elopment started early in February' and pro-
ceeded continuous and synchronously throughout the annual cycle
in both male and female gonads, showing two peaks of maximum

Seasonal Changes of Gametogenesis in Pinctada maz\tl\nica

129

U

o
LU

cc

3
I-

LU
Q.

LU

JAJOJAJOJ

1990 1991

A J 0 J A J 0

1992 1993

iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiHiiiiiiNiiiiiiiniiiiiiiiiiiiiiiiiiiiif

JAJOJAJOJAJOJAJOJAJOJAJO

1994 1995 1996 1997 1998 1999

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

Figure 1. Mean range of temperature variation at different sites of
Bahia de La Paz. A) Historical record from 1990 to 1999. Dotted lines
at the top and bottom of the graph are the summer and winter average
values, respectively: Bl \ alues obtained from the present study during
January to December 1999.

reproductive activity (defined in terms of tlie high incidence of
specimens in late-development and ripe stages). The first peak was
observed in spring (March to May), when water temperature was
21-23' C. However, no trace of spawning was detected during this
season. The second peak was seen in summer (July to August),
when water temperature rose to 25-26°C. In this case, a massive
spawning occurred in the following month, when temperature
reached its highest record (29-30°C). Organisms in spent stage
were observed from October to November, although some other
specimens were still spawning or starting a new gametogenic pro-
cess.

Sex Ratio

The analysis for the whole gametogenic cycle revealed a higher
frequency of males (497f ) than of females (38%). Therefore, the
female/male sex ratio was 0.78:1. The sample was also conformed
by indeterminate specimens {!%). hermaphrodites (5%). and a

small percentage of animals behaving as functional hermaphro-
dites (19f). Particularly, from January to May males completely
outnumbered females and reached a mean percentage of 63% vs.
24% of females. From June to August this trend changed to 54%
of males and 44% of females. Finally, from October on, the popu-
lation of males decreased considerably (25%) while females
strongly increased (51%).

Oocyte Examination

Figure 3 shows the variation of pre\itellogenic. vitellogenic.
and postvitellogenic oocytes over time. All types of oocytes were
observed throughout the annual cycle, although their frequencies
of appearance were variable each month. Vitellogenic oocytes
showed a relatively constant presence over time. An increase of
previtellogenic oocytes was clear in November (31%) and Decem-
ber (58%) after the main spawning was achieved. In comparison,
postvitellogenic oocytes showed higher incidences during March
(51%), May (49%), and August (48%), con'esponding to the same
peaks of maximum reproductive activity detected histologically.

Temporal variations in the size (area) of oocytes are showed in
Figure 4. Both decreases and increases in the volume of oocytes
observed over the annual cycle are probably associated with
changes in the temperature of water. Thus, small oocytes were
observed in February (eariy development: 19°C), June (resorption
process between both reproductive peaks: temperature dropped to
24°C), and October. November, and especially December (spawn-
ing and spent stage: 29.5, 28, and 26°C respectively). Large oo-
cytes were observed in March, April, and May and August during
the two reproductive peaks histologically detected. The

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
MONTHS

i INDET ; i E-DEV M M-DEV ^ L-DEV
^ RIPE I SPAWN ::B SPENT

Figure 2. Sexual gametogenic stages in Pinctada mazatlaiiica over an
annual cycle. Indet = Indeterminate stage; E-Dev = early-development
stage; M-Dev = mid development; L-Dev = late-development; spawr, -
spawning.

130

Saucedo et al.

>

0

z

UJ

D

0

LU

I
u_

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

MONTHS

i I PREvrr

VITEL

POSTVIT

Figure 3. Temporal variations In the mean frequency of occurrence of
different t>pes of oocytes in Pinclada mazutUmica o>er an annual ga-
met()j>enic cycle. Previt = previtellogenic oocytes; Vitel = vitellogenic
oocjtes; Postvit = postvitellogenic oocytes. Bars denote standard de-
viation.

ANOVA detected highly significant differences in the mean area
of oocytes over time (F = 102.97; P < 0.001).

Biochemical Analysis

Gonadal Tissue

Preliminary tests made to measure the amount of carbohydrates
in the gonadal tissue, digestive gland, and muscle indicated that
over 80% was glycogen. Thus, glycogen is reported in this study
as total carbohydrates (CHO).

When water temperature rose from 20 to 2.*)' C. CHO were
actively depleted during the first half of the year and decreased
from 12 mg/g in January to 4 mg/g in July (Fig. 5A). Afterwards,
CHO were not used for the production of gametes, yielding a slight
increase in their concentration between 5 and 6 mg/g. The varia-
tion in the concentration of this component showed significant
differences over time (F = 35.83; P < 0.001 ), but not according to
the sex of specimens (F = 0.414; P > 0.05). The decrease in the
levels of CHO in the gonadal tissue was not significantly corre-
lated with the increase in the area of postvitellogenic oocytes (/• =
0.102; P>0.05).

Proteins (PRO) accumulated mainly in the female gonadal tis-
sue during the first half of the reproductive cycle, resulting in a
.sharp rise in their concentration from 52 mg/g in January to 100
mg/g in May (Fig. 5B). After a drop in June to July, the concen-
tration varied between 58 and 65 mg/g for both sexes during the
second half of the year. There were not significant differences in the
PRO content over time (F = 0.954: P > 0.05), but differences were
significant in relation to the sex of specimens (F = 8.16; f < 0.05).

There were two peaks depicting the temporal variations in the
concentration of total lipids (LIP; Fig. 5C) and triacylglycerides
(TG; Fig. 5D) in the female gonadal tissue. The first peak was seen

in April for LIP (20.4 mg/g) and in May forTG ( 13.5 mg/g). while
the second peak was observed in August to September for both
components (13.6 mg/g for LIP and 9.4 mg/g for TG). There were
significant differences in the temporal variations of LIP (f =
13.23; P < 0.001 ) and TG (F = 2.84; P < 0.05) and also according
to sex (f = 14.24; P < 0.001 for LIP; F = 43.58; P < 0.001 for
TG). There was a positive and significant correlation between the
variations of both elements over time (/■ = 0.973; P < 0.001).
Similarly, variations in the concentration of LIP were significantly
correlated to the increase in the area of postvitellogenic oocytes
(,■ = 0.64; P < 0.005).

Digestive Gland

There was a condnuous accuiiiulalion of CHO m this tissue
from 23.4 nig/g in March to over 32 mg/g until November (Fig.
6A). After the spawnmg. the concentration of CHO decreased to
22 mg/g in December. There were significant differences in the
concentration of this component over time {F = 27.8; P < 0.001 ).
However, differences were not significant in relation to the sex of
oysters (F = 0.434; P > 0.05).

PRO showed a gradual decrease from January (156 mg/g) to
October (96 mg/g), especially in males (Fig. 6B). In females, this
trend .showed two minimum peaks in May and October. After the
spawning in late September, a shift in the concentration of PRO
was recorded again. The variations in the concentration of PRO
denoted significant differences over time (f = 24.86; P < 0.001 ),
while differences were not significant according to the sex of
specimens [F = 0.093; P > 0.05).

LIP and TG in this tissue presented two peaks of concentration
throughout time (Figs. 6C and D). The first peak occurred in April
(25 mg/g for LIP and 15 mg/g for TG). The second peak was
observed in August, with values of 17 mg/g (LIP) and 10 mg/g
(TG). Unlike gonadal tissue, the concentration of both components
during winter ( spent stages ) was the same or higher than that of the
summer. There were significant differences in the variation of the
LIP and TG content over time (f = 56.28; P < 0.001 for LIP;
F = 26.82; P < 0.001 for TG), but differences were not significant

7C00

5000
"^ 4000
UJ 3000

a:
<

2000

cti

□p

IE ±Sld Oev
□ ±Sld Err,
a Mean

m

_U C^Il

m

nan

m

m

np

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
MONTHS
Figure 4. Box and Whisker for temporal variations In the mean area
of oocytes in Pinclada mazallanica iner an annual gametogenic cycle.

D5

Seasonal Changes of Gametogenesis in Pinctada mazatlanica

A B

131

20

16

12

JAN FEB MAfl APR MAY JUN JUL AUG SEP OCT NOV DEC

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

MONTHS

MALE

FEMALE

Figure 5. Temporal and sexual variations in the mean levels of total carbohydrates (A), proteins (Bl. total lipids (C'l. and triacylglycerides lU)
in the gonadal tissue of Pinctada mazallaiiica over an annual gametogenic cycle. Bars denote standard deviation.

between sexes (F = 0.849; P > 0.05 for LIP; F = 0.267; P > 0.05
for TG). The temporal variations of both components were sig-
nificantly correlated (r = 0.963; P < 0.001 ).

Adductor Muscle

CHO in the muscle showed high concentrations during the first
half of the gametogenic cycle, varying between 95 mg/g (January)
and 1 15 mg/g (June) (Fig. 7 A). Afterwards, a sharp drop to 50
mg/g in October was observed mainly during the mid and late-
developing and ripe stages. After the spawning, a recovery in the
content of CHO was noticed. The ANOVA found significant dif-
ferences in the concentration of this component on time (F =
47.95; P < 0.001). Neither sex showed significant differences in
their CHO content over time (F = 1.39; P > 0.05).

During the first reproductive peak, the le\el of PRO in the
muscle decreased from 235 mg/g in February to 128 mg/g in May.
After a short period of accumulation in June, such levels gradually
rose and reached 227 mg/g in December (Fig. 7B). The variations
in the concentration of PRO denoted highly significant differences
over time {F = 103.61; P < 0.001), but differences were not
significant between sexes (F = QMS: P > 0.05). The decrease in
the PRO content of the muscle was significantly correlated to the
increase in the area of postvitellogenic oocytes ir = 0.632; P <
0.05).

DISCUSSION

Seasonal changes in energy storage and depletion in relati i- •■
gametogenesis have been well documented durins the las' liirct;

132

Saucedo et al.

O 0

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV OEC

JAN FEB MAB APR MAY JUN JUL AUG SEP OCT NOV DEC

FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

MONTHS

MALE

FEMALE

Figure 6. Temporal and sexual variations in the mean levels of total carbohydrates (A), proteins (B). total lipids (C), and triacylglycerides (D)
in the digestive gland of I'iiiclmla mazatkmica over an annual gametogenic cycle. Bars denote standard deviation.

decades for some commercial-edible species of marine bivalves.
such as scallops, mussels, and oysters (Giese 1969; Sastry & Blake
1971; Ansell 1974; Comely 1974; Gabbott 1975. 1976. 1983.
Bayne 1976; Taylor & Venn 1979; Zandee et al. 1980; Barber &
Blake 1981. 1983. 1991; Robinson et al. 1981; Bayne et al. 1982;
Epp et al. 1988; Couturier & Newkirk 1991; Martinez 1991; Pazos
et al. 1997; Racotta et al. 1998). Nevertheless, most of the aspects
concerning the storage capacity and mobilization of nutrients to
satisfy metabolic needs related to gametogenesis seem to be spe-
cies-specific (Barber & Blake 1981. 1991) and .some important
interspecific differences have been reported (Pazos et al. 1997).
The present work couples for the first time both gametogenic and
biochemical cycles for a species of pearl oyster.

Both histological and biochemical analyses showed the exis-
tence of two peaks of intense reproductive activity over the annual
cycle; one in spring (March to May) when water temperature was
rising from 21 to 23°C. and other in summer (August to October)
when water temperature was about to reach a maximum value of
28-29°C. The first reproductive peak was clearly defined in terms
of its high incidence of ripe specimens, high frequency of postvi-
tellogenic oocytes, and high levels of protein, lipids, and triacyl-
clycerides within the gonadal tissue. However, the histological
analysis did not identify any spawning activity in the sample. This
was confirmed by very low spatfall recorded in parallel field moni-
toring (CIBNOR-Pearl Oyster Research Program database). Such
result was probably ascribed to the low and abnormal water tem-

Seasonal Changes of Gametogenesis in Pinctada mazatlanica

133

JAN FEB MAR APR MAV JUN JUL AUG SEP OCT NOV DEC

<

EC

O

o

250

200

150 -

100 -

JAN FEB MAR APR MAY JUN JUL AUQ SEP OCT NOV DEC

MONTHS

MALE

FEMALE

Figure 7. Temporal and sexual variations in the mean \e\eh of total
carbohydrates (A) and proteins (B) in the adductor muscle of Pinctada
mazatlanica over an annual gametogenic cycle. Bars denote standard
deviation.

perature registered during March to May (21-23°C) and also to the
drop-out in the same parameter detected in June (22°C) as a con-
sequence of the presence of "La Nifia" phenomenon in waters of
Bahi'a de La Paz during the first half of 1999, which caused gonads
to undergo a partial lysis and resorption. Similar processes have
been reported to occur in the scallops Argopecten irradians cou-
centricus (Sastry 1966; Barber & Blake 1981. 1991: Epp et al.
1988) and Placopecten magellanicus (Robinson et al. 1981) under
low temperatures conditions or during times of food shortage.

In this study, the three major tissues related to gametogenesis
(gonad, digestive gland, and adductor muscle) were utilized dif-
ferently over time, but showed a clear seasonal cycle of gonadal
development, spawning, energy storage, and mobilization of nu-
trients. Gametogenesis started early in February, when food is
commonly abundant from the phytoplankton (Signoret & Sanloyo

1980; Lechuga-Deveze 1997). and wild specimens had stored
enough energy reserves in the adductor muscle (mainly) and di-
gestive gland (secondarily). When gametogenesis had already
reached the early-development stage in March, the first previtel-
logenic and vitellogenic oocytes started to differentiate within
acini. Because proteins in the muscle and digestive gland showed
a downward trend from January to May, it seems evident that
gonad growth took place at their expense during the first repro-
ductive peak. Therefore, a substantial growth in the size of oo-
cytes, an increase in the weight of gonadal tissue, and their levels
of protein, lipids, and triacylglycerides was recorded. This was
particularly evident in the female gonad, where the content of the
last three elements doubled that of males, at least during the first
reproductive peak, and partially during the second reproductive
peak. On the contrary, as gametogenesis proceeded into the mid
and late-developing and ripe stages (June to October), carbohy-
drates (glycogen) from the muscle were the only substrate mobi-
lized and used for the build-up of gametes. Following the spawn-
ing in October-November, when gonadal tissue decreased in
weight and their basic levels of lipids and triacylglycerides fell
sharply, the condition of the digestive gland and muscle improved
again.

Carbohydrates obtained from ingested food were used as an
energy-rich fuel for the build-up of gametes, via their conversion
into lipids and triacylglycerides reserves (lipogenesis). This pro-
cess has been reported to occur in some bivalves to satisfy the
metabolic demands derived from vitellogenesis (Gabbott 1973.
1976, 1983), especially because lipids and triacylglycerides are the
basic energetic reserves for sustaining embryonic and larval de-
velopment of most species of marine bivalves (Holland 1978;
Fraser 1989). However, there was a lack of correlation between
carbohydrates of the gonadal tissue and the total area of postvitel-
logenic oocytes. On the contrary, the correlation was significant
between the area of oocytes and the content of lipids in this tissue.
Several authors have discussed the relative contribution of food
intake vs. energy reserves to satisfy the metabolic demands of
growth and gonadal production in marine bivalves (Bayne 1976;
Gabbott 1976; Barber & Blake 1981, 1983; Robinson et al. 1981;
Epp et al. 1988; Racotta et al. 1998). However, because these
processes are highly dependent on several e.xogenous and endog-
enous factors, no pattern has yet been established. For example, in
Pecten maximus (Comely, 1974), Chlamys opercidaris (Taylor &
Venn, 1979), and Myiilus edulis (Gabbott, 1975), energy reserves
are required for both the initiation of gametogenesis and subse-
quent gonadal growth, while in others such as A. irradians con-
ceiitricus. food intake is necessary to sustain gonadal growth since
reserves from the muscle, digestive gland, and mantle are inad-
equate.

Parallel to their utilization for gametogenesis, ingested nutri-
ents were also incorporated into the adductor muscle and digestive
gland for storage. The muscle stored high levels of protein both at
the beginning and end of the annual cycle to sustain the start of
gametogenesis. No other study with bivalves has reported protein
values as high as those presented here for the adductor muscle of
P. mazatlanica. neither for scallops whose adductor muscle is
associated with locomotion. Because the decrease in protein con-
tent of the muscle was significantly related to the increase in the
size of oocytes during vitellogenesis, the role of this organ as the
most important site of energy storage is evident. In fact, a signifi-
cant loss of weight in the muscle to less than half its maximun-,
value was detected as both protein and carbohydrate reserves were

134

Saucedo et al.

rapidly consumed in benefit ofganietogenesis. The cDnlnbiitiini ol
the muscle has also been emphasized for other bivalves, such as
ChUimys sp (Ansell 1974; Taylor & Venn 1979). M. ediiUs (Gab-
bott 1975; Bayne 1976). A. inadians concentricus (Barber &
Blake 1981, 1991; Epp et al. 1988), P. maximus (Faveris & Lubet
1991 ), A. purpumtus (Martinez 1991 ). and A. vciuriiiisus (Racotta
et al. 1998).

Unlike the muscle, the digestive gland appears to have a sec-
ondary role in gonadal development of P. mazatlanica. This seems
evident since protein was the only fuel transferred to the gonadal
tissue during the first half of the year. On the contrary, carbohy-
drates were progressively stored from March to November, indi-
cating that despite the advancement of gametogenesis. these nu-
trients are not used for the cycle in course and rather accumulated
for further energy needs. This result is in agreement with the
finding of Barber and Blake (1981. 1991 ) that the digestive gland
acts as a short-term storage and transfer site of carbohydrates to
meet the reproductive events in M. ediilis. Regarding the storage
function, Saucedo et al. l2001b) found specialized vesicular con-
nective tissue (VCT) cells — filled mainly with carbohydrates —
surrounding excretory conduits among the digestive gland and
gonadal tissue. We believe that VCT cells from both tissues are
probably communicated via the interconnective tissue matri.\.
which may be the vehicle for the transpoil of stored carbohydrates
between the digestive gland and the gonadal tissue, and vice versa.
Lipids and triacylglycerides reserves were stored and rapidly ino-

bilized after each of the reproductive peaks detected. Therefore.
P. muzalliuucu may be considered a species that uses a strategy of
storing large quantities of energy for starting and sustaining ga-
metogenesis. However, our knowledge about the metabolic control
of reproduction in pearl oysters is still limited. More studies on
seasonal cycles of energy storage and mobilization are required,
especially involving the participation of the mantle tissue, which
has been proposed to be an important site of storage of glycogen
and lipids in some bivalves. It would also be interesting to obtain
data similar to that from this study for tropical species of Pincladci.

ACKNOWLEDGMENTS

This study was done as part of two institutional projects of
CIBNOR on pearl oyster culture and pearl induction in Bahia de
La Paz (Projects ABM-7 and ABM-39). Additional grants were
provided by the Consejo Nacional de Ciencia y Tecnologia
(CONACYT-Me.xico, as a Ph.D. scholarship), the Consejo Nacio-
nal para la Biodiversidad (CONABIO), and the Fondo Mexicano
para la Conservacion de la Naturaleza (FMCN). The authors thank
the following staff of CIBNOR: Diana Carreno and Carmen
Rodriguez (for support during the biochemical and histological
analyses, respectively) and to Horacio Bervera and Juan Jose
Ramirez (for SCUBA diving assistance and collection of oysters).
We also thank Dr. Paul Southgate (James Cook University, Aus-
tralia) for his comments and suggestions during the revision of the
paper and for editing the English-language text.

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HYBRIDIZATION OF TETRAPLOID AND DIPLOID CRASSOSTREA GIGAS (THUNBERG) WITH

DIPLOID C. ARIAKENSIS (FUJITA)

HUAYONG QUE' AND STANDISH K. ALLEN. JR."*

'institute of Oceanology, Chinese Academy of Sciences, Qingdao. Shandong 266071, P.R. China:
'Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Science, Rutgers University,
Port Norris, New Jersey 08349

ABSTRACT Three replicates of hybrid cros.ses ot tetraploid and diploid C. gigas (ThunbergI with diploid C ariakensis (Fujita) were
produced with controls. Larval survival and growth were documented. Cytological events were also monitored in oocytes from hybrid
crosses following insemination. Among the four types of hybrid crosses, diploid C. gigas (female) x diploid C. uiiakensis (male) (GA)
was the most successful. Survival of GA was about the same as that of controls in two of three replications, although its growth rate
was 25-30% lower. Crosses of tetraploid C. gigas (female) and diploid C. ariakensis (male) (GGA) had poor yield at day 2
post-fertilization (0.05%). but grew nearly as well as controls subsequently. The other two types of hybrids (i.e.. diploid C. ariakensis
[female] and tetraploid C. gigas [male] [AGG]. diploid C. ariakensis [female] and diploid C gigas [male] [AG]) suffered very low
yield at day 2 (0.01% and 0.003%) and grew very slowly. Spat were obtained from all replicates of GA crosses and one of three
replicates of GGA, and proved to be hybrids by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP)
diagnosis. GGA hybrids were confirmed to be triploid by flow cytometry. No larvae survived to eyed stage in AGG or AG crosses.
Cytological examination revealed that the vast majority (>99%r) of oocytes from hybrid crosses had a prolonged meiotic prophase I or
metaphase 1 at least through 180 min post-insemination.

KEY WORDS: Crassostrea gigas. Crassostiea ariakensis. diploid, hybrid, tetraploid. oyster, breeding, polyploidy

INTRODUCTION

There are numerous reports of attempted interspecific hybrid-
ization in the genus Cra.'isnsirea (Gaffney & Allen 1993). How-
ever, most should be viewed with caution because these reports
were unaccompanied by genetic confirmation of putative hybrids.
Even a modest amount of contamination may account for the ma-
jority or all of surviving progeny in hybrid crosses in which fer-
tilization rate and viability are normally low or nil (Allen &
Gaffney 1993), One case seems clear: Pacific oyster C. gigas
(Thunberg) and Suminoe oyster C. ariakensis (Fujita — formerly
C. rividaris Gould) can be crossed to produce viable hybrids
(Allen & Gaffney 1993).

The production of hybrids is interesting because they may pos-
sess qualities that improve commercial traits. Hybrids also could
be back-crossed to introgress certain traits into either of the
parental species; for example, disease resistance. Introgression
of disease resistance into C. virginica (the Eastern oyster) from
C. gigas was the rationale for the extensive hybrid trials under-
taken by Allen et al. (1993). Later, many more hybridization trials
were attempted, using bridging crosses between races of C. vir-
ginica. using (only slightly) fertile C. gigas x C. ariakensis hybrids
(GA), and using polyploidy (Lyu 1996). However, under no cir-
cumstances tested in the lab did C. virginica hybridize with C.
gigas or C. ariakensis.

Although they failed as a bridging cross to C. virginica, GA
hybrids are still of interest for several other reasons. First, no work
has been done on the qualities of diploid GA hybrids as an aqua-
culture product, although this work might more appropriately be
carried out where there is on-going commercial culture of these
two Asian species. For the East coast, they are nonnative. Second,
because diploid hybrids are possible, production of polyploid hy-

*Corresponding author. Fax: (804) 684-7717; E-mail: .ska@vims.edu
Present address: Aquaculture Genetics and Breeding Technology Cen-
ter, Virginia Institute of Marine Science, College of William & Mary,
Gloucester Point, VA 23062.

brids should also be possible. Polyploid hybrids are potentially
useful for improvement of commercial traits (Longwell 1986).
Virtually no work on polyploid hybrids of shellfish has been done.
Third, and most apropos to research on the East Coast, is the issue
of testing nonnative species as an alternative to the native Eastern
oyster because of the decline in the fisheries there.

Trials of nonnatives were begun in Delaware Bay several years
ago (Allen 1993) and have been conducted for C. gigas in the
Chesapeake Bay (Calvo et al. 2000). Trials with C. ariakensis have
been (Calvo et al. 2001) and continue to be conducted. C. gigas
seems more suitable for higher salinity environments and C. ari-
akensis seems suitable for more estuarine conditions. In all field
trials up to this point, triploids have been used to effect population
control because of their sterility (Allen & Downing 1990, Gaffney
& Allen 1992, Guo & Allen 1994a).

Triploid hybrids then are of interest because they are expected
to be sterile, more so than diploids because of the added burden of
gametogenesis in hybrids (Thorgaard & Allen 1986, Thorgaard &
Allen 1992). Triploid hybrids may also have characteristics inter-
mediate to the two parental species, for example, salinity prefer-
ence. The genotypes that might be available for culture in an
estuary as varied as the Chesapeake Bay, for example, could range
from triploid C. gigas (GGG) through two types of triploid hy-
brids— either tetraploid C gigas x diploid C ariakensis (GGA) or
diploid C. gigas x tetraploid C. ariakensis (GAA) — to triploid
C ariakensis (AAA), with phenotypes potentially encompassing
the full range of estuarine and marine conditions.

To date, all hybrid crosses between Crassostrea species have
been made between diploids. Diploids are also used in the pro-
duction of triploid hybrids using ploidy induction techniques
(Allen et al. 1989). For example, triploid hybrids were attempted
between C. virginica and C. gigas by inhibiting polar body 2 with
cytochalasin B treatment (Allen et al. 1993). However, this hybrid
seems to be inviable in any form. Triploid hybrids were also at-
tempted (S. K. Allen, Jr., unpublished data) between C. gigas and
C ariakensis. These, too. were unsuccessful for another reaso;;.
fertilization in this cross is protracted, taking more than 3 h, .\s a

137

138

Que and Allen

consequence, polar body 2 formation is asynchronous and treat-
ments to inhibit polar body 2 are useless. The availability of tet-
raploid C. f^i.i^as (Guo & Allen 1994b), tetraploid C. ariakeiuis. or
both, provides a new opportunity to produce triploid hybrids by
crossing (Guo et al. 1996), rather than induction and to further
investigate the fitness of them. As a first step, we examined the
feasibility of hybridizing tetraploid C. gii^as and diploid C. aria-
kensis. At the same time, we repeated crosses of diploid C. gii;cis
with diploid C. ariakensis as controls and also examined early
development of eggs cytogenetically.

MATERIALS AND METHODS

Oyster and Gametes

Sexually mature oysters used in this study were 2 years old and
obtained from stocks held at the Cape Shore Laboratory, Haskin
Shellfish Research Lab. Ploidy of tetraploid Pacific oysters was
confirmed in all individuals by flow cytometry prior to spawning.
Gametes were obtained by strip spawning. All surfaces and instru-
ments contacting the oysters were cleaned with dilute bleach and
rinsed with fresh water between handling and opening of different
individuals. Sex was determined by gonad biopsy under a light
microscope. Once the sex was determined, the animals from dif-
ferent sexes were removed to separate containers. Gametes from
each oyster were dissected into individual beakers. Eggs were
passed through a 60-(ji.m Nytex screen to remove the large tissue
debris and rinsed on a 25-jjim screen, then suspended in filtered
(2 |j,m) seawater at 23-25°C for at least 30 min to confirm that the
eggs were not self-fertilized. Sperm were separated from debris by
passing the suspension through a IS-fjim screen.

Experimental Design

Abbreviations for gamete contributions of the two oyster spe-
cies are as follows: G = diploid C. gigas: GG = tetraploid
C. gigas: and A = diploid C. uriakensis. v\ ith female listed first.
Eight types of crosses were conducted (Table I) o\'erall, although
not all crosses were possible in all three replicates. For each rep-
lication, an individual female and male were used. After spawning,

TABLE L

Experimental Design for Crosses Among 4n C. gigas. 2n C. gigas,
and 2n C. ariakensis.

G

GG

A

2

G

GG

GA

GG

GGA

Rep 1

A

AG

AGG

AA

9

G

GG

G/GG

GA

GG

GG/G

GGA

Rep 2

A

AG

AGG

AA

9

G

GG

G/GG

GA

GG

GGA

Rep .1

A

AG

AGG

AA

Individual females and males were used for each replication, and three
replicates were made. Gamete contribution is represented by GG. G, or A,
respectively, with female listed first.

parents were frozen at -80°C for subsequent genetic confirmation
of the progeny.

Embryonic and luirval Development

Insemination was conducted at 23-25°C and for hybrids, high
densities of sperm were used (Lyu & Allen 1999). Fertilization rate
was assessed by directly examining at least 100 oocytes under the
light microscope at 60-90 min post-insemination for controls and
up to 180 min post-insemination for hybrid crosses. After deter-
mining fertilization rate, oocytes were transfeired to culture ves-
sels whether fertilization was observed or not. Fertilization was
considered successful if the oocyte was at or beyond polar body
I formation.

Yield at 48 h post-inseinination was estimated by directly
counting straight-hinge larvae with normal appearance. Yield was
calculated as

(no. of straight-hinge x l(X))/no. of eggs incubated

Temperature and salinity for lar\al cultures of crosses of GG,
GA, GGA, G/GG, and GG/G, where C. gigas was the egg source,
were 25°C. 22-23 ppt (Breese & Malouf 1975). For crosses of AA,
AG. and AGG, where C. ariakensis was the egg source, tempera-
ture and salinity were 26°C and 20 ppt (Breese & Malouf. 1977).
Seawater in the larval cultures was renewed every 2 days. In all
hybrid cultures, densities of larvae were sufficiently low to prevent
density-related growth effects; densities in parental culture were
within those used in standard larval culture, beginning at 10/mL
and winnowing out to 1-2 /niL. During water changes, numbers of
remaining larvae were estimated and shell length was measured for
20 individuals for each cross. When larvae reached eyed stage,
eyed larvae were collected and treated with a solution of 10" M
epinephrine for 16 h (Coon et al. 1986). Following treatment,
melamoi-phosed larvae were held in a downweller system until
they reached a shell length of approximately I mm, when they
were transferred to an upweller silo. We took great care to elimi-
nate all sources of contamination throughout the culture process.

For cvtologlcal observations, eggs from each hybrid cross were
sampled and fixed with Carnoy's solution ( 1:3 glacial acetic acid
and absolute methanol) at 90. 120, 150, and 180 min post-
insemination. Fixatives were changed twice following light cen-
trifugation. Chromosomes were observed by acetic orcein stain
(Guo et al. 1992).

Genetic Confirmation

We randomly sampled 28 spat from each replicate of GA
crosses and all GGA spat. In progeny, the whole body was pre-
pared for DNA extraction, whereas mantle tissue (2-8 mg) from
corresponding parental species was prepared using a commercial
kit (PureGene, Centra, Minneapolis, MN). An additional gill tissue
sample from GGA spat was taken and stored in DAPI/DMSO
(Sigma, St. Louis, MO) solution at -80°C for flow cytometric
analysis.

An approximately 550-bp region of the nuclear rDNA genome
was amplified via polymerase chain reaction (PCR) using primer
ITS-I. (The primer pair was designed by Dr. Patrick M. Gaffney.
University of Delaware [Hedgecock et al. 1999]). Reaction volume
of 25 jxL contained 50 MM MgCI,. 0.2 niM of each dNTP, 0.2 |j.M
of each primer, 5 U/p.L polymerase (Taq DNA polymerase.
Sigma) and 1 p-L DNA extraction. DNA amplifications were per-
formed in a programmable thermal cycler (PTC- 100, M.J. Re-
search, Inc.. Walthram, M.A) using a 2-min initial denaturation at

Hybridization of Ckassostrea gigas

139

94°C and then 34 cycles of 45-sec denaturation at 94°C. 1-min
annealing at 52"C, 1-min extension at 72°C. and finally a 5-niin
extension at 72"C.

Restriction enzyme digestion of PCR products was made with
restriction endonuclease Hinf I (Sigma). Digestion volume of
20 fjiL contained 1 x buffer (supplied by New England Biolab Inc..
Beverly. MA). 5 units Hinf I. and 8 |jlL PCR product. Digestion
mix was incubated at 37°C for 3—1 h. followed by 3 (xL lOx
loading dye to stop the digestion.

All PCR products and restriction digest fragments were elec-
trophoresed in a 3% agarose (Sigma) gel in Ix TBE (0.089 M
Tris-borate. pH 8.3. 0.002 M ethylenediamine tetraacetic acid
(EDTAll buffer. A molecular weight marker (pUC 18. digested
with Hae III. Sigma) was loaded along with the product of interest.
The gel was run at 60-90 V. stained with ethidium bromide
(0.2 mg/mL) for 10-13 min. and visualized by transillumination.

Statistical Analyses

All data were analyzed with the computer program SYSTAT
(Wilkinson 1990). Fertilization rates and yield data were arcsine
transformed prior to statistical analysis (Sokal & Rohlf 1981 ). To
compare the performance of hybrids to their controls, a two-way
ANOVA was used. Paired /-tests were conducted to compare cer-
tain crosses to their reciprocals.

RESULTS

Larval Siinival and (irowth

After 48 h post-insemination, survival of GA crosses was about
equal to controls in two of three replicates (Fig. 1 ). Larvae of GGA
crosses had high survival, although the number of eyed larvae was
small (of 450 larvae on day two. 280 survived to eyed stagel. For
AGG and AG crosses, mortality was severe and steady for 1 3 days,
with no survival to eyed stage. In GG crosses, mortality was gen-
erally density related, moderating at lower densities at about day
6-10. For AA crosses, survival was generally poor, making this
cross a poor control. In general, survival ranked GG. GA. and AA.
in descending order. Sur\ i\'al of G/GG was variable: better than
GG in replicate 2 but worse than AA in replicate 3. Survival of

Fertilization Rate and 48-h Yield

Mean fertilization rates in the parental (nonhybrid) crosses
were 94<7r (GG). 779f (AA). 88% (G/GG). and 83% (GG/G)
(Table 2), with no statistically significant difference among them
(F = 3.1 18, P = 0.132) by ANOVA. In hybrids, signs of fertil-
ization did not appear until 180 min after insemination, precluding
estimates of fertilization rate.

Yield at 48 h post-insemination varied significantly among
crosses (Table 2) (F = 3.964, P = 0.018). Yield in GG was
significantly greater than that in AA (/ = 4.162, DF = 4,
P = 0.014) but there was no difference between other parental
crosses. Yields were similar in GGA and AGG (r = 1.010, DF =
4, F = 0.369), whereas GA had higher yields than AG (r = 3.364.
DF = 4. F = 0.006). Yields of GA and AA crosses were about the
same. GGA and AGG crosses produced many fewer larvae than
controls. AG crosses suffered extremely low yield (0.003%).

TABLE 2.

Mean fertilization rates and yields + SD (h) at 48-h in parental and
hybrid crosses combined from three replicates.

Cross

Fertilization Rate ( % )

Yield ( % )

GG

GA

GGA

AA

GG/G

G/GG

AGG

AG

94 + 4.9(3)
ND
ND

77 ± 13.9(3)

85 1 1 )
8X + . "5.0(21

ND

ND

21 ±7.5(3)
4± 1.2(3)
0.05 ±0.01 (2)
3 ±2.5 (3)

4(1)
18 ±24.4 (2)
0.01 ±0.02(3)
0.003 ±0.006 (3)

ND = no data.

' Fertilization rate was observed at 60-90 min post-insemination for pure

crosses; 180 min post-insemination for hybrid crosses.

DAY

Figure 1. Mean survival of hybrid larvae and their respective controls
from day 2 up to day 16 in crosses of diploid and tetraploid ('. gi^as
with diploid C. ariakinsis. (A) Six matings were made in replicate 1. No
larvae survived to day 2 in .AG, AGG, and GtiA. (B) Eight matings
were made in replicate 2, No larvae survived to day 2 in AG, .AG(;, and
A A. (Cl Seven matings were made in replicate 3. No larvae survived to
day 2 in GGA. Counts were terminated when harvesting of eyed larvae
was begun. GG ( ♦ ), GA (■), G/GG (A). GG/G (x), GGA (D), AG';
(A), AA (O), AG (•).

140

Que and Allen

G/GG crosses was better than its reciprocal, GG/G. Larvae sur-
vived to setting in all replicates of GG and GA. 2 of 3 replicates
of G/GG and 1 of 3 replicates of GGA. Spat were obtained from
all of these.

Larvae of GG, G/GG. GG/G, and GGA crosses grew at similar
rates. Crosses of GA grew slower than GG controls but faster than
AA (Fig. 2). GA larvae were generally smaller than GG larvae.
Both AG and AGG crosses grew very slowly (AG larvae died at
day 10). AA crosses grew slowest of all controls. Mean size of
eyed larvae was 350 |xm for GG, 336 |jim for GA, 360 \x.m for
GGA, 361 iJLni for G/GG. and 361 |xm for G/GG. ANOVA showed
no significant difference in eyed larvae size among crosses
(F = 1.712, P = 0.199j. After 90 days post-setting, spat from

GGA reached 12.0-17.5 mm in shell size compared with 2.87-8.0
mm in the corresponding GA cross (r = 8.49. DF = 6. P < 0.001 ).

Cytological Observation of Eggs from Hybrid Crosses

The vast majority of eggs from hybrid crosses were delayed at
prophase I or metaphase I at least through 180 min post-
insemination (Fig. 3A-E). In fact, of all eggs examined at 180 min
post-inseminalion (a 150 observations from each of GA. AG.
GGA. and AGG). only 2'7f of eggs from GGA had entered
anaphase I (Fig. 3F). In GA. AG. and AGG. 10 bivalents were still
observed at this time. Chromosome aggregation was much more
complicated in eggs from GGA crosses. In general, eggs contained
an average of 10 quadrivalents. although other types of synaptic
chromosomes were also present, i.e.. univalents, bivalents. and
trivalents (Fig. 3D. E).

Genetic Confirmation

Agarose gel electrophoresis of the PCR products consistently
revealed two bands, one at around 587 base pairs (bp) and the other
at around 527 bp (Fig. 4. top). Restriction digest fragments re-
solved distinct bands in both parental species. Two bands were
resolved, one at around 434 bp and the other around 138 bp in
C. gigas females, both tetraploid and diploid. In contrast, two
bands were resolved at around Iblllfil bp and 174 bp in C. ari-
akensis males (Fig. 4, bottom). The hybrids (GA and GGA) ex-
pressed all four bands corresponding to their parental species. An
additional band was detected at around 458 bp in hybrids. Identical
band resolution was observed in replicates 1 and 2 of GA crosses,
whereas an extra band was resolved at 587 bp consistently in both
parental species and hybrids in replicate 3 (Fig. 4. bottom, last four
lanes). All progeny samples (28 individuals from each replicate of
GA and a total of 4 individuals from GGA) were hybrids. Three of
four GGA hybrids were Iriploid as confirmed by flow cytometry;
the other hybrid was diploid.

DISCUSSION

In hybrid crosses, fertilization was not apparent even as late
as 180 min post-insemination. These same observations were
reported previously (Miyazaki 1939. Imai & Sakai 1961). both

B

Figure 2. Mean size (nm) of hybrid and control larvae from day 2-16
in crosses of diploid and tetraploid C. gigas with diploid ('. ariakensis.
(A) Larvae from GG, GA, GGA, and \X in replicate 1; (B) larvae
from GG, GA, GG/G, and G/GG in replicate 2; and (C) larvae from
GG, GA. G/GG, AGG. AA, and AG in replicate }. Measurement of
surviving larvae was made until eyed lar»ae appeared. QQ ( ♦ ), G.4
(■), G/GG (Al, G(;/(; (X), GGA (□). AGG (A). AA (O), .\G (•).

Figure 3. Chromosome ohser\ations In crosses of diploid and tetra-
ploid C gigas with diploid C. ariakensis at 180 min post-insemination.
(A) Prophase I in oocytes from GA; (B) prophase I in oocytes from
AG; (C) prophase I in oocytes from M'.C: (D) and (El prophase I in
oocytes from (;(;.\: and (F) anaphase 1 in oocytes from (JGA. Scale
bar on iDl: 10 fim.

Hybridization of Crassostrea gigas

141

M123456789

llllllilL

^yyygyyy^

M 1

FiKure 4. Top: Electrophoretic separation of undigested DNA prod-
ucts resulting from PCR ampliflcation of genomic DNA. using primer
of ITS-1. rDNA originated from parental species and their hybrid
progeny. Row 1: lane I = diploid ('. gigas I ; I; lane 2 = diploid C.
ariakensis ( :' ); lanes i—X = G x A progeny: lane 5 = diploid C. gigas
( I ), lane 6 = diploid C. ariakensis ( 6 ), lanes 7-9 = (; x A progeny . Row
2: lane 1 = tetraploid C. gigas ( 9 ), lanes 2-5 = GG x A progeny: lane
6 = diploid C. gigas i J 1, lane 7 = diploid C. ariakensis ( :' ). lanes 8-9 =
G X A progeny. Lane M = molecular weight marker. Bottom: Elec-
trophoretic separation of PCR ampliried rDNA products from GA and
GGA crosses digested with restriction endonuclease Hinf I. M = mo-
lecular weight marker: lane 1 = tetraploid C. gigas I l ; lanes 2-5 = GG
X A progeny, lane 6 = diploid C. gigas ( i ), lane 7 = diploid C. ariak-
ensis ( d I, lanes 8-9 = G x A progeny. The molecular weights of bands
resolved from the marker are. from largest (slowest migrating! to
smallest: 587, 458, 434, 298, 267/257, 174, and U)2 base pairs, respec-
tively.

of whom found little or no fertilization between C. gigas and
C. ariakensis in their studies of Japanese oysters. However, we
observed 8%-9% fertilization rate in GA crosses in other experi-
ments (data not shown). In a previous study, a mean fertilization
rate of 12% was reported for GA ct'osses (Allen & Gaffney 199.^).
Zhou et al. (1982) revealed that fertilization rates in C. gigas ( 2 )
X C. ariakensis { 6 ) were 0-52.6% and its reciprocal, 2.3^-1 8.8%.
These data indicate that the fertilization rate in these hybrids varies
widely. Success of hybridization should not be assessed solely on
the fertilization rate. In contrast, fertilization between other species
of Crassosirea occurred readily, and often showed relatively high
rates, but larvae survived for only a short time before complete
mortality (Menzel 1986; Allen et al. 1993).

Despite the apparent lack of fertilization (observed up to 180

min post-insemination), replicates of GA yielded viable spat.
which reconfirms the compatibility of the gametes from C. gigas
(2) and C. ariakensis (6). Buroker et al. (1979) reports a rela-
tively high genetic similarity between these two species. Other
studies also demonstrate the feasibility of hybridization between
these two species (Allen & Gaffney 1993; Downing 1988; Down-
ing 1991; Zhou et al. 1982). although only Allen & Gaffney (1993)
confirmed hybrids genetically. In contrast to the success of GA.
the reciprocal AG failed to produce any spat, although no mor-
phological deformities were observed in the larvae. AG larvae
were previously shown to be much less viable compared with the
reciprocal although a few spat were obtained (Allen & Gaffney.
1993). The diploid control AA (three replicates) consistently sur-
vived poorly and grew slowly in this study. This may partly ac-
count for the failure to obtain any spat from the AG cross. Ac-
cording to published accounts (Breese & Malouf 1977; Langdon &
Robinson 1996). the survival and growth of C. ariakensis are
similar to C. gigas under appropriate culture conditions. It is not
clear why larvae of C. ariakensis performed so poorly in this
study, because we have routinely cultured C. ariakensis on other
occasions.

Only one of three replicates of GGA yielded spat. The failure
of the other two replicates might be attributable to low fecundity of
tetraploid C. gigas used here (data not shown), although generally
tetraploids have shown high fecundity (Guo et al. 1996; B. Eude-
line, Taylor United. Inc. and S.K. Allen. Jr.. unpublished data). In
all three replicates, yield at day 2 was low. We suggest that the
major barrier for GGA production on a pilot- or production-scale
is low yield at day 2. Afterward, larvae of GGA crosses survived
well (virtually no mortality). While it is clear that more GGA
progeny could be obtained by using more parents, the real chal-
lenge is to find factors that lead to high levels of fertilization for
the gametes that are available. We also suggest that there is a
difference in growth rate between triploid (GGA) and diploid (GA)
hybrids. First, GGA eyed larvae appeared 5-7 days earlier than
GA and right after those of controls GG. G/GG, and GG/G. Sec-
ond, the size of spat from GGA was greater than the size of GA at
90 days post-insemination, although the number of GGA spat was
small.

All 28 progeny sampled from each of three replicates of GA
were hybrids. With respect to GGA progeny, some eyed larvae
attached to the culture containers, leading to loss of eyed larvae.
Consequently, only four culchless spat were obtained. However,
the fact that three of the spat were triploid hybrids demonstrates
that hybridization between tetraploid C. gigas ( 2 ) and diploid
C. ariakensis (S ) was successful. Triploid hybrids of C. gigas and
C. ariakensis cannot be obtained in any other way. In particular,
the use of cytochalasin B (or other polar body inhibitor! is pre-
cluded in GA crosses because of the prolonged period leading to
syngamy and polar body fomiation. It is not possible to create
triploids without some level of predictability and synchrony
among developing eggs (Allen et al. 1989).

PCR restriction fragment length polymorphism (RFLP) diag-
nosis was an effective means to verify putative hybrid progeny.
ITS-1 ampIification/Hinf I digestion successfully distinguished
among C. gigas. C. ariakensis. and hybrids, which show bands
present from both parental species. Application of this method is
based on availability of an appropriate primer: ITS-1 in this study.
Other methods have been used to confirm hybrid status (Allen &
Gaffney 1993: Allen et al. 1993; Nakamura et al. 1990; Jiang et al.
1988). Karyotype analysis has been useful for hybrids amonc ■.'■.

142

Que and Allen

pearl oyster genus PinciaJa (Jiang et al. 1988). For species within
the genus Crassusirea. the great similarity in their karyotype ren-
ders this type of analysis useless in hybrid documentation until
more discriminating markers are designed. Flow cytometry was
equivocal in discriminating between hybrid and pure crosses using
dissociated cells of pooled larvae (Allen et al. 1993).

Cytological examination of newly fertilized eggs from hybrid
crosses revealed that either fertilization was severely delayed or
early development of oocytes was extremely slow. The duration of
meiotic maturation in hybrid crosses was at least triple that of
controls. The delay of meiosis was probably not due to the quality
of gametes because eggs in controls were normal and reached first
cleavage at around 6(J niin post-insemination consistently (data not
shown). Another hybridization attempt between C. gigas and
C. ariakcnsis indicated there was no apparent difference in the
time of development from eggs to the straight-hinge larvae (Zhou
et al. 1982). Apparently, fertilization was also highly successful in
the study by Zhou et al. leading us to suspect contamination. An
alternative explanation is that Zhou et al. used a different popula-
tion of C. iiricikensis. The C. ariakensis used here are from a
population derived from Ariake Bay in Japan. Those of Zhou et al.
are likely from mainland China. In this study, despite the delay in
fertilization, healthy larvae were obtained and cultured through
metamorphosis in all replicates of GA and one replicate of GGA.
Furthermore, hybrid spat were obtained. Success in obtaining lar-
vae suggests that meiosis in GA and GGA crosses must have
resumed at some time, despite being delayed for an uncommonly
long time. A similar observation was made in C. angulata oocytes
fertilized with sperm from C virginica. where 47% of them ex-
hibited metaphase 1 nearly 4 h after insemination. However, no
parental crosses were made for comparison (Stiles 1973). Oocytes
in hybrid crosses of C. gigas and C. ariakcnsis with C. virginica
proceed through meiosis and early mitosis in fairly normal fashion
(Scarpa & Allen 1992). The timing of meiotic and mitotic land-
marks was the same for all crosses, parental and hybrid. However,
hybrids of C. gigas and C. ariakcnsis with C. virginica were in-
viable (Allen et al. 1993) after a short larval period despite normal
meiotic and early mitotic behavior. C. gigas x C. ariakcnsis
crosses were not included in Scarpa and Allen's work.

Overall, this study reveals a new potential application for oyster
breeding: triploid C. gigas x C. ariakensis hybrids. We were lim-
ited to relatively few tetraploid brood stock for this study, but since
that time, tetraploids have become commercialized on the West
coast of the United Staes. We were also limited to making the
triploid hybrid in one direction; that is, using only C. gigas as the
tetraploid, enabling experiments on GGA (4n$ ) or AGG (4nd ).
We were unable to explore GAA (4nc5 C. ariakensis) or A AG
(4n$ C. ariakcnsis). Presumably the former would behave like
GA and GGA cultures, and the latter would behave like AG and
AGG cultures. These experiments will have to wait for the tetra-
ploid C. ariakensis that are currently under development. At
present, GGA hybrids can be made only at a high cost of gametes
from the parental species. Even with high fecundity, the losses are
so severe that commercial production would be a problem. Studies
on factors that promote higher fertilization rate, syngamy. devel-
opment rates, or all of these would contribute to efforts to produce
pilot-scale quantities of GGA for future testing.

ACKNOWLEDGMENTS

We thank Greg A. DeBrosse for care ol' brood stock, larval, and
nursery cultures. We are especially grateful to Dr. Patrick M.

Gaffney and Dr. Ziniu Yu for technical assistance in PCR/RFLP.
This project was partly sponsored by the NOAA U.S. -China Ma-
rine and Fisheries Science and Technology Agreement in Living
Marine Resources. This is NJAES Publication Number D-32ll)()-
1-02 and VIMS Publication Number 2465.

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Journal of Shellfish Research. Vol. 21. No. 1, 14.'i-155. 2002.

MICROSCOPIC ANATOMY OF GONADAL TISSUE AND SPECIALIZED STORAGE CELLS

ASSOCIATED WITH OOGENESIS AND SPERMATOGENESIS IN THE CALAFIA

MOTHER-OF-PEARL OYSTER, PINCTADA MAZATLANICA (BIVALVIA: PTERHDAE)

PEDRO SAUCEDO, CARMEN RODRIGUEZ-JARAMILLO, AND MARIO MONTEFORTE

Cenlw de Investigackmes Biulugicas del Noroesle. S.C. La Puz. Buja Cidifoniia Sin: Mexico

ABSTRACT As part of a major experiment of (he reproductive biology of wild Phwtada muzatlanica. this study was aimed to
characleri/.e the microscopic anatomy of the gonadal tissue and storage cellular elements involved in the seasonal advancement of
oogenesis and spermatogenesis. Tissue samples were collected every fifteen days over an annual cycle and processed with histological
and histochemical techniques. The heniato.xylin-eosin (cytology description). Blue Alcian-PAS (PAS. for carbohydrates), and Black
Sudan (BBS) and Oil Red (OR. for lipids) techniques were employed. Temporal variations of the sex ratio and sexuality of specimens
were also analyzed. Gonadal tissue developed synchronously over time at the expense of a matrix of interconnective tissue and reserves
stored in the adductor muscle and digestive gland. The interconnective tissue communicates the gonadal tissue with the digestive gland
and also serves as substrate for the differentiation of vesicular connective tissue cells and auxiliary cells. In fact, gametes (particularly
oocytes) were exogenously nourished by both kinds of cells as gametogenesis advanced. The vesicular connective tissue cells, very
abundant among acini, adenomeres, and peari sac, were observed surrounding excretory conduits in the first two tissues, and were
identified as highly PAS++ and moderately BBS+ and 0R+. The auxiliary cells were found attached to previtellogenic and vitellogenic
oocytes. The endogenous synthesis of lipids during vitellogenesis was associated to the Balbiani body, a storage compartment of the
oocyte ooplasm not previously described for any species of peari oyster. The average female/male ratio is 0.35: 1 when specimens were
obtained from cultured conditions, but females outnumbered males when collected from the wild. Several cases of protoginic specimens
and a few functional hermaphrodite oysters were seen.

KEY WORDS: pearl oysters. Pinctci<hi mazatkmica. histology, oogenesis, spermatogenesis, storage cells

INTRODUCTION

When dealing with endangered or protected species, like the
majority of members of the genus Pinctada. the success of aqua-
culture requires a proper knowledge of the biology and ecology of
broodstock. and in particular, a clear understanding of their repro-
ductive biology and physiology (Southgate & Beer 1997), In ex-
periments focusing on gonadal conditioning, larval rearing, and
hatchery spat production under controlled conditions, such knowl-
edge is especially important for two reasons: (1 ) to assure the
formation of spermatozoa and oocytes of the best quality, that will
develop into viable and competitive larvae and, later, into settled
spat; and (2) to maintain the continuity of the spat production
process throughout the year, avoiding the geographic limitations
that prevail in subtropical environments like Bahi'a de La Paz, At
such localities, the tnain spawning season for most species of
marine bivalves is restricted to a short period of one to two months
of the annual cycle (Saucedo et al. 2001a; .Saucedo et al. 2001b;
Saucedo et al. 2001c).

Pearl oysters are tnarine bivalves having considerable eco-
nomic and coinmercial value because of their natural capacity to
produce pearls of high quality (Monteforte 1990. 1996). Despite
this, the factors affecting reproductive success in these species are
poorly studied and understood, although their basic reproductive
biology has been well documented histologically (Tranter 1958a:
Tranter 1958b; Tranter l95Kc; Wada 1959; Sevilla 1969; Chellam
1987; Rose et al. 1991 ; Wada et al. 1995; Garcia-Dojni'nguez et al.
1996; Saucedo & Monteforte 1997; Behzadi et al. 1997; Saucedo
et al. 2001a; Saucedo et al. 2001b; Saucedo et al. 2001c). These
studies reveal that most aspects of the gametogenic cycle are com-
mon to all members of the genus Pinvtadii. with differences as-

Corresponding author. Pedro Saucedo, Centro de Investigaciones Biologi-
cas del Noroeste, S.C. Mar Bermejo 195, Col. Playa Palo de Santa Rita, La
Paz, Baja California Sur. Mexico. E-mail: psaucedo@cibnor.mx

cribed basically to the geographic location of each species. The
gonad is absent as an anatomically discrete organ, and instead,
gonadal tissue gradually develops as part of the visceral mass,
intermingled with the digestive gland and interstitial connective
tissue. Gametogenesis occurs continuously and synchronously
throughout the annual cycle, but shows a variable number of
spawning peaks. Adults behave as protandrous hermaphrodites,
maturing as males and changing to females at a certain size/age. A
female/male sex ratio of 1:1 is reached with increasing age.

In Mexico, there have been some studies of the reproductive
biology of the Calafia mother-of-pearl oyster, Piiuiiuhi imizat-
laiiica (Hanley 1856) (Sevilla 1969; Garci'a-Dominguez et al.
1996; Saucedo & Monteforte 1997; Saucedo et al. 2001a; Saucedo
et al. 2001b; Saucedo et al. 2001c). Little is known, however,
about the step-by-step advancement of the gametogenic cycle and
the role of accessory tissues and cells upon the seasonal build-up
of gametes. This study describes the microscopic anatomy of go-
nadal tissue and specialized storage cellular elements involved in
the annual cycle of oogenesis and spermatogenesis in P. mazat-
kmica. This experiment was conducted as part of a major study of
the reproductive biology of the species.

MATERIALS AND METHODS

Twenty adult specimens averaging 139 (±9.7 mm SD) shell
height were collected every fifteen days over a year (January to
December 1999) from Caleta El Merito. Bahi'a de La Paz. Baja
California Sur, Mexico (24"16'N. 1 10°19'W). Cultured specimens
coming from the Experimental Marine Farm of the Centro de
Investigaciones Biologicas del Noroeste (CIBNOR) were collected
from January to August. Wild specimens were collected from Sep-
tember through December because of the scarcity of cultured
specimens remaining in the farm during the last third of the year.

After dissecting each specimen, a sample of the visceral mass
(where gonadal tissue develops concomitantly with the iige^i-'

145

146

Saucedo et al.

gland) was excised and preserved in Davidson's solution for 48 li.
Samples were dehydrated in ascending ethanol series, embedded in
Paraplast at 56"C. and sectioned with a rotatory microtome at 4-5
p.m along the anteroposterior axis. To characterize the general
morphology of tissues and the advancement of gametogenesis. thin
sections were stained with the conventional hematoxylin-eosin
technique (Howard & Smith 1983). To identify the presence of
carbohydrates (CHO) and lipids (LIP) in storage tissular and cel-
lular elements, additional sections were stained with blue Alcian-
PAS (referred to as PAS for CHO) and black Sudan (BBS) and o\\
red (OR), both for LIP (Prophet et al. 1992). Finished slides were
then examined with an Olympus BX-41 compound microscope.
The resulting images were stored in the computer using a digital
Cole-Snap camera (I-media Cybernetics) and processed with
Sigma Scan Pro software (vers. ,^.5).

To study the seasonal changes of oogenesis and spermatogen-
esis, a modified version of Tranter's original scheme (Tranter
1958a; Tranter 1958b; Tranter 1958c) for pearl oysters was used.
Gametogenesis was characterized by eight stages: (a) inactive or
resting); (b) sexual activation; (c) early development; (d) mid de-
velopment; (e) late development; (f) ripeness; (g) partially
spawned; and (h) spent. For oogenesis in particular, gametes were
classified according to their vitellogenic stage as oogonias (corre-
sponding in time to the sexual-activation stage), previtellogenic
oocytes (for the early-developing stage I, vitellogenic oocytes
(mid-and late-developing stages during yolk formation), and
postvitellogenic oocytes (ripe stage), adopting the criterion defined
by de Gaulejac et al. (1995) for the bivahe Piivni nohilis. Apart
from de Gaulejac's work, detailed descriptions of the cytological
characteristics of each type of oocyte may be found in Saucedo et
al. (2001a, b).

In addition, total area variation was recorded for thirty oocytes
in each developmental stage measured at three randomly-selected
areas of the ovary (Grant & Tyler 198.3). The sex ratio and sexual
condition of specimens were also recorded. One-way ANOVA was
applied for differences in the mean area of oocytes over time.

RESULTS

Gonadal Tissue

Pearl oysters lack a true gonad organ. Instead, gonadal tissue
develops gradually as part of the visceral mass by means of sea-
sonal soma-germline interactions. Macroscopically. this tissue is
formed by two symmetric lobules (a lobule corresponding to each
valve) that grow asymmetrically toward the dorsal region of the
visceral mass as gametogenesis proceeds, thus constituting an un-
paired gland when specimens are ripe. When observed under the
light microscope in a transversal, anteroposterior section (Fig. 1 A),
gonadal tissue appears delimited from the central region by the
digestive gland, and from the pallial cavity (along the periphery)
by a layer of neutral-acid mucopolysaccharides and collagen fi-
bers. A thick matrix of interstitial connective tissue — appreciated
in the form of a complex circulatory system running among
acini — gives support, communication, and substrate for the differ-
entiation of gonadal tissue and one kind of somatic nutritive-
storage cells, named as vesicular connective tissue (VCT) cells.
The size and grade of diffusion of the interconnective tissue matrix
shows an inverse relation to the advancement of gametogenesis.
The mucopolysaccharides layer is formed by eosinophilic highly
PAS-l-l- cells, with both fractions clearly contrasted, the neutral-
inactive (with a pink-magenta color) from the acid-active (with an

intense blue color) (Fig. lA). This tissue is also BBS-t- and OR-I-,
although its low reaction to both colorants suggests the presence of
relatively few lipids, which are present only in the form of esters
of cholesterol.

The gonadal tissue is initially formed as a series of small and
compact granular bags that latter enlarge and transform into a
complex network of branched tubules as gametogenesis advances
(Fig. lA). These bags, representing the structural units of the go-
nadal tissue, are called acini or follicles. Indistinct for the male
testis or the female ovary. The sex cells, either oocytes or sper-
matozoa, develop within these units by permanent mitosis of oo-
gonial or spermatogonial stem cells producing cloned-daughter
cells by centripetal moxements.

Digestive Gland

This tissue is formed by a large number ot blind-end granular
tubules that are connected to the stomach by branched conduits.
When observed microscopically in transversal section (Fig. IB),
tubules are composed of structural units called adenomeres. each
one having a typically round shape that conforms the blind-end of
the tubule. Adenomeres are formed from an external layer of ep-
ithelial cells that have a round basophilic nucleus resting upon a
thin, acidophilic basal membrane. A thin layer of interconnective
tissue suiTounds and supports the blind tubules. The digestive
gland is involved in a double function; the first one, as tissue, is
related to the digestion process of the oyster, while the second, as
endocrine gland, is for storage and release of nutrients. Intracel-
lular digestion takes place in the cells located in the basal mem-
brane. \ ia ciliated vesicles acting as carriers of the ingested food
and their pinocytosis to enter the cell (processes observed only
with an electron microscope). With light microscopy, the diameter
of the blind tubules varies with regard to the light beam that can
pass, indicating which digestive phase (assimilation or digestion)
is being observed. The glandular function of the digestive gland is
described in the section below.

Vesicular Connective Tissue (VCTI Cells

The glandular function of the digestive gland, which is closely
related to that of gonadal tissue, is associated directly with the
presence of a dense matrix of small, round or oval pleomorphic
somatic VCT cells. As an integral part of the connective tissue
network in which development and morphogenesis of the gonadal
tissue takes place, these eosinophilic cells are widely distributed
among adenomeres and acini (particularly in the former tissue)
surrounding wide excretory conduits (Fig. IC and D). These con-
duits are probably communicating with each other via the vascular
interconnective tissue matrix, and may be the vehicle for trans-
porting stored nutrients and energy reserves from the gonadal tis-
sue to the digestive gland, or vice versa. When specimens reach the
late-developing or ripe stage, this tissue network can grow and
invade the intestinal loop (known as the pearl sac for pearl culture
purposes), constituting an impoilant storage site for nutrients. VCT
cells are strongly PAS-i-h (Fig. IC and D) and moderately BBS-l-
and OR-l- (Fig. IE), confirming a high proportion of carbohydrates
(mainly glycogen), but also lipid droplets in a lower and variable
percentage. Transverse sections through the gonad indicate that the
proportion of VCT cells versus germ cells appears to be approxi-
mately the same in ovaries and testis.

Auxiliary Cells (AC)

These specialized intragonadal cells are observed only ni fe-
male acmi. always in intimate relation with developing oocytes, to

Gonadal Tissue and Storage Cells in P. maza

TLANICA

147

JBf^'

:9ni

fVI

^

Wt

M

P

Bs»

GT

^'W

Cm^s

^ ICT

W

DG

"?.

-*

H— 1

AOE

V

Figure 1. Color photomicrographs of accessory tissues and cells associated «ith oogenesis and spermatogenesis in Pinctada mazatlanica. A)
transversal section of the visceral mass at lOx, shov^ing. from the periphery to center, the neutral (magenta) and acid (bluel highlv PAS++
mucopolysaccharides layer (MPS), collagen Hbers (CF). and interconnective tissue (ICT) supporting and communicating the male gonadal tissue
(GT) wuh the digestive gland (DG); B) Microscopic anatomy of the DG at 40x. in which adenomeres (ADE) show several lavers of eosinophilic
ce s (EC) resting upon a basal membrane (BM) and the blind part (BP) in the center: C) and D) Highlv PAS++ vesicular connective tissue (VCT)
cells at 4(Jx. observed surrounding excretory conduits (EXC) among ADE and acini (ACI). respectively; E) Moderatelv BBS+ VCT cells at lOx
found in the boundaries between female GT and DG; F) Basophilic auxiliary cells (AC) at 40x, observed in different positions in relation to
developing oocytes, and the Balbiani body (Bb). Scale bar = 50Mm.

which they are attached by desmosome-hke gap junctions (ob-
served only with the electron microscope). AC range in size from
20-30 \x.m, and because of their basophiUc character and strong
affinity to hematoxylin, are easily distinguishable from oocytes
(except those previtetellogenic that are also basophilic) by their
dark purple color. AC exhibit an enormous plasticity to change
their morphology according to the stage of gametogenesis and to
their exact location with respect to growing oocytes. This plasticity
is most likely due to the disposition and arrangement of the mi-
crotubule skeleton (cytoarchitecture) of these cells. Thus. AC may
be observed basally (near the stalk region). laterally, or eccentri-
cally (Fig. IF). AC serve a nutritive function during oocyte devel-
opment, especially during the construction of the lipid fraction of

the yolk in the early-, mid-, and late-developing stages. In ripe
specimens, these cells gradually disappear or. if still appearing, are
detached from the oocytes.

Oogenesis

The step-by-step advancement of the annual oogenic cycle in P.
mazathmica is depicted in Figure 2. Here, the stages are described.

Inactive or Resting

There is no evidence of gonadal development and specimens
are undifferentiated by sex. Acini are observed collapsed and
empty, with some granulocytes and phagocytes remaining from the
gamete resorption process. On the contrary, the vascular inlen or

148

Salichdo et al.

Figure 2. Photomicrographs of female gonadal tissue (4()\). showing the sexual stages of oogenesis in Pinclada mazatlanica. A) lnacti>e stage, with
empty and collapsed ACI containing some phagocytes (Ph) and abundant ICT; Bl Activation stage, in which only stem cells (Sc» and oogonia
(Ogl are attached to ACI walls, and. among them, large amounts of VCT: Cl Early development, dominated by previtellogenic oocytes (PrO)
still attached to the acinus wall by the stalk region (Sr) and accompanied by AC and a few vitellogenic oocytes iVOl; Hi Mid-developing stage.
with only a few Og and PrO present, while more VO and postvitdlogenic oocytes (PO) start dominating the ACI. The I5h and the mitochondrial
clusters (Mc) are common during this stage: E) l.ate development, showing free PO tilling the ACI lumen, hut still leaving a wide inleroocyte
space; F) Ripe stage, with large, free PO and the interoocyte space reduced to its mininuuii: Gl Partial spawning, presenting many scattered
residual oocytes (RO), resorptive material (RMl. and a few Ph; H) Spent stage, showing empty ACI with atresic oocytes (AOl. RM, and more
signs of Ph. Scale bar = 50 pm.

Gonadal Tissue and Storage Cells in P. mazatlanica

149

nective tissue grows and appears distended and greatly diffused
among acini (Fig. 2A).

Sexual Activation

Acini look small, round or oval, and easily distinguished from
other acini. They are supported by interconnective tissue and nour-
ished by VCT cells. Each acinus is lined up exclusively with small
(3^ (xm diameter) spherical stem cells and larger oogonias (5-6
\x.m), either attached in clusters to the acinus wail or progressively
expanding toward the lumen (Fig. 2B). Apart from these cells no
other developmental stage has yet appeared.

Early Development

Acini begin to grow and enlarge, but their individuality is still
clear. Oogonias rapidly proliferate by mitosis and give rise, by
centripetal inovenients. to the first previtellogenic oocytes (Fig.
2C). These oocytes are easily distinguished from other types of
oocytes by the dark blue-purple color of their ooplasma. They
enlarge to a diameter of 14—23 (jim in diameter, lack yolk, and are
still connected to the acinus wall by the stalk region, in which
some AC are commonly observed. At the end of this stage, a few
\ ilellogenic oocytes appear in the acinus, but the interoocyte space
is still wide.

Mid Development

Acini continue to grow, adopting a more complex anastomosed
shape. The proportion of interconnective tissue and VCT cells
decreases, but AC are still observed. All developmental stages are
present within acini, but vitellogenic oocytes — characterized by
the growth of the yolk ooplasm and increase in diameter to 30 to
55 |j.m — are the dominant stage (Fig. 2D). This oocyte is still
connected to the acini wall, presents a peduncle or pear shape, and
shows a large nucleus and one or two dense, blue-stained nucleoli.
Two different basophilic structures are observed in the ooplasm for
the first time: the Balbiani body (seen as a black spot of variable
size and shape) and many smaller, irregularly scattered black spots
that probably correspond to mitochondrial clusters (Figs. IF and 2D).

Late Development

Acini continue to grow and stratify and interconnective tissue
and VCT cells to decrease. AC tend to disappear or. if still occur-
ring, they appear separated from oocytes. The interoocyte space
reduces as previtellogenic oocytes diminish and vitellogenic and
postvitellogenic oocytes progressively develop (Fig. 2E). Postvi-
tellogenic oocytes are seen free in the lumen, have a polyhedral or
polygonal shape, and increase ni diameter to 60-70 \xm. A dense
ainorphous mass (probably chromatin) is observed in the nucleo-
plasm. The nucleolus is dense, compact, and usually in a marginal
position. The Balbiani body and mitochondrial clusters may still be
present.

Ripeness

This stage marks the end of vitellogenesis. Acini are strongly
anastomosed and packed almost entirely with mature, free postvi-
tellogenic oocytes that attain a maximum diameter of 70-75 (xm
(Fig. 2F). VCT cells in gonadal tissue almost disappear and inte-
roocyte space is reduced to the minimum. The nucleus now occu-
pies a large area in the middle of the oocyte. Many oocytes begin
to enter the germinal vesicle stage, in which a gap between the
nuclear envelope and the ooplasm appears, indicating a readiness
for spawning.

Partially Spawned

After the germinal vesicle breakdown, oocytes are released
outside the acini, which look distended and partially empty (Fig.
2G). Some types of phagocytes and granulocytes appear in the
space between the free residual oocytes, which now look rounded
or pear-shaped. Large amounts of resorptive material are notice-
able. The nucleolus and chromatin disaggregate, but if still present,
they may be associated with a new developmental process. This
may be a strategy to recycle nutrients (proteins and lipids of the
yolk) destined for the new oocyte.

Spent

Acini are collapsed and empty, with clear signs of phagocytic
activity, oocyte degeneration, and much resorptive material. De-
pending on the duration of this phase, residual oocytes, especially
those of small diameter whose nucleus is not polylobed. undergo
atresia (Fig. 2H). This process represents deterioration and disin-
tegration of most of the major cellular constituents and thereby the
breakdown of the oocyte.

Spermatogenesis

The step-by-step advancement of the annual spermatogenic
cycle in P. mazatlanica is depicted in Figure 3. The stages are
described as follows:

Sexual Activation

Acini initially are small, round or oval, and supported by vas-
cular interconnective tissue and VCT cells. The germinal epithe-
lium is formed exclusively by a single layer of small (3-4 {xm
diameter) spherical stem cells that are strongly attached to the
acinus wall and by some spermatogonias starting to differentiate.
Development into spermatocytes, spermatides. or spermatozoa has
not yet appeared (Fig. 3A).

Early Development

Acini grow and look enlarged and slightly branched, hut their
individuality is still recognizable. Spermatogonias rapidly prolif-
erate in a centripetal direction, giving rise to several layers of small
(2-2.5 p.m diameter) primary and secondary spermatocytes ex-
panding toward the lumen, which are the doininant stage present
(Fig. 3B). A few isolated pockets of spermatids and spermatozoa
can also be observed. The proportion of interconnective tissue and
VCT cells decreases.

Mid Development

Acini begin to show stratification, causing a reduction of the
interconnective tissue and storage cells. Starting from the outer
layers to the center, all developmental stages are present: sper-
matogonia, abundant spermatocytes, spermatids, and ripe sperma-
tozoa (showing their acidophilic tails as pink lines radiating from
the center of the lumen) (Fig. 3C). Cell diameter decreases to
approximately 1-0.5 ixm.

Late Development

Acini continue their growth and stratification, appearing highly
anastomosed. As a consequence, the matrix, previously occupied
by interconnective tissue and VCT cells, has almost disappeared.
Spermatogonia and spermatocytes have been reduced in thickness
to a few layers of cells located at the periphery. Acini are p,hV,\i

150

Saucedo et al.

Figure 3. Photomicrographs of male gonadal lissiu' i4(l\i, showing the sexual stages of spermatogenesis in Piinlada muzatlanica. A I Activation
stage, showing AC! filled only with stem cells (St) and sperniatogonias (SPG) proliferating toward the lumen. Ahundant ICT with \ CT cells can
he observed; B) Early development, with many SPG and primary and secondary spermatocytes (SPI and SP2| rapidly differentiating into the
first pockets of sperniatides (Spl and spermatozoa (S); C) Mid development, characterized by the presence of all type of cellular stages, but more
mature S showing their acidophilic tails (T) radiating from the center; Dl Late-developing stage, in which SPCi decrease in number and instead
S sharply increase their frequency; E) Ripe stage, evidencing a dense volume of ripe S packing the acini: F) Partial spawning, showing distended
but empty acini, residual spermatozoa (RS) and some Ph starting to appear: G) Spent stage, with collapsed ACI and evident signs of RS
undergoing cytolysis. Scale bar = 50 nm.

Gonadal Tissue and Storage Cells in P. mazatlan/ca

151

with a dense, dark-blue band of
deep (Fig. 3D).

Ripeness

ripe spermatozoa several eel

Acini look like a complex network of branched tubules that
make distinguishing the boundaries between them difficult. The
dominant stage is now the spermatozoa, which strongly pack the
acini. Stem cells and spermatogonia are latent and restricted to a
thin layer at the periphery of the acini (Fig. 3E). Only a small
amount of interstitial connective tissue is evident at this stage.

Partially Spawned

After spermatozoa are expelled into the surrounding environ-
ment, acini walls look broken but still distended (Fig. 3F). Many
residual spermatozoa are observed scattered, with the first signals
of phagocytic activity. Much residual material is also noticeable. A
gap between acinus walls and the mass of residual spermatozoa
appears. In some cases, redevelopment or sex reversal processes
occur.

Spent

Because spawning is never complete, the spent stage may be
skipped when new gonadal replenishment occurs off the main
reproductive cycle. However, when gamete resorption starts, acini
look collapsed and empty, with no evidence of active spermato-
genesis taking place. This phase is characterized by the rapid pro-
liferation of different kinds of phagocytes, granulocytes, and
amoebocytes surrounding and destroying residual spermatozoa

(Fig. 3G). The matrix of interconnective tissue starts to grow and
develop again.

Bisexuality (Hermaphroditism)

This condition was observed in 8.3'7f of the sample |4() of 480
specimens). Bisexuality was classified as either consecutive sexu-
ality or functional hermaphroditism.

Consecutive Sexuality

The individual functions as one sex when young and later
changes to the opposite sex. As for bisexuality. two manifestations
of this condition were observed: ( 1 ) Protandrous hermaphroditism:
when sex reversal occurs, this is the normal condition reported for
many bivalves and pearl oysters. In this situation both germinal
lineages overlap in the same acinus, but the male gonadal tissue,
which developed initially, undergoes regression and lysis. Only a
few residual spermatozoa accompany oocyte development (Fig.
4A). This condition, which corresponds to the relation between
stage 7 of spermatogenesis and stages 1. 2. or 3 of oogenesis, was
observed in 5.89r of the sample (28 of 480 specimens). (2) Pro-
toginic hermaphroditism: observed in only {.1% of the total
sample (8 of 480 specimens). Again, both germinal lineages over-
lap in the same acinus, but now the male gonadal tissue proliferates
as female gametes gradually disappear. Therefore, only atresic
oocytes are found in the acini together with developing male ga-
metes (Fig. 4B). This condition corresponds to the relation of stage
7 of oogenesis and stages 1. 2. or 3 of spermatogenesis.

Figure 4. Photomicrographs of hermaphrodite specimens of Pinctada mazatlaiiica stained with hematoxylin-eosiii. A) Protandrous hermaphro-
dite, with active Ph surrounding and destroying RS. while PrO and VO present active development: B) Prologinic hermaphrodite, showing a
few RO undergoing regression among mature spermatozoa (MSl filling the ACI; C) Functional hermaphrodite, presenting MS with tails and
growing oocytes (PrO and VOl in equal proportion and apparent functionality; D) Functional hermaphrodite, in which both male and female
lineages spawned at the same time, and only RS and RO can be seen within ACI. Scale bar = 50 pm.

132

Saucedo et al.

Functional Hermaphroditism

This condition was detected in 0.87f of tlie sample (4 oysters).
Both sexes are present concomitantly within the same acinus in
apparently equal proportion and display no sign of gonadal i-egres-
sion in either gamete (Fig. 4C). Both lineages may be observed
spawning and undergoing regression at the same time (Fig. 4D).

Temporal Variation of Gamctogenesis, Oocyte Area, and Sex Ratio

Temporal variations of the main developmental stages ot ga-
metogenesis. oocyte area, and sex ratio of Pinctada imizatlanica
are shown in Table !. Gonadal development started early in Feb-
ruary and proceeded synchronously throughout the annual cycle,
showing two reproductive peaks, one in March to May ( in which
no trace of spawning activity was detected) and other in July to
August (concluding with a massive spawning in September and
October). Small oocytes were observed in February. June, and
October to December. Large oocytes were observed in March.
May. and August, corresponding to the two reproductive peaks
detected histologically. There were significant differences in the
mean area of oocytes over time (F = 102.97; P < 0.001 ). During
most of the annual cycle (January to August), when cultured speci-
mens were collected, males outnumbered females and led to an
average female/male ratio of 0.6:1. From September until Decem-

ber, when wild animals were collected, females doubled in number
relative to males, yielding an average female/male ratio of 2:1.

DISCUSSION

In this study of P. mazatkmica, oogenesis and spermatogenesis
were analyzed using a scheme of eight stages that adequately
describe developmental variations in the microscopic anatomy of
the gonadal tissues and storage cells. A 15-day sampling interval
was selected as the best procedure for obtaining detailed docu-
mentation of the step-by-step advancement of gametogenesis. The
timing of sampling also avoided the major problem of missing
partial spawns or sex reversal phenomena, and allowed us to ob-
serve many unreported phenomena of the reproductive process ot
P. mazatkmica. We introduced, for the first time, the activation
stage to classify acini in which no other developmental stage rather
than oogonias or spemiatogonias can be distinguished. This stage —
not pi-eviously described for any marine bivalve — clearly marks
the moment of sexual differentiation of germ cells and the very
incipient commencement of gametogenesis. Both events occurred
early in February to March, when phytoplankton is usually abun-
dant in Bahia de La Paz (Lechuga-Deveze 1997) and wild oysters
had stored sufficient energy reserves in the adductor muscle and
digestive gland for starting gametogenesis (Saucedo et al. 200 Ih).

TABLE L

Temporal variations in the main developmental stages, mean oocyte area (± standard deviation), and sexual condition oi Pinctada

mazatlanica during an annual gametogenic cycle.

Oocyte

Sexual Condition

Shell

M

F

I

B

F/M

Height
(mm ± sd)

ORI

Cult
Cult

Dev

Stage

TIM

Area (*)
(Jim ± sd)

(%)

(%)

(% )

(%»

SR

142 ± 7.4
151 + 11.8

Inactive
Activ

Jan
Jan

4004 ± 928

60
50

25
25

15
5

0
20

0.4:1
0.5:1
0.2:1
0.7:1

140 ± 9.9
1 39 + 9 6

Cult
Cult

E-Dev
M-Dev

Feb
Feb

3020 ± 649

80
50

15
35

0
10

5
5

146 ± 6.5
142 + 6.2

Cult
Cult

M-Dev
L-Dev

Mar
Mar

4644 ± 868

50

45

25
40

5
0

20

15

0.5:1
0.9: 1

150 ± 10.7
143 -^ 9.5

Cult
Cult

L-Dev
ML-Dev

Apr
Apr

4318 + 959

60
65

25
25

0
0

15
10

0.4: 1
0.4:1

130 + 7.6
139 + 8.5

Cult
Cult

ML-Dev
L-Dev

May
May

4566 ± 1 085

50
65

40

25

0
0

10

10

0.8:1
0.4:1

141 +8.1

Cult

L-Dev

Jun

3009 ± 644

70

30

0

0

0.4:1

149 + 9.3

Cult

M-Dev

Jun

45

55

0

0

123 + 9.1

Cult

L-Dev

Jul

3806 ±931

75

25

0

0

0.3:1

148 ± 10.6
139 + 9.8
140+ 10.1

Cult
Cult
Cult

L-Dev

Ripe

Ripe

Jul

Aug

Aug

4037 ± 1132

45
50
50

45
40
40

0
0
0

in

10
10

1 :1
0.8:1
0.8:1
1.3:1
2.8:1
2.4:1

134 ± 12.2
148 + 10.3

Wild
Wild

Spawn
Spawn

Sep
Sep

3259 ± 786

40

25

50
70

0
0

10

5

144 + 11.0

Wild

Spawn

Oct

2981 ±724

25

60

5

10

127 + 10.9
133 ± 10.2
137 + 9.6

Wild
Wild
Wild

Spent
Spent
Spent
Spent

Oct

Nov
Nov

2602 ± 587

20

25
35

55
40
50

20
25
10

5

10
5

2.8:1
1.6:1
1.4:1
1.6:1
1.7:1

129 ± 12.3
125 ± 11.6

Wild
Wild

E-Dev
E-Dev

Dec
Dec

1608 ±825

25
30

40
50

20
20

15
0

* Oocyte area was evaluated on a monthly basis only. c r, - i

ORI = origin of specimens: DEV. STAGE = developmental stage: TIM = timing correspondence (Activ = activation stage: E-Dev -- early
development: M-Dev = mid development: L-Dev = late development: M = male: F = female: 1 = inactive onesting: B = bisexual or hermaphrodite:
F/M SR = female/male sex ratio).

Gonadal Tissue and Storage Cells in P. mazatlanica

153

The developing stage was divided into three subcategories
(early, mid. and late) as a strategy to identify, especially in previ-
tellogenic, vitellogenic. and postvitellogenic oocytes, the presence
of specific cellular structures whose expression is short lived. The
Balbiani body and the mitochondrial clusters observed in the oo-
cyte ooplasm are examples of these structures. The Balbiani body
was initially described by Sastry (1968) in Aequipeaen irradians
concentricus. and thereafter, it has been rarely cited (Guraya 1979;
Dohem 1983; as cited in de Gaulejac et al. 1993 for the bivalve
Pin. nobilis). This structure, not previously described for any spe-
cies of pearl oyster, was mostly seen in vitellogenic oocytes and
seldom in postvitellogenic oocytes of P. mazatlanica. According
to Pipe (1987a. 1987b) and de Gaulejac et al. (1995). this body is
probably filled with lipid droplets arising from the degeneration
and transformation of mitochondria and other membranous or-
ganelles, such as the Golgi complex. However, the mechanisms for
the endogenous supply of lipids and other materials needed by the
growing oocyte are poorly understood, although pinocytosis was
proposed (Pipe 1987a; Pipe 1987b; de Gaulejac et al. 1995). The
mitochondrial clusters (whose presence cannot be confirmed using
light microscopy, but suggested because of their strong basophilic
character), appeared initially near the stalk region and later scat-
tered in the vicinity of the nucleus during the previtellogenic and
vitellogenic stages of oogenesis. Certainly, their expression is as-
cribed to the intense respiratory rate of early and mid-developing
oocytes and the active synthesis of energy related to the formation
of the yolk molecule.

Several bisexual speciinens were detected during the annual
cycle. Although sex reversal in pearl oysters is basically protan-
drous (Gervis & Sims 1992), many protoginic and functional her-
maphrodite specimens were found in this study, especially from
September through December, when wild oysters were collected.
This alteration in the sexual behavior of specimens was previously
considered by Coe (1945) an accidental or abnormal mode of
embryonic development, resulting from the failure of the heredi-
tary sex-differentiating mechanism to function normally. How-
ever, environmental influence is presently recognized as one of the
most important factors controlling sexual differentiation of germ
cells in the direction of maleness or femaleness. We believe that
feinales appear more frequently in the wild population, while males
predominate under culture conditions. Thus, oyster's age (as a biotic
factor) and the density in which individuals are kept within the culture
baskets (abiotic factor) may be important factors regulating sex re-
versal from female to male in P. mazatlanica. Taylor ( 1999) reported
a similar expression of sexuality in cultured P. iiuirgaritifera.

In subtropical areas, such as in Bahia de La Paz. the range of
temperature variation over an annual cycle is 1 1-1 2"C (Pearl Oys-
ter Project database). Therefore, the energy to fuel gametogenesis.
parallel to that arising from food intake, have to come from spe-
cialized somatic cells involved in the storage of nutrients (Lubet
1959). According to Mathieu and Lubet (1993), there are three
types of cellular elements commonly recognized as participating in
these processes in bivalves: specific storage cells (adipogranular
cells and VCT cells), intragonadal cells (follicular cells, Sertoli
cells, and AC), and muscular cells. We identified the presence of
two of them in P. mazatlanica: VCT cells (constituting an integral
part of the interconnective tissue matrix, observed in the gonadal
tissue, digestive gland, and pearl sac) and AC (within acini, always
attached to developing oocytes). The muscular cells were not con-
sidered in this study, since their active role upon gametogenesis
has been clearly demonstrated for several bivalves (Ansell 1974;

Gabbott 1975; Bayne 1976; Taylor & Venn 1979; Barber & Blake
1981. 1991; Epp et al, 1988; Faveris & Lubet 1991; Martinez
1991; Racotta et al. 1998), including the pearl oyster, P. mazat-
lanica (Saucedo et al. 2001b). Follicular and Sertoli cells were not
observed with light microscopy. Storage cells are expressed dif-
ferently between families. For example, Mytilidae possess both
types, VCT and ADG cells (Lubet 1959; Bayne et al. 1982; Pipe
1987a; Pipe 1987b; Peek & Gabbott 1989a; Peek & Gabbott
1989b). Glycymeridae only one type corresponding to ADG cells
(Mathieu & Lubet 1993), and Ostreidae only VCT cells (Swift et
al. 1988), but with an intermediate structure between ADG and
VCT cells as described for Mytilidae. In contrast, Pectinidae. Ven-
eridae. Cardidae. and Pinnidae possess none of them (Yongqiang
& Xiang 1988; Dorange & Le Pennec 1989; Dorange et al. 1989:
Rodn'guez-Jaramillo et al. 2001) since they rely mostly on energy
stored in the adductor muscle for the synthesis of gametes.

Storage cells also show important differences in their bio-
chemical composition and ways of releasing nutrients. In this
study. VCT cells were strongly PAS-h- and moderately BBS-H and
OR-I-. demonstrating that although specialized in the storage of
carbohydrates (glycogen), they also contain lipids. In M. edulis.
nutrients are released by a progressive autophagic mechanism that
involves the sequestration of small membrane-bound vacuoles
from the large, stored glycogen vesicle, which causes a reduction
in cellular volume (Bayne et al. 1982; Mathieu & Lubet 1993). As
proposed by Pipe ( 1987a. 1987b). these vacuoles fuse with the cell
membrane and release their glycogen content by eccrine secretion.
VCT cells were commonly observed surrounding excretory con-
duits in the gonadal tissue and digestive gland, indicating that both
tissues must be communicating by the interconnective tissue ma-
trix, which probably serves as the vehicle to transport the stored
carbohydrates from the gonadal tissue to the digestive gland, or
vice versa. Although it is difficult to determine the timing when
carbohydrate reserves flow from one tissue to another, we believe
that carbohydrates obtained from ingested food are initially used as
an energy-rich fuel for the immediate build-up of gametes, and
later incorporated into the digestive gland for storage. This pro-
posal is in agreement with results of Saucedo et al. (2001b). who
observed progressive accumulation of carbohydrates in this tissue,
despite the advancement of gametogenesis in the same species (at
least during the developing and ripe stages and later during the
spawning occurring in October to November). This trend indicates
that carbohydrates from the digestive gland are not transferred to
the gonadal tissue for usage during the reproductive cycle in course,
but instead stored and used to cover further energy demands. This
argument also coincides with the finding of Barber and Blake (1981,
1991 ) that the digesti\e gland acts as a short-term storage and transfer
site of nutrients to meet the reproductive events in M. edulis.

In this study, another cellular storage component associated
with the growing oocyte was the auxiliary cells. Although their
presence has been associated mainly with the nutrition of previ-
tellogenic and vitellogenic oocytes, an alternate function related to
the resorption of residual oocytes was hypothesized by de Gaulejac
et al. (1995). The first function was attributed to the intimate relation
of these cells with oocytes by means of desmosome-like junctions and
to their rich content of glycogen granules and lipid droplets. In con-
trast, the presence of an active Golgi complex and large autophagic
vacuoles suggests these cells may also have the ability to phago-
cytize. and as such, auxiliary cells might be implicated in the growlli
of oocytes by recycling nutrients originating by phagocytosis.

With the results previously reported in Saucedo et al. (2001b.

134

Saucedo et al.

2001c) and those presented in this study, we believe that successful
trials for the continuous production of P. imi:allaincii spat can be
conducted if nutrients with the correct biochemical composition,
energy profile, and quantity are supplied to broodstock during their
gonadal conditioning. This is especially important to allow the
vital energy storage process, either extracellular (in VCT cells and
auxiliary cells) and intracellular (in the Balbiani body) to be ac-
complished. More studies on seasonal cycles of expression, mo-
bilization, and depletion of VCT cells and AC are needed to con-
firm these findings, especially for the role of mantle tissue, which
is an impt)rtant site of storage of glycogen and lipids associated
with gametogenesis in bivalves. Ultrastructure studies of gameto-
genesis are also required.

ACKNOWLEDGMENTS

This study was done as part of tv\(i institutional projects of
CIBNOR on Pearl Oyster Culture and Pearl Induction in Bahi'a de
La Paz (Projects PAC-7 and PAC-.^9). Additional grants were
prinided by the Con.sejo Nacional de Ciencia y Tecnologia
(CONACYT-Mexico. as a Ph.D. scholarship), the Consejo Nacio-
nal para la Biodiversidad (CONABIO). and the Fondo Mexicano
para la conservacion de la naturaleza (FMCN). The authors are
grateful to the following .staff of CIBNOR: Horacio Bervera Leon
and Juan Jose Ramirez Rosas for SCUBA diving assistance and
collection of oysters. Teresa Arteche for histological processing of
samples, and Mr. Ira Fogel for editing the English-language text.

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opercidaris. from the Clyde Sea area. J. Mar. Biol. Assoc. UK 59:605-

621.
Taylor, J. J. 1999. Juvenile production and culture of the silver-lip pearl

oyster Pimtada maxima (Jameson). Ph.D. Thesis, James Cook Univer-
sity, Australia.
Tranter, D. J. 1959a. Reproduction in Australian pearl oysters (Lamelli-

branchia). 1. Pinctada albina (Lamark): Primary gonad development.

.Aiistr. J. Mar. Freshwater Res. 9:135-143.
Tranter. D. J. 1959b. Reproduction in Australian pearl oysters (Lamelli-

branchia). II. Pinctada albina (Lamark): Gametogenesis. Aiistr. J. Mar.

Freshwater Res. 9:144-158.
Tranter. D. J. 1959c. Reproduction in Australian pearl oysters (Lamelli-

branchia). III. Pimtada mar)>aritifera (L.). .Austr. J. Mar. Freshwater

Res. 9:509-523.
Wada. S. 1959. Biology of the silver-lip pearl oyster Pinctada maxima

(Jameson). 2. Breeding season. Margarita 1:15-28.
Wada. K, T,. A. Komaru, Y. Ichimura & H. Kurosaki. 1995. Spawning

peak occurs during winter in the Japanese subtropical population of the

peari oyster, Pinctada fucata (Gould. 1 850). Aquacidture 133:207-214.
Wright, W. G. 1988. Sex change in the Mollusca. Trends Eco. Era. 3:137-

140.
■^'ongqiang, F. & Q. Xiang. 1988. Studies on ultrastructure of oocyte in

process of maturing in pen shell. Ada Oceaiiol. Sin. 7:459^72.

Journal of Shellfish Ri'si'tinh. Vol. 21. No. I, 157-162, 2002.

GROWTH AND GAMETOGENIC CYCLE OF THE BLOOD ARK, ANADARA OVALIS
(BRUGUIERE, 1789) IN COASTAL GEORGIA

ALAN J. POWER AND RANDAL L. WALKER

Marine Extension Service. Shellfish Research & Aqmwulttire
Ocean Science Circle, Savannah, Georgia 3141 1-101 1

Lahoraiory, University of Georgia, 20

ABSTRACT We collected two cohorts ( 1994, 1995) of blood iirks, Anadara avails (Bruguiere 1789) from sets that had occurred on
surf clam cages in Wassaw Sound. Georgia. Subsequent to transferal into pearl nets, the arks were suspended from the main dock at
the Skidaway Institute of Oceanography on the Skidaway River, Georgia, between January 1995 and April 1997. To determine the
reproductive cycle and growth rate, we collected arks monthly, measured each individual for shell length, and took a gonadal sample
for histological analysis. Arks from the 1994 cohort grew from a mean shell length of 17.8 mm to 39.9 mm in 15 months, a rate of
1.47 mm per month. Arks from the 1995 cohort grew from a mean shell length of 9.9 mm to 42.6 mm m 16 months, a rate of 2.04
mm per month. Growth was most rapid during the spring and summer months. Of the 747 arks sectioned for histological examination,
males dominated the population (66'7r) and 7.2% were sexually indeterminate. A minor spawning event occurred in the winter months
of the first year for each cohort (0+ years) followed by a major spawning commencing in April and continuing through the summer
months. In Georgia, it appears that blood arks recruit in summer-early fall, and that juveniles grow rapidly to reach sexual maturity
at an early size {10-12 mm) and age (-8 months).

KEY WORDS: Anadara oralis, arks, gametogenesis, growth, sex ratio, spawning

INTRODUCTION

A member of the ark shell family (Arcidae). the blood ark,
Anadara oralis (Bruguiere 1789) is reported lo inhabit estuarine
waters from Cape Cod, Massachusetts, to the West Indies and
Brazil, at depths ranging from the low-tide line to >3 m (Abbott
1974; Anderson et al. 1984; Rehder 1981 ; Walker & Gates 2001 ).
This bivalve is found over a variety of substrate types, but is most
commonly found in sandy deposits (Alexander 1993), The blood
ark inhabits Chesapeake Bay waters in area where salinity is above
15 ppt (Chanley & Andrews 1971 ). Blood arks are equivalved, and
somewhat oval in shape, ranging in size (shell length) from 28 to
76 mm (Rehder 1981 ). Shell height is slightly less than the shell
length, with a reported length/height ratio of 1.16 (Alexander
1993); shell depth reaches approximately 70% of height (McGraw
et al. 1996). The blood ark is a short-lived species, which has up
to 80% mortality in the third year of life, and a maximum life span
of five years (McGraw et al. 1996; Walker 1998).

Several ark shell species form the basis of economically sig-
nificant molluscan fisheries and extensive culture operations
throughout the world (Baqueiro et al. 1982; Broom 1985; Baqueiro
1989; Manzi & Castagna 1989; Nie 1990; Umezawa 1992). The
1997 worldwide harvest of arks iScaiiluirca. Area, and Anadara
spp.) was 97,296 metric tons, landed in Cuba. Fiji, Indonesia.
Korea. Japan. Mexico, Philippines, and Venezuela (FAO 1999). In
the United States, ark resources have been largely ignored by the
fishing industry until recently. Some interest was expressed in
South Carolina in the 1980s, however, there were problems iden-
tifying viable markets (Anderson et al. 1984; Anderson and Ever-
sole 1985). Since then a small fishery, primarily for the blood ark.
Anadara oralis (Bruguiere 1789), and the ponderous ark. Noeiia
ponderosa (Say 1822), has developed in Virginia. Meats are sold
primarily as an ethnic food in Chicago, New York. Los Angeles,
and Washington D.C. or exported to Mexico (McGraw &
Castagna 1994; McGraw et al. 1996; McGraw et al. 1998).

The demand for arks has recently outpaced the numbers that
can be supplied by the Virginia fishery. Since blood arks have been
reported to grow up to twice as rapidly as ponderous arks in the

first 2 years after settlement in Virginia waters, it has become the
principal aquacultural species of interest (McGraw et al, 1996).
Growth rates of Georgia stocks have been reported to almost
double those observed in these more northern waters, reaching a
marketable size in a 1-year period (Walker 1998). Consequently,
the great potential of Anadara oralis as a new commercial re-
source in Georgia has been recognized, and an aquacultural fishery
is currently being investigated (Power & Walker 2001). Unfortu-
nately, little life-history information other than from Virginia
(McGraw & Castagna 1994; McGraw et al. 1996; McGraw et al.
1998) exists for this species in the United States. This study de-
scribes the growth and gametogenic cycle of the blood ark from
the coastal waters of Georgia,

MATERIALS AND METHODS

We collected blood ark cohorts in January 1995 ( 1994 cohort)
and December 1 995 ( 1 995 cohort ) at the mouth of House Creek,
Little Tybee Island. Wassaw Sound, Georgia. The clams had at-
tached themselves to surfclam (Spisida solidissima similis. Say
1822) grow-out cages (6 mm vinyl coated, 1 x 1 x 0.6 m) that were
partially buried (0.3 m) al the spring-low-water mark on a sand
flat. Cages had been deployed in September of both years and
when we stocked with surfclam seed in October, we noted that ark
wild spat had settled and attached with byssal threads to the wire
mesh sides of the cages. In the laboratory on Skidaway Island, we
measured for shell length (longest possible measurement, i.e., an-
terior-posterior) using Vernier calipers. Arks from the 1994 and
1995 cohorts had a mean shell length of 17.8 ± 1.52 (SE) mm
(range: 1 1.9-28.3 mm) and 9.9 ± 0.19 mm (range: 4.3-17.7 mm),
respectively. Each cohort was placed in a separate 3 mm mesh
pearl net, and suspended from a floating dock on the Skidaway
River. The 1994 recruitment was a small set (approximately 20-30
arks per cage), while the 1995 recruitment was a massive set with
over 500 arks collected per cage.

Between January 1995 and April 1996 we randomly collected
arks (N = 10. 1994 cohort) from the pearl nets. We repeated this
procedure for the 1995 cohort collecting arks (N = 30) moniliK
between December 1995 and April 1997. We measured ihesc .nk^

157

158

Power and Walker

for shell length, and dissected a mid-lateral gonadal sample (ca. I
cm") from each animal. Smaller speciinens with meats <l cm"
were preserved and sectioned whole. We also noted the coloration
of gonads during the dissections. Gonadal tissue was fixed in
Davidson's solution, refrigerated for 48 h, washed with 50% etha-
nol. and preserved in 707r ethanol until processing. We processed
tissues according to procedures outlined in Howard and Smith
( 1983). The examination of prepared gonadal slides was conducted
with a Zeiss Standard 20 microscope (20X). Each section was
sexed. and assigned to a developmental stage as described by
Walker and Heffernan (1994) and Spruck et al. (1994). A staging
criteria of 0 to 5 was employed for Early Active (EA = 3), Late
Active (LA = 4). Ripe (R = 5). Partially Spawned (PS = 2), Spent
(SP= I ), and Inactive (lA = 0). The determination of monthly go-
nadal index (G.I.) values was obtained by averaging the number of
specimens ascribed to each category score. We tested sex ratios
against a 1:1 ratio with Chi-square statistics (Elliott 1977).

Surface water temperature and salinity data were taken daily
froin the dock of the Marine Extension Service, adjacent to the
grow-out site at 0800 h (Monday-Friday) from January 1995 to
April 1997.

RESULTS

Monthly mean water temperature and salinity data for the Skid-
away River are given in Figure I . Water temperatures were coolest
during the months of January and February 1995, 1996, and 1997
ranging from a mean low of 9.34"C to a high of I3.32T. Water
temperatures reached their peak during the months of July and
August 1995, and 1996 ranging from 28.6 1 °C to 30.46°C. Mean
salinity values were less cyclical but typically reached their lowest
in the spring months each year (14.63 ppt in March 1995). The
highest salinity recorded over the study period was 28.00 ppt in
December 1996.

Both cohorts exhibited similar growth trends (Fig. 2). increas-
ing in size rapidly during the spring-summer months with growth
rates diminishing in the fall of the first culture year (i.e.. l-l- aged
individuals). Arks from the 1994 cohort grew from a mean shell
length of 17.8 mm to 39.9 mm in 15 months, a rate of 1 .47 mm per
month. Arks from the 1995 cohort grew from a mean shell length
of 9.9 mm to 42.6 mm in 16 months, a rate of 2.04 mm per month.

Of the 747 arks examined, 54 (7.2%) were sexually indetermi-
nate, 493 (66%) were male, and 200 (26.7%) were females. The

Dec-94 Feb-95 Apr-95 Jun-95 Aug-95 Oct-95 Dec-95 Feb-96 Apr-96

Jan-95 Mar-95 May-95 Jul-95 Sep.95 Nov-95 Jan.96 Mar-96 May-96 Jul-96 Sep-96 Nov-96 Jan-97 Mat-97

Figure L The mean monthly water temperature and salinity of the
Skidaway River, Georgia from January 1995 to April 1997.

Dec-95 Feb-96 Apr-96 Jun-96 Aug-96 Oct-96 Dec-96 Feb-97 Apc-97

Figure 2. (a) The mean monthly shell length (mm ± standard error! of
the 1994 cohort of hlood arks, Anadara oralis, grown in pearl nets
suspended in the Skidaway Ri\er, (korgia from .lanuary 1995 to April
1996. (bl The mean mimthly shell length (mm ± standard error) of the
1995 cohort of blood arks, Amiilant <ivalis. grown In pearl nets sus-
pended in the Skidaway River, Georgia from December 1995 to .\pril
1997.

Chi-square test revealed that the overall male/female ratio of 2.44
was significantly different from parity (x" = 123.9; P < 0.001).
Males dominated in both cohorts (1994: 1 .00:0.36; and 1995: 1 .00:
0.42) and in every monthly sample. Histological examination and
visual observations of the gonads revealed that all orange-red col-
ored gonads were late active or ripe females, while those showing
white coloration were typically ripe males.

Blood arks reached sexual maturity at an early age, exhibiting
ripe specimens in the winter months of the first year in both co-
horts with major spawning commencing after April-May and con-
tinuing to September-December (Figs. 3-5). A coinparison of the
gonadal stages for the two cohorts shows a striking difference for
the cohorts in December 1995 (1994 cohort age l-i- years, 1995
cohort age O-i- years) indicating that the 0+ year arks develop
gametes more easily during the winter months of their first year.
For the 1994 cohort, 22% were ripe, 22% were partially spawned
and 56% were spent in January 1995. In February and March, arks
were either spent or in the early active stage. By April, late active
(37.5%), ripe stages (25%) and spent stages (25%) were present.
The gonadal index increased between March ( 1 .9) and April (3. 1 )-
May (3.2) as more animals became reproductively active, with ripe
individuals dominating in May (60%) and June (42.9%). The go-
nadal index decreased to 1.4 by June, reflecting the occurrence of
partially spawned individuals. Partial spawning dominated from

Thh Blood Ark in Coastal Georgia

159

i I

1

Dec-94 Feb-95 Apf-95 Jjn-95 Aug-95 Oct-95 Dec-95 Feb-95 Apr-96

Figure 3. The relative frequency (percentage) of eacli gonadal devel-
opmental pliase (KA = early active, LA = late active, R = ripe, PS =
partially spawned, and S = spent! for the 1994 Aiiadara oralis cohort
from January 1995 to April 1996,

July (40%) through September (50%), with spent individuals oe-
curring most frequently through the latter part of the year. This
resulted in low gonadal index values until the initiation of game-
togenesis again in January (G.I. = 1 .5 in December to 2.36 in
January).

For the 1995 cohort, 229c of arks were ripe in December 1995,
with 28% in the early active and 50% in the late active stage (G.I.
= 3.93). Most arks were in the early active stage (50%) with
41.7% exhibiting the spent stage by January (G.I. = 2.25), indi-
cating a minor spawning event having taken place. Ripe individu-
als occurred again in March (9.4%) with most (76.7%) being ripe
in April (G.I. = 4.53). Ripe individuals were present through
August, but partially spawned and spent individuals dominated
until December 1996. As in previous years, early active and late
active individuals began occurring in December 1996 with ripe
individuals again dominating by April (69.27r) (G.I. = 4.82).

For the 1995 set, 225 arks in December 1995 were histologi-
cally processed to determine the size at sexual maturity for the
blood ark. Of the 225 arks examined. 26 ( 1 1.6%) were sexually
indeterminate, 156 (69.6%) were males and 43 (19.2%) were fe-
males. The sex ratio was 1.00:0.28 (M:F), which significantly

(b) 5

I

■
1

Dec-95 Feb.96

Jiin-96 Aug-96 Oc|.96 Dec-96 Feb-97 Acf-97

Figure 4. The relative frequency (percentage) of each gonadal devel-
opmental phase (KA = early active, LA = late active, R = ripe, PS =
partially spawned, and S = spent) for the 1995 Anadara ovalis cohort
from December 1995 to April 1997,

Dec-95 Feb-96 Apr.96 Jun-96 Aug-96 Oct-96

Feb-97 Apr-97

Figure 5. (a) The monthly gonadal index values of the 1994 cohort of
blood arks. Aiwdara ovalis, from the coastal waters of Georgia hetween
January 1995 and .Vpril 1996, Monthly gonadal index (G.l.) values
were determined by averaging the number of specimens ascribed to
each category score (EA = 3, LA = 4, R = 5, PS = 2, SP = 1), (b) The
monthly gonadal index values of the 1995 cohort of blood arks, Ana-
dara ovalis. from the coastal waters of Georgia between December
1995 and .\pril 1997. Monthly gonadal index (G,l.) values were deter-
mined by averaging the number of specimens ascribed to each cat-
egory score (EA = 3, LA = 4, R = 5, PS = 2, SP = 1),

differed from parity (x^ = 64.17; P < 0.001). Males averaged 9.9
± 0.26 mm and ranged from 4.3 mm to 16.2 mm in shell length.
Females average 1 1 .5 ± 0.37 mm and ranged from 7.4 mm to 17.7
mm in shell length. Indeterminate animals averaged 6.5 ± 0.29 mm
and ranged from 4.5 mm to 8.9 mm. Of the 199 arks that had
started gametogenesis, most were in the late active stage for males
(54.5%, 10.2 ± 0.27 mm, range: 6.3-16.2 mm) and the ripe stage
for females (44.2%, 1 1.8 ± 0.50 mm, range: 8.2-16.6 mm). At this
time, males also had 30.1% in the early active stage (7.3 ± 0.25
mm, range: 4.3 mm-13.4 mm), while females had 20.9% (9.6 ±
0.68. range: 7.-1-14.2 mm).

DISCUSSION

In Georgia, bloods arks recruit during the summer and early
fall; the spat grow rapidly and reach sexual maturity by the end of
the year. A minor spawning event occurs at this point, followed by
the major spawning period in the subsequent summer months. As
observed with one-year-old arks from the 1995 cohort, the minor
winter spawning event is not repeated in the subsequent year.

Rapid growth rates recorded for arks (<2-years-old) in li-

160

Power and Walker

TABLE 1.
Size at initial gametogenesis and sexual niaturlt\ for various marine bivalves species tn>ni (he Kamiiv Arcidae.

Shell Length at Initial Gametugenesis (mm) Shell Length at Sexual Maturity I mm)

Species

-Male

Female

Not Stated

Male

Female

Not Stated

Source

Broom. 1985

21

Narasimhum. 1968

24-25

Broom. 1983

This study

Natarajan & John. 1983

30

Baron. 1992

Tmg et al.. 1972

Walker & Power (in press)

Broom. 1985

Yolove. 1974

Amiduni cornea
Amuiiini i^ranosit
Anadara grunosa
Anadara avails
Anadara rhombea
Anadara scapha
Anadara siibcrcnala
Anadara transversa
Anadara mberctdosa
Senilia senilis

4
19

20
17.5

22
15
10

10-17

10
21-25

!l-25

36

present study (1.47 and 2.04 mm/mo) were comparable to earlier
reported growth rates for natural populations in Virginian waters,
and cultured arks in the coastal waters of Georgia. In Georgia, arks
that were similarly grown in pearl nets for 36 ntonths exhibited
rapid growth in the first year (<10 mm size class: 2.58 mm/mo;
10-20 mm: 2.01 mm/mo) with decreased annual growth for years
two (0.83 and 0.89 mm/mo) and three (0.19 and 0.18 mm/mo)
(Walker 1998). In Virginia, blood arks grew from an initial size of
14 mm to 30 mm in 1 1 months, a lower rate of 1.45 mm/mo
(McGraw & Castagna 1994). Arks were noted in the present study
to grow faster in the spring and summer months, therefore the
slower growth rates in Virginia may result from colder water tem-
peratures.

Sexual maturity was attained at an approximate mean size of 10
mm in shell length for males and 12 mm for females (age 7 to 8
months) however: gametogenesis was noted to occur at minimum
sizes of 4 mm and 7 mm, for males and females, respectively.
Animals from the 1995 cohort were known to be at least three
months old when gathered in December, since the cages were
deployed in September. At this time. 20% of the 225 collected
were ripe and 44% were in the late active stage. Because major
spawning for blood arks starts in April-May. the maximum age of

the new recruits on the cages is about 8 months. We believe that
blood arks had already recruited to the sand flat prior to the place-
ment of the field grow-out cages. Once the cages were in place, we
postulate that the arks migrated up the sides and attached them-
selves with byssal threads. Blood ark juveniles are commonly
found attached to the upper ends of Dioptera tubes in the estuary
(Walker, personal observations). An upward migration of juvenile
arks has also been observed with transverse arks. Anadara trans-
versa within the laboratory. Transverse arks contained within a
northern quahog. Menenaria menenaria. seed shipment were ob-
served to migrate to the top of the quahog seed mass and even up
the sides of the holding tray to the surface/water interface (Walker
& Power, in press). Members of the Arcidae are reported to initiate
gametogenesis and reach sexual maturity at a small size (Table I ).
The blood arks mature at a similar age to Anadara granosa (Lin-
naeus 1758: Narasimham 1968) and Senilia senilis (Lamarck
1758: Yoloye 1974). but typically at a smaller si/e than most of the
family (Table I ).

Temperature is one of the main exogenous factors controlling
reproduction in marine invertebrates (Giese 1959; Sastry 1975). In
Virginia, blood arks are reported to spawn in the summer months
when water temperatures reach abo\e I7°C (Chanley & Andrews

TABLE 2.

The sexual ratios and percentage hermaphroditism of various marine bivalves species from the Family Arcidae.

Species

Sample

Sexual Ratio

Size

(Female:Malel

1040

1.00:1.00

300

1.00:1.00

185

1.00:1.98

693

1.00:2.44

1155

1.(10:1,27

235

1.00:1.47

100

1.00:0.34

1.00:1.00

1.00:1.00

199

1.00:0.53

1.00:1.00

218

1.00:1.25

1094

1.00:1.00

1.(10; 1.00

181

1.(10:1.26

Hermaphroditism

Source

Anadara anlicjuala
Anadara granosa
Anadara ovalis
Anadara ovalis
Anadara rhombea
Anadara scapha
Anadara senilis
Anadara senilis
Anadara subcrenata
Anadara transversa
Anadara trapezia
Anadara tuberculosa
Anadara tuberculosa
Anadara tuberculosa
Noetia ponderosa

0.003

0
0

0
0.004

Toral-Barza & Gomez. 1985

Broom. 1983

McGraw et al.. 1998

This study

Natarajan & John. 1983

Baron, 1992

Yoloye, 1974

Broom. 1985

Broom. 1985

Walker & Power (in press)

Broom. 1985

Cardenas & Aranda. 2000

Cruz. 1984

Dzyuba & Maslennikova. 1982

McGraw et al.. 1998

The Blood Ark in Coastal Georgia

161

1971; McGraw et al. 1998). In Georgia blood ark^ spawned earlier,
from late spring ihrough summer, presumably due to the coastal
waters warming earlier in the year than in Virginia. By April-May
1995 and 1996, water temperatures were already above 20°C in the
Skidaway River. In general, spawning periods and gametogenesis
in marine bivalves start earlier and last longer in southern geo-
graphical areas than in northern ones (Eversole 1989; Thompson et
al. 1996).

In this study, males dominated the population of blood arks
with an overall sex ratio of 2. 38; 1.00 (M:F). This is in agreement
with the observed sex ratio (1.98:1.00 M;F) reported by McGraw
et al. (1998) for a Virginia population of 1-year-old blood arks
(Table 1 ). An equal sex ratio has been observed in Andara gnmosa
(Pathansali 1966; Broom 1983) and Anadaia siibcreiiata (Lischke
1869; Ting et al. 1972): however, males dominated Senilia senilis
populations (Yoloye 1974). For Anadara scapha (Linnaeus 1758),
males were reported to dominate in the smaller size-classes while
females were more frequent in the larger-size classes (Baron

1992). This may indicate a sex change but may also be a conse-
quence of the differential energy requirements of males vs. fe-
males. No hermaphroditic Anadara oralis were observed in the
present study. Hermaphroditism is rare in the Family Arcidae
(Table 2); Anadara scapha and Senilia senilis have been classified
as a protandric hermaphrodite (Baron 1992; Yoloye 1974). The
present study examined newly recruited blood arks, while in Vir-
ginia 1-year-old indi\ iduals were examined. Males typically domi-
nate protandric bivalve species in the first year; whereas older age
classes are generally equal. Blood arks can reach a maximum of 5
years and thei'efore these older age classes need to be examined to
determine if this species is protandric.

ACKNOWLEDGMENTS

This work was supported by the University of Georgia Marine
Extension Service. The authors thank Ms. Dodie Thompson for
processing the histological samples.

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Jounuil of Slu'lifish Research. Vol. 21. No. I. 16.V17I, 2002.

TEMPORAL FLUCTUATIONS (1989-1999) IN THE POPULATIONS OF HALIOTIS FULGENS
AND H. CORRIJGATA (GASTROPODA: HALIOTIDAE), AT ISLAS SAN BENITO (BAJA

CALIFORNIA, MEXICO)

J. A. RODRIGUEZ-VALENCIA* AND F. CABALLERO-ALEGRIA

Institiito Naciunal dc la Pesca. CRIP-Euseiuuki. Carrctcni Tijiuiini-Eiisciuiclci Km 1(17. Paiyiic Iiulustriul
Fondeport, El Sauzal. Baja Cullfornln. Mexico

ABSTRACT In our study area, green abalone (Haliotis jidi^cns) was more abundanl than pink abalone (H. conui^atu) until the end
of the 1980s, but nowadays, both .species have similar density levels and are close to values were recruitment failures may occur. In
compiirison to pink abalone, green abalone showed higher abundance of legal-sized stock and condition factors. Abundance of the
legal-sized stock of both species has significantly decrea.sed. but the density extracted by fishing remained the same. Overfishing of
legal sizes and recruits, or poaching, may have not significantly contributed to the observed patterns. Only the proportion of recruits
of pink abalone decreased significantly along the time and its fluctuation patterns support the idea that non-severe "El Nino" events
may have a positive effect on recruitment. The lack of significant changes in recruitment of green abalone suggests that this effect may
be species-specific. On the other hand, our results indicate that "El Niiio" events lead to poor physiological condition. Mean condition
factors of both species decrea.sed significantly along the time, and are significantly related with the mean annual surface temperatures.
The proportion of sexually mature organisms of both sexes, in both species, also decreased significantly along the time and could be
a by-product of impoverished condition. Additionally, the sex ratio of green abalone varied significantly along the time, in favor to
males.

KEY WORDS: abalone surveys, long-term dynamics. Baja California. Hiilioii\ fi(li;eii.\. H. ctirnigala

INTRODUCTION

The quanlificatioii of abalotie and the assessment of its tempo-
ral fluctuations are often difflcult tasks. Aggregated spatial distri-
butions difficult the obtainment of precise abundance estimates at
large spatial scales (McShane 1995), and even at small spatial
scales estimates may have with high variance and low precision
(McShane 1994).

The first attempts to quantify abalone in Baja California were
done between 1968 and 1970, covering wide spatial scales along
the coast (Guzman-del-Proo 1992). Mean density estimates de-
rived from these attempts had very low precision, due to habitat
heterogeneity (Guzman-del-Proo 1992), After 1988, abalone sur-
veys were restricted to each fishing area, in order to calculate local
yearly catch quotes, and regulate the fishery. We present the re-
sults of abundance surveys made between 1989 and 1999, for
green abalone {Huliotis fulgens. Philippi 1854) and pink abalone
{H. corntgata. Gray 1828), at Islas San Benito. These three islands
are located at the current main abalone tlshing area of Mexico.

This work brings information about one of the few long-term
monitoring efforts applied to natural abalone banks in North
America. Additionally to density values, we bring data that are
rarely collected during surveys: namely, sizes, condition factors.
sex ratios, and gonadal maturity of both species. We tried to assess
the contribution of commercial extractions, overfishing of legal
sizes, recruitment overfishing, and local climate forcing to the
observed fluctuations.

MATERIALS AND METHODS

Study Area

Islas San Benito are located at the Pacific coast of Baja Cali-
fornia, 40 km northwest from Cedros Island (Fig. la). They are

*Corresponding author. J. A. Rodriguez- Valencia. Alfred-Wegener-
Institute, Wadden Sea Station Sylt., D-25992 List., Germany. E-mail:
jrodriguez@awi-bremerhaven.de

away from continental runoff and pollution, and strong seasonal
upwelling favor the growth of Macrovyslis pyrifera, Eisenia
arhorea, and Egregia menzie.ssi ( Gonzalez- Aviles & Shepherd
1996). The extension of the abalone fishing area is 6.862.155 m~
(Arano-Castanon & Uribe-Osorio 1998). The fishermen co-
operative "'S.C.P.P. Pescadores Nacionales de Abulon" is the
unique concessionaire of the abalone and lobster fishery in the
area.

Survey Methodology

Surveys were conducted in the whole fishing area of the is-
lands, when the fishery is closed (October to January), between

1988 and 1999. The coastline of the three islands has always been
divided into 35 sections (=500 m width) placed in the same loca-
tion during all the surveys, using floats as markers. The offshore
extension of each section has been fixed at the 30 in isobath;
therefore, the length of each section varied between 88 m and 1473
m. perpendicular to the shore (Arano-Castanon & Uribe-Osorio
1998) (Fig. la).

Abalone divers from the fishermen cooperative collected all
visible abalone inside each section, but the sampling procedure
varied along the time (Fig. lb):

1989 to 1995. Six to 12 sampling stations were randomly distrib-
uted inside each section, depending on the number of divers. A
4 nr-quadrat was used as a sampling unit from:

1996 to 1999, Each section was divided into three bathymetric
strata: 0-10 m, 10-20 m, and 20-30 m. Three, five, and two
sampling stations were randomly located inside each bathymet-
ric strata, respectively. A 10 nr-quadrat was used as sampling
unit.

Density Estimations

Only sampling stations located on rocky bottom were used for
density estimations. The mean density was calculated consider!'':

163

164

Rodriguez-Valencia and Caballero-Alegria

1989-1995

1996-1998

6-12 sampling
points randomly
distributed Inside
each section

I-

3 sampling points
randomly distributed
inside this bathymetric
strata

Sampling unit: 4 m"! quadrats

t 5 sampling points

' randomly distributed ,"~ - ^ « " 1^ -„ • ,. .^
\ J .J t. .1. _ . • ' \ « •'■*■*• 20-m isobath
inside this bathymetric I x " \

^^ " ^ '^30-m isobath

strata ^^f__

2 sampling points

randomly distributed Sampling unit: 10 m*2 quadrats

inside this bathymetric

strata

Figure. 1. (a) Location of the study area. The dashed line represents iVi-va isobath. (b( Scheme of the sampling method.

all sizes. Estimations of the mean density of the legal-sized stock
were done considering only sizes larger than the minimum legal
size of capture (green abalone: 150 mm of shell length; pink aha-
lone: 140 mm of shell length). Ninety-five percent confidence
limits and standard errors of the total mean density, as well as 959r
confidence limits of the mean density of the legal-sized stocks are
shown. Significant temporal differences in the mean values were
tested with ANOVA, in both species. We estimated the statistical
power for the total mean density comparisons, at the given level of
replication, and a = 0.05 (Zar 1999).

Recruitment Estimations

Our survey methodology is inadequate to estimate recruitment
accurately, but serious recruitment failures due to recruitment
overfishing or environmental changes should be reflected in our
data (Shepherd et al. 1998). From the 1989 to 1998 for
size-frequency data, we calculated the percentage of recruits of
both species, using the size criteria of Shepherd et al. (1998).
Overall, significant differences between percentages (inside
each species) were tested with Chi-squared analyses (Zar 1999).
Paired comparisons are shown, to estimate statistical power (Zar
1999).

Assessing the Effect of the Fishery

The density extracted by fishing, between 1989 to 1996. was
estimated for each species. The following data were used: local
captures provided by the fishermen cooperative, legal-sized stock
densities, local area of suitable habitat for both species (Rodriguez-
Valencia et al. 1998). and mean weight of muscle (as described
later). Significant temporal changes were tested by Chi-squared
analyses.

Biomelric Data

Biometric data of all captured abalone are available for the
1989 to 1998 surveys.

Sizes and Weights

Shell lengths of all captured abalone were measured to the
nearest millimeter and muscles were weighed to the nearest gram.
Each 5-mm size category contained at least three individuals.
Since measuring and weighing is time-consuming, abalone were
maintained in shadowed places and were covered with wet algae to
prevent desiccation. Significant temporal differences in mean shell
sizes were tested with ANOVA. Significant ascendant or descen-
dant trends were tested by the significance of the slope (Beta) of
simple linear regressions using the time as independent variable.
Modal and maximum sizes of each species, at each survey, were
compared using Chi-square tests.

Condition Factor

The condition factor of each sacrificed abalone was obtained
dividing the muscle weight by the shell length, and the mean
condition of each species at each survey was estimated. Significant
temporal changes between mean condition factors were tested with
ANOVA.

Sex Ratio and Gonadal Maturity

Sexes and gonadal maturity were determined to randomly se-
lected abalone at each survey. Significant temporal changes in both
variables were tested using Chi-square analyses.

Fluctuations of H. fulgens and H. corrugata

165

Haliotis fulgens

Haliotis corrugata

a)

Q TO
<

b)

Isla Natividad (Shepherd at al. 1998)
Islas San Benito (This work)

05

07

>, E

w 0)

c 5

n ?R

0) o

Q CO

n

<

0

c)

uu/

'cO ^ 0 035
<

d)

0)

c
Q.

1988 1990 1992 1994 1996 1998 1988 1990 1992 1994 1996 1998

1989 1991 1993 1995 1997 1999 1989 1991 1993 1995 1997 1999

Year

Year

Figure. 2. (a) Temporal patterns of total mean densities and their 95 "7^ confidente limits. In) represents the number of sampling stations located
on rocky bottom: (b) Temporal patterns of the mean density of legal-sized stock and their 95% confidence limits; (c) Temporal patterns of the
density extracted by fishing; (d) Percentages of recruits.

RESULTS

Patterns of the Total Mean Density

Green abalone was 2.5 times more abundant than pink abalone
at the end of the 1980s, but at the beginning of the 1990s its density

decreased, while that of pink abalone increased. After 1995. both
reached their lowest density levels and followed comparable abun-
dance trends (Fig. 2a). Temporal fluctuations are significant (Table
1 ). Modifications in sampling methods were more precise after
1995. because the confidence limits and standard eiTors of the
means decreased (Fig. 2a and Table 2).

166

Rodriguez-Valencia and Caballero-Alegri'a

TABIE 1.
Assessment of significant temporal changes in thf loUil iman {lensit\ of //. fiilgeiis and //. coiniifula and istimates of the statistical poHcr.

H. fiilgeiis

H. iiirnigata

Comparison

^(df.Kflett. dr.Error)

Effect MS

El

rror MS

*

Power

r,di.l-.rfi-

Ll. dl.I-;rr.)n

Effect MS

Error MS

<>

Power

1989 10 1991

15.3

0.7

2.3

0.9

F,,,4.M

= 5.5 =

1.3

0,2

1,0

0.6

19X9 to 1993

F,,,„„„ = 16.9**

9.9

0.6

2.8

1.0

F„,,„„,

= 14.9**

8.4

0.6

2.6

1.0

1989 to 1995

F,,,,„ = 24.7**

12.0

0.5

4.2

1.0

•"11,785)

= 23.1**

11.1

0.5

4.2

1.0

1989 to 1996

F,4.M60, = 27.7**

11.3

(1,4

5.2

1.0

^(4.1 Ihll)

= 34.0**

11,3

0,4

5.1

1.0

1989 to 1997

F„.„c„, = 25.0**

9.4

0.4

5.5

I.O

F|?.l4y7(

= 30.3**

10.2

0.3

5,5

1.0

1989 to 1998

F„,„„ = 28.6**

9.9

0.3

6.4

1.0

P(h. 17491

= 25.7**

8.6

0.3

6.0

1.0

1989 to 1999

•|7,207til -''--

9.6

0.3

7.0

1.0

F,7,:,m„

= 11.9**

1,9

0,2

3.9

1.0

* = P < 0.05.

*" P <0.0\.

Patterns of the Mean Density of Legal-Sized Slocks and Density
Exlrueted by Fisliing

The pfopoi'tmn of har\ esl;ible sizes of green iibalone has been
twice as high as that of pink abalone (Table 3), and its density of
legal-sized stock is significantly higher (f, 1.3499, = 189.3**). The
mean density of the legal-sized stock of both species has signifi-
cantly varied and decreased along the time (green abalone:
^(6, 1744, = 34.0**; pink abalone: F,f,.i744, = 8.0**) {Fig 2b). On
average, the fishery extracted 24% and 14% of the legal-sized
stocks of green and pink abalone. respectively, coiresponding to
15% and 7% of their total mean densities. The extraction by fish-
ing has remained constant, since the densities extracted by fishing
of both species remained constant along the time {green abalone:
^-^^^ = 0.02: pink abalone: x',^, = 0.004). although that of green
abalone seems to have increased {Fisi 2c).

Patterns of Sizes and Condition

Mean sizes of both species showed significant temporal varia-
tions {Table 3) without trend (green abalone: F,, j,, = 1.2: pink
abalone: f , , ,,, = 3.5). Modal and maximum sizes showed non-
significant tempoial variations (Table 3).

The mean condition of green abalone was significantly higher
than that of pink abalone during the studied period (1.6 vs. 1.1;
f(i 11771 = 196.7**). No significant differences were detected
between sexes, inside each species. Significant temporal changes
in the mean condition of both species were detected (green aba-
lone: f,,,.io.i2, = 10- 1**: pink abalone: F,f,^^^, = 3.0**). and both
species showed significant decreasing trends (Fig. 3a). The mean
condition of both species was significantly related with the mean
annual surface temperature (Fig. 3b). which increased 2.rC be-
tween 1989 to 1998. in the studv area.

Recniilment Estimations

Recruits of pink abalone have been more abundant than green
(Fig. 2d). Overall comparisons between the percentages of recruits
of green abalone indicated non-significant changes (x"(6i = 1-8).
but those of pink abalone varied significantly (x'tb) = 19.5**).
with increasing percentages until 1995 and decreasing percentages
after. Paired comparisons indicated non-significant changes for
green abalone, and significant changes for pink abalone (Table 4).
The average power of the paired comparisons was 0.81 and 0.83.
for green and pink abalone. respectively.

TABLE 2.

Standard errors of the total mean density estimations
(Abalone*m"-).

Haliolis eornigala

0-030
0.039
0.042
0.027
0,017
0,017
O.OIX
0,021

urvey

Haliolis fnlgens

1989

0.06 1

1991

0.045

1993

0.046

1995

0.028

1996

0.026

1997

0.028

1998

0.025

1999

0.027

Patterns of Sex Ratio and Gonadal Maturity

The sex ratio of green abalone showed significant temporal
changes in favor of males, while that of pink abalone remained
close to 1:1. without significant changes (Table 5). The proportion
of mature organisms of both sexes, inside each species, decreased
significantly along the time (Table 4). In 1995. a conspicuous peak
of mature organisms of both sexes was detected, in both species.
Relationships between the proportion of mature organisms of
green abalone and its mean condition factors and mean annual
surface temperature were suggestive, but non-significant (Fig. 3
c-d). On the other hand, these relationships were significant for
pink abalone (Fig. 3 c-d).

DISCUSSION

The Surveys

We have tried to follow the survey approach proposed by Hil-
born and Walters (1992), Van der Meer ( 1997), and Gorfine et al.
( 1998). distributing sampling points in the whole capture area and
applying low sampling effort at each point. The method used to
estimate abalone abundance seems to be appropriate. However,
main disadvantages are: (a) The small portion of potential habitat
sampled (().01%-0.02% between 1989 to 1995; 0.05%^^-0.07% be-

Fluctuations of H. fulgens and H. corrugata

167

TABLE 3.

Sizes (minimum, maximum, mean and its 95% confidence limits, and m<ide: all in mm). Minimum size of capture = MSC.

P<i\M\.

= P < 0.05: ** =

Haliolis fulgciis

Hatiotis corrugata

n

Min.

Max.

Mean

Mode

> MSC

Min.

Max.

Mean

Mode

> MSC

1989
1991
1993
1995
1996
1997
199S
1999
Temporal

721
303
269
137
1030
1089
364
721

comparisons

47
31
.56
99
23

S-)

40
87

2113
196
193
190
197
200
197
212
M = 0,6

152, 7± 1

148 8 ± I
152,3 ±2,
151.2H
151.9± 1.
153,2 +
150,2 ±;
146,7 ±;

,4,47, = 5,4**

15(1
140
150
160
150
160
156
138
,,, = 1,2

66,4

269

73

169

51 8

311

65

173

64 9

350

(i6

175

65,0

159

75

180

63,9

827

29

187

66.7

847

46

190

55,2

598

54

180

42,4

269

48

177

= 19 F,.:

131, 8± 1,8

130

125,9± 1,8

120

1 3 1 ,2 ± 1 6

130

133,0 ±2,6

130

131, 6± 1,2

135

134,2 ± III

140

131,1 + 11

135

135.5 ±2.0

128

,,,74, = 11.3**

X^M =

6.0

28.3
19.6
29.7
38.4
30.5
39.6
23.4
42.4

tween 1996 to 1998); and lb) The lack of use of specific methods
to properly quantify Juveniles (e.g.. anesthetics (Prince & Ford
1983); sub-aquatic amplifiers (Shepherd & Turner 198.3); suction
method (McShane & Smith 1988). Differences in efficiency
among divers were surely low. since only on-duty abalone dixers
participated, and they are highly efficient locating abalone (Prince
& Gu/.man-del-Proo 1993). Additionally, the use of quadrants and
di\ ing transects prevents over estimations and competition among
divers (Findlay & Willerton 1996. McShane 1996). Shepherd et al.
(1998) stated that Mexico is the only country making serious ef-
forts to quantify the abundance of its abalone banks; nevertheless,
surveys are frequently criticized by local cooperatives and aca-
demics.

TABLE 4.

Paired comparisons of the percentage of recruits of H. fulgens and
H. corrugata belween surveys and their statistical power.

Pair

iA
isons

H.

fulgens

H. corrugata

Compar

Z-value

Povier

Z-value

Power

1989 vs.

1991

0.21

0.96

0.73

0.86

1989 vs.

1993

1.03

0.76

1.70

0.60

1989 vs.

1995

0.06

0.62

4.23*

0.99

1989 vs.

1996

1.40

0.68

0.42

0.92

1989 vs.

1997

0.24

0.94

0.72

0.85

1989 vs.

1998

1.70

0.60

1.26

0.70

1991 vs.

1993

0.62

0.81

0.86

0.84

1991 vs.

1995

0.21

0.95

3.62*

0.95

1991 vs.

1996

0.93

0.81

0.40

0.91

1991 vs.

1997

0.11

0.95

1.96*

0.55

1991 vs.

1998

1.05

0.71

2.42*

0.71

1993 vs.

1995

0.60

0.87

2.86**

0.83

1993 vs.

1996

0.22

0.95

1.70

0.60

1993 vs.

1997

0.89

0.74

3.34**

0.91

1993 vs.

1998

0.01

0.94

3.70**

0.96

1995 vs.

1996

0.74

0.90

4.91**

1.00

1995 vs.

1997

0.01

0.96

6.50**

1.00

1995 vs.

1998

0.89

0.82

6.60**

1.00

1996 vs.

1997

1.55

0.60

1.90

0.55

1996 vs.

1998

0.26

0.91

2.43*

0.73

1997 vs.

1998

1.61

0.63

0.66

0.88

* = P < 0.05.
** = P < 0.01.

Interpretation of Abalone Dynamics

Both species reflect the decline of the Mexican abalone fishery.
observed since 1984 (Guzman-del-Proo 1992; Shepherd et al.
1998). and their abundance patterns are similar to those reported
for Isla Natividad (Shepherd et al. 1998). Their most recent density
levels are close to values where recruitment failures may occur
(Shepherd & Partington 1995) and a clear tendency to reco\er is
still absent.

Vega et al. ( 1997) and Shepherd et al. ( 1998) proposed causing
factors for the decline. Here we discuss the local role of some of
these as follows: (a) overfishing of legal sizes and/or recruits; (b)
negative effect of "El Nino" events over recruitment (indepen-
dently of their intensity); and possible increases of recruitment
during non-severe "El Nino" events; (c) physiological weakening
durino "El Niiio" events.

Overfishing of Legal Sizes and/nr Recruits

Overfishing can happen by fishermen cooperatives not respect-
ing authorized fishing quotes, poaching, cooperatives fishing at
erroneously calculated quotes, or the mixture of all of them. The
first two choices have not occurred at Islas San Benito, since the
local cooperative has been exemplarily adhered to the regulatiims
and authorized quotes. Additionally, the location of the islands,
and strict surveillance programs applied to owners and strangers
resulting in rare poaching. The absence of significant temporal
changes in the recruits of green abalone support our suppositions,
and although that of pink abalone changed significantly, there is no
reason to believe that only this species has been affected by poach-
ing or recruitment overfishing. Changes in the percentage of re-
cruits of pink abalone may be related to climatic changes, (see
later). It seems that fishing quotes were eiToneously estimated,
since densities extracted by fishing remained constant, while the
abundance of the legal-sized stock decreased significantly in both
species.

Catches of green abalone have been higher than that of pink
abalone. at Islas San Benito (Fig. 4). Pink abalone was the dom-
inant species in the local catches between 1957 to 1984 and green
abalone became dominant until 1985 (S. A. Guzman-del-Proo.
pers. comm.). Nowadays, green abalone is preferably fished for
having a higher proportion of harvestable sizes and being more

168

Rodriguez-Valencia and Caballero-Alegri'a

a)

o

C

o
o

c

CC

b)

c)

d)

Haliotis fulgens

Mali Otis corrugata

1.4

0.8

n=471

-3:

n=65

n=137

|n=93

I

•I'

n=97r

n=62

n=115

,x. '■

f^= 0.53
b= -0.034 (F(, .5)=7.9; p<0.05)

0.8

b=-0.02(F,i.

0,70
5,=15.1;p<

0.05)

n=268

n=110

n=109

n=59

"X-

- i-

-^ n=159

.::

n=103

n=44

--

1989 1991 1993 1995 1996 1997 1998 ' 1989 1991 1993 1995 1996 1997 1998

Year

c

O 1.5

§ 1
O

i 05

/^= 0.94
b=-0.2(F,, 5)=75.9;p<0.01)

1.5

0.5

Year

i^= 0.93
t)=-0.15(F|i.s)=45.5;p<0.01)

18 19 20

Temperature (°C)

21 18 19 20

Temperature (°C)

21

50

0)

s

§ 25
O

q5

CL

i^= 0.60
b=0.78(F(i 4,=6.1;p>0.05)

50

25

r^=0.74
t)=0.86(F,i.4)=11.5;p<0.05)

2 0

Mean condition

50

Q)
O)
CO

^ 25

O

0)
Q.

i^= 0.56
b=-0.74(F,i,4,=5.1;p>0.05)

50

25

Mean condition

i^= 0.80
b=-0.89(F,i,4,=16.6:p<0.01)

18 19 20

Temperature (°C)

21

19 20

Temperature (°C)

21

I eriipeidiuic V ^; ' > ^ '

Fiaure ^ (a) Temporal patterns of the mean condition factors. (») represents the number of analv/.ed organisms; (b» Relationship bet«
condition factors and mean annual surface temperatures; (c» Relationship between the percentage of sexual mature organisms .
condition factors; (d) Relationship between the percentage of sexual mature organisms and mean annual surface temperatures.

een mean
and mean

revenue yielding by having larger sizes and shells that are more
valuable. Fishing has been surely more important tor the decline ot
green abalone than for that of pink abalone.

It would be desirable to have estimations of mortality by fish-
ing, but this is out of our reach. We can only speculate that they
would be lower than those at Isla Natividad (Shepherd et al. 1998),

since captures of both species have been much higher at Isla Na-
tividad (Fig. 4). but Isla Natividad and Islas San Benito have
similar density levels. Additionally, quotes at Isla Natividad have
been fixed considering an extraction of 30% of the legal-sized
stocks (Shepherd et al. 1998) and this percentage at Islas San
Benito has been lower.

Fluctuations of H. fulgens and H. corhi'gata

169

TABLE 5.
Sex ratio ( 9 : c? ) of //. fulgens and H. cornigata. at Islas San Benito.

Surve>

H. fulgens

H. cornigata

1989
1991
1993
1995
1996
1997
1998
Temporal variations

1:0.8

1:0.7

1:0.7

1:1.3

1:1

1:1

1:1.5

= 20.7*

1:1

1:1

1:0.9

1:1

1:1.5

1:1.5

1:0.9

S.5

"" P <0.Q\.

Negative Effect of "El Nino" Events over Recruitment (Independently
of their Intensity); and Possible Increases of Recruitment During
Non-severe "El Nii'io" Events

According to Vega et al. (1997), increased water temperatures
during "El Nifio" events reduce the extensions of Icelp beds and
negatively affect the recruitment, because larvae are taken away
from the banks by the currents. The frequent occurrence of "El
Nifio" events during the last decade (Fig. 5) should have continu-
ously affected the recruitment, but our results of green abalone do
not support this. The recruitment patterns of pink abalone support
the hypothesis about a possible positive effect of non-severe "El
Nino" events (Shepherd et al. 1998). Increasing recruitment was
detected until 1995. because between 1981 and 1988 larvae settled
under the influence of non-strong "El Nino" events (Fig. 5). but the
1981 to 1986 "El Nifio" was one of the strongest ever observed
(Amtz & Fahrbach 1996). Decreasing percentages were detected
after 1996. because after 1989 larvae settled under the influence of
frequent strong "El Nirn)" events (Fig. 5). It seems that the "El
Niiio" events determine a positive or negative effect over recruit-

ment, but differences between species suggest that this could be
species-specific. Data series longer than a decade are still neces-
sary in order to have evidence that is more reliable.

Physiological Weakening During "El Nii'io" Events

Vega et al. (1997) and Shepherd et al. (1998) suggested that the
reduction of food during warm periods could have lead to poor
physiological condition and greater susceptibility to predation. The
association between decreased physiological condition in both spe-
cies and increased water temperatures support this. Local fisher-
men affirm that the local extensions of kelp beds declined due to
the incidence of warm events, but we have no data to prove this.
Kelp beds are also exploited by the local chemical industry, but
records about the crop are rare.

The relationship between impoverished physiological condition
and reduced proportions of sexually mature organisms was sig-
nificant for only one species and suggestive for the other. Again,
longer time series are needed. The remarkable peak in maturity
detected in 1995 could have been produced by delayed spawning
and/or shift in the reproductive season. Changes in the sex ratio of
green abalone could be a by-product of changes in physiological
condition, but it is hard to find a clear explanation. Guzman-del-
Proo (1992) also reported inter annual variations and deviations
from the normal 1:1, in favor of females, in both species at other
locations. This indicates that the sex ratio in abalone is also a
dynamic parameter (Table 6).

Finally, as Shepherd et al. ( 1998) proposed, climatic variability,
represented in our case by the surface water temperature, contrib-
uted to the decline of the populations by their weakened physi-
ological condition. These effects have been underestimated, and
although the environmental effect of sea temperature anomalies is
considered in using the modified version of the Schaefer surplus
production model (Shepherd et al. 1998). it seems that this caution
has not been enough. Fluctuations in physiological condition
should be taken into account when deciding the fishing quotes.

300

250

«■ 200
<s>

c
o

t 150 -

O 100

50

H. fulgens Islas San Benito (This work)

^ -H. corrugata Islas San Benito (This work)
■••»■■ H. fulgens Isia Natividad (Shepherd et al. 1998)
•x- H. corrugata Isia Natividad (Shepherd et al. 1998)

■jfc^.-^iP".-'^'^--.ijji_ _

— 1 r-

"b^ "b^ -^ <b* -^ "^ <^ Ci^ Ci^ ^ ^ qj» 4" 4" 4^

•& <&> <^ 4> <§"' <^' %*' <§>' 4"' 4-' cF 4^ q^ <P 4"

Fishing season
Figure. 4. Captures of Haliotis fulgens and H. corrugata.

170

Rodriguez-Valencia and Caballero-Alegri'a

Year

1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998

Month
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

-A

„EI Nino" weak

„EI Nino" moderate

„EI Nino" strong

„La Nina"

Witliout influence

A

-A

-A

A

Survey

Figure. 5. Occurrence nf warm and cold events during 1981 to 1998 ifrom htt|)://\v««.cpc.ncep.noaa.gov/producls/analysis_nionitoring/
ensostuff/ensovears.litml ).

since they are fixed considering only abundance estimations and de Ahuiiin" For their help and resources offered during the sur-

negotiation (Ponce-Diaz et al. 1998; Shepherd et al. 19981. veys. especially to D. G. Romero-Arce. We appreciate the sup-
port of F. Uribe, J. Cordova, J. L. Rivera, J. Castro. R. Sanchez.

ACKNOWLEDGMENTS L. Velez. M. Navanete. J. Talavera. and O. Pedrin-Osuna dur-

We dedicate this work to the memory of our colleagues ing the surveys. We thank S. A. Shepherd. S. A. Guzman-del-

F. Salgado. G. Perez, and A. Lelevier. We thank all members Proo. G. Davis, M. Strasser, and K. Reise for their valuable sug-

and directors of the cooperative "S.C.P.P. Pescadores Nacionales gestions.

TABLE 6.

Percentage of sexual mature organisms of H. ftilgeiis and H. cornigata, at Islas San Benito

Survey

H. fiilgeiis

H. cornigata

1989
1991
1993
1995
1996
1997
1998
Temporal variations

54.3

44.4
193
96.0

5.3

3.2

4.9
= 206.7*

37.6

22.2

19.5

92.9

7.7

0.0

5.0

;3i.5*

21S.9
31.3
16.7
85.2

7.5

3.0

0.0
= ZOS.O'-

16.0
28.4
13.6
77.8
24.3

9.1

0.0
= 160.4*

P <0X)\.

Fluctuations of H. fvlgens and H. corrvgata

171

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Joimud of Shellfish Research. Vol. 21. No. 1. 173-183. 2002.

DEVELOPMENT OF THE NERVE GANGLIA OF ABALONE, HALIOTIS ASININA LINNAEUS

M. KRUATRACHUE.' P. LAIMEK." C. WANICHANON," V. LINTHONG,^ P. SRETARUGSA,"
E. S. UPATHAM,' AND P. SOBHON"

' Departiuenr of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand:
-Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand;
^Department of Biology, Faculty of Science, Mahidol University', Bangkok 10400 and Department of
Medical Science, Faculty of Science, Burapha University, Chonburi 20131. Thailand

ABSTRACT The development of cells in the ganglia during various ages of the ahalone, Haliolis usinina. was studied. There were
three types of neurosecretory cells (NS|_,). four types of neurons (NR|_,) and three types of neuroglia (NG,_,). In the cerebral ganglia,
NS| and NR, (giant neurons) first appeared in 1-month-old abalone while NS, and NS, first appeared in 3-month-old and 4-month-old
abalone, respectively. These cells increased in number in 5- and lO-month-old abalone. reaching a maximum number at 12 months,
and thereafter remained constant. In the pleuropedal ganglia, NS, and NR, first appeared in 1-month-old abalone, while NS, and NS,
first appeared in 2-month-old and 4-month-old abalone. respectively. They increased in number in 4- and 7-month-old abalone,
reaching a maximum at II months, and thereafter remained constant. In the visceral ganglia, NS, and NR, first appeared in
2-month-old abalone while NS, and NS, appeared later in 3-month-old and 5-month-old abalone. respectively. They increased in
number at 4 months, reaching a maximum number at 1 1 months, and thereafter remained constant. NR,, NR,. NR_, and NG were
present in all ganglia early in development from one inonth onwards, and their numbers increased rapidly with age.

KEY WORDS: development, nerve ganglia, abalone, Haliolis asinina

INTRODUCTION

During neurogenesis of gastropods, the central ganglia of gas-
tropods arise by proliferation and later delaminalion and/or invag-
ination of the ectoderm. Cell division continues in the peripheral
proliferative zones throughout embryogenesis, and post-mitotic
cells then migrate inwardly to join the central ganglia which are
formed nearby (Jacob 1984). Gangliogenesis in gastropods pro-
gresses from anterior to posterior with the cerebral ganglia devel-
oping first, followed by the pedal ganglia and then the more pos-
terior ganglia of the abdominal loop (Kerkut & Walker 1975). The
pattern of neurogenesis in the gastropod central nervous system
resembles the proliferation of cells in the neural tube and the
migration of the neural crest and ectodermal placode cells in the
vertebrate nervous system but differs from the pattern described
for other invertebrates (Jacob 1984).

Although the nervous systems of more derived species of gas-
tropods as well as the development of neurons and individual
transmitter systems have been studied (Lever et al. 1965; Cogge-
shall 1967; Kerkut & Walker 1975; Van Minnen and Sokolove
1984: Roubos et al. 1988; Carroll & Kempf 1994; Kruatrachue et
al. 1994; Keinpf et al. 1997; Marois & Carew 1997), the nervous
development of prosobranchs has not been investigated in detail.
Most studies of ganglia development in gastropods were con-
ducted on opisthobranchs and pulmonates. In pulmonates. varia-
tion in morphology and lobulization of the ganglia is related to age
and development (Keikut & Walker 1975). Roubos et al. (1988)
studied the development of neuroendocrine centers of Lymiiaea
stagnalis (Linnaeus) and found that the dorsal bodies and light
green cells were already present in snails of 1 mm in shell length
and that the caudo-dorsal cells first appeared in snails of 3 mm in
shell length. The dorsal bodies and caudo-dorsal cells increased in
nuiTiber and size with increasing shell length. In Achatina fiilica
(Bowdich), the size of the ganglia and the number of nerve cells in
the ganglia increased with increasing age. The prominent nerve
cells in the ganglia were large cells and giant cells. The large cells
were already present in all ganglia of the newly hatched snails,
while the giant cells first appeared in 1-month-old snails. The

neurosecretory cells in the cerebral ganglia of A. fiilica first ap-
peared in 2-month-old snails and increased in number and size
with increasing age, reaching a maximum number in 8-month-old
snails and thereafter remained constant in 9- to 1 2-month-old
snails (Kruatrachue et al. 1994).

In general, the number of nerve cells in the ganglia of gastro-
pods increases with age (Kerkut & Walker 1975; Leveret al. 1965;
Coggeshall 1967). Coggeshall (1967) studied the opisthobranch
snail. Aplysia californica (Cooper), and found that, during its
maturation, the number of nerve cells in the ganglia increased by
40V(- and that the greatest number of large neurosecretory cells
occurred in full grown animals. In the stylommatophoran snail.
Lima.x maxinms (Linnaeus), the morphology of the dorsal body
cells also changed during maturation (Van Minnen & Sokolove
1984). The neurosecretory cells were small and released little
secretory product in the immature and early male-phase animals.
In contrast, these cells became larger and released large amounts of
secretory product in the later female-phase animals (Van Minnen
& Sokolove 1984). To the best of our knowledge, there is little
information on the development of ganglia in prosobranch snails.
Hence, the aim of the present investigation was to study the de-
velopment of nerve ganglia of Haliotis asinina (Linnaeus), a com-
mon abalone species found along the coastal waters of Thailand.

MATERIALS AND METHODS

Ten adult H. asinina (average shell length 66.58 mm) (five
males and five females) were obtained from the Marine Biological
Station. Chulalongkom University. Chonburi Province. Thailand.
They were relaxed with 5% MgCL for 3^ hours prior to dissec-
tion. Dissections of the nervous system were done under an Olym-
pus stereoscopic binocular tnicroscope with a fiberoptic dissecting
light and drawings were made with the aid of a camera lucida.

To study the histological development of the nervous system of
H. asinina. adult abalone and those from 1 to 12 months old were
obtained. Ten animals from each age class were examined. They
were relaxed and the cerebral, pleuropedal and visceral ganglia
were dissected out and fixed in Bouin's fluid in 0,14 M NaCI for

173

174

Kruatrachue et al.

24 hours, and washed with 70% ethyl alcohol. Then, they were
dehydrated through a graded series of ethanol. cleared in dioxane.
infiltrated and embedded in paraffin. Serial frontal sections of 5
(xni thickness were cut and alternating sections were stained with
hematoxylin and eosin. chronie-hematoxylin-phloxine (Gomori
1941) and paraldehyde-fuchsin (Gomori 1950). Sections were ex-
amined under an Olympus Vanox light microscope. Measurements
(width, length and thickness) of the ganglia were taken from the
median frontal sections (10 sections per ganglion; 10 animals for
each age group). Neurons and neruosecretory cells in all ganglia
were identified based on their histological characteristics (cell size
and shape, nuclear size and shape) and staining affinities (Upatham
et al. 199S; Kruatrachue et al. 1999; Thongkukiatkul et al. 2000).
In addition, the numbers of cells in each ganglion of each age
group were counted. For each animal, the cell count was done on
the median frontal sections (10 sections per ganglion). The cell
type and number were scored as follows:

Neurosecretory Cell (per section)

Neuron (

per section)

(-)

= 0 cell

(-1

= U cell

(+)

= 1-5 cells

( + )

= 1-10 cells

(++)

= 6-10 cells

(++)

= 11-20 cells

(+++)

= 11-15 cells

(+++)

= 21-30 cells

(++++)

= >I5 cells

I++++)

= >?0 cells

RESULTS

Figure 1 shows a diagraniatic drawing of the gross anatomy of
adult H. asiniini. The nervous system consists of a pair of cerebral
ganglia, a pleuropedal ganglion and a visceral ganglion.

Figure 2 shows the frontal sections of the cerebral (Fig. 2A),
pleuropedal (Fig. 2B) and visceral ganglia (Fig. 2C) in adult H.
asinina. There are 10 types of nerve cells in all ganglia of H.
asinina (Figs. 2D. 3A), i.e., three types of neurosecretory cells
(NS,_3), four types of neurons (NR,„,) and three types of neuro-
glia (NG,_,) (Upatham et al. 1998; Kruatrachue et al. 1999;
Thongkukiatkul et al. 2000). The NS cells were identified using
special stains, i.e. chrome-hematoxylin- phloxine (Gomori I '■Ml)
and paraldehyde-fuchsin (Gomori. 1950).

The shape, size, and type of cells and their number in ganglia
during various ages of developing abalone are summarized in
Tables 1 . 2 and 3.

Cerebral Ganglia

In 1 -month-old abalone, the cerebral ganglion appeared as an
elongated bean shape whose size was approximately 121 x 47 1 x
100 \xm (Fig. 3B). Most of the ventral, dorsal and lateral parts of
the ganglia had a thick cortex that contained 3-4 cell layers, while
the medial part contained only 0-1 cell layers (Table 1 ). NS, cells
first appeared in 1 -month-old abalone; there were 1-2 cells per
section. These cells were concentrated in the dorsal horn of the
ganglion (Table 1 ). Most types of neurons (NR, 4) were present,
but NR, were the most numerous. NR, and NRj were moderate in
number, while the NR, or the giant neurons were rarely found but
when present were usually located in the dorsal horn similar to NS
cells (Table 1). All types of NG were present but in a small
number.

At 2—1 months, the ganglia appeared bean shape similar to
those in I -month-old abalone. The number of cell layers increased
with age. The number of NS cells increased to about 2-5 cells per
section (Table 1 ). Most of these were NS,. while NS, were ob-

served in 3-month-old and NS, in 4-month-old abalone. Most NS
cells were concentrated in the dorso-lateral and dorso-medial, ven-
tral and ventro-medial parts of the ganglia (Table I ). NR cells were
similar in type and number to those in 1 -month-old abalone. NG
cells increased in number from one month onwards.

At 5 months, the size of the ganglion increased to 203 x 632 x
200 |j.m (Table 1 ). From 5 months onwards, the ganglion assumed
a sickle shape (Fig. 3C). The number of cell layers in the cortex
increased, especially in the ventral and dorsal parts. The number of
NS cells increased to about 10 cells per section, and although all
types of NS were scattered in all parts of the ganglia, most were
still concentrated in the dorsal and ventral horns (Table 1 ). The
number of NR increased with age. and the NR, count was ap-
proximately 11-20 cells per section (Table 1). They were present
in the dorsal and ventral areas. At this age, the number of NG
slightly increased.

From 6 to 10 months, the ganglia appeared sickle shaped but
were larger and more elongated than those of 5-iTionth-old abalone.
The cortex in all areas thickened and the quantities and distribution
of NS and NR cells were similar to those of 5-month-old abalone
(Table 1).

At 1 1 months, the cerebral ganglia increased in size to about
377 X 810 X 300 ptm (Fig. 3D). Other appearances were similar to
those of 5- to 10-month-old abalone. However, the numbers of NS
and NR, cells increased (Table 1 ). NG also increased with increas-
ing age. When abalone were 12 months old, their ganglia (377 x
901 X 325 p.m in size) were fully developed and appeared similar
in all aspects to those of the adult abalone (Fig. 2A).

Pleuropedal Gaiialia

111 1 -month-old abalone, the pleuropedal ganglion appeared
butterfly-shaped and about 189 x 418 x 150 (xm in size (Fig. 4A).
In the ventral and lateral parts of the ganglia, the cortex was thick
and contained 2-5 cell layers (Table 2). The remaining parts of
cortex were relatively thin. There were only about 1-2 NS cells per
section. These cells were confined to the dorsal-sulcus of the gan-
glion; most of them being NS, (Table 2). There were all types of
NR, but a few NR, and NR4 were present in the dorso-medial part
(Table 2). All types of NG cells were found in the ganglion at this
age.

At 2-3 months, the size of the ganglia increased from 273 x
497 X 155 to 289 x 522 x 170 |jim, but the shape was not altered
(Fig. 4B). The number of cell layers in the cortex. NS and NR cells
appeared to increase, and most cells were found in all parts of the
cortex. However. NR cells were concentrated in the dorsal and
dorso-lateral parts, while NS cells were concentrated in the dorso-
medial and lateral sulci (Table 2).

At 4-6 months, the pleuropedal ganglia was still butterfly-
shaped but increased in size from 337 x 556 x 200 |xm to approxi-
mately 488 X 707 X 250 p.m (Fig. 4B). and the cortex became
much thicker. The number of NS cells (mostly NS,) increased to
about 20 cells per section and a larger number were found in the
dorso-lateral and ventro-lateral parts (Table 2). NR cells increased
in number with increasing age and were found in the \entro-
medial. ventro-lateral and ventral sulci (Table 2).

At 7 months, the ganglia were H-shaped and increased in size
to about 544 x 1 6 1 5 x 3 1 5 p.m (Fig. 4C). The number of cell layers
increased and NS cell (mostly NS, ) number was about 30-40 cells
per section; these cells were distributed in all areas (Table 2). NR
cells (mostly NR,) increased in number in comparison to earlier
stages. From 8 to 10 months, pleuropedal ganglia were similar in
shape to those of 7-month-old abalone (Table 2).

Development of the Nerve Ganglia of Abalone

Cerebral commissure

175

Appendage tentacle
Appendage tentacular nerve- _

Cerebro-pedal connective ^ _
Cerebro-pleural connectives^

Osphradium nerve-

Osphradium-

Esophagus--"

Gill

Intestine-

Visceral ganglion

Digestive gland

Cephalic tentacle
Cephalic tentacular nerve

-Eye
- Optic nerve

% Cerebral ganglion

^i- — Buccal ganglion
.>>^. -Buccal nerve

■Vj-Pleuropedal ganglion

Epipodium tentacle

Pedal ner\e

^S^- -Epipodium
■=^ tentacular nerve

Pedal ganglion
Pedal nerve cord

Gonad

0-4cm

Figure 1. Diagramatic drawing of the gross anatomy of adult abalone showing the ganglia and their peripheral nerves (modified from Crofts,
1929).

At 1 1 months, the ganglia increased in size to 589 x 2508 x 470
|j.m; the ventral and dorsal horns were elongated (Fig. 4D). The
number of cell layers in the cortex increased. The number of NS
cells was about 60 cells per section (Table 2). NR cells and NG
cells were distributed in all areas; their numbers increased with
increasing age. At 12 months, the pleuropedal ganglia (589 x 2543
X 500 p.m in size) were fully developed and appeared similar to
those of the adult abalone (Fig. 2B).

Visceral Ganglia

In 1 -month-old abalone. the visceral ganglion was as small as
37 X 72 X 30 p.m and bean-shaped (Fig. 5A). The cortex had only
one layer of cells (Table 3). NS cells and NR, cells had not yet
appeared. In contrast, the remaining types of NR (NR^j) were
present but still few in number (Table 3). All types of NG were
observed.

From 3 months onwards, the ganglion was dumbbell-shaped
(Figs. 5B-5D) and its size increased with increasing age (Table 3).
The cortex was thicker, especially the lateral part. NS, cells first
appeared in 2-month-old abalone, and their number was about 1-2
cells per section (Table 3). They were present in the left lateral, left
lalero-dorsal and left latero-ventral parts. There were all types of
NR, but NR, and NRj were rarely observed.

At 3-10 months, the visceral ganglion increased in size from
1 18 X 488 X 50 (xm to about 160 x 770 x 110 ixm (Fig. 53). The
number of cell layers in the cortex increased. The number of NS
cells increased to about 20 cells per section, and they were dis-
tributed in the right lateral part (Table 3). NR cells were similar in
number and distribution to those at 3 months (Table 3).

At 1 1 months, the ganglion ( 160 x 889 x 1 10 \xm) increased in
length, but still had a similar width to that of 10-month old abalone
(Fig. 5C). NS cells (mostly NS,) increased in number and were

176

Kruatrachue et al.

Figure 2. Photomicrographs of the frontal sections of ganglia of H. asinina. I A) A low-power micrograph of a cerebral ganglion, showing thick
cell layers on the ventral (\ ) and dorsal (D» sides. Ca-capillary. t'o-cortex, 1,-lateral. M-medial. Me-medulla. Mu-niuscle. (Bl A low-power
micrograph of a pleuropedal ganglion showing thick cell layers on the dorsal (Hi and lateral (I,) sides. Co-corte\. M-medial, Me-medulla,
V-ventral. (C) A low-power micrograph of a visceral gangli(m showing thick cell layers on the ventral side. 1,1. -left lateral, Rl. -right lateral. (J-
gill, Co-cortex, Me-medulla. (D) .\ high-power micrograph of a pleuropedal ganglion showing various types of nerve cells in the cortex region.
NGi-type 2 neuroglia, NG,-type 3 neuroglia. NS,-type 1 neurosecretory cell, NS,-type 2 neurosecretory cell, NS,-type 3 neurosecretory cell.

distributed in all areas of thie ganglia (Table }<). but were concen-
trated mostly in the right lateral part (Fig. 5D). At 12 months, the
ganglion ( 163 x 939 x 150 jxm in size) appeared similar to those
of adult abalone (Fig. 2C).

DISCUSSION

During postembryonic development of the nervous system of
gastropods, the nerve cells and neurosecretory cells increase in size

Development of the Nerve Ganglia of Abalone

177

NR2-|PSV^

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cell (NS,). (B) Frontal sections of cerebral ganglia in I- to 4-month-old abalone that appeared bean-shaped. (C) In 5- to l(l-nionlh-old abalone,
the ganglion appeared as a sickle shape. iDl In ll-month-old abalone, the ganglion greatly increased in size. D-dorsal, L-lateral, M-medial,
V-ventral

and number (Bullock & Horridge 1995). for example, the neuro-
endocrine cells of L. stagiuilis increase in number and size with
increasing shell length (Roubos et al. 1988). In addition. Lever et
al. (1965) showed the same result in the cerebral and parietal
ganglia of Bioinphalaria i;lahralu (Say). Similarly. Kruatrachue et
al. (1994) reported that the number and size of neurosecretory cells
in the cerebral ganglia of A. fiilica increased with increasing age.

A similar trend was observed in H. asinina in the present stud\.
Furthermore, our histological study indicates that the cerebral,
pleuntpedal and visceral ganglia appeared as definite organs with
specific shapes in 1 -month-old abalone. Later there were changes
in the size but not so much shape of these ganglia; and the numbers
of neurons and neurosecretory cells in all ganglia markedly in-
creased with increasina aae.

178

Kruatrachub et al.

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Development of the Nerve Ganglia of Abalone

181

Figure 4. Low-ixiuir mkni<;r;iplis of frontal siiiiiins <ir pkiiropudal na[ij;lla. ( \i In I- Id i-month-okl abaloiu-, I Ik pliiiKipedal ganglion had a
butttrfly shape. (Bl In 3- to 6-month-old abalone, the ganglion still had a butterfly shape. (C) In 7- to 10-month-old abalone, the ganglion had
an H shape. (D) In II -month-old abalone, the ganglion was larger in size but still had an H shape. Co-cortex, D-dorsal, L-latcral, M-niedial,
Me-meduIIa, V-ventral

There have been many reports on the functions of neurosecre-
tory cells in the cerebral ganglia of snails. It has been found that
some snails have factors that stimulate growth rate (Geraerts &
Algera 1976) and shell regeneration (Dillaman et al. 1976).
Thongkukiatkul et al. (1998) reported that neurosecretory cells in
the cerebral ganglia of H. asinina were positively stained with
anti-human GH and anti-human insulin. In this study, it was ob-
served that the neurosecretory cells in the cerebral ganglia first
appeared in 1 -month-old abalone and increased in number with

increasing age. A large number of neurosecretory cells were found
in 5- and 10-month-old abalone that are assumed to be juvenile and
pre-adult stages, respectively. They formed ma.ximum numbers
that were observed and appeared adult-like in 12-month-old aba-
lone that reached the adult stage. It seems, therefore, that the
increase in the number of NS cells is correlated with the increase
in abalone growth.

Similar studies of other species of gastropods suggest that the
number and staining properties of neuroendocrine cells in plcu-

182

Kruatrachue et al.

1L i** '-'•'! J ji

Mu

..^M '-I

Figure 5. Frontal sections of \iscer;il ganglia. (A( In 1- to 2-nionth-old ahalone, the visceral ganglion "as small and had a bean shape. (B) In
3- to-10-month-old ahalone. the ganglion was large and assumed a dunihhell shape. (C) In t l-monlh-okl abalone, the ganglion increased only
in length. (0) High magnification of (C) showing thick cortex containing all types of nerve cells (arrows). D-dorsal. (iu-gut, LL- left lateral, RL-
right lateral, Mu-muscle, V-ventral

ropeUal and visceral ganglia are related to the gonadal maturation
(Coggeshall 1967; Dogterom et al. 1983; Van Minnen & Sokolove
1984; Smith 1967). In our study it was observed thai the neuro-
secretory cells in the pleuropedal and visceral ganglia first ap-
peared in 1 -month old-ahalone. The number of neui'osecrelory
cells in the pleuiiipedal ganglia inci'eased in 4- and 7-moiilli-old

abalone. while in the visceral ganglia it increased in 4-month-old
abalone. They reached a maximum number in 1 1 -month-old aba-
lone. The development of these neurosecretory cells may correlate
with the development of the reproductive organs. Thongkukiatkul
et al. (199S) leported that neurosecretory cells in the pleuropedal
and visceral uanalia of W. asiiiina were stained by anti-human LH.

Development of the Nerve Ganglia of Abalone

183

while only those in the pleuropedal ganylimi were stained by anti-
human FSH.

It was reported that early spermatocytes and spermatids of H.
asinina appeared at 4 months, and early oocytes (0C|_2) at 6-7
months (Sobhon et al. 1999). while fully mature spermatozoa ap-
peared in the gonads as early as 6-7 months. At this age. there
were already a large number of neurosecretory cells in the visceral
and pleuropedal ganglia of the abalone. Moreover. Sobhon et al.
(1999) showed that a large number of mature oocytes of the re-
productive cycle of//, asinina occurred at 10 to 11 months, the age
at which the neurosecretory cells in the pleuropedal and visceral
ganglia reached a maximum number and appeared adult-like. Our
observations were supported by Yahata ( 1973) who demonstrated
that the pleuropedal and visceral ganglia might produce and re-
lease factors that could induce spawning.

In the present study, the number of giant neurons (NR,) in-
creased following the development of the ganglia. In the cerebral
ganglia, they increased in number in 5- and 10-month-old abalone
that were assumed to be the juvenile and pre-adult stages. They
reached a maximum number and appeared adult-like in 12-month-
old abalone. NR, first appeared in the dorsal horn of the pleurope-

dal ganglia, later they were regularly found ui both dorsal and
ventral horns. Thus. NR, may proliferate relatively later than the
other types of neurons (NR-,_4) that are abundant at an early age.
This may be related to active movement due to muscular activity
exhibited by abalone, as they become older.

NR, were more abundant in the pleuropedal ganglia than in the
cerebral ganglia of H. asinina (Upatham et al. 1998). These cells
are very large, multipolar and pyramidal in shape. Compared with
the classification of neurons in the nervous system of higher ver-
tebrates. NR| are similar to such large motor cells as the ventral
horn motor cells of the spinal cord and Purkinje cells of the cer-
ebellum in vertebrates. As such, they may be involved in control-
ling and coordinating motor activities, especially that of the pedal
muscle.

ACKNOWLEDGMENTS

This research was supported financially by the Thailand Re-
search Fund (Senior Research Scholar Fellowship to Prasert Sob-
hon) and BRG/04/2343. We thank Dr. Padermsak Jarayabhand of
the Marine Biological Station, Chulalongkorn University. Chon-
biiri Province, Thailand, for providing the abalone specimens.

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Thongkukiatkul, A.. P. Sobhon, C. Wanichanon. P. Laimek. V. Anupun-
pisith. P. Jarayabhand. M. Kruatrachue & E. S. Upatham. 1998. Lo-
calization of GH-. insulin. FSH. and LH-liked hormones in the nerve
ganglia of Haliotis a.\inina Linnaeus. Chiangmal. Thailand: Proceed-
ings of the Fifth International Congress on Medical and Applied Ma-
lacology, December 27-30, 1998.

Thongkukiatkul. A.. E. S. Upatham, P. Sobhon. M. Kruatrachue. Y. P. Chil-
ramvong. C. Wanichanon. T. Pumthong and J. Nugranad. 20(X). Histo-
logical studies of the pleuropedal ganglion, visceral ganglion and pedal
cord ganglia of Haliotis asinina. J. Med. & Appl. Malacol. 1 0: 1 1 1 - 1 20.

Upatham. E. S., A. Thongkukiatkul. M. Kruatrachue, C. Wanichanon. Y. P.
Chitramvong, S. Sahavacharin & P. Sobhon. 1998. Classification of neu-
rosecretory cells, neurons and neuroglia in the cerebral ganglia of Haliotis
asinina Linnaeus by light microscope. ./. Shellfish Res. 17:737-742.

Van Minnen. J. & G. Sokolove. 1984. Galactogen synthesis-stimulating
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Yaliata, T. 1973. Induced spawning of abalone (Nordotis discus Reeve) injected
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Journal of Shfllfish Research. Vol. :i. No. I. 185-191. 2002.

INDUCTION OF SPAWNING AND EARLY DEVELOPMENT IN Fissurella picta
(MOLLUSCA: ARCHAEOGASTROPODA) FROM SOUTHERN CHILE

INGRID BAHAMONDES-ROJAS' AND MARTA BRETOS'

^Instituto dc Biologi'a Marina. Universidad Austral de Chile. Casilla 567. Valdivia. Chile:
'Departamento Ciencias Bdsicas. Facultad de Medicina. Universidad de La Froutera. Casilla 54-D.
Temuco. Chile

ABSTRACT Specimens of Fissurella [ucta were collected from the rocky intenidal coast near Valdivia. southern Chile, and used for
studies of spawning. Males responded to anificial stimuli more frequently than females. Potassium chloride was the most effective
agent for inducing spawning, whether injected into the mantle cavity, added to the seawater, or a gonad macerate, or combined with
a period of exposure to air. Injection of hydrogen peroxide or addition of prostaglandin to ultraviolet-irradiated seawater in the
experimental containers also induced males and females to spawn. Oocytes off. picta (mean diameter 178 |xm) were surrounded hy
two membranes, the vitelline and albumen membranes, and a gelatinous outer coat, which was lost a few minutes after spawning. The
first polar body was released 15-20 minutes after fertilization, and various cleavage stages were observed during the following 7 hours.
Trochophore larvae were observed after 15 hours and early veligers 21 hours after fertilization (temperature 17.5 ± TC, salinity
29-30'*t). The hatched veliger larva began torsion and a free-swimming mode of life after 40 hours. The results showed the possibility
to obtain the larval development in F. picta under laboratory conditions, providing a significant progress for its future culture.

KEY WORDS: development, spawning, gastropod, Fissurella. induction, reproduction

INTRODUCTION

Species belonging to the genus Fi.s.siirclla (Bruguiere 1789).
known locally as "lapas". sustain a multispecies artisanal fishery in
Chile. In spite of the economic impoilance of this resource, there
is very little published inforination about the biology of the genus.
During the last years, research on Fissurella species in Chile has
been directed to establish the scientific and technological founda-
tions to culture them, by studying their reproductive biology and
embryo-larval development. In this sense, trials of gamete release
under laboratory conditions have been conducted in view of cul-
ture. Spawning induction in F. cioiiitigi (Vega & Osorio 1995;
Vega et ai. 1996) has had relative success in males, by using low
concentrations of hydrogen peroxide. Huaqui'n et al. (1998) ob-
tained gamete emission in F. crassa by intravisceral injection of
KCI. The spawning induction methods used in F. picta (thermal,
electric, and osmotic shock) have not produced positive results
(Gonzalez et al. 1999). Considering the ecological (Moreno &
Jaramillo 1983) and economic (Bretos 1988) importance of F.
picta at southern Chile, the objective of this study is to obtain
gametes from F. picta Gmelin ( 1791 ) by various spawning induc-
tion techniques, and to describe embryonic and larval development
in this species, in a population from southern Chile. Such infor-
mation will greatly facilitate attempts to grow this species in cul-
ture, and contribute to efficient management of aquaculture sites in
the future.

MATERIALS AND METHODS

A total of 451 specimens of Fissurella picta were collected
from a wild population from the rocky intertidal shore at Playa
Rosada and La Mision. near Valdivia (39°48'S. 73°24'W; Fig. 1 ),
when gonads were fully mature, between October 1996 and De-
cember 1997. Only individuals of shell length greater than 40 mm

Corresponding author. Ingrid Baramondes-Rojas. Depanamento Ciencias
Basicas, Facultad de Medicina. Universidad de La Frontera. Casilla 54-D.
Temuco. Chile. E-mail: mbretos@ufro.cl

were taken, to ensure that most of them were sexually mature
(Bretos et al. 1988).

In the laboratory the shells were scrubbed and thoroughly
washed with filtered seawater to retnove epibionts. and the limpets
were transfen-ed to .30-liter plastic tanks containing continuously
aerated filtered seawater (0.45 |j.m). The tanks were maintained
under ambient conditions of photoperiod. temperature ( I0-20°C)
and salinity (29-30%r), according to the season. In order to keep
the temperature constant, the plastic tanks were placed in a 500-
liter fiberglass container filled with circulating, temperature-
controlled freshwater.

The experimental animals were fed ad libitum with the algae
Macrocystis pyrifera (Linnaeus) C. Agardh 1820 and Ulva lac-
tiica Linnaeus 1771. collected from the same place as the limpets.
These algae are known to support growth in F. picta (Castro &
Iglesias 1995). Occasionally Mazzaella laminarioides (Bory) Fre-
dericq 1993. was also added, because it forms part of the natural
diet of the limpet.

In order to determine the sex of the specimens of F. picta, a
sample (0.1 ml) of gonad tissue was removed by aspiration through
an intravisceral-pedal puncture. The sex of the individual is easily
established from the color of the gonad tissue (Bretos et al. 1983).

The stage of gamete development was determined by optical
microscopy and characterized as follows: oocytes mature or un-
dergoing vitellogenesis. presence of gelatinous coat, amount of
yolk, motility and appearance of sperms. The diameter (|jim) of the
oocytes was measured with an ocular tnicrometer (Carl Zeiss) in
4-7 females taken at random from each sample within the size
range collected. The counting of sperms and estimation of motility
was done with a blood-cell counting camera (Malassez).

Iitductiun uf Spawning

Limpets were maintained for 7 days in the laboratory, without
food for 24 hours before the following stimuli were applied:

Temperature Stimuli

The animals were submitted to water temperatures from 10 to
24°C in increments of 3''C (12-15-I8-21°CJ each 15-20 min.

185

186

Bahamondes-Rojas and Bretos

p

MISION

Figure 1. Map i)f N'aldivia reyiiin. showing the loiations (if (he t"(i
sites used to collect Fissurellu piclii during this study.

Chemical Stimuli

(a) Potassium chloride: an inlravisceral-pedal injection (0.5 ml
of 0.5. 10. 20, and 100 mM), or added to the seawater
(concentration 10 niM).

(b) Hydrogen peroxide: an intravisceral-pedal injection (0.5 ml
of a 50 mM solution) or added to the seawater (concentra-
tion 8 mM).

(c) Dopamine: an intravisceral-pedal injection (0.5 ml of a
2-mM solution).

(d) Dopamme plus hydrogen peroxide: added to the seawater
(concentrations 2 niM and 8 niM. respectively).

(e) Dopamine plus seawater treated with UV radiation: added
to the seawater (concentration 1 and 2 mM; irradiation
period 40 minutes).

(f) Synthetic prostaglandin (a postrol. PG): an intravisceial-
pedal injection (0.5 ml of a 0.04 mg/ml solution).

(g) Synthetic prostaglandin plus seawater irradiated with UV
light: added to the seawater (concentration 0.04 mg/iT)l;
irradiation period 40 minutes).

Fh>sical Stimulation

The ani)iials were exposed to air for 15-20 minutes.

Other Stimuli

A combination of chemical and physical stimulation was used,
tiigether with the addition of macerated gonad tissue to the sea-
water. Stimuli for the induction of spawning were applied to
groups of 4 to 10 limpets, males and females separately, each
group being maintained in 4 liters of filtered seawater gently cir-
culated (0.5 1/min).

Any liiTipet failing to respond to a spawning stimulus within
two or three days was replaced. The nonparametric Wilcoxon test
^P < level 0.05) was used to compare responses of male and
female limpets to the various spawning stimuli, using the program
Statistica (Windows version 4.2),

Ferlilizalion and Devclopiiient

After the initiation of spawning. i)idi\ idual limpets were placed
in small containers with 1 liter filtered seawater. Triplicate samples
of 1 ml were taken from each suspension of gametes. Oocytes were

TABLE 1.
Effect of ditTerent inductors on spawnings in Fissurella picta.

Inductor

No Successful

No Successful

No of

No of

Trials with

Trials

Individuals

Trials

Males

with Females

78

Temperature Stimuli

Temperature ( 10-24"C)
Chemical Stimuli

KCl (injection)

0.3 mM

10 mM

20 mM

100 mM

8 mM (seawater)

50 niM (injection)

Dopamine 2 mM (injection)
Dopamine -i- H^O-, (seawater)
Dopamine + seawater treated with UV

PG (injection)

PG (injection) + seawater treated witli UV

PG (seawater) -^ seawater treated with UV
Other stimuli

KCl (seawater) + macerated gonad

KCl (seawater) -i- exposed to air

Seawater treated with UV
TOTAL

Date

Oct 1^)96. Dec 19%. Feb 1997

22

3

0

0

Feb 1997. Apr 1997

40

5

3

1

Oct to Dec 1996, Oct 1997

16

-)

0

0

Oct 1997

16

2

0

0

Oct 1997

Dec 1996. Oct Nov Dec 1997

96

12

1

1

Jan to Apr 1997

16

->

1

1

Nov 1997

9

1

0

0

Nov 1997

5

1

0

0

Nov 1997

26

4

1

0

Nov 1997

24

3

1

0

Apr to Nov 1997

^2

4

0

0

Feb to Nov 1997

24

3

1

1

Nov 1997

24

3

2

1

Nov 1997

8

1

1

0

Nov 1997

15

->

0

0

Dec 1996, Oct to Nov 1997

451

57

13

5

Spawning and Early Development of F. picta

187

Figure 2. A. Oocyte in \i\(i ol I'issunlki picui Hith its membranes, at
the light microscope, am: albumen membrane; gc: gelatinous coat; o:
oocyte; vm: vitelline membrane. B. ■"Micropyle" inside the gelatinous
coat of the oocyte of Fissurella picla. am: albumen membrane; m:
micropyle; o: oocyte; vm: vitelline membrane; gc: gelatinous coat.

counted under a stereomicroscope. a mean value obtained, and the
total number of gametes calculated. Sperms were counted with a
Malassez camera.

Sub-samples of oocytes taken during spawning were fixed in
3% glutaraldehyde. washed in phosphate buffer and distilled wa-
ter, and mounted under cover slips for examination and photomi-
crography (Zeiss Axiomat microscope).

The remaining oocytes were screened (150 |jLm mesh), washed
several times in sterile filtered seawater and maintained in the
1-liter containers to await fertilization. The sperm suspensions
were also screened (45 )xm) before use to get fertilization, gamete
suspensions from individual limpets were used in ratio 10-15
sperms per oocyte.

After fertilization had been confirmed by the appearance of the
first polar body, each batch of eggs was washed several times in
sterile seawater to remove the excess of sperms, then transferred to
a 2 liter container of sterilized filtered (0.45 |j.m) seawater and kept
in darkness with constant aeration. Every 24 hours afterwards, two
thirds of the water was replaced. Temperature was maintained at
17,5°C ± V'C and salinity at 29-30%6. Replicate samples were
taken every 15 minutes for the first 3 hours after fertilization, every
30 minutes from 3 to 24 hours post-fertilization, and every hour
thereafter until the liberation of the larvae.

The embryos and larvae were screened with different mesh
sizes appropriate for their size. The samples were examined under
a light microscope (Olympus) and obtained from a camera (Sam-
sung NF-E80SN) which replaced one of the eyepieces of the mi-
croscope. Selected images were captured (ATI program), digitized
(BMP format) and processed by image analysis (Scion Image PC).

The criterion for determining the stage of development for any
given sample was that at least 60% of the individuals should have
attained the stage in question.

RESULTS

liidiiclion of Spawning

Data from the 57 spawning induction trials, undertaken princi-
pally in October and November 1997 are presented in Table 1.
More males responded than females (Wilcoxon test; N = 16; Z =
2.201; P < 0.05). Potassium chloride was the most effective in-
ducing agent (10 mM), whether administered by injection, added
to the medium with macerated gonad tissue, or combined with air
exposure. A combination of UV-in-adiated seawater with either
dopamine or prostaglandin (PG) also induced spawning in males.

On one occasion with hydrogen peroxide injection, and on
another with PG added to the medium together with UV-
irradiation of the seawater. as many males responded as females
did. Only once did a male spawn in response to PG. Dopamine was
not effective for inducing spawning in males, except when com-
bined with UV-irradiated water.

Characteristics of Gametes

Mature oocytes had two external membranes, the vitelline
membrane and the albumen membrane, outside which was a ge-
latinous coat (Fig. 2A). The gelatinous coat was often lost, but
when it was present the micropyle originating from the albumen
membrane was clearly visible {Fig. 2B). The albumen membrane,
which in Figure 28 had yet to expand, was derived from the oocyte
and had the appearance of a clear gelatinous fluid.

Oocyte diameter varied from 1 1 7-327 p.ni (mean 1 78 \^.m). but
those with a gelatinous coat reached a diameter of 385 p-in.

Fertilization and Development

During December 1996, October and November 1997, a total
of 10 trials of artificial spawning were successfully completed
(Table 2); these were of three types: (a) use of gamete suspensions
obtained by dissecting the gonads (OT) of 10 F. picta individuals;
(b) oocytes from dissected ovaries together with sperm from in-
duced spawning (01) of 10 animals; and (c) oocytes and sperm
both obtained from spontaneous spawning (SS) in 10 specimen.

The spawned oocytes of F. picta frequently required several
seconds before complete hydration and expansion of the albumen
membrane. During this time eggs were observed to be surrounded
by large numbers of sperm, and occasionally the sperm head could
be seen within the micropyle.

At 17.5"C. fifteen to twenty minutes after fertilization the first
polar body was visible, having the appearance of a small, translu-
cent, refringent granule (Fig. 3A). The polar body was located
immediately adjacent to the vitelline membrane within the ex-
traembryonic fluid of the albumen membrane of the egg.

After 90 minutes the first two holoblastic segmentation divi-
sions had given rise to four blastomeres of equal size (Fig. 3B,Ci.
The third oblique plane of division resulted in the fonnation o

188

Bahamondes-Rojas and Bretos

TABLE 2.

Fissurella picla. Stages of development obtained in laboratory at
17.5 ± rc and 29-30 9,, salinity.

Diameter

Fertilization

stages

Time

(fim)

OT

Egg

First minutes

180

OT

First polar body

15-20 min.

ISO

OT

2 Blastomeres

<60 min.

180

OT

4 Blastomeres

80-90 min.

180

01. s.s

4-8 Blastomeres

80-90 min.

180

OI

Blastula

4—7 hours

180

ss

Trochophore

15 hours

180

ss

Early veligers

21 hours

180

ss

Hatched veligers

40 hours

200

OT = Dissected ovary and testicle

01 = Dissected ovary plus sperm from induced spawnings

SS = Spontaneous spawning in females and males.

eight blastomeres (Fig. .^D). of which four were very large inac-
romeres. located at the vegetative pole of the embryo and contain-
ing yolk reserves, and the remaining four were small micromeres
located at the animal pole.

After 4 to 7 hours successive divisions of the blastomeres had
given rise to a blastula, a compact, multicellular sphere that gently
rotated within the membrane, suggesting the presence of cilia on
the e,\ternal surfaces of the blastomeres. Embryonic development
was highly synchronized, but became less so during the larval
stages. Fertilized eggs obtained by OT and 01 (Table 2 1 developed
only until blastula stage.

The trochophore larva, exhibiting a ring of prototroch cilia,
appeared 15 hours after fertilization, and was completely envel-
oped by the albumen membrane (Fig. 4A). Survivorship was about
5iV'c of fertilized eggs in SS trials (Table 2).

Larvae became an early veliger. turning round within the al-
bumen membrane. 21 hours after fertilization. Forty hours after
fertilization, the albumen membrane had disappeared and the ve-
ligers had emerged (Fig. 4B) to spend a short period moving
through the water column. The pretorsional larva exhibited the
protoconch, an expanded velum, the foot, and the rudimentary

P

s

B

50 (jm

50 pm

mac

50 Mm

Figure .V Kmbryonic development stages of Fissurella pichi within the albumen membrane. A. Fertilized egg where a polar body (pb» is visible.
B. Two-blastomere stage. Arrow shows remnants of micropyle in the albumen membrane. C. Kour-blastomere embryo. D. Embry o « ilh unequal
eight blastomeres. View from animal pole, mac: macromeres; mi: micromeres.

Spawning and Early Development of F. picta

189

Figure 4. Larval development stages in Fissiirella picta. A. Tro-
cliopliore larva. Arrov^ shows ring of prototrochal cilia. B. Pretor-
sional hatched veliger larva.f: foot; pr: protoconch; rrm: rudimtntarv
retractor muscle; v: velum; vm: vitelline membrane.

retractor muscle. The visceral mass was well developed and the
yolk reserves and velar cilia diminished.

About a 30^0% of embryos obtained by SS trials survived to
veliger stage. Development did not continue, and larvae ultimately
died.

DISCUSSION

tion methods tested (thermal, electric, and osmotic shock) by
Gonzalez et al. (1999) in F. picta did not produce positive results;
this report suggests that a combination of various inductive stimuli
should be required to obtain positive responses, as has been shown
in the present study.

Potassium chloride injections, either alone or combined with
other stimuli (exposure to air, gonad maceration), and hydrogen
peroxide induced spawning in F. picta. Potassium chloride acts at
the cellular level and has been extensively used to induce meta-
morphosis in marine invertebrate larvae (Yool et al. 1986; Baha-
mondes-Rojas & Tardy 1988; Bahamondes-Rojas 1990) as well as
for the induction of spawning.

Hydrogen peroxide induces spawning in gravid male and fe-
male abalones Haliotis rufescens and Nordotis gigantea. the
mechanism possibly being direct stimulation of the enzymatic syn-
thesis of prostaglandin endoperoxidase (Morse et al. 1977; Tanaka
1979; Hahn 1989). Our data support those of Vega et al. (1996).
who successfully induced spawning in male Fissiirella cumingi
with low concentrations of hydrogen peroxide (3 mM when added
to the medium, and 5 niM when combined with gonad macera-
tion).

It is also known that seawater irradiated with ultraviolet light
induces spawning in many mollusks (Kikuchi & Uki 1974; Ka-
gawa & Nagahama 1981). but our data are not in agreement.
Nevertheless, we found ultraviolet irradiation to be effective when
combined with PG or dopamine, presumably as a result of syner-
gistic action. Moss et al. (1995) induced spawning in Haliotis iris
with two agents, hydrogen peroxide and seawater irradiated with
ultraviolet light, the former being more effective.

The role of prostaglandin remains unclear, although its pres-
ence and biosynthesis have been established (Ogata et al. 1978;
Nomura & Ogata 1976). We found PG to be a successful agent for
inducing spawning in male F. picta, but in females the mechanism
may be different. Thus Martinez et al. ( 1996) suggested that in the
hermaphrodite scallop Argopecten purpiiiatits. dopamine and PG
may be involved in the release of oocytes.

The fact that most of the trials were successful only in males
may be attributable to different mechanisms or maturation rates in
male and female gonads. Our microscopic observations demon-
strated that gonad maturation was almost continuous in males,
whereas in females there was a latent period during the vitello-
genic phase.

One outcome from this experiment should nevertheless be em-
phasized. Spawning inducers such as KCl and H^O-, are inexpen-
sive, easy to use and are widely used for the control of reproduc-
tion in molluscs such as fissurellids.

Induction of Spawning

Sex and Gametes

The spawning stimuli used in this study were similar to those
used by other authors (Morse et al. 1977; Morse & Morse 1984;
Uki & Kikuchi 1984; Pechenik 1986; Martinez et al. 1996) to
obtain gainetes from various marine invertebrates. In the case of F.
picta. the maximum maturity condition in the gonads detected in
this study during the spring (September to December) would fa-
cilitate the spontaneous gamete release to get fertilization and de-
velopment in a more successful way.

Our results suggest that induction of spawning in F. picta was
successful, but more effective with males than with females. Simi-
lar data have been obtained in previous studies on F. maxima and
F. cumingi (Vega & Osorio 1995). The artificial spawning induc-

Fissiirella picta is an archaeogastropod which, like all fissurel-
lids, does not exhibit external sexual dimorphism (McLean 1984).
There is no evidence for hermaphroditism or sex reversal (Bretos
et al. 1983). The technique we used for determining the sex of each
specimen was non-invasive, successful and simple.

There have been few studies of the life cycle (e.g.. spawning,
fertilization, and age of sexual maturation) in fissurellids. Ward
(1966) described the reproductive cycle of F. barbadensis, and
demonstrated the presence of oocytes of 80-180 |j.m in diameter
(including gelatinous coat). This species is known to have a pe-
lagic phase of two to three days duration (Lewis 1954; Lewis
1960). In Diodora aspeia spawned eggs are 160-198 \xm in di

190

Bahamondes-Rojas and Bretos

ameler. williout the gelatinous coat (Hadtleld & Strathiiiann 1996).
In /•'. ni(i.\iiiiti (Bretos et al. 1983) the oocyte diameter lies between
120 and 2S0 (xni. and that of F. crassa is approximately 300 |xm.
excluding the gelatinous coal (Huaqui'n et al. 1998). Our values for
oocyte diameter in F. piclii are similar to the maximum values
recorded for other Chilean species. Nevertheless, differences ob-
served from tropical little sized species such as F. barhadensis and
Diodoni apertitm may be related to differences in reproductive
strategy e.g., time to reach sexual maturity.

In Diodoni aspeni the external gelatinous coat which envelops
the oocyte appears to break open only in response to a mechanical
action (Hadfield & Strathmann 1996), whereas the internal mem-
brane disappears as a result of the action of enzymes produced by
the larva. In Patella (Fretter & Graham 1962) the gelatinous coat
of the oocyte disappears a short time after spawning, as in F. picki
(this study).

The presence of a canal, the micropyle, in the gelatinous coat of
the oocyte, and the presence of sperm cells within it. has been
observed in F. crj.vwj (Huaqui'n et al. 1998). Diodoni aspeni (Had-
lield & Strathmann 1996) and F. picta (Fig. 2B of this article).

Fertilizution and Dcveliipiiwiil

Knowledge of larval development of Chilean fissurellids is
limited mainly to a few unpublished observations; all of them
obtained by spontaneous gamete release and fertilization at the
laboratory. Vega and Osorio (1993). and Vega et al. (1996) deter-
mined that the duration of the pelagic larval phase in F. cwningi is
3-5 days under controlled conditions ( 16-19. 8°C), whereas in F.
latiimirginata at 13°C the veliger stage and metamorphosed larva
occur 6-7 days after fertilization (Pereira & Quezada 1996).

According to Gonzalez et al. (1999), who also got spontaneous

gamete release and lertili/ation, the initial trochophore stage was
observed in F. picia at 72 hours, and swimming trochophore
hatched at 96 hours at IOC. In the present study at 17.5°C. tro-
chophores of F. picta were obtained \fi hours after fertilization
within the albumen coat, and veligers hatched out of this coat after
40 hours. Our data are not consistent with those of Gonzalez et al.
(1999). probably owing to the difference in temperature. It has
been described for Hatiotis and other species, that the teinperature
can hasten or delay development (Hahn 1989). and this has prob-
ably happened in F. picla.

The veligers which were liberated in this study showed mor-
phogenetic movements associated with torsion before settlement
and metamorphosis took place. In this regard, our observations
agree with those of Page (1997) for the archaeogastropod Hatiotis
kamtscliatkami. demonstrating cephalo-pedal and viscero-pallial
rotation in pretorsional larvae.

The fact that development of F. picta took place in the labo-
ratory during this study suggests the potential for future cultivation
of this species. Nevertheless, it will be necessary to elucidate the
principal biological factors and ontogenetic mechanisms required
to improve survival rates and to identify the conditions required for
settlement and growth of a large number of larvae.

ACKNOWLEDGMENTS

This research was supported by Canadian International Devel-
opment Agency, and Memorial University of Newfoundland
awards, and by the Direccion de Investigacion y Desarrollo, Uni-
versidad Austral de Chile (Grant F-96/0I ). We thank Dr. Ray
Thompson for translating this manuscript. Dr. Oscar Chaparro for
his comments on the manu.script. and Jaime Oyarzo for assistance
and help with photography.

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Journal of Shellfish Reseanh. Vol. 21. No. 1. 193-200. 2002.

TYRIAN PURPLE FROM MARINE MURICIDS, ESPECIALLY FROM PLICOPURPURA PANSA

(GOULD, 1853)

LUDWIG C. A. NAEGEL' AND CHRIS J. COOKSEY"

^Ct'iitro Interdisciplinario de Ciencias Marinas, lustitittoPoUtecnico Nacional La Paz. B.C.S. 23000
Mexico; '59 Swiss Avenue. Watford, Herts WD] 8 7LL. United Kingdom

ABSTRACT A review of the literature disclose.s that most inarine snails of the family Muricidae produce in the hypobranchial gland
a viscous secretion containing, besides mucus and biologically active compounds, minute amounts of chromogens. These chromogens
develop enzymatically and under the influence of light and o.xygen into a purple pigment known as "Tyrian Purple". "Royal Purple"
or shellfish purple. In the hypobranchial gland the enzyme purpurase is kept apart from the chromogens. so that no pigments are formed
under normal conditions. Different species of muricids produce different pigments, depending on the number and concentration of
different chromogens and on the varying light intensity and oxygen availability during pigment formation. The main pigments obtained
from the hypobranchial gland from muricids are indigoids. The pigment of P. pansa is mainly 6.6'-dibromindigo with smaller amounts
of 6-bromoindigo and 6,6'-dibromoindirubin. similar to that of A7»/r.v hrandaris.

KEY WORDS: "Tynan Purple", gastropoda, muricidae. Plicopurpuia pansa. hypobranchial gland

INTRODUCTION

A review of the hypobranchial gland of muricids. its secretions,
including "Tyrian purple", seemed justified in view of the growing
interest in natural dyes and marine products with pharmacological
properties.

The majority of purple producing marine snails belong to the
family of Muricidae and most, if not all. produce a colorless se-
cretion in the hypobranchial gland, which turns purple on exposure
to air and light (Fretter & Graham 1994).

In antiquity, the purple from the muricids Miire.x tninculits, M.
brandaris. and Purpura haemastoma was produced in the eastern
Mediterranean countries - now called, Crete, Lebanon, and Israel.
Through the Phoenicians the art of purple production was spread
from the Mediterranean to West Africa and Ireland (Jackson
1917). Purple dyes were used extensively by Egyptians and sub-
sequently by Greeks and Romans. In view of the enormous quan-
tity of marine snails needed to produce a minute amount of the dye,
the scarcity of the animals, and the high costs of production. Tyr-
ian purple was at that time a most expensive luxury article. In
addition there was the symbolic importance of purple as a sign of
royalty, power and wealth, and the belief that it could possess
magic and supernatural powers (Reinhold 1970). At that time it
was the only known fast vat dye. other than indigo. With the Arab
conquest of Palestine in 638 A.D., and finally with the fall of
Constantinople in 1453 A.D. the use of Tyrian purple became, with
a few exceptions, extinct in the Old World (Herzog 1919; Bom
1936b; Clark et al. 1993). Through archaeological studies it was
confirmed that during the Middle Ages on the west coast of France
the muricid Nucella lapillus was used as a source for purple (Gruet
1993). From the I6th to the 18th century the artisanal use of purple
for marking linen was widespread in Ireland. South Wales and
Cornwall, as well as in Scotland, France, Norway and other parts
of Europe (Cole 1685; Jackson 1917).

In Japan, the muricid Rapana bezoar was of importance in
ancient dyeing processes (Baker 1974). On the Japanese peninsula
Shima. professional seafood collectors stained their diving suits,
made of cotton, with the purple from marine snails believing that
it contained supernatural powers (Yoshioka 1974).

Corresponding author. Ludwig C. A. Naegel. E-mail: lnaegel@cibnor.mx

The use of muricids for dyeing on the pacific coast of the
Americas dates at least from pre-Columbian times. In the same
way as the now extinct Mediterranean purple industry the exploi-
tation of the dye of marine snails led also on the pacific coast of the
Americas to a product of high economic value.

Today, however, there is not much general interest existing in
Tyrian purple derived from marine snails, since similar pigments
can be obtained from synthetic substitutes at much less cost (Bom
1936c). However, two remarkable exceptions have to be men-
tioned: (a) the dark violet-blue tekhelet color, which is relevant to
Jewish religious rituals derived from the Mediterranean muricids
Murex erinaceus and M. trunculus. and b) on the Pacific coast
from Peru to Mexico, the hypobranchial secretion of the muricid
Plicopurpura pansa (Gould 1853) has been exploited since pre-
Columbian times by Indians for dyeing cotton yam, which until
now is subsequently woven into traditional dresses (Martens v.
1874. 1898; Schunck 1980a; Nuttall 1909; Jackson 1917; Bom
1936c; Gerhard 1964; Turok et al. 1988; Yoshioka 1974; Thomp-
son 1994; Garay 1996; Sandberg 1997). v. Martens (1898) pointed
out that the use of the pigments from P. pansa for dyeing in
Central America must have had a very long and pre-Columbian
tradition and were not brought by the Spanish conquistadors from
Europe to the New World. Its presence in archaeological textiles
and pictures confimied his finding.

The camivorous muricid Plicopurpura pansa (Gould 1853).
according to Kool ( 1993) conspecific with Purpura pansa (Gould
1853). inhabits intertidal rocks exposed to the open sea with high
impact waves. The range of P. pansa extends at the Pacific from
the north-west coast of Mexico (Baja California Sur) (Clench
1947; Keen 1971) to northem Pern (Pena 1970; Paredes et al.
1999).

Hypobranchial or Mucous Gland

Since the mid 1 8th century the hypobranchial gland of muricids
has attracted the interest of natural scientists, investigating its func-
tional role, and the astonishing production of Tyrian purple. Fretter
and Graham (1994) consider the main function of the hypobran-
chial gland to be a secretor of mucus for trapping and cementing
particulate matter sucked into the mantle cavity with the respira-
tory water current, prior to its expulsion.

193

194

Naegel and Cooksey

The hypobranchial or niuciis gland is an elongated epithelial
struclLire located in the dorsal mantle cavity between the gills and
recto-genital organs, immediately below the shell. In the gland
three distinct anatomical and functional areas have been described:
two lateral regions composed of eight distinct cell types, among
them many active secretory cells (mucocytes). ciliated on the ven-
tral surface, and possessing pores through which the mucus and
other secretory products are released into the mantle cavity, and a
central area where the formation of the '"purple" precursors takes
place and where secretory products accumulate prior to their re-
lease from the snail (Bolognani Fantin & Ottaviani 1981; Roller et
al. 1995). The purple precursors (tyrindoxyl sulphate) and the en-
zyme (arvl sulfatase = purpurase) that induce the transformation
of the purple precursors into pigments are only localized in the
median zone of the hypobranchial gland (Erspamer 1946) and are
kept separate, so that no reaction occurs. Mollusk purple as such
does not occur in the live animal, but it is formed during a se-
quence of chemical reactions from the secretions produced by the
animal. When the animal contracts vigorously the cells are mas-
sively liberated, burst open by mechanical or osmotic pressure, and
their contents dispersed into the mucus (Lacaze-Duthiers 1859).
These observations were later refined by the histological work of
Bernard (1890). who found a well-developed innervation in the
gland, suggesting a role in perception (Verhecken 1989).

The pharmacological action by extracts of the hypobranchial
gland was discovered by Dubois (1909), and he described for the
first time their toxic and paralyzing action in both wann- and
cold-blooded species. The secretion of the hypobranchial gland
from a large number of muricids contains, besides mucus, the
precursors of the purple dye, proteins (aryl sulfatase, purpurase),
and toxins and narcotizing agents, like serotonin (5-
hydroxytryptamine), murexine (urocanylcholine,), choline ester
and biogenic amines (Erspamer 1952: Erspamer & Benati 1953;
Whittaker 1960; Malaszkiewicz 1967; Huang & Mir 1971; Roseg-
hini et al. 1996; Shiomi et al. 1998).

The secretion from the hypobranchial gland of P. pcinsa can be
obtained by "milking"' without harming the annuals. It is a milky-
white liquid, which turns on exposure to air and light, at first
yellow, then greenish, bluish and finally purple ("Tyrian purple").
During personal field observations (unpublished) we observed that
P. pcinsa uses the secretion to immobilize prey {Nerira sp.. Lii-
lorina sp.) in the intertidal zone, and does not resort to drilling
through the shells of other snails. Additionally interesting to notice
is the fact that during the predation no purple color is formed on
the prey, despite the presence of oxygen and intense light radia-
tion.

The chromogens containing the hypobrancial secretions seem
to be purely incidental, and their functional role, if any, is presently
unknown (Clench 1947). The volume of secretion obtainable from
P. puusi.1 depends not only on the size and sex of the animals, the
time interval between the each "'milking", but also on the season.
Its production and use may be in proportion to the type of food the
snails feed on. From small animals of less than 2 cm shell length
can be obtained about 0.5 ml of secretion, from 5-6 cm large
animals up to 4 ml (Rios-Jara et al. 1994). It has to be kept in mind
however, that in this volume only a minute proportion consists in
the dye precursors.

Chemistry of Tyrian Purple Formation

Several preliminary studies on the chemical composition of the
pigments of P. pansa are available. The comparison between the

chemical composition of the hypobranchial secretion of other mu-
ricids will lead to a better understanding of the metabolic pathways
that lead to the final production of Tyrian purple.

Since the re-discovery by Cole (1685) of "Tyrian Purple" from
Nidfllii iPnrpiini} lapillus a number of researchers have worked
on the determination of the chemical composition of the secretion
of the hypobranchial gland. Most remarkable, considering the lim-
ited knowledge of organic chemistry at the beginning of the 19th
century, is the analytical work by Bartolomeo Bizio about the
origin and properties of Tyrian purple from the Meditenancan
muricids Murex tniiiciiliis and M. hniiuUiris (Ghiretti 1994). When
collecting the glandular secretion of the snails he made the impor-
tant observations, first, that as soon as the coloriess fluid is ex-
posed to light and air it becomes immediately yellow and greenish,
and soon afterwards it turns into deep emerald green, blue, deep
blue and finally reaches the purple color. Next, that during the
production of the purple dye, a highly odorous compound is re-
leased. In comparing the color differences between the purple
from Murex tninciihis and M. bnmdaris he discovered that they
are species specific. Bizio also determined that Tyrian purple is
a substance with chemical properties similar to indigo. Schunck
(1879) isolated and crystallized the pigment from the ""ink" of
Nucflhi (Purpura} lapillus. and determined the chemical proper-
ties. He called the pigment punicin. To obtain 7 nig of punicin
he extracted the hypobranchial gland of 400 animals, after which
he reports "'my patience was exhausted". Friedlander (1909)
isolated 1.4 g of the pure pigment from 12.000 hypobranchial
glands from Murex hrandaris. and showed that it was 6.6'-
dibronioindigo.

Recently, using ad\anced analytical methods, Fouquet (1970).
Baker and Duke (1973), Michel et al. (1992) and Koren (1994,
1995) among others, have confirmed that the major pigment from
all studied muricids is 6,6'-dibromoindigo.

Different species of muricids produce different color qualities
of the dye, depending mainly on the number and concentration
of the different chromogens. Fouquet (1970) found four differ-
ent chromogens in the hypobranchial gland of M. trunculus: I)
indoxyl sulfate, II) 2-methylthio-indoxyl sulfate. III) 6-bromoin-
doxyl sulfate, and IV) 6-bromo-2-methylsulfonyl-indoxyl sul-
fate, and he described the chemical pathway leading to Tyrian
ptirple: The first step in the purple production is hydrolysis of
the sulfate group with purpurase (aryl sulfatase). Indoxyl sulfate
(I) and 6-bromoindoxyl sulfate (III) are then oxidized by oxygen
to give indigo and 6,6'-dibromoindigo, respectively. With 2-me-
thylthio-indoxyl sulfate (II) and 6-bromo-2-niethylsulfonyl-
indoxyl sulfate (IV) oxidation is followed by dimerisation and the
dimer is photolysed in light to give indigo and 6.6'-dibromoin-
digo respectively together with methanethiol or dimethyl disul-
fide. These reactions as described by Foquet (1970) are shown in
Figure 1 .

At the time of Fouquet's studies the possibility of cross-
coupling of the indoxyls which accounts for the large percentage
of 6-bromoindigo in the pigment of M. rruiuuluswdn was un-
known.

The composition of the chromogens of other muricids is less
complicated. Thais clavigera. T. hronnii. Dicathais orbita. M.
brandaris and N. lapillus contain 6-bromo-2-methylthio-indoxyl
sulfate (IV); M. erimiceus contains a single different chromogen
and Purpura luwmasuiuui and Rapana bezoar contain two other
different chromogens, but the chemical structures are not known
(Baker 1974; Hiyoshi & Fujise 1992). The reaction pathways of

Tyrian Purple

195

(i) (ii)

(I) —^ indoxyl -> indigo

r

(Hi) (ii) 2-

<— dimer 1 <— methylthio-

indoxyl

l/ 6-bromo- (iii) dimer 3 <^ -^^""^^
J\ indigo <- <]

ni

(i)

(II)

(i) ^ (ii) 6,6'- (iii) dimer 2 (ii) 6-bromo-2- (i)

(III) -^ 6-bromo- -> dibromo- <— <— methylsulfonyl- <— (IV)

indoxyl indigo indoxyl

Reagents: (i) aryl sulfatase, (ii) oxygen, (iii) light.

oso.

X=H, Y=H, (I) indoxyl sulfate

X=H, Y=SCH3. (II) 2-methylthio-indoxyl sulfate

X=Br, Y=H, (III) e-bromandoxyl sulfate

X=Br. Y=S02CH3, (IV) 6-brofno-2-methylsulfonyl-indoxyl sulfate

Figure 1. The chroniogtns from Miirex iriiiiculiis and their reactions to give indigoid pigments (Kouquet, 1970).

6-bromo-2-methylthio-indoxyl sulfate (tyrindoxyl) to give indi-
goid pigments are shown in Figure 2.

The composition of the different chromogens is not only de-
pendent on the species of muricids. but also environmental, and
physiological condition of the animals. The light intensity and
oxygen availability also play a role during pigment formation.
According to historical reports the best seasons to exploit the
purple snails in the Mediterranean are autumn and winter. During
summer the animals are hidden and in spring they lay eggs, at
which time the hypobranchial secretion presumably is losing its
coloring power and is not suited for color production (Bom 1936:
Cardon & du Chatenet 1990). Fouquet (1970) cites Schaefer (1941
"Neuere Ansichten iiber den antiken Purpur" Chemiker Zeitung.
273) and O. von Fiirth (1903 "Vergleichende chemische Physiolo-
gie der niederen Tiere". Verlag G. Fischer. Jena, page 377) who
stated that there are seasonal changes in the chromogens of snails
from the "trunculus" and "brandaris" types, due to age. gender, and
food.

Chemistry of Tyrian Purple from P. pansa

For more than a hundred years the chemical composition of
"Tyrian Purple" from P. paiisa has attracted the interest of chem-
ists. Edward Schunck (I88()a) obtained a sample of cotton yarn
dyed on the west coast of Nicaragua with the extract of Purpura
patuhi (now P. pansa). From 24 g of dyed material he obtained 99
mg of pure crystalline pigment with all the properties of punicin.
which he had earlier obtained from Purpura capillus (Nucella
lapillus) (Schunck 1879). Thirty years later it was shown by Fried-
lander (1909). that Schunck's punicin was 6.6'-dibromindigo. In
1922 Friedlander obtained from Mexico a sample of yarn dyed
with the excretion of P. aperta (the zoological description has to be
P. pansa. since P. aperta does not occur on the Pacific coast of the

Americas). The analysis of the dye showed no differences in solu-
bility, color, and absorption spectrum between the dye from M.
brandaris. which he had analyzed before and for this reason he
concluded with some certainty the dye of P. pansa consists mainly
of 6.6'-dibromoindigo (Friedlander 1922).

Saltzman ( 1992) showed that the reflectance spectrum of cloth
dyed with the "ink" of P. pansa had a maximum absorption at 520
nm. Very similar results obtained Withnall et al. (1993) and Clark
et al. ( 1993) for synthetic 6,6'-dibromoindigo. Mass spectrometry
was used by McGovern et al. (1991) to confirm that the major
colorant of the cotton sample from Saltzman. was 6.6'-
dibromoindigo with traces of 6-bromoindigo. It was found that
direct introduction of the cotton sample into the mass spectrometer
led in addition to the detection of large amounts of 6-bromoindigo
also to some indigo. This artifact arose from interaction of the
cotton fiber and 6.6'-dibromoindigo at the high injection tempera-
tures, leading to debromination and the formation of indigo. The
problem can be avoided by extraction of the dye from the fiber
with hot quinoline. or with dimethyl sulfoxide (McGovern et al.
1991). Using this extraction methodology, it was observed that
apart from the major 6.6'-dibromoindigo component there were
also smaller amounts of monobromoindigo and indigo which have
been previously obscured by other low molecular weight materials.

The analytical technique of choice for the characterization of
mixtures of indigoid dyes is the application of the High Perfor-
mance Liquid Chiomatography (HPLC). pioneered by Wouters
and Verhecken ( 1991 ). This technique allows the characterization
of pigments by retention time and absorption spectrum. Koren
(1994) applied this technique to a sample of Dr. Saltzman's ma-
terial containing the Mexican purple fiom Oaxaca. He could only
detect 6.6'-dibromoindigo. A recent chemical study of the pig-
ments of P. pan.sa confirmed the finding that the main component
of the dye is 6.6'-dibromoindigo (90'7r); with 9% monobromoin-

196

Naegel and Cooksey

SCHj

SCH,

SCH,

tyrindoxyl sulfate

tynndoxyl

tyrindoleninone

tynndoleninone

I

0 H

H 0

tyriverdin

6-bromoisatin

i

light

0 H

tynndoxyl

6.6'-dibromoindigo

6,6'-dibromoindirubin

Figure 2. The production of indigoids from t>rindoxyl sulfate in "brandaris-type" nuillusks.

digo, and I'X dibromoindirubin (Withnall et al.. unpublished). Re-
sults obtained by different authors (Wouters 1992; Cooksey et al.
1992; Keren 1993, Withnall et al. unpublished) using HPLC to
determine the chemical composition of the indigoid constituents of
the purple dye from various muricids are shown in Table 1.

Since the values in Table 1 were obtained using a variety of
HPLC protocols, close comparison is not justified, but some trends
can be noted: P. pansa belongs to the M. bmndaris group con-
taining no indigo in the pigment, some 6,6'-dibromoindirubin, and
showing a higher propoilion of 6-bronioindigo than the average.

TABLE 1.
The composition (in %, obtained through HPLC analysis) of the indigoid constituents of the purple dye from various muricids.

6-Mono

6,6'-

6.6'-

Bromo

Dibromo

nibromo

Indigo

Indirubin

Indigo

Indigo

Indirubin

Reference

Murex bmndaris

0

0

3

83

14

Wouters (1992)

Thais liaemastoma

0

0

3

91

6

Wouters (1992)

Niicella lapillus

0

0

3

88

9

Cooksey et al., (IW2)

Nucella lapillus

g

0

1

77

14

Withnall et al.. (unpublLshed)

P. pansa

0

0

9

90

1

Withnall et al.. (unpublished)

P. pansa

0

0

16

77

7

Wouters (pers. com).

Murex rruiiiutit.s

55

7

35

3

0

Wouters (1992)

Murex irumiiliis

3

0

15

63

T

Koren (1995)

Tyrian Purple

197

Textile Dyeing with "Tyrian Purple"

In using the "ink" for dyeing materials two significant differ-
ences have to be mentioned between the Mediterranean muricids
and P. pansa: (a) the Mediterranean snails have to be killed to
obtain the chromogens, whereas P. pansa can be "milked" to ob-
tam the dye withotit harming the animals; and (b) the "milk" from
the P. pansa can be applied directly on textiles where the final
pigments are formed in the presence of light and oxygen.

In textile dyeing, there are two methods for dyeing with niol-
lusk purple. The most simple is to have the chromogens react in the
presence of light and air to obtain directly the final pigment on the
fiber, as is the case with P. pansa. Since the molecular structure of
mollusk purple is indigoid, there exists also the possibility of start-
ing the dyeing process with the final oxidized purple colorant by
reducing it in an aqueous alkaline bath, and applying the highly
water soluble, and almost colorless leuco-form as a vat dye. just
like indigo. Exposure to air oxidizes the leuco-form back to the
indigoid pigment (Verhecken 1993; Clark et al. 1993; Schweppe
1998). In sunlight, the brominated leuco-indigoids can be photo-
debrominated, leading to 6-bromoindigo or indigo after aerial oxi-

dation and changing the purple color to blue. This chemistry of
reduction and photodebromination of 6.6'-dibromoindigo, was
first described by Driessen (1944), as shown in Figure 3.

DISCUSSION

Different species of muricids produce different color qualities
of the ink (Born 1936a), depending on the number and concentra-
tion of different chromogens. According to Verhecken ( 1 993 ) de-
pending on the precursors and chemical reactions for the formation
of the pigments, two groups of dyes from marine muricids can be
distinguished: the "trunculus type" where light is not necessary,
and the "brandaris type" requiring both light and oxygen. Since for
the formation of the dye of P. pansa light and oxygen is necessary,
the dye of P. pansa according to this definition belongs to the
"brandaris type". Another fact supports this characterization: the
pigments of the "brandaris type" contain mainly 6,6'-
dibromoindigo. The pigments of P. pansa contain 90% 6,6'-
dibromindigo, confirming that the snails are forming part of the
"brandaris type". This is in contrast to the purple pigment derived
from M. tnmcnhis, which is exceptional in containing non-

reduce

oxidise

6,6'-dibromoindigo (purple)

leuco-6,6'-dlbromoindlgo

light

0 H

reduce

oxidise

H 0

6-bromoindlgo (purple/blue)

leuco-6-bromoindigo

light

reduce

oxidise

'"*90(blue) leucoindigo

Figure 3. The reduction and photodebromination of 6.6'-dibromoindigo.

198

Naegel and Cooksey

brominaled precursors, leading to widely varying mixtures of in-
digoid pigments, including indigo and indiruhin (Malaszkiewicz
1967).

The biosynthesis of the chromogens in the hypobranchial gland
of muricids originates from tryptophan, an essential amino acid for
animals, which is enzymatically split into indole and pyruxate.
After a sequence of chemical reactions different intermediates are
formed from indole, which lead finally to the colorless precursor of
indigoid pigments tyrindoxyl sulfate, and subsequently through an
enzymatic reaction with aryl sulfatase to give the yellowish inter-
mediate tyrindoxyl (Fouquet 1970). In the presence of oxygen the
red tyrindoleninone is fomied. which reacts together with tyrin-
doxyl to give the greenish tyriverdin. In the presence of light
tyriverdin is photolysed to give dimethyl disulfide and the purple,
insoluble pigment 6.6'-dibromoindigo (Verhecken 1989). Addi-
tionally, from photolysis of tyrindoxyl or tyriverdin in the presence
of oxygen. 6-bromoisatin can be formed which reacts with tyrin-
doxyl to 6.6'-dibromoindirubin (Withnall et al.. unpublished).
Oxygen and the light intensity during the chemical reactions of the
intennediate substances determine the final composition of the
pigments. The higher content of 6-bromoindigo than average in the
pigments of P. pansa may result from the development of the dye
under conditions of high light intensity. Under these conditions,
any leuco-6,6'-dibromoindigo would be photodebrominated to
give leuco-6-bromindigo. which on aerial oxidation would give
6-bromindigo. Under high intensity light conditions, the photo-
elimination of dimethyl disulfide from tyriverdin to yield 6.6'-
dibromindigo will proceed rapidly, leading to a low concentration
of tyriverdin. Consequently, oxidative cleavage of tyriverdin, a
bimolecular reaction, to give 6-broinoisatin is a minor reaction
pathway, leading to a low concentration of 6.6'-dibroinonidirubin
in the purple pigments of P. pansa (Withnall et al.. unpublished).

The importance of the chromogens in the metabolism of the
pui-ple snails is unclear. The presence of the enzyme aryl sulfatase.
which presumably occurs in all muricids (Erspamer 1946). sup-
ports the hypothesis that the chromogens could serve as a storage
for the highly unstable indoxyls. which are formed enzymatically
by the aryl sulfatase from sulfate esters (Fouquet 1970). Additional
attention needs the question about the biological function of the
indo.xyls and their substituted bromo and methylthio analogs. It
could be possible that these bromo and thio substituted indoxyls.
like the iodine derivatives of tyrosine could act as hormones in the
metabolism of the snails. Since the chromogens. besides mucus
and bioactive substances, have their origin in a specialized area of

the hypobranchial gland, it is feasible that the gland could ha\e
additionally inner secretory acti\ities (Fouquet 1970).

We obser\ed during field work that P. pansa. above sea lex el
uses the secretion to immobilize their prey, without the formation
of purple pigments (unpublished personal observations). This sup-
ports the finding that under normal circumstances the enzyme
purpurase is kept apart from the chromogens. and therefore no
pigments are formed, despite the presence of oxygen and light
(Verhecken 1989). Additionally, in preliminary, yet unpublished
personal studies, we could show, that the secretion from P. pansa
is toxic to nauplii of Artemia, and has gram negative and gram
positive antibacterial properties.

From snails of less than 2 cm shell length can be obtained about
0..^ ml of secretion and from large animals 5-6 cm up to 4 ml (Rios
Jara et al. 1994). About one liter of secretion is required to dye
about 200 g of cotton (Acevedo Garcia et al. 1993; Michel Morfin
20()()). Since the average size of P. pansa is about 3 cm and a
collection of more than 1 ml secretion per animal seems difficult,
the enormous number of at least 1 .000 snails has to be "milked" to
obtain 1 liter of secretion to dye only 200 g of material. Since too
frequent "milking" does harm to the animals it was the right de-
cision of the Mexican government to permit only Indian commu-
nities the traditional exploitation of P. pansa for its pigments and
to declare it a protected species.

In contrast with the Mediterranean region, where the use of
purple from marine snails has long been forgotten and the craft of
dyeing today cannot exactly be reconstructed, in remote Pacific
regions of Mexico (in the States of Oaxaca and Michoacan) and
with the Indian community of the Borucas in Costa Rica (Turok
1999) its use is continuing now and represents the survival of a
knowledge of considerable antiquity. However, as Thompson
(1994) observed that this old tradition will be lost in the future. As
Thompson (1994) notes "In the eariy 20th century in Mexico shell-
fish purple was in much more widespread use than it is now\ The
beliefs, languages, and crafts of the Mexican Indians are fast dis-
appearing. The progressive "westernization" of rural Mexico has
led people in many villages to abandon their traditional textiles and
customs, in favor of factory-made cloth and western-style clothes
which are readily axailable everywhere. Cultural and social decay
is continuing to the point that the demand for traditional textiles
has almost vanished. Weavers in a few \ illages fomieriy noted for
their excellent textiles ha\e turned to making more 'commercial"
articles, for sale to people, such as tourists, outside their culture — a
classic manifestation of the "aiiport art' phenomenon.""

Acevedo Garcia. J.. M. Turok Wallace & M. A. Escalante Cavazos. 1993.
El caracol Purpura. Su uso y manejo en Mexico. Resumen de la III
Reunion Nacional para el estudio del caracol P. p. pansa. Sociedad
Mexicana de Malacologia. Universidad Autonoma de Nuevo Leon.
35 pp.

Baker, J. T. 1474. Tyrian purple: an ancient dye. a modem problem. En-
deavour 13: 1 1-17.

Baker, J. T. & C. C. Duke. 1973. Cliemistry of the indolenmones. II.
Isolation from the hypobranchial glands of marine molluscs of
6-bronio-2.2-dimethylthioindolin-3-one and 6-bromo-2-methylthio-
indoleninone as alternative precursors to Tyrian purple. Aii.stralinn
Joumut Chemisin: 26:2153-2157.

Bernard. F. 1890. Recherches sur la organes palleaux de gasteropodes
prosobranches. Annates des Sciences NaUirelles. Panic Zooliigic/ue 9:
296-305.

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Joiinml of Shellfish Rt'si'arch. Vol. 21, No. I. 201-210. 2002.

A CYTOLOGIC AL STUDY OF THE MANTLE EDGE OF HALIOTIS TUBERCVLATA L.
(MOLLUSCA, GASTROPODA) IN RELATION TO SHELL STRUCTURE

DOMINIQUE SUD,' * JEAN-MARC PONCET," ARMELLE SAIHI,' JEAN-MARC LEBEL,^
DOMINIQUE DOUMENC,' AND EVE BOUCAUD-CAMOU"

^ Museum Naliunal d'Histoire Naturelle. Laboratoirc de Biologic des Invertebres Mariiis et Malacologie,
CNRS ESA - 8044. 57 rue Cuvier. 75 231 Paris Cedex 05, France: -(Jniversite de Caen. Laboratoire de
Biologie el Biotechnologies Marines IBBA. Esplanade de la pai.x. 14032 CAEN Cedex. France

ABSTRACT The mantle cytology and ultrastructure of the abalone Halioiis mherciikiw were investigated and different structures are
detailed in relation to shell structure. The mantle edge of the abalone O-i mm near the shell edge) was chosen because it corresponds
to the most active area in shell formation. The mantle is composed of a thin lamella of connective tissue wrapped in monolayered
epithelia. the inner epithelium (facing the body cavity) and the outer epithelium (facing the shell). At the edge, the mantle ends with
the division of the edge in two folds. The main features of glandular and nonglandular cells were characterized. The inner epithelium,
responsible for ion exchanges with the external environment, is composed of three glandular cell types and ciliated or unciliated
nonglandular cells. The periostracal groove, where the periostracum originates, shows high metabolic activity, with many secretions
inside both from glandular and nonglandular cells. Numerous cilia have been observed in relation to these secretions. The outer
epithelium could be divided into two distinct areas: a first short area in the outer part surface of the outer fold at the mantle edge
(approximately 250-300 p.m long), called the tubular area, and a larger area which extends on the whole shell surface beyond the
tubular zone. These two areas are both folded, hut in the tubular area folds form tubules. Moreover, some alimentary pigments have
been observed to be associated with the tubular zone. Both structural characteristics of these two distinct areas as well as features of
the cells indicate that they are responsible for the formation of the two distinct layers of the shell: the tubular zone (the calcite prismatic
layer) and the outer epithelium (the aragonite nacreous layer), respectively.

A£} WORDS:

nantle. ultrastructural study, shell formation, marine gastropod. Halioiis

INTRODUCTION

The shell of the mollusc abalone. Halioiis Uiherculata. is made
up of three distinct layers (Nakahara et al. 1982; Wilbur & Saleud-
din 1983): the outer layer or periostracum. which is a tanned
proteinaceous layer, a middle prismatic layer (calcite). and an in-
ner nacreous layer (aragonite). The calcified layers are a tight
association of calcium carbonate minerals with organic matri.x.
Despite numerous studies of the mineralization processes in mol-
luscs (reviewed by Wilbur 1964; Wilbur 1972). the precise mode
of foimation of shell is far from clear. The organic matrix plays an
important role in mineralization, providing sites for crystal nucle-
ation and a template for crystal growth and orientation. Each layer
of the shell corresponds to a different composition of this organic
matrix. The precursors of this organic matrix are first secreted by
the mantle into a small cavity between the mantle and the shell (the
extrapallial space). Some studies on mollusc mantle structure show
that a regionalization of the mantle exists in relation with the
secretion of the three shell layers (Beedham 1958; Kawaguti &
Ikemoto 1962b; Wilbur & Saleuddin 1983).

Whereas the ultrastructure of the molluscan mantle edge, es-
pecially the bivalves, has been studied in great detail, the epithe-
lium of the inner (facing the mantle cavity) and the outer (facing
the shell) surface of the mantle has received considerably less
attention. In molluscs, the structure of the mantle, and more par-
ticularly the mantle edge, shows a high degree of variation. In
bivalves, the mantle edge is generally composed of three main
folds (Jabbour-Zahab et al. 1992; Morrison 1993): an inner mus-
cular lobe, a middle lobe believed to be sensory, and an outer lobe
(Richardson et al. 1981 ). It is generally held that the periostracum
in bivalves is secreted by the cells lining the surface of the outer

*CorTesponding author. E-mail: sud@mnhn.fr

lobe. In some gastropods, the mantle is also arranged into lobes,
however, in numerous species the mantle lobes can be reduced in
size and number (e.g.. Biainplialaria glalyrata. Bielefeld et al.
1993a; Bielefeld et al. 1993b). or are virtually lacking (e.g.. Lit-
torina littorea. Bevelander & Nakahara. 1970). An additional fea-
ture of gastropods is the presence of one or more groups of mar-
ginal glands embedded in the connective tissue (Bevelander &
Nakahara 1970; Saleuddin 1975).

Because no study of abalone mantle has been undertaken to our
knowledge, we investigated the histology and ultrastructure of the
whole mantle, and more particularly near the shell edge, which is
considered to be the more active part of the mantle in the bio-
mineralization processes. Then we attempted to relate features of
the cells to formation of the different shell layers: the perios-
tracum. the calcitic prismatic, and the aragonitic nacreous layers.

MATERIALS AND METHODS

Source and Maintenance of Animals

Adult (6-7 cm in length) and juvenile (<l cm in length)
abalones. H. tiiheniilata. were purchased from a French farm
(ECHINOXE. West coast of Cotentin, Manche, France). Animals
were maintained at the laboratory in tanks, in artificial, aerated
seawater at 18-20°C.

Histology

Pieces of the mantle (ca. 5 mm") were cut near the edge of the
shell, after the central foot of the abalone was removed. For this
study, the most active parts of the mantle were used: the two
frontal lobes and the right mantle near the shell edge of the aba-
lone. For fixation of the mantle, tissue was placed in Bouin's
mixture overnight (Martoja & Martoja 1967). Sections of 5 p.m ir.
thickness were made according to the usual paraffin method. Siain-

201

202

Slid et al.

ing with hemalum picro-indigii carmine was found to be excellent
in differentiating epithelial cells, muscle, and collagen fibers.

Identificalion of Cell Types: Transmission Electron Microscopy (TEM)

For TEM studies, different parts of the mantle near the edge
were used. Preparations were fixed in a cocktail containing 3%
glutaraldehyde. sodium cacodylale buffer at pH 7.0, and 0.35 M
saccharose. o\eniight at +4°C. Then preparations were postfixed
in a solution of 2% OSO4 for 1 h at room temperature and rinsed
twice in double-distilled water (2 x 10 min). Preparations were
then placed in uranyl acetate aqueous solution 1 19r) during 1 h at
room temperature and in the dark. Preparations were then rinsed
twice in double-distilled water (2 x 10 min). Preparations were
dehydrated through a graded ethanol series and then embedded in
Araldite. Thin sections were cut on a Reichert ultramicrotomc.
stained with uranyl acetate (5% in ethanol 5<()C. \\/\') and lead
citrate, and examined in an electron microscope (Philips EM 201
under 80 kV, Centre de Microscopic Electronique de Jussieu-
Piene et Marie Curie University. Paris. France).

RESULTS
Gross Morphology of the Mantle

The mantle of abaione is a thin lamella that originates at the
base of the foot muscle. The mantle covers the visceral mass and
extends to the shell edge so as to cover the shell surface. As we
investigated the structure of abaione inantle in relation with the
biomineralization processes, we focused on the mantle near the
shell edge. Indeed, scanning electron microscope (SEM) study of
the shell surface has demonstrated that the more active area of
shell growth (in length and in width) corresponds to a band of 2—1
mm in length near the shell edge (unpubl. data). For this study,
mainly adult abalones were used, except for Figure 9. which shows
a juvenile abaione.

Figure la corresponds to a transverse section through the
mantle edge of H. tiihcrcuhiiit. At the shell edge, the mantle is
composed of a thin lamella of connective tissue covered by a
monolayer of epithelial cells. In the connective tissue, hemolymph
lacunae are found; hemocytes are known to participate in the
biomineralization processes, and more precisely in shell regenera-
tion. A particular feature with the muscle fiber repartition is the
formation of a dense network of fibers parallel to the outer epi-
thelium (OE) mantle surface, whereas muscle fibers, mixed with
collagen fibers, remain with no specific orientation near the inner
epithelium (IE). Such muscle repartition is probably responsible
for the tight contact with the inner shell and for the mantle's
retractive ability in response to mechanical stimuli. Because the
extrapallial space (between the shell and the mantle) is very thin
and contact with the external medium may be limited, this par-
ticular organization of muscle fibers may contribute to the adher-
ence of the mantle onto the shell. The epithelium is separated from
the connective tissue by a basal membrane. The epithelium in
contact with the shell is called the OE. whereas the IE. in contact
with seawater, faces the visceral mass. The mantle edge ends with
the periostracal groove (PG) separating the mantle into two folds:
the inner fold (facing the visceral mass) and the outer fold (facing
the shell). For a better understanding, we have divided the mantle
epithelium into different functional areas (Fig. lb].

The Inner Mantle Epitlieliiiin: Area I

The inner mantle epithelium generally consists of palisadic
cells. The apical border of the cells exhibits a proiuinent border of

Figure 1. (a) Light micrograph of the mantle edge structure in Haliutis
luhcrculata. CT, connective tissue; IK, inner mantle epithelium (facing
the body): OE, outer mantle epithelium (facing the shelll; P(;, perios-
tracal groove. The hemol>mph lacunae are designated b) the black
arrows (xl60). (b) Subdivision of the mantle edge into different func-
tional areas: 1, inner epithelium: 2, epithelium of the outer face of the
inner fold: 3, bottom of the periostracal groove: 4, epithelium of the
inner face of the outer fold; 5, tubular area of the outer epithelium;
and 6, outer epithelium, which extends on the «hole shell surface
bevond area 5.

luicrovilli. 2-5 \x.n\ high (Fig. 2a). Cells are interconnected by
intercellular junctions comprising an apical desmosome and sep-
tate junctions. A distinguishing feature of the inner epithelial cells
is the highly convoluted interdigitalions of the plasma membranes
below the apical desmosome (Fig. 2b). Under the basal lamina, a
thin sheet of collagen fibers is observed (orientated parallel to the
basal lamina). Two major cell types can be identified, depending
on the secretions of their cytoplasm: epithelial glandular cells
(where the cell cytoplasm is filled with secretory granules) and
epithelial nonglandular cells.

Epithelial nonglandular cells are generally similar in size. Cilia
are occasionally observed, interspersed between the microvilli
(Fig. 2a). but the general characteristics of these cells remain simi-
lar along the luantle IE. These epithelial cells contain a basal
nucleus in which luost of the heterochromatin is concentrated in
the periphery. A Golgi complex is often observed in the vicinity of
the nucleus. Mitochondria are mainly located near the nucleus as
well as in the apical part of the cell cytoplasm, even though some
have been observed in the whole cytoplasm. Numerous vacuoles
are located in the apical part of the cells. Some vacuoles contain
some dark-stainiui; contents, which are sometimes heterogeneous

Mantle Ultrastructure of a Marine Gastropod

203

Figure 2. Area 1. (a) limer iiuintle epithelium iTEMi with nontjlaii-
dular cells. Cilia Ihlack arrowsl are interspersed with niicrntilli (M\ I.
Some vesicles with pigments are observed in the apex of the cells (white
arrows! 1x4,50(11 lb) Detail (TEMi of highlv convoluted plasma mem-
branes below the microvillar border. Cells are separated from each
other by intercellular junctions comprising a desmosome (white star)
and septate junctions (black arrowheads) (x20.0(l()).

aniJ couW be related to some kin(i of pigments. Tonotllament hunehes
are observed mainly orientated in the axis of the cell (Fig. 2b).

A general feature of the glandular cells (or secretory cells) is
their goblet shape. The narrow openings of glandular cells can
sometimes be seen. The main features of these cells are the oc-
currence of numerous secretory granules in their cytoplasm and a
basal small nucleus surrounded by a more or less developed rough
endoplasmic reticulum (Figs. 3. 4. and 5). Three glandular cell
types could be distinguished depending on their secretions. Type A
glandular cells (Fig. 3) contain clear ovoid granules, closely
packed and homogenously stained, which tend to coalesce. Type B
glandular cells (Fig. 4) contain distinct, darkly staining granules,
which have a denser core in some cells. The membrane of type B
granules is generally still distinct when they are discharged. Type
C glandular cells (Fig. 5) contain some distinct, membrane-bound
granules, with rosette-like mucous granules. Type C cells have
peculiar secretory granules of heterogeneous stages: in a clear and
finely granular matrix an array of complex chains of electron-
dense material is found. The basal nucleus is surrounded by a
well-developed rough endoplasmic reticulum of enlarged cisternae
filled with finely granular material. Between the rough endoplas-
mic reticulum and the secretory granules. Golgi stacks occur. The
immature granules from the trans Golgi only contain finely granu-
lar material, then the dense complex progressivelv condenses and
develops.

Figure 6 resumes the main features of the IE: a monolayered
epithelium which constitutes ciliated (Ci). or nonciliated. nonglan-
dular. and glandular cells (A. B. C types) with microvillous border
(Mv).

I iniue .^. \rea I. Detail of type A glandular cells in the inner mantle
epithelium (TEM): The cytoplasm is filled with ovoid granules, clo.sely
packed, homogeneously stained. N, nucleus (xl4,(l(IO).

Tlw Periostracal Groove: Areas 2a. 2h, .?, 4a, 4h

We divided the periostracal groove, which runs parallel to the
mantle edge, into three different parts of spatial and equal impor-
tance, depending on the structure of the cells. The first part (a)
corresponds to the opening of the periostracal groove directly in
contact with external seawater: areas (2a) and (4a) are, respec-
tively, the epithelium of the inner and outer folds of the periost-
racal groove. Principal features of the cells are very similar to
those of cells of the IE: in the 4a area, characteristics of the IE are
less marked (plasma membrane is not as convoluted as in the 2a
area, and pigment granules are less representated than in the 2a
area). The second part (b) corresponds to the middle of the peri-
ostracal groove: areas (2b) and (4b) are. respectively, the epithe-
lium of the inner and outer folds of the periostracal groove. A
principal feature of this area is the presence of a group of ciliated
cells of the epithelium 4b (outer fold. Fig. 7) and the presence of
numerous secretions from nonglandular cells of the epithelium 2b
(inner fold. Fig. 7). All cilia are orientated to the opening of the
periostracal groove and some secretions are often found associated
with the cilia. Principal characteristics of the cells are quite similar
to that of cells previously described.

In the periostracal groove, the microvillous border is not
equivalent in the inner and outer folds. In the epithelium of the
outer fold (4a. 4b). microvilli are dense and ananged in a brush
border, whereas the cells of the inner fold (2a, 2b) are lined by
microvilli irregularly arranged. A lot of material seems to be dis-
charged by these cells, as clear vesicles with a fine granular con-
tent. We can also observe some secretions from nonglandular cells
in the microvilli in area 2b (in the inner fold).

204

Slid et al.

Fisure 4. Arta I. Detail dI t\|)t B glandular cells in the inner mantle
epithelium (TEM): The cytoplasm is filled with distinct, darkly stain-
ing granules, which have a denser core in some cells |x30.0(l()).

In areas (a) and (b) of the inner and outer fold, we can notice
the presence of numerous B-type glandular cells. A-type glandular
cells have been observed in some sections in the epithelium of both
outer and inner fold. However, C-type glandular cells have only
been found in the epithelium of the inner fold.

At the bottom of the periostracal groove (area ?<), the cells
become gradually cuboidal. Essentially, nonglandular cells are ob-
served. Cilia and cell secretions could not be observed (Fig. 8).
The inner and outer folds appear morphologically equivalent.

Fine Structure of the Outer Mantle Epithelium: Areas 5 and 6

In H. tiiheniilata. two distinct areas of the outer mantle epi-
theliuin can be distinguished: a short area near the periostracal
groove (area 5) and the other part of the outer mantle epithelium
(area 6). The structure of the cells and the structure of the folds in
the OE determine this separation. Area 5 corresponds to a very
folded epithelium, which forms some tubules when this area is
quite developed. In scanning electron micrographs of the outer
mantle epithelium, this area corresponds to an alveolous structure
in external view: this area has been called the tubular zone (TA.
Fig. 9). In area 6. the OE displays folds that are quite parallel to the
mantle margin (Fig. 9).

In area 5 the tubular /one is more or less developed, depending
on the collected specimens in a same size class. To observe the
well-developed tubular zone, some juveniles have been observed
by TEM (Fig. 10). The principal features of these cells are their
cuboidal shape, a high nucleoplasmic ratio, and numerous round-
shaped mitochondria. In Figure 10. tubules are cut transversally.

Figure 5. .\rea 1. Detail of type C glandular cells in the inner mantle
epithelium (TEM): The cytoplasm is Tdled with distinct, membrane-
bound granules, which have a heterogeneous content. N, nucleus
(xl4.(H)0).

and we can observe in the lumen of each tubule some small mi-
crovilli and some secretions. In this area, pigments are found trav-
elling from hemolymph lacunae to tubules; they correspond to an
obvious red and/or green line along the border of the OE in live
specimens. The pigments are carried by "migrating cells" (likely
hemocytes). which have a central nucleus and a cytoplasm filled
with four or five vacuole sites containing a large central core ot
heterogeneous granules (Fig. 1 1 ).

Figure 12 summarizes the main features of the tubular zone: a
monolayered epithelium (which is very folded) composed of
cuboidal cells and tubules (T) containing microxilli (Mv). alimen-
tary pigments (Pi) in vacuoles, and soine secretions in the lumen
(Lu).

In area 6. a different structure than the OE has been observed:
cells are low columnar (<I0 |jim) (Fig. 13) to very high columnar
(>I5 (xm) (Fig. 14). depending on studied specimens in the same
size class. Meanwhile, the OE is always folded. In some specimens
with low columnar cells (Fig. 13), numerous "empty" cells have
been observed and the other cells have a cytoplasm where few
organelles are present, corresponding to low cell activity. In other
specimens, the OE is very high and narrow columnar (Fig. 14):
numerous mitochondria, Golgi apparatus, and inclusions are found
in the cytoplasm of these cells (Fig. 15). In such cells, all or-
ganelles, and in particular, all Golgi complexes, are found orien-

Mantle Ultrastructure of a Marine Gastropod

205

Figure 6. Area 1. Schematic drawing of the main features of the cells of the inner epithelium. In this area, three types of glandular cells are
present: types A. B, and C (encircled A, B, C), with nonglandular cells, with or without cilia (Ci). BL, basal lamina: Ci, cilia; GS, Golgi stacks;
M, mitochondria; Mv, microvilli; N, nucleus; RER, rough endoplasmic reticulum; Pi, pigments; TB, tonofilament bunches; V, vesicle (not to
scale).

tated in the longitudinal axis of the cells (Fig. 15). The microvil-
lous border is well developed in very high columnar cells and
numerous protruding cell processes are found associated with this
microvillous border (Fig. 14). In low columnar cells the microvil-
lous border is quite nonexistent excepted in fold of the IE; how-
ever, in the cells of the OE, some distensions of the membranes are
generally observed and they are often associated with mitochon-
dria (Fig. 15).

The schematic Figure 16 resumes the main characteristics of
the OE far from the periostracal groove: very high columnar cells
containing numerous mitochondria (Mi), Golgi apparatus (G). and

Figure 7. Area 2b-4b of the periostracal groove (TEM): Nuniirous
cilia are interspersed in the microvillar border of the epithelium of the
outer fold (4b). The microvillar border of the inner fold (2b) is irregu-
lar and some cell protruding processes can be observed (black or white
arrowheads). Some secretions of the B type glandular cells are asso-
ciated with cilia (*). IE, inner mantle epithelium: OE, outer mantle
epithelium (x9,0UO).

microvillar border (Mv| with protruding processes, orientated in
the longitudinal axis of cells.

DISCUSSION

Many publications describe shell formation in molluscs and
some of them reported the structure of the mantle and its role in
shell formation. But the majority of these studies dealt with bi-
valves (Kawaguti & Ikemoto 1962a; Kawaguti & Ikemoto 1962b;
Neff 1972;Bubel 197.^a, 1973b, 1973d; Garcia-Gasca et al. 1994).
Most of the work on gastropods has been done on the terrestrial
snail Heli.x and on freshwater gastropods (Zylstra et al. 1978.
Bielefeld et al. 1993a, Bielefeld et al. 1993b); only one reference
to a marine gastropod was found: Lillorina tittorea (Bevelander &
Nakahara. 1970). Although shell production is often assumed to be
quite similar among molluscs, there are important morphologic

Figure 8. Area 3. lioltiiju of the periostracal groove (TEM). The cells
are cuboidal. Only few secretions and no cilia can be observed in this
part of the periostracal groove (x4,()00).

206

SUD ET AL.

Figure 9. Areas 5 and 6. External structureof the outer nianlle epithelium (SEM). Near the periostracal groove (PG), the tubular area (TA) (area
5) appears as honeycomb lobe at the surface of the mantle (double black arrow I, whereas the outer epithelium (OEl beyond area 5, covering the
whole shell surface (area 6) (single black arrow) ha\e some folds parallel to the periostracal groove. This tubular area (area 5) can be
distinguished from the outer epithelium covering the remainder of the mantle (area 6| (x35(l).

differences of the mantle between bivalves and gastropods due to
different shell structure and morphology (Hedegaard, 1497).

As Martin et al. (1983) have demonstrated with their study of
the gonads in Haliotis ntfescens. two cell types can be defined
depending on their cytoplasm content: glandular (or secretory)
cells and nonglandular cells. In H. tiihercuhita. ultrastructural stud-
ies have allowed us to determine three types of glandular cells with
regard to their secretion xesicles content. Type A and B glandular
cells are present in the whole epithelium, whereas type C cells only
occur in the IE and in the inner fold epithelium of the periostracal
groove. Type A glandular cells correspond to a typical mucous
cell. Type A and B glandular cells are present in various species
(for example: Lymnaea stagnalis, Zylstra et al. 1978). Type C
glandular cells are rarely represented in molluscs and have already
been described in the gastropod Rhodope sp. (Has/prunar & KCin/
1996). Glandular cells, and )iotably type B. aie highly represen-

tated in the periostracal gi'oove. While in the IE glandular cells
play a role in the protection of the mantle (Lemaire-Gony & Bou-
dou 1997), various authors repon the presence of glandular cells.
which might facilitate the movement of the periostracum out of the
groove or protect the site of periostracum formation against invad-
ing water (Bielefeld et al. 1993a; Bielefeld. 1993b; Garcia-Ga.sca
et al. 1994). Some ulti'astructural studies demonstrated that glan-
dular cells play a role in periostracum formation (Tsujii 1968a;
Tsujii 1968b; Bevelander & Nakahara 1969a; Bevelander & Na-
kahara 1969b; Nakahara & Bevelander 1971): this role is to be
assumed in H. litherculala. The role of glandular cells in mineral
deposition is controversial in the studies; Tsujii (I960) suggests
that in Pincuula manensii. glandular cells in the OE do not par-
ticipate in shell forirtation. whereas Beedham (1958) (studying
Aiunlonhi cygnea. Mytilus ediilis. and Ostrea edulis) believes that
secretion may be incorporated in the organic matri.x of the shell.

Figure 10. ,\rea 5. Tubular area of the outer nianlle epithelium (TEMl in a juvenile abalone. .Structure in tubules are shown transversally cut.
Cells contain a central nucleus (N) and numerous mitochondria can be observed (arrow heads). In the lumen of the tubule some .secretions are
present (*) (x4,000).

Mantle Ultrastructlire of a Marine Gastropod

207

Figure 11. Area 5. Tubular area of the outer mantle epithelium
(TEM). Alimentary pijjments (AP) are found in \acuole.s of wandering
hemocyte-like cells. HI,, hemolymph lacunae; hemocyte (*) (x3,000).

Hillmun (1961) suggests that not all glandular cell types could
interfere with the biomineralization processes. The organic matrix
of the shell mineral layers (which are calcite and aragonite) con-
tains some mucopolysaccharides (Garcia-Gasca et al. 1994). As a
consequence, the participation of glandular cells in shell formation
could not be ruled out in H. niberciilata. and further studies need
to be undertaken to determine the nature of each glandular cell and
the role of glandular cells in shell formation.

In H. tubercidata. the mantle edge ends with a periostracal
groove, parallel to the mantle surface, which divides the mantle
into two folds: the inner and outer folds. In all molluscs, the
periostracum is secreted by the mantle edge, but the mode of

^\i

•^

Figure 13. Area 6 of the outer mantle epithelium (TK.Ml. Oetall of one
fold: Numerous empty cells can be obser>ed (*) (x3,000).

formation and the morphological structure of the mantle edge vary
from species to species. Generally, in molluscs, the periostracal
groove divides the mantle into folds, although it is virtually lacking
in some species such as the gastropod L. litlorea (Bevelander &
Nakahara 1970). The mantle edge of bivalves generally consists of
three folds compared with the usual two folds among (freshwater
or terrestrial) gastropods. In bivalves, the periostracum originates
from epithelial cells lining the inner surface of the outer fold of the
periostracal groove and/or from a row of basal cells at the bottom
of the groove (Kawaguti & Ikemoto 1962a; Dunachie 1963; Neff
1972; Bubel 1973a; Bubel 1973c; Bubel 1973d; Saleuddin 1974;
Petit et al. 1979). In some gastropods, such as Helix sp.. the pe-
riostracal groove is described as a single layered, flattened

Figure 12. Area 5. Schematic drawing of the tubular area of the outer epithelium (OE). The epithelium is folded and when well developed, it
forms tubules (T). Some alimentary pigments (AP) are observed near the hemolymph lacunae (HL). H. hemocyte; M, mitochondria: .Mv
micro\illi: N, nucleus (not to scale).

208

Figure 14. Area 6 of the outer mantle epithelium (TEM(. \ ery high
columnar cells are seen; numerous protruding processes (arrows) can
be observed within the microvillar border 1x2,00(1).

glandular epithelium called the "periostracal gland" (Beedhani
1958), although it does not form a compact body or have a duct.
The cells forming the gland seem to be equivalent to the basal cells
described in other bixalves (Saleuddin 1974). In some gastropods
(L. suignalis and Biamphalaria pfeifferi). some different types of
gland cells are found which have their cell bodies sunken into the
underlying connective tissue at the base of the groove (Zylsti'a et
al. 1978).

In gastropods, the periostracal groove is generally shallow, but
it is deeper in H. niherculata and morphologically similar to that
in the bivalves: this could be due to the primitive status of this
gastropod (Stasek & McWilliams 197.^). Periostracal gland and
basal cells, described respectively in gasti'opods and in bivalves
(Hillman 1961; Kawaguti & Ikemoto 1962a'. Kawaguti & Ikeinoto
1962b: Bubel 1973a: Bubel 1973b: Petit et al. 1979). have not been

Figure 15. Area 6 ol the outer mantle epithelium ( TK.M). Detail ol'one
very high columnar cell: Inclusions of different content are present in
vesicles (V). GS, Golgi stacks; M, mitochondria (x40,00()).

Figure 16. ,Vrea 6. Schematic drawing of an active outer epithelium.
Palissadic cells are tall and narrow, tilled with numerous organites
arranged in the longitudinal avis of the cells; (iolgi stacks ((iS), mito-
chondria (M). rough endoplasmic reticulum (RER), The microvillar
border (Mv) is well developed and some protruding processes (black
arrows) are observed (not to scale).

found in H. tuberculata. Secretions of material have been observed
in the outer surface of the inner fold, whereas it is generally ac-
cepted that, in bivalves, the cells lining the inner surface of the
outer fold contribute to the periostracum (Bubel 1973b). A group
of ciliated cells have been observed in the inner surface of the
outer fold of the periostracal groove of H. tiiberciikita. Such cilia
have been described in some bivalves {Pinctada iiiargariiifeni.
Jabboui-Zahab et al. 1992). but are generally located at the bottom
of the groove. These cilia may help to conduct and aggregate the
periostracum material outside the periostracal groove as well as
microvilli in the brush border along the inner surface of the outer
fold.

A distinct membrane-like lamellar layer has not yet been ob-
served in anv preparation or fixation in H. mbercukiui periostracal
groove, although such a membrane-like layer is reported for other
gastropods and bivalves (Saleuddin 1 979; Saleuddin & Petit 1983).
This could be explained by the fact that animals have not been
anesthetized and mantle contractions could tear up the perios-
tracum. Nevertheless, the peiiostracum has also not been found in
luveniles fixed in lolo with their shell. Anesthetized animals with
3-aminobenzoic acid ethyl ester (MS222) or 1% MgCI, could be
useful to answer this question. Moreover, no periostracal units
described in other bivalves or gastropods (Saleuddin 1976;
Bielefeld et al. 1993b; Schaefer & Haszprunar 1997) have been
observed in cells of//. tuht'icuUita. It seems that the secretions and
the formation of the periostracum differ from those previously
described, and a more detailed study of periostracum formation
must be undertaken to determine how the periostracum is secreted.

In H. mbeiTiilata. two distinct areas have been identified in the
OE. These two areas are morphologically different; both epithelia
are folded, but in the outer surface of the outer fold, the epithelium
can form tubules, whereas beyond this, the OE folds are quite
similar and are almost orientated parallel to the periostracal groove
side. In the first area (5). located in the outer surface of the outer

Mantle Ultrastructure of a Marine Gastropod

209

fold, cells are cuboidal. and numerous mitochondria have been
observed. According to Istin and Masoni (1973i. in bivalves, the
number of mitochondria underlying the epithelial border of the
outer fold indicates a metabolic activity for such cells. This activity
is not linked only with calcium movements, but may be linked with
matrix components synthesis. In the vicinity of this area, in the
connective tissue, and near the hemolymph lacunae, some accu-
mulations of partially digested pigments have been found. These
pigments originate from the algae diet, giving red or green color of
the prismatic layer, depending on the algae consumed as food. In
Haliotidae, these pigments are incorporated into the calcitic layer
of shell (Leighton 1961 ). Thus, this particular area called here the
tubular zone (5), can be related to the secretion of the calcitic layer
in H. tubeicuhiia. This area has already been described by Crofts
( 1929) as the "glandular acini." This tubular area is always present
in juveniles of H. nihcrciilaia. but it could be less developed or
even absent in adult specimens. A lower development of tubular
area in adult specimens could be related to a decrease in shell
growth rate compared to juveniles. The presence or the absence of
this area may be considered as a cyclic shell secretion, whereas the
other area (6) of the OE is likely related to the formation of the
aragonitic (nacreous) layer.

This regionalization of the outer mantle epithelium exists in
other molluscs: according to Nakahara and Bevelander (1971 ), in
P. radiata. the prismatic layer of the shell is derived exclusively
from the tall columnar cells lining the outer surface of the outer
mantle fold. Although some authors found a distinction between
the secretion of the different shell layers (Beedham 1958, Ostrea
ediilis: Kawaguti & Ikemoto 1962b, Mitscidus senlioiisia: Jabbour-
Zahab et al. 1992. Pinciada maxima: Schaefer & Haszprunar 1997,
Laevipilina antanlica). other authors found this distinction not to
be effective (Petit et al. 1980 - Amblema sp.). In H. tuberculuta.
this regionalization is in accordance with the differences in the
organic matrix components of each shell layer and with the pres-
ence of alimentary pigments incorporated into the calcitic layer.

In area 6 of the OE in relation with the nacre secretion, two
different structures of these epithelia have been found: a tall co-
lumnar and a very high, narrow columnar epithelium, depending

on the activity of the mantle. These two epithelial structures have
been observed, depending on studied specimens. Such morpho-
logical differences may be correlated with seasonal and/or indi-
vidual variations. In the tall columnar epithelium, organelles are
not in great number and numerous "empty" cells have been ob-
served, whereas in the very high, narrow columnar epithelium,
numerous organelles are found (mitochondria. Golgi complexes,
rough endoplasmic reticulum, numerous inclusions of various
types). It seems that the last epithelium is much more active than
the first. A large number of cytoplasmic vesicles as well as nu-
merous mitochondria were found, indicating an involvement in the
mechanisms of shell formation attributed to this epithelium (Wil-
bur 1964; Istin & Masoni 1973). Moreover, the very high, narrow
columnar epithelium is associated with protruding cell processes,
which can be related to the secretion of the organic matrix com-
ponents of the shell (Wilbur 1964; Jabbour-Zahab et al. 1992). In
the very high, narrow OE. some distensions of the intercellular
spaces have been found: these could act as ion pumps, particularly
for calcium (Zylstra et al. 1978: Richardson et al. 1981).

The structure of the mantle margin in H. nihercidata is very
peculiar and could not be related morphologically to the mantle of
other gastropods and bivalves. A spatial organization of the mantle
exists in relation with the secretion of the different shell layers: the
periostracal groove in relation with the secretion of periostracum.
the tubular area (in the outer surface of the outer fold) in relation
with the deposition of the prismatic layer, and the OE (beyond the
previous area) in relation with the secretion of the nacreous layer.
This spatial organization can be related to a temporal variation,
because the OE seems to have different structure depending on the
stage of secretory activity of the mantle. This temporal variation
could be related to the incremental bands deposited annually,
monthly, daily, or during another shorter period (Wilbur 1972).
because shell formation is incremental rather than continuous.

ACKNOWLEDGMENTS

This study was supported by the French Ministry of National
Education. Research and Technology (Grant 97 CO 230).

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Bevelander. G. & H. Nakahara. 1969b. An electron microscope study of
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Bevelander. G. & H. Nakahara. 1970. An electron microscope study of the
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Joiinuil of Shellfish Research. Veil. 21. No. 1. 21 1-217. 2002.

DIETARY BIOTIN REQUIREMENT OF JUVENILE ABALONE, HALIOTIS DISCUS HANNAI INO

GETIAN WU, KANGSEN MAI, BEIPING TAN, AND WEI ZHU

Manciilture Research Laboratory. College of Fisheries, Ocean University of Qingdao. Qingdao 266003.
Shandong. People 's Republic of China

ABSTRACT The experiment was conducted to quantify the biotin requirement of juvenile abalone, Haliolis discus hannai Ino. The
po,s.sible biotin-synthesizing capacity of intestinal microflora was also examined. Seven purified diets were formulated to provide a
series of biotin (0, 0.5, 1.0, 3.0, 6.0, 12.0, 20.0 mg/kg diet). An antibiotic diet was supplemented with tretracycline hydrochloride
(4-g/kg diet) in basal diet to suppress possible intestinal bacteria synthesis. To reduce leaching, dietary biotin and other water-soluble
vitamins were encapsulated by calcium alginate. Abalone juveniles of similar size (initial mean weight 173.1 ± 3.6 mg; mean shell
length 1 1.66 ± 0.14 mm) were distributed in a flowing-through system using a completely randomized design with eight treatments
and three replicates per treatment. They were fed the appropriate diet once every day for a 1 10-day period. Results of this study clearly
showed the necessity of dietary biotin for juvenile abalone as the daily increment in shell length (DISL). visceral pyruvate carboxylase
and acetyl-CoA carboxylase activities were significantly influenced by the dietary biotin levels (ANOVA. P < 0.05). However, the
survival, specific growth rate (SGR) and carcass proximate compositions were not significantly influenced by dietary biotin at the end
of the experimental period. The viscera biotin concentration ( VBC) of abalone increased as the biotin supplementation level increased
(r = 0.91). Compared to those of other groups, SGR, DISL, VBC and two carboxylase activities of antibiotic group were obviously
depressed. It indicated that the intestinal microflora probably contribute to biotin nutrition for juvenile abalone. Based on measurements
of DISL and two carboxylase activities, the optimum biotin requirement was estimated to be 0.42 mg/kg and 0.67-0.70 mg/kg for
maximum growth and carboxylase activities, respectively.

KEY WORDS: Halintis discus lumnai. biotin, carboxylases, microflora, bio-synthesis, mollusk. nutrition

INTRODUCTION

Biotin is a water-soluhle vitamin included in \ilaniin B com-
plex. As a coenzyme for several carboxylases, such as pyruvate
carboxylase (EC 6.4.1.1), acetyl-CoA carboxylase (EC 6.4.1.2),
propionyl-CoA carboxylase (EC 6,4,1.3), and methylcrotonyl-
CoA carboxylase (EC 6,4,1,4), biotin participates in the reactions
of relevant enzymes, and affects the metabolism of amino acids,
carbohydrates, nuclear acids and lipids.

Since biotin is one of the most expensive vitamins to add to
artificial feed rations, it is necessary to quantify the minimum
requirement for the vitamin in order to reduce feed cost. However,
several factors have been proven to influence the need for dietary
biotin in animals, for example, dietary factors such as the presence
of high dietary fat has been shown to obscure the effects of biotin
in rats, chicks, brook trout, and rainbow trout (Jacobs et al. 1970;
Marson & Donaldson 1972; Poston & McCarteney 1974; Walton
et al. 1984). In addition, biotin was found to be synthesized by
intestinal bacteria in considerable amounts in some land animals
(Victor & Rachel 1945) and freshwater fishes (.Sugita et al. 1992).
Thus, these factors should be taken into account during estimation
of biotin requirements.

Until now, the quantitative requirement of dietary biotin for
maximum growth has been studied in only a few species of aquatic
animals. Kitaniura et al. (1967) reported that 0.01-mg biotin/kg
diet was sufficient to support optimum weight gain in rainbow
trout fry. The biotin requirement of lake trout appears to be lower
than 0.1 mg/kg (Poston 1976). Shiau and Chin (1998. 1999) re-
ported that grass shrimp and tilapia require 2.0-2.4 mg/kg and 0.06
mg/kg of diet, respectively. Giinther and Meyer-Buorgdorff ( 1990)
concluded that optimum biotin supply for mirror carp was 2.0-2.5
mg/kg of diet. The deficiency signs of biotin included anorexia.

Corresponding author. Kangsen Mai. Mariculture Research Laboratory,
College of Fisheries, Ocean University of Qingdao, Qingdao 266003.
Shandong, Peoples Republic of China. E-mail: kmai@ouqd.edu.cn

poor food conversion, poor grow th, "blue slime" disease, light skin
and lower carboxylase activity (Phillips et al. 1950; Ogino et al.
1970; Halver 1972; Poston & McCartney 1974; Robinson & Lov-
ell 1978; Halver 1979).

Abalone is a large algivorous marine mollusk of genus, Haliotis
iCcLstropoda. Prosohranchia. Archaeogasiropoda. Haliotidae).
They are the most commercially important gastropod in aquacul-
ture. At present, limited information has been reported on the
essentiality or quantitative requirements of vitamins for abalone.
Only the effect of dietary vitamin C on the growth, survival and
level of ascorbic acid in the tissues of the abalone H. tuberculata
and H. discus hannai had been investigated (Mai 1998a). For the
sake of safety, biotin is generally supplemented in excess to diets
for abalone at levels of about 12 mg/kg of diet (Ogino & Kato
1964; Uki et al. 1985; Viana et al. 1993; Mai et al, 1995a; Mai et
al. 1995b; Mai 1998a). This dietary level of biotin may not provide
maximum efficiency of nutrient utilization and maximum profit
margins.

The objective of this study was to quantify the biotin require-
ments for juvenile abalone. H. discus hannai Ino. The possible
biotin-synthesizing capacity of intestinal microflora was also in-
vestigated.

MATERIALS AND METHODS

Preparation of Vitamin Microsphere

To reduce leaching from diets, dietary biotin, and other water-
soluble vitamins were encapsulated with sodium alginate. The
method of preparation was modified from Bodmeier and Wang
(1993). One hundred milliliters of sodium alginate solution (2%,
w/w) containing 2% (w/w) of water-soluble vitamins were mixed
with 150 ml of oil containing 6.74 g/L SpanSO, stiired at 400 rpm
for 15 min. The emulsified solution was slowly poured into 1%
(w/w) CaCl, solution, with continuously stirring for 1 min, then
filtered below normal pressure. Harvested microcapsules were
washed with cyclohexane and absolute alcohol, in turns, to remove

2i;

WU ET AL.

oil and water, dried below norniul pressure, tlien. kepi m -20 C rally, then the tlakes were sealed in a sample bag and stored at
until use. -20"C until use.

Feed Formiilalion and Manufacture

The basal diet formulation is given in Table 1. The basal diet
contained negligible intrinsic biotin. Dietary treatments were pre-
pared by replacing the dextrin with graded levels of biotin. 0-20
mg biotin/kg diet, in the form of crystalline biotin encapsulated
with sodium alginate, was used to prepared seven experiment di-
ets. These diets were designated DO. D0.5. Dl. D.^. D6. DIO and
D20. respectively. The biotin concentrations of the seven diets
were determined by HPLC method (Hudson et al. 1984). They
contained 0. O.-Sl. 0.91. 3.04, 6.0,3. 10.1 1. and 19.89 mg/kg diet,
respectively. A treatment with tetracycline hydrochloride (A. P..
Japan) (4 g /kg of diet) was included to ascertain the biotin-
synthesizing capacity of intestinal microflora.

Procedures for diet preparation were modified from those de-
scribed by Mai et al. (199.'ia. 1995b). Casein, gelatin and some
minerals that were in the form of small grains were ground indi-
vidually using a Pascal Mill and then passed through a mesh with
200-p.m pore size. Dry ingredients were weighed on an electronic
balance and thoroughly mixed. After adding water (about 120%,
v/w) to the mechanically mixed ingredients containing 20% so-
dium alginate, a paste was made. The paste was shaped into 0.5-
mm thick sheets, which were cut into 1-cm" flakes. The flakes
were dipped into an aqueous solution of CaCU (5%. w/v) for 1
niin. By this treatment, sodium alginate was converted to an in-
soluble calcium alginate gel, in which the nutrients were bound
(Uki & Watanabe 1992). The surplus solution was drained natu-

TABLE 1.

Composition of the basal diet (g/kg, dry weight basis)."

Ingredients Content

Casein (vitamin-free. .Sigma Chemical. St. Louis. MO. USA) 260.0

Gelatin (Sigma Chemical. St. Louis. MO. USA) 65.0

Dextrin (Shanghai Chemical Co., Shanghai, China) 300.0

Carboxymethylcellulose (Sigma Chemical. St. Louis, 50.0

MO, USA)

Sodium alginate (Shanghai Chemical Co.. Shanghai. China) 200.0

Vitamin mix (biotin-free)'" 20.0

Choline 5.0

.SO/MFO (Food grade)' 35.0

Mineral mix'' 40.0

Filler'- 25.0

" The basal diet contained (dry weight basis): crude protein. 29.67%; crude

lipid. 3.68%; gross energy. 18.48 kJ/g.

'^Vitamin mix. each 1.000 g of diet contained: thiamin HCI. 120 mg:

ribotfavin. 100 mg: folic acid. 30 mg; PABA. 400 mg; pyridoxme HCI. 40

mg; niacin. 800 mg; Ca pantothenate, 200 mg; inositol, 4,000 mg; ascorbic

acid, 4,000 mg; vitamin E, 450 mg; menadione, 80 mg; B,-,, 0.18 mg;

vitamin A, 100 000 lU; vitamin D, 2,000 lU; ethoxyquin, 400 mg. All

water-soluble vitamins coated with calcium alginate.

' Soybean oil and menhaden fish oil (1:1) with 0.001% ethoxyquin.

'' Mineral mix. each 1.0(30 g diet contained: NaCI, 0.4g; MgSOj ■ 7H,0.

6.0g; NaH_,POj ■ 2H,0. lO.Og: KH^POj, 12.8g; Ca(H,P04), ■ H,0, 8.0g;

Fc citrate. I.Og; Ca-lactate. I.4g: ZnSOj • 7H,0. I4L2 mg; MnSO^ • H,0.

64.8 mg; CuSOj ■ 5H,0. 12.4 mg; CoCI, ■ 6H,0, 0.4 mg; KIO,. 1.2 mg:

Na,SeO„ 0.4 mg.

"Dextrin replaced with biolm supplements.

Leaching

The leaching test of dietary biotin was modified from the
method used by Marchetti et al. ( 1999). Ten-gram aliquots of each
feed, stored in nylon bags (mesh size, 149 p.m). were immersion in
a beaker containing 2 L of sea water maintained at 20.0 ± 1 °C and
mechanically stirred. Thimerosal was added at 100 ppm to reduce
bacterial activity. At the end of allotted time (3, 6, 12 hours,
respectively), the remained food was removed from the bags and
dried overnight at 60 'C in an oven. Dried food was submitted for
analysis of total dietary biotin by HPLC. The leaching of dietary
supplemented biotin was reflected as retention efficiency (RE),
which defined as:

RE:

(Biotin retained in diet after immersion)
(Biotin contained in diet before immersion)

X 100

Animal Rearing

Juvenile abalone (//. discus hannai Ino.) used in this experi-
ment was derived from a spawning in June 1999. at Mashan Fish-
eries Co.. Shandong, China. Before the trial, shell lengths were
measured with calipers to the nearest 0.02 mm and the animals
were weighed to the nearest 0.1 mg using an electronic balance.

Animals were kept in acrylic square cages (35 cm x 28 cm x 20
cm). Each rearing unit was stocked with 40 abalone juveniles.
Similar sized juveniles (mean weight 175.1 ± 3.6 mg; mean shell
length 1 1 .66 ± 0. 14 mm) were assigned to the rearing system using
a completely randomized design with eight treatments and three
replicates per treatment. The system was flow-through, with water
filtered to 30-p.m by primary sand filters, then to 10-|jLm by sec-
ondary composite sand filters. The flow rate was about 0.5 L per
min per cage. Cages were kept in dim light by screening with black
plastic drapes. During the experimental period, water temperature
ranged from 18.2 to 22.0°C. salinity 30-34%r. pH 7.6-7.9. Dis-
solved oxygen was not less than 7.0 mg/1, and there were negli-
gible levels of free ammonia and nitrite (AOAC, 1995).

Prior to initiation of the experiment, the abalone underwent a
1-week conditioning period during which they readily acclimated
to environmental conditions. The feeding trial was run for 16
weeks. Abalone were hand-fed with the test diets at a rate equaling
5-10% of abalone wet body weight once daily at 17:00. E\ei-y
morning, uneaten food and feces were cleaned to maintain the
water quality.

Sample Collection and Analysis

At the termination of the feeding trials, to deplete the digestive
canal, animals were not fed for 3 d, then all abalone were removed
from the rearing system, weighed, measured and ctiunted. Then. 30
abaknie from each replicate were quickly frozen (-70°C) for sub-
sequent analysis. Growth was reported as specific growth rate
(SGR, %/day) and daily increment in shell length (DISL. p.m/d).
The formulae are as follows:

I (InWt - lnWi)/tl X 100
I (InSLt -SLi)/tl X 1000

SGR (%/d)
DISL (p.m/d)

Where, Wt, Wi are final and initial mean weights (mg), respec-
tively: SLt, SLi are final and initial mean shell lengths (mm),
respectively: t is the feeding trial period (days).

Dietary Biotin Requirement of Juvenile Abalone

213

The frozen samples were finely cut. Then, an aliquot of the
whole visceral tissues was hand homogenized with 18 ml of ice-
cold phosphate buffer (pH 3.5). The homogenate was centrifuged
for 20 min at 3.000 rpm. Then, the biotin was estimated using
HPLC method (Hudson et al. 1984). Another aliquot of viscera
was homogenized on ice in 5 volumes of Tris-HCI (pH 7.3) buffer.
The homogenate was pre-centrifuged (2,000 rpm, 10 min) to re-
duce the foam and sediment debris. A subsequent centrifugation
was carried out at 20,000 rpm for 30 min. The protein concentra-
tion of supernatant was assayed following the method of Lowry et
al. (1951) using bovine serum albumin as the standard. Then,
pyruvate carboxylase (EC 6.4.1.1). and acetyl-CoA carboxylase
(EC 6.4.1.2) activities were measured as described by Zempleni et
al. (1997). The NaH['-'C]03 (specific radioactivity. 30.1 MBq/
mmol; NEN, USA) was used as substrate. Preliminary work with
each assay also ensured that enzyme-saturating conditions were
achieved. '"'C were counted in 5 ml of Ultima Gold XR scintilla-
tion fluid (Packard Instrument, Meriden. CT) in a liquid scintilla-
tion analyzer Winspectral-1414 (Wallac). Pyruvate carboxylase
activity was expressed as units per milligram of protein, where I
unit equals 1 |j,mol of H| '"*C]0, incorporated into oxaloacetate per
minute at 30°C. Similarly. acetyl-CoA carboxylase activity was
expressed as 1 unit equaling 1 pmol of HI '""CIO, incorporated into
malonyl-CoA per minute at 37°C.

Proximate analyses to determine carcass protein, lipid, and
moisture contents were conducted using conventional procedures
(AOAC. 1995).

Statistical Analysis

All percentage data were arcsine square-root transformed prior
to analysis. Data from each treatment were subject to one-way
ANOVA. When overall differences were significant at less than
5% level. Tukeys" test was used to compare the mean values
between individual treatments. Statistical analysis was performed
using STATISTICA'^' package (StatSoft, Inc.. USA).

RESULTS

Leaching

The results of the 1 2-h leaching test of the biotin-suppleniented
diets are presented in Figure 1 . The supplemented biotin contents
in all diets decreased with the increasing of the immersion time.
After 3 and 6-h of immersion in seawater, the retention efficiencies
(RE) were approximately 90.3-96.1% and 76.8-84.2%. respec-
tively; and this value decreased to 42.5-69.2% after 12-h of im-
mersion in all diets. In the first 6-h. there were no differences in the
RE among all levels of the biotin supplementation (ANOVA. P >
0.05). After 12-h immersion, the retention efficiencies of all biotin-
supplemented diets were significantly different and showed a
negative correlation with the biotin supplemental content {r =
0.87). By taking into account the leakage rates in the first 3-h
immersion and the amount initially encapsulated, the real amounts
of biotin delivered to the abalone were about 0, 0.49. 0.87, 2.88.
5.75. 9.37, and 17.96 mg biotin/kg diet for DO. D0.5. Dl. D3. D6.
DIG, and D20, respectively.

Survival and Growth

Survival, specific growth rate (SGR) and daily increment in
shell length (DISL) data are shown in Table 2. During the 1 lO-day
experimental period, there were no significant differences in sur-

g 80

96.1 95.8 94.6 95.3

E]:!h

84. 2 J., T

EF^ S3 1 fir

69. 2a ^ in ^

92. 7 90. 3

81. 4

rii

Jl

k

J,

DO. 5

Figure 1. Tht retention efficiency (RE) of dietary biotin in six biotin
supplemented diets ut different interval (3, 6 and I2h, respectively)
immersed in seawater in triplicate. Error bars are SE. Means with the
same letter are not significantly different [P > 0.05). In the first 6h
immersion. RE were not significantly dilierent among all diets. After
12h, the values were significantly dilTerenl and show a negative rela-
tionship with the biotin supplemental content (/' = 0.87).

vival (89.2-96.2%.) with any of dietary treatments (ANOVA. P >
0.05). The SGR of the antibiotic group (0.83%/day) was signifi-
cantly lower than those of biotin treatinent groups (1.10-1.28%/
day) (P < 0.05). There were no differences observed in SGR
among biotin treatment groups during the experimental period
(ANOVA. P> 0.05).

Similarly. DISL were significantly higher for dietary biotin
treatment groups (60.0-69.1 p.m/d) than for the antibiotic group
(50.9 |xm/d) (P < 0.05). Among all dietary biotin treatments, di-
etary biotin levels influenced DISL significantly (ANOVA. P <
0.05). DISL increased from 60.0 to 69.1 |a.m/d with dietary biotin
levels increasing to I mg/kg. There was a decline in DISL observed
in abalone fed diets with biotin supplementation higher than 3
mg/kg. Thus, the broken-line regression model (Robbins et al.
1979) was used in this study to express the relationship between
DISL and dietary biotin content. The regression equations are
shown in Figure 2. As the break point at 0.42 mg/kg gave the least
mean square enor. the adequate amount of dietary biotin for ju-
venile abalone is estimated to be 0.42 mg/kg.

Carcass Composition and Biotin Concentration

As seen in Table 3. all the moisture (75.30-77.91%). crude
protein (55.87-58.35%). and crude lipid content (5.16-6.37%) of
abalone were not affected by dietary treatments (ANOVA, P >
0.05). However, the visceral biotin concentration (VBC) signifi-
cantly responded to dietary treatments (ANOVA, P < 0.05). The
differences among the eight groups were all significant. By addi-
tion of 0.4% tetracycline hydrochloride in the basal diet, the VBC
was significantly lower than those of other groups were. A linear
increase in the VBC was observed when the biotin supplementa-
tion le\el increased (r = 0.91).

Carboxylase Activity

The activities of pyruvate carboxylase (EC 6.4.1.1 ) and acetyl
CoA carboxylase (EC 6.4.1.2) in abalone fed test diets are ;ire-
sented in Table 4. Both enzyme activities showed similar trends lo

214

WU ET AL.

TABLE 2.
Effect of dietary biotin on survival and grovvtli of abalone. H. discus haiinai Ino. (mean [s.e.), n = 3l.

Dietary

Initial Shell

Biotin

Initial Weight

Length

Final Weight

Final Shell Length

Survival

SGR'

DISL-

Diet

(mg/kg)

(nig)

(mm)

(nig)

(mm)

C^f )

(%)

(um/d)

DO

(1

171.2 (27. .S)

11.5(0.71

576.9(18.1)"'

18.1 (0.2)''"

90.01 7.5)

1.10(0.15)"

60.0 (5.3)"

D().5

0.5 1

173.3(27.5)

11.6(0.4)

599.3 (38.9)'"-

18.5(0.2)-*

95.3(1.4)

1.13(0.13)"

62.7 (4.0)"

Dl

0.91

173.4(5.0)

11.8(0.2)

711.8(29.2)''

19.4(0.4)"

96.2(1.4)

1.28(0.05)"

69.1 (4.0)'"

D3

3.04

I7S.6(12.5)

11.6(0.2)

629.0 (85.0)'=''

18.7(0.9)-"'

94.2(5.2)

1.14(0.08)"

64.5(6.5)"'

D6

6.03

181.1 (22.5)

11.9(0.6)

613.5(43.4)'^"

18.7(0.9)-'"

90.8(3.8)

1.11 ((.).08)"

61.8(5.6)"

D12

10.11

175.0(5.0)

11.7(0.3)

599.0(27.2)"'

18.5(0.5)""

92.5(2.5)

1.12(0.04)"

61.7(3.2)"

D20

19.89

177.3(22.5)

11.7(0.4)

629.0(71.6)'

18.0 {e.g)-""

89.2 (7.6)

1.15(0.17)"

62.0(1.7)"

Antihiolir

0

170.7(15.0)

11.5(0.4)

424.0(39.4)"''

17.1 (0.3)"

92.5(2.5)

0.83(0.01)-"

50.9 (2.3)"

ANOVA

F

0.27

0.46

5.70

4.75

2.52

2.74

3.84

P

0.956

0.S51

0.019

0.005

0.059

0.044

0.012

' Specific growth rate.

- Daily increment in shell length.

^ Supplemented with 0.4'7f tetracycline in basal diet.

"^ Means in the each column sharing the same letter are not significantly different based on Tukeys' test (P > 0.05).

(dietary treatments. Compared to the basal group, two carboxylase
activities in viscera wei'e significantly depressed when animals
were fed the antibiotic diet (93.8 U/mg protein and 7.31 U/mg
protein for the activity of pyruvate carboxylase and acetyl-CoA
carboxylase, respectively). Apart from the antibiotic diet, the ac-
tivities of pyruvate carboxylase activity ( 155.9 U/mg protein) and
acetyl-CoA carboxylase (8.33 U/mg protein) of basal group were
the lowest among the biotin treatment. The activities of both en-
zymes generally increased with increasing dietary biotin up to
3-mg/kg diet then leveled off. The differences between the lowest
group and the groups fed diets with biotin higher than 1 mg/kg diet
were significant iP < 0.05). It is obvious that the relationships
between both enzyme activities and dietary biotin levels were in
accord with the broken-line regression model (Robbins et al.
1979). Based on pyruvate carboxylase and acetyl-CoA carboxylase
activities, the regression equations were Y = 8.97 + 0.03 x (X -
0.70) (r = 0.93") and Y = 171.1 +03S x {X-0.61} {r = 0.85).
respectively. Therefore, the biotin requirements were estimated to
be 0.67-0.70 ma/ka that based on two carboxylase activities.

DISCUSSION

Abalone are known to be slow feeders or nibblers. They can
take seseral hours before consuming one feed flake. Thus, it is
necessary to reduce leaching to precisely quantify the requirement
of water-soluble micronutrients including water-soluble \ itamins.
Microencapsulation is one of the most potential methods to reduce
leaching by sheltering encapsulated materials from outer environ-
ment (Louis 1970; Gupta & Rao 1985: Shun et al. 1988). Accord-
ing to Marchetti et al. (1999). the retention efficiencies of lipid-
walled \itamin B, in pelleted food were 87.5* and 72.5'7f for 1
and 2-h immersion, respectively. In the present study, the RE of
biotin was up to 90.3-96. 1'/r after 3-h immersion. It is evident that
leaching out of biotin froni the experimental diets was reduced in
a great degree. A previous study indicated that the guts of most

TABLE 3.

The effects of dictarv biotin on carcass composition and VBC' of
abalone. H. discus haiinai Ino. (mean |s.e.|, n = 3.

Protein"

Lipid'

.\Ioister

VBC

Diet

('7f)

C^f)

{'7c)

(ng/g)

DO

56.30(0.14)

5.74(0.78)

75.30(3.85)

202.2(17.6)"

D0.5

56.88(0.10)

6.37 (0.38)

75.51 (0.81)

292.3(21.9)'=

Dl

56.73(1.60)

5.64(0.51)

75.73 (0.28)

369.4 (27.4)"

D3

58.16(0.59)

6.15(0.64)

77.91 (1.11)

555.2 (40. !)■-■

D6

58.35(1.92)

5.79(0.25)

76.03 (0.42)

936.2(22.2)'

D12

56.39(0.83)

5.16(0.44)

76.60(1.22)

1068.4(31.3)^

D20

56.42(0.67)

5..59(0.28)

77.31 (2.77)

1208.7(59.2)"

Aniibioric^

55.87 (0.97)

6.04(0.04)

76..50(0.47)

75.8(7.2)"

ANOVA

F

2.21

1.22

0.75

566.23

P

0.089

0. 1 35

0.636

0.000

0 5 10 15

Dietary biotiti(mg/)tg)

Figure 2. The effect of dietary biotin on daily increment in shell length
(DISL) of abalone. Each point represents the mean of three groups o
abalone (n = 3), with thirty abalones per group. Based on the broken-
line model, the biotin requirement is estimated to be 0.42 mg/kg.

' Viscera biotin concentriition.

" Dry weight basis.

' Dry weight basis.

■• Supplemented 0.4<7r tetracycline in basal diet.

' " Means in the each column shanng the same letter are

different based on Tukeys' test iP > 0.05).

not signiticanllv

Dietary Biotin Requirement of Juvenile Abalone

215

TABLE 4.

Effect of dietary hintin on carboxylase acti> ities in viscera of
abalone H. discus haniiai (mean |s.e.l, ii = i).

Pyruvate Carboxylase'

Acetvl-CoA Carboxviasc-

Diet

(U/nij; Protein)

(ll/nig Protein)

DO

155.9 (4.07 )*"

8.33(0.19)'"

DU.5

167.5 {6.63)"'^

8.80(0.14)'""

Dl

173.2 (5.59f

9.36(0.21)'

D3

175.0(7.72)'

9.45(0.331'

D6

171.8(8.33)'

9.20(0.29)'

D12

174.7(9.25)'

9.32(0.14)'

D20

178.3(5.31)'

9.40 (0.24)'

Antibiotic^

93.8 (6.22)"

7.31 (0.21)-'

ANOVA

F

93.88

41.12

P

0.000

0.000

' Per unit equal 1 (jimol of H['''C]0, incoiporaled into oxaloacetale per

minute at 30°C.

" Per unit equal 1 pmol of H|'''C]0, incorporated into nialonyl-CoA per

minute at 37°C.

' Supplemented with 0.4% tetracycline in basal diet.

■" Means in the each column sharing the same letter are not significantly

different based on Tukeys' test (/> > 0.05).

abalone were full of food after 2 to 3-h feeding with premium
quality diets (Mai et ul. 1998b). There were no significant differ-
ences observed in retention efficiency when all diets immersed in
seawater for 6-h (ANOVA, P > 0.05). Thus, in this study, the
leaching may not influence the result of biotin requirement for
abalone by encapsulating biotin and other water-soluble vitamins.
On the other hand, wall-forming material calcium alginate is apt to
be digested by abalone as high level of alginase appears in the
digestive tract of abalone (Oshitna 1931: Nakado & Sweeny 1967;
McLean 1970; Toshio 1985; Hugo & Maria 1998). In comparison
to those reported by other authors (Uki et al. 1985; Uki & Wa-
tanabe 1992; Mai et al. 1995a; Mai et al.l995b). the satisfactory
abalone growth indicates encapsulated vitamins can be efficiently
utilized by abalone and supports our conclusions as follows.

Until now. the essentiality of dietary biotin for any mollusk
species was unknown. Results of this study clearly show that di-
etary biotin is necessary for juvenile abalone as the shell growth
(DISL) was significantly influenced by dietary biotin. The shell
growth of abalone fed lower levels of dietary biotin was depressed
implying that biotin probably affects the course of biomineraliza-
tion of shell. This is in accord with the findings of Bain et al.
( 1988) that biotin deficiency affects bone growth in broiler chick.
During the 110-day experimental period, dietary biotin did not
significantly affect SGR. However, there still was an obvious trend
that lower biotin levels reduced SGR. As we know, biotin is a type
of micronutrient in diets and the signs of biotin deficiency are
usually produced under specific conditions. Thus, a 110-day ex-
perimental period is probably not long enough to significantly
affect SGR in abalone. In juvenile animals, body length growth is
usually faster than the body weight gain. This agrees with DISL
being a more sensitive parameter to dietary biotin than SGR. and
DISL may be a responsive criterion for estimating dietary biotin
requirement for other mollusk juveniles. Based on the daily incre-
ment in shell length (DISL). the optimum biotin requirement for
the maximum growth is estimated to be 0.42 mg/kg diet by the
broken-line regression analysis. Compared with other reports, the

optimum requirement for juvenile abalone is higher than that of
brook trout (0.05 and 0.25 mg/kg) (Poston & McCartney 1974).
lake trout (0.1 mg/kg) (Poston 1976). rainbow trout (0.05-0.14
mg/kg) (Woodward & Frigg 1989; Castledine et al. 1978). com-
mon carp (0.02-0.03 mg/kg) (Ogino et al. 1970), tilapia (0.06
mg/kg) (Shiau & Chin 1999), but less than the requirements re-
ported for the mirror carp (2.0-2.5 mg/kg) (Gijnther & Meyer-
Buorgdorff 1990) and grass shrimp (2.0-2.4 mg/kg) (Shiau & Chin
1998). The different biotin requirements probably attribute to the
differences of experimental procedures and species.

The presence of high dietary fat has been shown to obscure
effects of biotin in rats, chicks, brook trout, and rainbow trout
(Jacobs et al. 1970; Marson & Donaldson 1972; Poston & Mc-
Carteney 1974; Walton et al. 1984). In the present study. 3.68% of
lipid was measured in the basal diet. According to Mai et al.
(1995a). optimum dietary lipid content was 3-5% to maintain
maximum weight gain for abalone. Therefore, 3.68% of lipid is
just sufficient to meet requirements of the abalone and lipid effects
on biotin requirements can be omitted here.

As a coenzyme for several carboxylases, biotin affects the me-
tabolism of amino acids, carbohydrates, and lipids. Thus, some
studies have demonstrated that the proximate composition of ani-
mals usually respond to dietary biotin treatment. Poston (1970)
reported the carcass fat content of lake trout was depressed when
animals were fed biotin-free diets. The crude ash, fat and protein
contents of mirror carp were significantly affected by dietary bi-
otin (Giinther & Meyer-Buorgdorff 1990). Similar results were
also obtained with common caip (Ogino 1970) and tilapia (Shiau
& Chin 1999). However, in the current study, the crude protein and
lipid contents in the soft body tissues, which consist of mantle, foot
muscle, and viscera that include all inner organs, of juvenile aba-
lone remained independent of biotin supplementation. This effect
may be manifested by a longer experimental duration. It is also
generally believed that carboxylase activities are depressed in bi-
otin deficient animals such as mammals, fishes and shriinp
(Deodhar & Mistry 1969; Arinze & Mistry 1971; Poston & Mc-
Carteney 1974; Castledine et al. 1978; Walton et al. 1984; Shiau &
Chin 1998. 1999). In the present study, the pyruvate carboxylase
and acetyl-CoA carboxylase activities of abalone visceral tissues
positively responded to dietary biotin levels. This implies that
abalone did experience biotin deficiency, which would lead to
depressed enzyme activities. Thus, both carboxylase activities are
useful criteria in estimating dietary biotin requirements for aba-
lone. Based on two carboxylase activities, the optimum biotin
requirement for the maximum enzyme activities is estimated to be
0.67-0.70 mg/kg diet by the broken-line regression analysis. In the
present study, the recommended biotin requirement for maximum
carboxylase activities is higher than that for maximum growth
(0.42 mg/kg diet). This means that the biotin level resulting in
maximum enzyme activities and the level resulting in maximum
growth are not necessarily the same.

In some species of land animals, such as calves and cows,
biotin has been found synthesized in considerable amounts by
intestinal bacteria (Victor & Rachel 1945). In the present study,
when tetracycline was incorporated into basal diet as an antibiotic
group, a deficiency developed. The SGR DISL of antibiotic group
were significantly lower than those of the basal group (P < 0.05).
Similarly, the visceral biotin concentration (VBC) and the carbox-
ylase activities were also obviously depressed by addition of tet-
racycline. Sugita et al. ( 1992) examined the biotin-producing abil-
ity of intestinal microflora in freshwater fishes and proved (he

!16

WU ET AL.

requirement of freshwater fishes for hiolin was significantly influ-
enced by intestinal microflora. Thus, we believe that the poor
growth of the abalone in the treatment with antibiotics could at-
tribute to the suppression of intestinal microflora that can probably
synthesize biotin. Further investigation is needed to provide direct
evidence.

Another interesting phenomenon is that VBC increased from
292.3 to 1208.7 ng/g with increasing biotin supplemental levels
(r = 0.91). Theses results indicate that biotin can be stored in
abalone viscera in amounts corresponding to the dietary supply.
Therefore, the VBC can be used to monitor biotin status in aba-
lone. To our knowledge, the toxicity of biotin in animals is still

unccrtaui. however, depressed growth was observed when abalone
were fed high levels of dietary biotin and this would require further
studies.

ACKNOWLEDGMENTS

This study has been supported by grant No. 39925029 from
National Science Foundation for Talented Youths of the People's
Republic of China and the Excellent Young Teachers Program of
MOB of the P. R. China. We thank Dr. Q. F. Ye (Institute of
Nuclear Agriculture. University of Zhejiang. China) for his excel-
lent technical assistance.

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Dietary Biotin Requirement of Juvenile Abalone

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Joiinwl of Shellfish Rfsciirch. Vol. 21. No. 1. 219-226, 2002.

IDENTIFICATION OF SOUTHERN HEMISPHERE ABALONE (HALIOTIS) SPECIES BY
PCR-RFLP ANALYSIS OF MITOCHONDRIAL DNA

NICHOLAS G. ELLIOTT,' JASON BARTLETT,' BRAD EVANS," AND NEVILLE A. SWEIJD^

^CSIRO Marine Research. GPO Box 1538, Hobart. Tasmania 7001. Australia: 'Department of Zoology,
University of Cape Town. Private Bag. Rondesboscli. 7701. Cape Town. South Africa. Current address:
BENEFIT Secretariat. Box 912 Swakopmund. Namibia: School of Zoology. University of Tasmania.
GPO Box 252-05. Hobart. Tasmania 7001, Australia. Current address: School of Marine Biology &
Aquaculture. James Cook University. Townsville. Queensland 481 1 , Australia

ABSTRACT Illegal fishing and species-substitution of abalone (genus Haliotis). a highly valuable marine gastropod, are of world-
wide concern. A mitochondrial DNA PCR-RFLP analysis of fragments of the cytochrome oxidase I (mtCOI) and II (mtCOII) genes
was developed for the identification of 1 1 Southern Hemisphere species of abalone. These included five temperate and one tropical
species from Australian waters, three temperate species from New Zealand and two temperate species from South Africa. All species,
with the exception of the Haliolis ruhralH. conicopora complex, can be unequivocally identified using the combined profiles from four
individual restriction enzyme digests {Ddel. Hhal, HinFI and Hpall) on a 1 93 base pair fragment of mtCOI. Six species each displayed
a unique profile for a single restriction enzyme. A one hundred and fifty-nine base pair fragment of mtCOlI allowed individual
identification of six of the species using the combined profiles from five individual restriction enzyme digests {Dilc/. EcoRV, Hhal.
Hpall. and R.sal). These primers failed to amplify in H. iris. Again H. rubra and H. conicopora could not be separated, and neither
could H. australis and H. spadicea. No DNA sequence variation in either fragment was observed between H. rubra and H. conicopora;
the latter may be a subspecies of//, rubra. The use of both fragments and a minimum of two restriction enzymes is recommended for
species differentiation. DNA was successfully extracted, PCR amplified and identified from canned tissue and mucous samples of //.
rubra. A conformational mutation in the mtCOI fragment was observed in H. miilac. but in no other species nor in the mtCOII
fragment.

KEY WORDS; Haliotis. abalone, mitochondrial DNA, identification

INTRODUCTION

Abalone. genus Haliotis Linnaeus, are a highly valuable com-
mercial marine univalve niollusk. There are over 55 recognized
species worldwide (Geiger 1998). of which nearly half are ex-
ploited by commercial or recreational divers. Abalone generally
inhabit rocky reefs to depths of 65 m, but more are usually found
in shallower waters to 30 m. The foot muscle of abalone attracts
high prices in Asian markets, with species differential. Once the
distinguishing shell and mantle tissue have been removed, it is
very difficult to differentiate the commercial product of one spe-
cies from another. The high price, market demand, ease of harvest
and similarity of processed product between species makes aba-
lone very suitable targets for illegal marketing and both highly
organized and small scale poaching.

Abalone poaching and species-substitution of abalone products
is of concern to many countries, including the USA (Daniels &
Floren 1998), Mexico (Ponce-Di'az et al. 1998). South Africa
(Sweijd et al. 1998) and Australia. The value of the illegal trade is
difficult to quantify. Conservative estimates in Australia alone are
over $US25M annually. The legal Australian abalone fisheries.
dominated by Haliotis rubra, account for about half the annual
world abalone harvest of ca. 10,500 mt (FAO 2000) and is worth
around $US80M per year. The South African abalone fishery (H.
midae ca. 500 mt/yearl is worth approximately $US 1 5M with legal
sales of confiscated (poached) abalone from just one area fetching
over $USlm (Sweijd et al. 1998). In New Zealand the main com-
mercial species is H. iris and the illegal harvest is estimated at

Corresponding author. Nicholas G. Elliou, CSIRO Marine Research, GPO
Box 1538. Hobart. Tasmania 7001. Australia. E-mail: nick.elliott@
marine.csiro.au

about 33% of the annual commercial catch of 1 ,300 mt (Roberts et
al, 1999), The high but unknown level of illegal harvesting of
abalone creates major problems for fishery managers endeavoring
to maintain viable and economic fisheries.

Whilst H. rubra is the dominant commercial species within
temperate Australian waters, both H. laevigata and H. roei are
subject to significant levels of commercial fishing under indepen-
dent quota systems, and a H. scalaris fishery is under consider-
ation, A problem for fisheries enforcement is the overlapping
ranges of these and non-commercial species. Such species richness
is common with abalone (Geiger 1999). and after removal of the
characteristic shell and mantle species identification of the foot
muscle is obscure. The need exists for a definitive means of iden-
tifying tissue and by-products (e.g., mucous in instances of sus-
pected poaching when tissue has been disposed) of individual aba-
lone species.

Identification of plant and animal species when morphological
characters have been removed is possible using either protein or
DNA-based methods (Palumbi & Cipriano 1998; Toro 1998; Jo-
hannesson & Stenlid 1999; Hare et al. 2000; Sweijd et al. 2000).
The protein based methods are very dependent on tissue quality;
generally requiring fresh or frozen material. Often identification
for commercial needs may require analysis of processed (dried or
canned) tissue or degraded tissue. DNA-based methods are rela-
tively independent of tissue quality, and those that rely on ampli-
fication of small DNA fragments are less likely to be affected by
degradation (Mackie et al. 1999). A number of techniques are
available for species identification including; random amplifica-
tion of polymorphic DNA (RAPD) (Martinez & Malmheden
Yman 1998). restriction fragment length polymorphism (RF-LP)
analysis (Innes et al. 1998; Wolf et al. 2000). direct DNA sequenc-
ing (Quinteiro et al, 1998) and single-stranded conformation poly-

219

220

Elliott et al.

morphism (SSCP) (Mackie et i\\. 1999) of PCR (pdlymerase chain
reaction) amplified fragments.

A PCR-RFLP analysis of a 1,300 base-pair (bp) fragment of the
nuclear lysin gene was devised for identification of two South
African abalone species. Haliotis midae and H. spadicea (Sweijd
et al. 1998). Generic PCR primers that amplify across the intron
differentiated between species based on the size of the intron.
Preliminary analyses found that the size of the lysin intron varied
greatly between other Haliolis species (generally 500 to 1.100 bp).
but the intron in the Australian greenlip abalone H. laevigata was
over 4,000 bp (unpublished data). Products of such size are not
ideal for species identification tests with the likelihood of unreli-
able PCR products due to tissue and DNA degradation.

To differentiate between the more common Southern Hemi-
sphere abalone species a PCR-RFLP method was developed using
short fragments (less than 200 bp) of the mitochondrial DNA
(mtDNA) molecule. To satisfy potential legal scenarios in Austra-
lia and South Africa, 11 species were included. Within species
variation and potential non-Haliotis amplification of our designed
primers were examined in addition to testing the primers with
canned abalone tissue and abalone mucous samples.

MATERIALS AND METHODS

Sample Collection and DNA Extraction

Whole individuals (live or frozen) or alcohol preserved tissues
were obtained for 1 1 purported Haliotis species: Haliotis asinimil
Linnaeus, Queensland, .^ustralia (30 individuals); Haliotis aiistra-
lis Ginelin, New Zealand (10); Haliotis conicopora Peron, West-
em Australia, Australia (11): Haliotis iris Gmelin, New Zealand
(10); Haliotis laevigata Donovan, Tasmania & Victoria, Australia
(62): Haliotis midae Linnaeus, South Africa (10); Haliotis roei
Gray, Western Australia, Australia (10); Haliotis rubra Leach.
Tasmania, Victoria & New South Wales, Australia (50); Haliotis
scalaris Leach, Tasmania & Western Australia, Australia (22);
Haliotis spadicea Donovan, South Africa (10); Haliotis virgiiiea
Gmelin. New Zealand (10).

Total genomic DNA was extracted from ca. 25 mg of foot
muscle or gill tissues using a modified CTAB (hexadecyltrimeth-
lammoniumbromide) protocol (Grewe et al. 1993). Tissue was
incubated overnight at 50' C instead of 30 to 60 min at 60X.

To verify the use of our PCR primers on processed product.
DNA was extracted from commercially canned H. rubra. Approxi-
mately 0.5 g tissue was digested for 30 min at 65°C in 5 mL
digestion buffer ( 100 mM Tris. 50 mM EDTA. 400 niM NaCl, 1%
SDS). 50 (iL proteinase K ( 10 mg/mL) was then added and the
solution was incubated overnight at 55''-C. 150 \x.L NaCl (5 M) and
520 jjiL of 10% CTAB were added and the solution incubated at
65°C for 1 h with regular mixing. Samples were then extracted
once with equal volumes of chloroform/isoamyl alcohol (24: 1 ) and
precipitated with 2 volumes of 100% ethanol. Precipitated DNA
was washed twice with 70% ethanol. once with 100% ethanol,
air-dried and re-suspended in 200 |xL TE.

PCR amplification was also tested using DNA extracted from
H. rubra mucous. Two mucous samples were obtained by placing
individual freshly captured H. rubra in separate plastic bags for
approximately 2 h, removing the abalone and placing the bag and
fluid contents at 4°C, Tissue samples were taken from the indi-
vidual abalone as positive controls for DNA extraction. DNA was
extracted from mucous swabs taken from the sides of the bags and
from the control tissue samples using the modified CTAB protocol

described above. In addition, a 600 |iL sample of fluid (mixture of
seawater and mucous) from the bottom of each plastic bag was
taken, incubated overnight in 20 jjlL proteinase K (10 mg/niL) and
5% SDS, and then genomic DNA extracted using the same modi-
fied CTAB protocol.

Genus specificity of the PCR amplification was tested on total
genomic DNA extracts (using above CTAB protocol) from a va-
riety of marine organisms. These consisted of an alga (unidentified
red alga), an anemone (unidentified), a crustacean (Antarctic krill
Euphausia superha). mollusks (unidentified chiton and Pacific
oyster Crassostrea gigas) and teleosts (bigeye tuna Thunnus obe-
sus. southern bluefin tuna T. maccoyii, pink ling Cenypterus bla-
codes. Patagonian toothfish Dissostichus elegiiioides. school shark
Galeorhinus galeits and gummy shark Musteleus antarcticus).

PCR Primers and Amplification

Generic PCR primers were designed for the mitochondrial cy-
tochrome r oxidase subunit I gene (mtCOI) by alignment of either
our own unpublished or published Haliolis sequences (Metz et al.
1998). DNA sequences used for the design of the mitochondrial
cytochrome c oxidase subunit II gene (mtCOII) were either our
own or other unpublished sequences (Sandy Degnan. University of
Queensland).

The primers designed to amplify a 193 bp fragment of the mtCOI
gene were designated HALCOi-NGI (5'-ClGACATRGCITTYC-
CICGACT-3') aTid HALC01-NG2 (5'- CCGGCTARGTGIAGIGA-
RAAAAT-3'). Those designed for a 159 bp fragment of the intCOII
aene were designated HALC02GENA (5'-CAATYTGAACYAT-
TCTMCCAGc''-3') and HALC02GENB (5'-CCTTAAARTCT-
GAGTATTCGTAGCC-3'). (Degenerate nucleotide lUB codes; 1.
Inosine = A, C. G or T; M, aMino = A or C; R, puRine = A or G;
Y. pyrimidine = C or T).

PCR reactions consisted of 50 to 100 ng of total genomic DNA,
2.5 mM MgCU, 200 |xM each dNTP, 10 pmoles of each primer,
and 0.55 U Taci DNA polymerase (Biotech) in a buffer supplied by
the manufacturer. PCR amplifications were carried out in a 50 ^.L
final volume using a Perkin Elmer GeneAmp® System 9600 with
hotlid. The cycling parameters were as follows: denaturation at
95°C for 3 min, 10 initial amplification cycles (94°C for 30 s,
60-55''C for 30 s, 72°C for I min. with a decrease in the annealing
temperature of 0.5°C per cycle), a further 25 amplification cycles
(94°C for 30 s, 55°C for 30 s, 72"C for 1 min) and final extension
at 72°C for 5 min. Negative controls, without DNA template, were
prepared for each series of amplifications to exclude the possibility
that PCR reagents and buffers were contaminated with template
DNA. Amplification products were examined by electrophoresis
through a 2% agarose gel (GIBCOBRL) made up in 1 X TBE.
Gels were stained in ethidium bromide at a concentration of
0.5 (j.g/mL and visualized under UV light. A one-hundred bp lad-
der (GIBCOBRL) was run concurrently to facilitate sizing of am-
plification products.

DNA Sequencing

PCR products were sequenced to confirm variation in restric-
tion fragments and sizes, and to improve PCR primer design. PCR
products were purified using Wizard™ PCR purification columns
(Promega) according to manufacturers instructions, and sequenced
using an ABl Prism '^' BigDyeT" Terminator Cycle Sequencing
Read\ Reaction Kit (Perkin Elmer). Cycle sequencing reactions

Southern Hemisphere Abalone Identification

were electrophoresed on an ABI377 automated DNA sequencer
(Perkin Elmer) and analyzed using ABl Prism '^' Sequencing
Analysis Version 3.3 (Perkin Elmer).

RFLP Analysis

For each individual of the I I species, four separate restriction
digestions of the mtCOI fragment were performed using the four
enzymes Ddcl, Hhal. HiiiFI and Hpall (New England Biolabs.
Genesearch). For the mtCOII fragments five separate restriction
digestions were performed for each species individual using the
enzymes Dclel. EcoRV. Hhal, Hpall and Rsal (New England
Biolabs, Genesearch). Restriction digestions were carried out in a
15 (jlL total volume consisting of 5 jjlL of PCR product, 1.5 p,L
digestion buffer supplied by the manufacturer, 0.5 [xL enzyme, and
8 (jlL ddHoO for all enzymes except Hlial. Digestions for Hlial
were carried out in a 15 \i.L total volume consisting of 5 p-L of
PCR product. 1 .5 |j.L digestion buffer supplied by the manufac-
turer, 0.5 p-L enzyme, 1.5 |j.L 10 X Bovine serum albumin (BSA)
and 6,5 |xL ddH.O.

Mitochondrial haplolypes were scored by electrophoresis of 10
|xL of digested PCR product in a 3% agarose gel made up in 1 X
TBE at lOOV for 3 h, stained in ethidium bromide (0.5p,g/mL) and
visualized under UV light. Electrophoresis of restriction digestions
was also performed on 12% polyacrylamide (Austral Scientific)
gels made up in 1 ,\ TBE and run for 2 h at 100 V.

RESULTS

DNA Exlraclioii and PCR Amplification

DNA e.xtraclions from fresh, alcohol preserved and canned
tissue, resulted in high yields of high molecular weight total ge-
nomic DNA. Amplification of these extracts consistently produced
high quality PCR products.

Extractions from mucous scrapings and fluid samples from
plastic bags produced a small amount of high molecular weight
genomic DNA. PCR amplification of these extracts failed at times
to yield a product when undiluted, however when diluted 10 fold,
produced a strong PCR product in all samples (Fig. 1).

PCR amplification of non-Haliotis DNA with the designed
primers was only observed in the tuna samples. Both tuna species
amplified ( 160 bp fragment) with the mtCOl primers. Sequencing
of the tuna mtCOl products confirmed that the observed product
was not contamination from abalone DNA, While nucleotide dif-
ferences and RFLP cut site differences existed to separate these
teleost products from abalone products, high levels of nucleotide
sequence conservation suggests that the amplified product was part
of the tuna COI gene.

Restriction Digests intCOI

The expected 193 bp fragment was generated in each abalone
species following PCR amplification with the HALCOl-NGl/
HALC01-NG2 primers. Comparison of the DNA sequences indi-
cated suitable restriction sites for discrimination between species
using four restriction enzymes (Fig. 2).

Intraspecies restriction digest polymorphisms were observed in
four species, but in each case for a single individual for only one
enzyme (Table 1 ). Two of the observed polymorphisms were the
result of a loss of a restriction site and two the result of a gain. All
individuals showing a different restriction fragment profile for the
species were sequenced to confirm the profile. All other digestions

10 11 12 13 14 15 16 M

Figure 1. Electrophoretic analysis of undiluted (lanes 1 to 61 and di-
luted (xlO. lanes 9 to 141 193 bp mtCOl (upper image) and 159 bp
mtCOII (lower image) PCR fragments for H. rubra tissue and mucous
samples. M = UK) bp ladder. Samples in lanes are as follows: 1, 2, 9 and
10 muscle tissue: 3. 4, 1 1, and 12 fluid sample from plastic bag: 5, 6, 13,
and 14 mucous swab from plastic bag; 7 and 15 positive H. rubra DNA
(x20 dilution) control: 8 and 16 negative H,0 control.

returned a single restriction pattern in all individuals examined for
each species.

At this 193 bp fragment, six of the eleven species had a unique
species-specific restriction pattern for at least one enzyme, and so
could be individually identified (Table 1 ). With the exception of
the H. rubra and H. conicopora pairing, all species are discernible
from each other using the four restriction enzymes, regardless of
all but one observed polymorphism. The exception polymorphism
was a single H. nihni individual that had gained a Dilel cut site,
and therefore had a profile similar to H. scalaris. The restriction
profiles for the canned tissue, mucous and fluid samples all
matched that expected for H. rubra.

One purported H. scalaris individual returned a different pro-
file at three enzymes to all other H. scalaris individuals. This
particular individual displayed the expected cut pattern for H. lae-
vigata for all four enzymes; three of which are diagnostic between
the two species for all other specimens analyzed. Laboratory con-
tamination was ruled out and the results confirmed with repeated
tissue sampling. DNA extraction and PCR amplification for this
one individual.

The observed fragment lengths produced in this study were all
examined on agarose and (non-denaturing) polyacrylamide gels
and confirmed by sequence analysis. A fragment mobility change
was observed in the mtCOI fragment for H.inidae when run on a
polyacrylamide gel (Fig. 3). This assumed conformation-induced
mutation was only observed in H. midae.

Restriction Digests mtCOII

The expected 159 bp fragment was generated in each abalone
species following PCR amplification with the HALCOIGENA/

222

Elliott et al.

H. rubra

1
CTGACATGGC

TTTTCCTCGA CTAAATAATA

TAAQATTCTG
HinFI

50
ACTACTCCCA CCCTCACTAA

CCCTTCTATT

100
AACATCGGGT GCTGTAGAAA GTGGTGrrqr;

Hpall

.C.
.C.

. . . .A. .

C.

C.

.C.

.c.
.c.

.A

C.

, .c. .c.

HmFI

G

T

. .T

T

. GT . . . .

G

. . .A C.

Hpall

H. scalaris

HmFI
-G

T

. .T

T

.G

. . .A

H.asinina

HinFI

-G

HinFI

linFI
. . .T.

. . .T.

C ,

.T. .C ,

, .A. .C. .C.

, .T

. .A. .TT.G.
. -T. . .T. . .

.T.
.T.

.TT

.T.
.T,

. A . . TC .
.GT. . . .
.A. .T. .

r.A. .C. .

. . ,r G . . . .

Hpall
. . .A

. . .A. .G . . . .

.c.

.G

Hpall
. . .A. .

H.midae

.A A. .

.0

.A A. .

.A. .T

.G

CIGACATRGC

A.
C.
G.

.c.
.c.

.N.
'TYC

T.

HinFI
.C

...„ ^^.

HinFI
.G T.

"CICGA CT -

c.

c.

c.

.c. .c.

.c. . . .

HinFI I

Hpall
. . .A. .

HinFI
-G , .

H.australis

H.iris

H. virginea

HALCOl NGl

HmFI
T. .

T. .

T. .

G

. .T.
. .T,

.T. .C .

.T. .C ,

T .

. .A. .T. .C.
. .A. .C. . . .
. .G. .CT. . .

TC .

CC .

. A . . TC .

G. .

C. .

^ — ..

Hhal
. . .A. .A . .0.

. . .A. .G . .C.

. . . .c

Hpall
. . . .C A. .

.C..A..

Hhal

HALC01-NG2
H . rubra

101
GACAGGATGA

ACAGTCTACC CCCCACTATC

CAGCAACCTA

150
GCCCATGCCG Gf^QQATCAGT
Hpall Hhal

AGACTTGGCA

ATTTTTTCAC TTCACCTAG£ CGG
Hpall

200

H. laevigata

H. scalaris

Hpall

Hhal
.T

Hhal
.T

.A

G. .

.C

A

Hpall

Hpall

. . .T

Hpall

H.asinina
H. roei

T. .

. . .G. . .

C. .

A G. .

c

G .!..

Ddel
T. .T C

. . .T

T. .G C

T. .T T

T. .T T

. . .T T

-

Hpall

G.__ ,

Hpall

Ddel
ICC. . T

A

..CC. .A..

c

Hpall
Ddel Hpall

T

A. . ,
A. . ,
A. . ,

A. . .

. . .C. . .

, . .C. .G

, . .G. . .
. . .C. . .

. . .T. .A. .
. . .T. .G. .

A. .

T T. .

T. .

. . .T. .

. . .T. .
TT. . . .
TT . G . .
C

.A. .
.A. .

T

Hpall

Hpall
C. .T. ,

. . .A. .

Ddel

Hhal

Hpall

Hhal
G

C. .A. . .

Hpall

H.iris

.T. .

...A. ,
C. .G. ^

• * ■ c

C T

C C A

T

Hpall

H . virginea

Hhal
,.,... .r. .

.CT

NO C

Hpall

HALCOl-NGl
HALC01-NG2

Hhal

Hpall

TAAAARAGIG AIGTGRATCG GCC

Figure 2. Stquence alignment ol the 193 bp mtCOI fragment for eleven abalone species. Primer sequences and cut sites for the four restriction
enzymes Ddel, Hhal. HinFI, and HpaH are included. (N = sequence data unclear whether (.' or T).

TABLE 1.

Expected restriction fragment lengths for eleven Haliulis species for the 193 hp mfCOI fragment when cut with restriction enzymes Ddel.

Hhal. HinFI and Hpall. The number in parenthesis represents the total number of individuals examined for each species that displays the

given restriction pattern. Unique restriction profiles are shown in bold. H. laevigata numbers include the misidentified H. scalaris individual.

COI Restriction Digestion Patterns

Ddel

Hhal

HinFI

HpaH

H. Lisimna

H. aiislrulis

H. t onictipora

H. in.s

H. hii'\'igiita

H. midae

H. roei

H. ruliro

H. .sdiltiri.',

H. spudicea

H. virginea

7.5I).I37(29)
7,187 (X)
193(10)
193(11)
193(10)
193(63)

193(10)
29,164(10)

193(49)
29.164(1)
29,164(21)

193(10)

193(10)

193(30)

79,114(10)
42.151 (ID
42.1.51 (10)

193 (62)
94,99(1)
42.151 (10)

193(10)
42.15! (50)

42.151 |21)
42,151 (10)
42,57,94(9)
42.151 (1)

-M.159(.Wl

193(10)
34,159(11)
19,174(10)
34,159(62)

193(1)
.U.159(10)
6,34,97(10)
34.159(49)

193(1)
34,159(21)
15,19,159(10)

193(10)

3.42.72.76(30)

3,93,97(10)

3,42.51.97(11)

3,190(10)

3,42,148(63)

3,42.148(10)

45.51.97(10)

3.42.5 L97 (50)

3.42,51.97(21)
3.190(10)
3.190(10)

Southern Hemisphere Abalone Identification

223

Ddel
1 2 3

Hhal
2 3

Hpall
1 2 3

Figure 3. RFLP patterns on a non-denaturiny polvacrylamide gel of
the 193 bp nitCOI fragment for three abalone species produced with
four restriction enzymes. Species \ - H. midiie. species 2 - H. rubra,
species 3 - H. laevigata. M - 100 bp DNA ladder. Reduced mobilty in
H. midae fragments suspected to be due to a conformational mutation.

HALC02GENB primers, except H. iris that failed to amplify
for all ten individuals examined. Comparison of the DNA se-
quences for the other species indicated suitable restriction sites for
discrimination between species using five restriction enzymes
(Fig. 4).

All restriction digestions for the five enzymes resulted in a
single restriction pattern for each species, except for two enzymes
for H. rubra (Table 2). The two polymorphisms were each ob-
served in two different individuals, all were sequenced to confirm
the observed RFLP. None of these four individuals was respon-
sible for the polymorphisms observed at the mtCOI fragment, and
the individual H. nihra with a mtCOI profile similar to H. scalaris

was clearly identified as H. rubra at this fragment. The restriction
profiles for the canned tissue, mucous and tluid samples all
matched that expected for H. rubra.

As with the mtCOI RFLP analysis, an unusual species profile
was observed for three enzymes with a single H. scalaris indi-
vidual (the same individual), and again all three profiles match that
recorded for//, laevigata. Sequence data showed a 100% similar-
ity to H. laevigata across the 159 bp fragment, while three other //.
scalaris samples each differed at 9 nucleotides from the H. laevi-
gata sequence.

At this 159 bp fragment, three of the ten species (excluding H.
iris that did not amplify) had a unique restriction pattern for the
enzyme Ddel and so could be individually identified (Table 2). In
addition to the //. ruhra/H. coiiicopora complex it was not possible
to separate H. australis and H. spadicea using the five enzymes on
this fragment. All remaining species combinations were separable
from each other using one to five of the enzymes (Table 2).

Fragment mobilities on polyacrylamide gels were all consistent
with known fragment lengths; no conformation induced mutations
were observed in the mtCOIl fragment.

DISCUSSION

The ability to identify abalone species from tissue samples
and/or mucous is important to the continued survival of significant
abalone fisheries. The tests described in this article will provide
one more tool in the fight against illegal fishing, which has the
potential, along with commercial over-fishing and environmental
variables (Davis et al. 1998, Shepherd et al. 1998), to lead to the
decline and collapse of fisheries. The methods are straightforward

H. rubra
H.conicopora

H. laevigata

1
CAATTTGAAC

CATTCTACCA

50
GCCATTATCC TTATTTTCCT CGCCCTACCA

TCCTTGCGGC
Hhal

TCCTTTACCT

ACTAGACGAA

GTCGGTATAT

100
CGTGCCTTCT

c

-C.C.

Hhal

.T. .A

.T

.CI

Ddel
CT. .......

. .A.
. .A.

.G

EcoRV
.A. . . .

_._C

C.
T

. . . .c. . .

c

C. .c.

... .G. -C.

. . . . A. . .

Ddel
... A

. .G. . .

. .T. . .
. .C. .

Hhal
A. . . .

. .TCC. .A.

. . . .A . . .

.C

Ddel

. .T.
. .A.

.G

EcoRV

_._T CT.

T.
C.

. . . .c. . .
c. . .

. .T. . .

C. .C.

A. .0.

.C C. .

H. midae

H. spadicea

H. australis

H.Virginia

HALC02GENA
HALC02GENB

H. rubra

H. conicopora

H. laevigata

. .0. . .

. . TC A . . A .

. . . .C

C

. .G.

. . . .G.

.A. .T. .CT.

CAATYTGAAC

1 2. . .

. .C

YATTCTMCCA

C

C

. .TT .A

A. .C.

A. . .

■.,1... ^.■
Ddel

. .G. .G

..C.C

. . .CA. .A.
. . AC T . . A .

.T

C T. . .

T T. .G

.A

.A CT .

.A A. .

101
AACAATCAAG

GCAACTGGTA

ArrAGTGATA CTGAGG
Ddel

CTAC GAAl

150
^ACTCAG
Ddel

159
ACTTTAAGG

. . .G.

.T.

T

.A

. . .G. . . .

. . .A. . . .

...C..A.
Hpall
. . .C. . . .

.T. . . .
.T. .A.
... .A.

Ddel
-G

Rsal Ddel
-G G. .

Ddel
DdeJ

Rsal
G. .

Ddel

.T. .A.
... .A.

. G G . .

Ddel

. . .G.

T

Hpall

Rsal

Ddel

.T.
.T.

.A
-A
.A

. . .r . .c.

Hpall
...C..A.

Hpall
...C.c.

Hpall

... .A.
... .A.
... .A.

Ddel

Ddel

Ddel
T

Ddel

Ddel

Ddel

HALC02GENA
HALC02GENB

DdeJ

--

--

--

CCGATG CTTATGAGTC

TRAAATTCC

Figure 4. Sequence alignment of the 159 bp mtCOII fragment for ten abalone species. This fragment did not amplify for H. iris. Primer
sequences and cut sites for the five restriction en/ymes [)del. EcoRV, Hhal. Hpall, and Rsal are included. (1 = sequence data unclear whether
C or T: 2 = sequence data unclear whether C or .A).

224

Elliott et al.

TABLE 2.

Expected restriction fragment lengths for 10 Haliotis species for the 159 bp mtCOII fragment when cut with restriction enzymes Ddel.

KcoRV, Hhal. Hpall, and Rsal. H. iri\ did not amplify with these primers. The number in parenthesis represents the total number of

individuals examined for each species that displays the given restriction pattern. I nique restriction profiles are shown in bold. H. luevigala

numbers include the misidentified H. scalaris individual.

con Restriction

Digestion Patterns

Ddel

EcoRV

Hhal

Hpall

Rsal

H. asinina

13.3H,108(30)

71.88(30)

159(30)

44.115(30)

159(30)

H. australis

13.15.131 (10)

159(10)

159(10)

44.115(10)

159(10)

H. coiiicopora

13.15.131 (11)

1 59 ( 1 1 )

58.101 (11)

159(11)

159(11)

H. iris

H. Icievigahi

13,15,60,71 (63)

71.88(63)

58.101 (63)

159(63)

30.129(63)

H. midae

13.15,131 (10)

159(10)

159(10)

159(10)

159(10)

H. roei

13,146(10)

159(10)

159(10)

44.115(10)

30.129(10)

H. rubra

13.15.131 (50)

159(50)

58.101 (48)
159(2)

159(48)
44.115(2)

159(50)

H. scalaris

13.71,75(21)

159(21)

1 59 1 2 1 1

159(21)

30.129(21)

H. spadicea

13.15.13! (10)

159(10)

159(10)

44.115(10)

159(10)

H. virginea

13.38.108(10)

159(10)

159(10)

44.115(10)

159(10)

and suitable for use in any laboratory with basic DNA analytical
equipment. The PCR-RFLP tests utilize short DNA fragnients that
can be amplified from processed products and slightly degraded
material, and therefore are of potential forensic use.

Care has been taken in this study to include e.xantination of
intraspecies variation as well as possible non-Hiiliolis amplifica-
tion with our PCR primers. Samples from different geographic
locations were examined for the two main Australian commei'cial
species (H. rubra, five locations and H. laevigata, three locations).
While not exhaustive, the results suggest that what limited in-
traspecies variation exists can be accounted for using the two
fragments and multiple restriction enzymes. Both PCR primer sets
devised for the test are relatively degenerate and so cross genus
amplification was not unexpected. However, of the groups we
have exaniined only DNA from the tunas {TIntnnus spp.) anipli-
fied, and it was possible to easily differentiate these from Haliotis
species.

The restriction patterns produced by Dciel for the intCOIl frag-
ment would discriminate three of the species, while five other
species would be differentiated by a single restriction pattern at the
mtCOI fragment. Such species-specific patterns are useful, how-
ever as rare polymorphisms may exist it would be wise to confirm
identification with multiple enzyiT)es and/or both short fragments.
None of the rare polymorphisiT)s observed occurred in niore than a
single individual, and no individual displayed (nore than one varia-
tion. With the exception of the H. riihra/H. coiiicopora pairing, all
other combinations of the 1 1 abalone species can be differentiated
from each other using two or more of the restriction profiles shown
in this study. We therefore reconimend using both fragments and
at least two of the restriction enzymes included here to differen-
tiate species.

The ability of our test to differentiate between species was
inadvertently put to the test during the intraspecies examinations.
Of 20 purported H. scalaris individuals, one was found to display
a different restriction profile at six of the nine profiles examined.
The combined profile of this individual matched completely the
expected profile for H. laevigata: and was confirmed by DNA
sequence analyses. Although occupying different niicrohabilats,
these two species have overlapping distributions and co-occur in

the saiT)e area (Shepherd 1973). Shell and mantle morphology did
not separate the abeirant individual from other H. scalaris indi-
viduals. This individual is either a H. laevigata and morphological
characters between the two species are more plastic than currently
recognized, or a hybrid between the two species.

Naturally occurring hybrids between abalone species with
overlapping ranges, although relatively rare, have been reported
(e.g., Talmadge 1977; Sasaki et al. 1980: Aral et al. 1982; Messier
& Stewart 1994). The two Australian species H. rubra and H.
laevigata, also show evidence of backcrossing and introgression
(Brown 1995). There are no records of hybrids between H. laevi-
gata and H. scalaris. but H. laevigata is more closely related to H.
scalaris than to H. rubra (Brown & Murray 1992), and hybrids
would not be unexpected. Allozynie analysis of the abenant indi-
vidual could not confirm nor refute its putative hybrid status as
there are no known diagnostic loci between the two species
(Brown 1991).

The possible existence of hybrids, albeit at low frequencies,
does not minimize the validity of our mtDNA-based test for aba-
lone. However, the possibility of hybridization and backcrossing
between species does question the legal "species identity" of an
individual. If hybrids were infertile and only Fl hybrids were
possible, then a single diagnostic nuclear DNA marker would con-
firm the individual as a hybrid, and the mtDN.A marker would
confirm the maternal species. Such individuals could be legally
classed as hybrids. However at least some abalone hybrids appear
to be fertile and backcrossing occurs (Brown 1995), and identify-
ing the "nuclear lineage" of a potential backcross offspring would
require multiple nuclear DNA markers. Even then it could never be
proved that an individual was not the offspring of a backcrossing
event, except based on probability. A suite of nuclear DNA mark-
ers could never disprove a claim of backcrossing, although making
it impi'obable. On the other hand using a mtDNA-based test, the
maternal lineage of the individual can always be validated. We
suggest that for legal purposes where hybrid backcrossing may
exist between abalone species that the genetic "species identity" of
an individual be classified as its maternal lineage, which can be
confirmed from its mtDNA. Hybrid individuals (those with
nitDNA of one species and nuclear DNA wholly or partly of

Southern Hemisphere Abalone Identification

225

another species) while biologically acknowledged should not be
legally recognized as the existence of backcross hybrids cannot be
disproved except by probability based on a large number of diag-
nostic nuclear DNA markers. Mitochondrial DNA in abalone as in
most organisms appears to be only maternally inherited (Conod
2000). The aberrant individual in our study therefore is classed as
H. laevigata.

The advantage of the tests described here to previous studies
(Sweijd et al. 1998) for abalone is the smaller size of the DNA
fragment; an advantage when examining processed or slightly de-
graded material (Mackie et al. 1999). The lysin gene protocol
described by Sweijd et al. (1998) did aim for fragments less than
300 bp. but the presence of an intron increased this at least three
times, and for H. laevigata by about ten fold (unpublished data).
The authors did however successfully use PCR primers for a
smaller 146 bp fragment to discriminate between canned H. luidae
and H. rubra products.

PCR inhibition was observed when testing our primers on the
mucous samples of H. rubra. Dilution ( 10 fold) to a lower con-
centration did not have the same inhibitory effect. Similar PCR
inhibition due to high levels of polysaccharides is common in plant
tissue extracts (Fang et al. 1992), and inhibition due to muco-
polysaccharides in the abalone mucous may have caused the ob-
served PCR failure.

No DNA sequence variation was observed between H. rubra
and H. conicopora in either short mtDNA fragment examined in
this study. In an assessment of all recent taxa in the family Hali-
otidae, Geiger (1998) concluded that there was some justification
for sub-species recognition of conicopora under H. rubra. Allo-
zyme data suggested conspecifity but shell and geographic distri-
butions indicated distinct taxa. Fifteen of 22 DNA microsatellite
primers developed for use in H. rubra amplified a similar product
in H. conicopora (Evans et al. 2001). This compares to the con-
servation of only 1 2 of the 22 markers in other temperate Austra-
lian species (H laevigata. H.scalaris and H. roci). Our short DNA
sequences lend some support to the possibility of sub-species sta-
tus for conicopora. however further research is required to resolve
the issue.

The altered mobility of the H. midiu' mtCOI fragment run on
polacrylamide gels is most likely due to a conformation change.
Conformational mutations attributed to sequence-specific varia-
tions are restricted to polyacrylamide gels and not seen on agarose
gels (Singh et al. 1987). The location and conservation of this
conformation variant requires further investigation. Its presence,
however, raises a note of caution when using RFLPs as mobility
variation of fragments seen on polyacrylamide gels may be mis-
leading as they can be length or conformation polymorphisms. It is
therefore recommended that species differentiation using the
RFLP tests described here be run only on agarose gels.

The test described here fulfills the aim of our study to provide
a relatively straightforward and cost-effective means for identify-
ing several abalone species of commercial importance to Australia.
Costs for any DNA-based analyses are not insignificant, but the
PCR-RFLP technique is generally considered more cost-effective
for routine species identification than alternatives such as direct
DNA sequencing of the PCR product (e.g.. Asensio et al. 2000).
The opportunity to sequence a PCR product is of course still avail-
able for differentiation of individuals if problems arise following
PCR-RFLP analysis.

To increase the potential value of this study to the sustainability
and protection of abalone fisheries worldwide, additional species,
particularly from Northern Hemisphere waters, need to be incor-
porated either into this test or a modified one. so that a single test
is available for discriinination of all abalone species.

ACKNOWLEDGMENTS

This study was funded in part by the Australian Fisheries Re-
search and Development Corporation (Project 1999/164), with ad-
ditional financial assistance from the Tasmanian Abalone Council
and the Tasmanian Marine Police. The authors are grateful for
their support as well as that provided by the South African Police
Services. Phil and Audrey Critchlow. Sandy Degnan. Greg Magu-
ire, Elizabeth O'Brien, Rodney Roberts and Tasmanian Seafoods
Pty Ltd. Sharon Appleyard, Malcolm Haddon and Bob Ward pro-
vided useful comments on an earlier version of this paper.

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Jourmil of Shellfish Research. Vol. 21, No. 1, 227-231. 2002.

DISTRIBUTION AND ABUNDANCE OF STROMBUS COSTATUS (GMELIN, 1791) LARVAE AND
ADULTS AT THE BIOSPHERE RESERVE: BANCO CHINCHORRO, QUINTANA ROO, MEXICO

ALBERTO DE JESUS NAVARRETE

Dcpiirniiiieiito tie Pesqiierias Artesanales. El Colei(io de hi Fronrera Siir. Unidad Clictuiiuil. A. P. 424,
Chetuimd Q. Roo, Mexico. C.P.: 97UU0

ABSTRACT In order to study the distribution and abundance of Strombus costulus. larvae and adults, samples were collected
bimonthly from August 1997 to July 1998 at six sites at Banco Chinchorro. To collect larvae, replicate surface plankton tows were
made with a conical net. Larval density ranged from 0.0018 ± 0.026 veligers/10 m' to 4.77 ± 3.50 veligers/10 m'. Presence of all larval
stages suggested a complete development of veligers from egg stage to metamorphic competence in the reef lagoon; 8.529}- of the larvae
corresponded to stage II veligers. 18% to stage III and 72.65'7f to stage IV. At the bottom, all conch found within three 100-m- replicate
areas were counted, shell lengths were measured and egg masses recorded. Conch density varied from 0.0025 to 0.22 conch/m- with
a dominance of juveniles. The total population in Banco Chinchorro was estimated at 1.3 x 10* conch, but only 8% were estimated
to be of legal size. Larvae and adults were more abundant in Cayo Centro, the principal distribution site. Egg masses were abundant
from May to October with a greater abundance in May. Banco Chinchorro is an important source of S. costatus veligers and sustains
an adult and juvenile population within the reef lagoon. Nevertheless, this S. costatus population is not large enough to support a
commercial fishery. It is necessary to protect the reproduction sites to maintain larval supply downstream.

KEY WORDS: Caribbean, distribution, larvae. Strombus costatus

INTRODUCTION

In 1997. Mexico harvested 140.021 tons of mollusks. with a
total value of US$39.79 million. This harvest represents 10.06% of
the country's total fish and invertebrate catch and couesponds to
4.15% of the total economic value (SEMARNAP. 1998).

In the Yucatan Peninsula, the mollusk fishery is niultispecific;
catches principally consist of i|ueen conch iSlrdiiihiis fiigas Lin-
naeus. 1758). and on a minor scale, milk conch (SiroiiihK.'i antaliis
Gmelin, 1791), and the fighting conch (S. piigilis Linnaeus. 1758).
Other gastropods are also caught, such as the red conch. (Pleu-
roploca gigantea, Kiener, 1840) and the black conch. (Xancus
angulata. Lightfoot. 1786) (Sosa-Cordero et al. 1993; Perez
1997).

The milk conch (5. costatus) is widespread in the Caribbean,
but it is commercially caught only off the Yucatan (Stoner 1997)
where it has the same market value and demand as the queen conch
(Aldana-Aranda & Patiiio-.Suarez 1998). In the South of Quintana
Roo fishing regulations only allow harvesting mollusks in Banco
Chinchorro. The queen conch is the targeted species and the milk
conch are not harvested.

The decline of 5. gigax. between 1985 and 1990 in the Yucatan
Peninsula, led to the closure of the conch fishery off Yucatan State
and a ban in some areas off Campeche State and northern Quintana
Roo State. In addition, a system of capture quotas was established
in sourthern Quintana Roo.

With the descend of the queen conch fishery, alternative
sources such us milk conch could be caught to meet the growing
demand for conch meat earmarked for the local markets and the
Costa Maya's tourism industry in southern Quintana Roo. The
potential for the non-exploited resource milk conch. S. costatus,
needs to be evaluated.

There are only a few studies on milk conch in the Caribbean.
Percharde ( 1968) studied the distribution of the genus Strombus in
Trinidad and Tobago. Brownell ( 1977) repotted that S. gigas was
the most important fishery resource in Los Roques. Venezuela, but

E-mail: alberto@ecoosur-qroo,mx

that other mollusks such as S. co-ttatus and S. piigilis also contrib-
uted to the fishery, Appeldoorn (1985) studied the growth, mor-
tality and dispersion of laboratory reared S. gigas and S. costatus
in Puerto Rico, and Berg et al. ( 1989) described the abundance and
distribution of S. costatus in Bermuda. In Mexico. Aldana-Aranda
et al. (1989) studied the effect of temperature and algal food on
larval growth of milk conch. Recently. Aldana-Aranda and Patiho-
Suarez ( 1998) reviewed algal diets used in larviculture. of several
Strombid species, including the milk conch, and Shawl et al. (in
press) reared S. costatus juvenUcs raised from egg masses laid in
captivity.

There are no studies related to the milk conch at Banco Chin-
chorro, Consequently, the objective of this work is to describe the
distribution and abundance of 5. costatus adults, determine their
reproduction sites, and evaluate larval abundance. This study tests
the following hypotheses: ( I ) the lagoon reef is an important site
for reproduction and distribution of milk conch; and (2) there is
complete larval development (egg to metamorphosis competence)
of this specie in Banco Chinchorro,

MATERIALS AND METHODS

Study Area

Biosphere Reserve Banco Chinchorro is a false atoll on Quin-
tana Roo's South coast, within the Mexican Exclusive Economic
Zone (18°23'; I8°47'N. 87°14'; 87°27'W) (Jordan and Martin
1987) (Fig. I ). Chinchorro is 46 km long and 19 km at its maxi-
mum width, with a reef lagoon area of 560 knr. The reef lagoon
has an extensive sand bottom with patches of sea grass. In the
North these species are most abundant. Thalassia testiidiiuun
Banks ex Koning. Halodule wrigthii Ascherson. and Syringodium
fdifonne Kiitzing. Reef patches are common in the South region of
the lagoon.

Banco Chinchorrt) has four keys; in the north there are two
small keys known as Cayo Norte. In the central area, there is Cayo
Centro, which is the largest, and in the southern area. Cayo Lobos,
which is the smallest (Fig. 1).

227

"* '8

DE Jesus Navarrete

Kisure 1. Map of Banco Chinchorro showing six sampling sites within
the reef lagoon.

The dry season extends Ironi March to June, while the rainy
season is from July to October, and the cold season, characterized
by strong northerly winds ("nortes"). is from November to Feb-
ruary.

Sampling Methods

Samples were collected bimonthly from August 1997 to July
1998, within the reef lagoon, in six traditional queen conch fishing
sites: Cayo Lobos (I8°23'45, O'N, 87°2r 20.9'W), Isla Che
(18°30' 12.3'N. 87°26'13. I'W). Cayo Centro {18°33' 32.7'N.
87°18' 24..'^'W), Cayo Centro Oeste (18°33'24, I'N. 87°24';
56.6'W). Penelope (18°42' 47.6'N. 87°14'55.5'W). and Cayo
Norte (18°45'28. I'N. 87''47'()1. 1 "W) (Fig. 1). At each site, tem-
perature (°C) and dissolved oxygen (mg/1) were measured at sur-
face level using an oxygen meter (YSI model 58). Salinity was
measured with a thermoconductivity meter (OHALUS model 50).

Replicate surface plankton tows were made at each site using a
conical net. 0.50-m dia. with a 202-jjLm mesh size. Plankton tows
were conducted for 15 min at a velocity of -1.0 m/min. Water
\oluiTie was measured with a flow meter (General Oceanic model
203 IH) attached to the net. Plankton were fixed with a mixture of
neutralized 5% sea water-formalin (Stoner & Davis 1997a). Tows
were conducted diurnally and additional night collections were
made at Cayo Centro and Cayo Lobos.

Gastropod larvae were sorted from other plankton using a dis-
secting microscope (x20). Identification and developmental stages
(I to IV) were assigned following the descriptions of Davis et al.
( 1993). and larvae were counted and measured for maximum shell
length. Total larval abundance was standardized to larvae/10 m\

To determine the abundance of juvenile and adult conch, bi-
monthly samples were conducted at each site. All conch found in
each of the three circular unit replicates (100 nr) were counted.
The shell length and lip width were measured to the nearest mm
using calipers, after measurements all conch were returned to the
sea bottom. Egg masses within the unit samples were counted.

Conch abundance was compared among months and sites
sampled using a two-way Analysis of Variance ( ANOVA). on Log
(x -I- I) transformed data. The procedure considered independence
between sites and months.

RESULTS

Maximum mean temperatures were recorded during August
and October, 28.9 ± 0.55 and 29.3 ± 0.28°C (/! = 6), respectively.
whereas lowest temperature occurred in December (26.3 ± 0.94 n
= 6). Dissolved oxygen varied between 5.87 ± 0.37 mg/1 in July
and 7.01 ± 0.56 mg/1 (/; = 6) in August. Salinity ranged from 35.9
± 0.12% in October to 37.0 ± 0.9%f {n = 6) in March.

A total of 245 larvae were collected over the 1 2 months. Most
larvae were collected in July during night tows (158 veligers at
Cayo Lobos and 75 veligers at Cayo Centro). Diunial tows con-
tained a very low number of veligers. Six larvae were collected in
October; one at Isla Che. one at Cayo Centro. and four at Centro
Oeste. In May, six larvae were collected at Penelope. No veligers
were found in August. December, and March. Larvae were cat-
egorized into the following stages, 8.53% of larvae were stage II
(451-750 |jim). 18.77% corresponded to stage III (751-950 jxm)
and 72.65% were stage IV at Cayo Centro in October, to 4.77 ±
3.50 veligers/10 m" at Cayo Lobos in July (Table 1 ).

A total of 280 juvenile and adult conch were collected during
the sampling period. Conch abundance was significantly different
between sites [P = 0.0036), Cayo Centro had the highest density
and Isla Che had the lowest (Tables 2 and 3). A total 184 indi-
viduals were found at Cayo Centro; 89 conch were collected in

TABLE 1.

Total number of larvae and density (veligers/KI m') of Strombiis coslalus (all stages) collected at Banco Chinchorro. August 1997 through

July 1998. No ^eligers were found in August. December, and March.

Cayo Lobos

Isla Che

Cavo Centro

Counts

Density

Counts

Density

Counts

Density

October

May

July+ l.'^S

4.77 ± 3..M)

1 ().()]8±0.03 1 ().()26 + 0.0.^

7.S 1.62 ±1.07

i Night ccillections

C. Centro Oeste

Counts

Density

U.ll ±0.15

Penelope

Counts

Density

0.17 ±0.24

S. cosTATUs Larvae and Adults at Banco Chinchorro

229

TABLE 2.
Milk conch adults density (no. conch/ni'^) at Banco Chinchorro.

Cayo Lobos

Isia Che

Cayo Centro

C. Centro Oeste

Cayo Norte

August

0.0075

0

0.0075

0.0025

0.0100

October

0.0050

0.0025

0.0825

0.0025

0.0200

December

0.0125

0.0025

0.0025

0

0

March

0.0725

0

0.2225

0.0125

0.0125

May

0

0

0.1400

0

0.0025

July

0.0125

0.0025

0.0050

0.0050

0.0550

March, 56 in May. .^.^ in October, 3 in August, 2 in July, and 1 in
December. Juvenile and adult density varied from 0.0025 conch/
m~ to 0.222 conch/ni". Despite significant differences between
sites (Table 3), a uniform conch distribution was used to calculate
the population size at Banco Chinchorro. Based on the lowest
conch density (0.0025 ind/m"^), and a lagoon area of 560 km" for
Banco Chinchorro, a total of 1.3 x 10** conch were estimated.
However, only 100,000 conch were estimated to be harvest size
(8%). This low population size does not appear sufficient to sup-
port a commercial fishery.

The highest numbers of conch found in Banco Chinchorro were
collected in March, with a total of 128 organisms. Fifty-seven
conch were collected in May. 45 in October. 32 in July, 1 1 in
August, and 7 in December. The size-frequency distribution varied
from 30 to 1 80 mm shell length with 92% of total sample of conch
in the range of 81 to 160 mm shell length (Fig. 2).

Reproductive activity, copulation, and the presence of egg
masses in medium sands, were observed from March to Deceinber.
A total of 1 13 egg masses were counted with the majority at Cayo
Centro (39 egg masses in October, 58 in May, and 1 in December).
Fifteen egg masses were counted at Cayo Lobos in March.

DISCUSSION

Temperature has a direct influence on the beginning and ces-
sation of conch reproductive activity, egg-hatching time, and on
the duration of the larval phase (Berg et al. 1989; Appeldoorn et al.
1983; Stoner et al. 1992; Stoner & Davis 1997a; Pechenik 1999).

At Banco Chinchorro, egg masses of S. cosiatiis were first
observed in March and were found until December when bottom
water temperature decreased to 26.3"C. In Banco ChinchoiTO. the
reproductive season of S. costatiis is during a ten-month period,
with a peak in copulation and egg laying in May. This period is
longer than in Trinidad and Tobago where mating occurs from
November until June (Perchard 1968) and longer than in Venezu-
ela, where reproduction occurs from November to May (Brownell
1977). In comparison, the reproductive season for queen conch, S.
gigas. has great variation. It varies from 5 to 12 months, with the

shortest season duration in Bermuda and Florida and the longest at
Banco Chinchorro (Cruz 1984; Corral & Ogawa 1987; Stoner et al.
1992). A difference in reproductive cycles of S, gigas and S. cos-
tatiis as a function of temperature was mentioned by Brownell
(1977). He observed that S. costatus reproductive season began
when the season for S. gigas usually ended. Brownell (1977) as-
sociated this with a one-centigrade degree decline in mean water
temperature. However, in this study S. aisluttis reproduction sea-
son was from March to December and coincided with S. gigas
reproduction period at Banco Chinchorro.

In Banco Chinchorro. the number of S. costatus egg masses
exceeded that of S. gigas (113 vs. 19 egg masses) for the same
study period (de Jesiis-Navarrete 1999). This could be an effect of
fishing activities, because only S. gigas is collected not S. costatus.
Females constitute a high percentage (-65%) of the S. gigas catch,
(pers. obs.) and that might also explain why there were higher
number of S. costatus egg masses, juvenile and adults than S.
gigas. in some areas, like Cayo Centro.

Fishing practices are known to modify the structure of popu-
lations by reducing the overall biomass (Alcala, 1988; Roberts,
1995), decreasing age and size at sexual maturity (Harnielin et al.
1995), and altering sex ratios and genetic structure (Ryman et al.
1995). The displacement of target species due to the effect of
fishing pressure and a consequently higher abundance of non-
target species was mentioned by Seijo et al. ( 1997). This may be
happening in Banco Chinchorro, as the 5. gigas population de-
clines there may be an increase in the 5. costatus density, due to
habitat and food resources becoming available.

A greater quantity of 5. costatus larvae was found in July when
mean water temperature was at its maximum. It is possible that this
is related to higher egg mass production, and increase in food
availability for the larvae (Davis 1998). Local water circulation
might also influence number of larvae retained inside of Banco
ChinchoiTO. Larvae were collected from nearly all stages (II, III
and IV) indicating a complete larval growth process within the reef
lagoon. Although the current circulation pattern of Banco Chin-
choiTO is unknown, it is probably influenced by trade winds, and

TABLE 3.
Results of two way ANOVA of S. costatus abundance in Banco Chinchorro Quintana Roo, Mexico.

Source of
Variation

Sum of
Squares

D.F.

Mean
Square

F-ratio

p-level

Sites
Months
Residual
Total

20.5434
8.24020

21.7742
50.5578

5

5

25

35

4.|{J86
1 .6480
0.8709

4.7170
1.8229

0.0036*
0.1316

230

DE Jesus Navarrete

August

O

c
o

cr

0

'-4— •

40

30 ^

20

10

0

40
30 -
20
1 0
0

40
30
20
1 0 -
G

4 0
30
20

I 0
0

40
30 -
20
1 0 -
0

40
30 -\
20
10 H

«=11

■Ijjjij

October

■ ill. ■

«=45

December

M arch

,»=128

lllli-

May

«=57

111.

July

iIlo*

«=32

Siphonal length (mm)

Figure 2. Relative frequency (%) and size distribution of juvenile and
adults of Siniiiihiis costatiis at Chincliorro Bank.

this ciaulation would transport larvae tov\ard the iiUerlor of the
reef lagoon causing local retention. In Florida. Stoner et al. ( 1997)
found that larvae of 5. costatiis were very abundant in early stages
and rare in advanced development stages. They noted that the
abundance of later larval stages was associated with water flow
produced by the wind, which suggests local recruitment from
spawning populations that inhabit outer reefs. The same effect
could occur in Banco Chinchorro. Evaluating populations outside
iif the reef lagoon might explain why 6S9f of the larvae were
collected at Cayo Lobes, the southern site of Chinchorro. The high
number of larvae may indicate a contribution from deep-water S.
costatiis populations or populations external to Banco Chinchorro.

The density of .S'. cosiouis larvae found ui this study (4,77 ±
3.50 veligers/IO m') is higher than the values reported from other
sites in the Caribbean. In Florida, densities of 0.04 to 1 .40 veiigers/
10 m' have been found with maximum abundance peaks in June
(Stoner et al. 1997). In the Bahamas. Stoner and Smith (1998)
reported 5. costatiis densities ranging from 0.026 to 0.069 veiigers/
10 m\ In Banco Chinchono. the higher percentage of S. costatiis
larvae m night tows (9.'ip). may be due to lack of intense surges and
wind action that were prevalent during the day. Stoner and Davis
(1997b) documented vertical movement of S. gigas larvae toward
deeper zones due to wind and surge effect, and Stoner and Smith
( 1998) noted that wind and surge also produce horizontal trans-
portation of larvae.

Density of S. costatiis adults and juveniles found in Banco
Chinchorro is higher (0.22 conchs/m") than other values reported
in the Caribbean. In Bermuda. S. costatiis density was highest in
the basins, (0.00299 conchs/ni"). than on the platform, (1.9 x lO"'*
conchs/ni") (Berg et al. 1989). In Puerto Rico, the density of 5.
costatiis varied from 0.18 x lO""* conchs/nr in reef patches to
0.0032 conchs/m' in coarse sands to 0.0049 conchs/m" in rubble
areas (Appeldoorn 198.5). Although the densities found in Banco
Chinchon-o exceed these values, 92<7f of the population consisted
of individuals smaller than 170-mm shell length, which cannot
support commercial harvest. In Yucatan State. 5. costatiis shell
sizes varied from 65 to 225 mm shell length, and legal fishing size
(180 mm) represented over 65% of the relative abundance of
conch catches, (Perez 1997). In Banco Chinchorro, the large size
was 180-mm shell length and this represented only 8% or an
estimated population of 100,000 conch. Therefore commercial
catch of this species is not recommended.

Banco Chinchorro was designated as a Biosphere reserve in
1996. The main goals were to conserve biodiversity, protection,
and enhance overexploited species, such as queen conch (S. gigas)
and spiny lobster [Paiuilints argiis). It is now clear that other
species should be considered in the management plan, including
the milk conch, S. costatiis.

Many marine reserves like Banco Chinchon'o, have small
nucleus areas to protect fishing species. At Banco Chinchorro,
only 3.2'7f of the total lagoon area comprises of protected zones. It
is likely that these areas do not contain the full habitats needed to
protect or enhance species during their entire life cycle. Therefore,
the role of these nucleus areas in protecting species and connecting
different habitats together cannot be totally ascertained (Appel-
doorn & Lindeman, in press). In the establishment of management
plans, data on distribution and abundance of adults and larvae,
reproductive sites, egg mass numbers, and recruitment dynamics at
a regional level are required. For example, the data from this study
suggests that critical areas like Cayo Centro with its high distrib-
tuion of milk conch adult, juveniles, egg masses and larvae needs
to become a conch protected area within the Biosphere Reserve
Banco Chinchorro.

In general, marine reserves help to protect some fishing spe-
cies, and can be effective in the recovery of commercial exploited
species (Alcala 1988; Roberts & Polunin 1991; Roberts 1995;
Allison et al. 1998). However, to assure conservaton of the species
it is essential to design reserves with an understanding of the
ontogenetic requirements of target and non-target species. Re-
serves need to be placed in strategic locations to protect larval
production sites and settlement sites. This information on source
sites and sink sites will support metapopulation d\ nanucs. and help

S. cosTATus Larvae and Adults at Banco Chinchorro

231

in optimize fisliery benefits for many marine invertebrates species
like S. costatiis that have a pelagic larvae. In the Bahamas, a high
density of S. fiii^a.s larvae and adults ha\c been reported in a
protected area in Bahamas (Stoner & Ray 1996). It is possible that
both S. gigiis and 5. coshiln.s populations in Banco Chinchorro can
be maintained for conservation and fisheries if reserve areas are
expanded and management regulations to be practiced.

ACKNOWLEDGMENTS

El Consejo Nacional de Ciencia y Tecnologi'a (CONACyT)
grant 420P-N93()6 supported this research. The authors thank Jose
Oliva and A. Medina for help in the field. The comments of M.
Davis, E. Sosa, and S. Monks and one anonymous reviewer im-
proved the manuscript.

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Joiinml ofShfUflsh Re.secinh. Vol. 21, No. 1, 233-237, 2002.

VELAR CHARACTERISTICS AND FEEDING CAPACITY OF ENCAPSULATED AND PELAGIC
LARVAE OF CREPIDULA FECUNDA GALLARDO, 1979 (GASTROPODA, CALYPTRAEIDAE)

O. R. CHAPARRO,' A. E. SOTO,' AND C. E. BERTRAN'

'lii.stituto de Biologiu Marina Dr. J. E. Winter. Univcrsulad Austral dc Chile. Casilla 567,
Valdivia. Chile; 'Institiito de Zoologi'a, Universidad Austral de Chile. Casilla 567. Valdivia. Chile

ABSTRACT Veligers of Ciepiclulu fcciiiidii develop the cap;icity lor ingesting particukitc material during early stages of their
development within the egg capsules. The potential feeding rate of encapsulated larvae is low compared with that of hatched larvae
and pelagic larvae of other mollusks. Once the veligers emerge from the capsules, they increase the capacity for particle retention. This
situation could result from the increase in the velar area, length of the ciliated border, length of the preoral cilia, potential filtering area
and the width of the feeding canal, or to some combination of these, compared with encapsulated larvae of the same shell length.
Extra-capsular development of the velum is an adaptive feature of the species which promotes efficient feeding and active locomotion
during the pelagic phase, m preparation for settlement and metamorphosis on inshore rocky substrates.

KEY WORDS: Crcpuiula fecunda. veliger larvae, velar morphology, particle clearance rate

INTRODUCTION

The presence of a lobed velum is a common characteristic in
veliger larvae of the Mollusca (Strathmann & Leise 1979), where
in free living species the velar lobes carry out functions of swim-
ming and feeding (Hadfield & laea 1989). Veligers have external
borders on the velar lobes that include preoral and postoral ciliary
bands (Strathmann et al. 1972; Strathmann & Leise 1979). The
pre-oral band is formed of long cilia that produce water currents
used in locomotion and feeding. The postoral band consists of
shorter cilia that beat toward the preoral band. The combined
activity of these ciliary bands promotes capture and retention of
food particles. Between the two ciliary bands lies the food canal
covered with very small cilia that transport food particles to the
larval mouth.

In suspension-feeding niollusks the pailicle clearance rate (CR)
may be used to calculate ingestion rate when food concentration is
known (Sprung 1 984). Clearance rate in veliger larvae is partially
determined by the length of the velar margin and the length of the
preoral cilia (Strathmann et al. 1972). Strathmann and Leise (1979)
observed that although longer preoral cilia were related to higher
rates of water flow, they were less efficient in capturing particles
in the smaller size ranges. The latter may be compensated for by
an increase in total filtering area that increases the larval clearance
rate. In addition to ciliary size, the length of the velar margin is
important in larval feeding. Increase in ciliated area is accoinpa-
nied by an increase in CR (Strathmann et al. 1972). The rate of
clearance is not only influenced by the factors cited, but also by the
ability of the organism to transport captured particles away from
the primary sites of capture to the mouth. Thus, the rate at which
the cilia in the food canal transport particles to the mouth directly
affects the overall CR.

Crepiditla fecunda. a sedentary filter-feeding gastropod, inhab-
its intertidal and shallow subtidal zones on the Chilean coast. It is
a protandric hermaphrodite whose reproductive mechanism in-
cludes the deposition of egg capsules on rocky substrates, followed
by parental brooding (Gallardo 1976. 1977. 1979). Most eggs in

Corresponding author. O. R. Chaparro, Instituto de Biologia Marina Dr. J.
E. Winter. Universidad Austral de Chile. Casilla 567. Valdivia. Chile.
Phone: +56-63-221791; Fax: +56-63-221455; E-mail: ochaparr^mercuriii.
uach.cl

the capsules develop, and veliger larvae of approximately .'iOO p.m
in shell length are released from the capsules (Gallardo 1976.
1977. 1979) to complete their development in the inshore plankton
prior to settlement.

Encapsulated larvae have no apparent source of food, and it is
not known whether they are capable of suspension-feeding prior to
eclosion. Development of the ciliated velum within the capsule
(Gallardo 1977, 1979; Chaparro et al.. in press) suggests that these
larvae are capable of feeding and swimming before hatching, and
that these properties would improve upon eclosion. since the pe-
lagic larvae must actively feed and swim in preparation for settle-
ment and metamorphosis (Gallardo 1977. 1979. 1989). We hy-
pothesize that the ability of C. fetiiuda larvae to remove particles
from suspension is initiated during the intracapsular phase and
further developed during the pelagic phase, including increases in
the size of the cilia, filtration area, and capacity for particle trans-
port \Mthin the larval food groove,

MATERIALS AND METHODS

Stacks of adult specimens of C. fecunda were collected from
the intertidal at Yaldad Bay, Chiloe (43'08'S; 73"44'W). In the
laboratory, individuals were removed from the substrate in order to
expose the egg capsules from which embryos and/or larvae were
obtained for experimentation. Before each experiment, the cap-
sules were observed under a stereomicroscope to identify the de-
velopmental stage of the embryos.

Developmental stages (egg, morula, bhistula, trochophore, and
veliger) were identified by their morphology. Fifteen to 20 indi-
viduals of each group of larvae were observed microscopically and
videotaped as described below. The shell lengths of the larvae
were determined from images captured on videotape (see later).

Clearance Rale

Clearance rate (CR) was quantified in several encapsulated
stages and in pelagic veligers using the method of Coughlan
(1969). Several thousands of einbryos (egg. morula, trochophore)
and veliger larvae frotn different spawning masses were manually
removed from capsules and placed in individual 1-L glass aquaria
containing 500 ml 0.45 p,m filtered seawater (salinity 30 %c. tem-
perature 17°C). Embryos used in each aquarium came from the
same egg muss. Laboratory cultured Isochrysis galhana was added
to each experimental system (final concentration 3 x 10'' cells/ml^

233

234

Chaparro ht al.

Aquaria were stirred to ensure iidequiite mixiiii;. At intersals be-
tween one and two h. the concentrations of algae remaining in the
aquaria were determined using an ELZONE 180 XY particle
counter. Control aquaria were run during each experimental periiid
under the same experimental conditions but without lar\ ae.

At the end of each experiment, five samples of 1 .5 nil each
were obtained from each experimental system to determine the
number of embryos or larvae in each aquarium. For measurement
of shell length, larvae from each experiment were fixed in 5%
formaldehyde and stored in Eppendorf tubes to await analysis.
They were measured as previously described.

Larval cultures were maintained to determine the CR of pelagic
larvae of C. fccunda. Naturally hatched larvae were collected on a
100 (jil nylon screen as they emerged from the capsules and placed
in 100-L aquaria containing filtered seawater as described above,
with gentle aeration. Each day the larvae were fed ad libitum with
/. galluina. The seawater was replaced every two days. For deter-
mination of clearance rate, a known number of larvae were placed
in experimental aquaria as described above for the encapsulated
larvae, and the same procedures used to determine CR. Groups of
larvae from these experiments were also set aside for measurement
of shell length.

Velar Morphology

Larvae obtained from capsules as well as pelagic larvae were
videotaped by placing them in a plankton decantation chamber
with seawater under an inverted microscope fitted with a video
camera. Selected images were captured for subsequent processing
on a computer equipped with an ATI Corp. "All in Wonder" video
card and Scion Image 3.0 PC software. We obtained the velar area
(extended velar lobe), length of the ciliated boi-der, length of the
preoral cilia, and shell length. The width of the food canal was also
determined. The potential feeding area was calculated by multi-
plying the length of the ciliated border by the length ( = height) of
the cilia in the preoral band.

Velocity of Particle Transport

A suspension of red plastic particles 2-10 |xm m diameter was
offered to larvae in a plankton settling chamber as previously
described by ChapaiTO et al. (1993). Trajectories of these particles
along the food groove were videotaped as described above for
observing larvae, and the distances traversed by the particles were
calculated using the Scion Image PC program. Simultaneously, the
time taken by particles to traverse measured distances was deter-
mined (Ward 1996J for calculations of transport velocity. These

♦ Encapsulated larvae
o Pelagic larvae

E M T 200

400

Stage-Larva shell length (nm)

Figure 1. Clearance rate in different enibr>()nic stages, encapsulated
ill = 281 and pelagic (/; = 10) larvae of C. feciiiida. E = egg (» = 5l,
M = morula (;i = 5| ,T = trochophore in = 5).

determinations were cairied out on larvae obtained from capsules
and on pelagic (culttired) larvae.

RESULTS

Clearance Rate

No uptake of particles was observed in early developmental
stages, including the egg. morula, blastula and trochophore (Fig.
1 ). The clearance rate (CR) of encapsulated veligers of C.fcciiiula
increased with the size of the individuals. The initial CR was 0.31
jjil h'' larva"' in larvae with shells 20S p-iii in length, increasing to
5.13 |j.l h"' larva"' in pre-hatched larvae 353 ixm in shell length
(CR = 0.071e '""=i-'^^-'' -''■'" ''-■"-'^ r^ = 0.4564. n = 28) Figure 1.

Recently hatched veliger larvae showed much higher values for
CR than those of the same size that remained encapsulated (Table
1). CR values ranged from 45.3tJLl h"' larva"' in larvae 337 p.m in
shell length at one day post-eclosion to 464 p,l h"' larva"' in larvae
442 p-m in shell length at 13 days post eclosion (CR = 1.0952

\elar Morphology

Velar Area

The velar area of encapsulated larvae showed linear growth
during the developmental period, fluctuating between 0.012 mnr

TABLE L

Crepidala fecunda. Comparison of clearance rate iCRl, area of a velar lobe (VA). length of ciliated velar border (LCVB). length of preoral

cilia (CL), potential filtering area (I'FA). and width of food groove (VVF(;) in encapsulated and pelagic larvae. \ alues represent indi\iduals

of 340 urn in shell length and were obtained from appropriate regression equations. C^r I = percent increase in \ariable when comparing

pelagic stage (recently eclosed) with encapsulated larvae having the same shell length.

Stage

Shell length
(Mm)

(Ml

CR

h"' • larv"

VA
(mm")

LCVB

(mnrl

CL

(Mm)

PFA

( mm' )

WFG

(pm)

Encapsulate
Pelagic

<7c

.WO
340

2.28
74.21

0.037

688

66

0.034

19

0.049

739

83

0.061

21

32.4

7.4

25.8

79.4

10.5

Velar Characteristics and Feeding of Crepiduu Fecunda

235

in larvae 188 yim in shell length and 0.056 nmr in larvae 372 |j.m
in shell length (velar area = 0.0002 x larval shell length - 0.0312.
r = 0.8755. n = 52) Figure 2.

In recently liberated pelagic larvae of C. fecunda the velar area
was 32.4 % greater than in encapsulated larvae having the same
shell length (Table 1). The velar area was 0.054 mm" in recently
eclosed larvae 340 p-m in shell length, reaching 0.198 mnr in
larvae ready for metamorphosis at 16 days post-eclosion at 650
|xm shell length (velar lobe area = (6 x 10"^) x (larval shell
length)' "-"-'. r- = 0.9586, )i = 5) Figure 2.

Length of Ciliated Velar Border

The length of the ciliated border in encapsulated larvae showed
gradual growth with development. The initial length. 287 p,ni in
larvae 205 p.m in shell length, increased to 755 p.m in larvae 372
|xm in shell length (length of ciliated velar border = 858.36 x In
(larval shell length) - 4315.5 , r" = 0.9724, n = 19) Figure 3.

The length of the ciliated velar border in recently liberated
pelagic larvae of C. fecunda was 7,4% greater than that of encap-
sulated larvae having the same shell length (Table 1 ). The length of
the border varied from 772 ixm in larvae of 340 (jtm shell length,
to 1548 |xm in larvae of 650 p.m shell length (ciliated length of
velar border = 1.7826 x (larval shell length)' '"■", r" = 0.9307.
/I = 5) Figure 3.

Length of Preoral Cilia

These cilia showed rapid growth during intracapsular develop-
ment, reaching lengths of 80 (j.m in larvae with shell length 400
(xm (ciliary length = -0,0023 (larval shell length)" -i- (1,7307 x
larval shell length) -256.29, r^ = 0.8954. n = 29) Figure 4.

The preoral cilia of recently hatched pelagic larvae were ap-
proximately 25,8'7f longer than the cilia of encapsulated larvae
having the same shell length (Table 1 ). The length of these cilia in
recently eclosed larvae was about 82 p,m. This increased to 1 11 p.ni
in those larvae at 15 days post eclosion that were ready to undergo

^ 1

* Encapsulated larvae

" 1

_

1500

E

a.

0 Pelagic larvae J-

c

^^*f

f

I2U0 .

r ^"-"'^'^ "

ea

E

J

^ X

b.

^

WO

^^ y=l.7826x

■u

^i^^ R" = 0.9307

n

-. _P^

■3

M)U

tM^

0

iT

c

30(1 ■
0 -

^g;t ^ y= 858.36Ln(x) - 43 1 5.5
R" - 0.9724

Larva shell length (fim)

Figure 3. Length of ciliated velar margin (periphery of an extended
velar lobe) in relation to larval size in encapsulated (/( = 19) and pelagic
in = S) larvae of C. fecunda. When the SD bar is not shown, it is smaller
than the symbol size. Each data point represents the mean for at least
15 individuals for encapsulated larvae and 25 individuals for pelagic
larvae.

metamorphosis (ciliary length = 61.316e «*i="-vai sheii iengih_

r- = 0.9041. (1 = 5) Figure 4.

Potential Filtering Area

The potential filtering area increased during larval development
from 0.002 mnr in larvae of 205 [xm shell length to 0.054 mm' in
larvae of 372 |jLin shell length [potential filtering area = (-5 x
I0~^) X (larval shell length)" + (0.0006 x larval shell length) -
0.1 121, r- = 0.9417. n = \6] Figure 5.

In recently eclosed pelagic larvae, the potential filtering area
was about 79.4'7f greater than that of encapsulated larvae of com-
parable .shell size (Table 1 ), This value was 0.06 mm" in one-day-
old larvae, increasing to 0.18 mm" in larvae at 15 days post eclo-

♦ Encapsulated larvae

O Pelagic larvae

<

y = 0.0002x-0 03l2

R- = 0.8755 ^-^

^■^^

y = (6'lo-')x"^-'

II ai*^^

R' = 0.9586

ui^^^

— 1 1 1 1 1

Larva shell length (fim)

Figure 2. Area of an extended velar lobe in relation to size in encap-
sulated (n = 52) and pelagic (/; = 5) larvae ni C. fecunda. When the .SI)
bar is not shown, it is smaller than the symbol size. Each data point
represents the mean for at least 10 individuals for encapsulated larvae
and 25 individuals for pelagic larvae.

120 .

♦

Encapsulated larvae T

too -

o

Pelagic laj-vae _. ^— --"^ — ^

.2

m -

h^^^"""^^ y = 61.316e°»«»^

O

^=^W- R-= 0.9041

0£

e
-J

60 .
40 -

A I y = -0.0023x' + 1.7307X - 256.29

20 -

jfii_ r' = 0.8954

0 .

150 250 350 450 550 650 750

Larva shell length (^m)

Figure 4. Length of preoral cilia in relation to larval size in encapsu-
lated (H = 29) and pelagic (h = 5) larvae of C. fecunda. When the SD bar
is not shown, it is smaller than the symbol size. Each data point rep-
resents the mean for at least 15 individuals for encapsulated larvae and
25 individuals for pelagic larvae.

236

Chaparro et al.

♦

Encapsulated larvae

s

«

1

o
'C

0 16 .
012 -
0 08 .

o

Pelagic larvae ,

„r.T^T 0 003x /

v = O0221e /
' R= = 0 9505 y^

y o

1

B
U

1

0t)4 -
0 -

JT^ y = (-5'10 >'+00006x-() 1121
JC R' = 09417

^ , ,

3 20(1

!^ ISO

1_

0.

♦ Encapsulated larvae
^ Pelagic larvae -

y = 0.02X ' °"
R- = 0.3562

Larva shell length (fim)

Figure S. Potential filtering area in relation to larval size in encapsu-
lated (n = 16) and pelagic (/; = 5) larvae of C.feciinda. Kach data point
was calculated by multiplying the mean length of the ciliated velar
edge by the mean length of the preoral cilia.

.- I c-i^ • /\ mil , nnn3*larvi)l shell length ^2 _

sion (potential filtering area = ().()221c '^ . r -

0.9505. /? = 5) Figure 5.

Width of Food Groove

The width of the larval food groove increased with the size of
the individual. The groove of encapsulated larvae 205 (Am in shell
length was 6.22 [xm wide, increasing to 34.98 \x.m in pelagic larvae
of 650 (jLin shell length (Fig. 6). In recently eclosed larvae, the
width of the food groove was 10.5% greater than that of encap-
sulated larvae of the same shell length (Table I ).

Velocity of Particle Transport in the Food Groove

Particle transport velocity increased throughout the larval en-
capsulation period, with minimum values of 86 |j.m s"' in larvae
225 |ji,m in shell length, increasing to 270 [Lva s~' in larvae .?00 jxm
in shell length (approaching eclosion) [particle velocity = 0.02 x
(shell length)' "". r' = 0.3562. n = 16] Figure 7.

4(.) -

♦ Encapsulated larvae -

35 -

o Pelagic larvae ,

)

30 -

-

25 -

20 -

1

15 -

1

10 -

s _

11 -

♦

I

Larva shell length (fim)

Figure h. Width of larval feeding groove in relation to individual size
in ('. fcciiiida. Vertical bar = standard deviation.

Larva shell length (^nl)

Figure 7. Transport velocity of particles in the larval food groove in
relation to individual size in encapsulated in = 16) and pelagic in = 5)
larvae of C. fecitiida. When the SI) bar is not sho\\n. it is smaller than
the symbol size. Kach data point represents the mean for at least HI
individuals for encapsulated larvae and 20 individuals for pelagic lar-
vae.

In pelagic larvae the velocity of the particles remained rela-
tively constant with values near 250 jo-m s"' (Fig. 7).

DISCUSSION

All encapsulated stages of C. fecunda veligers are able to ingest
particulate material. This ability is related to the development of
the ciliated velum (Gallardo. 1977, 1979), and particulariy to the
presence of opposed ciliary hands (Chaparro el al.. in press). In the
pelagic phase of the life cycle, development of the velum is of vital
importance to swimming and feeding prior to settlement in this
species, thus a large and well developed velum appears to be
related to the mixed developmental strategy used by C. fecunda
(Gallardo, 1979).

Encapsulated larvae of C. fecunda develop the ability to ingest
particles before their transition to the planktotrophic habit. Chap-
arro et al. (in press) have observed particle capture and ingestion
by pre-eclosed larvae which were fed immediately after artificial
excapsulation, suggesting readiness for the planktt)trophic phase.

The low CR observed in veligers removed from capsules is
probably attributable to the early stage of development, as ob-
served in larvae of other molluscan species (Hawkins et al. 1984;
Welborn & Manahan 1990). Values for particle clearance by C.
fecunda veligers obtained from capsules are lower in all cases than
values reported in the literature for pelagic molluscan larvae (Mac-
Donald 1988; .Sprung 1984; Bayne 1965). although CR in C. fe-
cunda pelagic larvae is well above the rates cited by these authors.

The rapid growth of the velum in encapsulated larvae of C.
fecunda may be related to the mode of nutrition and the necessity
for efficient swimming in the pelagic stage. Once the larva is free
from the capsule, it is advantageous to have as large a velum as
possible (Fretter & Graham 1962).

The increase in the length of the ciliated border oi the velum in
C. fecunda is a result of enlargement of the bilobed velum, not the
formation of new lobes. The length of the preoral cilia in encap-
sulated and pelagic larvae of this species falls within the size range
reported by Strathmann ( 1987). i.e.. 30 to more than 100 |xm. and

Velar Characteristics and Feeding of Crkpidula Fecunda

237

also agrees with the 50 fj.m value given by Riedel ( 1992) for larvae
of the gastropod Cuheslana spcnfilcri (200 fxm shell length), which
also exhibits mixed development.

The potential filtering area of recently eciosed pelagic larvae is
nearly 80% larger than that of encapsulated larvae of the same size
(370 p.m shell length). This is due to the elongation of the ciliated
border of the velum and growth of preoral cilia, and provides the
larvae with a large surface area for particle capture, as suggested
by the CR of pelagic larvae in this study. This is consistent with
observations on the planktotrophic character of these larvae (Gal-
lardo. 1977. 1979. 1989). The transport velocity of particles within
the food groove of encapsulated larvae of C. fecunda increases
with developinental stage to values near 250 jjim s"'. In pelagic
larvae this velocity is more or less constant but the width of the
food canal continues to increase during pelagic development, sug-
gesting that pelagic larvae are able to capture and handle several
particles at the same time, or increasingly larger particles (or par-
ticle aggregates) as they develop. Together with the increase in
potential filtering area, this may explain the high values for CR in
pelagic larvae of C. feciiinla.

The greater capacity for particle clearance in pelagic larvae
compared with encapsulated larvae in C. fcciiiula may be related to
increases in the velar area, cilated length of the border of the
velum, length of the preoral cilia, potential filtering area, and the
width of the food groove, or to some combination of these vari-
ables. These changes are obviously adaptive as the larvae are
released into the environment, where they must be able to swim
and feed before settling. Some larvae remain in the capsule, yet
have a shell size similar to that of newly released larvae, suggest-
ing a differential rate of development between shell and velum, the
latter requiring more time to develop before eclosion.

ACKNOWLEDGMENTS

Financial assistance was provided by an operating grant to
ORC from the Fondo Nacional de Investigacion Cientifica y Tec-
nologica (FONDECYT 1980984) and the Direccion de Investiga-
cion of the Universidad Austral de Chile (S 200114). We also
thank the Canadian International Development Agency (CIDA) for
support during the preparation of the manuscript.

LITERATURE CITED

Bayne. B. L. 1965. Grovvtii and delay of nielanicirphosis (if the lar\ae of

Mylilii.s ediilis (L.). O/^lH-lla 2:1—17.
Chaparro. O. R.. R. J. Thompson & J. E. Ward. 199.'^. //) r/ivi observations

of larval brooding in the Chilean oyster. Ostrea citilensis Philippi.

1845. Biol. Bull. 185:365-372.
ChapaiTO. O. R., J. L. Charpentier & R. Collin. Embryonic velar structure

and function of two sibling species of Crepidulu with different modes

of development. In press.
Coughlan. J. 1969. The estimation of filtering rate from the clearance of

suspensions. Mar. Biol. 2:356-358.
Fretter, V. & A. Graham. 1962. Larval forms. In: British Prosobranch

Molluscs. London: Ray Soc. pp. 448^76.
Gallardo, C. S. 1976. Natural history and reproduction of Crepidulu

dilatata Lamarck in a Mehuin Bay population (Valdivia province.

Chile). Medio Ambiente. 2:44-50.
Gallardo. C. S. 1977. Two modes of development in the inorphospecies

Crepidulu dUatata (Gastropoda: Calyptraeidae) from Southern Chile.

Mar. Biol. 39:241-251.
Gallardo, C. S. 1979. Twin species of Crepidulu genus (Gastropoda. Ca-
lyptraeidae) in the Chilean coast; a redescription of C. dilatata Lamarck

and description of C. fecundu n. sp. Stud. Neotropical Fauna Environ.

14:215-226.
Gallardo, C. S. 1989. Reproduction patterns and vital cycle of bentic ma-
rine mollusks. an ecological and evolutive approach. Medio Ambiente

10:25-35.
Hadfield. M. G. & D. K. laea. 1989. Velum of encapsulated veligers of

Petulocoiuinis (Gastropoda), and the problem of re-evolution of plank-
totrophic larvae. Bull. Mar Sc. 45:377-386.

Hawkins. A. J. S.. P. N. Salked. B. L. Bayne. E, Gnaiger& M. Lowe. 19X4.
Feeding and resource allocation in Mytilus edulis: evidence for time
averaged optimization. Mar. Ecol. Prog. Ser. 20:273-287.

MacDonald. B. A. 1988. Physiological energetics of Japanese scallop Pu-
tinopecten yessoensis larvae. / £v/). Mar. Biol. Ecol. 120:155-170.

Riedel, F. 1992. A re-evalualion of the ontogeny of Cuhestana spengleri
(Perry. 1811) (Gastropoda: Tonnoidea: Ranellidae). Veliger 35:117-
121.

Sprung. M. 1984. Physiological energetics of mussel larvae (Mytilus edu-
lis). II. Food uptake. Mur. Ecol. Prog. Ser. 17:295-305.

Strathmann. R. R. 19X7. Larval feeding. In: A. C. Giese. J. S. Pearse & V.
B. Pearse. editors. Reproduction of Marine Invertebrates. Volume IX.
General Aspects: Seeking Unity in Diversity. Blackwell Scientific Pub-
lications & The Boxwood Press. Pacific Grove. CA. pp. 465-550

Strathmann. R. R. & E. Leise. 1979. On feeding mechanisms and clearance
rates of moUuscan veligers. Biol. Bull. 157:524-535.

Strathmann, R. R., T. L. Jahn & J. R. C. Fonseca. 1972. Suspension feeding
by marine invertebrate larvae: clearance of particles by ciliated bands
of a rotifer, pluteus. and trochophore. Biol. Bull. 142:505-519.

Ward. J. E. 1996. Biodynamics of suspension-feeding in adult bivalve
molluscs: particle capture, processing, and fate. Inv. Biol. 1 15:218-231.

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Jounwl iif Slu'llfish Rfxcinrh. Vol. 21. No. 1, 2.^4-241, 2002.

DISTRIBUTION, DENSITY AND LENGTH-WEIGHT RELATIONSHIP OF CHITON
ARTICULATUS SO WERE Y, 1832 ( MOLLUSC A-POLYPLACOPHORA) ON ISLA SOCORRO,

REVILLAGIGEDO ARCHIPELAGO, MEXICO

OSCAR EFRAIN HOLGUIN QUINONES AND JESUS EMILIO MICHEL-MORFIN

Centra Intenlisclplinario de Ciencias Marinas Insfttiito Politecnico. Nacional Apartado Postal 592. La
Paz. B.C.S. 23096. Mexico

ABSTRACT Chiton articidciuis is an abundant mollusk species on the Isla Socorro. In March 1992 C. articulatus populations were
sampled at four sites on the southwestern and northern coast of the island. Average densities of C arliculatus ranged between 3.7 to
8.5 ind/nr among sites. The length-weight relationship for all sampled nidividuals (N = 385) is W(g) = 0.00022 L"''.

KEY WORDS: Cliiimi uriiciilalus. mollusk, distribution, density. Isla Socorro

INTRODUCTION

Chitons are polyplacophoran niollusks that are common grazers
of intertidal epilithic and endolithic algae. Since their first appear-
ance in the late Cambrian, chitons have modified hard marine
substrates using their strong raduia (Glynn 1070; Rasmussen &
Frankenberg 1990).

Cliilon articulatus is a species that was recently very abundant
along the Pacific tropical coast of Mexico, today C. articulatus is
uncommon because of over exploitation by fishermen. The foot is
commercially utilized and sought in subsistence fisheries. In the
Revillagigedo Archipelago (Fig. 1 ) it is a potential resource that
has integral usage possibilities, it is still an important member of
the malacofauna since it has not been fished commercially (Vil-
lalobos 1960: as C. laevigatus. Keen 1971: Ferreira 1983: Holguin
et al. 1992: Holguin 1994: Bautista-Romero et al. 1994: Mille-
Pagaza et al. 1994; Emerson 1995).

Only one study has been conducted for the Mexican Pacific

continental shore population of C. articulatus. in the Acapulco
area (Rojas-Heirera 1988). Little is known about the Revillagigedo
Archipelago chiton populations, because of its distance from the
Mexican mainland. This article describes the distribution, density
and length-weight relationship of C. articulatus sampled on Isla
Socorro, Revillagigedo in March 1992,

MATERIALS AND METHODS

The Revillagigedo Archipelago (Fig. 1) is located approxi-
mately 350-650 km, southeast of the Baja California Peninsula
and 580 km west of the Colima coast (Llinas-Gutierrez et al.
1993). The Archipelago is comprised of four oceanic islands of
volcanic origin. Socorro, Clarion. San Benedicto and Roca Partida.

The surface area of Isla Socorro, the largest island, is 1 67 km"
with a maximum diameter of 16 km in a NW-SW direction. Geo-
graphically Isla Socorro is located at 18°4r57"N and
1 10^56'33"W (Troyo-Dieguez & Pedrfn 1994). The island is com-
posed primarily of braced igneous rock, with the presence of
smooth stones and sandy or stone-sandy beaches (Holguin 1994).

During March 1992, C articulatus were sampled in the follow-
ing rocky areas, from south to north (Fig. I): Bahia Braithv\aite
(La Braulia). Bahia Binners, Bahi'a Grayson (Palmasola), and
Bahi'a Academia (Playa Noite). Some of the areas are more acces-
sible than other areas where there is a greater abundance of or-
ganisms. At each one of the four satnpling locations, we took
samples along a transect of 50 m~ in the intertidal zone. We laid
out transects parallel to the coast line, using 25 m cord which
delineated a 2 m wide sampling swath. The entire sampling was
accomplished during ebb tides. The density of C. articulatus in

TABLE L

Mean densities for Chiton arliculatus at four sites on Isla Socorro,
Revillagigedo. Mexico, March 1992.

Figure L Map of the sampling sites on Isla Socorro, Revillagigedo,
March 1992.

Maximum

Number

Area

No. Total

Relative

Chitons

Locality

(m-)

Organism

Density (m')

Grouped

Bahia Binners

50

186

3.72

22

Baliia Braithwaite

50

424

8.48

35

Bahia Grayson

50

193

3,86

33

Bahia Academia

50

249

4.38

41

239

240

QUINONES AND MlCHEL-MORFIN

20 K 30 35 40 45 50

95 90 95 100 105

Length (mm)

Figure 2. Length frequency distribution of Chiton aiiiiiilaliis sampled
on Isla Socorro, Revillagigedo, March 1992.

each rocky shore are;i was determined from the coiinl of all Iimiisj
organisms present m the transect. The maxiniiim number of the
chitons grouped was determined.

A sample of 30-40% of chitons (N = 385) was taken from the
transects in order to be measured and weighed. The length and
weight measurements from selected chitons were recorded. The
length was measured using a vernier caliper, taking the major
distance between the front and the posterior parts of the body.
Weight, including the shell, was obtained with a three-arm scale
having an accuracy of ±0.5 g. We liberated all the animals in the
rocky intertidal zone after measuring them.

RESULTS AND DISCUSSION

90 •

/

80
70

y

= 0CI002x"'
r' = 0 86

n=385

09/
0/

~ 60
% 50
5 40

o

o

0 '^.^

cb

o

30
20

o

>

F'

10

,^

^

60

Length (mm)

o Braithwaite -Binners -> Grayson AAcademia

Figure 3. Length-weight relationship of Chiton articulatus sampled on
Isla Socorro, Revillagigedo, March 1992.

B Binners

y = 6E-05x^"

R^ = 0 96

n=55

On Isla Socono all rocky coasts exposed to strong waves are
populated by Chiton articiikiltis. The species is discontinuously
distributed along the reef line; in some areas adult animals over 4
cm length were found crowded within a narrow belt along the
coast, while in adjacent areas animals bigger than 3 cm were not
found at all. According to Rojas-HeiTcra (19X8) the size of the
mollusk in its first sexual maturity is 40 mm. We observed juvenile
C. arliculotiis «4 cm) in Isla Socorro, mostly in the rock fissures
and in the cracks of the intertidal belts occupied by the sea urchin
Echiiiomt'lni vaiilvunii. We observed that for C. anicidutiis the

TABLE 2.

Mean lengths and weights for Chiton articulatus at four sites on Isla
Socorro, Revillagigedo, Mexico, March 1992.

;i

Length

(mm)

Weight (g)

Locality

min

max

mean

s.d.

min

max

mean

s.d.

Bahia

Binners

5S

42

1(38.0

67.9

13.9

4.3

S6.3

23.3

19.1

Bahi'a

Biaithvvaile

\m

24

Q.^i..^

66.3

124

2.0

73-4

26.6

14.2

Bahia

Grayson

1^

44

102.0

73.6

10.6

9.0

S-^.3

33.3

14.9

Bahi'a

Academia

86

49

90.0

71.9

8.9

8.,^

49.0

27.4

9.3

TOTAL

.W.-S

29

lOS.O

69.6

12.5

2.0

S6.3

27.9

14.3

B Grayson

0/

y = 6E-05x>"

0 y

r' = 090
n=75

,^

> 0

B Academia
y = 0CI002x^"

0=86

Length (mm)

Figure 4. Length-weight relationship of Chiton articulatus for each
sampling site on Isla Socorro, Revillagigedo. March 1992.

Distribution, Density and Length-weight Relationship of C. articulatus

241

major feeding activity was nocturnal and food was generally com-
posed of crusty filamentous algae and diatoms.

Several other species of invertebrates coexist with C. aiiiciila-
nis. Some are chiton predators such as the gastropods Plicopitr-
pura pansa and Thais spp., as well as the cephalopod Octopus sp.
Other coexisting species are Littorina aspera, L. modesta and L
puUata. Nerita finuciilata, Diodnva iiuiequalis, Chama sqiianiulii;-
era. ColiseUa spp. and Fissiirella spp. Several crustacean species
can also be found in those areas, such as the decapod crab Grapsus
grapsus, the cirriped Tetraclita sp. and the isopod Ligia exotica.

We counted a total of 1,052 C. articulatus occurring in the
sampling transects (Table 1 ). The highest relative density average
(8.5 animals/m^) was found in Bahia Braithwaite and the lowest in
Bahi'a Binners (3.7 animals/m") with an average of 5.3 animals/ni"
for the four sampling sites combined. We observed the maximum
densities of up to 41 chitons/m" in small sections of the transect
(Bahi'a Academia. Table 1 ).

The abundance of chitons on Isla Socorro is greater than mi
Acapulco seaside (4.2 animals/m". Rojas-Herrera 1988). The high
density of the mollusk. that we observed in Bahi'a Academia, is
probably due to the fact that the bay is far away from any popu-
lated center. Bahia Binners which is most accessible to the navy
village, shows lower densities because of fishery activity.

The length frequency distribution of the 385 chitons collected
on Isla Socorro, ransed between 29 and 108 mm with a mode of

70 mm (Fig. 2). The smallest chiton sampled weighed 2.0 grams,
while the longest weighed 86.5 grams (Table 2). The average
values are of 69.6 mm and 27.9 g for length and weight respec-
tively (Table 2). The length- weight relationship W = 0.0022 L" ''
where W is total weight (g) and L= total length (mm) was derived
from all 385 C. articulatus measured in March 1992 on Isla So-
corro (Fig. 3 and Fig. 4).

There are no significant differences between length-weight re-
lationship in the sampling areas. The major length weight per unit
was found in Bahi'a Braithwaite and the minor weight was found in
Bahi'a Grayson.

It is important to point out that the predominance of Chiton
bigger than 6 cm. in the rocky intertida zone of the Isla Socorro, is
due mainly to its geographic location, this part of the island is
almost inaccessible by land or sea. The population of this species
in general, is unaltered and its partial exploitation under strict
protection measures is possible.

ACKNOWLEDGMENTS

We thank Direccion de Estudios de Posgrado e Investigacion
del Institute) Politecnico Nacional and Consejo Nacional de Cien-
cia y Tecnologia for funding this work. Thanks to the Comision de
Operacion y Fomento de Actividades Academicas. Thanks to Sil-
via Mille P. Alicia Perez and Ma. de Jesus Parra for their help
alone the field work.

LITERATURE CITED

Bautista-Romero. J.. H. Reyes-Bonilla. D. B. LIuch-Cota & S. E. Lliich-
Cota. 1994. Aspectos generales sobre la Fauna Marina. In: R. Ortega &
V. Castellanos. editors. La Isla Socorro. Reserva de la Biosfera. Ar-
chipielago de Revillagigedo. Mexico. Puhl. No. S. C/BNor. S.C.
Mexico, pp. 247-275.

Emerson. W. K. 1995. A Zoogeographic Summary of the Marine Mollusks
of the Revillagigedo Island (Tropical Eastern Pacific Ocean). The Ff.v-
tivus, American Mu.ieum of Natural History. 27(1);3-18.

Ferreira, A. J. 1983. The Chiton fauna of the Revillagigedo Archipielago.
Mexico. The Veliger. 25(4):307-322.

Glynn, P. W. 1970. On the ecology of the Caribbean chitons.- Acanihoft-
leura granulata Gmelin and Chiton tubercnUitus Linne; density, mor-
tality, feeding, reproduction and growth. Smithsonian Contr. Zool. No.
66. 21 pp.

Holguin. Q. O.. S. Mille-Pagaza & A. Perez-Chi. 1992. Resultado de las
campanasde muestreo de 1991 para el estudio del bentos marino de isla
Socorro. Revillagigedo. Colima, Mexico. Zool. Inf. 24:1-20.

Holguin. Q. O. 1994. Comunidades marinas bentonicas. In: R, Ortega & V.
Castellanos, editors. La Isla Socorro, Reserva de la Biosfera, Archi-
pielago de Revillagigedo, Mexico. Publ. No. 8. CIBNor. S.C. Mexico,
pp 225-245.

Keen, M. A. 1971. Sea Shells of Tropical West America. Marine Mollusks
from Baja California to Peru, 2nd Ed. Stanford: Stanford Univ. Press.
1065 pp.

Llinas-Gutierrez. J., D. LIuch. A. Castellanos. 1993. Isla Socorro. Revil-
lagigedo. In: S. I. Salazar-Vallejo & N. E. Gonzalez, editors. Biodi-
versidad Marina y Costera de Mexico. Mexico: CONABIO-CIQRO.
pp. 520-534.

Mille-Pagaza, S.. A. Perez-Chi & Q. O. Holguin. 1994. Fauna malaco-
logica bentonica del litoral de la Isla Socorro, Revillagigedo, Mexico.
Cienc. Mar. 20(4):467^86.

Rasmussen, K. A. & E. W. Frankenberg. 1990. Intcnidal bioerosion by the
chiton Acanthopleura granulata: San Salvador. Bahamas. Bull. Mar.
Sci. 47(3):680-695.

Rojas-Herrera. A. A. 1988. Analisis Biologico-Pesquero de la cucaraclia de
mar (Chiton articulatus. Sowerby 18321 de Acapulco, Gro.. Mexico.
In: Memorias IX Congre.so Nacional de Zoologi'a, Mexico. Univ. Jua-
rez Aut. Tab. & Soc. Mex. Zool. Villahermosa, Tab. Mex.. pp. 151-
156.

Troyo-Dieguez. E. & S. Pedn'n. 1994. Aspectos hidro-fisiogrdficos y geo-
logicos. pp. 43-53. In: R. Ortega & V. Castellanos. editors. La Isla
Socorro, Reserva de la Biosfera, Archipielago de Revillagigedo, S.C.
Mexico: Mexico. Puhl. No. 8. CIBNor.

Villalobos, A. 1960. Notas acerca del aspecto hidrobiologico de la parte sur
de la isla. In: A. E. Cobo, et al. editors. La Isla Socorro. Archipielago
de las Revillagigedo. Monog. Inst. Geofis. UNAM. 2 pp.

Journal of Shellfish Research. Vol. 21. No. 1. 243-248, 2002.

CELLULAR IMMUNOLOGICAL PARAMETERS OF THE OCTOPUS, OCTOPUS VULGARIS

BEATRIZ NOVOA, CAROLINA TAFALLA, ANGEL GUERRA, AND ANTONIO FIGUERAS

Institiito de Investigackmes Marinas. CSIC. Eduardo Cahello. 6. 36208 Vigo, Spain

ABSTRACT The white body is the main hematopoietic organ of cephalopods. In this study, we have investigated the capacity of the
octopus (Octopus vulgaris) white body cells to petlbmi common cellular defense parameters known to be done by heniocytes of other
mollusks such as phagocytosis of zymosan particles, respiratory burst activity and nitric oxide (NO) production. White body cells were
capable of respiratory burst and NO production, however, they exhibited a low phagocytic response. Similar capabilities were observed
in hemocytes withdrawn from the hemolymph. We have studied the effects of in vitro incubation with bacterial lipopolysacharide (LPS)
or zymosan for 24 hours on these two functions. Incubation of the white body cells with zymosan, hut not with LPS. resulted in a
significantly increased respiratory burst activity and NO production.

We have also investigated the capacity of circulating hemocytes and white body cells to increase their thymidine uptake (indicative
of DNA synthesis) in response to LPS and phytohemaglutinin (PHA). In some animals, both mitogens induced a significant increase
in thymidine uptake. If this thymidine uptake correlates with cell proliferation, this will be the first report of any proliferation of
hemocytes in mollusks.

In the hemolymph, we observed two different morphologies under the electron microscope, however, we cannot conclude that they
correspond to two distinct cell types. Among white body cells different morphologies that may correspond to intermediate stages were
observed. All these findings represent a baseline for future studies to elucidate mechanisms of host defense in this mollusk.

KEY WORDS:

Octopus vulgaris, hemocytes, white body, respiratory burst, thymidine uptake, nitric oxide (NO), phagocytosis

INTRODUCTION

Due to the decrease in Spanish cephalopod fishei'ies, the inter-
est in the commercial culture of cepahalopod species has gained
increasing attention. Diseases are one of the major obstacles in
achieving this goal. Several pathogens have been identified in wild
and aquarium-kept octopuses, including viruses, bacteria and
fungi, Farley (1978) described the presence of viruses in the
muscle of the octopus. Octopus vulgaris. Bacteria have been re-
ported to cause several disease outbreaks in laboratory reared oc-
topuses (Hanlon & Forsythe 1990; Hanlon et al. 1984). Parasites
have been blamed for several pathogenic problems. Hochberg

( 1990) described a flagellated parasite in cultured octopuses. Fungi
have been also described as causing problems in cephalopods
maintained in captivity (Polglase 1980; Polglase et al. 1984). De-
spite the threat these pathogens may cause to octopus populations,
elucidation of defense mechanisms in these species have received
little attention.

Humoral defense factors have been identified and are reviewed
by Ford (1992). Rogener et al. (1985) and Fisher and Dinuzzo

(1991) described hemaglutinating activity and aglutinnins in cell
free hemolymph of several molluskan species, including the octo-
pus. Octopus vulgaris. In this species, an antiprotease of the
a-macroglobulin family was also detected in the hemolymph
(Thogersen et al, 1992), Malham et al. (1998) reported lysozyme
and antiprotease activity in hemocytes and hemolymph of the
lesser octopus Eledone cirhhosa. Like in other mollusks. cepha-
lopod hemocytes are believed to play a role in host defense mecha-
nisms, however, the function of the hemocytes has been poorly
studied. Cowden and Curtis (1981) estimated that the phagocytic
capacity of octopus hemocytes was low while high phagocytosis of
carbon particles has been described in Eledone cirrhosa (Stuart
1968). Bayne (1983) reported a clearance of Serratia niarcescens
by hemocytes of the octopus. Octopus dofleini.

Corresponding author. Antonio Figueras, Instituto de Investigaciones Ma-
rinas. CSIC. Eduardo Cabello. 6. 36208. Vigo. Spain. E-mail: patol@
nautilus.iim.csic.es

The generation of hemocytes of cephalopods is believed to take
place in an organ situated around the optic nerve called the white
body, gland of Hensen or gland of Faussek (Stuart 1968; Cowden
1983; Bolognari et al. 1980). Hence, the aim of this study was to
determine whether white body cells from Octopus vulgaris are
capable of performing certain functions believed to be of relevance
to defense mechanisms like phagocytosis of zymosan, respiratory
burst and nitric oxide (NO) production and to compare their func-
tionality to that of circulating hemocytes. We have also examined
the ability of two mitogens, bacterial lipopolysacharide (LPS) and
phytohemaglutinin (PHA). to stimulate thymidine uptake, an in-
dicative of DNA synthesis. In the case of the respiratory burst and
NO production, we have also determined the effect of in vitro
activation with bacterial lipopolysacharide (LPS) and zymosan.

MATERIALS AND METHODS

Sampling

Adult octopuses (Octopus vulgaris) of both sexes, weighing
2-3 kg were caught from a raft in the Ri'a de Vigo (Spain ) and after
a day of adaptation under laboratory conditions, were anesthetized
with excess MS-222. When animals were fully anesthetized (iden-
tified by muscle relaxation and absence of movement), the visceral
cavity was dissected. Once the heart and its associated vessels
were reached, around 1 ml of hemolymph was drawn using a 27
gauge needle from the artery and subsequently from the heart,
placed in an eppendorf and kept on ice until used. The white body
was removed and kept on ice until used.

In order to obtain single cell suspensions, individual white
bodies were passed through a 100 |jLm nylon mesh using Leibovitz
medium (L-15, Gibco) supplemented with penicillin { 100 lU/mL).
streptomycin (100 |xg/mL) and 2% fetal calf serum (FCS). The
resulting cell suspension was centrifuged (500 x g for 15 min at
4°C) and the cells were resuspended in L-15 supplemented with
penicillin, streptomycin and 2% (FCS). Cell viability was deter-
mined by Trypan blue exclusion. Cells were resuspended in L-15
supplemented with penicillin, streptomycin and 0.1 "^r FCS at a
density of 1x10" cells/mL.

243

244

NOVOA ET AL.

Hemocytes were obtained by centrifuging octopus heiiiolyniph
(500 X g for 15 min at 4'C) and hemocytes were resuspended in
L-15 supplemented with penicillin, streptomycin and 29f FCS. The
ceil viability was determined by Trypan blue exclusion test. Cells
were resuspended in L-15 supplemented with penicillin, strepto-
mycin and 0.1% FCS at a concentration of 1 x 10" cells/mL.

Electron Microscopy

Pelleted cells (from white body and hemolymph) were fixed for
one hour in 1% osmium tetroxide in cacodilate buffer 0.1 M (pH
7.3). In some cases, a previous step of fixation with \':i glutaral-
dehyde in cacodilate buffer 0.1 M (pH 7.3) for 15 minutes was
performed. Following three washes in 0.1 M cacodilate. the cells
were dehydrated with increasing pei-centages of ethanol and em-
bedded in Araldite/Poly Bed (Polyscience). Ultrathin sections (50-
70 nm) were stained with uranyl acetate and lead citrate and ex-
amined using a Phillips electron microscope CM 100.

Phagocytosis Assay

To measure the phagocytic ability of octopus cells. 200 |J.L of
cell suspensions (derived from white body or hemolymph of six
animals) were incubated in chamber slides (Nunc) for 2 h at 18"C
in moist incubation chambers to allow the cells to adhere. Medium
was removed and the adherent cell layer was washed twice with
L-15. Zymosan A (Sigma) resuspended in sterile phosphate buf-
fered saline, PBS, at a concentration of 1 mg/mL. was heated at
100°C for 30 min, washed twice, and resuspended in the same PBS
volume. Zymosan was added to the cells at a final concentration of
250 jxg/mL. and the same volume of L-15 was added to the con-
trols. Slides were incubated in a moist chamber for one hour at
room temperature or 18°C to allow phagocytosis. Some slides
were kept up to three hours. Slides were then washed twice in PBS,
fixed in absolute ethanol, stained with Hemacolor, and mounted
with DePex. Two replicas were made for each octopus and at least
150 cells were observed in each replica.

Respiratory Burst Activity

Respiratory burst activity of octopus cells was assayed by the
reduction of ferricytochrome C (Cit C, Sigma) by released super-
oxide anion (O^-). following stimulation of the cells with phorbol
myristate acetate (PMA, Sigma) (Secombes 1990) in 4 octopuses.
White body adherent cells and circulating hemocytes were ob-
tained as described above, resuspended in L-15 supplemented with
penicillin, streptomycin and 0.1% FCS and dispensed into 96-well
tissue culture plates (Iwaki) at a concentration of 1 x 10*" cells/mL
(100 jjlL per well).

In a preliminary experiment, we determined the specificity of
the respiratory burst by assaying the response of the cells to PMA.
After 24 hours of incubation at I8°C, octopus cell monolayers
were washed twice in phenol red-free Hank's balanced salt solu-
tion (HBSS, Gibco). One hundred (jlL of HBSS containing Cit C (2
mg/mL) and PMA (1 ng/mL) were added to each well. As a
control for specificity, 300 lU/mL superoxide dismutase (SOD,
Sigma) was added to some wells. The optical density (O.D.) was
measured at 550 nm after 30 min in a multiscan spectrophotometer
(Labsystems). Triplicate wells were used in all the experiments for
each octopus and the mean ± SD was calculated.

Once the responsiveness of octopus cells to PMA had been
deterinined, the effects of stimulation with zymosan or Escherichia
coli serotype 0111: 84 lipopolysacharide (LPS) on the respiratory

burst of octopus cells triggered by PMA was also studied. After
three hours incubation of the cell monolayers at 18"C, LPS and
zymosan were added to a final concentration of 50 and 250 p-g/mL
respectively. After an additional 24 hours of incubation at 18"C
with these substances, the respiratory burst activity was measured
by adding 100 p.L of HBSS containing Cit C (2 mg/mL) and PMA
( 1 |j.g/mL) were added to each well. The O.D. at 550 nm was then
detemiined as described above.

NO Production

The ability of octopus cells to produce NO in response to LPS
and zymosan was also determined in 4 animals. Cells resuspended
in L-15 with 0.1% FCS were disposed into 96-well plates at a
concentration of 1 x 10'' cells/mL. After 3 h of incubation at 18°C.
LPS and zymosan were added at a final concentration 50 and 250
|j.g/mL respectively. After additional 24 h of incubation at 18°C.
the NO concentration present in the cell supernatants was assayed
through the Griess reaction (Green et al. 1982) that quantifies the
nitrite content of the cell supernatants. since NO is an unstable
molecule and degrades to nitrite and nitrate. Fifty (jlL of hemocyte
supernatants were reiuoved from individual wells and placed in a
separate 96-well plate. One hundred microliters of 1% sulfanil-
amide (Sigma) in 2.5% phosphoric acid were added to each well,
followed by the addition of 100 |a.L of 0.1% N-naphthyl-
ethylenediamine (Sigma) in 2.5% phosphoric acid. Optical density
at 540 nm was determined using a multiscan spectrophotometer.
The molar concentration of nitrite in the sample was determined
from standard curves generated using known concentrations of
sodium nitrite (100. 10. 5. 2.5. 1. 0.5. 0.25. and 0.1 |jlM).

Effect of Mitogens on (^H)-Thyinidine L'ptake by Octopus Cells

The thymidine uptake by octopus cells was assayed following
a modification of the method described by Marsden et al. ( 1994) in
six octopuses. Briefly, hemocytes derived from the hemolymph or
the white body were adjusted to a density of 5 x 10"' cells/mL in
RPMI 1640 medium (Gibco) supplemented with penicillin (100
lU/mL), streptomycin (100 |jLg/mL), 25 mM NaHCO, and 1 x 10"^
M 2-P-iuercaptoethanol. Aliquots of 100 |xL of cell suspensions
were added to wells of 96-well plates containing 100 |jiL of LPS or
PHA dilutions (Sigma) to make final concentrations of 50. 25 and
12.5 |j.g/mL or 5, 2.5, and 1.25 |jig/mL, respectively. Controls
without mitogens were also included. After 3 h of incubation at
18°C, FCS was added to the wells to give a final concentration of
10%. Following 48 h of incubation, the cells were pulsed with 0.5
(id of ('H)-thymidine (Amersham). After additional 24 h incu-
bation at 18"C. DNA v\as harvested onto glass filter mats. One niL
Xylofluor scintillant (Packard) was added to dried filter circles in
vials and counts per minute (cpm) were recorded using a Packard
liquid scintillation counter. Triplicate cultures were used in all
cases.

Statistics

The data were compared using a Student's t test. Results are
expressed as the mean ± standard deviation and differences were
considered statistically significant al P < 0.05.

RESULTS

Cell Morphology

After two hours of incubation of the cell suspensions at 18°C,
the white bod\ adherent cells were adhered to the bottom of the

Octopus Cellular Immunology

245

wells, tlutten and spread over the surface by extending pseudopo-
dia. Circulating hemocytes presented the same aspect.

When visualized under the electron microscope (Fig. I), what
seems as two different morphologies, that were distinct under the
inverted microscope, were observed among circulating hemocytes.
No differences were found in the quality of fixation when the
previous glutaraldehyde step was omitted and therefore the cells
were always directly fixed in osmium tetroxide. We identified in
the first cell type a kidney-shaped nucleus that occupied about 2/3
of the cell volume with a well-defined nucleolus and abundant
heterochromatin in peripheral positions. Their cytoplasm was rich
in vacuoles and electron-dense granules of various sizes. The sec-
ond cell type had a nucleus with faint chromatin, a round nucleus
that occupied about 1/3 of the cell volume. The cytoplasm was rich
in vacuoles, but had only scarce granules. However, these results
are not conclusive and we cannot assure that what looks as two
different morphologies corresponds to two distinct cell types.

In the case of white body cells, we were able of identifying
cells showing other morphologies that may correspond to interme-
diate stages between the two cell types found in the hemolymph.

Phagocytosis Activity of White Body Adherent Cells and
Circulating Hemocytes

The phagocytic activity detected after incubation of octopus
hemocytes and white body cells with zymosan was low. The per-
centage of phagocytosis observed in circulating hemocytes was
19.3% (SD = 14), while only 9.3% (SD = 8) of white body cells
contained zymosan particles in their cytoplasm. In all cases, varia-
tions among individuals were high, since in some samples no
phagocytosis was observed (0% of phagocytosis). The same results
were obtained with the two incubation temperatures (18°C or room
temperature) in both circulating and white body cells. No differ-
ences were observed when the hemocytes were incubated in their
own hemolymph (data not shown).

Respiratory Burst Activity

Octopus white body cells showed a significant increase in the
release of superoxide anion after stimulation with PMA compared
with controls, as depicted in Figure 2A. The specificity of the

Figure 1. Under the electron microscope, two distinct morphologies
were identified among circulating hemocytes. The first cell type (I) is
characterized by its kidney-shaped nucleus and the high number of
electron-dense granules. The second cell type (III possessed a round
nucleus with a few electron den.se granules. Bar = 5 fini.

A

-

T

"■i:W'

-

. T ,

-1-

Cit PMA PMA+SOD

Treatmenl

Conlml LPS ZyimtiJii

Ta-.itinenl

c

*

-

T

-

i

T

T

1

X

c

Control LPS Z\niosaii

Treatment

Figure 2. Respiratory burst activity of octopus white body cells. First,
the capacity of PMA to stimulate the respiratory burst activity and the
specificity of the reaction was assayed (A) (N = 2). Once, this was
determined, the effects of LPS and zymosan on the respiratory burst
activity of white body hemocytes (B) (N = 4) and circulating hemocytes
(Cl were studied through the reduction of Cit C, stimulating the cells
with PMA. Data are shown as the mean O.I), at 550 nm. *Respiratory
burst significantly higher than the respiratory burst obtained in con-
trols. P < 0.05.

reaction was demonstrated since SOD completely inhibited the
respiratory burst response of octopus cells in all cases. This re-
sponse was also observed with circulating hemocytes. The respi-
ratory burst activity of hemocytes incubated directly in their own
hemolymph was also assayed. In these conditions, some octopuses
did not respond to PMA and did not elicit a respiratory burst
response.

Figure 2B shows the effect of incubation for 24 hours with LPS
or zymosan on the respiratory burst triggered by PMA of white
body cells, compared to the respiratory burst observed in cells that
had been incubated with L-15 only. The pre-incubation of the cells
with zymosan, but not LPS. significantly increased the respiratory
burst of white body cells in response to PMA. The same response
was observed in cells obtained from hemolymph. showing a higher
respiratory burst after zymosan treatment than controls (Fig. 2C).

NO Production

The NO production of white body cells in response to LPS and
zymosan is shown in Figure 3. Zymosan significantly stimulated
the cells for NO production in all individuals, however, as in the
case of the respiratory burst activity, LPS failed to stimulate the
cells.

In the case of hemolymph cells, a similar response was ob-
served. The NO production in the cultures treated with zyinosan
(S.4; SD = 0.8) was higher than the response observed in controls
(6.7; SD = 0.3).

246

NOVOA ET AL.

Control LPS Zymosan

Treatment

Figure 3. NO production of cells from the wliile bodj after incubation
witli LPS or zymosan. Data are presented as the mean nitrite concen-
tration obtained with 4 octopus. 'Nitrite concentration si}>niricantl>
hifjher than the one observed in controls onlj treated with 1,-15. P <
0.05.

('H)-Thymidiiie Uptake

Not all animals studied showed a significant response. Only
two out of six individuals assayed responded with a significant
increase of the thymidine uptake in response to mitogens. The
individual responses observed in these two animals are shown in
Table I . When means were compared, concerning white body
cells, a maximum thymidine uptake was observed with the higher
LPS dose (687.3: SD = 6.7). In this case, the PHA dose that
significantly stimulated the thymidine uptake was 2.5 p.g/ml (495;
SD = 20) in comparison to controls (.'^78.3; SD = 2.5).

In these two responsive animals, hemocyte thymidine uptake
was also significantly affected by the mitogens. All concentrations
of LPS significantly increased ('H)-thyniidine uptake compared to
non-stimulated controls, although the higher response was ob-
served with the highest LPS dose. However, hemocyte thymidine
uptake was only significantly increased with the higher dose of
PHA.

TABLE 1.

Thymidine uptake by circulating hcmocytes and white body cells in

two responsive animals. Data are presented as the mean cpni

obtained in the replicas (N = 3l ± SD.

Octopus 1

Octopus 2

White

White

Circulating

Body

Circulating

Bodv

Mitogens

Hemocytes

Cells

Hemocytes

Cells

LPS 50 |xg/ml

630.6 ± 242

694 ± 35 1

515.3 + 37

680.6 ± 1 04

LPS 25 (jLg/ml

340 ± 1 1 S

.%6± 144

457 ±217

370 ± 70

LPS 12.5 jjig/ml

458 ± 98

761 +418

427 ±41

524 ± 142

PHA 5 Jig/ml

340 ± 92

241 ± 171

615 ±128

253 ± 58

PHA 2.5 |xg/m]

23S± 12

475 ± 147

141 ±32

515 ± 154

PHA 1.125 |j.g/ml

178 + 52

313 + 79

222 ± 42

290 ± 55

Control

204 ± 64

381 ± 105

294 ± 94

376 ± 1 2

DISCUSSION

The findings in this study suggest that both octopus white body
cells and circulating hemocytes are capable of performing func-
tions associated with host defense mechanisms. This is particularly
important to determine since scanty data is available on the im-
mune response of this octopus species. This is the first work in
which reagents and techniques usually used in vertebrate immu-
nology have been successfully applied to study cellular responses
of cephalopods.

Under the electron microscope, two distinct morphologies
among circulating hemocytes were identified, although it had been
described as only one cell type of the hemolymph of Oclopus
vulgaris (Bidder et al. 1989). Our results are not conclusive and
more work should be done to determine whether these two mor-
phologies correspond to different states of activation or they con-
stitute two different cell types. Previous studies in bivalve mol-
lusks have identified two main hemocyte types in the hemolymph
(Fisher 1986; Lopez et al. 1997) that have been subdivided (Auf-
fret 1988; Nakayama et al. 1997). In the white body, it is well
known that there are cells, refened to as hemocytoblasts. with a
large cytoplasmic volume and abundant rough endoplasmic reticu-
lum and nucleoli (Ford 1992). Hemocytoblasts transform to leu-
koblasts by reducing its cytoplasmic volume and decreasing its
nuclear size, whereas secondary leukoblasts (mature hemocytes)
are cells with a larger size and a folded nucleus. Both under the
light and electron microscope, we were able to identify different
morphologies that may correspond to these intermediate stages
among white body cells. As well, the two different morphologies
that we describe in this work among circulating hemocytes seem to
correspond to these two cell types (hemocytoblasts and leuko-
blasts). It may be possible that in these individuals maturation ends
in the hemolymph or as will be discussed later, that these two cell
types are functionally different.

The first cell type may correspond to what has been called
granulocyes in bivalves (Auffret 1988). These cells possess a kid-
ney-shaped nucleus that resembles those of vertebrate granulo-
cytes. The second cell type had a round nucleus and a cytoplasm
that was also rich in vacuoles, but had only scarce granules. This
cell type may correspond to what in bivalves has been called
hyalinocytes (agranular hemocytes) (Auffret 1988).

Results concerning in vitro phagocytosis by octopus hemocytes
are in accordance with those previously reported (Crowden &
Curtis 1981) where it was suggested that gill tissue or phagocytes
from gill tissue could be responsible for clearing foreign sub-
stances and that circulating hemocytes had low activity (Crowden
& Curtis 1981: Bayne 1983). However, studies in other cephalo-
pod species, like Eledone cirrhosa reported bacterial phagocytic
rates of even 40% (Malham et al. 1997). The low phagocytic
activity that is observed in the octopus. Octopus vulgaris, circu-
lating hemocytes also contrasts with the high phagocytic rate de-
tected in bivalves (Mortensen & Glette 1996; Ordas et al. 1999).
animals in which phagocytosis is a critical defense mechanism.
Many factors can affect phagocytic rates in mollusks such as tem-
perature (Carballal et al. 1997), time and pH (Abdul-Salam &
Michelson 1980), size and nature of the particle presented for
phagocytosis (Bayne 1983). In some cephalopod species, phago-
cytosis is not possible in the absence of hemolymph (Stuart 1968).
It may be possible that on the contrary to what happens with the
respiratory burst (that is inhibited by the presence of hemolymph).

Octopus Cellular Immunology'

247

phagocytosis in this species needs humoral factors present in the
hemolymph.

Octopus hemocytes, however, were able to respond to PMA
and release significant amounts of superoxide anion. Respiratory
burst activity is an interesting non-specific defense mechanism that
vertebrate macrophages use to avoid bacterial and parasitic infec-
tions due to the potent microbiocidal effects of oxygen radicals.
The reason why the respiratory burst activity was not detected in
some of the octopuses while incubated in their own hemolymph is
unknown. The immune system is regulated by multiple signals that
can up or down modulate the animal responses. Maybe regulating
factors, affecting this immune response, are present in the
hemolymph of these animals, in the same way that other humoral
factors such as lysozyme or antiproteases have been detected in
cephalopod hemolymph (Malham et al. 1998).

We have also demonstrated that octopus cells deri\ed from
hemolymph and white body produced NO. In \'ertebrates. NO
mediates many functions including neurotransmission, vasodilata-
tion, as well as several immune functions. It is known that this
molecule when secreted by macrophages is microbicidal against
viruses, parasites and bacteria (Nathan & Hibbs 1991; Tafallaet al.
1999). In the octopus, NO production was significantly increased
when the cells were incubated with zymosan for 24 hours. How-
ever, incubation with LPS did not have an effect on the NO se-
cretion. This is the first report on NO production by cells of any
cephalopod. NO production in other mollusks has been shown, in
which NO production was demonstrated by indirect methods
(Maninez 1995: Otaviani & Franchini 1995).

Octopus hemocytes were also able to increase their thymidine
uptake after stimulation with LPS and PHA. although this response
was not found in all indi\iduals. maybe because this immune
function is more related to a specific response not known to be
present in cephalopods. Thymidine uptake implies DNA synthesis.
so this increase may mean that the hemocytes are proliferating. If

so. this will be the first report of any proliferation in mollusks.
outside the hematopoietic organ. More work must be done to de-
termine if this thymidine uptake detected coirelates with cell pro-
liferation. In higher vertebrates, these two mitogens correlate to B
and T lymphocyte mitogens, respectively. Since we observed ul-
trastructurally two morphologies among hemocytes in responsive
animals, it may be possible that LPS and PHA-induced prolifera-
tion are being supported by different hemocyte populations, and
therefore imply a certain grade of heterogeneity, as in vertebrate
immune cells.

In conclusion, we have effectively applied immunological tech-
niques designed for vertebrates to evaluate cephalopod hemocyte
functions. Among mollusks, cephalopods are highly evolved ani-
mals with a body design, nervous system and sense organs that
often reach vertebrate standards (Budelmann et al. 1997). It seems
possible that immune system may also be more developed than
other mollusks in which modulation of immune responses by
pathological agents or other factors has been reported (Ordas et al.
1999). We have set up different in vitro techniques, such us de-
termination of respiratory burst activity, NO production and cell
proliferation, which will let us further evaluate the octopus im-
mune response against infections or the influence of environmental
conditions. This latter aspect is especially relevant because of in-
creasing interest that cephalopod aquaculture has had in the past
years (Osako & Murata 1983; Guerra & Rocha 1994; Guerra et al.
1994). More work must be done to clarify the regulation of these
immune functions in cephalopods and determine their role in the
defense against pathogens.

ACKNOWLEDGMENTS

The authors thank Dr. Guerra"s team. Begona Villaverde and
Jose Ramon Caldas for their technical assistance and Dr. M. Faisal
(Virginia Institute of Marine Science. USA) for reading and re-
viewing this article.

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Journal nf Shellfish Rt-seanh. Vol. 21, No. 1, 249-253. 2002

ESTIMATING GONADO-SOMATIC INDICES IN BIVALVES WITH FUSED GONADS

H.-JORG URBAN'* AND JOSE M. RIASCOS V.-

Alfred Wegener Institute for Polar and Marine Research, Section for Comparative Ecosystem Researcli,
Postfacli 12 01 61, 27515 Bremerhaven. Germany: 'Mnseo de Ciencias Naturales "Federico C.
Lehmann " A. A. 5660, Cali - Coloml^ia

ABSTRACT A new method was developed to estimate the gonado-somatic index in bivalves, taking into account that in most
bivalves gonad and non reproductive tissues are fused. As examples, four tropical bivalve species ^Dollux demifer. Cardita ajfinis.
Pmctada imbricara and Pteria colymhus) from the Colombian Pacific and Caribbean were used. The fused gonad regions were replaced
by geometric bodies, thus the gonad volumes were estimated with geometric equations (corresponding to geometric bodies) and linear
measures obtained directly on frozen soft-body samples. The very good correlation between the gonado-somatic index and the
gametogenetic cycle of the stage "ripe", leads to the conclusion that this method is a suitable tool to estimate gonad indices of bivalves
with fused gonads.

KEY WORDS: method, gonado-somatic index, bixalves. Doiiax demifer. Cardihi affhus. Pmctada imhricaia. Pteria cohmbi(s

INTRODUCTION

A standard procedure in marine biological reproductive .studies
is to determine gametogenetic stages (Guillou et al. 1990) or lo
estimate indices (Moore 1934; Giese 1967; Pearse 196.'5). Among
many existing indices the so called gonado-somatic indices, cal-
culated from the relationship between a variable depending di-
rectly on reproductive processes and a variable independent from
reproductive processes, are most frequent used. Examples for the
variables used in gonado-somatic indices are the gonad volutne
and the total volume. Such a gonado-somatic index, followed pe-
riodically over a year, is a valuable tool because it is related
directly to reproductive activities, such as spawning events.
Among marine species, the estimation of gonado-somatic indices
in bivalves often presents a problem because most species of this
group have fused gonads, such that reproductive and non-
reproductive tissues cannot — or are difficult to be separated.

Based on four tropical bivalve species, the objective of this
article is to present and explain a new method, that permits the
estimation of gonado-somatic indices in bivalves with fused go-
nads. In order to evaluate the new method, the gonado-somatic
index is compared with gametogenetic stages obtained from smear
samples.

MATERIALS & METHODS

Investigations were carried out in three areas: the Tayrona Na-
tional Nature Pari:, close to the city of Santa Marta. Caribbean of
Colombia (1 1°20'N. 74°10'W, Fig. la), the "Cal?o de la Vela", a
Peninsula located in the Guajira province. Caribbean of Colombia
(I2°I0'N. 72'20'W. Fig. lb) and the "Bahia Malaga', a bay
close to the harbor town of Buenaventura, Pacific Coast of Co-
lombia O'-Se'N. 77°19'W. Fig. Ic). In Tayrona Park the pearl
oyster Pteria colyitibus. which lives attached to octocorals is a
dominant species, while in Cabo de la Vela a characteristic Thalas-
sia testudinnm community gives a suitable substrate for a variety
of bivalve species, especially a dense PiiictaJa imhricaia popula-

*Corresponding author. H.-Jorg Urban. Alfred Wegener Institute for Polar
and Marine Research. Section for Comparative Ecosystem Research. Post-
fach 12 01 61. 27515 Bremerhaven. Germany. Phone: -H49-471 4831-1315;
Fax: ■H49-471-4831-1119; E-mail: jurbanCgawi-bremerhaven.de

Figure 1. Study areas a) Cabo de la Vela, Colombian Caribbean; 'i
Tayrona Park: Colombian Caribbean; and c) Bahi'a Malaga. Colom-
bian Pacific

249

250

Urban and Riascos

Diinax denlijer
12 3 4 5 6

Curiiifa ajpnis

12 3 4 5 6

Figure 2a shows the sotl parls (mantle, siphons and gills removed)
on the left shell of D. dcnlifcr and C. uffhn.s demonstrating the
fused character of somatic and gonad tissues. Figure 2b shows the
six transversal sections. In D. dentifer the visceral mass is located
in the middle-dorsal body part, partially enclosed by the gonad
region. The pedal muscle occupies the major part of the total body
volume extending in anterior-posterior direction and enclosing the
gonad region. Contrary, in C. affinis the visceral mass is located in
the posterior-dorsal part, decreasing towards the anterior end. The
gonad region is located in the posterior- ventral zone, increasing its
volume towards the middle part (section 3) and then extending

3) Donax dentifer

Cardila a/finis

Figure 2. a) Morphological description of body tissues (on left valve,
mantle, siphons and gills remo\ed) of Donax dentifer and Cardita af-
finis. Location where transversal sections were taken is indicated. b»
Transversal section according to Figure 2a. Gonadal tissue is given in
black, visceral tissue in gray and muscle tissue in white.

tion. Finally, at Bahki Malaga the infaunal Doiiax dentifer found
in the intertidal of sandy beaches and the borer clam Cardita
ajfinis from rocky shores were studied.

Sampling for Dmiax dentifer was earned out in 1997/98 as well
as in 1999/00, while for the latter three species the study period
lasted one year, but at different dates (Pteria colyinhtis: 1994/95.
Pinctada iinbricata: 1997/98, Cardita affinis: 2000/01). At
monthly intervals, samples were taken and the shell length (ante-
rior-posterior axis) of all specimens was recorded with vernier
callipeis. Two subsamples of 30 specimens per species each were
taken. One subsample was used for length-weight relationships:
soft parts were removed and dried at 70°C to constant weight to
determine shell free dry weight (SFDW). Monthly length weight
relationships were used to estimate a shell free dry weight ( = con-
dition) cycle of a standard individual ( = 667f of maximum length)
based on Equation ( I );

SL = a- SFDW

(I)

where SL is the shell length |mm| and SFDW is the shell free dry
weight [g]. The second subsample was deep frozen and used to
obtain linear measures of gonad regions with a vernier callipers in
order to estimate the gonado-somatic index (details are given un-
der "Results" later). Finally, after taking the linear measures, ga-
metogenetic stages of smear samples were determined according
to a microscopic scale (Urban 2000): "developing 1 (= early de-
veloping)", "developing 2 ( = late developing)", "ripe" and
"spent".

RESULTS

Using frozen samples, six transversal sections (thick slices)
were taken at different locations of the body: these were used to
identify the gonad legion geometrically. As an example only the
morphological sections o'i two of the four species are presented.

m

-if-ii""

b) Cardita affinis

's-K

triangle
prism ^^

^

a

■be

-\-

a-b-c

2

4

area

C)

d)

cylinder

y.r, = "

e) Pinctada imbricata & Pteria colymbus

pyramid

1 fe-t

Figure 3. Geometric bodies defined according to the morphological
examinations of the transversal sections (Fig. 2bl. The linear measures
necessary to calculate the volume as well as the formulas are given (see
text), a) Ellipsoid of Ihmax dentifer. h) Two fused triangle prisms of
Cardita affinis. c, d. and e) Fused cylinder and pyramid of Pinctada
imbricata and Pteria colynihns. Simple small letter correspond to linear
measures of somatic and reproductive tissues, while gonadal tissues
are given as small letters with "'" (e.g. a').

Estimating Gonado-Somatic Indices in Bivalves

251

a) Donax dentifer

b) Cardita affinis

c) Pinctada imbricata &
Pteria colymbus

Figure 4. Body tissues (upper row ) and geometric bodies replacing gonad region and or body tissues (lower row), a) Donax dentifer. b) Cardita
affinis and c) Pinctada imbricata and Pteria colymbus

towards the anterior-dorsal part where its volume decreases gradu-
ally. The pedal muscle is reduced, corresponding to a small ex-
tension of the posterior-ventral end. The volume of the abductor
muscle increases from the middle ventral zone, extending towards
the anterior end.

Based on the morphological examination of the gonad region
(Fig. 2) for all species three-dimensional bodies of the gonad re-
gion were defined. In other words, the gonad region was replaced
with geometric bodies. The volumes of these bodies and thus the
gonad volume, can be estimated using the following geometric
equations and linear measures [mm] taken directly on the bivalves
from frozen samples. Based on macroscopic criteria such as tex-
ture, color, etc.. gonad tissue could be easily distinguished from
somatic tissue. Thus, for each species during the study period the
gonad volumes |ml) of approximately 30 specimens were obtained
at monthly intervals.

Donax dentifer

The gonad morphology corresponds to an ellipsoid half (Figs,
3a & 4a). located between the foot and the visceral mass. The
equation below Figure 3a corresponds to the volume of an ellip-
soid half. It is clear, however, that a small visceral region is located
in the dorsal part of the "gonadal ellipsoid" (Fig. 2a). On average,
this visceral tissue occupied 10% (max: 19%, min: 6%) of the total
ellipsoid volume. Thus, in order to yield the gonad volume, the
formula was corrected multiplying with 0.9 (Eq. 2):

V„

a b

TT • - • - • c I /2

■0.9

(2)

Measures are indicated in Figures 3a & 4a; "a" is the gonad
breadth (lateral to the body axis), "b" is the gonad length (anterior-
posterior body axis) and "c" is the gonad height (dorsal-ventral
body axis at transversal section number 3.

Cardita affinis

The geometry of the gonad region suggests the form of two
fused triangle prisms (Fig. 3b & Fig. 4b). Thus, the formula of a
single triangle prism has to be multiplied by 2 (Eq. 3):

V = [A,

base "prismJ ■"

a-h,,

(3)

Where A^,^„^. is the area of the base, which corresponds to a triangle
with "a" being one side and "h " the heiehl of the triangle corre-

sponding to "a", finally "h

the height of the prism. The

gonad volume was calculated as the sum of these two prisms
(Eq. 4):

a-b-c] Tab-c

(4)

Where "a" is the gonad length (anterior-posterior body axis), "b"
is the gonad height (transversal to gonad length corresponding lo
transversal section number 3. Fig. 2) and "c" is the gonad breadth
at the fusion point of both prisms. (Note: "a" corresponds to the
length of both fused prisms, therefore for the two bodies "1/2 a"
has to be used yielding Eq. 4.)

Pinctada imbricata and Pteria colymbus

These two species of the family Pteridae are different from the
latter two because the gonad tissue covers the somatic tissues. In
D. dentifer and C. affinis the total body volume was estimated with
the water displacement method and gonad volume corresponds
directly to the geometric abstractions, while in P. imbricata and P.
colymlnis first the total body volume (visceral mass and foot in-
cluding gonads) and then in a second step the "covering" gonad
volume was estimated. The total body volume can be defined by a
cylinder (Fig. 3c & Fig. 4c. Eq. 5a) fused with a pyramid (Fig. 3d
& Fig. 4c. Eq. 5b). The cylinder corresponds to the visceral mass

252

Urban and Riascos

Fig. 5a & c

— •— OSl

■ - G- - npe
Fig. 5b & d
— • — dry weiglil
D developing I
D developing 2

■ npe

■ spent

Figure 5. a) Overlay of gonado-somatic index and gametogenetic .stage
"ripe" cycle, b) Overlay of shell free dry weight cycle and distribution
of gametogenetic stages of OoHa.v deiuifer from Bahia Malaga, Colom-
bian Pacific, c & d) Cardila affinis from Bahi'a Malaga, Colombian
Pacific.

and the pyramid to the basal region of the foot. These two bodies
have the general formula:

a) V,.„, = -IT • r" • h.

b)V„

= 3A,

(3)

Where "r" is the radius, h^.^,, is the height of the cylinder (Fig. 3c).
"Abase" 's 'hs ^'■'S^ of the base of the pyramid and "hpy/" is the
height of the pyramid (Fig. 3d). So the total body volume of these
two species coiresponds to the sum of the \ olume of a cylinder and
a pyramid (Fig. 3e. Eq. 6 & 7):

'body.tulpi^r.j^j ■

^v,

.yl

pyr

b + c\-

(6)

Where "a" is the length, "b" is the height and "c" is the breadth of
the visceral mass (including gonad region) and "d" is the length.
"e" is the height and "f" is the breadth of the foot (including gonad
region). In the second step the somatic volume was estimated using
the satiie equation (Eq. 6). but with linear measures where gonad
regions were excluded. Gonad volume was estimated subtracting
the somatic volume from the total volume (Eq. 7);

V = V,

gonptcniliK-

liudy 1

„" ^b,.Jy ..,„„,.,

(b + cy-

■n ■

I 4 ) -J

+

L

e ■ t

— 55 ■

Pwria tolymhiiy

■S 50-

\ ■' '•

a 45 ■

\ ■' '

i 40.

\'^"''-. ■■«■■ ■"'■

1 "■

»—,■.,■'/>, ■' y^\ A '

S 30

^* V/ \ /' V'

g

No^ ■

—

~ 80"o-
st

5 60%-

.a

e

it .(. .

\

1

f!

t

A^

V

=«^

1

1

L

1

II

'fi

50 S.

X

40 I

30 !

20 =

u

10 s

0 5 ,if

<i£

H^V^-

I-

A

V

Fig. 5a & c

— •— GSI

. ■ C- - npe
Fig. 5b & d
— • — iity weight
□ developing 1
D developing 2

■ npe

■ spent

t07 |-
I

Mji Ap! May Jun Jul Aug Sc-p Oci Nov Dec Jan Feb Mar

Figure 6. a) Overlay of gonado-somalic index and gametogenetic stage
"ripe" cycle, b) Overlay of shell free dry weight cycle and distribution
of gametogenetic stages of Pteria ciilymbus from Tay rcma Park. Co-
lombian Caribbean, c «& d) Pinctadu imhricata from Cabo de la Vela,
Colombian Caribbean.

b'+c'

Where "V,„,j^ ,^„"' is the total body volume and '"Vb„jy .

(7)
is the

volume of the somatic tissues [ml], "a", "b", "c". "d". "e". and "f
are identical to the measures of Eq. (6), while a', b'. c'. d'. e'. and
f are linear measures parallel to "a", "b". "c". "d". "e". and "f
coiTesponding only to somatic tissues (Fig. 3e).

Figure 4 shows in the upper row for all four species the soft
body parts on the right shell and below the geometric abstractions
defined to estimate the gonad volume. Note that in D. denlifer and
C. affinis only the gonad region corresponds to the (gray) geomet-
ric abstractions, while in P. imhriaila and P. colyinhus all soft
parts were replaced with the geometric abstraction, and the gonad
volume is covering non-reproductive tissues (in black).

TABLF. 1.

Spearman correlation matrix of gonado-somatic index and
gametogenetic stage "ripe" as well as shell free dry weight cycle

P. colyinhus

P. imhricata D. denlifer

C. affinis

Gonado-Somatic Index (GSI)

Ripe
SFDW

0.811
0.?i.)4

0.501 0.669
0.170 0.4,SS

0.627
0.264

Estimating Gonado-Somatic Indices in Bivalves

253

Based on the total body and the gonad \iilume a gonado-
somatic index (GSl. Eq. 8) was estimated.

V..„„

GSJ:

V.

100

(81

Table 1 gives the Spearman correlation matrix of the gonado-
somatic index (GSI) and the gametogenetic stage "ripe" as well as
the condition ( = shell free dry weight. SFDWi cycle. For all spe-
cies a high correlation between GSI and the amount of ripe gonads
is indicated (p > 0.5). For the correlation between GSI and SFDW
only for Pterin colyinhiis and Donax clciitifer high \alues can be
observed.

This result is confirmed in Figure 5a & c and Figure 6a & c
where GSI and ripe cycles are plotted in overlay graphs. In all
species the pattern of GSI and ripe cycle is very similar. Figure 5b
& d and Figure 6 b & d show the SFDW cycles together with the
distribution of gametogenetic stages.

DISCUSSION

In order to evaluate a new method it is most convenient to
compare the results with alternative information obtained com-
pletely independent. This can easily be accomplished for the GSI.
It can be assumed thai the GSI is positively correlated with the
gametogenetic stage "ripe". GSI gives the mean gonad volume/
total volume relationship per month. In other words if a large part
of the population has voluminous gonads, the GSI is high. On the
other hand the gametogenic stage "ripe" gives the percentage of
specimens with ripe gonads (shortly before spawning). In this
developinent stage, egg and sperm size is large, resulting in volu-
minous gonads. The results clearly confirm this assumption and
thus prove the applicability of the GSI method. Graphically (Fig.
5a and c and Fig. 6a and c) as well as statistically (Table 1 ) a high
correlation between the GSI and the gametogenic stage "ripe"
exists. It should also be noted that GSI and stage "ripe" are com-
pletely independent measures; The GSI is a quantitative index

based on linear measures, whereas the "ripe" cycle is a semi-
quantitative measure obtained from microscopic observations of
smear sample.

Together with the GSI and the "ripe" cycle, the shell free dry
weight (SFDW = condition) cycle as well as the distribution of all
gametogenetic stages is presented (Fig. 5b and d and Fig. 6b and
d). Here, the typical pattern of tropical species with a very long or
permanent spawning season and continuous reproduction strategy
is demonstrated. All four species studied have ripe ovaries through
out the year with no resting or inactive gonads found. This char-
acteristic pattern is in contrast to cold temperate species. As an
example. Urban and Campos (1994) studied the gonad cycles of
the infaunal clam Gari solida from Chile at 37°S. In this species
during 9 months of the year, developing I and/or spent stages
dominate (85'7f ). Only during the latter 3 months did ripe and/or
developing 2 stages doininate indicating a short spawning period.

Contrasting to the "ripe" cycle only for Pteiia colymhus and
Donax dentifer. a high correlation between GSI and SFDW was
observed. However, a high correlation between these two variables
cannot be expected generally in a tropical environment with little
annual variability of biotic and abiotic factors such as temperature,
nutrients or phytoplankton. Thus, under rather continuous feeding
conditions a condition cycle might be independent from the gonad
activities, contrasting to cold temperate latitudes with typical
spring/summer phytoplankton blooms.

ACKNOWLEDGMENTS

Data were obtained in the context of two larger projects: "Bi-
valve Culture in the Colombian Caribbean. 01/94-04/99" and "Ef-
fects of El Nino on Biological Resources of Latin America 1999-
2001". The first author thanks the following students for their
collaboration in the field, as well as working on the samples:
Angela Maria Perez, Maria Cristina Murillo, Juan Pablo Assmus
and F. Marcos Ablanque.

LITERATURE CITED

Giese. A. C. 1967. Some methods for study of the biochemical constitution
of marine invertebrates. Oceanogi: Mar. Biol. Ann. Rev. 5:159-186.

Guillou, J., G. Bachelet. M. Desprez, J. P. Ducrotoy, I. Madani. H. Rybar-
czyk. P. G. Sauriau. B. Sylvand, B. Eikaim. & M. Glemarec. 1990. Les
modalites de la reproduction de la coque (Cerastodenna edule) ser le
littoral franfais de la Manche et de I'Atlantique. Aquulic Living Re-
sources 3:29—41.

Moore, H. B. 1934. A comparison of the biology of Echinus esciilentiis in
different habitats. Part I. / Mar Biol. Ass. U. K. 19:869-885.

Pearse. J. S. 1965. Reproductive periodicities in several contrasting popu-
lations of Odontasler validiis Koehler. a common Antarctic asteroid.
Anwrcric. Res. Ser. 5:39-85.

Urban. H.-J. 2000. Aquaculture potential of the Caribbean Pearl Oyster
Pinclada imbricaki I. Gametogenic activity, growth, mortality and pro-
duction of a natural population. Aquaculture 189:361-373.

Urban. H.-J. & B. Campos. 1994. Population dynamics of the bivalves
Gari solida. Scmele solida and Protothaca tluua from a small bay in
Chile at 36'S. Mar. Ecol. Prog. Ser. 1 15:93-102.

.loiimal ,'f Shellfish Research. Vol. 2 I . No. I. 255-2?8, 2002.

COMPARATIVE FECUNDITY OF THREE PROCAMBARUS SPECIES

ARNOLD G. EVERSOLE* AND YAVUZ MAZLUM

Department <if Acjuacultiirc, Fisheries and Wiicllife. Clemson University.
Clenisoit. Soiilii Carolina 29634-0362

ABSTRACT Adult female Proemnbarus acinus acinus. P. zonangulus. and P. clarkii {« = 64) were held in simulated burrows for
160 days. Mean percentage hepatopancreas moisture for a subsample (/i = 12) of P. zonangulus was significantly less than that for
the other two species. Procambarus clarkii oviposited eggs 30 days earlier than the other two species; however, a significantly lower
proportion of the P. clarkii (33.3%) oviposited eggs than did P. a. aciiliis (95.3%) and P. zonangulus (71.9%). The number of extruded
eggs increased with crayfish total length (TL). Similar-sized P. clarkii oviposited significantly more eggs than P. zonangulus and the
eggs/feinale for P. a. acutus were significantly less than for the other two species. The dry weight, protein, and lipid content per egg
for P. a. acutus and P. zonangulus were similar and significantly greater than those for P. clarkii eggs. The smaller eggs oi P. clarkii
yielded shorter instars than P. a. acinus and P. zonangulus instar TL. A standard-sized P. a. acinus ( 104.6 mm TL) allocated 10.2%.
13.6%. and 22.2% of total-body dry weight, protein, and lipid to extruded eggs, respectively.

KEY WORDS: crayfish, fecundity, Procanihariis clarkii, Procambarus zonangulus. Procambarus acutus acutus

INTRODUCTION

Red swamp crayfish, Procambarus clarkii (Girard), dominates
crayfish aquaculture and wild harvests in the southeastern LInited
States (Eversole & McClain 2000). Frequently, these harvests eon-
tain the white river crayfish, originally classified as Procambanis
acutus acutus (Girard) until Hobbs and Hobbs (1990) revised the
taxonomy. Upon revision, the white river crayfish, which over-
lapped P. clarkii's range in Louisiana and in other states along the
Gulf of Mexico, was named Procambarus zonangulus Hobbs and
Hobbs. P. a. acutus. whose range extends from Maine to Georgia,
occurs sympatrically with P. clarkii only in locations where the
latter has been introduced.

Comparative studies of P. clarkii and P. zonangulus indicate
significant differences in life history traits between the two spe-
cies. For example. P. zonangulus produces fewer large eggs.
whereas P. clarkii produces a greater number of small eggs
(Noblitt et al. 1995; Noblitt & Payne 199,5). Noblitt et al. (1995)
suggested that P. zonangulus followed a prudent reproductive
strategy that was characteristic of species adapted to an environ-
ment where nutrients are low or pulsed at an unpredictable rate. In
contrast, P. clarkii' >i reproductive strategy is consistent with exis-
tence in a nutrient-abundant, predictable environment. P. zonan-
gulus also appears to be more conservative in mobilizing nutrients
during starvation than is P. clarkii (Powell 2001). Other compari-
sons include length-weight relationships, production characteris-
tics, and population studies in aquaculture systems (Romaire &
Lutz 1989; Deng et al. 1995; Huner 1994).

P. clarkii was introduced into South Carolina for aquaculture in
1978 (Pomeroy & Kahl 1987). Although P. clarkii is the inainstay
of the South Carolina aquaculture industry (Whetstone, pers.
comm.), there are concerns about its and other crayfish introduc-
tions (Taylor et al. 1996). Comparative research on P. clarkii and
P. a. acutus has been limited to investigations of the two species
in experimental culture ponds. Denson and Eversole (1996) re-
ported a dramatic shift in species composition from P. clarkii- to
P. a. rtfKrii.v-dominated culture ponds over a relatively short pe-
riod. Follow-up 2 years of supplemental stockings (59-1 14 kg/ha)

of P. clarkii failed to increase its long-term production in these P.
a. «<:7(f((i-doniinated ponds (Eversole et al. 1999). Although re-
tlooding of drained culture ponds in September increased P. clarkii
percentage in the harvest in comparison to October and November
reflooding dates, it did not improve production (Mazlum & Ever-
sole 2000).

A series of investigations have been designed to gain a better
understanding of P. clarkii and P. a. acutus interactions. The focus
of this study was to provide comparative reproductive data for P.
a. acutus. P. zonangulus. and P. clarkii. Specific objectives were
to determine the number of extruded eggs, egg size and composi-
tion, egg development lime, and instar size for the three species.

MATERIALS AND METHODS

Experimental Animals

Adult female P. a. acutus and P. clarkii were collected from
harvests at two locations in South Carolina; Clemson University
Aquaculture Research Facility at Clemson and a commercial farm
in Fountain Inn on May 24 and June I. 1999, respectively. Female
P. zimangulus were collected from Ben Hur Farm. Louisiana State
University. Baton Rouge. Louisiana, air shipped, and arrived at
Clemson on May 19. 1999. Individuals Ui = 76) of each species
with a complete set of appendages were either placed in simulated
burrows (?i = 64) or sacrificed for hepatopancreas moisture de-
terminations in = 12).

Hepatopancreas Moisture

The total length (TL) of 12 individuals of each species was
measured to the nearest millirneter. The hepatopancreas was ex-
cised, weighed to the nearest 0.01 g after blotting (//„„,) and dried
in a convection oven (80°C) to a constant weight (Hj^). Moisture
content of the hepatopancreas (HM) was calculated using the fol-
lowing formula;

HM = -

H„

H,,

X 100

*Corresponding author. E-mail: aevrsl@clemson.cdu
Technical Contribution No. 4728 of the South Carolina Agricultural Ex-
periment Station, Clemson University, Clemson, South Carolina.

Reproductive Success

Individual crayfish {n = 64) were measured (TL) and placed in
1-L plastic containers (simulated burrows). Although these con-

255

256

EVERSOLE AND MaZLUM

tainers were not intended ti) duplicate conditions in earthen bur-
rows, they do represent a technique to compare fecundities across
species under more uniform test conditions. The simulated bur-
rows were suspended in restaurant glass racks held in four race-
ways receiving dechlorinated aerated water. Each raceway con-
tained a rack of 16 individuals of each species. Holes drilled in the
bottom and along the side of the container allowed water exchange
and maintained 5-cm water depth. Crayfish were held at ambient
water temperatures and not fed over the 160-day experimental
period. Crayfish were checked 5 days/wk and every fifth day the
water was exchanged in the raceway. Water temperature and dis-
solved oxygen were checked by meter (Model .S?. Y.Sl). Survival
and the presence of extruded eggs were noted by date. A propor-
tion of the ovigerous females were removed from burrows, usually
within 24 h of oviposition. and the extruded eggs removed v\ ith
forceps, counted, and fixed in 10% neutral formalin. Eggs on the
other ovigerous females were allowed to develop and hatch. De-
velopment time was recorded and the TL of third instars measured.

Egg Characteristics

Egg subsamples (/i = 4-5) from 12 individuals of each species
dried at 80'C were weighed to the nearest 0.001 mg to estimate
mean egg dry weight. Following drying, the nitrogen content of
egg samples was determined by the Dumas method using a Carlo-
Erba NA 1800 (Series 2) nitrogen analyzer. Nitrogen content (per-
cent) in an egg sample was multiplied by 6.16 to estimate the
percentage protein per egg on a dry weight basis (Noblitt & Payne
1995). Lipid content was determined on duplicate subsamples of
10 eggs each from six ovigerous females per species. Egg lipid
content (percent) was determined as described by Mann and Gal-
lager (1985). Egg lipid content (percent) was calculated on a dry
weight basis as a mean of the duplicate subsamples.

Statistical Analysis

Data were analyzed with the analysis of variance (ANOVA).
analysis of covariance (ANCOVA), and linear regression using
Micro-SAS Statistical Software System Version 8 (SAS 1999). A
split-block design with trays representing blocks was used to ana-
lyze the effects of raceways on the reproductive performance of
the three species. The general linear model for ANOVA was used
to compare species differences in initial TL, instar TL, hepatopan-
creas moisture content, time to ovipositing, hatching time, and egg
size (dry weight, lipid, and protein content). The differences be-
tween ovipositing and nonovipositing female crayfish within spe-
cies were compared by ANOVA. Linear regression was used to
determine the relationship between crayfish TL and number of
extruded eggs per ovipositing female. The ANCOVA procedure
was used to compare slopes and intercepts of the regressions and
eggs/female. Differences in means were detected with least-
significant difference (LSD) test (Ott 1993). Level of significance
was set at P < 0.05.

RESULTS

Experimental Animals

The TL of the species stocked in simulated burrows were sig-
nificantly different; P. a. acinus was the longe.st, P. zoiuingiiliis the
shortest, whereas P. clurkii was intermediate in TL (Table 1 ). P.
zonaiifiiiliis hepatopancreas contained the least amount of moisture
and was assumed to be in better conditions than the other two

TABLE I.

Mean (±SD) and range of tola! length ( TLl of the females used in

the experiment, ovipositing and n(>n<i\ipositing cravfish,

hepatopancreas moisture, time from the start of experiment to

ovipositing, and number of extruded eggs per female.

Variable*

P. a. acutus

P. zonangulus

P. clarkii

Initial female TL

109.1 ±6.89"

9i.l±19'i'"

100.5 ± 9.44'-

(mm)

(90-127)

(80-120)

(83-125)

u = 64

n = 64

n = 64

Hepatopancreas

55.1 +8.31''

39.1+6.17"

55.3 ± 5.93-

moisture {%)

(41-69)

(29-50)

(48-64)

n = 12

II = 12

,; = 12

0\ipositing femule

109.3 ±6.99^

97.4 ± 7. 22'^

103.0 ±7.5(.)^

TL (mm)

(90-127)

(85-120)

(88-115)

II = 61

n = 46

n = 24

Nonovipositing TL

104.7 + 1.15'

91.1 ±8.38"

99.1 ± 10.51'^

(mm)

(104-106)

(80-112)

(83-125)

/( = 3

n = 16

n = 38

Time to ovipositing

108.4 + 6.87-'

114.0 ± 8.00-

99.0 ± 19.72"

(days)

(89-122)

(92-146)

(58-131)

n = 61

n = 46

n = 24

Eggs/female

304.9 ± 50.34-

367.2 ± 36.48"

586.5 ± 58.89'

(234-555)

(189-420)

(282-764)

n = 40

n = 24

/; = 12

* Those means sharing lower case superscripts within rows or uppercase
superscripts within columns are not significantly different iP > 0.05).

species (Huner et al. 1985; Huner et al. 1990). Mortality in the
simulated bunxiws was low; only two individuals each ot P. zi>-
iHiii.tiKliis and P. clarkii died over the 16()-day experimental period.
Mean (±SD) water temperature and dissolved oxygen was 24.5 ±
2.2 T'C and 4.0 ± I. .38 mg/L. respecti\ely. o\er the period.

Percentage of crayfish ovipositing was similar among the four
raceways. Because of the lack of difference among the raceways,
blocks (raceways) were pooled in subsequent statistical analysis of
reproductive performance measures among species.

Reproductive Success

A significantly higher proportion of P. a. acutus (953%) ovi-
posited eggs than P. zoiuiiiguhis (74.2%). and the proportion of
ovipositing P. clarkii (3SJ9c} was significantly less than the other
two species. In contrast. P. clarkii was the first species to oviposit
eggs, more than .30 days earlier than the other two species (Table
I ). No crayfish were observed ovipositing eggs within the last 4
weeks of the experiment; however, glair gland development was
not assessed in these crayfish.

Individuals of P. a. acutus entire size range (90-127 mm TL)
oviposited eggs, whereas some smaller individuals of the other two
species did not oviposit (Table 1 ). Although the ovipositing indi-
viduals of each species were larger than the nonovipositing indi-
viduals, the onl\ significant difference in TL was observed with P.
Z(>iiaui;iilus.

Number of extruded eggs ranged from 189 on a 85-mm TL P.
zonangulus to 764 on a 1 14-mm TL P. clarkii (Table 1 ). Extruded
eggs per female varied significantly with increased TL in the three
species (Fig. 1 ). These linear relationships explained 80%-90% of
the variation among individuals of the species. Using TL as a
covariant. ANCOVA indicated significant differences in extruded
egg counts among the three species, with P. clarkii being most

Comparative Procambarid Feci'ndity

257

fecund (Table 1 1. The slopes and intercepts of the regressions were
also significantly different among species.

Egg Charuclcristics

Eggs of P. a. acutiis and P. zonangulus were similar in weight
and significantly heavier than P. clarkii eggs (Table 2). The eggs
of these two species also contained a significantly greater propor-
tion of lipid and protein than did those of P. clarkii. The smaller
eggs of P. clarkii hatched approximately 3 days earlier than those
of the other two species; however, the TL of the third instars were
significantly shorter than the instars of P. a. aciilus and P. zoiuiih
gulus (Table 2).

DISCUSSION

P. a. aciilu.s and P. zoiuingnliis share many morphological,
ecological, and leproductive characteristics. Both species belong
to the subgenus Ortmannicus. diagnosed by the terminal element
of the first pleopod (Hobbs 1972; Hobbs & Hobbs 1990). Although
this characteristic clearly distinguishes P. zonangulus as a species
and distinct from P. a. acutus. these two species have similarly
shaped chelipeds. carapace, and overall body proportions (Hobbs
1981; Hobbs & Hobbs 1990, pers. observations). Crayfish with
similar body forms frequently occur in similar habitats (Hobbs
1975; Holdich & Reeve 1988). P. a. aciilus and P. zonangulus
occur in sluggish streams with aquatic vegetation and lentic habi-
tats, including those with fluctuating water levels such as borrow
pits, roadside ditches, sloughs, and farm ponds (Hobbs 1981;
Hobbs & Hobbs 1990; Deng et al. 1993). Adults of both species
retreat to burrows in drying habitats and also to oviposit eggs,
which is characteristic of tertiary burrowers (Hobbs 1981).

P. a. acutus and P. zonangulus shared several of this study's
measured reproductive characteristics with the exception of
hepatopancreas moisture content and the number oi extruded eggs
per female (Table 1 ). Crayfish store large amounts of energy
(lipid) in the hepatopancreas for oocyte development and survival
while in the buiTow (Huner 1989). For this reason, relative mois-

P. clarkii

Eggs/Female = -1498.9t19,94TL

r'- 0.8997

n= 12 / P. zonangulus

Eggs/Female = .426.34*7-60TL

r'= 0 7938

n= 24

A p. a, aculus

Eggs/Female = -962.92+1 2,1 2TL

r'= 0,8033

n=40

80 90 100 110 120 130

Total Length (mm)

Figure 1. Relationships between total length (TL, mm) and number of
extruded eggs per female for Pnicainhanis clarkii. (closed circles), P. a.
acutus (triangles), and P. zonangulus (open circles).

TABLE 2.

Mean (-fSD) and range of egg dry weight, lipid, protein, hatching

time from ovipositing dale to the appearance of instars and the total

length (TL) of instars.

Variable*

No. P. a. aculus P. zonangulus

P. clarkii

Dry weight 12

(mg/egg)

Egg lipid (%) 6

Egg protein O ) 12

Hatching time 9

(days)

Instar TL (mm) 200

3.49 ± 0.379'

(2.83-4.20)
26.0+ 1.16'

(23.6-28.2)
58.9 ± 3,67-'

(47.4-60. 1 )

21.1 ±0.60-'

(20-22)

9. .5 ± 0.55-'
(9-11)

3.44 ± 0.443'

(2.64-+.29)
26.6 ± 2.06-'

(22.0-28.6)
57.6 ± 4.37"

(43.4-58.8)

21.6 ±0.88"

(20-23)

9.6 ± O.SS"
(9-11)

1 .94 ± 0.245'"
(1.46-2.27)
17.6 ± 1.75*'
(19.2-20.7)
45.4 ±3.21"
(40.5-50.9)
18.6 ±0.73"

(18-20)

7.6 ±0.51"

(6-8)

* Those means within rows sharing lowercase superscripts are not signifi-
cantly different (/'>0.05).

ture content of the hepatopancreas has been suggested as an indi-
cator of crayfish condition (Huner et al. 1985; Huner et al. 1990).
It was expected, based on hepatopancreas moisture content, that P.
zonangulus fecundity would be greater than a similar-sized P. a.
acutus. which was the case. The "better conditioned" P. zonangu-
lus oviposited more lipid as eggs (349.9 mg on a dry weight basis)
than a comparable-sized P. a. acutus (271.6 mg lipid).

The reproductive characteristics of P. a. aculus and P. zonan-
gulus contrasted that of P. clarkii (Tables 1 and 2). For example,
P. clarkii fecundity was higher and the extruded egg size was
smaller than that observed for P. a. acutus and P. zonangulus.
Also, the hatching time and TL of hatchlings (instars) was shorter
for P. clarkii than for the other two species. Our observations are
consistent with the hypotheses of Noblitt et al. (1995) in that the
reproductive characteristics of P. zonangulus and P. a. acutus
follow a prudent strategy where more resources (energy) are in-
vested in each egg, improving the survival or fitness of offspring
in less predictable environments. Instar fitness is more dependent
on size in this reproductive strategy scenario, and those species
with bigger eggs yield larger instars. The larger instar P. a. acutus
(10 mm TL) in allopatric and sympatric competition experiments
survived at a higher rate than the smaller P. clarkii instars (8 mm
TL) (Mazlum & Eversole. unpubl.).

The fraction of dry weight, protein, and lipid extruded in eggs
were computed from linear regressions for whole-body female P.
a. acutus dry weight, protein and lipid levels (calculated from data
of Turker 1997) and for our fecundity estimates in terms of each of
these parameters. A standard-sized P. a. acutus (104.6 mm TL)
with an average fecundity of 249.4 eggs oviposited 1,065 mg dry
weight, 573.7 mg protein, and 271.6 mg lipid. The fraction of the
whole-body dry weight, protein, and lipid used for extruded eggs
was 10.2%. 13.6'7r. and 22.2%. respectively. The average repro-
ductive lipid output (extruded eggs) of 22.3% total-body lipid
computed for 3 1 ovipositing P. a. aculus (Eversole et al. 2000) was
almost identical to the estimate derived in this study. Similarities
in the proportion of lipid devoted to reproduction are explicable in
terms of fecundity, egg size, and the total-body content. Because of
the differences in systematics and reproductive strategies among
the species, it might be expected that the allocation of energy
(lipid) to reproduction differs as well. Unfortunately, this compari-
son will have to wait until compatible data are available for tho
species or reproductive strategies.

258

EVERSOLE AND MAZLUM

ACKNOWLEDGMENTS

Funding was provided by the S.C. AqLuiciilliire Reseaixh Ini-
tiative. S.C. Agricultural Experiment Station, and the Turkish Min-

istry of Education. Dr. L. W. Grimes provided statistical advice.
Dr. Robert Romaire provided the P. zoiiaiifiulKS. and Ms. J. Richard-
son suffered through multiple drafts of this manuscript. We also thank
.Shane M. Welch and Danny R. Jones for reviewing the manuscript.

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studies of two sympatric Prncamharu.s crawfishes. ./. Shellfish Res.

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crayfish production in South Carolina. Freshwawr Cniyfish 11:550-

565.
Eversole, A. G. & W. R. McClain. 2000. Crawfish culture. In: R. Stickney,

editor. Encyclopedia of aquaculture. New York: John Wiley, pp. 188-

Eversole, A. G., Y. Mazluni & C. Erkebay. 1999. Effects of stocking
Procambants clarkii (Girard) in culture ponds dominated by Procam-
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Eversole, A. G., Y. Mazlum. Q. C. Fontenot & H. Turker. 2000. Evaluation
of a non-invasive technique for predicting reproductive success in
white river crayfish. Int. Assoc. Astacology Syinp. 13:(abstract).

Hobbs. H. H. Jr. 1972. Crayfishes (Astacidae) of North and Middle
America. Biota of freshwater ecosystems. Identification Manual No. 6.
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Journal of Shellt'ish Reseunh. Vol. 21. No. 1, 259-265. 2002.

CONFIRMATION OF TWO COMMON MUD CRAB SPECIES (GENUS SCYLLA) IN THE
MANGROVE ECOSYSTEM OF THE MEKONG DELTA. VIETNAM

D. J. MACINTOSH/* J. L. OVERTON,' AND H. V. T. THU"

^ Centre for Tropical Ecosystems Research. Department of Ecology and Genetics. Universir\- of Aarhiis.

Aarhits. Denmark

'Department of Biotechnology and Aquacultitre. Institute of Oceanography. Nhatrcmg. Vietnam

ABSTRACT Adult and iiivenile mud crahs (genus Scxlla) were collected from two sites in the Mekong Delta. Vietnam. The
specimens were examined morphologically and analyzed by starch gel electrophoresis to confirm the species present based on a
recently published revision of the genus Scylla that recognizes four species from adult morphological characters and electrophoresis.
The Mekong Delta samples contained two species. S. olivucea ("red crab") and S. paianuunosiiin ("green crab"). Ratios derived from
measurements of the carapace width and the frontal spines, which are species diagnostic in adult mud crabs, did not separate juvenile
S. panwmmosain and S. olivacea reliably. However, in combination, differences in the shape of the frontal spines, the spination on the
cheliped. and cheliped shape and color, make it possible to distinguish juveniles of these two species down to a size of about 1 .5 mm
carapace width. This is the modal body size of new recruits entering the mangroves of the Lower Mekong Delta. The diagnostic
morphological features of adult and juvenile S. olivacea and S. paramamosain from the Mekong Delta are based on the architecture
of the frontal lobe spines and the number of dorso anterior propodal spines and ventral carpal spines. These are described, together with
features relating to their coloration and habitat preferences. The recognition of two named species of Scylla within the mangrove
populations of mud crab in the delta is important to fisheries management and aquacullure development, as both adult and juvenile mud
crabs are fished heavily for marketing and for stocking in aquaculture systems, respectively.

KEY WORDS: Scylla. mud crab, species identification, mangrove. Mekong Delta

INTRODUCTION

Mud crabs of the genus Scrlla are the only swimming crabs
(Family Portuiudae) that are found habitually in tropical and sub-
tropical mangrove forest ecosystems; their habitat extends to man-
grove estuaries, embayments and surrounding coastal waters
(Macnae 1968). As well as providing shelter from predation and
desiccation, especially during moulting, mangroves also provide
mud crabs with a plentiful supply of food in the form of other
crustaceans and molluscs (Arriola 1940; Hill 1979).

Mud crabs have significant economic importance to artisanal
fisheries throughout the Indo West Pacific region. They are also
contributing increasingly to aquaculture production in several
countries, most notably Vietnam and the Philippines (Johnston &
Keenan 1999; Fortes 1999). Their rapid growth and high market
value, coupled with easy post-harvest handling, make them an
attractive alternative to farming shrimp within coastal areas (Over-
ton & Macintosh 1997). The growing importance of mud crab
fanning is also supported by a rapidly expanding demand for crab
products, both regionally and internationally.

The mangrove forests of Vietnam originally covered an area of
up to 400.000 ha (Maraund 1943), of which about 250.000 ha
flourished in the Mekong Delta. The greatest concentration of
mangrove ( 150.000 ha) was in the Minh Hai Peninsula, which is
now divided into Ca Mau and Bac Lieu provinces (Fig. 1 ). Despite
heavy exploitation in recent decades, mangroves are still the dom-
inant habitat in the extensive saltwater-influenced regions of the
delta.

Within the coastal provinces of the Lower Mekong Delta
(LMD), rural communities are heavily dependent on mangrove-
based fisheries and aquaculture to support their livelihoods. These
include crab fishing and crab culture as a secondary activity to

^Corresponding author. D. J. Macintosh. Institute of Aquaculture. Univer-
sity of Sliding. Stirling FKI4 7EB. Scotland. United Kingdom. Phone:
+44-1786-467785; Fax: +44-1786-472133: E-mail: d.j.macintoshta
stir.ac.uk

shrimp farming within the mangrove forest ecosystem. Average
mud crab production in the LMD ranges from 504 to 839 kg/ha/y
(World Bank/Danida 2000). Nearly all the available brackishwater
areas of the delta have been converted to extensive aquaculture
ponds for shrimp and/or crab production integrated to varying
degrees with the mangrove forest (these fanning systems are de-
scribed by Binh et al. 1997; and Johnson et al. 1999). To date,
there are no commercial hatcheries in Vietnam to support mud
crab culture and, consequently, the natural recruitment of juvenile
mud crabs into the mangrove ecosystem provides the only source
of seed available to crab farmers.

A previous study using morphonietric analysis confirmed the
existence of two common moiphs of Scylla within the South China
Sea region (Overton et al. 1997; Overton 2000), while Keenan et
al. ( 1998) have proposed a total of four species of mud crab based
on genetic data from a wider geographical range. They also de-
scribe morphological characters to distinguish the four species.
Although Keenan et al. (1998) attribute tv\'o species to Vietnam,
only one species is mentioned from the Mekong Delta, called
Scylla paramamosain Estampador. Moreover, the study is based
entirely on large adult specimens (carapace width greater than 95
mm), because the distinguishing characteristics of each species are
more clearly defined in the adult stages. Until now, no species
identifications have been reported for the juvenile stages. The
people who catch or rear juvenile mud crabs in the Mekong Delta
recognize two morphs of Scylla on the basis of color, using the
local names "cua xanh" (green crab, due to its greenish carapace)
and "cua lua" (red crab, due to dark red coloration on the lower
chelae). These color differences are not distinct in the smaller
juveniles, however, especially after capture.

In the present study, adult and juvenile mud crabs were col-
lected from the Lower Mekong Delta. They were then examined
morphologically and analyzed using electrophoresis to determine
which species are recruiting into the mangrove crab fishery. The
main objective was to produce scientific descriptions of both ju-
veniles and adults of the species identified, which confirm Iheir

259

260

Macintosh et al.

10°N

g^N

60 Km

105 °E 106 °E 107 "E

Figure 1. The Mekong Delta, Vietnam sliovving the location of the
sampHng sites in Bac Lieu and Kien Giang provinces.

local identities based on color and behavior. This knowledge is
vital for mud-crab stock assessment and for the future develop-
ment of mud crab culture using selected, known species of Scylla.
Recruitment and growth studies on juvenile mud crabs are par-
ticularly needed in this regard.

MATERIALS AND METHODS

Crab Collection

Study sites were chosen within two of the most southerly
coastal provinces of the Mekong Delta; Bac Lieu (9°00'N:
105°14'E) and Kien Giang (9°22'N; 104°26'E) (see Fig. 1). The
crabs were collected from mud crab fishers or traders. They were
interviewed to confirm that the crabs obtained were all fished from
the selected locality.

In the fiist investigation. 2.^ adult crabs. 84 to 125 mm carapace
width (CW), including both common morphs of Scylla. were
sampled from the commercial mud crab fishery in Bac Lieu Prov-
ince. Their species identities were confirmed by comparing their
morphology and allozyme mobility with those published by
Keenan et al. (1998). In the second investigation, juvenile mud
crabs (CW 45-62 mm) from Bac Lieu and Kien Giang provinces
(62 and 58 individuals, respectively) were studied. The majority of
specimens from Kien Giang were the "red" morph, whereas all
those collected from Bac Lieu in the second sample were typical of
the "green" morph. In addition, a larger number of smaller juve-
niles (CW 10—4^5 mm) were available for examination from the
commercial crab seed fishery in Ca Mau.

Crab Measurement

Three physical measurements were made on each sampled mud
crab using digital calipers, namely Frontal Lobe Width (FLW).
Internal Carapace Width (ICW) and Frontal Median Spine Height
(FMSH) (Fig. 2). Measurements were recorded to the nearest 0.1
mm. From these measurements, two ratios described by Keenan et

ICW

Figure 2. Illustration of the three measurements forming the ratios
used to discriminate hetween the two Scylla species. FL\\ : Frontal
l.ohe Width: FMSH: Frontal Median Spine Height; ICW: Internal
Carapace Width.

al. (1998) as discriminating between the four known species of
Scxila were used to compare the red and green morphs of Scylla
from the LMD. These ratios were FLW/ICW and FMSH/FLW.

Allozyme Electrophoresis

Use of genetic markers, including allozyme electrophoresis, are
the most reliable in identifying Scylla species (Keenan et al. 1998;
Overton 2000). Other mud crab specimens representing the four
Scylla species proposed by Keenan et al. (1998). which had been
identified earlier (Overton 2000). were used as reference material
to help interpret the results from electrophoresis using the mud
crabs obtained from the Mekong Delta.

Muscle tissue was extracted from each crab specimen and
stored at -80°C. The allozymes extracted from the muscle tissue
were separated using starch gel electrophoresis. The electropho-
retic techniques used followed those described by Hanis and Hop-
kinson (1976). Shaklee and Keenan (1986) and Pasteur et al.
( 1988). Four allozymes. using two buffer systems that distinguish
the four species of Scylla identified by Keenan et al. ( 1998). were
used to identify the two common species collected from the LMD.
These enzymes were alanine aminotransferase (ALAT), argenine
kinase (ARGK). mannose phosphate isomerase (MPI) and phos-
phoglucomutase (PGM). They were visualized using standard
staining procedures (Shaw & Prasad 1970; Harris & Hopkinson
1976; Shaklee & Keenan 1986; Pasteur et al. 1988).

RESULTS

The results of the allozyme electrophoresis confirmed that the
two coinmon species o( Scylla in the Mekong Delta are S. olivacea
Herbst (red morph) and S. panuiumiosain Estampador (green

Mud Crab Species in the Mekong Delta

261

morph). as described by Keenan et al. (1998). Figure 3 and Ta-
ble 1 illustrates the fixed mobility observed between the LMD
specimens and the typed samples of the four species (also based on
Keenan et al.).

The frontal spine morphology represented by the ratios FLW/
ICW and FMSH/FLW did not prove to be conclusive in distin-
guishing the two species identified from the allozyme data. Both
adults and juveniles of the green morph iS. puramamosain) have
narrower frontal lobe widths (compared to their internal carapace
width) and taller frontal lobe spines, than crabs of the red morph
(5. olivacea). This conforms with the spinal architecture of sharp
frontal spines on the green morph and smooth, obtuse frontal lobe
spines on the red morph (Fig. 4). However, the ranges obtained for
each of the ratios show that there is a large overlap between the
two species and that it is not possible to identify an individual crab
solely from either of these ratios.

Although both juveniles and adults of the two mud crab species
from the Mekong Delta could be distinguished from each other
using the above ratios, it is less clear which species they represent
when they are compared to the equivalent ratios for the four ScylUi
species proposed by Keenan et al. ( 1998) (Table 2). Mean values
of FLW/ICW for the juvenile red and green morphs of Scylla lie
out with the ratios reported by these authors. The mean values for
the adult specimens from Bac Lieu are within the published ranges
for the four Scylla species recognized by Keenan et al. 1998. but
they lie between the values of 0.037/0.038 for S. serratalS. para-
mamosain and 0.412/0.415 for 5. tntnquebarica IS. olivacea
(Table 2).

Mean values for the ratio FMSH/FLW for adults and juveniles
of both Vietnamese morphs also fall within the ranges set by
Keenan et al. 1998 for the four Scylla species they recognize
(Table 2). FMSH/FLW ratios positively identified the adult green
morph from Bac Lieu as S. paramamosain and the juvenile red
morph as S. olivacea. but the other two sample groups cannot be
classified reliably on the basis of this ratio. This is not surprising
as there is also considerable overlap in the ranges of the FMSH/
FLW values for each species as reported by Keenan et al. 1 998.

DISCUSSION

The results from allozyme electrophoresis were conclusive in
identifying the two common species of Scylla in the coastal man-
grove ecosystem of the Mekong Delta. Based on the recent review
of the genus Scylla by Keenan et al. (1998). the morphs recognized
by fishers and crab farmers as "red crab" and "green crab" are S.
olivacea and 5. paramamosain. respectively. These confirmed
identifications help to overcome the long-standing confusion about
the identity of mud crab species when their fisheries biology, or
use in aquaculture is described. As noted by Fortes (1999). several
authors have assumed a single species. Scylla serrata. in aquacul-
ture. despite evidence that this mud crab is actually rare in the
Southeast Asian region compared to the other three species of
Scylla now recognized (Keenan et al. 1998; Overton 2000).

It is not surprising that the morphometric ratios based on cara-
pace measurements (FLW/ICW and FMSH/FLW) were inconclu-
sive for the identification of juvenile mud crabs, or that the values
obtained do not lie within the equivalent ranges for adult speci-
mens. Developmental changes in exoskeletal dimensions between

juvenile, sub-adult and adult crabs are well documented (Hartnoll
1982). Indeed the relative allometic changes in the dimensions of
the body are often used to define the molt of maturity in crustacean
species (e.g.. Somerton 1980; Paul & Paul 1995). However, the
same ratios obtained from the adult mud crabs sampled also did
not conform to the equivalent values reported by Keenan et al.
( 1998). This apparent discrepancy may be explained by (a) opera-
tor differences when taking carapace measurements with calipers;
or (b) natural variation in the morphology of these closely related
species. It is notable that there is a high degree of overlap between
the ratios recorded by Keenan et al. (1998). and clearly morpho-
metries alone cannot be used to identify individual mud crabs
reliably.

The morphological characters used to identify the species in
adult samples can also be extended to juvenile crabs down to a size
of 1 .5 cm carapace w idth. the modal size of new recruits into the
Mekong Delta. In particular, the architecture of the frontal lobe
spines and the number of spines on the carpus are reliable char-
acters which can be used to distinguish juveniles of S. olivacea and
S. paramamosain, even in the field.

The presence of two species in the Mekong Delta mud crab
fisheries is important from a fisheries management and aquacul-
ture development perspective, as natural recruitment of juvenile
mud crabs into the mangrove ecosystem is the only source of seed
available to crab farmers in the delta. It will now be possible to
investigate and compare ecological differences between these two
species, especially their potential in aquaculture.

The yield of mud crab in Ca Mau Province was 5000 tonnes in
1999, of which 1000 tonnes came from aquaculture production
(Xuan 2001). In the ten-year fisheries development plan for Ca
Mau Province (2000 to 2010), the production of cultured mud crab
is expected to increase to 3,500 tonnes per annum. This increased
emphasis on aquaculture has important implications regarding the
sustainable level of exploitation of the mud crab seed recruiting
into the coastal mangroves. From observations of the crab seed
fishery in Ca Mau, it is clear that there are significant seasonal
fluctuations in the supply and species composition of juvenile mud
crabs caught by local fishers. It also appears that the recruitment
dynamics of S. paramamosain and S. olivacea vary from province
to province in the delta (Macintosh: personal observation). By
being able to identify these two common species of Scylla down to
the early juvenile stages entering the mangroves, a more accurate
assessment of this important fisheries resource can now be at-
tempted.

Scylla paramamosain and S. olivacea are farmed throughout
the brackish water regions of the Mekong Delta, usually in earthen
ponds constructed within the mangrove forests. The farmers gen-
erally stock juveniles of both species purchased from fishermen or
crab dealers. Their general opinion is that S. olivacea is the more
aggressive species. It also likes to burrow, which weakens the
pond structure and making harvesting very difficult. In contrast,
Scxlla paramamosain is regarded as more suited to pond culture as
it seldom burrows. However, female S. olivacea are very popular
as "egg crab" (crabs with mature ovaries) because they develop
large ovaries at a comparatively small body size. Differences in
size at maturity and gonosomatic index have also been recorded in
sympatric populations of S. olivacea and S. paramamosain in tlie
Gulf of Thailand (Overton 2000). By confirming their species
identity at the juvenile stage, it will now be possible to research the

262

Macintosh et al.

107
100

Origin

Genotype

100
95

Ongin

100/100
SP./S-S./S.T.

Genotype

Genotype

Figure 3. Illustration of allozyme mobilites for Scylla species using a) mannose-6-phospiiate isomerast (MPI): h) pliosplioglucomutase (PGM);
c) argenine kinase (ARGK); and d) alanine aminotransferase (ALAT, using ultra violet stain); from Overton tunpublished).
Note: S.O. = Scylla olivacea; S.S = S. serrata; S.T. = S. Iranquebarica and S.P. = S. paramamosain.

Mud Crab Species in the Mekong Delta

263

TABLE 1.

Results of mobility for four allozymes used to compare two Scylla morplis from the Lower Mekong Delta with typed specimens of the four
species of Scylla recognized by Keenan et al. ( 19981 used as markers (from Overton, 20(10).

Adults

Juveniles

Specimens from Overton (2000)

Bac Lieu

Bac Lieu

Bac Lieu

Kien Giang

Scvlla

Scylla

Sc\lla

,Sfv//fl

Locus

Green

Red

Green

Red

serrala

traiiquebahca

paramamosain

olivacea

Numbers of indi\iduals

11

i:

s

S

6

5

5

b

ALAT

100

95

nd

nd

100

100

100

95

ARGK

75

75

nd

nd

100

75

75

75

MPI

100

95

100

95

100

100

KJO

95

PGM

100

85

100

85

100

107

100

85

biological basis for the farmers' observations about red crab (5.
olivacea) and green crab (5. paramamosain) in aquaculture. Stud-
ies of their comparative tolerance to different combinations of
salinity and temperature, their comparative growth rates and matu-
ration under different pond conditions, and the possible affect of
interspecific interaction between S. olivacea and S. paramamosain
in mixed culture, could yield valuable information to improve mud
crab farming in the Mekong Delta.

Diagnoses of the Two Common Species of Scylla in the Mekong Delta
Scylla olivacea Herbst

Dorsal carapace brown to dark green. Dorsal chelae and pereio-
pods range from dark brown to green, no reticulation present.
Ventral propodus orange/red. Carapace rounded with blunt, obtuse
frontal lobe teeth all equal in size, with shallow interspaces. Short,

Ictn

1cm

Figure 4. Drawings of the frontal carapace spination and left cheliped of adult Scylla paramamosain (a and b) and Scylla olivacea (c and d) based
on specimens collected in the Mekong Delta, Vietnam.

264

Macintosh et al.

TABLE 2.

Means, standard deviations (S.D.) and ranges for three morphonu-tric characters (measured in mm I and two rati.ms used to discriminate

between four species of mud crab, Scjila described by keenan et al. ( 1998).

Adults

Juveniles

Keenan et al. (1998)

Bac Lieu

Bac Lieu

Bac Lieu

Kien Giang

Scylla

Scylla

Scylla

Scylla

Character

Green

Red

(Jreen

Red

serrata

puramamosmii

trunquehurica

olivacea

Numbers of
individuals

II

12

62

58

68

9

25

66

ICW (mm)
Mean
S.D.
Rantie

104.84

10.24

84.39-34.24

107.64

13.67

85.51-1333.54

51.07

3.81

45.00-61.30

55.71

3.87

47.11-61.65

138.40

23.20

95.50-191,70

114.70
9.00

104.80-134.10

113.70
11.40

97.10-137.80

107.50

10.10

95.00-133.90

FLW (mm)
Mean

4(1.S8

42.82

22.62

25.30

nd

nd

nd

nd

S.D.

3.57

3.99

1.94

1 .65

Range

34.24-15.68

38.84-48.41

I9.8(J-3I.70

21,70-27.85

FMSH (mm)
Mean

2.37

1.75

0.90

0.58

nd

nd

nd

nd

S.D.

0.44

0.2S

0.24

0,17

Range

1.72-2.92

1.16-2.05

0.40-1.40

0.40-1.20

FLW/ICW
Mean
S.D.
Range

0,39
0.01
0.38-0.41

0.40
0.02
0.36-0.42

0.44
0.03

0,41-0.63

0.45

0.01

0.42-0.48

0,37
0.02
0.34-0.41

0.38
0.01
0.36-0.39

0.41
0.02
0.38-0.44

0.42
0.02
0.37-0.45

FMSH/FLW
Mean
S.D.
Range

0.06
0.01
().()4-0.()7

0.04
0.01

0.03-0.05

0.04
0.01

0.(11-0.05

0.02

0.01

0,02-0,05

0.06
0.01
0.04-0.10

0.06
0.01
0,04-0,08

0.04
0.01
0.03-0.05

0.03
0.01
0.02-0.04

obtuse anterolateral spines uniform in size and shape. None to two
vestigial spines present on dorsal propodus of the chelae. Pro-
nounced nodule present on inner lateral propodus. Spination on
ventral carpus of chelae ranges from two vestigial, nodule-like
spines to a total absence of spines. Abdominal flap of mature
females dark brown to purple with a pale lateral stripe on each
abdominal segment.

Coloration of juveniles similar to that of the adults. Chelae are
elongate, with a distinctive dark red flash on the outer propodus
and fixed dactyl. Chelae bear blunt, obtuse frontal lobe teeth that
are equal in size, with shallow interspaces. Two sharp spines on
dorsal propodus. and one pronounced nodule on inner lateral pro-
podus. Single spine present on ventral carpus.

Scylla paramamosain Estampador

Adult carapace pale olive green. Dorsal chelae and pereiopods
pale green with prominent dark green/brown reticulation. Reticu-
lation breaks up and forms brown dots on outer upper half of
propodus. Lower half of outer lateral propodus pale yellow to
cream with orange to red flashes on tips of propodus and dactylus.
A black patch is present on ventral surface of caipus.

Carapace flattened dorso-ventrally, with short, triangular fron-
tal lobal teeth with angular interspaces. Central pair of frontal lobe
teeth slightly protruded in some individuals. All anterolateral
spines uniform in morphology, and compressed with small spaces
between spines. Two sharp spines on dorsal propodus with ridges
following posteriorly behind spines. One small spine present on
inner lateral propodus. Two sharp spines on ventral carpus, on
some individuals the anterior spine is vestigial, or missing.

Juveniles have similar body coloration to adult form. Carapace
bears short frontal lobal teeth, sharp and triangular with angular

interspaces. Two pronounced spines on the dorsal propodus. No
nodule on inner lateral propodus. Ventral carpus usually bears two
spines, but 6% of crabs examined have only one spine.

Hahilat

Scvlla olivacea is associated with mangrove forests, in particu-
lar estuarine and lower salinity areas associated with the river
systems well within the mangrove zone or deltas where there is a
high amount of freshwater runoff. S. olivacea prefers to burrow in
the soft embankments during low tide. S. olivacea has an extensive
distribution, populations of this species having been identified
from the coastlines fringing the western Indian Ocean to northern
and western Australia and islands of the Pacific Ocean (Keenan et
al. 1998). Another species. Scylla inmqnelnirica. is often associ-
ated with S. olivacea. These two mud crabs occupy similar niches
in the mangroves of East Malaysia and the Philippines (Macintosh,
personal observation).

Scvlla paramamosain inhabits a more subtidal environment
than Scxila olivacea. This species is found in the mouth of estua-
rine areas and shallow subtidal mudflats and along the coastal
fringes of mangrove areas. Keenan et al. 1998 also remarks that the
habitat of this species extends to coral reefs areas, as in Singapore.
Scvlla paramamosain is widely distributed along the coastlines of
the South China Sea particularly along the coast of Vietnam and
the eastern seaboard of the Thai-Malay peninsula (Overton 2000).

ACKNOWLEDGMENTS

The authors thank the staff of the Aquaculture and Fisheries
Sciences Institute of Can Tho University. Vietnam for providing
facilities and manpower support for this study. Financial assistance

Mud Crab Species in the Mekong Delta

265

was provided by Daiiida through the Can Tho University - Aarhus
University Linl< in Environmental Sciences (CAULES) project.

and ill the form of a Danida scholarship awarded to Mr H.V.T.
Thu.

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Paul. A. J. & J. M. Paul. 1995. Changes in chela heights and carapace
lengths in mature male red king crabs Paraliihoides camthschaiicus
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versity. 28 pp.

Joiinuil ,if Shellfish Research, Vol. 21. No. 1. 267-272. 2002.

PHYSICO-CHEMICAL CHANGES IN ACID SULFATE SOIL DURING SEMI INTENSIVE
CULTURE OF PENAEUS MONODON FABRICIUS, IN CLEARED MANGROVE AREAS OF THE

CHAKARIA SUNDARBANS, BANGLADESH

BIPLOB DAS,' YUSUF SHARIF AHMED KHAN,' KHAN TOWHID OSMAN," PRANAB DAS,'
AND MD. NURUL AMIN'

Institute of Marine Sciences, University of Cbittagong, Chittagong-4331 . Bangladesh: 'Department of
Botany, University of Chittagong, Chittagong-4331, Bangladesli

ABSTRACT The present study analyzed physico-chemical properties of pond soil during Penaeus monodon culture in a semi-
intensive shrimp farm in Cox's Bazar district. Bangladesh during winter 1996 to 1997. Soils were acidic with high concentrations of
SOj-S. extractahle Fe and AI. Soluble salt concentration were in order Na>Mg>Ca>K. The physico-chemical properties changed with
flooding both under simple inundation and inundation with shrimp culture. The most noticeable change was soil P". SOj-S and
extractable Al. Extractable A! decreased a minimum value at 145 days after preparation of the culture ponds. Extractable Na and K
were increased gradually while, Ca and Mg remained fairly constant throughout the entire period. The extent of change was different
for the culture and the inundated ponds. Results of the present study indicated a positive effect on the improvement of acid sulphate
soil in the cleared mangrove forest area because of shrimp culture.

KEY WORDS: Peiuieti.s moiuiJon. shrimp pond, culture
INTRODUCTION

Pond soil plays an important role in the balance of an aquacul-
tural system and consequently on the growth and survival of
aquatic organisms. Sediment/soil can function as a buffer. It pro-
vides water nutrients and serves as a biological filter through the
adsoiption of organic residues (Ray & Chien 1992). Soil thus
playing a vital role in aquaculture practices is considered the
"Chemical Laboratory'" of the pond (Felix 1988). In ponds there is
an intense interchange of organic and mineral compounds between
the soil and the water (Wrobel 1983). Accumulation and decom-
position of organic matter take place on the pond floor. As a result
macro elements are eliminated from tlie pond water while the
decomposition of organic matter releases inorganic substances and
acids, and dissolutes minerals. These go into solution with water
and influence aquatic biota.

A number of conditions are conducive to the formation of acid
sulfate soils (Pons & Van Breemen 1982). Vast tracts of acid
sulfate soils abound in Asia and Africa (Singh 1980; Brinkman &
Singh 1982). In Bangladesh about 0.23 M ha of land constitute
acid sulfate soils (FAO 1988). However, these are considered
problem soils everywhere resulting in severe limitations for agri-
cultural use (Nhung & Ponnamperuma 1982; Van Breeinen &
Pons 1978; Ponnamperuma & Solivas 1982; Hechanova 1983;
Simpson et al. 1983).

Severe acidification of pond water in acid sulfate areas has
been reported by many workers (Webber & Webber 1978; Lin
1986; Boyd 1989). The low P" exerts its effects through the in-
fluence on most aspects of pond water chemistry. The main prob-
lems arising after construction or deeper excavation of fish ponds
in acid sulfate soils comprise insufficient growth of algae, poor
condition and consequent slow giowth of phosphate fertilizers
(Camacho 1977; Bay Ion 1981; Brinkman & Singh 1982; Poer-
nomo & Singh 1982). Low P" of the acid sulfate soils causes
dainage to gill tissues of fish (Ferguson 1988) and influences the
impact of toxins (Alabaster & Lloyd 1980; Colt & Armstrong
1981) and heavy metals (Boyd 1989). In acid waters crustaceans
and fish may experience impaired ionic regulation. P" <4.8 is

Corresponding author. Yusuf Sharif Ahmed Khan, Institute of Marine
Sciences, University of Chittagong, Chittagong-4331, Bangladesh

lethal for the Penaeids (Tsai 1990). Additionally, a high organic
matter content in the bottoin soil depletes O, of water (Wrobel
1983).

Characteristics of acid sulfate soils in some areas of Cox's
Bazar have been reported by Rahman (1990), Rahinan et al.
(1993), and Mahmood and Saikat (1995), But no study has .so far
been done on the changes of pond bottom acid sulfate soil that take
place by the amendments done for shrimp culture. Keeping these
views in mind, this work was undertaken in a semi-intensive
shrimp farm at Cox's Bazar. Ponds constructed on acid sulfate
soils revealed physico-chemical changes in soils of shrimp {Pe-
naeus monodon) culture ponds as well as inundated ponds.

MATERIALS AND METHODS

Location of the Farm

The seini-intensive shrimp farm namely "Chakaria Chingri
Khamar" is situated on the western bank of the Matamuhuri River,
about 5 km upstream from the estuary and about 65 km south of
the Chittagong City.

Sampling

P. monodon was cultured in 14 ponds during winter of 1996 to
1997. Among those, three ponds (P-1, P-2 and P-3) were selected
randomly on the basis of the previous years production data and
soil P" values to accommodate the maximum variability for the
present study. From the rest of the fallow ponds (temied as inun-
dated ponds), three others (P-4. P-5 and P-6) were selected where
pond preparation was done similarly to the culture ponds but
stocking was not.

Soil samples from 0-15 cm and 15-30 cin depths of pond
bottom were taken from three spots diagonally of each pond (cul-
tured and inundated), so that one spot was in the center and two
were in the corners. Samples were drawn underwater with a spe-
cially designed sampler. Soil sampling was done 30 days before
(DBP) and 7, 50. 95 days after completion of pond preparation
(DAP). Soil samples were also taken at 145 DAP from the culture
ponds.

Analysis of Soil Samples

Soil texture was determined according to Bouyouco's Hydrom-
eter method (Piper 1950), P" from 1:2 soil water suspensions and

267

268

Das et al.

TABLE 1.
Textural classes of different soil samples collected from culture ponds and inundated ponds.

Pond Type

Pond No.

Depth (cm)

Sand ( f/r I

Silt ( % 1

Clay ( % )

Textural Class

Culture Ponds

Inundated Ponds

p = 1

00- 1 3

L'^-30

P = 2

00-15

15-30

P = 3

00-15

15-30

P = 4

00-15

15-30

P = 5

00-15

15-30

P = 6

00-15

15-30

65
49
67
52
48
66
70
45
65
55
69

14
13
25
12
23
24
13
14
28
13
19
12

34

Sandy Clay

22

Sandy Clay Loam

26

Clay Loam

21

Sandy Loam

25

Sandy Clay Loam

28

Clav Loam

21

Sandy Clay Loam

16

Sandy Loam

27

Clav Loam

22

Sandy Clay Loam

26

Sandv Clav Loam

19

Sandv Loam

organiL- matter I'roiii loss ol ignition. Total nitrogen was deter-
mined by micro-Kjeldhal digestion and distillation procedures.
Available phosphorus was extracted with Bray and Kurtz No. 2
extractant (0.03 N NHjF in 0.10 N HCl) followed by Spectropho-
tomelric determination according to SnCl, reduced molybdophos-
phoric blue color method (Jacl\son 195SI. Extraclable Ca. Mg. Na.
K and Mn were determined by 1 N NH^OAC. P" 7.0 ± 0.1 satu-
ration and FE by 1 N NHjOAC. P" 4.6 saturation followed by
Atomic Absorption Spectrophotometry (Allen et al. I9S6).

RESULTS AND DISCUSSION

Physico-chemical parameters of the culture ponds and inun-
dated ponds are tabulated in Table 2 and Table 3 respectively. The
textural classes of the soil samples are shown in the Table 1 .

Soil samples of the present study were sandy clay to clay loam
in texture. The texture has been inherited from the deposition of
the sediments. Such compositions of acid sulfate soils are also met
with the saline soils of Chakaria Sundarbans (Rahman et al. 1990).
The textural variation could have been created by the sedimenta-
tion processes during past mangrove swamp systems.

The soils were found rather poor in total nitrogen in compari-

son to the organic matter content resulting in high C/N ratio, that
may inhibit the decomposition of organic matter. This feature
shows the characteristics of peat layers. A high organic matter poor
in nitrogen is also conducive to reduction processes. Similar total
nitrogen values were reported earlier from other acid sulfate soils
of the area in association with low organic matter (Rahman et al.
1993).

Values of soil P" remained neutral. After submergence, the P"
of acid sulfate soils gradually increases due to reduction and in-
activation of SOj-S through microbial activities (Singh 1982). This
reduction rate depends on the presence of sulfur reducing bacteria,
P" of the system, energy source (organic matter), etc. (Van Bree-
men 1976; Pons et al. 1982). In the present investigation, the P" of
soils was found to be reduced by aii-drying with the average
values ranging from 3.68 to 6.2. Further reduction could have been
achieved by slow oxidation with prolonged drying. Additionally,
there were significant differences in P" values of the soils of the
same pond at different spots. It might be due to the fact that the
sulfidic horizon had not been uniformly parallel to the land surface
and during excavation residual sulfidic materials were left. Evi-
dence indicates that the pond soils had ucid sulfate characters. In

TABLE 2.
Phsico-chemical parameters of different soil samples collected from culture ponds.

Pond

Deptll

pH

I

EC

Salinit>

OM

N,

P.,

S,

s„

Na.,

K

Ca„

Mg.

Fe,

Mn,

\l

No.

Parameters

(cml

.Mi>ts(

Dry

(ds/m)

(ppll

1 percent)

(percent)

(ppml

ippti

ippt)

(ppl)

(ppt)

(ppt)

(ppti

(ppni)

(ppnil

(ppni)

P-1

BP

n-l.s

5.72

5.16

8,92

11 42

7 90

0.16

62.98

I 40

1 ()4

7.78

1 (6

0.92

(57

88.20

58.87

66.30

I.S-30

6.64

5.08

16.93

21.67

10.42

0.13

5.^.50

1,26

3.80

8.30

0.8(

0.84

2,()(

115.87

45.00

128.(3

AP

0-15

6.06

5.59

1 1 34

14..57

9.70

0.16

63,20

0,57

5.23

6.88

1.05

0.89

(..50

128.44

48.70

43.37

l-^i-.TO

6.43

5.39

16.55

21.17

12.68

0.17

58,73

0.79

12.29

8.97

(.23

1.06

2.(2

176,15

68.72

83.59

P-2

BP

tn.';

ft.7S

4.79

12.88

16.48

10.51

0 19

69.30

0,92

3.97

6.33

0.48

0.89

(.94

124,33

73.33

99.40

l-'i-.IO

6.13

4.36

20.92

26.69

12.73

0.17

53.(5

2,07

S..39

8.7S

0.76

OSS

2.24

169,4(1

73.33

2(5,43

AP

0-15

6.65

5.72

14.59

18.69

10.44

0 18

60,93

0.73

5.98

8.24

(.16

1.02

1.58

133,75

50,83

3 (.04

15-30

6.38

5.14

18.65

23.86

12.26

0.15

55.04

0,89

1141

9.77

1.04

0.94

1.99

172.13

73.08

58,47

P-3

BP

0-15

6.87

5.28

9.64

12.33

8.87

0.18

58.77

0,68

3.51

5.82

1.18

(..^6

2.05

105,33

93.80

65.46

L5-30

6.54

4.57

13.69

17.52

10.84

0.17

48.95

0.58

6.58

6.95

0,79

0.70

2.04

122.46

52.00

(.W.63

AP

0-15

6.50

5.53

14.82

18.98

10.53

0.17

62.3 (

0.78

6.42

8.27

1,37

0,97

1.78

1.54.42

.50.48

30.33

1.5-30

6.41

5.19

19,96

25.54

13.35

0.16

57.(1

1.07

12.54

10.67

1,31

0,96

2.21

192.24

71.88

69.87

EC = E(eclrica( Cdnduciivity: OM = Organic Matter; N, = Tota( Nilrogcn; P^ = Avai(ab(e Phosptioms; S^ = Su(phate Su(fur; S„ = Oxidizab(e Su(fur: Na„ = Extraclab(e
Sodium; K,. = Extractab(e Poias.sium; Ca.. = Extractable Calcium; Mg.. = Exlractable Magnesium; Fe.. = Extraciable Iron; Mn.. = Extraclable Manganese; A!., =
Extractable Aluminum; BP = 30 days Before Production & AP = Mean value of 5 days, 50 days. 95 and 145 days Adcr production.

Physico-Chemical Changes in Acid Sulfate Soil

269

TABLE 3.
Phsico-chemical parameters of different soil samples collected from inundated ponds.

Pond

No.

Parameters

Depth

(cm)

PH

Moist Dry

FX

(ds/ml

.Salinit>
ippti

OM

(percent)

N,
(percent)

P.,

ippm)

(ppt)

(ppt)

Na,
(ppt)

(ppt)

Ca,
(ppt)

Ms.,
(ppt)

Fe,
(ppm)

Mn„
(ppm)

Ale
(ppm)

P-4 BP 0-15 5.6<) 4..13 19.50

15--TO 4.79 i.bH 31.28

AP 0-15 5.82 5.29 19.73

15-30 5.67 4.86 29.30

P-5 BP 0-15 5.93 4.77 14.77

15-30 6.75 4.27 37.51

AP 0-15 5.78 5.77 13.56

15-30 6.12 5.U 20.22

P-6 BP 0-15 5,81 4,67 12,05

LS-.W 6,44 4 48 20,42

AP 0-15 5,92 5,51 14.07

15-30 5.98 4.87 18.76

24.96

15.75

0,22

36.66

2,50

5 52

1 1 10

0.52

0 66 2

:s

4U.04

19.56

0.09

32.28

2,93

12.42

14.06

1.76

0.79 2

25

25.25

16.08

0.13

29.77

1.78

7.64

9,29

0.78

1,28 1

70

37.22

16.62

0.12

26.31

1.83

13.87

12,71

1.14

1.61 2

56

18.90

15.60

0.19

40.88

1.27

5.54

7,26

0.43

0.73 1

96

48.00

12.99

0.15

41.05

1.53

11.57

7,778

0.57

0.62 2

23

14,59

10.02

0.17

57.48

1.07

3.75

7.88

1.15

1.17 1

68

25.89

14.99

O.IS

50.23

1 27

13.67

9.75

0.92

1.11 2

20

15.43

11.00

0.16

39.30

1 .07

4,51

6.09

0,54

060 1

66

26.14

12.87

0 19

41.23

1.55

6,65

6.81

0.55

0.56 1

94

18.01

11.38

0 17

49.37

0,96

549

7.31

1.11

1.07 :

56

24.00

13.19

0.16

49,41

1.43

11.59

8.27

0.66

0.87 1

9S

177.53 71.13 219.50
326.00 92.60 602,03
143,29 49.19 101.29
212.58 87.39 216.69

185.00 127.93 148.87

178.54 107.33 262.33
110.33 68.33 37.25
225,18 87,04 92,00

190,73 71.40 141.67

252.46 59.73 216.37

126 57 63.27 47.96

21004 93.18 126.48

EC = Electncal Conductivity; OM = Organic Matter; N, = Total Nitrogen; P^ = Available Phosphorus; S, = Sulphate Sulfur; S„ = Oxidizable Sulfur; Na^. = Extractable
Sodium; K,. = Extractable Potassium; Ca, = Extractable Calcium; Mg^ = Extractable Magnesium; Fe^ = Extractable Iron; Mn^ = Extractable Manganese; Al^ =
Extractahle Aluminum; BP = 30 days Before Production & AP = Mean value of 5 days, 50 days, and 95 days After production.

TABLE 4.
Correlation and regression co-efficient between chemical parameters of soil In culture ponds.

Independent

Dependent

St.

Variahle

Variable

No.

>

^

1

P"(airdiy)

EC

2

pH

Salinity

3

pH

Organic Matter

4

pH

Total-N

5

pH

Availahle-P

6

pH

Sulfate-S

7

pH

Oxidizable-S

8

pH

Extractable Na

9

pH

Extractable K

10

pH

Extractable Ca

11

pH

Extractable Mg

12

pH

Extractable Fe

13

pH

Extractable Mn

14

pH

Extractable Al

15

Organic

Matter

Total-N

16

Organic

Matter

Available-P

17

Organic

Matter

Sulfate-S

18

Organic

Matter

Oxidizable-S

19

Organic

Matter

Extractable Ca

20

Organic

Matter

Extractable Mg

21

Organic

Matter

Extractable Fe

22

Organic

Matter

Extractable Al

23

Organic

Matter

Extractable Mg

24

Extractable Ca

Extractable K

25

Extractable Ca

Extractable Fe

26

Extractable Ca

Extractable Fe

27

Extractable Ca

Extractable Mn

28

Extractable Ca

Extractable Al

29

Extractable Ca

Total-N

Correlation
Co-efficient

Regression

Co-efficient

a

b

33.07123

3.34.353

44.82607

-4.68567

12.35626

-0.14819

0.17374

-0.00101

29.36178

5.57392

253.2928

-38.70149

10.10835

-0.37784

12.-36769

-0.73409

2.02837

-0.16984

0.66337

0.05491

3.41039

0.28362

162.7490

-2.09753

119.7637

-10.91958

389.0405

-60.73047

0.1595

0.(.)0I415

66.77517

-0.62038

295.15233

-21.40631

-4.2054

1 .06366

-3.3018

1.061 10

0.94343

0.00102

1.24631

0.05671

50.22998

8.76288

42.89656

2.00984

1.67808

0.23581

0.87904

0.06789

132.4095

20.08978

23.6488

39.83474

131.01246

-67.90904

0.62448

1 .96549

-0.34109"''^

0.38810*

-0.02294'^^

-0.02367"^^

0.35322"^=^

-0.06742'"'''

-0.04 140^^

-0.19885"^^

-0.11505"^^

0.16318'^=

0.48109**

-0.02689^^

-0.38776'"^

-0.55449**

0.21277"^^

-0.25392"^^

-0.24085'^^

0.75288***

0.76297***

0,01974^^

0.62131**

0.71889**

0.11847'^'*

0.13485'"'^

0.29724^^

0.08586"^^

0.41462*

-0.20863^^

0.25 100^'^

NS: Not Significant

* Significant at 57c leyel

** Significant at \'i level

*** Highly Significant at 5'* level

270

Das et al.

TABLE 5.
Correlation and regression co-efficient Ijelwten chemical parameters ol soil in inundated ponds.

Independent

Dependent

Correlation

Regression

Co-efficient

St.

\ ariable

\ ariable

Co-efficient

No.

X

^

r

a

b

1

P" (air dry)

EC

-0.51448*

50.91311

-6.11398

2

P"

Salinity

-0.57717**

71.19709

-9.09594

3

pH

Organic Matter

-0.45927*

25.03221

-2.19786

4

pH

Total-N

0.23651^^

0.08123

0.01552

5

pH

Available-P

0.47452*

-1.68086

8.69177

6

pH

Sullale-S

-0.76008**

5.12290

-0.72512

7

pH

Oxidizable-S

-0.273 le'^'*

18.66437

-1.92903

8

pH

Extractable Na

-0.25399^^^

14.18430

-1.0025

9

pH

Extractable K

-0.19285^^

0.17974

0.14421

10

pH

Extractable Ca

0.23543'^^

0.13667

0.18206

11

pH

Extractable Mg

0.44329*

3.26257

0.24920

12

pH

Extractable Fe

-0.55978**

473.3843

-57.52583

13

pH

Extractable Mn

-0.37716''^

143.671

-12.96930

14

pH

Extractable Al

0.81063***

1040.365

-177.6317

15

Organic Matter

Total-N

-0.39531^^

0.23594

-0.00544

16

Organic Matter

Avaikible-P

-0.71448**

80.31563

-2.7357

17

Organic Matter

Sulfate-S

0.58190**

-0.15350

0.12600

18

Organic Matter

Oxidizable-S

0.64261**

-4.29046

0.94829

19

Organic Matter

Extractable Ca

0.70217**

-4.57551

1.07604

20

Organic Matter

Extractable Mg

0.18387''^

0.64117

0.02791

21

Organic Matter

Extractable Fe

0.78295**

0.72273

0.09197

22

Organic Matter

Extractable Al

0.60919**

0.71976

13.08166

23

Organic Matter

Extractable Mg

0.59527**

-236.0092

27.2572

24

Extractable Ca

Extractable K

0.29912^^^

1.82904

0.16691

25

Extractable Ca

Extractable Fe

0.41430*

0.66661

0.42843

26

Extractable Ca

Extractable Fe

-0.29640"^^

224.666

-39.3892

27

Extractable Ca

Extractable Mn

-0.19698"^^

87.4361

-8.7412

28

Extractable Ca

Extractable Al

-0.26591^^

223.517

-75.3507

29

Extractable Ca

Total-N

-0.34532'"^

1.70459

-4.t)679

NS:

Not Significant

* Significant at 5% level

** Significant at

1% level

***

Highly Signi

ificant at 5'7r level

the Chakana Sundarbaiis zone, dry P" of surface soil was found as
low as 3.15, which increased gradually with depth (Mahmood &
Saikat 1995). The wet soil P" in the surface soil of the present
study was less than the sub-soil. Excavating a depth greater than
1 m for pond construction might have exposed the less acid sub-
soil. Aeration of water might have caused some oxidation. The
higher reduction in P" of the sub-soil due to drying might, on the
other hand, be responsible for downward leaching of sulfate. Air
dry soil P" was found to range from 2.6 to 4.3 in some acid sulfate
soils of Cox's Bazar and Chakaria (Rahman et al. 1993). Liming
during the past cropping might have also caused an increase in P"
value. Electrical Conductivity (EC) values of the presently studied
pond soils are quite high. ECe values greater than 80 ds/m were
reported from Senegambia (Vieillefon 1977). Some of the present
values (ECe = EC 1:2 x 4 approximately) appear to be still higher
and soil salinity may be responsible for the salinity of inundated
water because of the similar trend of variation with ponds.

In the present findings, available P concentration was found
frequently above 24 ppm. In acid sulfate soils, low available P is
a result of solubility in acid reaction, insolubiliz.ation of fixation by
Fe. Al and Mn. and low release from organic matter (Banerjea &
Ghosh 1970; Andriesse et al. 1973; Subosa & Bautista 1991).

Addition of phosphate fertilizer to acid sulfate soil is ineffective
because of fixation of added P in the form of irreversible iron and
aluminum phosphates. In alkaline conditions, colloidal materials in
mud and organic matter may be inactive P (Tisdale & Nelson
1975; Singh 1982; Poernomo & Singh 1982). Available P in pond
soil might have increased due to application of 25 kg TSP/ha.

The problems of extreme acidity of soils in agriculture and
aquaculture arise from toxicities of H,S, SO4, Fe. Al, and Mn (IFP
1974: Singh 1982). In the present study SO4-S values ranged from
0.26 to 2.93 ppt. oxidizable S from 1.04 to 15.41 ppt. extractable
Fe from 88.2 to 326.0 ppm. extractable Al from 0 to 602.03 ppm
and extractable Mn from 37.2 to 127.93 ppm. Extractable Ca was
found to be low and the concentration followed the sequence
Na>Mg>Ca>K. Another study of Rahman (1990) on acid sulfate
soils of Cox's Bazar and Chakaria, the ranges of soluble Fe. Al and
Mn were found to be fiom 52 to 75 ppm. from 3 to 260 ppm and
from 2 to 22 ppm respectively (Rahman 1990). There was. how-
ever, a basic difference between the nature of the two studies and
soil type. Concentrations of iron and aluminum in the present study
were found lesser than the acid sulfate soils of many other coun-
tries (Baylon 1981; Hechanova 1983; Andriesse et al. 1973). The
extractable Mn values in the present study were found correspond-

Physico-Chemical Changes in Acid Sulfate Soil

271

ing to the active Mn concentrations reported from the Chakaria
Sundarbans (Rahman 1990). In acid sulfate soils of Thailand, Viet-
nam, and Philippines active Mn ranges from 5 to 400 ppm (Pon-
namperuma 1972), Actual acid sulfate soils have smaller amounts
of active Mn (Tanaka & Yoshida 1970; Van Breemen 1976). Con-
centration of Mn in the present study were found higher than the
flooded acid sulfate soil (Rahman 1990; Attanandana 1971 ).

From the present study it was observed that during shrimp
culture or keeping the ponds inundated without stocking, soil P"
increased gradually while SO4-S and extractabie Al decreased rap-
idly. Such phenomena are characteristic of acid sulfate soils under
flooding and may be exploited in their reclamation and utilization.
Extractabie Al was found negatively correlated (Table 4 and .'i)
with the soil P", which was also observed by some other workers
(Van Breemen 1973. 1976; Baylon 1981; Rahman 1990), On the
other hand, oxidizable S and extractabie Fe were positively cor-
related with the organic matter. It appears that transformation of
aluminum was P" dependent and transformation of .S and Fe in
submerged systems were organic matter dependent. Both S and Fe
transformations are microbial processes, the agents of which may
utilize organic matter as energy source. So. submergence of many
acid sulfate soils may eliminate two most important problems, low

P" and high Al. but the removal of S seems to be inadequate if the
soil/sediment contains high organic matter. Therefore, flooding
these soils is a temporary relief. Together with liming, this might
keep the soils more tolerable. To remove pyrite. drying of pond
bottom with subsequent repeated flushing has been advised (Felix
1988) but prolonged drying, or intense pyrite oxidation may render
the soils more acidic,

CONCLUSION

The extents of increase in P" and decrease in extractabie Al and
SO4-S were higher in the culture ponds than those under simple
inundation. This suggests that shrimp culture had a positive effect
on the improvement of acid sulfate soils under submergence. This
could be due to fish fed. mechanical aeration of water, frequent
water exchange, shrimp excretion, mixing of surface soil by bur-
rowing, leaving Ca rich in shells during molting, etc. In the light
of the above discussion, it seems that clearance of mangrove for-
ests would lead to destruction of environmental balance. In cleared
areas, however, cultivation of shrimp under careful management
should be preferred to other forms of land use because of the better
scope of soil improvement. Research on integrated soil-water and
crop management is necessary to address the.se problems.

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Joiinml of Shellfish Research. Vol. 21. No. 1. 273-27S. 2002.

PHYSICAL, CHEMICAL AND BIOLOGICAL VARIATION AMONG FRESHWATER CRAYFISH
(CHERAX ALBIDUS CLARK, 1936) RESEARCH PONDS

CRAIG S. LAWRENCE,' - NOEL M. MORRISSY,' PHILIP E. VERCOE," IAN H. WILLIAMS,"
AND YUK W. CHENG'

^Department of Fisheries, WA Marine Research Laboratories, North Beach. WA 6020. Western
Australia: 'Animal Science Group. Faculty- of Agriculture. The University of Western Australia.
Nedlands. WA6907. Western Australia

ABSTRACT Previous workers pertbrming classical agricultural field e.Kperiments and aquaculture pond trials have emphasized that
in order for results from experiments to be analyzed and evaluated, it is important to either demonstrate that the experimental units are
homogeneous or quantify any variation between plots, ponds or blocks of these experimental units. Moreover to ensure that results of
aquaculture experiments are applicable to industry, research ponds must have similar characteristics to those of industry. Physical,
chemical and biological characteristics of 24 research ponds were recorded at the Avondale Research Station. Western Australia. This
demonstrated that: (a) The ponds behaved in a manner smiilar to farm dams typical of the Western Australian wheat belt; (b) The water
chemistry and turbidity profiles of the ponds were within the range recorded for wheat belt farm dams; (c) The average % organic
matter of the sediments was 3.49'7r. which was within the range recorded for farm dams in Western Australia (d). The coefficient of
variation for growth of yabbies [Cherax albidus) among the ponds at the Avondale Research Station was 9.26%. The comparatively
low level of variation among the ponds was attributed to a number of factors including the age and design of the facility, and the
homogeneous water supply for all ponds. Power analysis has been applied to determine the number of replicates required for
experiments in these research ponds.

A'£l' WORDS: variation, ponds, crayfish. Clienix albidus. power analyses

INTRODUCTION

Agricultural plot experiments on field research stations provide
a bridge between glass house experiments and industry paddocks
(Fisher 1958; Fisher 1960). Similarly, aquaculture research pond
experiments provide both a bridge between aquarium studies and
commercial ponds, and a more realistic estimate of production than
laboratory aquarium or tank studies that tend to underestimate
yields (Shell 1983).

For the results of aquaculture experiments to be applicable to
industry it is essential to be able to measure production character-
istics both accurately and in a system that mimics the natural
environment in ponds as closely as possible. Aquarium studies
provide an opportunity to measure single characters accurately in
a tightly controlled environment. However, they do not simulate
production in ponds well because they do not provide the same
natural biota, or algal and clay turbidity. Consequently, long-term
growth performance and nutritional health of freshwater crayfish,
such as yabbies {Clwra.x albidus Clark 1936). is usually so poor in
clean aquaria that experimental comparisons are compromised
(Morrissy 1984a). Generally, aquaria studies tend to underestimate
yields (Shell 1983). Furthermore, laboratory tank experimentation
is impractical at industry densities of 1-5 yabbies/m". Since den-
sity and growth of freshwater crayfish are related inversely (Mor-
rissy 1992; Mills & McCloud 1983; Brown et al. 1995; McClain
1995a; Morrissy et al. 1995), tank experiments in small containers
at high densities are unrealistic and give poor growth and survival
( Ackefors et al. 1 989; Verhoef & Austin 1 999a; Verhoef & Austin
1999b).

Another alternative is to estimate production characteristics of
yabbies (C albidus) directly from commercial ponds. In contrast

Corresponding author. Craig S. Lawrence. Department of Fisheries. WA
Marine Research Laboratories. PO Box 20, North Beach. WA 6020. Aus-
tralia. E-mail: clawTence& fish. wa. gov. au

to the smaller Eastern Australian yabby (C destructor) industry
that relies on wild caught and pond reared animals, the much larger
farmed Western Australian yabby (C albidus) industry is based on
large "commercial ponds" that are argillotrophic. clay-based, pad-
dock catchment dams filled by rainfall runoff to provide drinking
water for sheep (Lawrence 1998; Lawrence & Jones 2001). How-
ever, it is difficult to obtain useful infonnation from the large
ponds and farm dams used for rearing yabbies (C. albidus) because
of the variability in physical and biological characteristics (Mor-
rissy 1974; Lawrence, et al. 1998). In addition there are other
limitations of farm dams for efficient research. Management prac-
tices differ between dams, they are difficult to drain and hence
measure the total population, and they generally contain so many
yabbies (C albidus) that the labor required to measure production
traits like growth and population size is excessive.

Research station experiments include most of the advantages of
true ecological studies, where the population of animals is influ-
enced by many uncontrolled natural factors, as is experienced in
commercial ponds, and classical experimentation, usually single
factor, under highly controlled conditions in the laboratory.

To carry out yabby (C albidus) experiments a research facility,
consisting of 25 ponds supplied by a homogeneous water supply
from a header dam. was built near Beverley Western Australia
(32°7'S. 116°55'E). This was capable of supporting replicated,
randomized and reproducible experiments, with controls, in an
environment that simulated farm dams.

Large variability among ponds in aquaculture experiments of-
ten leads to imprecise estimates of treatment effects. Previous
workers in classical agricultural field experiments such as at Roth-
amsted (Fisher 1958; Fisher 1960) and aquaculture pond trials at
Auburn (Shell 1983) emphasized that in order for results from
experiments to be analyzed and evaluated, it is important first to:
(a) demonstrate that the experimental units are relatively homoge-
neous; or (b) to quantify any variation between plots, ponds or
blocks of these experimental units.

Cross-over (Change-over) designs, which make comparisons

273

274

Lawrence et al.

directly w ithin the same pond instead of between ponds, have been
proposed for use in aquaculture experiments to eliminate the varia-
tion between ponds (Smart et al. 1997). The construction of an
appropriate cross-over design is challenging (Cheng 1996; Cheng
& Street 1997) as: (a) due to environmental factors most species in
aquaculture respond significantly differently according to seasonal
variations over a year; and (b) the interaction between seasonal and
other treatment effects are unknown. It is likely that seasonal varia-
tion and the effect of the interaction between seasons and other
treatments in each pond within a year may be greater than the
variation among ponds within the same time frame. Consequently
cross-over designs require a longer experimental period and the
analysis may involve a more complex statistical model. To in-
crease the degree of precision for estimation of treatment effects,
the most efficient way to account for between pond variation is to
minimize the variability between ponds and quantify that level of
variation.

By measuring the level of variation between ponds prior to
commencing a field trial program it was possible to (a) take varia-
tion between experimental units into account when planning the
randomization and replication of treatments in future experiments.
and (b) detennine whether results recorded from future experi-
ments will be due to the application of treatments or merely a
result of naturally occurring variability between ponds.

This study aimed at testing the homogeneity of experimental
units, quantifying the variation between these units, determining
the number of replicates required when planning experiments and
confirming that the ponds had similar characteristics to wheat belt
farm dams.

MATERIALS AND METHODS

The site for ponds was selected according to clay profiles from
the region and to ensure that all ponds were placed as close as
practicable to each other. All 25 ponds were constructed v\ ithin the
same soil type with the same dimensions (10 m x 10 m water
surface area, 1.5 m deep and 3:1 side, or batter slopes). The 25
ponds all received water from the same supply dam.

The twenty-five 0.01 ha ponds were filled from the water stor-
age dam four weeks prior to stocking. Two weeks prior to stocking
each pond with yabbies (C. atbidus) for the first experiment, the
ponds received the addition of 50 L of sheep manure to condition
pond sediment and increase organic matter to the level commonly
found in farm dams.

Twenty-four of the 25 ponds were divided into 6 blocks ac-
cording to possible sources of variation (clay type, location —
north, south, east, west, upper row, lower row); a randomized
block design was used, each block contained 4 ponds which re-
ceived the same four treatments (yabbies 4.5/nr unfed, yabbies
4.5/m- fed lupins at the rate of 2.5 g/m-/week, yabbies l/m" unfed,
yabbies 1/nr fed lupins at the rate of 2.5 g/tn-/week). The remain-
ing pond received yabbies 4.5/nr fed lupins at the rate of 2.5
g/m~/week. Prior to stocking each yabby (C ulhidus) was
weighed, sexed and 10% of animals were tagged by tail punching
(Morrissy 1980; Getchell 1987). The ponds were stocked with
7.050 yabbies (C. ulbiclus) (mean weight 19.41 g ± 0.22 SE) at a
sex ratio of 1 male: 1 female on the 29th November 1 994 and the
trial was harvested between the Xth to the 15th of March 1995.

At the commencement and conclusion of the experiment
samples were collected for water chemistry analyses. Samples
were submitted to the Chemistry Centre of WA for analyses of Ca,

N-NO,, N-NO3, CI, Cu. Fe (dissolved), Fe-total (unfiltered
sample). HCO,. K. Mn (dissolved). Mn-total (unfiltered sample).
Na. P-SR (Phosphorous, soluble reactive). SO4-S (Sulphate, ex-
pressed as sulphur). Zn-total (unfiltered sample). CO,. pH. Elec-
trical conductixity (25°C) (Econd.). Alkalinity and Hardness.
Max-Min thermometers were used to record water temperature in
the ponds.

At fortnightly intervals subsurface water samples (lO-cm
depth) and benthos core samples were collected from each pond.
The benthos corer removed a 2-cm diameter core of sediment and
clay to a depth of 15 cm. The percentage organic matter of water
samples and sediment was determined by drying samples in pre-
dried and weighed crucibles and then ashing out. As the loss in
weight is due to combustion of organics, percentage organic matter
was calculated according to the formula:

Organic Matter = [weight of organic matter/di-y weight] x 100%

At fortnightly intervals Secchi disk depth was measured in each
pond as an index of turbidity.

All data in the randomized block design were analyzed using
analysis of variance (ANOVA) to determine significant differ-
ences among treatment means. Data were considered significantly
different at the 0.05 lexel of significance. The coefficient of varia-
tion (C.V.) is a measure of variation and was calculated according
to the formula:

C.V. = standard deviation/mean ( Shell 1983).

Power analyses was applied using the methods described by
Searcy-Bemal (1994) to determine the number of replicates re-
quired for a given number of treatments to measure a difference of
5. 10, and 20% in the growth of yabbies (C albidits) from the
research ponds.

RESULTS

VlV((er Chemistry

Water chemistry parameters at the commencement and conclu-
sion of the experiment are presented below (Table 1 ). Using the
nonparametric Wilcoxon-Mann-Whitney test (Wilcoxon 1945;
Mann & Whitney 1947) to examine paired observations, there was
no significant difference between the initial chemical parameters
and final chemical parameters (P = 0.73). During the experiment
water temperature ranged from 13 - 35°C (mean 22'=C ± 0.6 SE).
There was no significant difference in minimum (P = 0.51 ) (C.V.
= 9.63%) or maximum (P = 0.21) (C.V. = 12.38%) water
temperature among blocks of ponds.

Turbidity

There was no significant difference in turbidity among blocks
of ponds at either the commencement (P = 0.67) (C.V. = 4.74%)
or conclusion of the experiment iP = 0.73) (C.V. = 11.75%).
However, there was a significant decrease in turbidity over the
course of the experiment across all ponds (P < 0.0001) by two
tailed f-test with paired observations (Fig. 1). As there was no
variation in salinity (measured as electrical conductivity Table 1 ),
it is probable that the decrease in turbidity was due to a reduction
in suspended organic matter (see Fig. 3).

Variation Among Crayfish Research Ponds

273

TABLE 1.
Water (.hi'iiiistr) parameters of Avondale research ponds at commencement and conclusion of experiment (;i = 25 1.

Units

Commencement

Conclusion

Parameter

Mean

SE

Min

Max

Mean

SE

Min

Max

Alkalinity

mg/L

186

9

L30

200

172

15

130

200

CO,

mg/L

II

2

<2

22

13

4

<2

18

Ca

mg/L

33

2

26

38

30

3

26

38

CI

mg/L

918

49

662

1 1 50

936

103

662

1150

Cii total

mg/L

0.05

0.02

<0.02

0.21

0.09

0.04

0.04

0.21

l;ccind

mS/m

343

14

259

404

346

31

259

404

I-L-

mg/L

0.07

(1.(11

<0.05

0. 1 3

0.08

0.02

<0.05

0.13

He total

mg/L

0.8

0.2

0.1

1.4

0.6

0.3

0.1

1.4

HCO3

mg/L

206

10

160

230

185

12

160

210

Hardness

mg/L

348

18

270

430

365

35

270

430

K

mg/L

7.8

0.5

6.0

10.0

8.8

0.6

7.0

1 0.0

Mn

mg/L

0.02

0.00

<0.02

0.02

0.02

0.00

<0.02

0.02

Mn total

mg/L

0.02

0.00

<0.02

0.02

0.02

0.00

<0.02

0.02

N-NO,

mg/L

0.46

0.24

0.02

2.00

0.52

0.49

0.02

2.00

Na

mg/L

593

28

412

678

578

58

412

678

P-SR

mg/L

0.05

0.01

0.03

0.08

0.06

0.01

0.04

0,08

SO4-S

mg/L

139

6

121

173

146

11

121

173

Zn total

mg/L

0.41

0.24

0.03

2.00

0.10

0.05

0.03

0.23

PH

8.4

0.1

7.5

8.8

8.4

0.3

7.5

8.8

Organic Mailer

The organic matter of the pond sediment (Fig. 2) was not
significantly different among bloclcs of ponds at the commence-
ment {P = 0.16) (C.V. = \9M9c). conclusion {P = 0.07)
(C.V. = 31.03%) or during the experiment (P = 0.09.) by two
tailed r-test. All ponds ranged between 1 .6% and 53% sediment
organic matter content.

There was no significant difference in the suspended organic
matter in water among blocks of ponds at either the commence-
ment iP = 0.10) (C.V. = 24.87%) or conclusion (P = 0.13)
(C.V. = 21.13%) of the experiment (Fig. 3). However, there was
a significant decrease in suspended organic matter o\er the course
of the experiment across all ponds (P < 0.0001 ) by two tailed Mest
with paired observations (Fig. 3). The low levels of variation in

organic matter during this trial may be largely attributed to the low
industry based feeding rates (0-2.5 g/m"/week) in this experiment.

Yabby (C. albidus) Survival, Growth and Biomass

There was no significant block effect on yabby (C. albidus)
production determined as either survival (65% ± 2 SE) {P = 0.24),
change in biomass of adult yabbies (C. albidus) stocked (P =
0.26), total change in biomass of all yabbies (C albidus) harvested
(including juveniles) (P = 0.50) or final mean weight of yabbies
(C albidus) (P = 0.47) (Fig. 4). The coefficient of variation for
yabby (C albidus) growth (expressed as final mean weight-initial
mean weight) between the blocks of research ponds was 9.26%
in = 6).

These results indicate that there was no trend across the ponds

rh

*

*

*

*

70

60

'E 50

•S 40

Q.
U

!S 30
u

8 20

10
0

Bbck
Figure I. Turbidity of research ponds (cm ± SE) at the commencement and conclusion of experiment - the higher the Secchi disk depth, the lower
the turbidity of the water.

0

U

ui

u

U

^

^

»

u

u

U

^

U

U

>
0

z

H

H

H

H

^

S

K

X

8

8

8

8

_]

1

_j

_i

,j

J

J

J

-1

_i

-3

_i

-5

m

m

CQ

ca

CQ

an

•s

m

03

0

03

0

03

276

Lawrence et al.

2

u

1

E?
o

10
9
g

7
6
5
4
3
2

rih n

*

*

*

Btock

3^

U.

u

U

^

Ui

o

^^

^

^

U!

u

^

>

o

CJ

u

u

O

^

U

(J

y

y

o

y

o
Z

o

O

s

o

R

o

S

_l

h^

y

o

-J

ii

o

■s

m

03

CQ

CQ

CD

ca

■5

03

m

oa

ca

Figure 2. Organic matter (% ± SE) of pond sediment al the commencement and conclusion of experiment.

70-,
60

? 50

W

e 40

CO

o 30 -I

1

£? 20
O

10

0

n,n,n,Pl,n^D-

^ - f^

o U U.

I 8 8

■^ _i J -1 -J

g CQ 03 oQ oa

^ »/-l 'O

;^ ui u: ui:

8 8 8 8

^

s

— <s m

^^ :^ 1^

8 8 8

>j -1 ^

P3 CQ oa

^ u: :^

U CJ o

o o o

J -J -1

oa BQ CQ

Bbck

op

Figure 3. Suspended organic matter (% ± SE) of pond water at the commencement and conclusion of experiment.

40

35

30

^ 25
'So

20
15
10

5

0

BLOCK 1 BL0CK2 B1jOCK3 BLXXX4 BLOCKS BL0CK6 INrrL\L

Block
Figure 4. Mean ,val)b> (C alhidiis) weight (g ± SE) at commencement of experiment (Initial) and variation in final vabhv (C. alhidus) weights
(g ± SE) among pond blocks at conclusion of experiment (Block 1-6).

Variation Among Crayfish Research Ponds

277

for change in yabby (C albichis) growth due to inherent pond
factors at the site. The low variation in yabby (C. albidiis) growth
(C.V. = 9.26%) between individual ponds treated identically
showed a highly acceptable homogeneity in the pond site for future
experiments.

DISCUSSION

To ensure that results of aquaculture experiments are applicable
to industry, research ponds must have similar characteristics to
those of commercial ponds. In addition the variation (C.V.l in
yabby (C. alhidus) growth among ponds due to inherent site fac-
tors needs to be as small as possible to minimize the replication
needed for future experiments to be effective in showing effects
due to different treatments. If differences between treatments are
smaller than the coefficient of variation then the observed differ-
ences are likely to be due to chance variation rather than a treat-
ment effect.

The coefficient of variation for yabbie growth among the ponds
at the Avondale Research Station was 9.26'7f.

Experiments within earthen ponds at the Auburn University
aquaculture research station in Alabama have produced coeffi-
cients of variation for fish production ranging from 4.2-34.6%,
with an average of 20%' (Shell 1983). Previous research using
adjacent ponds for marron {Cherax tenuiinaniis) experiments at-
tributed 1 1% of variation in growth to differences between ponds.
(Morrissy 1992; Morrissy et al. 1995).

The comparatively low level of variation between the ponds at
the Avondale Research Station may be attributed to a number of
factors including: ( I ) the age of the facility: (2) the planning and
design of the facility to ensure a homogeneous environment: and
(3) the homogeneous water supply for all ponds.

Comparative trials between ponds, cages and lined tanks have
shown that the primary causes of variation are differences in en-
vironmental conditions (including water quality, productivity, soil
types, and water source) (Shell 1983). This experiment has shown
that the pond site was homogeneous and although environmental
parameters (i.e.. turbidity and % suspended organic matter which
showed an inverse relationship due to the initial algal bloom from
pond fertilization declining) change over time, all ponds followed
similar patterns of change. The ponds were therefore suitable for
testing treatments (such as density, diet, monosex culture etc.)
because any observed difference in yabbie growth greater than
9.26% (C.V. ) was likely to be due to the effect of the experimental
treatment.

For a given number of treatments, power analysis can be used
to determine the number of replicates required. Power analyses are
therefore considered to be a basic tool in experimental and sam-
pling design (Searcy-Bernal 1994). In general, aquaculture experi-
ments can only detect relatively large effect sizes with a reasonable
power. This is particularly true with pond experiments that are
often characterized by high within-treatment variability and low
numbers of replicates (Searcy-Bernal 1994; Shell 1983). A power
value of 0.80 has been proposed as the minimum desirable to avoid
committing a Type II error (i.e.. accepting a false null hypothesis)
(Searcy-Bernal 1994). Applying the methods described by Searcy-
Bernal (1994) a power table (Table 2) using the data from this
experiment shows the miniinum number of replicates (n) required
for treatments (k) to measure a difference of .S. 10. and 20% in the
growth of yabbies (C alhidus) from the research ponds, using the
a = 0.05 level of significance, with a power of 0.8. As would be

TABLE 2.

Number of replicates (n) required to identify a difference in growth
of 5, 10. and 20% with mean 60g and standard deviation 4.5g for
a = 0.05 level of significance with a power of 0.8 for (k) treatments.

Difference

No.

Treatments

No.

Replicates

in Growth

(k)

(n)

5%

3

4
5

20
30
60
70

10%

2
3
4
5

3
3
4
5

20%

2
3
4
5

2
2
2

2

expected from the C.V. of 9.26% an unrealistically high number of
replicates (>20) would be required to measure a difference in
yabby (C alhidus) growth of 5% between 2 or more treatments
(Table 2). In contiast. to record a difference in growth of 10%
requires considerably less replicates (i.e.. 3-5 replicates depending
on number of treatments under investigation).

The condition of the ponds was similar m manner to typical
farm dams of the Western Australian wheat belt. The water chem-
istry of the ponds was within the range recorded for farm dams in
the Western Australian wheat belt (Morrissy 1980; Lawrence et al.
1998; Cheng et al. 2001 ), which is dominated by sea saU ions, (Na
and CI), rather than by salts from catchment erosion. (Ca, Mg,
SO4). as found in other world freshwaters (Francesconi et al.
1995b). The high salinity recorded 346 mS/m (1885 mg/1), is typi-
cal of cleared catchments in the Western Australian wheat belt
(Lawrence et al. 1998; Cheng et al. 2001). The values of a number
of parameters (i.e.. CI. Na. SO4-S and hardness) were higher than
those recorded from most wheat belt farm dams but within the
range suitable for yabbies (Moirissy 1980: Lawrence et al. 1998:
Cheng et al. 2001) (Table 1). This may be attributed to initial
disturbance of the catchment due to dam construction and subse-
quent flushing of the catchment.

The turbidity of the ponds was within the range recorded for
farm dams in the Western Australian wheatbelt (Lawrence et al.
1998). Similarly, the percentage of organic matter in the sediments
of the experimental ponds, mean 3.49%. was also within the range
recorded for farm dams in the Western Australian wheat belt,
which range from 0.27-12.13% organic matter (Lawrence et al.
1998).

The research ponds were therefore suitable for conducting ex-
periments on yabbie farming and the results were likely to be
directly applicable to the farm dam environment.

ACKNOWLEDGMENTS

This work was supported by funding from FRDC Project No.
94/75. We thank R. Allison and M. Stuckey technical officers.
Fisheries WA for assistance in maintaining the experiments. We
also thank Dr. G. Maguire. Dr. N. Hall, and Dr. N. Caputi for their
coinments.

278

Lawrence et al.

LITERATI

Ackefors. H.. R. Gydemo & L. Weslin. 1989. Growth and survival of
juvenile crayfish. Astacus astacus in relation to food and density. In: N.
De PuLiw, E. Jaspers, H. Ackefors & N. Wilkins. editors. Aquaculture
- A Biotechnology in Progress. Bredene, Belgium: European Aquacul-
ture Society, pp. 365-373.

Brown, P. B., K. A. Wilson, J. E. Wetzel & B. Hoene. 1995. Increased
densities result in reduced weight gain of crayfish Onoiiectes virilis. J.
World Aqiuuult. Soc. 26:165-171.

Cheng, Y. W. 1996. Construction of optimal change-over designs. PhD
thesis. University of NSW.

Cheng, Y. W. & D. J. Street. 1997. Constructions for optimal non-strongly-
balanced change-over designs. Comm. Stars. 26:1073-1082.

Cheng, Y. W., C. S. Lawrence, N. M. Morrissy & J. E. Bellanger. 2001.
The statistical correlations and implied causal relationships between
physical, chemical and biological parameters and yabby (C/icra.v alhi-
ihis) production in Western Australian farm dams. Freshwater Cray-
fish. 13:

Fisher, R. A. 1958. Statistical methods for research \v<u±ers. 13th edition.
Edinburgh: Oliver and Boyd. 356 pp.

Fisher. R. A. 1960. The design of experiments. 7th edition. Edinburgh;
Oliver and Boyd. 248 pp.

Francesconi, K. A., N. M. Momssy, C. J. Fellows & C. Bird. 1995. Survey
of marron farms and the marron recreational fishery in Western Aus-
tralia for pesticides and water chemistry, with related laboratory ex-
perimentation. Fisheries Research Bulletin No. 30. Perth: Fisheries De-
partment of Western Australia. 14 pp.

Getchell, R. G. 1987. Effects of V-notching on the lobster, Homarus amen-
caints. Can. J. Fish. Aquat. Sci. 44:2033-2037.

Lawrence, C. S. 1998. Yabbies. In: K. Hyde, editor. The new rural indus-
tries - A handbook for farmers and investors. Canberra: Rural Indus-
tries Research and Development Corporation, pp. 147-152.

Lawrence, C. S., N. M. Morrissy, J. Bellanger & Y. W. Cheng 1998.
Enhancement of yabby production from Western Australian farm dams.
Final Report Fishenes Research and Development Corporation Project
44/075. FRDC. Canberra. Australia. 134 pp.

Lawrence, C. S. & C. Jones. 2001. Cherax. In: D. M. Holdich. editor.
Biology of Freshwater Crayfish. Oxford: Blackwell Science, pp. 635-
670.

Mann, H. B. & D. R. Whitney. 1947. On a test of whether one of two
random variables is stochastically larger than the other. .Aim. Math.
Stat. 18:50-60.

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McClain. W. R. 1995. Growth of crawfish Procamhanis clarkii as a func-
tion of density and food resources. J. World Aquacult. Soc. 26:2-1-28.

Mills, B. J. & P. I. McCloud 1983. Effects of stocking and feeding rates on
experimental pond production of the crayfish Cherax destructor Clark
(Decapoda: Parastacidae). Aquaculture 34:1-74.

Morrissy. N. M. 1974. The ecology of marron Cherax tenuimanus (Smith)
introduced into some farm dams near Boscabel in the Great Southern
area of the w heatbelt region of Western .Australia. Department of Fish-
eries and Fauna, Australia. Perth: Western Australia Fisheries Research
Bulletin No. 12, Fisheries WA. 55 pp.

Morrissy. N. M. 1980. Production of marron in western Australian farm
dams. Fisheries Research Bulletin No. 24. Perth: Fisheries Department
of Western Australia. 80 pp.

Morrissy. N. M. 1984. Assessment of artificial feeds for battery culture of
a freshwater crayfish, marron {Cherax tenuimanus) (Decapoda: Parast-
acidae). Research Report No. 63. Western Australia: Department of
Fisheries and Wildlife. 42 pp.

Morrissy, N. M. 1992. Density-dependent pond grow-out of single year-
class cohorts of a freshwater crayfish Cherax tenuimanus (Smith) to
two years of age. J. World Aquacult. Soc. 23:154—168.

Morrissy, N. M., P. Walker & W. Moore 1995. Predictive equations for
managing semi-intensive grow-out of a freshwater crayfish (marron).
Cherax tenuimanus (Smith 1912) (Decapoda: Parastacidae). on a com-
mercial farm. Aquaculture Res. 26:71-80.

Searcy-Bemal, R. 1994. Statistical power and aquacultural research. Aqua-
culture 127:371-388.

Shell. E. W. 1983. Fish farming research. Auburn University. Auburn. 108

PP-

Smart. T. S.. T. S. Riley & G. Haylor 1997. Eliminating pond differences
with cross-over designs. Aquaculture Res. 28:621-627.

Verhoef G. D. & C. M. Austin. 1999a. Combined effects of temperature
and density on the growth and survival of juveniles of the Australian
freshwater crayfish. Cherax destructor Clark. Part 1. Aquaculture 170:
37^7.

Verhoef G. D. & C. M. Austin. 1999b. Combined effects of shelter and
density on the growth and survival of Juveniles of the Australian fresh-
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Wilcoxon. F. 1945. Individual comparisons by ranking methods. Biomet-
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Journal of Shellfish Reicunh. Vol. 21, No. 1. 27y-2SS, 2002.

POPULATION DYNAMICS OF THE SPINY LOBSTER PANULIRUS GUTTATUS (LATREILLE)
IN A CORAL REEF ON THE MEXICAN CARIBBEAN

FERNANDO NEGRETE-SOTO, ENRIQUE LOZANO-ALVAREZ, AND
PATRICIA BRIONES-FOURZAN

Instituto de Ciencias del Mar y Limnologia. Unidad Academica Puerto Morelos, Universidud Nacional
Aiitoiioiiia de Mexico. Ap. Postal 1152. Canciin. Q. R. 77500 Mexico

ABSTRACT The dynamics of a population of adult spiny lobsters. Paimlirus guttauis. was studied in a group of coral reef patches
in Puerto Morelos, Quintana Roo (Caribbean coast of Mexico), from October 1986 to November 1987. Lobsters were extracted by
divers from traps deployed in the perimeter of reef patches for 10-13 days every month. In total, 778 P. gutmtus were caught. Males
(size range: 42.4-87.5 mm carapace length (CL), were significantly larger than females (45.5-73.5 mm CL). The highly biased sex ratio
(2.6 males: I female) was partly due to a differential catchability in traps of males and females. We tagged 227 males and 90 females
with spaghetti-type tags and recaptured 62 males and 12 females, some on multiple occasions, yielding 1 19 recaptures of males and
26 of females. The monthly population size in the patch complex, as separately estimated for each sex with the Fisher-Ford
multiple-recapture model, was higher from June to November 1987, when both the percentage of ovigerous females and the mean size
of individuals was smaller, indicating a possible recruitment of young adults into the trappable population during the summer and early
autumn. The average density of lobsters was 126 lobsters ha"'. Growth data were scarce, but suggest that growth rates decrease as size
increases, and that males can molt at least 2-3 times per year. The egg-incubation period of recaptured females was 2-3 weeks. Females
>50 mm CL may produce 3^ broods per year. In addition to P. giittcitus. 120 individuals of P. argus. mostly sub-adults (<80 mm CL)
were also caught. Size of male and female P. argus was similar, but the size of P. argus was significantly larger than that of P. gunatus.
Time at large of recaptured individuals suggests that the reef patches are a temporary habitat for P. argus. but a more permanent
residence site for P. giinaius.

KEY WORDS: Panulinis gunatus. Pauulirus argus. population dynamics, spiny lobster, abundance, growth, reproduction, Mexico

INTRODUCTION

Spiny lobsters are ainong the most \ altiahle fishing resources in
the world. Population studies provide information on the abun-
dance, movements, growth rates, reproductive dynamics, and sur-
vival of these species, and are therefore useful to improve their
fishery management (Morgan 1980). However, such studies are
also important to increase the knowledge on the ecology of the
species, whether they support major fisheries or not. In the Carib-
bean Sea and adjacent Western Atlantic coasts, two sympatric
species of spiny lobsters occur, Panulinis argus (Latreille) and P.
gullcitus (Latreille). Pauulirus argus is a migratory, large-sized
species that undergoes several ontogenetic changes in habitat. The
postlarvae (pueruli) of P. argus settle in shallow, vegetated areas,
where they remain throughout the so-called "'algal juvenile"" phase
(6-15 mm carapace length (CD). The ""postalgal juveniles" (15—1.5
mm CL) move to crevice-type shelters, also in shallow areas,
whereas the "subadults"" (45-80 mm CL) move to coral reef habi-
tats. Further on, the adults (>80 mm CL) migrate to deeper, more
diverse habitats (Butler & Hermkind 1997), In contrast, P. gunatus
IS a small, rather sedentary species. The pueruli of P. gutlatus are
believed to settle directly on the coral reef habitat, and remain in
this habitat throughout their entire benthic life (Briones-Fourzan &
McWilliam 1997; Sharp et al. 1997).

Pauulirus argus supports major fisheries throughout its geo-
graphic range, but P. gunatus is mostly a by-catch or secondary
catch in most areas, with specific fisheries only in Bermuda and
the French West Indies (Evans & Lockwood 1994). Consequently,
numerous and extensive population studies of P. argus have been
conducted in locations such as Florida (e.g., Lyons et al. 1981;
Forcucci et al. 1994), Cuba (review in Baisre 2000), Jamaica (Mun-
ro 1974), and Mexico (review in Briones-Fourzan & Lozano-
Alvarez 2000), In contrast, most population studies on P. gutlatus
have been conducted in Martinique (Farrugio 1975, 1976; Farrugio

& Saint-Felix 1975; Marfin 1978), and Bermuda (Sutcliffe 1953;
Evans & Lockwood 1994; Evans & Evans 1995, 1996; Evans et al.
1995, 1996). but also in Florida (Caillouetetal. l971;Chitty 1973;
Sharp et al. 1997) and in Mexico.

In the Caribbean coast of Mexico (coast of the state of Quintana
Roo), P. guttatus amounts to -6% of the lobster catch (Padilla-
Ramos & Briones-Fourzan 1997), and has been the subject of
specific studies on size distribution (Briones-Fourzan 1991).
movement patterns (Carrasco-Zanini 1985; Lozano-Alvarez et al.
unpubl. data), reproductive dynamics (Briones-Fourzan & Con-
treras-Ortiz 1999), and the description of its puerulus (Briones-
Fourzan & McWilliam 1997). Also, comparative studies have been
conducted on biological, ecological and fisheries aspects of P.
guttatus and P. argus (Colinas-Sanchez & Briones-Fourzan 1990;
Briones-Fourzan 1995; Padilla-Ramos & Briones-Fourzan 1997;
Briones et al. 1997), and on the den choice and occupation patterns
of shelters by these two sympatric species (Lozano-Alvarez &
Briones-Fourzan 2001).

The present paper provides information on a field investigation
into the population dynamics of adult P. guttatus in a coral reef of
northern Quintana Roo. Monthly changes in the population size of
adult P. guttatus were explored, by means of capture-recapture
techniques, in a group of coral reef patches separated, but not
entirely isolated, from adjacent coral patches. Because P. guttatus
is a sedentary species, we hypothesized that population additions
would be due mostly to recruitment of young adults, and popula-
tion losses to predation-induced mortality. We also aimed to de-
tennine the growth rates of P. guttatus and to compare them to
those reported for P. argus. as well as to obtain direct evidence for
repetitive breeding of females throughout the year. Although our
study was focused on P. guttatus. our samplings also yielded in-
dividuals of P. argus. providing an opportunity to compare the size
ranges and time at large of individuals of both lobster species in
this reef patch habitat.

279

280

Negrete-Soto et al.

MATERIALS AND METHODS

Sliidv Area

The study was conducted in the coral reef at Puerto Morelos. in
northern Quintana Roo (Fig. 1 ). Puerto Morelos is located in the
northern portion of a barrier-fringing reef tract that extends from
Belize to the Yucatan Strait. Rather than a continuous barrier, the
coral reef in Puerto Morelos consists of a series of reef patches,
separated from the coast by a reef lagoon 300-1000 m in width.
The reef lagoon (<5 m in depth) is covered by seagrass meadows,
a habitat where juveniles of P. argus dwell, but where P. guriatKs
does not occur (Briones-Fourzan 1995). Along the reef tract, the
sloping fore-reef has relatively few high-relief features, but hard
coral cover is dense at the reef crest and in the back-reef zone
(Ruiz-Renten'a et al. 1998). providing an intricate habitat with
numerous crevices and caves where both P. gunanis and P. aigiis
occur (Briones-Fourzan I993-. Lozano-Aivare/ & Briones-Fourzan
2001).

Lobster Sampling and Tagging

The main study site (site 1 ) consisted of a group of several reef
patches, close to each other (maximum distance between adjacent
patches: 50 ni). but relatively separated (-200 m) from the rest of
the reef tract. In addition to their relative isolation, we chose these
reef patches because they are not fished for lobsters, owing to their
proximity to the navigational channel to the port. The area of the
patch complex was 2.5 ha, excluding the areas between patches,
which consisted mostly of sand and sparse sea grasses. Average
depth around the leef patches was 4 to 5 m. From October 1986 to
November 1987, 20 lobster traps (mesh size: 5 x 2.5 cm) were
deployed by divers in the underside of ledges or coral formations
around these patches. The distance between adjacent traps ranged
from -20 to 100 m. The traps remained fixed for 10-13 days every
month, during the dark portion of the lunar cycle, after which the
traps were recovered and relocated the following month. The
divers carefully extracted the lobsters from within the traps every

20° 50' —

Figure 1. Location of study sites on the coral reef tract at Toerto
Morelos, Mexico.

morning during each sampling period, unless impeded by bad
weather. Lobsters were tagged with modified Australian spaghetti-
type tags (Lozano-Alvarez et al. 1991; Lozano-Alvarez 1992) that
were manually assembled in the laboratory as described by Chittle-
borough (1974). These tags consist of an individually numbered
vinyl "spaghetti" and a small plastic toggle, joined by a thin nylon
thread. The toggle is inserted in the dorsolateral muscle of the
lobster, between the cephalothorax and abdomen, with a stainless
steel applicator. The original toggles measured II x 3 x 0.5 mm,
but we shortened them to 9 mm in length with a grinding machine
before assembling the tags, in order to reduce their possible del-
eterious effects on the relatively small-sized P. guttatus.

All lobsters were measured (carapace length. CL, in mm. from
between the rostral horns to the posterior margin of the cephalo-
thorax) with digital calipers (±0.1 mm), and injuries (i.e. number
and type of missing appendages) were recorded. The reproductive
stage of females was determined according to the following scale
(Briones-Fourzan & Contreras-Ortiz 1999): ( I ) clean carapace, no
extruded eggs or traces of spermatophore; ( 2 ) new and intact sper-
matophore on sternum; (3) newly extruded eggs (bright orange);
(4) dark orange eggs, with eyespots visible; (5) brown eggs, em-
bryo and eyes cleariy visible; (6) remnants of empty egg capsules
and/or eroded spermatophore. After tagging, the lobsters were re-
turned immediately to natural crevices in the coral patches. To
reduce tag loss, individuals that were about to molt or recently
molted ("soft-shelled") were not tagged. All lobsters were care-
fully examined to ascertain whether they had lost a tag, as evi-
denced by a distinctive scar in the site of tag application.

Additional information on size composition and sex-ratios was
obtained from a large, elongated patch (site 2) located 4 km north
of site 1, where 13 traps were used in a similar fashion as in site
1. Lobsters from site 2 were not tagged because they were sacri-
ficed to study their diet (Colinas-Sanchez & Briones-Fourzan
1990) and the fecundity of females (Briones-Fourzan & Contreras-
Ortiz 1999).

Size Distribution

We compared the overall size distribution of P. argus and P.
guttatus; of P. giittatiis between sites I and 2, and of males and
females of each species, with Student's /-tests for unequal sample
sizes (Zar 1984). When necessary, data were log-transformed to
homogenize variances. To explore temporal changes in the size
distribution of P. giitlcitus in site 1. monthly data were grouped in
2-mm size classes and analyzed with a one-way repeated measures
ANOVA, with time as the repeated factor, followed by a Tukey's
test for unequal sample sizes (Winer 1971).

Population Size and Survival

The population size of P. guttatus was monthly estimated by
means of the Fisher-Ford model (Fisher & Ford 1947). which
relies on several tagging occasions and several recaptures. We
chose this model because the capture-recapture data were rela-
tively scarce and the survival rate was fairiy constant (see Results).
In these circumstances, the Fisher-Ford model tends to yield more
reliable results than other models based on multiple-recapture data
(Bishop & Sheppard 1973; Begon 1979; Lozano et al. 1982). while
still providing estimates of population losses (death -I- emigration),
and population additions (recruitment -i- immigration). The Fisher-
Ford model assumes a constant survival rate (ct>), but this assump-
tion can be analyzed by means of two goodness-of-fit tests (Begon

Population Dynamics of Panul/rus guttatus in Mexico

281

1 979); a test of the period-to-period differences in <i> (observed vs.
expected periods survived) (test 1 ). and a test of the independence
of <b from the age of tags (observed vs. expected recaptures of
various ages) (test 2). To avoid bias due to a possible "trap addic-
tion" of lobsters, individuals that were recaptured more than once
within a sampling period were only considered to have been cap-
tured once (Bishop & Hartley 1976). Owing to differences in
catchability between sexes (see Results), we applied the Fisher-
Ford model separately to males and females, and then summed the
monthly estimates of both sexes to obtain the monthly and average
population sizes and sex ratios. Estimates of population size were
further standardized as lobster densitv (number of adult lobsters
ha-').

Growth

Because of possible measurement errors, only lobsters whose
CL increased over I mm between recaptures were considered as
having grown (Forcucci et al. 1994). Growth rate (mm CL week"' )
was calculated by dividing the increase in CL of recaptured indi-
viduals by the number of weeks between recaptures. To calculate
molt increments (increment in CL per molt), we followed the
technique proposed by Forcucci et al. (1994), in which each ob-
servation in change in CL is plotted against time at large (in
weeks). In these plots, the data points cluster in groups represent-
ing single and multiple molting events. Since growth may vary as
a function of sex, size, and injury condition (Hunt & Lyons 1986),
we produced different plots for uninjured and injured males and
females, and analyzed the data for each sex by size class (<60 mm
CL. 60-70 mm, and >70 mm CL). Only single-molt observations
were used to estimate molt increments. The intermolt period (in
weeks) was then calculated by dividing the average molt increment
(mm CL) by the average growth rate. The results were then sum-
mari/ed in a table.

Reproductive Aspects

The monthly percentage of ovigerous females was obtained
from the total sample to determine the main reproductive season.
Data from females of P. gnrtatiis that were recaptured in different
reproductive stages allowed for a preliminary estimate of the egg-
incubation period, and of the duration of a complete breeding
cycle.

RESULTS

In total, 920 lobsters (including recaptures) were obtained. 778
(85%) P. guttatus and 142 (15%) P. argus. Results on the issues
explored are given for each species separately.

Paniilirus guttatus

Size Distribution

In all, 551 P. guttatus were caught in site 1 (410 males and 141
females), and 227 in site 2 ( 160 males and 67 females). Mean size
of P. guttatus was similar between sites U = 0.782, df = 777, P
= 0.495). Overall, male P. guttatus ranged in size from 42.4 to
87.5 mm CL (mean ± SD; 64.5 ± 7.6 mm CL), significantly larger
than females (range: 44.5 to 73.5 mm CL; mean ± SD: 59.3 ± 5.2
mm CL) (log-transformed data. ; = 8.748, df = 774, P < O.OOOI )
(Fig. 2A).

(a) Panulirus guttatus

20 0

.^ <^^ C? fot. ^ ^t. ^q, ^^ ^- ^^ ^ ^
.. .. ., ,, ^<^, ^ „, ,. ., ,.

Carapace length (mm)

HFemales(N= 175) BMales (N= 456)

(b) Panulirus argus

20 0

(A
0)

in
o

0)
O)

ra

c

0)

u

9)
Q.

150

100

<^ .0, q, q, q, q, a ^'^ ^^ ^'^ J^ r?>
i-^ <^- «?• <c^• <§>- ^^ a'* -l}" <§> q,*" qQ N^
<b^' <P' <§>' <&' \^' ^ <&' 'P' ^' ^' ■"

Carapace length (mm)

H Females (N= 82) BMales (N= 59)

Figure 2. Size distribution of the total sample (including recaptures) of
(a) Panulirus guttatus and lb) Panulirus argus caught in traps in coral
reef patches at Puerto Morelos, Mexico.

Tagged and Recaptured Individuals

Individuals of P. guttatus (excluding recaptures) in site 1 were
331 males and 127 females (sex ratio 2.6:1 ). Of these, we tagged
227 males and 90 females, and recaptured 62 males (27.3%) and
12 females (13.3%). Individual males remained at large for 1-50
weeks, and females for 1—43 weeks (Fig. 3a). No significant dif-
ferences were found in time at large of males and females (x" =
1.22, df = 5, P > 0.95). Some individuals were recaptured on
multiple occasions (from two to seven), yielding a total of 119
recaptures of males and 26 recaptures of females. Fifteen lobsters
( 10.3% of recaptures) lost their tags, but were taken into account
when estimating the population size by calculating the "average
age" of their tags, based on the average age of the tags of the
individuals concurrently lecaptured (Lozano et al. 1982). Of the
recaptured individuals, 53% were recaptured within a distance of
20 m from their site of release, 45% at distances from 20 to 50 m,
and 2% at distances from 50 to 100 m. Predation-induced mortality
within the traps was high (28%^), accounting for 94 males (69
untagged and 25 tagged) and 33 females (28 untagged and five

282

Negrete-Soto et al.

(a) Panulirus guttatus

1-7

^ , ^ I I "^ I ,

-15 16-23 24-31 32-39 > 40
Total weeks at large

I Males (N = 62) HFemales (N = 21)

(b) Panulirus argus

u> 80 0 -1

^

0)

^^H

+-»

(/)

^ 60,0 -

^H

o

|fl

»^

^^^

S 40.0 -

^^^

D)

^^BSs

TO

S 20 0 -

^K^

vv*

<j

^^K^

i$:

0)

^^■VV

^^

°- 00 -

-^Ki;:!.;:)^

i,M^

1-7

8-15 16-23 24-31 32-39
Total weeks at large

>40

I Males (N = 10) HFemales (N = 14)

Figure 3. Ptrcentage of indhidual lobsters recaptured in different
periods at large (In weeks), (a) Panulirus guttatus, (b) Panulirus argus.

tagged). Predators found in traps included groupers (Epineplwhis
spp.). triggerfish (Batistes vetida). snappers (Liirjiiniis spp.). and
moray eels {Cymiuithorax spp.).

Population Size and Survival

A lower catchability of female P. liiittiilus in traps was reported
by Evans and Lockwood ( 1994) in Bermuda. A differential catch-
ability of males and females in our traps would contradict one of
the principal assumptions of the Fisher-Ford model, namely that all
individuals are equally catchable. Therefore, before applying the
Fisher-Ford model, we explored whether the biased sex ratio of P.
guttatus was (a) the actual sex ratio over the size range of trappable
lobsters, caused by a lower number of females in the largest size
classes due to a differential growth rate of males and females
(Wenner 1972), or (b) an artifact due to a differential catchability
of males and females in traps. To test (a), we plotted the percent-
age of males in each 5-mm size class of the total catch. In species
where males reach significantly greater sizes than females, such as
in palinurids (Morgan 1980), male percentages tend to fluctuate
around 50 in the small size classes, then decrease as adult females
accumulate in one or a few size classes, and further rise as size
increases until approaching 1009f in the largest size classes (Wen-
ner 1972; Herrnkind & Lipcius 1989). However, our curve showed
higher percentages of males in most size classes, indicating that
females were undersampled. and suggesting a differential catch-
ability of males and feinales. This was further confirmed by a test
of difference in catchability between population sub-groups (i.e..
males and females! (Begon 1979), which was highly significant
(X" = 28.756, df = 12, P< 0.001).

The most likely cause for this differential catchability is a dif-
ferent beha\'ior of males and females (Lozano et al. 1982). Ap-
parently, the protracted reproductive season of P. guttatus (Farru-
gio 1976; Sharp et al. 1997: Briones-Fourzan & Contreras-Ortiz
1999) results in females being overall less active than males, as
evidenced by the higher occunence of male P. guttatus in fixed
gear, such as nets and traps compared to samples obtained by
divers, who search actively for lobsters (Table 1 ). Sex ratios, then,
may depend on the sampling method, the location, and the size

TABLE 1.
Panulirus guttatus. Size-ranges and sex-ratios of individuals sampled with different methods in several locations of its geographic range.

Sampling

Sample

Sex-Ratio

Size Range (C

L. mm)

Location

Method

Size

(M:Fl

Males

Females

Source

Florida

Divini;

S94

1.2:1

32-S5

36-7 1

Caillouet et al. (1971)

Florida

Divmg

1477

0.7:1

32-S4

26-71

Chitty (1973)

Florida

Diving

U2

(.).5:l

1 X-75

21-63

Sharp etal. (1997)

Mexico

Diving

212

1.7:1

40-89

44-78

Padilla-Ramos & Briones-Fourzan (1997)

Martinique

Nets

234

2.8:1

33-70

40-60

Farrugio ( 1975)

Martinic|iie

Nets

772

1.7:1

36-66

30-66

Farrugio ( 1976)

Martinique

Nets

1461^

1 .9: 1

37-74

34-68

Marfin (1978)

Bermuda

Traps

\52

10.0:1

64-88

58-74

Sutcliffe (1953)

Bermuda

Traps

919

13.0:1

51-81

49-69

Evans & Lockwood (1994)

Bermuda

Traps

1656

16.0:1

51-83

51-69

Evans etal. (1996)

Jamaica

Traps

114

2.1:1

43-61

4.3-70

Munro{1974)

Mexico

Traps

136

1.9:1

55-82

54-65

Carrasco-Zanini (1985)

Mexico

Traps

631

2.6:1

42-88

45-74

Present paper

CL is carapace length: M is males: F is females

Population Dynamics of Panulirus guttatus in Mexico

283

range of individuals in the samples. Allowing tor a higher propor-
tion of males in the largest size classes, the 1.7:1 sex ratio of the
P. i^itttaiiis sampled by Padilla-Ramos and Briones-Fourzan ( 1997)
from fisher divers in Puerto Morelos over a fishing season (Table
1 ), appears more realistic than ours. Consequently, we applied the
Fisher-Ford model separately to the male and female data, and
obtained separate estimates of population sizes and survival for
each sex (Begon 1979).

The estimated survival rates (4)) were 0.727 for males and
0.867 for females. The two tests for the constancy of <J) showed
that this assumption was not violated in any one period (month) for
either sex, so the monthly x"s were added and further tested for
their significance. For males, the overall result of test 1 was x" =
18.504 (df= 12. P> 0.10) and of test 2 x" = 6.901 (df = 12, /•
> 0.75). For females, the overall result of test 1 was x" = 4.770 (df
= \2.P> 0.95) and of test 2 x" = 13.840 (df = 12. P > 0.25).

Hence, the assumption of a constant survival rate for both males
and females was reasonably supported.

Male population size ranged from 97 to 373 individuals, with
higher values in November 1986. and from April to September
1987 (Table 2a). The size of the female population varied between
69 and 435, with higher values in April, and from June to Novem-
ber 1987 (Table 2b). When adding the male and female population
estimates, higher abundances of adult P. giiltaliis occurred in
April, and from June to November 1987 (Table 3). In contrast to
the more heavily biased sex ratios in our samples, monthly sex
ratios in the population varied from 0.3 to 2.8 males: 1 female, with
an overall sex ratio of 1.3:1 (Table 3).

The monthly population size was converted to density (number
of lobsters ha~') and confronted with the monthly mean size of
lobsters (Table 4). The mean size of P. guttatus was significantly
different in time (F = 2.637, df = 13, 208; P = 0.002), with

TABLE 2.

Panulinis guttatus. Statistics of the Fisher-Ford model for (a) males: and (b) females in the reef patches. Losses include mortality and

emigration: additions include recruitment and immigration.

(a) Males

Number of Males

New Tags
in Period

Male Population

Sampling Period

Caught

Released

Size (N)

Losses

Additions

Oct. 1986

31

28

28

Nov.

42

41

38

219

60

-62.2

Dec.

23

20

13

97

27

43

Jan. 1987

28

19

14

114

31

68

Feb.

46

28

17

Lsn

41

-4.3

Mar.

18

14

9

KI.S

29

218

Apr.

34

23

19

294

80

-47

May

24

14

11

167

46

156

Jun.

33

21

20

278

76

171

Jul.

21

14

12

373

102

-89.5

Aug.

26

15

11

182

50

83

Sep.

18

18

15

215

59

14

Oct.

25

23

19

171

47

-8.8

Nov.

2^

0

0

115

Total

391

278

226

(b) Females

Nui

Tiber of Females

New Tags

Female Population

Sampling Period

Caught

Released

in Period

Size (N)

Losses

Additions

Oct. lysh

8

6

h

Nov.

14

14

14

7S

10

49

Dec.

6

6

6

117

16

-32

Jan. 1987

6

5

5

69

9

20

Feb.

14

11

8

80

11

8

Mar.

10

9

7

77

10

94

Apr.

14

7

7

161

21

-38

May

5

2

2

102

14

45

Jun.

16

10

8

133

18

28

Jul.

7

6

5

143

19

93

Aug.

5

3

3

217

29

-52

Sep.

11

10

8

136

18

60

Oct.

13

12

11

178

24

281

Nov.

9

0

0

435

Total

138

101

90

284

Negrete-Soto et al.

TABLE 3.

Pamiiinis guttatus. Total population size in the reef patches estimated h\ the Fisher-Ford model, and comparison of sex ratios of individuals
sampled in the reef patches «ith those obtained from the model population estimates.

Number of Lobsters Sampled (n)

Population

Size Estimates (N)

Males

Females

Total

Sex Ratio

Males

Females

Total

Sex Ratio

Period

(Hm)

(n,,)

(n.4, + n,.l

(M:F)

(N\,l

(N,.-)

(N„ + Nf)

(.M:Fl

Oct. 1986

.M

9

40

3.4:1

Nov.

46

14

60

3.3:1

219

78

297

2.8:1

Dec.

27

6

33

4.5:1

97

117

214

0.8:1

Jan. 1987

29

6

35

4.8:1

114

69

183

1.7:1

Feb.

51

14

65

3.6:1

150

80

230

1.9:1

Mar.

20

10

30

2.0:1

105

77

182

1.4:1

Apr.

35

14

49

2.5:1

294

161

455

1.8:1

May

25

5

30

5.0: 1

167

102

269

1.6:1

Jun.

33

16

49

2.1:1

278

133

411

2.1:1

Jul.

21

7

28

3.0:1

373

143

516

2.6:1

Aug.

26

6

32

4.3:1

182

217

399

0.8:1

Sep.

19

11

30

1.7:1

215

136

351

1.6:1

Oct.

25

13

38

1.9:1

171

178

349

1.0:1

Nov.

22

9

31

2.4:1

115

435

550

0.3:1

Total

410

140

550

2.9:1

Average

191

148

.?.?9

1.3:1

smaller values from June t(i October 1987 than throughout the
remaining months (Table 4). In this period, some of the highest
population density values also occurred. The overall average popu-
lation density of P. guttatus was 126 indisiduals ha"'.

Reproductive Aspects

We recorded the reproductive stage of 202 female P. guttatus
in the total sample. Of these. 83 (42.19;-) were ovigerous. Oviger-
ous females occurred every month, except in August 1987. hi
general, the occurrence of ovigerous females was higher (39.3-

TABLE 4.

PanuUrus guttatus. Population characteristics over the study period.

Mean carapace lengths were grouped with a Tukey's test for

unequal sample sizes after a repeated-measures ANO\ A. Population

density was derived by dividing the total population size (males +

females) estimated with the Fisher-Ford model by the surface area

of the reef patches (2.5 ha).

Populatitm

Carapace

Den.slt\

Percentage

Sampling!

Sample

I.engtti (mm)

Groups

( Lobsters

of Ovigerous

Period

Size (n)

(Mean ± SD)

of Means

ha-')

Females

Oct. iys6

411

68.5 ± 6.5

c

33,3

Nov.

60

65.1 ±5.8

abc

111)

35.3

Dec.

33

67.1 ±6.4

be

79

42.9

Jan. 1987

35

65.5 ± 6.3

abc

68

54.5

Feb.

66

63.6 ± 8.4

ab

85

52.2

Mar.

30

62.0 ±5.2

ah

67

53.8

Apr.

49

63.1 ±6.8

ah

169

65.4

May

30

61.6±7.9

ah

100

42.9

Jun.

49

60.4 ± 6.8

a

152

39.3

Jul.

28

59.9 ± 6.3

a

191

50,0

Aug.

32

60.6 ± 6.5

a

148

!),()

Sep.

30

61.1 ±4.9

a

130

8.3

Oct.

38

61.2 ±5.7

a

129

33.3

Nov.

31

62.3 ± 5.9

ab

204

27.3

Average

63.1 ± 7.4

125

42.1

65.47r) from December 1986 to July 1987. and kiwer (0-359i-) in
August to November 1987 (Table 4).

Of the 21 recaptured females, three changed from reproductive
stage 3 to 6 in 13. 13, and 21 days, indicating an egg incubation
period of 2-3 weeks. This agrees with Chitty (1973). who esti-
mated the egg-incubation period of P. guttatus in less than 30 days.
Evidence for repetitive breeding occurred in six females, which
had different broods v\hen captured and when recaptured after 25.
31. 46, 97, 101, and 240 days. Repetitive breeding of female P.
guttatus has been reported before (e.g., Chitty 1973; Farrugio
1976: Sharp et al. 1997; Briones-Fourzan & Contreras-Ortiz 1999)
and, in Puerto Morelos. large females (>50 mm CL) breed more
times in a year than small females (<3() mm CL) (Briones-Fourzan
& Contreras-Ortiz 1999). Based on the short incubation period, the
large size (>50 mm CL) of all our recaptured females, and the
repetitive breeding evidence from recaptured females, we conser-
vatively propose an average duration of 90-120 days for a full
breeding cycle in large females, i.e., up to 3— f broods per year.

(irowth

In total, 1 19 recaptures of males (37 uninjured and 62 injured)
and 26 recaptures of females (19 uninjured and 7 injured) were
considered in the growth analyses. Si/e range at capture of tiiales
was 47.8-81.1 mm CL, and of females 53.7-69.0 mm CL. Time
between subsequent captures was 1-24 weeks in males, and 2-34
weeks in females. Only 19 males (10 uninjured, 9 injured) and
three females (1 uninjured, 2 injured) grew betv\een recaptures
(Table 3). In general, itijured individuals had smaller molt incre-
ments than uninjured individuals. Small uninjured males showed
higher growth rates and molt increments than larger males, but
their intermolt periods were similar. The uninjured female had a
growth rate of 0.32 mm CL week"'. Thirteen injured males and
two injured females molted and regenerated between 1 and 5 ap-
pendages in periods of 4 to 1 7 weeks, but showed no increment
inCL.

Population Dynamics of Panulirus guttatus in Mexico

285

TABLE 5.

Panulirus guttatus. Summary of groHth data for males (size range: 47.5-81.0 mm carapace length. CL) and females (size range: 5.^.7-65.3

mm CL) by size class and injury condition at time of release (uninjured: complete individuals: Injured: individuals missing one or more

appendages). For each sex and size class, average growth rate (mm CL week ') was estimated by dividing the increase in CL of recaptured

individuals by the number of weeks at large. Average molt increments in CL were calculated as in Forcucci et al. ( 1994), by plotting each

observation of change in CL against weeks at large and considering only those individuals that underwent one single molt. Interniolt

interval was then calculated by dividing the average molt increment by the average growth rate.

Number

(a) Uninjured

Individuals

That Did Not Grow

That Grev»

Size Class

Time at

Average Intermolt

Average Molt

Av(

erage Growth

(CL, mm)

Recaptured

N

l>arge (weeks)

N

Period (weeks)

Increment (CL, mm)

Rate (

mm CL week"')

Males

<60

11

8

5-18

3

10

4.0

0.40

60-65

21

16

1-17

5

8

2.8

0.37

65-70

14

12

1-14

2

12

2.2

0.19

>70

11

11

1-24

0

Total

57

47

10

Females

<60

10

9

2-18

1

7

2.1

0.32

>60

9

9

2-34

0

Total

14

18

1

Number

(b) Injured Indii

.iduals

That Did Not Crow

That Grew

Size Class

Time at

Average Intermolt

Average Molt

Av

erage Growth

(CL, mm)

Recaptured

N

Large (weeks)

N

Period (weeks)

Increment (CL, mm)

Rate!

mm CL week"')

Males

<60

9

5

1-16

4

10

2.4

0.16

60-65

20

19

1-24

1

4

1.2

0.30

65-70

IS

16

1-21

2

8

1.1

0.14

>70

15

13

1-12

2

10

1.2

0.12

Total

62

53

9

Females

<60

4

3

3-8

1

14

1.6

0.11

>60

3

2

2-7

1

9

1,4

0.17

Total

7

5

1

Panulirus argus

The mean size of P. cirgiis (range: 49.0-123.6 mm CL; mean ±
SD: 69.8 ± 12.7) (see Fig. 2b) was significantly larger than that of
P. i^uttatKs (range: 42.4-87.5 mm CL; mean ± SD; 63.1 + 7.4 mm
CL) (log-transformed data; l = 8.055. df = 914. P < 0.0001).

We caught 120 P. argus in site 1 (including recaptures) and
only 22 in site 2. Overall, there were 48 males (size range; 49.0-
123.6 mm CL) and 72 females (size range; 50.1-98.4 mm CL).
Mean size of males and females was similar (mean ± SD of males;
70.3 ± 14.2 mm CL; of females; 69.5 ± 1 1.6 mm CL; r = 0.362,
df = 138, P = 0.716).

In site I, new individuals of P. argus (excluding recaptures)
accounted for 35 males and 41 females (sex ratio 0.85;l). We
tagged 33 males and 39 females, and recaptured 9 males {2T^/c ) and
14 females (369f). Most recaptures of P. argus were obtained
within 15 weeks; only four individuals remained at large for longer
periods (up to 31 weeks) (see Fig. 3b). No significant differences
in time at large were observed between males and females (x" =
2.92. df = 3. P > 0.25). Distance of recapture varied from 20 to
150 m. Some individuals were recaptured more than once, yielding
a total of 12 recaptures of males and 30 of fernales. Of these, four

males (60.8-75.8 tiini CD and seven females (50.1-78.0 mm CL)
grew. The average molt increment, intermolt period, and growth
rate of males were, respectively, 5.2 mm CL. 8.4 weeks, and 0.67
mm CL week"'; whereas those for females were 4.3 mm CL. 6.5
weeks, and 0.68 mm CL week^'. These data were too scanty to
attempt any further analyses on population size or growth, but
indicate that individuals of P. argus were less abundant, had higher
growth rates, and retiiained in the reef patches for shorter periods
than individuals of P. guttatus. Also, in contrast to P. guttatus.
there was no evidence of reproductive activity of P. argus in the
coral patches; all female P. argus were in reproductive stage I.

DISCUSSION

In the reef patches surveyed in this study, we caught over five
times as many P. guttatus as P. argus. The underlying causes for
these disparate results could be interspecific competition, a differ-
ential catchability in traps, or a real difference in abundance of
both species. Competition is not likely to occur, because P. argus
and P. guttatu.s do not seem to compete for food or shelter re-
sources (Sharp et al. 1997; Lozano-Alvaiez & Briones-Fourzan
2001 ). Although differences in catchability of both species cannot

286

Negrete-Soto et al.

be discarded, we believe that our results reflect a true scarcity of
subadult P. argu.s recruiting to the reef habitat. In Puerto Morelos.
densities of algal juveniles of P. urtiHs in the shallow vegetated
reef lagoon are high (146-263 individuals ha"'. Briones-Fourzan
& Lozano-Alvarez 2001a). but the density of the subsequent pos-
talgal juveniles is drastically reduced (0-31 individuals ha"'), ow-
ing to a lack of crevice-type shelters throughout the reef lagoon
(Briones-Fourzan & Lozano-Alvarez 2001b). This would further
result in low numbers of subadult P. argiis moving to the reef
habitat. Over 80% of the individuals of P. argiis in our sample
were subadults (<80 mm CL) as opposed to the individuals of P.
guttatus, which were all adults. In addition, each species appears to
make a differential use of the reef habitat: subadult P. argus shelter
in the reefs during the day. but forage at night in rubble areas or
seagrass meadows adjacent to the reefs (Cox et al. 1997). whereas
individuals of P. giitlnliis forage on the reef itself (Sharp el al.
1997) and never abandon this habitat completely.

Our estimates on population sizes and survival rates of P. gut-
tatus were similar to those estimated in the Florida Keys (Sharp et
al. 1997). but. although we did acknowledge the tag loss, our
estimates may be biased owing to an unquantified tag-induced
mortality. When analyzing the effects of the Australian tag on
juvenile P. cygiuis (40-75 mm CL). Chittleborough (1974) esti-
mated an overall ""tag loss" of 23% over 39 weeks, but he could not
separate tag-induced mortality from tag loss. In addition, although
the size range of Chittleborough" s P. cygnus and our P. guttatus
was similar. Chittleborough's tags had considerably larger toggles
( 14 mm long) than ours (9 mm). Mortality of Hauuirus aiueiicauus
(43.5-97.5 mm CL) induced by sphyrion tags (similar to the Aus-
tralian tags but with a steel anchor instead of a plastic toggle) was
significantly lower (s 5%) in lobsters tagged during intermolt or
premolt than in those tagged during postmolt (soft-shelled) (Mori-
yasu et al. 1995). a precaution we observed in our study. Lozano-
Alvarez (1992), using exactly the same type of tag as ours on a
population of juvenile and young adults of P. argus (25-90 mm
CL). estimated a tag-induced mortality of 5%. If a similar tag-
induced mortality occurred shortly after tagging in our individual
P. guttatus. it might have not affected the estimates of survival, but
it may have contributed to an underestimation of the population
size (Begon 1979, Moriyasu et al. 1995).

Therefore, our population size values are probably underesti-
mates. However, the estimates of survival rates for males (0.73)
and females (0.87), and the average density of adult P. guttatus in
our coral patches ( 1 26 lobsters ha" ' ), were comparable to the mean
densities of 1 18 and 1 15 adult P. guttatus ha"' estimated by Sharp
et al. (1997) in two patch reef areas in a lobster sanctuary in
Florida, based on nighttime samplings by divers, with overall sur-
vival rates of 0.87 and 0.67 in each area. In contrast, in reef-crest
areas of Bermuda, where a specific fishery for P. guttatus exists,
Evans and Lockwood (1994) obtained a mean density of 29 ± 7.6
trappable P. guttatus ha"', but with an overall niale:female ratio
of 7: 1 .

The largest population sizes of adult P. guttatus in our study
site were obtained in June to November. Within this period, the
lowest percentages of ovigerous females and the smallest mean
size of lobsters afso occurred, suggesting a recruitment of small
adults to the trappable population during the summer and early
autumn. The juveniles of P. guttatus presumably live in the same
habitat as adults (Shaip et al. 1997), but we have never observed
individuals <34 mm CL during daytime divings in these reefs.
However, the coral reef is a very intricate habitat, and the small

phases of P. guttatus may hide deep within small recesses and
crevices during the day, warranting future extensive surveys by
nighttime divings to reveal their occurrence and their contribution
to the whole population size.

Because P. guttatus is a rather sedentary species, we hypoth-
esized that the population additions would be mostly ascribed to
the recruitment of small adults. However, we cannot rule out en-
tirely the contribution to the population additions and losses of
movements of males and females throughout the reef habitat, and
from and to our limited study area. Most of our individual P.
guttatus were recaptured from distances <50 m, and our reef
patches were relatively separated from adjacent reef habitats, but
low-relief hard grounds between these and other patches may have
been traversed by some individuals. In displacement experiments
with tagged adult P. guttatus. Can'asco-Zanini ( 19<S5) and Lozano-
Alvarez et al. (unpubl. data) estimated the home range of adult P.
guttatus to lie within a 100 m radius along the reef tract, but some
of their individuals were able to return to their initial shelters from
up to 200 m. Evans and Lockwood ( 1994) documented an autum-
nal offshore migration, associated with reproductive activities, of
females and young males of P. guttatus in an extensive reef habitat
in Bermuda. However, the fore-reef habitat in our study area is
neither as extensive nor as developed as in other coral reef areas
(Ruiz-Renten'a et al. 1998). so offshore migrations of P. guttatus
are not likely to occur.

Sharp et al. (1997) proposed that the availability of suitable
shelters in the reef habitat may be the primary factor controlling
the abundance of P. guttatus. In our study patches, in addition to
P. guttatus and P. argus. many other crevice-dwelling species
(e.g., Mithrax spmosissimus. moray eels, groupers, triggerfish, oc-
topuses, etc.) occur. However, some of these species are also po-
tential lobster predators, as well as other species associated to the
reef habitat. Predation-induced mortality was high within our
traps, where confined lobsters were unable to escape predators, but
the true magnitude of this type of mortality in the natural reef, to
which we ascribe most of the population losses, will remain un-
determined until specific studies on predator-prey dynamics are
conducted in these reefs.

Protracted reproductive periods, indicative of repetitive breed-
ings, are common in tropical palinurid species (Quackenbush
1994). In the coral reefs of Puerto Morelos. Briones-Fourzan &
Contreras-Ortiz (1999) found that female P. guttatus <50 mm CL
breed mostly during the winter and spring, whereas females >50
mm CL breed during the four seasons. These authors estimated the
index of reproductive potential of P. guttatus by conservatively
assuming one brood per year for all size classes. But if females >50
mm CL can breed three to four times per year as suggested by our
results, their contribution to the overall egg production may be
much higher than that estimated by Briones-Fourzan & Contreras-
Ortiz (1999).

An effect of repetitive breeding on females is a decrease in their
growth rales, due to a reduction in the molt increments and an
increase in the intermolt periods (Hunt & Lyons 1986). but our
data on female growth were insufficient to explore this assump-
tion. Injuries have no clear effect on growth in some lobster spe-
cies (Davis 1986; Forcucci et al. 1994). but in others injuries
reduce molt increments (Brown & Caputi 1986). In our study,
those injured P. guttatus that grew showed, in general, lower molt
increments than uninjured individuals. Moreover, other injured
individuals molted, as evidenced by their regenerated appendages,
but without any increase in CL. Injured lobsters were mostly

Population Dynamics of Panulirus guttatus in Mexico

287

males, and the occurrence of detached appendages in our traps
suggests that some were injured while in the traps, perhaps through
aggressive interactions (Lozano-Alvarez & Briones-Fourzan 200 1 )
or when trying to deter the predators that entered the traps. The real
incidence of injuries among the population is undetermined.

Our male growth data, although scant and inconclusive, suggest
that growth rates of male P. guticitiis decrease as size increases, but
few males grew during their time at large. In Martinique, most P.
guttatus molt at least twice a year (Marfin 1978). whereas the
average intermolt periods of our 10 uninjured males that grew (8-
12 weeks) suggest the possibility of four to six molts per year.
Applying the average growth values of uninjured males in Table .'i.
it would take a 48 mm CL male about 2.5 years to reach 81) mm
CL. However, many individuals did not molt during similar or
longer times at large (up to 24 weeks), and if this portion of males
is also taken into account in the growth estimates, the results
suggest that uninjured adult male P. guttatus may molt at least 2-3
times per year. The average molt increment of males over the
entire size range of recaptured individuals was 2.8 mm CL. With
these values, a 48- mm CL male P. guttatus would grow to 80 mm
CL in about four years. More data are needed to improve these
results, which nevertheless indicate that P. guttatus has lower
growth rates than P. argus of similar sizes, because it would only

take one year for a male P. argus to grow from 48-80 mm (Lo-
zano-Alvarez et al. 1991b).

Most of the recaptures of P. argus were obtained within 15
weeks, suggesting that these patch reefs are a transitory habitat for
these subadults, which would presumably emigrate further on to
deeper offshore areas where the large adults dwell (Lozano-
Alvarez et al 1991a). In contrast, some individuals of P. guttatus
were recaptured over periods up to 50 weeks, suggesting that these
patch reefs are a more permanent site of residence for P. guttatus.

Our results show that the coral reef patches at Puerto Morelos
support a sizeable population of P. guttatus and are also an im-
portant habitat to the subadults of P. argus. In the future, more
refined studies focused on the population dynamics of P. guttatus
should include sampling by nighttime diving, a direct estimation of
tag-induced mortality, tagging in a broader area, and increasing the
sampling effort to obtain a larger set on grovMh data.

ACKNOWLEDGMENTS

We thank Felix Colinas, Jorge Siiiionin, Martha Fonseca,
David Gutierrez, and Gabriela Contreras for their help in field
and/or laboratory activities. This study was funded by Universidad
Nacional Autonoma de Mexico.

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RESPONSES OF MIGRATING WESTERN ROCK LOBSTERS PANULIRVS CYGNUS (GEORGE,
1962) TO TWO DIFFERENT TAGGING RELEASE PROCEDURES

ROY MELVILLE-SMITH' AND YUK WING CHENG^

^Western Australian Marine Research Laboratories, P.O. Box 20. North Beach. Western Australia.
Australia 6020: 'Western Australia Centre of E.xcellence in Industrial Optimisation. Curtin Universirs' of
Technology. GPO Box U1987 Perth. Western Australia. Australia 6845

ABSTRACT In the 1997/1998 season. 3.412 migrating 'white' sub-legal sized western rock lobsters were tagged at Jurien Bay
(42—19 m depth), and Cervantes (92-133 m depth) on the Western Australian coast. Most lobsters in = 2.245) were brought ashore
to be tagged, held overnight in coffs. and released 1 8-24 hours after capture within 6 km and + 1 0 m depth froiT) where they were taken.
A smaller number (n = 1,167) were tagged and released at sea irnmediately after capture. Over three seasons, more animals tagged
at sea were recaptured than those tagged ashore (P < 0.01). At both sites, lobsters tagged ashore moved significantly further and faster
than those tagged at sea {P < 0.01 ). The correlation between the angle of movement and the speed of movement ranged from 0.25 to
0.42 {P < 0.05 ) for the two tag-and-release procedures and two sites. However, all the lobsters moved in a northerly to north-westerly
direction. Growth increments in the first moult after release were unaffected by the different tag and release procedures (P = 0.08);
loss of a single limb was. however, likely to result in a 229c decrease in the growth increment.

KEY WORDS: Pamilinis cygiuis, tagging, movement, mortality, growth, migration

INTRODUCTION

Over the last three decades, tens of thousands of western rock
lobsters {Paniilinis cxi^niis. George, 1962) ha\e been tagged to
estimate movement, growth, natural mortality and discard mortal-
ity. Tag and relea.se programs are generally expensive, as they
usually require deploying research vessels, chartering commercial
fishing vessels, or purchasing part of a cominercial catch. Often
only selected size classes are required for tagging, therefore it
would be more economical for commercial fishers to retain indi-
viduals with the required characteristics and in this way accumu-
late large numbers of the animals being targeted for tagging.

However, accumulating animals for later tagging would inevi-
tably mean displacing theni from their area of capture and previous
re,search suggests that this can affect subsequent growth (Brown &
Caputi 1983, 1984), recapture rates (Chittleborough 1974: Brown
& Caputi 1983) and movenient patterns (Chittleborough 1974) and
may lead to nomadism (Hennkind 1980).

The object of this investigation was to compare the results of
two tag-and-release procedures: one where migrating lobsters were
released to the waters where they were caught straight after cap-
ture: and the second to bring the lobsters ashore to be tagged, and
return them to roughly the saine area the next day. In both cases
recapture rates, distance speed, direction of movement and growth
increments have been compared for the two release procedures.

The lobsters tagged in this study were all migrating animals,
known colloquially as 'whites' because they are paler than the dark
red resident animals. For most of their lives, adult western rock
lobsters show only limited movement: only during the white phase
when the lobsters are 4—5 years old, do they move for any distance,
migrating from the shallow inshore coastal reefs to the offshore
breeding grounds (Morgan 1977: Phillips 1983). The migration
takes place each year between late November and January. Tag-
ging data have shown that, while most migrating animals move
directly offshore, a significant number make more extensive long-

Corresponding author: Roy Melville-Smith. Western Australian Marine
Research Laboratories. P.O. Box 20. North Beach, Western Australia, Aus-
tralia 6020. E-mail: rmsniith@fish.wa.gov.au

shore migrations, generally in a north-westerly direction (Phillips
1983: Cheng & Chubb 1998).

METHODS

Sampling

A total of 3,412 migrating sub-legal size ""white" lobsters in the
size range 65 mm to 77.9 mm carapace length were tagged and
released west of Jurien Bay between 29 December 1997 and 4
January 1998 in 43— +9 in and south-west of Cervantes between 29
December 1997 and 6 Januar> 1998 in 92-133 m depth (Fig. 1.
Table I ). The depths at Jurien Bay and Cervantes were chosen
because they represented two different stages of the offshore mi-
gration, the animals in 43^9 m range being in the process of
moving to deeper depths offshore and those at the Cervantes site
being at the offshore limit of the migration run.

All of the lobsters used for tagging were caught during the
course of commercial tlshing. The experimental lobsters (Cer-
vantes. ;) = 1.400 and Jurien Bay. /; = 845) were kept alive in
tanks with flow-through water circulatory .systems and brought
ashore to be tagged. After tagging, they were kept overnight in
plastic fishing baskets tied to the side of the fishing boats. The next
day. they were placed in tanks, taken to the fishing grounds and
released { 18 to 24 hours after capture) together in a single batch on
suitable substrate within 6 km and ±10 m depth from the respective
sites where they were caught (Table 1 ). GPS details of their release
positions were recorded. The control lobsters (/; = 1, 167) were
tagged and released at sea within 12 minutes of capture and within
500 m of where they were caught (Table 1 ).

All lobsters were tagged ventrally with individually numbered
Hallprint type TEA- 1 internal anchor tags, as described by
Melville-Smith and Chubb (1997). The tag number, size, sex.
number of appendages missing, dates of capture and release, and
position and depth of capture and release were recorded for each
tagged lobster. Tagging at Cervantes and Jurien Bay was done by
different people, both experienced at tagging lobsters: at each site
the same person tagged at sea and ashore.

Tagged lobsters were generally recaptured by commercial fish-
ers, although small numbers were caught by research and recre-

290

Melville-Smith and Cheng

30°00'S

30°20'S

30°40'S

31°00'S

INDIAN
OCEAN

114''30'E 115"0U'h I15°20'E

Figure 1. The Western Australian coast, showing where tagged lob-
sters were released in this study.

ationul fishers. All tag recaptures were made during the fishing
season, which extends from mid-November to 30 June. Fishers
were encouraged to provide details of tagged lobsters by offers of
a AUD$2 instant lottery ticket for information relating to any
tagged animal recaptured. They were asked to record size sex.
number of limbs lost. date, position and depth of capture.

by their times (one or two and three .seasons) at large and any
recaptured within M) days of release were excluded from analyses
calculating the mean time at large and distance from release sites,
to avoid biasing the results by including migrating individuals. The
second and third seasons were combined instead of being treated
separately because published and unpublished data show that once
western rock lobsters have undertaken their migration and have
settled on the deep water spawning grounds, that thereafter their
movements are limited. Combining the second and third years
increased the power of the statistical tests.

Recaptured animals were analyzed for defined time periods at
large by student-r tests to estimate the power of the test. General
linear modeling was used to model the size of the first growth
mcrement after release, with distance moved and loss of limbs
treated as covariates; and sex. site of release and release procedure
as factors.

The uniformity of the directional movement of lobsters tagged
at sea and ashore was examined by a Rayleigh test (Zar I99S) and
the mean angle of movement of the animals tagged by the two
procedures relative to 0° (i.e., True North), was tested by a Wat-
son-Williams test (Zar 1998). Angular-linear correlations for lob-
sters tagged at sea and ashore were determined by the method
described in Mardia (1976) and Johnson and Wehrly (1977). Re-
gression methods were used to establish the relationship of the
angular movement to the maximum speed of movement.

RESULTS

Recapture Rate

The mean percentage of tagged rock lobsters that were recap-
tured from the two release sites and tagging procedures ranged
from 7.4% to 12.6% (Table 1).

A generalized linear model showed that recapture rates were
unrelated to the two release locations (P = 0.38). but that they
were significantly correlated with the two tagging procedures (P
= 0.03). A binomial test on two proportions (Zar 1998) produced
a similar outcome. The overall recapture rate of lobsters tagged at
sea was about 4Vr hiaher than those that were tagged ashore.

Analysis

The percentage of tag recaptures to numbers tagged were com-
pared for the two tagging sites and their release sites by a gener-
alized linear model with binomial family (logit link). The mean
time at large, distance from release sites and speed of movement
were calculated. The last two were based on the shortest distance
between the point of release and recapture. Animals were grouped

Time at Large

The time at large before recapture can influence comparisons of
different tag-release procedures. For example, one group of lob-
sters may move further than another, not because of the experi-
mental procedure, but because they were at large for longer. In the
first season (to the end of June 1998) after release, animals that
were tagged ashore were recaptured significantly later (P < 0.03:

TABI.K 1.

Western rock lobsters tagged at sea and on shore at Cervantes and .lurien Bay on respectively 29 December 1997 to 6 January 1998 and 29

December 1997 to 4 January I99S, and recaptured by .V) June 20(11.

Tagging

Procedure

Ashore

At Sea

Release Sites and Depths

Release

(n)

1 400

S45

224.5

Recaptured

(n)

Percentage
Recaptured

Released

(n)

Recaptured

(nl

Percentage
Recaptured

Cervantes (92-133 m)
Jurien Bay (43-49 in)
Total

103
79

IS2

7.36
9.35
S.ll

564
603

1167

71
72
143

i2.5y

1 1 .94
12.25

Migrating Lobsters and Tag Release Procedures

291

TABLE 2.

Days at large (Days) of western rock lobsters released at Cervantes and Jurien Bay in 1997/98 after tagging either at sea (h = 11671 or
ashore (;; = 22451. Results are for the sexes combined, recaptured by the end of the first, and second and third fishing seasons at large.

Days Before Recapture

End of First
Season (30 < days <210 After Release)

Second and Third Seasons
(21)) < days <(210 + 730) Days After Release)

Days (mean)

SE

P Value

Days (mean)

SE

P-value

Tagged at sea
Tagged on shore

.SI
?()

103.12
I 11.70

2.41
2.96

0.0.^

72
60

414
4.^0,4

9.14
y.24

().3S

conihined sexes) than those that wefe tagged at sea (Table 2).
However, in the second and third seasons at large, there was no
significant difference between the two groups (P = 0.38: com-
bined sexes) (Table 2). Although the data have not been presented
here, neither the release sites nor the sex of the lobsters produced
different results.

Distance Moved

Lobsters that were tagged ashore inoved greater distances on
the whole than those tagged and released at sea. This difference
was significant (P < 0.01) for recaptures at both Cervantes and
Jurien Bay made by the end of the first season (Fig. 2). The trend
remained clear when only recaptures made in the second and third
seasons were considered, but was only statistically significant for
the Cervantes data (Fig. 2). The substantial differences in the dis-
placement of recaptured lobsters at the two sites and depths have
precluded the data being combined in analyses comparing their
movement.

Depending on whether they had been tagged at sea or ashore,
lobsters that were tagged at Cervantes and that were at large for
two or three seasons had moved about the same distances as those
that had been at large for only one season (means ranging from
38.23-45.53 km for those released ashore, compared to 8.98-17.01
km for those released at sea. Fig. 2). This did not apply to lobsters
released at Jurien Bay, where the one-season lobsters had moved
shorter distances than the two or more season lobsters (means of
10.87 and 5.03 km for those released ashore and at sea respectively
by the end of the first season, compared to 30.06 km and 15.78 km
for those released ashore and at sea respectively at the end of the
combined second and third seasons at large. Fig. 2). However, the
proportional differences moved by the at-sea and ashore release
groups at Jurien Bay was similar for those at large for one com-
pared to two or more seasons, in all cases those tagged ashore
moving two to three times further than those released at sea.

Speed

As there is little movement by western rock lobsters after their
"whites" migration phase which ends by late January, only data for
recaptures made from 1 February to 30 June 1998 (i.e., within the
first season after release) have been presented in Table 3. Bearing
in mind the differences in distance moved between lobsters tagged
at sea and ashore (Fig. 2), it is not surprising that the two groups
showed significant differences in their speeds of movement at both
tagging locations (Table 3).

Direction of Movement

Compaiisons using a Rayleigh test of uniformity of movement
showed that the lobsters did not migrate uniformly in one direc-

tion, regardless of whether they were tagged at sea or ashore. A
Watson test to compare the mean angles of movement from the
two treatments showed no significant difference for either those
animals released in the intermediate depths at Jurien Bay (P =
0.2), or those released in the deep water at Cervantes (P = 0.6). In
both cases the mean direction of movement was north to nor-nor-
westerly.

Relationships Between Direction, Distance and Speed of Movement

Angular-linear conelations (Mardia 1976; Johnson & Wehrly
1977) used to determine whether distance or speed of nio\ement is
correlated with directi(.)n of movement, were determined separately
for the two tagging sites and for the animals released at sea and
ashore. Some, but not all, of the correlations comparing angle of
movement to distance moved were significant (Table 4); however,
angle of movement was significantly correlated (P < 0.05) with
speed of movement in all cases.

A regression analysis comparing speed and angle of movement
showed that, while the general direction moved was northerly,
recaptured lobsters that had been tagged ashore had a more west-
erly component to their movement. Since the correlation between
angle and speed of movement was higher for lobsters tagged
ashore (0.34, 0.42) than at sea (0.25, 0.33). this indicates that
migrating western rock lobsters moved fastest when heading in a
north-westerly direction.

Growth Increment

The results from a general linear model of the growth incre-
ments of lobsters that were likely to have moulted once between
release and recapture (i.e.. only animals recaptured between Feb-
ruary 1998 and July 1998) are shown in Table 5. Distance moved
is treated as a covariate (P = 0.15). and sex (P = 0.46) and

TABLE 3.

Mean speed of movement of recaptured yvestern rock lobsters

tagged at Cervantes and Jurien Bay in December 1997 and January

1998 and recaptured between 1 February and 30 June 1998 (i.e.,

within the first season after release).

Release

Site and

Tagging

Recaptu

red

Speed

Depth (m)

Procedure

(H)

(m/day)

S.E.

P = value

Cervantes

Ashore

27

392. 1

55.64

92-133 m

At sea

26

142.1

30.35

<0.0I

Jurien Bay

Ashore

23

103.7

28.18

43^9 m

At sea

s5

-S2..S2

4.50

0.01

292

Melville-Smith and Cheng

p<0 01

(a)

p<0 01

n=23

n=55

Cervantes Cervantes
Ashore At-sea

Jurien Bay Jurien Bay
Ashore At-sea

50 1

J p<001

40

1

30

20

n=30

n=64

10
0

Cervantes Cervante

Ash

ore

At-sea

(b)

p=0 32

n=30

i

Jurien Bay Jurien Bay
Ashore At-sea

Figure 2. Mean distance western rock lobsters moved after tagging at
Cervantes and Jurien Bay in December I**"}? and January 1998 (a) by
the end of the first season excluding the first month after release and
(b) in the sec<md and third seasons.

release procedure (f = 0.08) as factors; none had a significant
effect on growth increment. However, region of release (P = 0.03)
and the number of limbs lost at tagging (P = 0.01) did have a
significant effect on growth increment. As only three recaptured
lobsters had more than two lost limbs at tagging, nothing can be
inferred about the effect of multiple limb loss on growth. However,
it can be seen (Table 5), that the loss of a single limb is likely to
result in a 22'7r decrease in growth increment.

DISCUSSION

The lower recapture rates of lobsters tagged ashore and re-
leased a day later, suggest that their mortality rates were higher
than lobsters tagged and released at sea within 12 minutes of
capture. Brown and Caputi (1983) noted that the length of time
between capture and release had a detrimental effect on survival
and growth of sub-legal size lobsters. However, in the present
study every effort was made to limit the time of exposure out of
water. After capture, the animals were stored on board in baskets
submerged in dark, aerated wells in the hull as is commercial
product. Once ashore, the lobsters were tagged immediately and
then kept overnight in plastic baskets hung over the side of the
boats in well-aerated surface water. For the return trip to the fish-
ing grounds, they were stored in the same way as after capture.

Displacement of western rock lobsters from their home range
probably increases their vulnerability to predation (Chittleborough
1974; Brown & Caputi 1983). Brown and Caputi (1983) further
concluded that "when lobsters are migrating they are somehow
affected by displacement more than they are affected by it in their
non-migratory phase (p. 125)." When western rock lobsters make
their offshore migration, they are thought to move in groups or

"packs", rather than singularly (Gray 1992). There is some evi-
dence of a survival benefit in groups, rather than single lobsters
moving over the sand (Herrnkind 1969; Kelly et al. 1999). Dis-
placing migrating lobsters from their groups by bringing them
inshore to be tagged may. therefore, have increased their vulner-
ability to predation. at least until they could rejoin another group
of migrating animals.

Even though a higher percentage of lobsters tagged ashore were
recaptuied significantly sooner (within the first season) than those
tagged at sea. they had moved significantly further from their
release site than had those tagged at sea. This group of shore-
tagged lobsters had been at liberty for a shorter time, and therefore
had less time in which to migrate. There is evidence that Panulinis
arsons and P. cyi^iiiis. can retum to their resident homesite if dis-
placed over a few hundred metres (P. i vijiik.v; Chittleborough
1974. and P. artiKs: Herrnkind et al. 1975). Chittleborough (1974)
and Herrnkind et al. ( 197.5) both showed that displacing animals
from their home range or a familiar region results in abnormal
patterns of movement. Although he had no data on relocating
migrating lobsters. Herrnkind ( 1980) speculated that moving them
away from their characteristic migratory pathway could conceiv-
ably cause disorientation and wandering, which would distort tag-
recapture studies.

The present study does not confirm Hen'nkind's (1980) expec-
tations: although the animals tagged ashore, that had both a de-
layed and displaced release, migrated further, their mean direction
of movement was the same as that of the lobsters tagged at sea.
This observation does not support the suggestion they wandered in
a disorientated fashion. Furthermore, rock lobsters have been
found to use magnetic fields to orient themselves (Lohmann et al.
1995). It seems that, despite the unusual treatment of most of the
animals in this study, they retained their directional orientation
during their time in captivity. The question as to why shore-tagged
lobsters travelled further remains unresolved.

Previous western rock lobster tagging programs have shown
that while most lobsters migrate less than 10 km from their point
of release, many migrate up to several hundred kilometers, almost
always in a north-westerly direction (Gray 1992). Furthermore.
Cheng and Chubb (1998) report that migrating western rock lob-
sters tagged in deepwater move longer distances than those in
shallow water. It is commonly accepted therefore, that most
"white" migrating lobsters become resident in 40-90 m depths
west or northwest of the shallows from where they started their
migration, but that a minority that reaches deep water (>90 m)
follows the contours northward, before peeling off inshore along
the way to settle on the breeding grounds in depths of around
40-90 m.

The distance and direction moved by animals tagged in the
depths reported in this study are consistent with the scenario sug-
gested above. Those tagged in the middle depths (43—17 m) at
Jurien Bay generally moved shorter distances than those in the
deepwater (>90 m) at Cervantes. These middle depths (40-90 m)
is where the resident breeding stock population occurs. Interest-
ingly, lobsters at large for one season at Cervantes, whether tagged
at sea or ashore, moved roughly the same distances as those at
large for two or three seasons. In contrast, at Jurien Bay. lobsters
recaptured in the second and third seasons had moved further than
those recaptured in the first season. However, animals tagged at
sea and ashore both moved greater distances in the second than in
the first. The lack of difference in distances moved once the white
migrating lobsters become resident (within the first season) sug-

Migrating Lobsters and Tag Release Procedures

293

TABLK 4.

Correlations between displacement and angle of moement. and speed and angle of movement of western rock lobsters tagged either ashore

or at sea and released at one of two different sites.

Correlation Between

Correlation

Release

Tagging

Distance Moved

and

Between Speed

and

Location

Procedure

Tagged ashore

Angle

of Movement

P-value

Angle of Movement

P-valuc

Cervantes

0.18

0.3-'i

0.34

0.03

92-133 m

Tageed at sea

0.31

0.01

0.2.^;

0.05

Jurien Bay

Taased ashore

0.33

0.04

0.42

0.01

43-49 ni

Tagged at sea

0.27

0.10

0.33

0.03

gests that the tagaing-release procedure affects distance only in the
first season.

The differences between the distance travelled by displaced
lobsters (shore tagged) and those released where they were cap-
tured suggests that movement data from animals tagged during
their migration phase be interpreted cautiously. The spatial and
temporal displacement resulting from bringing the animals ashore
to be tagged and releasing them at a distance from their catch site
clearly has an effect. Smaller displacements, such as result from
the drifting of tagging platforms or being held overnight in a pot
when captured, might also affect the movement of lobsters tagged
at sea dunng their migratory phase. Results from this study would
therefore suggest that the effects of release procedures on the
behavior of migrating lobsters, must be considered before biologi-
cal conclusions are drawn.

No oceanographic data were collected, either at the tagging
locations, or at the anchorages where the animals were held over-
night. However, substantial amounts of environmental data have
been collected on the western rock lobster grounds in the past, and
it can be confidently assumed that differences in the salinity, tem-
perature and oxygen concentrations in the coastal waters w here the
lobsters were held, compared to the offshore waters where they
were caught, would have been well within the seasonal ranges
experienced by the species. This does not preclude the possibility
that the overnight acclimation of the animals to the changes in
water conditions may have contributed to the different behavior of
the at-sea and ashore tagged animals.

The mean speeds of movement recorded by the inigrating ani-
mals tagged in this study are substantially slower than those (1.2
km per day) recorded by Cheng and Chubb (1998). Our speeds
have been underestimated because all recaptures over the first
season at large were used in the calculation, but western rock
lobsters migrate only between late November and late January.
The strong correlation between angle of movement, and both dis-

TABLE 5.

Results of a general linear model of growth increments of lobsters
between February 1998 and July 1998 (one moult increment).

Name of Variable

Estimated

P-value

Intercept

4.28

0.00

Distance moved

0.00

0.15

Released procedure

-0.4.^

0.08

Released location

0.61

0.03

Sex

-0.19

0.46

Single limb loss at tagging

-0.94

0.01

tance traveled and speed of movement, is a reflection of the lob-
sters that moved furthest (and therefore recorded the fastest speed
of movement while at large) generally heading in the northwest-
erly direction almost parallel to the coastline, whereas those that
moved least (and therefore recorded the slowest speed of move-
ment while at large) generally heading in a northerly direction.

Single growth increments of lobsters at the end of the first
season (after their first moult) did not suggest that migrating ani-
mals tagged at sea grew either faster or slower than those held
ashore overnight and displaced from their point of capture (P >
0.05). This result differed from that of Brown and Caputi (1984).
who found that displaced undersize western rock lobsters of a
larger size range than those used in this study, had significantly {P
> 0.05) smaller growth increments than nondisplaced animals. Al-
though the animals in this study would presumably have been
stressed immediately after capture, during tagging and possibly
while being held overnight, these affects have previously been
shown (Melville-Smith et al. 1997) to be short-term and unlikely
to affect the moult increment later in the season. The significant
diffeience (P = 0.03) in growth increment between the two tag-
ging sites is likely to be due to regional differences in growth,
which are well documented in this fishery (Chittleborough 1976;
Joll & Phillips 1984).

This study investigated the effects of different handling meth-
ods and. on the basis of the results, drew some conclusions about
the scope for possible cost reduction in tagging programs by hav-
ing fishers accumulate selected size-classes of migratory lobsters
for tagging ashore. The result has shown that this approach would
not be feasible for studying movement patterns or for purposes
where recapture rates are critical to the result, such as estimating
mortality and/or population sizes. However, growth increments
were not significantly affected; if growth measurements were the
only requirement from the data, then this cooperative approach
between research and industry could result in affordable and reli-
able tagging projects. Comparative cost savings of tagging the
cumulative catch of a number of fishers ashore, as opposed to a
single fisher at sea is not possible, because the result is dependent
on catch rates of the animals being selected for tagging and on the
number of fishers accumulating that part of their catch.

ACKNOWLEDGMENTS

We thank Kim Brooks and Doug McCashney for tagging the
lobsters in this study, the skippers and their crews for their help
and the many fishers and rock lobster processors who returned the
recapture details of tagged lobsters. Sonia Anderton ran the tag-
ging data base, Mark Rossbach and Jim Christianopoulos did much
of the field organization of staff and fishers. Lynda Bellchambers.

294

Mklville-Smith and Cheng

Nick Caputi. Vivienne Mawson and Bruce Phillips otTered Liselul
suggestions lor improving earlier drafts of this manuscript. The

work was part funded by the Fisheries Research and Development
Corporation (Project 96/108).

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size western rock lobster Pamtlinis cygnus George returned by fisher-
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western rock lobster. Painilinis cyginis George, returned by fishermen
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Cheng. Y. W. & C. F. Chubb. 1998. Statistical analysis of the directional
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Chiuleborough. R. G. 1974. Home range, hommg and dominance m juve-
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Lohmann. K. J.. N. D. Pentchetf. G. A. Neviit. G. D, Stetten. R. K.

Zimmer-Faust. H. E. Jarrard & L. C. Boles. 1995. Magnetic orientation

of spiny lobsters in the ocean: experiments with undersea coil systems.

J. Exp. Biol. 198:2041-2048.
Mardia. K. V. 1976. Linear-angular correlation coefficients and rhythmom-

etry. Bionielrika 63:403—105.
Melville-Smith. R. & C. F. Chubb. 1997. Comparison of dorsal and ventral

tag retention in western rock lobsters. Paimlirus cygnus (George). Mar.

Fre.slmar. Res. 48:577-580.
Melville-Smith. R.. J. B. Jones & R. S. Brown. 1997. Biological tags as

moult indicators m Panulirus cvgnus (George). Mar. Freshwar. Res.

48:959-965.
Morgan. G. R. 1977. Aspects of the population dynamics of the western

rock lobster and their role in management. PhD. Thesis. University of

Western Australia. Nedlands. Western Australia. 341 pp.
Phillips, B. F. 1983. Migrations of pre-adult western rock lobsters. Panu-
lirus cygnus. in Western Australia. Mar. Biol. 76:311-318.
Zar. J. H. 1998. Bioslatistical analysis. Upper Saddle River. NJ: Prentice

Hall. 663 pp.

Journal ,>f Shfllfish Research. Vol. 21. No. I. 295-298, 2002.

MUSHROOM TYROSINASE AS A CONTROL MATERIAL FOR PHENOLOXIDASE ASSAYS
USED IN THE ASSESSMENT OF CRUSTACEAN 'HEALTH *

A. BATTISON.' R. CAWTHORN," B. HORNEY,' AND A. MACKENZIE^

^Departiiwnt of Pathology ciiul Microhiology, Athiiitic Veterinary College, University of Prime Edward
Island. Charlottetown, Prinee Edward Island. Canada: 'Atlantie Veterinary College Lobster Seienee
Centre. Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward
Island. Canada

ABSTRACT Mushroom tyrosina.se (E.C. 1.14.18.1 1 was evaluated as a control material for use in a plasma prophenolo.\idase assay
in the American lobster Homanis amerkanits as pan of the assay's validation procedure. Reconstituted, lyophilized tyrosinase solution
was stable for 72 h at 2— +°C. Percent recoveries were 94%. 120%, 113%, and 48% for "mid-range"" activity dilutions and 93%, 94%.
87%. and 38% for "low-range'" activity dilutions at 24. 48, 72, and 96 h, respectively. The activity of the reconstituted tyrosinase
solution was stable for 4 wk when stored at -80°C, and percentage recoveries, compared to activity of the freshly reconstituted solution,
were generally within 15% for both mid-range and "high"-activity dilutions. The between-run coefficients of variation (CVs) were
10,6% for the mid-range and 10.8% for the high-activity dilution over the 4-wk period. Enzyme activity was unstable when stored at
-20°C. Mushroom tyrosinase is an acceptable control material for use in assays of phenoloxidase activity. Inclusion of this control
material will provide a means to confidently compare results on a day-to-day or run-to-run basis when phenoloxidase assays are used
in the assessment of crustacean "health."

KEY WORDS: prophenoloxidase assay, control, tyrosinase, crustacean, health

INTRODUCTION

The prophenoloxidase activating system (PPA.S) and its active
en/.yme product, phenoloxidase (o-diphenoliO, oxidoreductase
E.C. 1.14.18.1 ) are integral components of the innate defense sys-
tem in crustaceans (Soderhiill et al. 1996; Soderhall & Cerenius
1998: Sritunyaiucksana & Soderhall 2000). Dark pigmentation
(melaninization) at sites of injury reflects prior generation of phe-
noloxidase, followed by the spontaneous formation of tnelanin
(Soderhall et al. 1996: Soderhall & Cerenius 1998: Sritunyaiuck-
sana & Soderhall 2000). Both the reactive quinone intermediates
and the final melanin products fomied during this reaction have
antimicrobial and protective properties (Soderhall et al. 1996: Ri-
ley 1997).

Determination of the activity of phenoloxidase in either whole
hemolymph or hetnocyte lysate supernatants has been used to as-
sess the "health"' or "'immune" status of crustacean species (Hauton
et al. 1997; Sritunyaiucksana et al. 1999; Rodriguez & Le Moullac
2000). Commercial test kits' are available to detect phenoloxidase
activity in whole hemolymph samples from shrimp. It is recog-
nized that assays used in the assessment of crustacean health
should be standardized (Bachere 2000).

Evaluation and validation of a laboratory test are required to
confirm that an assay is performing within its defined parameters
before it can be accepted for use in a diagnostic setting (Bellamy
& Olexson 2000). This includes determination of the assay's re-
portable range, precision, accuracy, and reagent stability. Control
materials are essential to monitor the performance of the assay
(Westgard & Klee 1999). Inclusion of a control with each group of
samples permits reliable comparison of the results obtained from
samples analyzed during different runs. Ideally, control materials
are derived from the same biological source as the samples that are
being evaluated. When this is not possible, materials with similar
properties can be substituted (Westgard & Klee 1999).

Most phenoloxidase assays are based on (or are modifications

'Spot On. DiagXotics hic. Wilton. CT 06897.

of) the method of Horowitz and Shen (Horowitz & Shen 1952;
Aspan & Soderhall 199.5). During development of an assay to
detect prophenoloxidase activity in plasma of the American lob-
ster, Homarus americanus H. Milne Edwards, a control material
was required. A purified source of lobster phenoloxidase having
consistent activity was not available. Lyophilized mushroom ty-
rosinase was evaluated as a control material because of its simi-
larities to phenoloxidase and commercial availability (Aspan &
Soderhall 1995).

MATERIALS AND METHODS

Mushroom tyrosinase (E.C. 1.14.18.1) stock solution was pre-
pared by adding 2.5 mL of sodium phosphate buffer (0.2 M
Na^POj. pH 7.5) to 13 mg of lyophilized mushroom tyrosinase
(Sigma Chemical Company. St. Louis, MO), giving a final con-
centration of approximately 10.700 units of tyrosinase activity per
niL of solution. Serial dilutions of this reconstituted stock solution
were used to detei'mine assay linearity, sensitivity, and within-run
precision as determined by calculation of coefficient of variation
(^rCV). A second stock solution, calculated to have similar activ-
ity per unit volume, was prepared from a different lot of lyophi-
lized tyrosinase. This second solution was used to conduct refrig-
erated and frozen stability trials. Eight 150-|ji.L aliquots were
placed in 0.5-niL polypropylene microcentrifuge tubes (Fisher Sci-
entific, Canada). Four aliquots were stored at -20°C, four aliquots
were stored at -80"C, and the remaining solution was refrigerated
(2^°C), for stability trials.

To complete linearity and precision assessments, serial dilu-
tions at 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400 of the
stock solution were made using a 0.2 M sodium phosphate buffer.
pH 7.5. Twenty microliters of each dilution was added to indi-
vidual wells of a flat-bottomed tissue culture plate (Falcon Mi-
crotest 3072, Becton Dickinson and Company, U.S.A.). There
were five replicates per dilution. Wells to be used as "substrate-
free blanks", one well for each dilution, also received 20 (xL of the
appropriate tyrosinase stock dilution. Twenty microliters of phos-
phate buffer was placed in wells used as reagent blanks. Eighty
microliters of sterile, distilled water was added to all wells using a

295

296

Battison et al.

multichannel pipette. One hundred microhters of the substrate so-
lution. 3.8 mM dopamine (3-hydro.xytyramine) (Sigma Chemical
Company ). was then added to all wells except for the substrate-free
blanks. These wells each received 100 |xL of sterile distilled water.
Production of the gold-orange quinone intermediate was moni-
tored every 1 I sec (wavelength 470 nm) during a 5-min period in
an automated spectrophotometer (SpectraMax. Molecular Devices
Corporation. U.S.A.) after a 3-sec mixing cycle. The software
package SoftMax^"^' (Molecular Devices Corporation, U.S.A.) was
used for calculation of maximum enzyme activity (V,^i„), defined
as the rate of maximal change in optical density (OD) of the
reaction solution per minute (A mOD/min) over a user-defined
time interval. The V^nax used for all further calculations for each
dilution was the mean V^nax of the five replicates. Within-run co-
etTicients of variation [ %CV = (standard deviation/mean) x 100]
were then calculated (Table 1 ).

Stability of the refrigerated stock solution was determined by
calculating the percentage recovery for two dilutions, identified as
having "low" and "mid-range" activities relative to the linear range
of the assay, over a period of 4 days. Percent recovery was cal-
culated as: [(measured activity at time ".v"/initial activity at recon-
stitLition) X 100] at 24. 48. 72. and 96 h (Table 2). Four replicate
wells were used for each dilution.

Long-term frozen stability of the stock solution, stored at
-20°C and -80°C, was evaluated by measuring enzyme activity in
serial dilutions of a thawed (room temperature. 20-22°C) aliquot
every week for 4 wk. Four replicate wells were used for each
dilution. Percent recovery calculations were made for the aliquots
stored at -20°C and -SOX (Tables -3a and 3b). Between-run (week
to week) %CVs were calculated for each dilution of the aliquots
stored at -80°C (Table 4),

The refrigerated stability of thawed stock solution, after storage
at -80°C, was also evaluated. Enzyme activity was measured at 48
and 72 h post-thawing for two dilutions (mid-range and high ac-
tivities). Percentage recoveries were calculated by coniparing the
activity after 2 and 3 days of refrigeration to the activity obtained
immediately after thawing (Table 5).

RESULTS

No significant absorbance was detected in the substrate-free
blank wells during the initial assays. Substrate-free blanks were
not used for the remaining assays. Reagent blanks were used for all
assays and also functioned as the plate blanks. The reaction curve

TABLE L

Sensitivity, linearity, and precision results for reconstituted
mushroom tyrosinase.

Expected" Activity

Observed Activity"

cv

Dilution

(mOD/min)

(mOD/minl

SD'

(%)

1:200

—

41.6

0.53

1.3

1:400

20.8

19.6

0.16

0.8

1:800

10.4

9.3

0.39

5.0

1:1600

5.2

5.0

0.32

6.5

1:3200

2.6

1.4

0.20

14.3

1:6400

1.3

0.4

0.23

60.7

"Calculated from the 1:200 dilution.

" Represents the mean of five replicates.

^' Standard deviation.

'' Coefficient of variation I^CV = (standard deviation/mean) x 10()|.

TABLE 2.
Refrigerated (2-4 C) stability of reconstituted mushroom tyrosinase.

... , Percent Reco\er\ (9c)"
Initial Activity" ^

(niOD/min) I day 2 days 3 days 4 days

Dilution no. I
Dilution no. 2

22.4
12,3

94
93

120
94

113
87

48
38

'Percent recovery = (measured activity/uiltial ,ii.ii\itvi x ]()(),
' Represents the mean of four replicates.

was linear during minutes 2—1 of the 5-min nnniitoring period,
^niav was determined froin this 2-min period. Initially, a gold-
orange product developed in all of the reaction wells containing
the dopamine substrate and the mushroom tyrosinase. Subse-
quently, a black product also appeared in the wells. At first, only
small amounts of the black product were present; however, with
time, the gold-orange product was no longer visible and only the
black product could be seen. The amount of this second product
seemed proportional to the concentration of enzyme solution in the
wells.

The catechol oxidase activity of mushroom tyrosinase, as mea-
sured in this assay system, was linear, and had acceptable precision
results, from 5.0 to 41.6 mOD/tnin when evaluated by serial dilu-
tion (/?- = 0.99) (Table 1. Fig. 1). Using the activity of the 1:200
dilution as a starting point, the expected and observed (in paren-
theses) activities (mOD/min) for each further dilution were 20.8
( 19.6) for the 1:400 dilution. 10.4 (9.3) for the 1:800 dilution, 5.2
(5.0) for the 1:1600 dilution. 2.6(1.4) for the 1 :3200 dilution, and
1.3 (0.4) for the 1:6400 dilution. The within-run CVs in = 5
replicates), for each dilution were 1.3% for the 1:200 dilution.
0.8% for the 1:400 dilution, 5.0% for the 1:800 dilution, 6.57c for
the 1:1600 dilution, 14.3% for the 1:3200 dilution, and 60.77f for
the 1:6400 dilution (Table 1).

The stability of the freshly reconstituted reagent was acceptable
(i.e., generally within 15% of the initial values) for up to 72 h after
refrigeration, but decreased to an average of 43% of initial activity
by 96 h (Table 2). Percent recoveries for a mid-range dilution at
24. 48. 72. and 96 h were 94%. 120%, 113%. and 48%. respec-
tively. Percent recoveries for a low-activity dilution at 24. 48. 72,
and 96 h were 93%. 94%. 87%, and 38%, respectively (Table 2).

The stock solution was unstable when stored at -20°C. The
enzyme activity, calculated as a mean of all dilutions, decreased to
37%, 28%, 27%, and 21% of the initial activity of the freshly re-
constituted reagent by weeks 1, 2, 3, and 4, respectively (Table 3a).

The stock solution was stable when stored at -80°C. The per-
cent recoveries, average of mid-range (dilution no. 2) and high-
(dilution no. 1 ) activity dilutions, by week, were 87% for week 1.
99% for week 2. 1 16% for week 3, and 106% for week 4 (Table
3b). The between-run CVs, over the 4-wk period, were 10.6% for
the mid-range dilution and 10.8% for the high-activity dilution
(Table 4). The refrigerated stability of the thawed stock solution
was generally satisfactory for weeks 1-3. but tended to deteriorate
by week 4 of storage at -80 C (Table 5).

DISCUSSION

The value of any assay resides in its ability to consistently
provide accurate and precise results. An incorrect result can be

Mushroom Tyrosinase: Control for Phenoloxidase Assays

297

TABLE 3a.

Frozen stability of mushroom tyrosinase stock solution stored at
-20'C for 4 weeks.

TABLE 4.

Weekly between-run coefficients of variation (CV I for mushroom
tyrosinase activity when stored at -80^C.

Initial Activity'
(mOD/min)

Percent RecoM

;ry ( ^r f

Mean'

' Activity (mOD/min)

Dilution

1 wk 2 wk :

1 wk 4 wk

Weeks at

-80C

Dilution No.

1 Dilution No. 2

No. 1

33.4

37 2,S

26 22

0

33.4

17.0

No. 2

17.0

37 2S

28 1^)

1

28.4

15.1

Mean

37 28

27 21

~)

31.1

17.7

° Percent recovery = (measured activity/initial activity) x

100.

3
4

37.2
35.7

20.2
17.9

*■ Represents

the mean of tour

replicates.

Mean
SD"

33.1

3.5

17.6
1.9

v\ orse than

no result at all.

Assay validation include

s quality con-

CV-C/f)

10,6

10.8

trol steps used to ensure that diagnostic tests pert'orm within pre-
determined specifications. Control materials are used to monitor
the assay's performance on a run-to-run basis. Ideally, such ma-
terials are derived from the same species as the samples to be
analyzed; however, this is not always possible. Steps in assay
validation usually include (but are not limited tol: determination of
the assay's reportable range; evaluation of assay precision by cal-
culating both within-run and between-run coefficients of variation
C/fCV = standard deviation/mean x I (.XT); and. determining the
stability of the assay reagents and the sample under different stor-
age conditions (Bellamy & Olexson 2000).

Phenoloxidase (d-diphenoliO, oxidoreductase E.C. 1.14.18.1)
assays have been used to investigate and assess the health status of
a variety of crustaceans in both research and commercial settings
(Sritunyalucksana et al. 1999; Rodriguez & Le Moullac 2000). A
stable source of enzyme to use as a control material was required
in the development of a plasma prophenoloxidase assay for use in
H. americanus. A commercial source of purified lobster (//. omeri-
caiuis) phenoloxidase is not available. Lobster hemocyte lysale
supernatant preparations represented a highly concentrated source
of prophenoloxidase; however, these were unsuitable due to their
poor long-term storage characteristics and the inherent, inconsis-
tent activity between preparations (unpubl. results, ALB).

Mushroom tyrosinase (E.C. 1.14.18.1) was evaluated as a po-
tential alternative because of its similarity to phenoloxidase. Both
enzymes have cresolase (monophenol monooxygenase) and cat-
echol oxidase (polyphenol oxidase) activity (Aspan & Soderhall
1995). Determination of the enzyme's copper content and location
of conserved histidine residues indicates that arthropod proph-
enoloxidase belongs to the tyrosinase group of enzymes found in
bacteria, fungi, and plants (Aspan & Sbderhiill 1995). The reported
long-term stability of frozen, reconstituted mushroom tyrosinase

TABLE 3b.

Frozen stability of mushroom tyrosinase stock solution stored at
-80 C for 4 weeks.

Initial Activity"
(mOD/min)

Percent Recovery ( % )"

Dilution

1 wk

2wk

3 wk

4 wk

No. 1

33.4

85

93

112

107

No. 2

17.0

89

105

119

105

Mean

87

99

116

106

' n = 4 replicates.
^ Standard deviation.
" Coefficient of variation

(mean/standard deviation) x 100.

and its commercial availability made this enzyme an attractive
alternative (Kertesz & Zito 1965).

Mushroom tyrosinase was readily soluble in the 0.2 M sodium
phosphate buffer (pH 7.5) used in this assay system. Other studies
have used a sodium cacodylate-based buffer. Sodium cacodylate is
listed as a hazardous material and is a recognized carcinogen
(Sigma-Aldrich 2001 ). The sodium phosphate buffer used herein
presents minimal hazards, is readily available, and was compatible
with this assay system. L-DOPA (3.4-dihydroxyphenyl-L-
alanine). a commonly used substrate in phenoloxidase assays, was
not compatible with the phosphate buffer. Dopamine (3-
hydroxytyramine) was chosen as an alternate catechol. Dopamine
has been identified as a preferred substrate for phenoloxidase from
Mciinltica se.xla (Sugumaran et al. 1999). This may also be true for
other invertebrates. Mushroom tyrosinase converted the dopamine
substrate to an orange-gold intermediate product, presumably the
o-quinone. 4-(2-aminoethyl)-l.2-benzoquinone. This product was
eventually replaced by a black precipitate (melanin), which is the
expected result of activation of the PPAS /;; vivo (Soderhall et al.
1996; Soderhall & Cerenius 1998; Sritunyalucksana & Soderhall

TABLE 5.

Refrigerated stability of thawed mushroom tyrosinase stock solution
stored at -SOX.

Dilution

Weeks
at -80 C

Initial activity''
(mOD/min)

Percent recovery ( % )"

2 days

3 days

No. 1

No. 2

33.4
28.4
31.1
37.2
36.7
17.0
15.1
17.7
20.2
17.9

nd^
110
106
106

85
nd
106

86
104

84

nd

135

110

102

75

nd

1.39

103

97

89

'Percent recovery = (measured activity/initial activity) x 100.
' Represents the mean of four replicates.

' Percent recovery = (measured activity/initial activity) x 100.
' Represents the mean of four replicates.
' Not done.

298

Battison et al.

> —

O c

ra t

-a Q

B O

" E

a: i,
a

0 10 20 30 40 50

Observed activity (mOD/min)

Figure 1. Linearity of catechol oxidase activit> of reconstituted mush-
room tyrosinase determined by serial dilution.

2000). An identical series of products is observed when either
lobster hemocyte lysate supernatant or lobster plasma, after acti-
vation with trypsin, is used as the enzyme source for the assay.

The reconstituted tyrosinase solution was stable, when refrig-
erated, for up to 3 days as indicated by the percentage recovery
studies (Table 2). Tyrosinase activity was stable for up to 4 wk
when the reconstituted solution was stored at -80°C. Reconstituted

mushroom tyrosinase was unstable when stored at -20°C, in con-
trast to a previous report (Kertesz & Zito 1965). The discrepant
results found in this study may reflect differences in the buffers
used for reconstitution. Overall, the refrigerated and frozen
(-80°C) stability of reconstituted mushroom tyrosinase was con-
sidered acceptable for its use as an assay control material.

Ideally, control materials contain a predetermined amount of
the enzyme being measured and are incorporated in a material
(matrix) similar to the matrix of the sample to be analyzed. In this
study, mushroom tyrosinase was reconstituted in a phosphate
buffer. This would be the optimal situation when phenoloxidase
activity is being measured in hemocyte lysate supernatants where
a similar buffer had been used to prepare the lysates. Similarly,
incorporation of the mushroom tyrosinase into whole hemolymph
or plasma collected from normal, apparently healthy animals
would be preferable when phenoloxidase activity is being mea-
sured in these types of samples. This was not evaluated in this
study. When reconstituted in a sodium phosphate buffer, mush-
room tyrosinase is a suitable control material for use in phenoloxi-
dase assays, providing assurance that the assay is working cor-
rectly and permitting comparison of within-laboratory sample re-
sults on a run-to-run or day-to-day basis.

Aspan. A. & K. Soderliall. IW5. The prophenoloxidase activating system
in invertebrates; Assays of the prophenoloxidase activating enzyme (a
serine proteinase) and phenoloxidase. In: J. S. Stolen. T. C. Fletcher. S.

A. Smith. J. T. Zelikot'f. S. L. Kaattari. R. S. Anderson. K. Soderhall &

B. A. Weeks-Perkins, editors. Techniques in tlsh immunology — 4. Fair
Haven, New Jersey; SOS Publications, pp. 161-17(1.

Bachere. E. 2000. Introduction; Shrimp immuniiy and disease control.
Ai/KMultiire 191;.^-! 1.

Bellamy, J. E. C. & D. W. Olexson. 2000. Evaluating laboratory proce-
dures. In: J. E. C. Bellamy & D. W. Olexson. editors. Quality assurance
handbook for veterinary laboratories. Ames; Iowa State University
Press, pp. 61-77.

Hauton, C, J. A. Williams & L. E. Hawkins. 1997. The effects of a live iii
vivo pathogenic infection on aspects of the immunocompetence of the
common shore crab. Cuniim.s nmenas (L.). J. Exp. Mar. Biol. Ecol.
211:115-128.

Horowitz, N. H. & S. C. Shen. |y.'i2. Neurosporo tyrosinase. J. Biol. Cliem.
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Kertesz, D. & R. Zito. 1965. Mushrooin polyphenol oxidase I. Purification
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Riley, P. A. 1997. Molecules in hicus: Melanin. Int. .1. Bioclwrn. Cell Biol.
29:1235-1239.

LITERATURE CITED

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Sigma-Aldrich Canada Ltd. 2001. Cacodylic acid sodium salt trihydrate
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Sritunyalucksana. K. & K. Soderhall. 2000. The proPO and clotting system
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Sntunvalucksana. K.. P. Sithisarn. B. Withayachymnarnkul & T. W. Fle-
ael. 1999. Activation of prophenoloxidase. agglutinin and antibacterial
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Suguinaran. M.. R. Duggaraju. F. Generozova & S. Ito. 1999. Insect mel-
anogenesis. II. Inability of Mandma phenoloxidase to act on 5.6-
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Westgard. J. O. & G. G. Klee. 1999. Quality management. In: C. A. Burlis
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Mmnuil of Shellfish Research. Vol. 21. No. 1. 299. 2U02.

ERRATUM

ARTIFICIAL ENVIRONMENTAL CONDITIONS CAN AFFECT ALLOZYME
GENETIC STRUCTURE OF THE MARINE GASTROPOD PATELLA CAERULEA

ANNAMARIA MAURO, NICOLO' PARRINELLO, AND MARCO ARCULEO*

Dipartiinemo ili Biologiu Aiilniale via Arcliirafi ]<S, 90123 Palerma. Ikd\
[Journal of Shellfish Research. Vol. 20, No. 3. 1059-1063, 2001]

After this article appeared in the December 2001 issue of the .lounuil of Shellfish Research, the following printer's errors
were noticed:

The author's name was misspelled in the author line of the title paye of the article.

Marculeo was misspelled in the title page footnote.
*Corresponding author: marculeotS'unipa.it

Enzyme was misspelled in the Table 1 title.

TABLE 1.
Enzyme commission number for the loci analyzed.

299

Journal of Shellfish Resfanh. Vol. 21. No. 1, 301-319, 2002.

ABSTRACTS OF TECHNICAL PAPERS

Presented at The 5th
INTERNATIONAL CONFERENCE

ON
SHELLFISH RESTORATION

Nanaimo B.C., Canada

September 18-20. 2001

301

Shellfish. Restoration. Nanaimo B.C.. Canada Abstracn. September 2001 303

CONTENTS

W. S. Arnold

Bay scallop [Argopecteu irradians) population restoration in Florida. USA: Culture versus management 303

W. S. Arnold

Testing alternative strategies for enhancement of hard clam (Mcrccnaria spp.) population in Florida. USA waters 305

Use M. Aiiffrey, Shawn M. C. Robinson and Myriam A. Barheau

Effect of macroalgal mats on burial depth of soft-shell clams 303

Leslie Banigan

Protecting and restoring commercial shellfish beds in Kitsap County, Washington 303

Helen A. Beadman, M. J. Kaiser and R. I. Willows

Increasing mussel shell thickness by relaying to improve resistance to crab predation 306

Brian F. Beat

Public stock enhancement of soft-shell clams. Mya areiuiria. in Maine. USA: The role of community-based

aquaculture, applied research, and technology transfer in shellfish restoration 306

Alan J. Birch, M. W. Luckenbach, P. G. Ross, R. Gammisch, J. Brubaker and J. A. Wesson

Design and construction of subtidal oyster reefs: Evaluating effects of reef scale 306

C. Boldt

The muck stops here a community decides 307

William T. Collins

Acoustic seabed classification for shellfish habitat mapping 307

Paul Cormier, T. Landry and J. F. Mallet

Oyster population restoration in Caraquet. N.B.; Phase II. Optimizing productivity 307

A. Craig and C. Downs

Abstract theme area 308

Dale Diaz, K. Caevas and W. S. Perret

Side scan sonar as a management tool in Mississippi 308

Craig A. Downes, Charles E. Robinson, Arnold Huang, Gary Shigenaka and John E. Eauth

Cellular-physiological assessment of bivalves after ten years of chronic exposure to spilled Exxon Valdez crude oil

using a Noval Molecular Diagnostic biotechnology 308

Brett Dumbault, D. Armstrong, G. Hosack and B. Semens

Oyster aquaculture as fish habitat in Pacific Northwest coastal estuaries 309

John William Ewart, J. Alderman and A. A/a////

Demonstration and evaluation of commercially important bi\alve culture and stock enhancement methods in

Rehoboth and Indian River Bay. Delaware (1998-2001 ) 309

A. G. Field

Molecular detection of fecal Bacteroides as source indicators for fecal pollution in water 309

Gef Elimlin and Michael P. Stringer

Habitat restoration and shellfish stock enhancement initiatives in New Jersey 309

Stuart D. Glasoe

New directions for shellfish protection in Puget Sound 310

Heather Goble

Semiahmoo Bay Water Quality Project: Phase I findings 310

William Goldsborough, Stew Harris, D. Jackson, D. W. Meritt and S. Tobash

Remote-set alternative substrate on leased ground: An innov ative approach to reef restoration 311

Kimberly A. Hamilton, D. LaDon Swann, Richard K. Wallace, Yolanda J. Brady, David B. Rouse, Scott Rikard and Holly Hall

Restoration of the eastern oyster (Cnissostrcii viri;iiiicii) through a xdlunteer program in Mobile Bay. Alabama 31 1

Matthew Hardy, T. Landry and A. Boghen

Reproductive sanctuary for the northern quahaug. Meneiiciria iiicrcciiaiia, in Prince Edward Island 311

J. Hetrick

Status of shellfish enhancement in Alaska 311

304 Ahsrruclx. September 2001 Shellfish. Restoration. Nanaimo B.C.. Canada

Barbara Joughin and William Heath

Innovative coniniunit\ processes prodtiee integratis e management tools 311

Brian Kingzett

Advances in shellfish safety assurance and participation in sanitary growing water monitoring programs hy the BC

shellfish aquaculture industry 312

Brian Kingzett

Development of intensive deepwater oyster culture systems in British Columbia 312

K. E. Landrum, K. M. St. Pe, B. Ache and F. Kopfler

Using innovative stormwater controls for water quality impro\ements and oyster habitat restoration in the

Barataria-Terrebonne National Estuary 312

Kevin L. LeBlanc

Water quality improvement; A focus on new technologies 313

A. T. Leggett, A. Blow, \\. Goldshorough and R. D. Brumbaugh

Commercial-scale oyster production for reef restoration and slock enhancement 313

Donald Lennartson

Protection and restoration: Washington State shellfish program 313

B. G. Lucas and A. Campbell

Abalone restoration through collaborative partnerships in British Columbia 313

B. G. Lucas, A. Campbell and D. Brouwer

Site assessment, selection and monitoring methods for abalone restoration near Kitkatla, British Columbia 314

Mark W. Luckenbach, J. Nestlerode, P. G. Ross and A. J. Birch

Effects of reef architecture and scale on t~ish utilization of oyster reefs in Virginia 314

Thomas B. McCormick

The role of aquaculture in the restoration effort to save white abalone {Hdlictis soreiiseiii) in California 314

Jim Mclsaac

Boat waste and shellfish cUisures: A comparison of Canadian and United States policies 315

C. W. McKindsey

Succession in mussel communities: The importance of what is measured 315

Eric McLay

An archeological perspective on the culture significance of shellfish resources to First Nations in the Georgia Basin... 315
Donald W. Meritt, S. Tobash, C. S. Frentz, W. Goldsborough and S. Reynolds

Restoring oysters to the Chesapeake Bay: A coordinated effort involving government universities, community groups.

and industry 315

Donald W. Meritt. S. Tobash. K. T Paynter and T. Koles

Oyster restoration in Chesapeake Bay: 1. Criteria for site selection 316

C. A. Milbuiy and P. M. Gaffney

Genetic monitoring of oyster stock enhancement in the Choptank River. Chesapeake Bay. Maryland. USA 316

Rob Miller

Open Saanich Inset shellfish beds: A collaborative approach 316

Marc Ouellete and T. Landry

Hydroacoustic seabed classification technology applied in shellfish productivity research 317

Paynter, Kennedy and Elizabeth Flynn

The living veneer: Characterizing habitat structure created by oysters 317

Paynter, Kennedy, T. Koles, D. Meritt and S. Tobash

Oyster restoration in Chesapeake Bay: II. Impacts of water quality and disease 317

P. G. Ross, M. W. Luckenbach and A. J. Birch

Early community dc\ elopmcnt of oyster reefs in Virginia: Effects of reef scale 318

Rejean Tremblay and T. Landry

Genetic and pathogenic aspects in shellfish restoration of scallop populations 318

Aswani K. Volety, S. Gregory Tolley and James T. Winstead

Water quality and oyster health (Cmssostrea virginica): An integrated approach to determining habitat restoration potential 318

Pamela Winquist

The nutrient value of shellfish and other traditional foods, their past and cuiTent contribution to the diet of First

Nations people 319

Melisa C. Wong, M. A. Barbeau, L. A. Grecian, L-A. Davidson, M. Miles and Donna Murray

Predation of juvenile sea scallops (Placopeclcn inagelliiiiici(s) during seeding trials in the Northumberland Strait 319

Shellfish, Restoration, Nanaimo B.C., Canada

Abstracts, September 2001 305

BAY SCALLOP {ARGOPECTEN IRRADIANS) POPULA-
TION RESTORATION IN FLORIDA, USA: CULTURE
VERSUS MANAGEMENT. W. S. Arnold, Florida Marine Re-
search Institute, Florida Fish and Wildlife Conservation Commis-
sion. 100 Eighth Avenue SE. St. Petersburg. FL 33701. USA.

Bay scallops have historically been widely distributed in
Florida waters, comprising discrete populations from West Palm
Beach on the Atlantic coast to Pensacola in the panhandle. In
recent decades, many of those discrete populations have collapsed
due to one or a combination of anthropogenic factors. In response
to that collapse, recreational and commercial harvest restrictions
have been implemented and a culture-based restoration program is
underway. The restoration program is experimental in nature and
includes a genetic component that is designed to provide informa-
tion on the relative contribution of cultured scallops to the subse-
quent year-class.

Preliminary results of this ongoing study suggest that, despite a
two-orders-of magnitude increase in mean scallop density within
the targeted restoration area, active restoration has contributed
little to this recovery. The lineage of fewer than 5% of the subse-
quent year-class can be traced back to the parental stock that was
originally cultured. Instead, it appears that reductions in harvest
intensity precipitated by a change in harvest regulations may be the
primary contributor to the observed resurgence in scallop popula-
tions. Apparently, removal of fishing pressure on already depau-
perate scallop populations allowed for increased reproductive suc-
cess and recruitment. If these results are verified through the final
year of the study, they indicate that active intervention is not
always required to instigate population recovery. Instead, effective
and ecologically based management may suffice.

TESTING ALTERNATIVE STRATEGIES FOR ENHANCE-
MENT OF HARD CLAM [MERCENARIA SPP.) POPULA-
TION IN FLORIDA, USA WATERS. W. S. Arnold. Florida
Marine Research Institute, Florida Fish and Wildlife Conservation
Commission, 100 Eighth Avenue SE, St. Petersburg, FL 33701. USA.

Although lacking the history of its northern counterparts, the
hard clam fishery in Florida became a significant contributor to US
clam landings during the 1980s and early 1990s. Most Florida
landings have been realized from the Indian River lagoon. In the
late 1990s, the clam population in the lagoon almost completely
collapsed in response to increased rainfall associated with ENSO.
Salinity has since "recovered" to a level suitable for hard clam
survival, but the clam population has shown no concomitant re-
covery. Active intervention may be required to increase clam
population abundance to a level at which the fishery is again
profitable.

Three methods have been compared for their suitability to in-
crease clam abundance. Spawner transplants involve the harvest of
widely dispersed adult clams followed by the release of those
clams in a relatively small area. This approach is designed to

concentrate reproductively active clams, thereby increasing fertil-
ization efficiency and the production of larval clams. Seeding in-
volves planting various size classes of clams under protected con-
ditions, again with the goal of increasing fertilization efficiency by
creating dense clumps of actively spawning clams. Larval injection
involves spawning clams in the hatchery, fertilizing the resultant
eggs, and releasing the developing embryos within a preselected
area of the lagoon. Preliminary results suggest that larval injection
can increase abundance of larval clams by several orders of mag-
nitude, but there is as yet no evidence that any of the three strat-
egies has actually led to increased recruitment of hard clams in the
lasioon.

EFFECT OF MACROALGAL MATS ON BURIAL DEPTH
OF SOFT SHELL CLAMS. Lise M. Auffrey,' Shawn M. C.
Robinson," and Myriam A. Barbeau," 'Department of Biology.
Uni\'ersity of New Brunswick. Bag Service 45111. Fredericton,
NB. E3B 6E1. Canada; "St Andrews Biological Station. 531
Brandy Cove Road. St Andrews. NB. E5B 2L9, Canada.

Green macroalgal mats are becoming prevalent on important
soft-shell clam (Mya uienaiia) harvesting beaches in southwestern
New Brunswick. Canada. We investigated the effect of macroalgal
mats on burial depth and body condition of clams in the field (2
sites with high algal cover) and laboratory. In the field, we located
patches covered with algae and clear of algae at each site. We
measured burial depth by digging trenches and measuring the dis-
tance between the anterior edge of the clam and sediment surface.
We then collected the clams for body condition analysis. Burial
depth was significantly lower for clams under macroalgal mats
than for those in clear areas. Clam body condition was generally
lower under algae than not under algae. In the lab. clams (4/
aquaria) were placed in mud ( 10 cm deep) and covered either with
0, 2 or 6 cm of macroalgae. Clams under algae de-burrowed after
a few days. Clam body condition and various abiotic variables are
being measured in the lab and will be analvsed in the near future.

PROTECTING AND RESTORING COMMERCIAL
SHELLFISH BEDS IN KITSAP COUNTY, WASHINGTON.
Leslie Banigan, Environmental Health Specialist. Bremerton-
Kitsap County Health District, 109 Austin Dri\e. Bremerton. WA
98312, USA.

Kitsap County, located in the central Puget Sound area of
Washington State, developed a Surface and Storm Water Manage-
ment (SSWM) Program that successfully identifies and corrects
nonpoint pollution affecting shellfish areas. The Program's success
is due to:

I. Political Support: In 1992, Washington passed legislation re-
quiring counties to address water pollution sources causing
shellfish closures. In 1993. the Health District received local

306 Abstnias, September 2001

Shellfish. Restoration. Nanaimo B.C.. Canada

pohtical support to develop Kitsap County's SSWM Program,
a comprehensive four-agency team to address noiipoint pollu-
tion.

2. Financial Support: SSWM piovides ongoing stable funding for
the Health District's Pollution Identification and Correction
(PIC) Program.

3. Effective Public Outreach: An effective outreach program was
developed to help inspectors gain access to private property.

4. Enforcement Capability: The Health District developed regula-
tions addressing failing on-site sewage systems and improper
animal manure management.

5. Standardized Procedures: The Health District uses an objective
system of ranking water quality problem areas and has a de-
tailed procedures manual.

In 1446. the State closed a portion of Port Gamble Bay to
shellfish harvesting. The Health District completed a PIC project
in the watershed. In 1499. the State upgraded the commercial
shellfish beds to "approved" status.

Barley Lagoon was closed to shellfish harvesting in early 1999.
Due in large part to PIC work conducted by the Health District,
one-half of Burley Lagoon was re-opened to shellfish harvest in
2001.

INCREASING MUSSEL SHELL THICKNESS BY RELAY-
ING TO IMPROVE RESISTANCE TO CRAB PREDATION.
Helen A. Beadman,' M.J. Kaiser' and R. 1. Willows,- 'School
of Ocean Sciences, University of Wales-Bangor. Menai Bridge.
Gwynedd. LL59 5BP. UK; "National Centre for Risk Analysis and
Options Appraisal. Steel House, 1 1 Tothill Street. London, SWIH
9NS, UK.

The mussel cultivation industry is cuirenlly the fastest expand-
ing and most valuable sector of the shellfish aquaculture industry
in the United Kingdom. One of the constraints of the potential
continued success and expansion of the industry is an unpredict-
able supply of seed mussels for relaying. It is suggested that a
possible solution to this problem is to lay seed in times of abundant
spatfall on high shore areas where they can ongrow. In subsequent
years when spatfall is low these mussels can then lie tiansferred
further downshore to allow faster growth to marketable size. A
further advantage of growth in high shore areas is the development
of a thicker shell that aids resistance to crabs predation.

The aim of this study is to determine how the difference in
mussel shell thickness achieved by growth at different heights on
the shore can be related to resistance to crab predation and how
this changes when mussels are relaid at different tidal heights. This
has been achieved through an experimental apprtiach relaying
mussels from high shore, intertidal and suhtidal areas to the inter-
tidal. Shell thickness is determined through direct means and in-
directly through crab predation experiments and shell compres-
sion. Results show a siunificant diffeience between mussels grown

at different shore heights and associated differences in shell com-
pression strengths and resistance to crab predation. Mussels grown
at lower shore levels initially displayed thinner shells, of lower
compression strength and were preferentially predated upon by
crabs.

PUBLIC STOCK ENHANCEMENT OF SOFT-SHELL
CLAMS, MYA ARENARIA, IN MAINE, USA: THE ROLE OF
COMMUNITY-BASED AQUACULTURE, APPLIED RE-
SEARCH, AND TECHNOLOGY TRANSFER IN SHELL-
FISH RESTORATION. Brian F. Beal, Division of Environmen-
tal and Biological Sciences. University of Maine at Machias.
Machias, ME 04654, USA.

In Maine. USA. the intertidal zone is legally owned by the
coastal community adjacent to it. For 50 years, commercially im-
portant soft-shell clams. Mya arenaria. residing in these tidal flats
have been co-managed by communities and the state's marine
resources department. Clam landings have been cyclical varying
from 3,100 metric tons (t) in 1950 to 658 t in 1959, to 3,500 t in
1 977 to 800 t in 1997. Historically, management has been based on
the vagaries of natural recruitment and the strength of a particular
0-year class.

Beginning in 1987. the first-ever soft-shell clam restoration
progi-am in the US was established through the Beals Island Re-
gional Shellfish Hatchery (BIRSH) whose mission is to enhance
Maine's soft-shell clam and other shellfish resources through
aquaculture. applied research, technology transfer and public edu-
cation. BIRSH produces 5-10 million 8-12 mm seed clams annu-
ally, and. over the years, these cultured individuals have been
planted in approximately 50 of the state's 100 coastal communities
that manage their shellfish beds.

BIRSH has been the focal point of Maine's research and out-
reach on soft-shell clam ecology during the past 1 5 years and has
been done at the request and in conjunction with coastal commu-
nities. Research projects have tested the interactive effects of
planting date, seed size and density, type and aperture of protective
netting and how these factors vary spatially and temporally. Gen-
erally, seed clams (>I0 mm) should be planted during April, at the
beginning of the growing season, and protected with flexible net-
ting (6.4 mm).

DESIGN AND CONSTRUCTION OF SUBTIDAL OYSTER
REEFS: EVALUATING EFFECTS OF REEF SCALE. Alan
.1. Birth, M. W. Luckcnbach, P. G. Ross, R. Ganiniisch, and
J. Brubaker, College of William and Mary. Virginia Institute of
Marine Science, and J. A. Wesson, Virginia Marine Resources
Commission. USA.

Restoration of oyster reef habitat in mid-Atlantic estuaries is
requisite for restoring oyster fisheries and recovering lost ecologi-
cal services. Recent evidence suggests the importance of estab-

Shellfish, Restoration, Nuiuiinio B.C., Canada

Abstracis, September 2001

307

lishing proper vertical relief and interstitial space in the initial
placement of substrate. A third component of reef architecture that
has yet to be investigated is the aerial extent or scale of reefs. We
are currently evaluating the effects of scale on various components
of communities that develop on high relief reef bases ranging in
size from 400 m^ to 8000 ". As a result, accurate physical descrip-
tions of the constructed reefs are important to meaningfully quan-
tify reef scale.

We present some preliminary design and ct)iistruction aspects
of three dimensional subtidal reefs in the Rappahannock River, a
tributary of the Chesapeake Bay. Because of their subtidal nature,
describing the reefs physical characteristics becomes difficult.
Subsequently, Side-scan sonar mapping, bathymetry surveys and
current velocity mapping were conducted during the first year after
construction and will be continued in subsequent years as the reefs
develop.

From a management aspect, where potential substrate is limit-
ing and expensive, any ecological advantages or disadvantages
derived from allocating resources to many small vs. several large
reefs can have direct economic impacts on restoration efforts. Fur-
thermore, identification of details of the physical characteristics
that may help achieve restoration goals can be extremely important
in providing resource managers with the information that can aid
future reef design criteria.

THE MUCK STOPS HERE A COMMUNITY DECIDES.
C. Boldt, Union Bay Liquid Waste Management Committee, P.O.
Box 32, Union Bay, British Columbia. Canada, VOR 3B0.
What does an ocean side community do when:

• residents cannot safely harvest shellfish from its beaches for
over 7 years'^

• a quiet, community walk lets you know who is washing clothes,
who is having a shower, who has flushed the toilet?

• its ditches are unsafe for child's play

• its ditches are overgrown with water cress costing $1000"s of
dollars to government to clean out?

• kids swimming on local beaches can be a health issue?

• over 60'^ of septic fields are failing?

Well, one community said "Enough is Enough!" While its
neighbour were saying NO to a 1 997 valley wide referendum on a
solution to liquid waste problems. Union Bay said "Yes".

Union Bay has worked hard to involve, inform, educate and
enlist residents, local credit union, local businesses, politicians and
government agencies from all levels to develop a solution.

Working with major funding from Georgia Basin Ecosystem
Initiative, Environment Canada and provincial grants. Union Bay
has developed a liquid waste treatment solution which will ulti-
mately turn waste to wealth, using liquid waste, including a cam-
pus for post-secondary institutions to support their environmental

program, enhance salmonid habitat, provide an intei"pretive centre.
Community process has been the key to significant funding for
this project and it has created a climate of acceptance and support
for rural grassroots, community-based planning. This process has
been accepted by the Regional District of Comox-Strathcona and
adopted in at least three other rural communities in the Comox
Valley.

ACOUSTIC SEABED CLASSIFICATION FOR SHELLFISH
HABITAT MAPPING. William T. Collins, Quester Tangent
Corporation, Sidney. BC V8L 5Y8, Canada.

The amplitude and shape of an acoustic signal reflected from
the sea floor is determined mainly by the sea bottom roughness, the
density difference between water and the sea floor, and reverbera-
tion within the substrate. Remote classification of the sea bottom
requires an acoustic data acquisition system and a set of algorithms
that analyze the data, determine the seabed type and relate the
results of the acoustic classification to the physical properties of
the marine sediments. Quester Tangent Corporation has success-
fully combined high-speed digital signal processing technology
with multivariate statistical analysis to create the QTC VIEW sea-
bed classification engine. The QTC VIEW captures and digitizes
the seabed echo from a conventional echo sounder, processes,
analyses, displays and records acoustic waveform data character-
izing the sea floor. The output is a set of point data representing sea
floor acoustic diversity. The georeferenced classifications can then
be objectively correlated with other data such as sediment prop-
erties or directly to fish census.

The technology has been applied world wide to map marine
sediments for a variety of applications. It has been used for map-
ping substrate in support of benthic habitat characterization. The
technology can also be configured for use in water depths less than
5 ni. Examples of oyster mapping projects in West Galveston Bay,
Texas and Chesapeake Bay, Maryland are highlighted. Strategies
for the integration of acoustic and nonacoustic data are included.

OYSTER POPULATION RESTORATION IN CARAQUET,
N.B.; PHASE II, OPTIMIZING PRODUCTIVITY. Paul
Cormier. T. Landry, and J. F. Mallet. Department of Fisheries
an Oceans, GFC, P.O. Box 5030, Moncton N.B., EIC 9B6, and
Department of Agriculture, Fisheries and Aquaculture, 22 Boul.
Saint-Pierre, Caraquet, N.B. EIW IB6, Canada.

A decrease in the productivity of oysters in Caraquet Bay, N.B.
is generating some interest in restoration projects. This bay repre-
sents the most northern location with a sustainable oyster (Cras-
sosrrea virginica) population. The first phase of this initiative pro-
vided invaluable information on the distribution, abundance and
population structure of oyster beds. Over 60% of the oysters found
in this bay are pre-recruits to the fishery (35-7.*imm). These oysters

308 Abstracts. September 2001

She

sh. Restoration. Nanaimo B.C., Canada

are mainly tound in the northern portion of the main bed, which
has a relatively low abundance of commercial size oysters
(>75mm) and. is locally renown for its "stunted growth oysters"".
Samples of oysters from this location where transferred at four
stations in the bay and monitored for grov\lh and conditioning.
Results are suggesting that the growth limitation of these oysters is
associated with the geographical location and even more to the
bottom conditions. Management strategies will be developed to
optimize the productivity of oyster from this highly successful
recruitment location, which offers limited possibility for commer-
cial production.

can cover large areas in less time, provide accurate measurements
of coverage, and simultaneously provide differential GPS coordi-
nates. The information acquired from side scan sonar provides a
long-term record, which can be used to detect changes due to
environmental catastrophes, vessel groundings and harvest pres-
sure. A side scan sonar survey was perf"ormed on two cultch plant
sites located in the western MS Sound, St. Joe Cultch Plant and
Pass Christian Cultch Plant. The St. Joe Cultch Plant site was
surveyed before and after cultch planting. On the Pass Christian
Cultch Plant only a post side scan sonar survey was conducted.
The data was processed and input in a mosaic software program
for further analysis. This study will demonstrate the abilities of
side scan sonar as a management tool for oyster reef management.

ABSTRACT THEME AREA: A. Craig, and C. Downs, EnVir-
tue Biotechnologies. Inc. 2255 Ygnacio Valley Rd., Suite H-1.
Walnut Creek, CA 94598, USA.

Using the tools of proteomics and molecular diagnostics, one of
our primary goals is to determine if environmental factors are
affecting shellfish susceptibility to infections. This stiategy of re-
search can also aid in answering other important issues to shellfish
husbandry such as why some shellfish cultivars or strains are more
susceptible to infection than others and the cellular mechanisms
responsible for disease resistance? Proteomics and functional ge-
nomics can be used to discover novel and appropriate molecular
biomarkers that can aid in diagnosing the health status of a crop,
develop powerful biomarkers to aid in predicting crop yield, and
aid in developing genetic markers for advantageous cultivar traits.
Finally, understanding the cellular and biochemical mechanisms of
shellfish disease may allow for the development of practical mea-
sures or treatments to limit disease.

SIDE SCAN SONAR AS A MANAGEMENT TOOL IN MIS-
SISSIPPI. Dale Diaz, K. Cuevas and W. S. Perret, Office of
Marine Fisheries, Mississippi Department of Marine Resources,
1141 Bayview Avenue, Suite 101, Biloxi, MS 39530, USA.

Oyster harvest from Mississippi reefs provide jobs for numer-
ous fishermen and contribute substantially to the economy. Proper
management of these reefs is vital for continued harvest. Enhanced
management techniques (i.e. cultch planting) should help to ensure
even greater future production. Cultch planting is a key component
of management measures conducted by the Mississippi Depart-
ment of Marine Resources (MDMR). The use of sounding poles,
tongs, dredges or scuba equipment gauged the effectiveness of
different cultch planting methods. These methods are not efficient
techniques when surveying large areas for coverage rates or dis-
tribution of materials.

LIsing side scan sonar to monitor cultch plants has a number of
advantages over these previously used methods. Side scan sonar

CELLULAR-PHYSIOLOGICAL ASSESSMENT OF BI-
VALVES AFTER TEN YEARS OF CHRONIC EXPOSURE
TO SPILLED EXXON VALDEZ CRUDE OIL USING A
NOVAL MOLECULAR DIAGNOSTIC BIOTECHNOL-
OGY. Craig A. Downes,' Charles E. Robinson." Arnold
Huang.' Gary Shigenaka." and John E. Fauth,' Envirtue Bio-
technologies, Inc., 2255 Ygancio Valley Rd., Suite H-l, "Walnut
Creek, CA 94598, USA; "Hazardous Materials Response Division,
US National Oceanic and Atmospheric Administration, 76(X) Sand
Point Way NE, Seattle, WA 981 15, USA; 'Department of Biology,
University of Charleston, 66 George Street, Charieston, SC 29422,
USA.

The objective of this study was to determine the cellular physi-
ological status of the bivalves Mya arenaria and Mytilus trossulus
in an area impacted by a ten-year chronic exposure of spilled
Exxon Valdez crude oil in Prince William Sound.

Bivalves were collected from a well characterized mipacted site
on Knight Island and from a nonimpacted site north of Bainbridge
Island. We used a novel biotechnology known as the Envirtue
Molecular Diagnostic System^M (EMDS) to determine if bivalves
were (I) physiologically stressed, (2) the nature of the altered
physiological state, and (3) whether the organisms were respond-
ing specifically to an exposure to polyaromatic hydrocarbons
(PAH). Molecular diagnostic analysis indicated that bivalves at the
impacted site were stressed, experiencing both an oxidative and
xenobiotic stress, resulting in increased protein turnover and chap-
erone activity. Further, bivalves from the impacted area were re-
sponding specifically to a PAH xenobiotic exposure and accumu-
lating DNA-PAH adducts. Finally, species-specific responses were
observed that were related to the spatial-habitation characteristics
of each species.

We conclude that bivalves inhabiting an area impacted by
crude oil from the Exxon Valdez spill of 1989 are still adversely
affected by the spill's remnants.

Shellfish, Restoration, Nanaimo B.C., Canada

Abstriicr.s. September 2(101 309

OYSTER AQUACULTURE AS FISH HABITAT IN PA-
CIFIC NORTHWEST COASTAL ESTUARIES. Brett Dum-
bault,' D. Armstrong.- G. Hosack," and B. Semens," 'Washmg-
ton State Department of Fish and Wildlife, Willapa Bay Field
Station, P.O. Box 190, Ocean Park, WA 98640, USA; "School of
Aquatic and Fishery Sciences, Box 355020, University of Wash-
ington, Seattle, WA 98195, USA.

Increased pressure on traditionally managed stocks of marine
and anadromous fish, calls for protection of essential fish habitat
under the Magnuson-Stevens Act. and recent listings of several
salmonid stocks under ESA have brought aquaculture activities
that take place in coastal estuaries under increased public scrutiny.

We initiated a study designed to examine the ecological role
that oyster aquaculture plays as habitat in coastal estuaries of the
Pacific Northwest. The goal of the project is to identify and quan-
tify beneficial and adverse impacts of shellfish farming on eel-
grass, juvenile salmonids, and other selected estuarine fauna and
flora and to develop farming practices and recommend manage-
ment protocols that protect or enhance those resources. We present
results of initial fish and invertebrate sampling in selected habitats
from Willapa Bay during Spring and Summer 2001 and an experi-
ment designed to examine the effects of oyster harvesting on
eelgrass habitat. We make a plea to managers to consider oyster
aquaculture areas as fish habitat on a broader estuarine scale.

DEMONSTRATION AND EVALUATION OF COMMER-
CIALLY IMPORTANT BIVALVE CULTURE AND STOCK
ENHANCEMENT METHODS IN REHOBOTH AND IN-
DIAN RIVER BAY, DELAWARE (1998-2001). John William
Ewart,' J. Alderman", and K. Maull," 'Delaware Sea Grant Ma-
rine Advisory Service. Graduate College of Marine Studies, Uni-
versity of Delaware, 700 Pilottown Road, Lewes. DE 19958. USA:
"Delaware Center for the Inland Bays, 467 Highway One, Lewes.
DE 19958. USA.

Delaware's coastal lagoons, known locally as "inland" bays,
have been experiencing the impacts of chronic eutrophication and
sediment erosion resultant from several decades of sustained nu-
trient input and development from within the sun'ounding water-
shed. The Delaware Center for the Inland Bays (CIB) was estab-
lished as a nonprofit organization in 1994 to oversee the imple-
mentation of the Inland Bays Comprehensive Conservation and
Management Plan and to facilitate a long-term approach for the
wise use and enhancement of the watershed. The James Farm
Ecological Preserve, a 150 acre property with frontage on Indian
River Bay, was established in 1998 to serve in part as a demon-
stration site for beneficial land use practices and similar watershed
based activities such as shoreline stabilization, habitat creation/
protection and shellfish stock enhancement to improve water qual-
ity. In 1998. the CIB initiated an ongoing program to evaluate and
demonstrate the technical feasibility of using aquaculture methods
to maintain or improve populations of commercially viable bi-

valves and to create additional habitat for shellfish and other spe-
cies. Current field demonstration activities include low density
plantings (1-2/square foot) of hard clams (Mercenaria merce-
naria). for stock enhancement and recreational fishery improve-
ment; monitoring oyster growth, survival and prevalence of MSX
and Dermo disease in eastern oysters (Crassostrea viigiuica) held
in off-bottom transient gear, submerged trays and Taylor floats;
construction and deployment of a Floating Upweller System
(FLUPSY) for nursery culture of bivalve seed; and establishment
of a 1/4 acre oyster reef to monitor growth, survival, disease preva-
lence and habitat development.

MOLECULAR DETECTION OF FECAL BACTEROIDES
AS SOURCE INDICATORS FOR FECAL POLLUTION IN
WATER. K. G. Field, Oregon State University, Department of
Microbiology, Corvallis. OR 97331, USA.

Fecal contamination of shellfish environments afflicts many
regions worldwide, and carries numerous risks to human health.
Fecal bacteria, pathogenic protista and viruses may be highly con-
centrated in shellfish. Often the problem can't be coiTCCted, be-
cause standard methods of measuring fecal pollution in water do
not distinguish its source. We have developed a PCR-based alter-
native method of fecal source discrimination. The method consists
of amplifying I6S rRNA gene fragments of the fecal anaerobic
Bciclemides-Prevolella group with specific primers. Because
Bacleroides is restricted to gut habitats, its presence in water in-
dicates fecal pollution. Molecular detection circumvents the com-
plexities of growing anaerobic bacteria. We identified Bacleroides
length-heterogeneity PCR (LH-PCR) and terminal restriction frag-
ment length polymorphism (T-RFLP) ribosomal DNA markers
unique to either cow or human feces, and recovered the same
unique fecal markers out of polluted natural waters. We cloned and
sequenced the markers and used the sequences to design PCR
primers that reliably discriminate human and ruminant sources of
fecal contamination. New primers under development will distin-
guish elk/deer, horse, pig. chicken, duck/goose, beaver, and harbor
seal fecal pollution. With this approach, sensitivity is greater than
fecal coliform assays. The method does not require culturing bac-
teria, allows for small sample size and easy sample handling, is
comparable in complexity to standard tests carried out in food
safety and public health diagnostic labs, and lends itself to auto-
mation and high-throughput.

HABITAT RESTORATION AND SHELLFISH STOCK EN-
HANCEMENT INITIATIVES IN NEW JERSEY. Gef Flim-
lin,' and Michael P. Stringer," Rutgers Cooperative Extension.
1623 Whitesville Rd.. Toms Ri\'er. NJ 08755. USA; "NY/NJ Bay-
keeper, Bldg. 18. Sandy Hook. Highlands, NJ 07732, USA.

New Jersey has been a major shellfish production state since
colonial times. The Raritan Bay area once boasted a 400-boat
oyster fleet from the town of Keyport alone. However, industrial

310 Abslracls. September 2001

Shellfish. Restoration. Nunuimo B.C.. Canada

polliilion. water qtiality degradation, and the onset of MSX and
Dermo ha\e practically eliminated natural oyster stocks in most
areas.

Efforts over the past three years have lead to the establishment
of two reefs, one on the Liberty Flats near the Statue of Libeity and
one off the shore of Keyport. These shellstocked reefs have had
cultured oyster seed placed on them and a monitoring program has
begun. Another effort is underway in the Mullica River, the last
area on the state's Atlantic Coast side to produce oysters, to re-
stock the public beds with natural oysters from the seedbeds up-
river. This area will be proposed for a larger scale project for a
Community Based Restoration Program in the year to come.

The Baykeeper and Rutgers Cooperative Extension have been
working with the Baynien's Protective Association and the New
Jersey Shellfisheries Association, two shellfish industry groups, to
propel these initiatives. Primary and secondary school education
projects explaining the benefit of oyster restoration and aquacul-
ture are being done to garner more support for the projects. It is
envisioned that eventually the industry will reap the benefit of
these projects v\hen the reefs may be harvested in the future.

NEW DIRECTIONS FOR SHELLFISH PROTECTION IN

PUGET SOUND. Stuart D. Glasoe, Office of the Governor.
Puget Sound Water Quality Action Team. PC Box 40900. Olvin-
pia, WA 98504. USA.

The Puget Sound Water Quality Management Plan serves as a
cornerstone for shellfish protection in Puget Sound. First adopted
in 1987, the plan employs a suite of strategies to raise awareness,
change behaviors and build and strengthen systems to protect the
health of Puget Sound.

Although the plan was designed with the dual goal of protect-
ing and reopening shellfish beds, early trends were daunting. Be-
tween 1987 and 1989 alone, nearly 33,000 acres of commercial
beds were downgraded as a result of declining water quality and
more intensive monitoring. Trends shifted dramatically in the
1990s as restoration efforts successfully upgraded over 13,000
acres, offsetting nearly identical acreages downgraded during the
period. These results show that the strategies are paying off e\en
in the face of rapid population growth, but they also reveal an
unsustainable emphasis on reactive, restoration techniques.

The region must now shift gears and make better use of proven
approaches to more effectively and permanently prevent contami-
nation of shellfish growing areas. These include better land use
planning under the Shoreline Management Act and Growth Man-
agement Act to preserve shellfish areas as critical areas and natural
resource lands: greater u.se of pollution prevention measures such
as low impact development practices and on-site sewage mainte-
nance programs; widespread creation of surface water utilities to
fund local services and projects: early detection and rapid response
to emerging water quality problems: and better communication

with all audiences to raise awareness on the impacts and tradeoffs
associated wilh pollution and urbanization in shellfish watersheds.

SEMIAHMOO BAY WATER QUALITY PRO.IECT: PHASE
1 FINDINGS. Heather Goble, Shellfish Project Coorduiator.
Georgia Basin Ecosystem Initiative. Clean Water Action Plan, 224
West Esplanade, North Vancouver, EC, V7M 3H7, Canada.

We have now completed Pha.se 1 of a three-year action plan to
identify the major sources of contamination to Boundary Bay and
propose subsequent actions required to mitigate and remediate the
prohibited status of the shellfish growing waters in this area. The
objective of Phase 1 of the Semiahmoo Bay Water Quality Project
was to characterize non-point source contamination patterns within
Semiahmoo Bay. We sampled 19 beach locations along the shore-
line as well as 32 natural and manmade storm water drainages into
Semiahmoo Bay.

Precipitation levels had a positive correlation with bacteriologi-
cal counts for both outfall and ocean sampling sites. This indicates
contamination in storm water outfalls had a direct impact on water
quality along the shoreline of Semiahmoo Bay. Ocean sites dem-
onstrated fecal coliform counts that consistently exceeded accept-
able standards for shellfish growing waters and occasionally ex-
ceeded accepted standards for recreational water quality. The ma-
jority of outfalls had fecal coliform counts that occasionally
reached values associated with sewage-impacted waters.

Boundary Bay sediments and shellfish were tested for common
metals and organics that may adversely affect growing areas. The
levels of heavy metal residues and organochlorines in shellfish
were found to be within acceptable limits. However the tests for
lead and arsenic levels were inconclusive and the shellfish will be
retested using different parameters.

Phase 2 of the Semiahmoo Bay Water Quality Project will
conduct contaminant investigations to reduce the impact of con-
taminated drainage systems on the shellfish growing waters of
Semiahmoo Bay.

REMOTE-SET ALTERNATIVE SUBSTRATE ON LEASED
GROUND: AN INNOVATIVE APPROACH TO REEF RES-
TORATION. William Goldsborough,' Stew Harris,' D. Jack-
son,' D. W. Meritt," and S. Tobash,' 'Chesapeake Bay Founda-
tion, 6 Herndon Avenue. .Annapolis. MD 21403, USA: University
of Maryland, Center for Environmental Science. Box 775. Cam-
bridge, MD 21613, USA.

Oyster reef restoration in Chesapeake Bay faces a variety of
challenges that will require new and innovative approaches.
Among the challenges are: identifying sanctuary locations, obtain-
ing suitable substrate, establishing oysters on the substrate, and
protecting the site from poaching.

Thousands of acres of Bay bottom are currently under lease
from the states of Maryland and Virginia. Much of this ground is

Shellfish. Restoration. Naiiiiimo B.C.. Canada

Abstmcls. September 2001

not being actively farmed and may be available for establishing
reef sanctuaries under agreement with leaseholders.

Oyster shell is considered the best reef material, but supplies of
shell are dwindling. The scale of reef restoration now anticipated
will require development of alternative materials. Marine lime-
stone rock (marl) has been shown to be a good setting substrate
and may prove to be a valuable substitute for shell in reef con-
struction. A second valuable function that marl may provide is that
of a physical obstruction to poaching with traditional harvest gear.

In a pilot project undertaken in the Nanticoke River, a tributary
of Chesapeake Bay in Maryland, marl rocks were set remotely
with hatchery-produced larvae and distributed on a one-acre area
of leased ground to create a field of mini-reefs. In the process a
new and promising approach to reef sanctuary creation was devel-
oped.

RESTORATION OF THE EASTERN OYSTER iCRASSOS-
TREA VIRGINICA) THROUGH A VOLUNTEER PRO-
GRAM IN MOBILE BA^ . ALABAMA. Kimberly A. Hamil-
ton, D. LaDon Swann. Richard K. Wallace. Yolanda J. Brady.
David B, Rouse, Scott Rikard. and Holly Hall. Department of
Fisheries and Allied Aquacultures, Auburn Uni\ersity. Aubuin.
AL 36849. USA. E-mail: hamilka@acesag.aubum.edu

Restoration programs for the eastern oyster. Crassostrea vir-
ginicu, in Mobile Bay, Alabama are used to increase the number of
oysters on remaining beds and to renew ecological functions such
as providing habitat and water filtration. To educate the general
public of the ecological importance of oysters and oyster restora-
tion efforts, an oyster gardening program was created for restora-
tion of natural and constructed reefs. The volunteer program was
implemented in Mobile and Baldwin counties along Mobile Bay
and monitored for 7 months beginning in May 2001.

A total of 30 volunteers representing sites located in unre-
stricted oyster harvesting waters were provided a Taylor float and
1.000 oyster spat per volunteer. The 36.2 mm spat were provided
to the first 15 volunteers and 12.5 mm spat were provided to a
second group of 13 volunteers. Spat were stocked into the Taylor
float (surface area of 0.74 m") and tied to a pier, piling, or weight.
Growth and condition of oysters in Taylor floats were compared
and correlated with water quality parameters (temperature, dis-
solved oxygen, salinity, and water clarity) between each site.

REPRODUCTIVE SANCTUARY FOR THE NORTHERN
QUAHAUG. MERCENARIA MERCENARIA. IN PRINCE
EDWARD ISLAND. Matthew Hardy,' T. Landry.- and A.
Boghen,' Universite de Moncton. Moncton. N.-B.. El A 3E9; "De-
partment of Fisheries and Oceans Canada. Science Branch, Mari-
time Region. Moncton. N.-B.. EIC 9B6. Canada.

West River was historically one of the most productive estu-
aries for quahaugs in PEL However, due to the construction and
subsequent removal of a causeway, in addition to extensive fish-

ing, stocks are now considered to be too low to sustain the present
level of commercial harvesting. Density estimates in West River
ranged from 1.37 to 2.52 quahaugs • m"'. which are low in relation
to other quahaug producing areas.

Several years of research lead to the establishment of a repro-
ductive sanctuary for large quahaug in the subtidal zone of West
Ri\er. This was based on results showing that reproductive success
could be optimized through low-level and inexpensive interven-
tions. Large quahaugs are advantageous as broodstock because
they have high fecundities and they are not significantly affected
by high stocking densities. In vitro fertilization experiments
showed that reproductive success increased significantly with
higher gamete concentration and lower gamete ages. These related
directly to the distance between a spawning pair of adults. The
subtidal zone also provides ecological and socio-economic ben-
efits.

This type of enhancement offers certain advantages over hatch-
ery produced seeding programs. Preliminary work tends to indicate
that the sanctuary concept could be an effective restoration or
enhancement technique for the quahaug. Plankton tows showed a
greater presence of quahaug larvae in West River, following the
establishment of the sanctuary, compared to previous years and
other estuaries. Ultimately, it might be possible to enhance overall
productivity to sustainable levels through properly managed
broodstock areas.

STATUS OF SHELLFISH ENHANCEMENT IN ALASKA.
J. Hetrick, Chugach Regional Resource Commission. 4201 Tudor
Center Drive. Anchorage. AK 99308. USA.

The Alaskan shellfish aquatic farm industry has been growing
steadily with an increase in the number of fanns and an instate
shellfish hatchery for production of indigenous species. Recent
attempts by native villages and local communities to enhance
beaches for subsistence and recreational groups have been mired in
bureaucratic gridlock. At issue is a directed use of a public re-
source, management of enhanced stocks and their interaction with
standing stocks and the lack of enabling legislation for shellfish
enhancement. An outline and review of projects completed and
underway will be presented.

INNOVATIVE COMMUNITY PROCESSES PRODUCE IN-
TEGRATIVE MANAGEMENT TOOLS. Barbara Joughin'
and William Heath." 'Comox Valley Project Watershed Society.
Box 3007. Courtenay, BC, V9N 5N3, Canada, "BC Ministry of

Agriculture. Food and Fisheries. 2500 Cliffe Avenue, Courtenay,
BC, V9N 5M6, Canada.

Baynes Sound, located in western Canada on the east coast of
Vancouver Island, is a highly productive area for shellfish culture,
salmon, herring and waterbirds. Over the past decade. Baynes
Sound has experienced increasingly degraded water quality from

312 Ahsiracts, September 2001

Shellfish, Restoration, Nanaimo B.C., Canada

non-point source pollution, as well as loss and degradation of
sensitive w ild habitat. The Baynes Sound Round Table was formed
in 1994 to improve water quality in Baynes Sound, and has guided
a series of collaborative community stewardship projects that ad-
dress threats to environmental and economic hcallh m Baynes
Sound. These projects create opportunities for citi/ens. govern-
ments and shellfish growers to work together to identify and re-
mediate non-point source pollution impacts in Baynes Sound.
Hundreds of citizen volunteers have participated in recent stew-
ardship programs. Community involvement and stakeholder col-
laboration have established a strong foundation for proactive and
integrated remedial planning and programming. An important tool
has evolved from the Baynes Sound process - the development of
the 'State of the Sound' Program. The State of the Sound Program
is a long term monitoring, reporting and planning process that
measures and reports the health of Baynes Sound. The program
uses a geographic information system (GIS) to gather and analyse
data for water quality and other indicators. Results are used to plan
actions, and are communicated to the community to help increase
public awareness and involvement. Information becomes acces-
sible, 'crisis control" is replaced with comprehensive management,
and an effective tool is available to assist with planning proces.ses
such as local liquid waste management programs.

ADVANCES IN SHELLFISH SAFETY ASSURANCE AND
PARTICIPATION IN SANITARY GROWING WATER
MONITORING PROGRAMS BY THE BC SHELLFISH
AQUACULTURE INDUSTRY. Brian Kingzett. Kingzett Pro-
fessional Services Ltd. .^21 St. Julian, St. Duncan B.C. V9L 3S5,
Canada.

The BC Shellfish Growers Association (BCSGA) represents
the majority of shellfish production in British Columbia. Promot-
ing shellfish safety and involvement in the growing water man-
agement have become significant aspects of the work of the asso-
ciation and its members. Decreases in regulatory monitoring bud-
gets and increased demands for service by shellfish aquacullurists
have led to proactive industry partnerships and programs. These
involve industry participation in monitoring for Vibrio paia-
bacinolxiiciis. an industry driven farm based Vp control program,
involvement in sanitary growing water monitoring and remedia-
tion, and maintenance of marine biotoxin sentinel sites. Currently
the BCSGA is participating in a national program developing stan-
dard tnethodologies and cuiriculum for industry growing water
sampling and farm based HACCP for shellfish culturists.

DEVELOPMENT OF INTENSIVE DEEPWATER OYSTER
CULTURE SYSTEMS IN BRITISH COLUMBIA. Brian

Kingzett, Kingzett Professional Services Ltd, .^21 St. Julian. St.
Duncan B.C. V9L 3S5. Canada.

Socio-political reasons have restricted the growth of the shell-
fish culture industry in British Columbia during the last decade.
The industry is relatively small and recent growth has primarily
come about on small deep-water sites. Shellfish growers in British
Columbia have developed a series of approaches to developing
intensive oyster production in deep-water (off bottom) culture sys-
tems. This has invohed adapting international techniques and de-
veloping unique technologies, which allow significant production
from small areas.

USING INNOVATIVE STORMWATER CONTROLS FOR
WATER QUALITY IMPROVEMENTS AND OYSTER
HABITAT RESTORATION IN THE BARATARIA-
TERREBONNE NATIONAL ESTUARY. K. E. Landruni,'
K. M. St. Pe.' B. Ache.- and F. Kopfler,-' 'Barataria-Terrebonne
National Estuary Program. P.O. Box 2663. Nicholls State Univer-
sity. Thibodaux. LA 70310. USA; 'Battelle. 191 East Broad Street.
Suite 315. Athens, GA 30601. USA; 'EPA/Gulf of Mexico Pro-
gram. Stennis Space Center, Building 1 103, Room 202. MS 39329.
USA.

The loss of nearly 22 square miles of emergent wetlands per
year in the Barataria-Tenebonne National Estuary represents the
imminent loss of a nationally significant wetland resource and
threatens the area's unique culture and local infrastructure. Oppor-
tunities exist for rerouting some of the estuary's 260 stormwater
pump station outfalls within the estuary to improve the water qual-
ity associated with these discharges prior to entering historically
productive oyster grounds. Runoff from rural and agricultural ar-
eas are collected in borrow canals within the existing levee sys-
tems and are generally pinnped into large man-made canals to
ensure rapid evacuation of stormwater.

The Barataria-Terrebonne National Estuary Program and
EPA's Gulf of Mexico Program are spearheading efforts to moni-
tor and assess changes in estuarine \'itality near rerouted pump
station outfalls to demonstrate the benefits of this unique process.
Qualitative evidence of the positive effects of the redistribution of
stormwater into adjacent wetland areas is visually evident through-
out both basins, although only limited quantitati\e assessment has
occurred. Redirecting discharges so that freshwater is retained in
adjacent wetlands rather than moved through them has been dem-
onstrated to maintain lower salinities, promote vigorous plant
growth through nutrient uptake, and lead to pathogen degradation.
Numerous studies provide evidence of wetland uptake of pollut-
ants and nutrients in constructed wetlands and riparian fringes, and
the enhancement of marshes adjacent to hurricane protection
levees would provide additional storm surge protection for prop-
erties and local infrastructure.

Shellfish, Restoration, Nanaimo B.C., Canada

Abstracts. September 2001 313

WATER QUALITY IMPROVEMENT: A FOCUS ON NEW
TECHNOLOGIES. Kevin L. LeBlanc, Fisheiies and Oceans
Canada. Gulf Fisheries Centre, 343 University Street, Moncton.
New Brunswick, EIC 9B6, Canada.

Water quality improvement requires effective monitoring, fi-
nancial support, partnerships, remediation and bio/socio-economic
planning. However, tools that clearly define sources of contanu-
nation remain the backbone for effective water quality improve-
ment. Sewage is typically measured through the use of an indicator
species such as faecal coliform bacteria, but effective remediation
requires a clear link to the source of pollution. The South-western
New Brunswick Clam Resource Committee (CRC), New Bruns-
wick, Canada, has improved water quality and monitoring for the
reclassification of shellfish growing areas since 1995 using the
above mentioned strategies. By 2001, the CRC has increased the
overall shellfish growing area by 277r. However, an additional
38% of shellfish growing areas, equivalent to 434 hectares ( 1 ,074
acres), could be available for harvest if the identification of the
actual source of fecal coliform was possible. Tools such as genetic
markers and gene sequencing of bacteria are examples of new
approaches in differentiating between different sources of fecal
coliform. These innovations will be critical in establishing feasible
and effective courses of remediation in shellfish growing areas.
Furthermore, such advances could lead to the development of mo-
lecular-based kits for monitoring activities, similar to those used
for biotoxin monitoring. The continued development of science in
these fields is necessary for the continued success of water quality
improvement initiatives.

COMMERCIAL-SCALE OYSTER PRODUCTION FOR
REEF RESTORATION AND STOCK ENHANCEMENT.
A. T. Leggett. A. Blow, W . Goldsborough. R. D. Brumbaugh,

Chesapeake Bay Foundation, 142 W. York Street, Suite 318, Nor-
folk, VA 23510, USA.

Oyster restoration in Virginia has focused on the construction
of three-dimensional reefs made from oyster shells since the early
1990s. Since 1997, the Chesapeake Bay Foundation (CBF) has
assisted with his effort by organizing and training citizens and
students to grow oysters (Crassostrea virginica) using small-scale
aquaculture techniques. To date, approximately I . I million oysters
have been grown using these small-scale techniques, and stocked
on Virginia Sanctuary reefs.

In 2000, CBF initiated the Virginia Oyster Aquaculture Pro-
gram to complement these volunteer-based efforts by producing an
additional one million oysters per year for Virginia reefs. A com-
mercial-scale oyster farm was designed and put into operation
growing oysters in ADPl mesh bags contained in commercial oys-
ter trays stacked on PCV racks. The oysters were placed directly in
the trays when they reached an appropriate size (>25 mm) with
approximately 1,500 oysters per tray. After eleven months, over
930,000 oysters (mean size = 52 mm) were harvested using vol-

unteer labor and transplanted onto eight sanctuary reefs. Survival
to transplanting for the three stocks used in the program was ap-
proximately 85%, and first year capital expenses were approxi-
mately $62,400. The oysters produced through this program were
used as in-kind match for newly appropriated federal funds dedi-
cated to Virginia oyster restoration. Future considerations of the
program include genetic aspects of the oyster stocks used and the
application of new techniques including the use of a floating up-
weller (FLUPSY) for the nursery phase of the grow-out.

PROTECTION AND RESTORATION: WASHINGTON
STATE SHELLFISH PROGRAM. Donald Lennartson, Office
of Food Safety and Shellfish Programs, Washington State Depart-
ment of Health, 7171 Cleanwater Lane, Building 4, Olympia, WA
98504, USA.

The Washington State Department of Health oversees 126
shellfish growing areas with over 1.400 marine water sampling
stations. The state shellfish industry produces over 80% of the
shellfish harvested on the west coast, not including geoducks. This
rich shellfish resource faces a range of threats, from rural land use
activities to rapid urban growth. Prevention of classification down-
grades and the restoration of water quality require careful plan-
ning, strong technical skills, tough regulatory tools, and creative
partnerships among local, tribal, state, and federal agencies.

The stimulus for shellfish restoration activities was the inno-
vative Puget Sound Water Quality Management Plan, whose pur-
pose is "to restore and protect the biological health and diversity of
Puget Sound". A major component of this ongoing Plan is the
Shellfish Protection section, which outlines seven action elements,
the first two of which address shellfish protection policy and the
restoration of commercial shellfish beds.

In keeping with the spirit of the Plan, the Department of Health
initiated an Early Warning System, a proactive measure which
alerts county governments to the threat of classification down-
grades. If preventative measures fail and a downgrade does occur,
state law requires the county to establish a Shellfish Protection
District, and a Closure Response group is convened to formulate
actions in the watershed to identify and correct the pollution
sources.

ABALONE RESTORATION THROUGH COLLABORA-
TIVE PARTNERSHIPS IN BRITISH COLUMBIA. B. G. Lu-
cas, and A. Campbell, Stock Assessment Division. Science
Branch. Fisheries and Oceans Canada. Pacific Biological Statio.
Nanaimo. BC V9R 5K6, Canada.

Diverse stakeholder groups are collaborating to test potential
methods to restore threatened abalone populations in British Co-
lumbia. Fisheries and Oceans Canada is working with six groups
throughout coastal BC in an attempt to halt further decline of
abalone stocks and restore populations to self-sustaining levels.

314 Abstracts. September 2001

Shellfish. Restoration. Nanainio B.C.. Canada

Five aquacuiture project.s are underway to develop the culture
technology required to provide northem abalone seed for pilot
restocking experiments. In addition, four projects are currently
promoting local community stewardship of abalone resources
through public education, increased awareness and, in some cases,
experimental manipulations of wild abalone populations. These
projects are providing training and employment in local commu-
nities which have been impacted by declining opportunities in the
fishing industry.

SITE ASSESSMENT. SELECTION AND MONITORING
METHODS FOR ABALONE RESTORATION NF:AR KIT-
KATLA. BRITISH COLUMBIA. B. G. Lucas, A. Campbell.
and D. Brouwer. Stock Assessment Division, Science Branch,
Fisheries & Oceans Canada, Pacific Biological Station, Nanaimo,
BC V9R 5K6. Canada.

Several pilot projects are currently underway to attempt to re-
store abalone populations in British Columbia (BC). Near Kitkatia,
on BC"s north coast, an extensive process of assessing, selecting
and surveying sites for pilot abalone rebuilding experiments was
recently completed. General areas for potential experiments were
selected in conjunction with local First Nation advisors and Fish-
eries & Oceans Canada staff.

In September 2000, five divers spent 7 days conducting pre-
liminary surveys to locate specific areas of suitable habitat that met
predetermined site selection criteria. After analysis of the survey
results, 16 potential sites with similar characteristics were chosen.
In April 2001, three dive teams spent 6 days permanently marking
15 sites and collecting detailed baseline information at those sites.

The proposed experiments will test the sur\ i\ al and recapture
rates of seeded juvenile abalone while examining the effects of sea
urchin sizes and densities in the experimental plots. Long teitn
monitoring of the sites is expected to continue for at least five to
ten years after the experiments begin.

EFFECTS OF REEF ARCHITECTURE AND SCALE ON
FISH UTILIZATION OF OYSTER REEFS IN VIRGINIA.
Mark W. Luckenbach. ,|. Nestlerode, P. G. Ross and A. J.

Birch, College of William and Mary, Virginia Institute of Marine
Science, Eastern Shore Laboratory, Wachapreague, VA 23480.
USA.

Current efforts to restore oyster reefs in the Chesapeake Bay
are directed toward establishing self-sustaining reef sanctuaries
that provide valuable ecological functions, such as benthic-pelagic
coupling and support of increased diversity and production of mac-
robenthos and finfish. Recent evidence has revealed the impor-
tance of two components of reef architecture — vertical relief and
interstitial space — on the development of oyster populations on

restored reefs. We will present data which show that finfish utili-
zation varies between "reefs" with and w ithout viable oyster popu-
lations. A third component of reef architecture — aerial extent or
.scale — is expected to affect both reef development and utilization
by fish. In conservation biology this topic has often been charac-
terized as the SLOSS (Single Large or Several Small) debate, but
is more generally about optimizing the scale of a bio-reserve or
sanctuary to support the desired species. To investigate this issue
for oyster reefs we have initiated a large-scale restoration experi-
ment in the Chesapeake Bay. In a replicated block design, we have
constructed high relief reef bases ranging in size from 400 sq ni to
8000 sq m and are characterizing the development of resident and
transient assemblages of organisms on the reefs. We will present
data from the first year of the study on finfish utilization of these
reefs.

THE ROLE OF AQUACULTURE IN THE RESTORATION
EFFORT TO SAVE WHITE ABALONE [HAUOTIS SO-
RENSENI) IN CALIFORNIA. Thomas B. McCormick. Chan
nel Islands Marine Resource Institute. 323 E. Matilija Street. Ojai.
CA 93023, USA.

Stocks of white abalone {Haliolis sdicnscni) in southern Cali-
fornia declined precipitously from an a\'erage of 2,000 to 10.000
abalone per hectare in the to 1.6 per hectare in the 1990s. A
short-lived fishery in the 1970s landed 270 metric 1970s tons from
depths of 20 to 50 m. The fishery was closed in 1997 and in 2001
the white abalone was listed as an endangered species, the first
marine invertebrate to be .so classified in the USA.

The White Abalone Working Group was formed by federal and
state agencies, scientists, universities, non-profit organizations,
and mariculturists as a proactive step towards preventing the ex-
tinction of white abalone. The group developed the following four-
step plan for recovery of the species: ( 1 ) survey historic fishing
grounds to locate survivors: (2) collect and hold adults as breeding
stock: (3) produce a new generation of young adult abalone in the
hatchery: and (4) introduce hatchery grown adult animals into
refugia to reestablish self-sustaining wild populations.

Success has been achieved in maintaining and spawning white
abalone in captivity. Abalone cultivation systems incorporate the
use of the red macrophyte. Pacific dulse {Pciliiniria mollis) as both
a nutritious feed and biofilter. Large numbers of ju\eniles are
being raised using commercial techniques. This program differs
from other enhancement efforts in that the abalone will be raised
to adult size (10 cm shell length) prior to release. Larger animals
such as this should be better able to resist predation and are ca-
pable of spawning immediately.

Shellfish, Restoration. Nanaimo B.C.. Canada

Abstracts. September 2001 315

BOAT WASTE AND SHELLFISH CLOSURES: A COM-
PARISON OF CANADIAN AND UNITED STATES POLI-
CIES. Jim Mclsaac, Coastal Enterprise and Resource Coopera-
tive Association. 202-1931 Mount Newton X Rd.. Sidney, BC
V8M 29A. Canada.

Diseases such as hepatitis, typhoid and cholera have been trans-
mitted by untreated boat sewage. Popular beaches and shellfish
areas attract boaters; the resulting contamination from boat sewage
creates health concerns for millions of shellfish lovers. Swimmers
that frequent contaminated waters have been known to develop
skin rashes, amoeba dysentery, and parastic worm diseases. Boat
sewage has closed beaches and shellfish harvesting areas around
the world.

In 1991. the Canadian government passed the Pleasure Craft
Sewage Prevention Regulations intending to protect the marine
environment from the negative impacts of sewage disposal. Since
passing the Regulations, nine west coast marine waterbodies have
been designated no-discharge zones (NDZl. A further 54 marine
sites have been recommended for NDZ and are under review.
However, no reduction in breach or shellfish closures has been
noted.

In contrast, the United States Congress passed the Clean Vessel
Act in 1992. Today, all vessels inside US waters are required to
have a marine sanitation device. Seven states have all their surface
waters designated as NDZ. An additional eleven other states have
segments of their waterbodies designated as NDZ. Washington and
Massachusetts have already identified a reduction in beach and
shellfish closures.

With intentions from both federal governments so evident, why
are the results so different? In the United States, the Clean Water
Act and the Clean Vessel Act clearly lay out areas of jurisdictional
responsibility and financing arrangements. Implementation plans
are in State control where boat waste disposal plans were devel-
oped. In Canada, boat waste policies are caught in multi jurisdic-
tional bureaucracies with overlapping acts and conflicting regula-
tions, resulting in no boat waste disposal being developed.

SUCCESSION IN MUSSEL COMMUNITIES: THE IMPOR-
TANCE OF WHAT IS MEASURED. C. W. McKindsey, En-
vironmental Sciences Division. Department of Fisheries and
Oceans, Maurice Lamontagne Institute, 850 Route de la Mer. PC
Box 1000; Mont-Joli, QCG5H 3Z4. Canada.

This study examines the community of organisms in crevices in
the St. Lawrence estuary, Canada, through succession and shows
how extrapolations from the study of a limited number of organ-
isms to the entire coinmunity of macro-invertebrates may not be
valid.

I first examined how varying the lower size limit of the organ-
isms considered in the analyses (organisms >0.5. I, 2, 4. and 8
mm) alters the observed trends in community structure (richness
and diversity) through succession. Diversity was maximal in mid-

succession for the >2 and >4 mm size groupings but continued to
increase through succession for the >1 and >0.5 mm groupings
whereas richness always increased through succession for all
groupings. Examining one of four distinct zones in crevices gave
the same result as examining the total community, improving sam-
pling efficiency. However, the other three zones did not show this
predictive power.

I then examined whether variation in richness and diversity
through succession was a function of the structural heterogeneity
(SH) provided by the dominant taxa or of the age of the commu-
nities per se. This was done by comparing control cre\ice com-
munities at four successional stages to ones that had been modified
such that only the dominant species remained following a brief
period to allow for colonization (mimic crevices). Within three
months, control and mimic communities resembled each other in
terms of both diversity and richness and the trends min'ored those
observed four months early, thus supporting SH. Nonparametric
multivariate analyses supported these conclusions.

AN ARCHAEOLOGICAL PERSPECTIVE ON THE CUL-
TURAL SIGNIFICANCE OF SHELLFISH RESOURCES TO
FIRST NATIONS IN THE GEORGIA BASIN. Eric McLay,

Archaeologist, Hurqumi'num Treaty Group.

Archaeology offers an invaluable perspective toward under-
standing the fundamental importance of shellfish resources to
coastal First Nations in British Columbia. This study examines the
relationship between archaeological site locations and shellfish
resources on Valdes Island, a large southern Gulf Island in the
Georgia Basin. British Columbia. A close association is observed
to exist between the location, size and content of archaeological
shell deposits and the distribution of local shellfish resources.

It is argued that Central Coast Salish settlement patterns on
Valdes Island demonstrate a precontact economic orientation to-
ward exploiting productive coastal resource zones, particularly
sandy intertidal habits, where populations aggregated to collect
localized, predictable and abundant bivalve shellfish and other
sandy foreshore resources. This archaeological study has important
implications for modelling strategies of precontact settlement and
subsistence in the Georgia Basin, and for considering the cultural
significance of shellfish resources to modem First Nations.

RESTORING OYSTERS TO THE CHESAPEAKE BAY: A
COORDINATED EFFORT INVOLVING GOVERNMENT.
UNIVERSITIES. COMMUNITY GROUPS, AND INDUS-
TRY. Donald W. Meritt," S. Tobash,' C. S. Frentz,' W.
Goldsborough,' and S. Reynolds,^ 'University of Maryland. Cen-
ter for Environmental Science, Box 775. Cambridge. MD 21613,
USA; "Maryland Oyster Recovery Partnership, Box 6775, An-
napolis, MD 21401, USA; ' Chesapeake Bay Foundation, 6 Hern-
don Avenue, Annapolis, MD 21403, USA.

Once the world leader Chesapeake Bay oyster harvests are near
all-time lows today. Overexploitation, disease and habitat loss

316 Ah.slnuts. September 2001

Shellfish. Restoration. Nanaimo B.C.. Canada

have all contributed to this decline. Traditional techniques of
spreading shell to collect spat are no longer reliable to produce
consistent numbers of high quality oyster seed because, oyster
diseases are highest in areas where natural spatfall is most abun-
dant.

Utilizing hatcheries and a strategy designed to minimize the
risk of infection, oyster seed have been produced with little or no
Dermo. Hatchery spat have been used to accomplish a wide range
of objectives including supplying citizens growing oysters for res-
toration, re-seeding harvest grounds in areas sensitive to disease,
involving commercial oystermen in restoration, and establishing
ecological sanctuaries. This program has grown dramatically over
the past decade and now is responsible for planting tens of millions
of oyster spat each year.

The Maryland Oyster Recovery Partnership along with the Uni-
versity of Maryland Center for Environmental Science, the Chesa-
peake Bay Foundation. Maryland Watermen's Association and
local community groups have been successful in producing the
seed oysters, preparing the bottom, and planting the seed in a
coordinated effort.

OYSTER RESTORATION IN CHESAPEAKE BAY: I CRI-
TERIA FOR SITE SELECTION. Donald VV. Meritt,' S. To-
bash,' K. T. Paynter," and T. Koles." 'University of Maryland.

Center for Environment Science, Box 775, Cambridge, MD.
21613. USA; "Department of Biology. University of Maryland.
College Park. MD 20742, USA.

Interest in restoring historical oyster reefs or in creating new
oyster reefs in Chesapeake Bay has heightened in recent years. The
state of Maryland has a long history of activities aimed at oyster
repletion for the public fishery. Additionally, private oyster culture
was once a major producer of market oysters in the Bay region.
Disease, overharvest, and habitat loss have all contributed to the
decline in oyster populations and the resultant harvests are al near
all time lows.

The economic importance of a healthy oyster fishery to water-
front communities is well documented. Of more recent interest is
the concern for the ecological role of healthy oyster communities
to the overall health of the Chesapeake Bay. Oyster restoration for
non-commercial uses is becoming more commonplace while the
more traditional repletion program activities are being modified in
an attempt to become more efficient.

Many community groups tmd it desirable to have an oyster reef
in their local area. Not every site is suitable for oyster reef con-
struction. Many exhibit some but not all of the characteristics
needed for successful oyster growth and survival. Success of any
given project depends upon accurate as.sessment of these charac-
teristics and the use of proper construction techniques.

GENETIC MONITORING OF OYSTER STOCK EN-
HANCEMENT IN THE CHOPTANK RIVER. CHESA-
PEAKE BAY, MARYLAND. USA. C. A. Milbury. and P.M.
Gaffney, Graduate College of Marine Studies, University of Dela-
ware, 700 Pilottown Road, Lewes, DE 19958, USA.

The increased spread of parasitic diseases (primarily MSX and
Dermo), in conjunction with overharvesting, has led to the rapid
decline of many Eastern oyster (Ciassostrea virginica) popula-
tions, especially in Chesapeake Bay. Regional variation in disease
resistance to these parasites may be useful in restoration efforts. In
collaboration with the University of Maryland Horn Point Lab. we
have proposed to assess the success of recent enhancement efforts
within the Chesapeake Bay using molecular genetic markers.

In 1997. oysters propagated from Louisiana broodstock were
planted at ten sites within the Choptank River. Maryland. C. vir-
,i;iiiica exhibits regionally diagnostic DNA profiles in the form of
North Atlantic, South Atlantic, and Gulf Coast 16s mitochondrial
haplotypes. The presence of the Gulf Coast haplotype in newly
settled spat confirms the survival and propagation of the Louisiana
broodstock. DNA sequencing techniques developed by Pyrose-
quencing Inc. were used to determine the mitochondrial haplotypes
of a large number of oyster spat collected at several bars through-
out the Choptank River estuary. This rapid, mass screening method
revealed that 94% of spat collected were of the North Atlantic
haplotype and approximately 59c were South Atlantic. Of 2,466
spat screened, four possessed the Gulf Coast haplotype (0.2%).
Haplotype identifications were confirmed using restriction frag-
ment length polymorphisms in other regions of the mitochondrial
genome. The use of these genetic markers has enabled us to as.sess
the survival, propagation, and dispersal of the Louisiana oyster
stock within the Choptank River. Chesapeake Bay.

OPEN SAANICH INLET SHELLFISH BEDS: A COLLABO-
RATIVE APPROACH. Rob Miller. RLN Cameron. Environ-
mental Programs. Capital Regional District. 524 Yates Street. Vic-
toria British Columbia, V8W 2S6, Canada.

The Saanich Peninsula and Inlet are located on the southern tip
of Vancouver Island near Victoria. British Columbia. The Saanich
Inlet shellfishery is an imponant food source to three Saanich
Peninsula First Nation Bands and the community. Many of the
shellfish beds on the east coast of Saanich Inlet are closed for
harvesting due to elevated levels of fecal coliform bacteria. The
primary method of fecal coliform transmission from the land to the
marine environment is stormwater.

The Capital Regional District (CRD) Stomiwater Quality pro-
gram works to limit the impacts of stormwater runoff to the envi-
ronment and public health and protect freshwater and nearshore
marine ecosystems. In 1999, Environment Canada and the CRD
established a project titled Open Saaiiicli Inlet Shellfish Beds
iOSISB). This collaborative project will run until 2003 and in-

Shellfish. Restoration, Nanaimo B.C.. Canada

Abstracls. September 2001 317

volves all levels of government and the community working to-
wards opening shellfish beds.

Under this project, the sources of fecal coliform contamination
responsible for shellfish closures are identified. The jurisdiction(s)
involved then work toward their reduction/elimination.

Limited nearshore marine sampling is also undertaken to de-
termine fecal coliform levels near shellfish beds and their associa-
tion with stormwater. The data are particularly relevant to moni-
toring changes over time and measuring the success of efforts to
open shellfish beds.

Since the project began there has been a significant decrease in
the number of stormwater discharges with high fecal coliform
le\els along this 27 kilometer coastline. Limited marine nearshore
sampling has also shown a significant reduction in fecal coliform
levels.

HYDROACOUSTIC SE.ABED CLASSIFICATION TECH-
NOLOGY APPLIED IN SHELLFISH PRODUCTIVITY RE-
SEARCH. Marc Ouellette and T. Landry, Fisheries and Oceans
Canada. Gulf Fisheries Centre. Science Branch. Moncton. NB.
EIC 9B6. Canada.

A good understanding of the relation between shellfish popu-
lation dynamics and their habitat is essential in order to develop
successful enhancement and/or restoration methods for our natural
populations through shellfish or habitat management. The physi-
cal, chemical and biological characteristics of the seabed are key
elements in shellfish productivity. In the past, this element has
proven to be difficult and expensive to evaluate in part due to our
inability to efficiently classify seabed on a large scale. With the
recent development of new data acquisition and analysis tools
(Global Positioning System. Geographic Information System, geo-
statistics and hydroacoustics), however, it is now possible to map
seabed areas within a reasonable time and financial framework.
Most importantly, these new technologies are capable of providing
accurate and repeatable measurements. This will provide us with
the ability to measure spatial and temporal variation of the benthic
assemblage in relationship to mollusc productivity.

A new shallow water seabed classification system, QTC View
(Series V), is being used for the first time to survey sites in the
Gulf of St. Lawrence and Fundy Bay were research is presently
being carried out on.oyster bed restoration, quahaug population
management and mussel farming.

THE LIVING VENEER: CHARACTERIZING HABITAT
STRUCTURE CREATED BY OYSTERS. Paynter, Kennedy.
and Elizabeth Flynn, Department of Biology. University of Mary-
land, College Park. MD 20742, USA.

Oyster reefs, like coral reefs, are biogenic structures. The settle-
ment and growth of oysters create them. Therefore, the density of
settlement, growth rate, and the mortality rate of oysters on a given

reef will greatly affect the structure of the reef and the habitat
created. Natural densities of oysters up to 1 .000 indi\ iduais/m"
have been reported while the mean density of exploited reefs in
Maryland is less than 3 oysters/m". Although the large-scale,
three-dimensional relief aspects of oyster reefs has receixed much
attention, the structure created by oysters themselves — the spaces
among and between living oysters — is less well studied.

We have attempted to characterize the habitat created by dif-
ferent densities of oysters both in the lab and in the field. A variety
of approaches are available for this kind of measurement including
estimates of the "space" created by oyster shells in various assem-
blages, a chain index, and fractal dimension. While interstitial
volume was not significantly different between clumps and loose
shell, chain indices and fractal dimension estimates were quite
different. Seabed roughness may also be a useful measure as it
takes into account shell height and nearest-neighbor distances.
Such characterization is important to understand the relationship
between physical structure and habitat value. We hope to associate
the structural differences between reefs with benthic fauna asso-
ciated with them as an as.sessment of habitat value.

OYSTER RESTORATION IN CHESAPEAKE BAY: II. IM-
PACTS OF WATER QUALITY AND DISEASE. Paynter,'
Kennedy,' T. Koles.' D. Meritt," and S. Tobash.' 'Department
of Biology, University of Maryland, College Park, MD 20742,
-Horn Point Laboratory, Center for Environmental Sciences, Uni-
versity of Maryland, Cambridge, MD 21613, USA.

The siting of oyster restoration projects is typically guided by
several considerations. Physical habitat present (ie.. "hardness of
the bottom") is typically the first consideration. Local disease
prevalence is sometimes assessed and water quality is usually as-
sumed. Dissolved oxygen (DO) levels are typically not measured
prior to restoration.

Oxygen dynamics in shallow zones of estuaries are not well
understood. While general trends typical of eutrophic systems are
apparent including surface overproductivity, stratification, and
deep anoxia, relatively little attention has been paid to water qual-
ity in shallow (<6m) nearshore. sub-littoral zones. Furthermore,
the effects of hypoxia (<4mg Oj/l) on benthic communities and
organisms are not well understood. We have observed chronic
hypoxia on many oyster reefs in Chesapeake Bay. One reef was
exposed to <3mg 0,/l for nearly 72 h with only 2 or 3 brief (<30
min) episodes of higher DO. DO lesels measured by continuous
monitoring at this reef were <5 mg/l 90% of the time throughout
August and September, 2000. and May and June. 2001. DO levels
measured in 2001 at approximately 30 other reefs throughout the
Maryland portion of the Bay was typically <5 mg/l and often <3
mg/l. These levels of hypoxia could have important detrimental
affects on restoration projects.

318 Alxsiracts. September 2001

Slielltish. Restoration. Nanuimo B.C.. Canada

EARLY COMMUNITY DEVELOPMENT OF OYSTER
REEFS IN VIRGINIA: EFFECTS OF REEF SCALE. P. G.
Ross, M. W. Luckenbach and A. J. Birch, College of William
and Mary. Virginia Institute of Marine Science. Eastern Shore
Laboratory. Wachapreague. VA 23480, USA.

Traditionally, oyster reef restoration in the Chesapeake Bay
region has primarily focused on oyster production. Increasingly,
however, efforts have included restoration of associated commu-
nities and the overall ecological function of self-sustaining reefs as
management goals. The communities that develop in association
with oyster reefs in Chesapeake Bay. VA have been shown to be
diverse and ecologically important. Additionally, reef architecture,
such as vertical relief and interstitial space, has been shown to be
important to developing and maintaining community diversity.
Another, previously unaddressed architectural component, aerial
extent or scale, is also expected to affect community development.
In a replicated block design, we have constructed high relief reef
bases ranging in size from 400 sq m to 8000 sq m and are char-
acterizing the development of the associated resident and transient
assemblages of organisms. We will present data evaluating early
community development on these different scale subtidal reefs.
We compare reefs of differing scales (i.e.. small versus medium
versus large size reefs), but also make comparisons within a reef
(e.g.. inner portions of reef base versus reef edge). Substrate for
constructing reefs is often limiting and expensive and resource
managers face tough decisions on how to allocate scarce resources
(e.g.. construct several large or many small reefs). When maxi-
mizing diversity and ecological function of reefs is a management
objective, knowing the impact of reef scale can be a valuable asset
for makin" such decisions.

GENETIC AND PATHOGENIC ASPECTS IN SHELLFISH
RESTORATION OF SCALLOP POPULATIONS. Rejean
Tremblay, and T. Landry, UQAR-MAPAQ. Centre Aquacole
Marin, Grande-Riviere, Que.. GOC IVO; Gulf Fisheries Center.
DFO. Moncton. N.B.. EIC 986. Canada.

With recent declines in the scallop fishery in Atlantic Canada,
several projects on stock enhancement are being conducted. The
success of shellfish population restoration is directly related to the
fitness of the scallop seed. Results obtained from restoration ac-
tivities of Giant scallops (Placopecten magellanicus) populations
will be used to discuss of the impacts of genetic and pathogenic
aspects on the fitness of scallops and the success of these activities.
Factors that will contribute to reduce the overall genetic variability
may compromise the capacity of a species to adapt to environ-
mental changes and to resist to pathogens. Thus, the long-term
survival of that species may be compromise. Indeed, if genetic
variation within individual populations is reduced, there will be
less of a basis for future adaptation within a given population.
Genetic changes often occur during the hatchery process. In Que-
bec, scallop enhancement activities are carried out using juveniles

produced in hatchery, or collected over scallop beds and that are
either re-released in the same area or transfer to other areas. The
impacts of all these practices on genetic variability of populations
are not known. The genetic of shellfish produced in hatchery are
frequently altered through inbreeding, selective breeding or do-
mestication with an overall reduction in genetic variability. This
can also be true for seed collected on artificial collectors. These
enhancement activities could result in hybridization, with the pos-
sibility of reducing the genetic variation and hence, the fitness of
the enhanced population.

WATER QUALITY AND OYSTER HEALTH {CltASSOS-
TREA VIRGINICA): AN INTEGRATED APPROACH TO
DETERMINING HABITAT RESTORATION POTENTIAL.

Aswan! K. Volety.' S. Gregory Tolley,' and James T. Win-
stead," 'Florida Gulf Coast University. Division of Ecological
Studies. 10501 FGCU Blvd. S.. Fort Myers. FL 33965. USA: "U.S.
Environmental Protection Agency, Gulf Ecology Division. 1 Sab-
ine Island Drive. Gulf Breeze. FL 32561. USA.

The influence of water quality and season on disease preva-
lence and intensity, gonadal condition, recruitment potential, and
growth of oysters was examined monthly at five locations along
the Caloosahatchee River estuary. Florida. Habitat suitability of
oyster reefs for fishes and decapod crustaceans was examined at
three of these sites. Higher temperatures and salinities favored the
parasite Perkinsiis mahuiis. and histological analyses revealed the
presence of several additional parasitic or comniensalistic organ-
isms.

Comparison of mortality among sites indicated that juvenile
oysters tolerated salinities of 15-38 ppt. Spat recruitment was
higher at sudtidal (1-5 spat/shell) than at intertidal locations,
where sparse oyster distribution and swift currents appeared to
limit settlement success. The late peak in gameiogenesis (August
to September) observed at all sites may have resulted from reduced
salinities during May to Jul) or may imply that oysters spawn
twice per season. Oyster-reef habitat supported a fish and decapod
assemblage averaging 77 individuals m~" or 14 individuals 1
oyster cluster.

Reef-resident fishes included Gobieso.x stnimosits, Chasmodes
sahurrae. Gobiosoma rohiisnim. and Opsamis beta: xanthid and
porcellanid crabs represented the dominant crustaceans. Species
diversity (H') did not vary among sites: however, significant dif-
ferences in density (individuals 1"' oyster cluster) were found in
the fishes C. strumosiis and C sabuirae and in the crabs Paiwpeus
herhstii and Petrolisthes arimitus. Overall, results suggest that pe-
riodic freshwater releases may benefit oysters by lowering the
salinity and thus the intensity of parasite infection. PerkinsKS imiri-
mis. It should be cautioned that the long-term effects of low sa-
linity on oysters have not been investigated.

Shellfish, Restoration. Nanaimo B.C.. Canada

Abstracts. .September 2001 319

THE NUTRIENT VALUE OF SHELLFISH AND OTHER
TRADITIONAL FOODS. THEIR PAST AND CURRENT
CONTRIBUTION TO THE DIET OF FIRST NATIONS
PEOPLE. Pamela Winquist. First Nations and Inuit Health
Branch. Pacific Region. Health Canada. #540-757 West Hastings
Street. Vancouver, EC V6C 3E6, Canada.

The nutrient value of shellfish and other foods traditionally
used by First Nations people in British Columbia. Canada will be
reviewed. Information on past and current consumption, and fac-
tors affecting use. will be discussed.

Shellfish and other traditional foods offer superior nutrient
value compared to commercial alternatives. These foods have con-
tributed significantly to the overall nutrient intake of First Nations
people, in the past. Seafood and other traditional foods continue to
contribute to the overall nutrient quality of the diet of First Nations
pet)ple. Traditional foods provide nutrients that are often low in the
commercial food diets of First Nations people, such as vitamin A,
calcium, iron, polyunsaturated oils and omega-3 fatty acids and
folic acid. When these foods are replaced by commercial alterna-
tives, fat. sugar and salt intakes increase. Diets high in saturated
fat. sugar and salt are one of the risk factors in the development of
chronic diseases such as obesity, diabetes and coronary heart dis-
ease. Higher rates of these illnesses are seen in First Nations
people, compared to the general population.

Factors infiuencing present use of traditional foods include ac-
cess to a commercial food source, knowledge and skills of family
members to fish, hunt and/or trap, amount of traditional food a\ ail-
able, fish and wildlife regulations, contamination of traditional
foods, and household income.

In conclusion, shellfish and other traditional foods are of high
nutrient \ alue. These foods should continue to be consumed as part

of a nutritious diet, when they are available for safe harvest and
preparation.

PREDATION OF JUVENILE SEA SCALLOPS (PLA-
COPECTEN MAGELLANICUS) DURING SEEDING TRI-
ALS IN THE NORTHUMBERLAND STRAIT. Melisa C.
Wong,' M. A. Barbiau," L. ,\. Grecian,' L-.\. Davidson," M.
Niles," and Donna Murray,"' 'Department of Biology, University
of New Brunswick. Fredericton. NB. E3B 6E1, Canada; "Depart-
ment of Fisheries and Oceans, Gulf Fisheries Centre, Moncton,
NB, EIC 9B6. Canada; 'Botsford Professional Fishermen's Asso-
ciation Inc.. 1696 Route 955. Little Shemogue. NB, E4M 3M6,
Canada.

The Botsford Professional Fishermen's Association Inc. and
the Department of Fisheries and Oceans (Moncton. NB) have con-
ducted a scallop enhancement project in the Northumberland Strait
since 1998. Predation of juvenile sea scallops by sea stars [Asterias
vulgaris) and rock crabs {Cancer irroratiis) is a concern in the
Northumberland Strait. Our goal was to quantify predator-induced
mortalitv of seeded scallops and to determine how quickly mor-
tality occurs after seeding. We deployed assays (consisting of scal-
lops tethered to lead-weighted lines) to monitor scallop mortality
at seeded and non-seeded sites, after seedings in October 1999 and
October 2000. In both years, shell remains on the tethered lines
indicated the presence of both sea stars (empty intact shells) and
rock crabs (shell fragments) at both sites. In October 1999. scallop
mortality did not differ between the seeded site and the non-seeded
site or show a significant change over time. In October 2000,
scallop inortality did not differ between sites, while scallop mor-
tality was significantly higher 10 days after seeding than 2 days
after.

Joiinitil of Shellfish Research, Vol. 21, No. 1. 321-328. 2002.

ABSTRACTS OF TECHNICAL PAPERS

Presented at the 55 Annual Meeting

NATIONAL SHELLFISHERIES ASSOCIATION

&

PACIFIC COAST OYSTER GROWERS ASSOCIATION

(PACIFIC COAST SECTION)
Silverdale, Washington
September 20-22. 2001

321

Pacific Coast Section, Silverdale, Washington Abstracts. September 2001 323

CONTENTS

Jeremy M. Breach and Jeimfer L. Ruesink

Population and behavioral characteristics of introduced oyster drills. Cenitnstoma innnwtiini. in Willapa Bay 325

Atnilee Caffey and Are Strom

Juvenile growth study on the geoduck clam, Panopea ahnipta 325

Hakan Calik, Michael T. Morrissey, Paul W. Reno, and Haejiing An

Effect of high-pressure processing on Vibrio panihat'iiuilyiiciis strains in pure culture and Pacific oysters 325

Elyse K. Cronin and Brent Vadopalas

Age determination in geoduck clams {Panopea abnipta) utilizing patterns in the shell annuli 325

Robyn M. Estes, Russell P. Herwig. and Ralph A. Elston

Characterization of pathogenic and nonpathogenic bacteria associated with bivalve larvae and shellfish hatcheries 326

Stephen P. Ferraro and F. A. Cole

Oyster grounds: A superior habitat for small, sediment-dwelling invertebrates 326

Graham E. Gillespie

Status of the Olympia oyster. Ostrea concliapinla. in Canada 326

Geoff Hosak, David Armstrong, Bryce Semens, Brett Dutnbauld, and Steven Rumrill

Oyster aquaculture as fish habitat in Pacific northwest coastal Estuaries 327

Chris Langdon, Ford Evans, John Brake, and Sean Matson

Survival and growth effects on yields of Pacific oysters. Crassostrea gigas 327

Cynthia S. Marshall and Russell P. Herwig

Research to examine use of high hydrostatic pressure to inacti\ate human enteric viruses in oysters 327

Sean E. Matson, Chris Langdon, Ford Evans, John Brake, and Dave Jacobson

Predictability of grow-out performance from nursery performance of Pacific oyster, Crassostrea gigas 327

Daphne M. Munroe and Leah Bendell-Young

The effects of clam aquaculture on intertidal ecosystem structure and function 328

Paul G. Olin and Jim Hobbs

Habitat \ alue of commercial oyster culture gear 328

Pacific Coast Section, Silverdale, Washington

Abstracts. September 2001 325

POPULATION AND BEHAVIORAL CHARACTERISTICS
OF INTRODUCED OYSTER DRILLS, CERATOSTOMA IN-
ORNATUM, IN WILLAPA BAY. Jeremy M. Breach and Jen-
nifer L. Ruesink, Department of Zoology. University of Wash-
ington. Seattle. WA 98195-1800.

Japanese oyster drills in Willapa Bay were studied to discern
population parameters including density, size-frequency distribu-
tion, food preference, and movement rates. Drills were collected
during the summer of 2001 from 0.0625 and 1 m" quadrats near the
Washington Department of Fish and Wildlife lab at Nahcotta,
Washington. Drill density averaged 31 m"". and the size distribu-
tion included a peak of small individuals suggestive of this year's
recruitment. Drills showed a preference for feeding on smaller
oysters, and oysters appeared to escape drill predation at shell
lengths exceeding 2 cm. Drills were labeled and released into three
different habitats to .study drill movement. Significant differences
were found between movement rates on eelgrass. oyster bed. and
mudtlat. with drills traveling up to 1 m/day on mudtlat and eel-
grass until reaching small patches of oysters. Risks of drill impacts
are highest for seed oysters placed on the bottom in infested areas,
particularly if few oysters are currently present.

JUVENILE GROWTH STUDY ON THE GEODUCK CLAM,
PANOPEA ABRUPTA. Amilee Caffey, Washington Department
of Fish & Wildlife, Point Whitney Laboratory. Brinnon. WA
98320: Are Strom, University of Washington, WA 98062.

The geoduck clam has proven to be a challenging species to
culture in the hatchery setting. Water temperature, salinity, sun-
light, food concentrations, and handling all affect levels of growth.
The goal of this study was to determine what effect temperature
and varying food concentration levels in the sunounding water had
on the juvenile geoduck clam.

The statistical analysis method was a two-way ANOVA in
which juvenile clams were introduced to four varying environ-
ments where temperature and food concentrations were aberrant.
Clams were placed in 1-in. square, plastic trays, layered with per-
meable fabric and sand. The stocking density was 100 juveniles
per tray. Smaller juveniles were chosen, ranging between 14 and
19 mm in shell length in order to determine rapid growth rates.
Three identical trays were subjected to each of the four treatments,
with a total of 12 trays. The first and second treatments were
subjected to a higher temperature, between 1 5 and 1 6°C. The third
and fourth treatments were at a lower temperature, 10-1 1°C. The
first and fourth treatment were held at a higher concentration of
food. 45 k/mL. The second and third treatments were held at a
lower food concentration of 15 k/mL. Flow rate was 6 L/min.

Shell length did not vary significantly between treatments,
however. Juveniles exposed to higher temperature (I5-16°C). at
higher food concentration (45 k/mL algal cells), demonstrated the
most growth.

EFFECT OF HIGH-PRESSURE PROCESSING ON VIBRIO
PARAHAEMOLYTICUS STRAINS IN PURE CULTURE
AND PACIFIC OYSTERS. Hakan Calik, Michael T. Morris-
sey, Paul VV. Reno, and Haejung An, Department of Food Sci-
ence & Technology. Oregon State University Seafood Laboratory.
2001 Marine Drive. Room 253, Astoria. OR 97103.

Several cases of Vibrio parahaemolyticiis (Vp)-induced gastro-
enteritis occurred in the Pacific Northwest due to consumption of
raw oysters. High-pressure process (HPP) technology has shown
good potential in reducing pathogens. Environmental and clinical
strains of Vp in broth cultures and Vp-inoculated live Pacific oys-
ters (Crassostrea gigas) were subjected to HPP at different treat-
ment settings ( 1-10 min at 241 MPa; 1-5 min at 276 MPa; 30-120
s at 310 MPa; 10-90 s at 345 MPa). Results showed that Vp
numbers were reduced by HPP in both pure culture and whole
oysters. Vp inactivation was dependant on treatment time and
pressure. Optimum conditions for reducing Vp in pure culture and
oysters from 109 to 101 CFU/mL were achieved at 345 MPa for 30
and 90 s. respectively. Resistance variations were detected be-
tween Vp in pure culture and in oysters. Further high-pressure tests
with clinical 03:K6 Vp strain isolated from an outbreak in Texas
( 1998) showed that 5 min at 310 MPa was necessary for complete
elimination, making the isolate the most baro-resistant of all strains
used in the study. HPP proved to be an efficient means of reducing
Vp in oysters.

AGE DETERMINATION IN GEODUCK CLAMS {PANO-
PEA ABRUPTA) UTILIZING PATTERNS IN SHELL AN-
NUEL Elyse K. Cronin and Brent Vadopalas, School of Aquatic
and Fishery Sciences. University of Washington, Seattle, WA
98105.

Geoducks (Ponupcti iihnipia) are deep-burrowing bivalves dis-
tributed from Southeast Alaska to Baja, California. This extremely
long-lived species supports a lucrative fishery in Washington state,
yet little is known of geoduck population dynamics. Age data are
being collected from various sites in Puget Sound to investigate
both population dynamics and temporal genetic structure in geo-
ducks. via collaborations between the Washington Department of
Fish and Wildlife (WDFW), Washington Sea Grant, and the Uni-
versity of Washington School of Aquatic and Fishery Sciences.

To age geoducks, the hinge plate of the right valve was thin-
sectioned using a diamond saw. polished, and finally etched with
19f HCl. Annual growth rings (annuli) were visualized via light
microscopy, and two independent observer counts were made on
three thin-sections for each clam. Annuli revealed year-specific
width variation consistent across samples. These patterns may cor-
relate with localized environmental factors or regional oceano-
graphic conditions, and are used to establish year reference points
in series of annuli.

326 Ahsinicls. September 2(1111

Paeitic Coast Section, Silverdale. Wasliuigtoii

Ages determined from 200 geoducks collected from a Hood
Canal site sampled in 2000 {N = 1010) ranged from 3 to 136 y.
When complete, this age data set will be used in analyses of
genetic population structure and population dynamics of the spe-
cies in Puget Sound.

CHARACTERIZATION OF PATHOGENIC AND NON-
PATHOGENIC BACTERIA ASSOCIATED WITH BI-
VALVE LARVAE AND SHELLFISH HATCHERIES. Robyn

M. Estes, Russell P. Herwig, School of Aquatic and Fishery
Sciences, Box 355020, University of Washington. Seattle. WA
98195-5020: Ralph A. Elston, AquaTechniques/Pacific Shellfish
Institute, P.O. Box 687, Carlsborg. WA 98324.

Bacterial diseases are a major cause of larval mortality in shell-
fish hatcheries. Even with proper sanitation measures, bacterial
pathogens cannot be eliminated. The proper identification of
pathogens and the application of probiotics may help control dis-
ease outbreaks.

More than 100 bacterial isolates were collected from larval
Pacific oysters, larval geoducks. and locations within hatcheries in
the Pacific Northwest and Hawaii. Isolates were characterized by
whole cell analysis and restriction fragment length polymoiphism
(RFLP) of 16S rDNA using three restriction enzymes. Both of
these methods show similar relationships between the isolates.
Pathogenicity tests of isolates collected from Pacific oyster larvae
from Washington and Oregon showed that 3 out of 33 isolates
were highly pathogenic. These as.says examined larval mortality
and the ability of larvae to remain suspended in a water column.
Pathogenic and nonpathogenic bacteria strains appear closely re-
lated to each other. These results could provide information for the
development of probiotics in shellfish aquaculture.

Future research includes sequencing the 16S rDNA of patho-
genic bacteria, declaring new species if they cannot be identified as
known species, and developing polymerase chain reaction (PCR)
primers for rapid detection of pathogenic bacteria. This will help in
early detection of pathogenic bacteria and in determining the
source and ecology of the pathogenic organisms in hatcheries.

OYSTER GROUNDS: A SUPERIOR HABITAT FOR
SMALL, SEDIMENT-DWELLING INVERTEBRATES.
Steven P. Ferraro and F. A. Cole, U.S. Environmental Protection
Agency. 2111 S.E. Marine Science Drive. Newport. OR 97365-
5260.

As part of a programmatic effort to determine estuarine habitat
values for ecological risk assessments, quantitative tleld studies ol
small, sediment-dwelling invertebrates were conducted in Willapa
Bay, Washington, in July 1998 and Tillamook Bay, Oregon, in

July 1999. The six habitats included in both studies were (1)
"grow-out" (2-3 y old) oyster ground culture, (2) eelgrass, Zosleni
marina. (3) mudshrimp, Upogebia piit-ctlciisis. (4) ghost shrimp.
Neotnpaea califonuciisis. (5) bare mud. and (6) subtidal. un-
dredged. About fifteen 0.01 nv x 5 cm deep. 0.5 mm mesh
samples were collected randomly in each habitat throughout bulh
estuaries.

Multix ariate analyses of the data revealed that the invertebrate
fauna on oyster grounds was much more similar to that in eelgrass
and mud shrimp habitat than that in ghost shrimp, bare mud. and
subtidal habitat. Among the six habitats studied, oyster grounds
consistently tanked either first or second in terms of the number of
species, abundance, and total biomass of invertebrates. Oyster
grounds, which have high economic value in terms of oyster pro-
duction, are also ecologically valuable because they provide a
superior habitat for small invertebrates upon which many larger
animals (e.a.. fish, crabs, waterfowl) feed.

STATUS OF THE OLVMPIA OYSTER. OSTREA CON-
CHAPHIIJi, IN CANADA. Graham E. Gillespie. Fisheries and
Oceans Canada. Pacific Biological Station. Nanaimo. British Co-
lumbia. V9R 5K6.

The Olympia oyster. Ostrea amcluiphila. is the only oyster
native to the Canadian Pacific coast. Olynipias supported commer-
cial fisheries in British Columbia from the late 1800s to about
1930. when focus of the oyster industry shifted to Pacific oysters.
Cnissostrea gigcis.

Olympia oyster distribution in British Columbia is limited by
specialized habitat requirements, and relatively low fecundity and
dispersal. Olympias are vulnerable to temperature extremes, and
are not resistant to haivests on a commercial scale. Habitats that
once supported large aggregations in Georgia Strait no longer do.
in part because of historic overharvests and en\ ironmental stresses,
and because development of large oyster reefs tnay require centu-
ries without disturbance. Small relict populations survive at low
tide levels and under floating structures. Olympias are locally com-
mon at sites on the west coast of Vancouver Island, and little
information exists on populations in Johnstone Strait or in the
Central Coast. They do not occur in the Queen Charlotte Islands.

Olympia oysters are not likely facing imminent danger of ex-
tinction or extirpation in Canada. Limiting factors have led to
significant reductions to population levels in the past. From the
limited data available, the Committee on the Status of Endangered
Wildlife in Canada (COSEWIC) assigned a status of Special Con-
cern in November 2000. Proposed federal legislation, the Species
At Risk Act (S.ARA). will require development of an Olympia
oyster management plan within 3 y.

Pacific Coast Section, Silverdale. Washincton

Abstracts. September 2001 327

OYSTER AQUACULTURE AS FISH HABITAT IN PA-
CIFIC NORTHWEST COASTAL ESTUARIES. Geoff Ho-
sack. David Armstrong, Bryce Semens, School of Aquatic and
Fishery Sciences. Box 355020, University of Washington. Seattle,
WA 98195: Brett Dumbauld, Washington State Department of
Fish and Wildlife. Willapa Bay Field Station. P.O. Box 190. Ocean
Park. WA 98640; Steven Rumrill, South Slough Estuarine Re-
search Reserve. P.O. Box 5417, Charleston. OR 97420.

Increased pressure on traditionally managed stocks of marine
and anadromous fish, calls for protection of essential fish habitat
under the Magnuson-Stevens Act, and recent listings of several
salmonid stocks under ESA, have brought aquaculture activities
that take place in coastal estuaries under increased public scrutiny.

We initiated a study designed to examine the ecological role
that oyster aquaculture plays as habitat in coastal estuaries of the
Pacific Northwest. The goal of the project is to identify and quan-
tify beneficial and adverse impacts of shellfish farming on eel-
grass, juvenile salmonids. and other selected estuarine fauna and
flora, and to develop farming practices and recommend inanage-
ment protocols that protect or enhance those resources. We present
results of initial fish and invertebrate sampling in selected habitats
from Willapa Bay during spring and summer 2001. and an experi-
ment designed to examine the effects of oyster harvesting on eel-
grass habitat. We make a plea to managers to consider oyster
aquaculture areas as fish habitat on a broader estuarine scale.

SURVIVAL AND GROWTH EFFECTS ON YIELDS OF PA-
CIFIC OYSTERS, CRASSOSTREA GIGAS. Chris Langdon,
Ford Evans, John Brake, and Sean Matson, Coastal Oregon
Marine Experiment Station and Department of Fisheries and Wild-
life. Oregon State University. Newport. OR 97365.

In Spring 1999. 29 full-sib families derived from crossing non-
selected "wild" oysters were planted at a subtidal site in Yaquina
Bay. Oregon. In addition, in Fall 1999. 32 full-sib. families from
crosses within two groups of six selected families were planted at
a an intertidal site in Tomales Bay. California. After about 1 y of
growth, each cohort was harvested and average yields, survival.
and individual growth rates determined for each family. These
parameters were adjusted for the effects of differences in average
initial weights of planted spat per family if a significant {P < 0.05)
effect was detected.

Adjusted survival explained 517c and 68%. and growth ex-
plained 25% and 21% of the variation in adjusted family yields at
the Yaquina and Tomales sites, respectively. Furthermore, up to
79%' of the variation in family yields at the Tomales site was
explained by differences in survival among families, if survival
was not adjusted for a significant (regression, r = 0.38; P <
0.0001 ) positive effect of initial spat weight. These results indicate
that both growth and. perhaps to a greater degree, survival should

be considered when designing breeding programs to increase oys-
ter yields.

RESEARCH TO EXAMINE USE OF HIGH HYDROSTATIC
PRESSURE TO INACTIVATE HUMAN ENTERIC VI-
RUSES IN OYSTERS. Cynthia S. Marshall and Russell P.
Herwig, School of Aquatic and Fishery Sciences, Box 355020,
University of Washington. Seattle. WA 98195-5020.

The desire for safe shellfish products in the retail market and
food service establishments is paramount to the success and
growth of the shellfish industry. Unfortunately, bacterial and viral
pathogenic organisms may be associated with fresh and processed
shellfish. These organisms can lead to seafood-borne illness that
may result in severe economic impacts on the shellfish industry,
causing shellfish bed closures, product recalls, and lost consumer
confidence. Human enteric viruses are the causative agents asso-
ciated with a large, but poorly understood, number of seafood-
borne illnesses per year. The principle viruses associated with
seafood include hepatitis A. Norwalk virus, Norwalk-like viruses,
and astrovirus. Viruses are typically not monitored in seafood
products and in shellfish growing waters because of the difficulty
and expense of quantifying active virus particles.

There is a large deinand for raw oysters in the market. Depu-
ration or relaying may be used to reduce the number of pathogenic
organisms present in live shellfish before they are harvested. In
recent years, high hydrostatic pressure (HHP) treatment has been
shown to reduce or eliminate bacterial pathogens from shellfish
while retaining sensory properties of the raw product. We are
beginning a new research project that will examine the effects of
HHP on human enteric viruses. Poliovirus will be used as a model
virus in our experiments. This virus was chosen because it can be
safely used in the laboratory and can be cultured using standard
protocols. In laboratory experiments, we will vary hydrostatic
pressure, time of exposure. pH. and temperature on preparations
containing poliovirus.

PREDICTABILITY OF GROW-OUT PERFORMANCE
FROM NURSERY PERFORMANCE OF PACIFIC OYS-
TER, CRASSOSTREA GIGAS. Sean E. Matson, Chris Lang-
don, Ford Evans, John Brake, and Dave Jacobson, Hatfield
Marine Science Center. Oregon State University. 2030 S. Marine
Science Dr. Newport. OR 97365-5296.

The predictability of Pacific oyster (Crassostrea gigas) grow-
out performance from nursery performance was investigated by
measuring yield, survival, and growth of pedigreed families of
Pacific oysters, in the nursery and at a grow-out site in Totten Inlet.
Washington. Early prediction of grow-out performance could sig-
nificantly reduce labor, materials, and space required to select
pedigreed families on the basis of performance in an oyster breed-
ing program. This research was conducted as part of the Molluscan

328 Abstracts. September 2001

Pacific Coast Section. Silverdale, Washington

Broodstock Program, a breeding program for the sustainable in-
crease in Pacific oyster yields through genetic selection. Yield was
measured as oyster bag weight in grams and is a function of both
survival and growth.

Unadjusted yield, yield adjusted for initial size, and unadjusted
individual growth of Pacific oyster seed in the nursery were used
as predictors of unadjusted yield, yield adjusted for initial weight,
and unadjusted mdividual growth in the field. Unadjusted nursery
yield was found to be a significant predictor of unadjusted grow-
out yield IP = 0.0009. r = 0.250). Initial planting weight, how-
ever, was also a significant predictor of unadjusted grow-out yield
(P < 0.0001. r = 0.283). Similarly, initial nursery weight was a
highly significant predictor of nursery yield (P < 0.0001. r =
0.703). Thus, adjustments were employed to both nursery and
grow-out yield, to remove the effect of initial weight. Adjusted
nursery yield was not a significant predictor of adjusted grow-out
yield (P = 0.9975, /" = 2.475 x 10" ). These data indicate that
heavy spat grew into heavy oysters because they were planted as
heavy spat. They also indicate that Pacific oyster families with
high yields at grow-out (adjusted for initial weight) are not easily
identified in the nursery using the methods described here.

THE EFFECTS OF CLAM AQUACULTURE ON INTER-
TIDAL ECOSYSTEM STRUCTURE AND FUNCTION.
Daphne M. Munroe and Leah Bendell- Young. Department of
Environmental Science. Simon Fraser University. 8888 University
Drive. Burnaby. BC V5A IS6.

Quadrat and core sampling was carried out to gather baseline
data regarding ecosystem structure and function from three
beaches on Denman Island. British Columbia. Ecosystem structure
was examined through biodiversity measures (species richness,
evenness, and heterogeneity I. community composition, and spe-
cies distribution. Ecosystem function was evaluated using percent
silt and percent organic matter from core samples. The three study
sites experienced different levels of commercial aquaculture. One
site was a recreational harvest beach and the other two were leased
for commercial aquaculture: one was 159c covered with nets, the
other was only 20% covered.

We observed differences in ecosystem structure and function
among the three sites. First, species richness was higher on the

beach where no commercial practices occurred. Second, there
were higher numbers of organisms per quadrat in the upper areas
of the recreational beach and considerably more surface species on
that beach. Third, we saw that the distribution of the most abun-
dant clam species was limited to the upper half of the beach where
no commercial clam culture was conducted; however, on the other
two beaches, the distribution of this clam species extended the
length of the study area.

This research indicates a high probability that commercial clam
culture causes changes in intenidal ecosy.stem structural and func-
tional components. Further research is imperative to provide man-
agers with the scientific information needed to develop sustainable
and environmentally sound management protocols.

HABITAT VALUE OF COMMERCIAL OYSTER CUL-
TURE GE.AR. Paul G. Olin. University of California Sea Grant.
Davis. CA 95616; Jim Hobbs. University of California, Davis,
CA 95616.

Oyster growers in Tomales Bay, California, produce deeply
cupped single oysters for the half-shell market using plastic mesh
socking attached to stakes, and plastic mesh bags on the bottom, on
racks or floating attached to longlines. This oyster culture gear and
the oysters that are grown form a complex three-dimensional habi-
tat that interacts in a variety of ways with the biological and
physical components of the estuarine ecosystem. This habitat is
utilized by a myriad of fish and invertebrates, which are often prey
for larger commercially important species such as halibut or
dungeness crab.

To document the habitat value of cultured oysters and gear, fish
and invertebrates from 36 culture bags were collected and all mac-
roorganisms were enumerated and identified to species. Ten phyla
and 11 taxonomic classes were represented by the 51 different
species identified. One oyster culture grow-out bag held more than
5.000 organisms, although more typically between 600 and 1.000
individuals were found in each bag.

In Tomales Bay approximately 8% of the intertidal and subtidal
bottom lands are leased for shellfish culture by the state. Of this
S^f, around 1% is actively farmed. Although this represents a
small portion of the Bay, it is highly productive and provides
complex intertidal habitat that has been lost in many areas due to
erosion and resultinc sedimentation.

Journal of Shellfish Research. Vol. 2!. No. I. 329-356. 2002.

ABSTRACTS OF TECHNICAL PAPERS

Presented at the 22'"' Annual

MILFORD AQUACULTURE SEMINAR

Milford, Connecticut
Fehruarv 25-27. 2002

329

Milford Aquaculture Seminar. Milford. Connecticut Abstracts. 2002 Annual Meeting. February 25-27. 2002 331

CONTENTS

Walter Blogoslawski

Overview, 22'"' Milford Aquaculture Seminar 335

Peter Adamik, Roxanna Andersen, Murray Croiis, Graham Mains, Vicke Starczak, Isabelle Williams, Diane Murphy, and
Dale Leavitt

Influence of qualiog grow-out nets on benthic diversity in Wellfleet, MA 335

John Aldred, hike Siinila, and Christopher Martin

Possible lar\ al mycosis as a cause of bivalve seed mortality in a production hatchery 335

Standish K. Allen, Jr.

New developments with nonnative shellfish species in the Chesapeake Bay 336

Brian J. Ball, Stephanie T. Rutkowski, Emily T. Griffiths, and Kim W. Tetraiilt

The effects of varying cold water temperatures, size and population density on the growth and mortality rates of bay

scallops. Argopecteii imidiaiis imidiiiiis. in an upweller 336

Amber L. Beitler and John J. Roy

Catch statistics of Htmuinis aiiiericaiuis. the American lobster, from a 3-wk study conducted by students from the

Sound School Regional Aquaculture Center aboard a commercial lobster vessel in the waters off Fishers

Island. New York 337

David A. Bengtson, Stephen Willey, Erin McCaffrey, and David Alves

Effects of water velocity on conditioning of summer flounder ParuUclitliys dciitatus for net pens 337

Luther Blount

Reminiscences of early pioneers in oyster culture 338

Diane J. Brousseau and Ronald Goldberg

"Crab sightings'" in Long Island Sound during 2001 338

Walter J. Canzonier

Some classical personalities in the shellfish research arena: Eclectic observations of an erstwhile observer 339

Diane Carle

Massachusetts Ocean Resource Information System (MORIS) 339

Joseph Choromanski, Sheila Stiles, Mark Dixon, and Christopher Cooper

Habitat suitability ascertained by growth and survival of bay scallops in tiered cages 340

Carmela Cuomo, Paul R. Bartholomew, Leslie Angelini, Brian King, and Jeffrey Byczko

Horseshoe crab aquaculture: Preliminary results from hatching and rearing studies 340

John J. Curtis, Sherry W. Lonergan, Paul J. Tnipp, Peimin He. Raqiiel Carmona, Charles Yarish, George P. Kraemer,
Christopher D. Neefns, Thierry Chopin, and George i\'ardi

A cooperative study on the aquaculture of Porphyra leiicosticta (Rhodophyta) for an integrated finfish/seaweed

recirculating aquaculture system in an urban application 341

Gef Flinilin

Update on shellfish restoration and review of local hatcheries in New Jersey 341

Michael J. Goedken and Sylvain De Guise

Flow cytometry as a tool to quantify the oyster phagocytosis, respiratory burst, and apoptosis 342

Josh Goldman

Cobia culture 342

Eric Goodman, Colleen Cook, and Michael Weiss

Creating opportunities for student aquaculture projects 342

Helene Hegaret, Gary H. Wikfors, Philippe Soudant, Maryse Delaporte, Jeanne Moal, and Jean-Franfois Sainain

An experimental investigation of dietary fatty acids and sterols and the immunology of the American oyster,

Crassostreci virginica: A well-fed oyster is a healthy oyster, n'est-ge pas? 342

Andrea Hsu, Erin Summers, James Estrada, and Roxanna Smolowitz

The role of three bacteria in shell disease of the American lobster [Hoiminis ainfiicanus) 343

Richard C. Karney, Amandine Siirier, David W. Grunden, and Thomas E. Berry

Dermo investigations, razor clam nursery trials, and preliminary bay scallop adhesion culture efforts 343

Robin Katersky, Barry Smith, Dean Perry, and David Nelson

Some culture strategies for growing rotifers (Brachioiiiis plii(itili\) as feed for aquaculture applications 344

Brian Kilpatrick, Joseph DeAlteris, and Robert Rheault

Assessing habitat value of modified rack and bag aquaculture gear in comparison with submerged aquatic vegetation.

in particular, an eelgrass (Zosteiu iiuiriini) bed 344

332 Ahslracls. 2002 Annual Meeting. February 25-27. 2002 Milford Aquaculture Seminar. Milford. Connecticut

.Suspended aquaculture development in Connecticut "^^

It takes a coinnuinity to yrow a scallop '^

Gordon King

The culture of black pearl oysters on subsurface longlines in Savu Savu. Fiji 345

Gordon King

Urban commuiiit\ meets aquaculture: A case study in the Northwest 345

Dale Leavitt, William Bnrt, Diane Murphy, and Rebecca Hanson

Progress with culturing the razor clam (£);.s(.s cliirctiis) 345

Kathryn R. Markey and John J. Roy

A comparison of sur\ ival m luvenile Argopccieii irnidicms iinulians using various culture techniques at the Sound

School Regional Aquaculture Center ■*"

Paul D. Mangle

Suspendei
Mary F. Morgan, Kathleen K. Becker, and Kim TetraiiU

It takes a coinnuinity to grow a scallop

Diane Murphy, Dale Leavilt, Bill Burt, and Bill Clark

Bay scallop (Argopectcn irnidicms irnidicms ) restoration on Cape Cod 347

Jennifer Mugg-Pietros and Michael A. Rice

Effects of CnissDsircii viri^inica populations on sedimentation, phytoplankton species composUion. and ammonia

348
cycling in experimental mesocosms

Steven Pitchford and Richard Rohohm

A review of diseases in the bay scallop {Argopectcn irnidiims irnidicms) and some observations on mortalities at the

348
Milford Laboratory

Perry Raso and Michael A. Rice

Shellfish aquacullure's effect on total organic carbon (TOC) in the benthos 349

Edwin Rhodes

Industrial-scale scallop culture in Chile— the C.M.I, experience 349

Karen Rivara, Amber L. Beitler, and John J. Roy

The development of an instructional shellfish hatchery: A collaborative effort between Aeros Cultured Oyster Co. and

the Sound School Regional Aquaculture Center "^^

Karen Rivara

The East Coast Shellfish Growers Association: A work in progress 350

Rene Sanz, Sherry Lonergan, Jennifer Sutorius. and Dania Lieberthal

Heavy metal survey of Fkciis spiralis collected from southv\esterii Long Island Sound 350

Martin P. Schreibman, Chester Zarnoch, John T. Tanacredi, Lucia Magliiilo-Cepriano, Jacob Raz, and Stefano Diomede

Aquaculture activ ilies in Brooklyn. New York?

Sandra E. Shumway

Shellfish aquaculture: Good for the economy, good for the em ironment. good for you! 351

Roxanna Smolowitz, Susan Find, and Lisa Ragone-Calvo

Health management guidelines for shellfish culture in the northeastern United States 351

Roxanna Smolowitz, hike Sunila, Nancy Stokes, and Lisa Ragone-Calvo

Prevalence and mortality associated with SSO and SSO-like infections of Crcissaslrca virginica in the Northeast 352

Karin A. Tanimi, Najih Lazar. Arthur Ganz, James G. Turek, and John G. Catena

Rhode Island's Shellfish Restoration Program in response to the North Cape oil spill 352

A7;// TetrauU, R. Michael Patricio, and Maty Morgan

SPAT (Special Proizrams in Aquaculture Tramins) update. 2002. Establishing a model for community-bused shellfish

: " 353

culture and restoration

John Wadsworth, Tessa Simlick, and Nancy Balcom

353
A new clam for Connecticut

Bethany A. Walton

Life in a trailer— development of a new shellfish hatchery at the Massachusetts Maritime Academy 354

Donald Webster

The new oyster wars: Policy perspectives on the introduction of Crassostrcci ciriakciisis in the Chesapeake Bay 354

Milford Aquacultiire Seminar, Milford. Connecticut Abstracts. 2002 Annual Meeting. February 25-27. 2002 333

Scott Weston. Mark Fregeau. and Joe Biittner

Developments in softsheli clam hatchery and nursery production on Massachusetts" North Shore 3.'i4

James C. Widman, Jr. and David J. Veilleux

Rapid grow th of ha\ scallops. Argopecteii irradiims irnulinns. in Long Island Sound 355

Gary H. Wikfors

Livestock domestication in the third mnienniiun: All wet? 355

iMwrence Williams and Tessa Simlick

Blue mussel aquaculturc m Long Island Sound 356

Stephen Willey, David A. Bengtson, and Moti Harel

Arachidonic acid requirements in lar\ al summer flounder. Paraliclitlns dematiis 356

Milford Aquaciilture Seminar. Milford. Connecticut

Absn-acts. 2002 Annual Meeting. February 2.S-27. 2002 .^.^.'^

OVERVIEW, 22"" MILFORD AQUACULTURE SEMINAR.
Walter J. Blogoslawski. U.S. Departinenl of Commerce. National
Oceanic & Atmospheric Administration. National Marine Fisher-
ies Service. Northeast Fisheries Science Center. Milford Labora-
tory. 212 Rogers Ave.. Milford. CT 06460.

With more than 19.^ registrants, the 22'"' Annual Milford Aqua-
culture Seminar was our largest gathering of industry, research,
and academic interests.

By blending both the theoretical and practical aspects of aqua-
culture, the meeting permitted attendees an exchange of technol-
ogy in aquaculture methods outside their own expertise and pro\ ided
a forum where the latest innovations were introduced and discussed.

Fifty formal papers and posters were presented by attendees
from 10 U.S. coastal states and France. Meeting attendees repre-
sented ? vocational aquaculture high schools. 16 universities. 6
marine labs, and several state and federal institutions involved in
shellfish and finfish aquaculture. A highlight of the meeting was a
set of papers reviewing the northeastern aquaculture pioneers,
making us aware of the difficulties and successes of those who laid
the foundation for our aquaculture ventures and studies. Other
topics included information on new hatcheries, education, disease,
nutrition, and culture techniques.

The seminar has developed a tradition of offering the latest
information available in the field in an informal atmosphere. This
has successfully promoted a free exchange among all with an
interest in the success and future of aquaculture. This seminar
continued that approach which allowed all attendees to enjoy and
learn from the formal presentations, and afforded informal oppor-
tunities to discuss the latest developments pertinent to this impor-
tant expanding field.

At this year's seminar. 45 separate aquaculture companies met
in an evening session to discuss the feasibility of forrning an in-
dustry group tentatively titled "The East Coast Shellfish Growers
Association." The proposed Association's goals are to promote
and protect association members' needs in state and regional con-
texts and involve all stakeholders in the task of enhancing the
shellfish aquaculture industry. The main reason for joining to-
gether is to promote industry unity and to counteract recent anti-
aquaculture litigation and publicity.

The meeting was sponsored by the National Marine Fisheries
Service, Northeast Fisheries Science Center, Milford Laboratory.
Milford. Connecticut.

INFLUENCE OF QUAHOG GROW-OUT NETS ON
BENTHIC DIVERSITY IN WELLFLEET. MA. Peter Ad-
amik. Roxanna Andersen. Murray Crous, Graham Mains,
Vicke Starczak, Isabelle Williams, Diane Murphy, and Dale
Leavitt, SouthEastern Massachusetts Aquaculture Center. Massa-
chusetts Maritime Academy. 101 Academy Dr.. Buzzards Bay.
MA 02532.

The question of environmental impacts tiom shellfish aquacul-
ture has been posed recently both locally and at an international

level. An often-used indicator of environmental health is the di-
versity of species within the ecosystem in question. The prevailing
assumption is that a decrease in biodiversity suggests a decrease in
the quality of the environment. To begin addressing the issue of
en\ ironmental impacts of shellfish aquaculture on local intertidal
Hats, we initiated a study investigating benthic diversity associated
with netted raceways used for quahog (Menenaria inerceiicuia)
grow-out as compared to uncovered, unimpacted intertidal flats.
The specific question asked was. "Does the presence of aquacul-
ture netting structures and high quahog planting densities in the
intertidal marine habitat alter the diversity of macrobenthic species
in proximity to the nets?"

The study plan was to visit two sites having a history of quahog
aquaculture and collect replicated core samples within and outside
of the netted raceway. The samples were preserved, stained, and
returned to the laboratory. In the laboratory, the samples were
sieved to 500 p.ni and all living tissue, as stained with rose bengal,
was sorted and identified to the lowest taxonomic level possible.

A total of ."^9 macrobenthic species were identified in the core
samples from sample site 1 (Town Landing. Welltleet. MA) and
27 from site 2 (Old Wharf. Wellfleet, MA). The dominant species
in all samples at Town Landing was the glassy tube worm. Spio-
chaetoptents ociilatiis: at Old Wharf, the dominant species were
the capitellid worm. Heieromastus fdifonmis and the mud snail.
Uxanassa ohsolela. The impact of shellfish aquaculture on envi-
ronmental quality was analyzed in terms of the species richness,
species diversity, and distribution of dominant species between
sites and treatments.

In general, there were minor or no differences between netted
and nonnetted treatments at the two sites investigated. Many other
factors can influence diversity, species richness, and evenness of
the intertidal macrofauna. including hydrography and fluid inter-
action under the netted area, human activities related to shellfish
farming, and the historical background of the netted site (long-term
culture, mid-term culture, or short-term culture). It was concluded
that further study was warranted to include a seasonal and long-
term component to the study.

POSSIBLE LARVAL MYCOSIS AS A CAUSE OF BIVALVE
SEED MORTALITY IN A PRODUCTION HATCHERY.

John Aldred, Town of East Hampton. Shellfish Hatchery. 159
Panligo Road, East Hampton. NY 11937; Inke Sunila, State of
Connecticut, Department of Agriculture. Bureau of Aquaculture,
P.O. Box 97. Milford. CT 06460; Christopher Martin, USDOC.

NOAA. National Marine Fisheries Service. Northeast Fisheries
Science Center. Milford Laboratory. Milford. CT 06460.

Since 1996. the shellfish hatchery at East Hampton has expe-
rienced sporadic unexplained mortalities in cultures of hard clams
{Mcrccnaria mcrcciiaiio) and bay scallops (Argopecten irradians
iiradiaiis). Typically, a population of larvae progressed normally
through metamorphosis and then, eariy in the juvenile stage (post-
set) stopped feeding. In such cultures, examination revealed many

336 Ahstmct.s. 2002 Anniiiil Meeting. February 25-27. 2002

Milford Aquaculture Seminiir. Milt'ord. Connecticut

dead or dying individuals. Within 2-4 days of the first indication
of the problem most animals were dead. Affected juveniles ranged
in shell length from 0.2 to \.5 mm. Eastern oysters {Cnissosrreci
virginiiu) reared at the same hatchery have not been affected.
Preventative measures have been unsuccessful. These included
switching algal diets, carelul monitoring of pH. changing water
more frequently, and giving special attention to cleaning and dis-
infecting the system. On occasion, it has been possible to rescue
some juNcniles from affected cultures by mo\ uiy them to a tlow ing
water system, with clams surviving better than scallops.

During the 2001 growing season, similar mortalities were ob-
served. Samples of ju\enile clams (approximately 10 mm in shell
length) from two cohorts were preserved in Davidson's fixative.
Scallop juveniles also from two cohorts (ranging from approxi-
mately 0.2 to O.-S mm in shell length) were similarly fixed. Fol-
lowing decalcification, the specimens were stabilized in agarose,
embedded in paraffin, sectioned to 6 (xm. and stained with hema-
toxylin-eosin according to standard histopathological procedures.

Microscopic examination revealed invasion of most tissues and
the shell by fungal mycelia. Mycelia were detected in the gill
filaments where they often completely obstructed the heniolymph
canals. Fungal hyphae were observed within the mantle cavity,
with invasion of all epithelia (mantle, gill, and foot). The hyphae
appeared to have some affinity to mucus. Cilia were entrapped. All
layers of shell were invaded, with penetration to the exterior. The
hyphae stained dark blue with hematoxylin, measured approxi-
mately 2-3 (jLin in diameter, and were rarely branched and septate.

Evidence of starvation was apparent. In most instances, food
particles were absent from the digestive system and adductor
muscles were atrophied. In adsanced stages of invasion, tissues
were disintegrated, with complete loss of vesicular connective tis-
sue in some specimens. We concluded that larval mycosis was the
cause of the observed mortalities. The fungus appears to be an
opportunistic pathogen causing significant morbidity and mortality
when conditions are optimal for its pathogenic development. Bi-
valve cultures in hatcheries often collapse and new cultures are
initiated. The cause or causes of mortality are rarely determined.
We suggest that larval mycosis may be a common, under-
recognized cause of such events.

NEW DEVELOPMENTS WITH NONNATIVE SHELLFISH
SPECIES IN THE CHESAPEAKE BAY. Standish K. Allen.
Jr., Aquacultuie Genetics and Breeding Technology Center. Vir-
ginia Institute of Marine Science. Gloucester Point. VA 23062.

VIMS has been systematically examining the potential of sev-
eral nonnative species to alleviate the serious decline of oyster
stocks in the Virginia portion of the Chesapeake Bay. One that has
emerged as quite promising is the Suminoe oyster, Crassostiea
ariakt'iisis. Field trials with sterile triploids have shown a general
resistance to disease, rapid growth rate, and high smvival. Formal
and informal test marketing of the product has been similarly

successful. As with any nonnative species, serious concerns exist
about the long-term implications of introduction. Some of these
issues are associated with simultaneous introduction of pests or
pathogens, and some are associated with the ecological effects of
sustained population growth in the Bay. By culturing this species
ill the hatchery, pest and pathogen issues are largely, but not
wholly, addressed, Aquaculture of triploid-only individuals miti-
gates, but does not eliminate, most of the ecological concerns.
Aquaculture of hatchery-raised sterile triploid seed represents an
intermediate solution to assisting the industry between abandoning
research on nonnative species and wholesale introduction of dip-
loids. The industry potential is enormous, but there are lingering
questions about how precisely this "revolution" will or won't pro-
ceed.

THE EFFECTS OF VARYING COLD WATER TEMPERA-
TURES, SIZE AND POPULATION DENSITY ON THE
GROWTH AND MORTALITY RATES OF BAY SCAL-
LOPS. ARGOPECTEN IRRADIANS IRR.ADIANS. IN AN UP-
WELLER. Brian J. Ball, Stephanie T. RutkowskI, Emily T.
Griffiths, Mattituck High School. LSI 2.5 Main Road. Mattituck,
NY 1 1952; Kim W. Tetrault, Cornell Cooperative Extension —
Suffolk County Marine Program, 3690 Cedar Beach Road,
Southold, NY 11971.

The growth of scallops has been known to slow in cold tem-
peratures. The objective of this experiment was to observe the
growth and mortality rates of larger and smaller sized bay scallops
sorted into silos of varying densities in an upweller with decreas-
ing coldwater temperatures.

To set up the experiment, five silos were filled with smaller
scallops (10-20 mm) and five silos were filled with larger scallops
(21-30 mm). Each silo of larger and smaller scallops varied in
population density. The five silos of smaller scallops had 300. 700,
SOO, 900. and 1 ,200 scallops per silo, respectively. The five silos
of larger scallops had 150. 1 80. 250, 330, and 350 scalk)ps per silo,
respectively.

Once a week, data were collected from a sample size of 30
scallops from each silo. Using a caliper, the size of the scallops
was recorded to observe any growth. Any mortality within the
sample population was also noted. Eight weeks into the experi-
ment, January 10, 2002, all of the scallops were counted in each
silo and all of the deaths were recorded. Using the data, mortality
ratios of the entire population for each silo were calculated. A total
count was done to verify the accuracy of our sample populations.
The scallops were then returned to the silos for further study.

The results of this experiment show that tor the varying popu-
lation densities that we measured, the larger scallops continued to
grow and had a low mortality rate in coldwater temperatures rang-
ing from 13.21 to 0.27°C. The population density of each silo for
the larger scallops had little effect on the growth and mortality
rates. The growth and mortality rates in the smaller scallops were

Milford Aquaculture Seminar. Milford, Connecticut

Abstracts. 2002 Annual Meeting. February 25-27. 2002 .\^7

greatly affected by the population densities in our experiment. The
silos with the fewest scallops (296 and 659) showed the most
growth, whereas the silos with the highest density of scallops
(1,162 and 844) showed less growth. For the smaller scallops, the
silos with the highest density had the greatest mortality rate. The
silo containing 1,200 scallops had a mortality rate of 10.4%,
whereas the mortality rates for the remaining smaller groups of
scallops had a mortality rate of approximately 5'yf . Experimental
testing will continue through April in order to reach valid conclu-
sions from this study.

CATCH STATISTICS OF HOMARUS AMERICANUS. THE
AMERICAN LOBSTER, FROM A 3-WK STUDY CON-
DUCTED BY STUDENTS FROM THE SOUND SCHOOL
REGIONAL AQUACULTURE CENTER ABOARD A COM-
MERCIAL LOBSTER VESSEL IN THE WATERS OFF
FISHERS ISLAND. NEW YORK. Amber L. Beitler and John
J. Roy, The Sound School. 60 South Water St.. New Haven. CT
06519

During the summer of 2001. from July 9 to August 1. six
students from the Sound School Regional Aquaculture Center kept
catch statistics on Hoiminis aniericaiuis. the American lobster,
while interning as deck hands aboard the UP FOR GRABS, a
commercial lobster vessel berthed in Noank. Connecticut. The
student time aboard the vessel was credited toward their Super-
vised Occupational Experience (SOE) compliance requirements.
The UP FOR GRABS fishes out of Noank and possesses both a
Connecticut and a New York State Commercial Lobstering Li-
cense. Eighty-five percent of the gear was fished on grounds that
lie between an east-west line that can be drawn through Ram
Island and Sea Flower Reef on the northern (Connecticut) side and
a line running east to west 0.5 miles north of Fishers Island on the
southern (New York) side of Fishers Island Sound. The remaining
15% of the gear was fished in an area just north of Ram Island. No
differentiation was made between the areas being fished. Trawls
were at times moved within this zone from areas of low produc-
tivity to areas where the catch was found to be higher, during a
given week.

The time of the students" internship corresponded with the peak
season for lobster capture in that area. Traditionally termed "the
run" in the commercial lobster community, the month of July is
considered to be the peak month for lobstering in the entire year.
Forty-seven pot trawls were fished each week. All of the pots that
were fished were wire. An average of 10 trawls, or 70 traps, were
hauled each day. 4 days each week. Each trawl was hauled once
each week. The students recorded the catch statistics from the 280
traps as each trawl was landed. The data that were taken included:
the number of legal lobsters that were caught in each trawl; the
number of sub-legal lobsters (or shorts) that were returned to the
water from each trawl; the number of male and female legal lob-
sters found in each trawl; the number of dead lobsters found in

each trawl; and the number of lobsters that exhibited shell-rot
disease in each trawl. All categories were totaled daily. The daily
totals were then compiled by week for 4 wk. A total of 2.018
lobsters were caught during the study.

Forty-five percent of the lobsters (191) landed in the first week
were of legal size. The number of legal lobsters (341) that was
landed in the second week increased by 56%. The third week of the
study had a 4%' drop in the number of legal lobsters (322) taken.
There was a 38% drop in the number of legal lobsters (201) landed
in the final week of the study. The percentage of sub-legal lobsters
varied between 44% and 54% of the total number of lobsters taken
each week during the study. More legal male lobsters were caught
in the first and second week, whereas legal female lobsters ap-
peared in greater numbers during the final 2 wk of the program.
The number of dead lob.sters found in the trawls ranged from
0.65% to 1.44%. All deaths were attributed to cannibalism, pre-
dation. or physical impact of the trap or trap components. The
number of lobsters exhibiting shell-rot disease was less than 1%
throughout the entire study. However, because of the proximity to
the molt, this percentage may become greater later in the season.

The students at the Sound School had first hand experience
with the dramatic declines in the lobster populations in western
Long Island Sound during the late 1990's. We believe that it is
becoming increasingly important to monitor accurately the exist-
ing stocks of Homants ameiicanus at all levels of the fishery, as
well as in the scientific community. A concerted effort will be
required to ensure the survival of this important natural resource.

EFFECTS OF WATER VELOCITY ON CONDITIONING
OF SUMMER FLOUNDER PARALICHTHYS DENTATUS
FOR NET PENS. David A. Bengtson, Stephen Willey, and Erin
McCaffrey, Department of Fisheries. Animal and Veterinary Sci-
ence. University of Rhode Island, Kingston. RI 02881; David
Alves, Coastal Resources Management Council, Stedman Govern-
ment Center, Wakefield. RI 02879.

Attempts in the late 199()s to rear summer flounder in net pens
suffered from high mortality during the month after fish were
transferred from the nursery facility to pens. As part of the Uni-
versity of New Hampshire's Open Ocean Aquaculture Demonstra-
tion Project (GOADP). we investigated whether exposing suinmer
flounder to increased (and constant) current velocity in the nursery
stage would condition them for better performance in cages placed
in Narragansett Bay. Rhode Island (not the OOADP site).

Three experiments, two of 60-day duration and one of 30-day
duration, were conducted with different water velocities in tanks,
using fish of 1 24 ± 4 g (exposed to 0. 1 5. or 30 cm/sec for 60 days).
257 ± 12 g (exposed to 0. 20. or 40 cm/sec for 60 days), and 387
± 13 g (exposed to 0. 15. or 30 cm/sec for 30 days), in a raceway
system with adjustable paddlewheels. For all of the size groups of
fish, survival was significantly reduced at the highest current ve-
locity. For the 124-g fish, survival in high velocity (26 ± 1%) was

338 Abstracts. 2002 Annual Meeting. Febriiury 25-27. 2002

Milford AquacLilture Seminar. Millord. Connecticut

significantly lower than that in medium xelocity (."^7 ± 7% ) and in
low velocity (67 ± 69f )• For the 2.^7-g fish, survival in high ve-
locity (35 ± 19%) was significantly lower than that in medium and
low velocities ( 1 00% in both cases). For the 387-g fish, survival in
high velocity (50 ± 11%) was significantly lower than that m
medium velocity (98 + 2%); all 387-g fish in the low-velocity
treatment were lost due to a system malfunction. For both 1 24- and
257-g fish, growth in the medium-velocity treatment was signifi-
cantly better than that in the low-velocity treatment, which in turn
was better than that in the high velocity treatment. For 1 24-g fish,
growth was 76 ± 1 2 g. 49 ± 8 g, and 39 ± 0 g in the medium, low.
and high velocities, respectively. For 257-g fish, growth was 47 ±
10 g, 25 ± 4 g, and -7 ± 17 g (weight loss) in the medium, low.
and high velocities, respectively. For 387-g fish, growth at medium
velocity (26 ± 6 g) was significantly greater than that at high
velocity (-22 ± 8 g). Food consumption data from the 257-g fish
showed that the fish in medium velocity grew most because they
consumed significantly more food during the experiment (1.622 +
128 g per tank) than did fish in low velocity (915 ± 65 g). which
in turn consumed significantly more than fish in high velocity (640
± 90 g). Nevertheless, there was no significant difference in food
conversion ratio (FCR) between fish at low velocity ( 1.54 ± 0.37)
and those at medium velocity ( 1.37 ± 0.23).

At the end of the experiment with 1 24-g fish, fish from the
low-velocity and medium-velocity treatments were moved to
cages in Narragansett Bay. where currents of about I knot (approx.
55 cm/sec) are routine. After 3 wk in the cages, no significant
differences in survival were observed (low velocity = 83% ±
12%; medium velocity = 81% ± 2%). Subsequent damage to
some of the cages and escapement of the fish precluded funher
statistical analysis of survival, as well as any growth measure-
ments. We conclude that cuirent velocities of 15-20 cm/sec in the
nursery improve growth of juvenile summer flounder, that current
velocities of 30— lO cm/sec are excessive, but that increased current
velocity in the nursery does not improve fish survival upon transfer
to cages.

REMINISCENCES OF EARLY PIONEERS IN OYSTER
CULTURE. Luther Blount, Blount Shipyards, Warren, Rl
02885.

In the late 1920s and early 1930s, there were about six oyster
companies in Warren, Rhode Island, which not only worked
closely with the Connecticut oyster people but also had connec-
tions in the nearby Taunton River and Assonet Bay areas in Mas-
sachusetts, where there was always a good oyster set.

In those days, oyster fishermen in Rhode Island were really into
summer clam bakes, where all the oystermen gathered. My uncle.
Byron Blount, of E.B. Blount and Sons, often invited Dr. Paul
Galtsoff there. In fact. Dr. Galtsoff would drive over from Woods
Hole to talk oysters with my uncle. So 1 knew him as a white-
haired scientist discussing good and bad oyster sets and this always

was his primary subject. Dr. Victor Loosanoff of the Milford
Laboratory, Milford. CT followed Dr. Galtsoffs visits to my uncle
and 1 happened to be there the day he showed us a paper egg crate
coaled not only with cement, but also literally loaded with thou-
sands of oyster set. For the past half century 1 found myself work-
ing with these shellfish pioneers and learning from them as I built
Prudence Island Farms.

Because we had previously bought spat on scallop shells from
Dr. George Matthiessen on Fishers Island and knew how he got
them, we looked for a marine biologist to help us get a set from
Green Hill Pond on the Rhode Island south shore. We hired a
young man who just graduated from the University of Rhode Is-
land, called him "John Oyster,"" and began to buy scallop shells
and get rafts made, which are used today in my salt water oyster
pond on Prudence Island.

"CRAB SIGHTINGS" IN LONG ISLAND SOUND DURING
2001. Diane J. Brousseau, Biology Department, Fairfield Univer-
sity, Fairfield. CT 06430: Ronald (Joldberg. USDOC. NCAA.
National Marine Fisheries Service. Northeast Fisheries Science
Center. Milford Laboratory. Milford. CT 06460.

During summer and fall of 2001. there were a number of in-
quiries to the Milford Laboratory about observations of crusta-
ceans in Long Island Sound. In July, swarms of niegalopae of the
Asian shore crab. Hemii>rapsiis sanguineus, were seen both in the
water column and rafting on floating rockweed near Charles Island
in Milford. Connecticut. These larvae were likely the result of an
early summer spawning. Recent increases in population size of
Hcuiigrapsus may account for greater abundance of the larvae than
in the past. In early fall, reports were again made of dense aggre-
gations of small crabs swimming close to the surface. Initially
mistaken for Asian shore crabs by many observers, they were later
identified by Nizinski (pers. comni.) as sub-adult pinnotherids,
Pinni.ui cluietoptenuui. This small crab is often found living as a
commensal within the tube of the polychaete worm Chaetopterus.
Pinnixa sightings were widespread, occurring in the Thimble Is-
lands, Milford Harbor, and Housatonic River in Connecticut, and
near Orient Shoals off Long Island. Fall swarming of Pinnixa prior
to settlement has been observed previously by Mroczka (pers.
comm.). but the early life history of this species has not been
described fully. In October, an adult male shame-faced crab. Cat-
(ippa tlaiuiuea. was collected in shallow water by a scuba diver in
Stoningtoii. Connecticut. Calappa is predominantly tropical to
subtropical, but larvae can drift as far north as southem New
England, accounting for occasional occurrence of adults in this
region. This molluscivorous crab spends much of its time buried in
the sand, making brief excursions to search for food. The obser-
vations described above are not unique, but nia\ ha\e been more
apparent during 2001 because of interannual variability in climate
or the population dynamics of the species involved.

Milf'oid Aquaculture Seininur, Milford, Ctniiiccticut

Abstracts. 2002 Anmiiii Meeting. Fehruaiy 25-27, 2002 339

SOME CLASSICAL PERSONALITIES IN THE SHELL-
FISH RESEARCH ARENA: ECLECTIC OBSERVATIONS
OF AN ERSTWHILE OBSERVER. Walter J. Canzonier,

M.R.O.C.F.. P. O. Box 662. Port Norris. NJ 08349.

The personalities of many of the scientists associated in the past
with the shellfish research and development community might be
of general interest to those currently engaged in this field. The
personalities of these indi\iduals. other than makuig them colorful
and intriguing characters, probably also had a considerable influ-
ence on the approaches they used in addressing problems requiring
scientific or technological intervention. Ergo, to better appreciate
the contributions of some of the members of the community in-
volved with the recent, and not-so-recent evokition of shellfish
research, it has been suggested that it might be both entertaining
and educational to have a glimpse at their personae. Using a cap-
sule review of some of the key players in the shellfish research and
development saga. I will attempt to share with the more recent
entrants in the game a few anecdotal sketches of some of those
individuals with whom I have had contact in my 45 years of
involvement with the academic and the industry elements of mol-
luscadom on the East Coast. This informal documentary, as dis-
torted as it might be as a result of the undoubtedly biased perspec-
tive of the reporter, is offered to the curious in hope that it will help
them gain a better insight into the personalities of thiise whom they
have encountered merely as names in the nondigitized literature.
Some of those mentioned were colleagues, others mentors, still
others known to me primarily through accounts by relatives or
close associates. Interest in documentation of personalities was
first stimulated by the efforts of Sewell Hopkins, who prepared a
set of three unpublished but extremely interesting and informative
memoirs, two of which (University of Illinois and Project Nine)
present enlightening and amusing sketches of his teachers and
colleagues.

In this rambling account of the members of the "Old Guard." 1
will attempt to reconstruct the images of some of the well-
recognized, as well as the lesser-known individuals. Certainly the
names of Julius Nelson (known to me through the stories from his
son); Galtsoff and Loosanoff, who worked in this immediate area;
T. C. Nelson; J. G. Mackin and Sammy Ray of dermo fame; J. D.
Andrews; and H. H. Haskin will probably be familiar to many
currently working in this field. Other, less commonly recognized
names include W. F. Wells, known to me only from exchanges of
vitriolic correspondence with T. C. Nelson; L. A. Stauber. a poly-
valent researcher more noted in the field of parasites of vertebrates,
but always available to his students and colleagues as a valid
anchor in the basic principles of shellfish pathology and physiol-
ogy; his students S. Y. Feng and M. R. Tripp, among others; Joe
Glancy. who doggedly pursued development of commercial shell-
fish hatchery technology despite strident attempts to rectify his
errant behavior by his neighbor across the Sound; Mike Castagna
who pursued a similar course in more southern waters; and
M. R. Carriker. Carl Shuster. Herb Hidu. Tom Cheng, and others

who at some point in their careers had close ties with the Depart-
ment of Oyster Culture of the New Jersey Agricultural Experiment
Station.

If asked what might be a common trait of these individuals, my
observations would lead me to conclude that it was a capacity to
mobilize their imagination to devise a plan of action that effica-
ciously utilized the often very limited resources available to
them — whether this he in the laboratory or in the field. This in-
trinsic ability to adapt their efforts to the resources at hand enabled
them to make significant contributions in the realm of academia.
and to cost-effectively assist the shellfish industry in its quest for
more productive culture practices and strategies. For this vanish-
ing breed, a lack of sophisticated facilities and equipment was
never considered to be an insurmounlablc limitation, but merely
a challenging impediment to be circumvented by innovative
tactics.

MASSACHUSETTS OCEAN RESOURCE INFORMATION
SYSTEM (MORIS). Diane Carle. Office of Coastal Zone Man-
agement, Executive Office of Environmental Affairs, 251 Cause-
way Street. Suite 900. Boston. MA 021 14.

Massachusetts Coastal Zone Management (CZM) has em-
barked on a long-term project to develop the Massachusetts Ocean
Resource Information System (MORIS). MORIS will be a com-
prehensive database and ArcView GIS extension providing access
to the broad range of information necessary to carry out the CZM
mission. The first phases of the MORIS project were completed in
2001 and focus on information and tools useful for screening for
potential aquaculture sites.

CZM contracted EVS Environmental Consultants to conduct
the initial data mining and GIS application development. CZM
also teamed with NOAA's Coastal Services Center (CSC). Mas-
sachusetts Division of Marine Fisheries (DMF), and Massachusetts
Department of Environmental Protection (DEP) to add a "georegu-
lation" tool that allows users to query an area for aquaculture-
related regulations. Users are presented with a list of relevant
regulations and can view summaries as well as the actual legisla-
tion. Users can also view the web sites of the relevant regulatory
agencies. The CSC is also creating new data layers for the project,
including benthic maps of selected areas of the Massachusetts
coast and maps of areas suitable for sustaining shelltlsh growth.
CZM is completing maps of existing aquaculture leases in Mas-
sachusetts. Future phases of the project will develop new water
quality data layers and enhance the applications' water quality
mapping capabilities. CZM is also pursuing funding to create an
Internet mapping site for the project.

The MORIS CD containing the ArcView extension and data-
base is available at no cost from CZM.

340 Ahstmcls. 2002 Annual Meeting. February 25-27, 2002

Milt'ord Aquaculture Seminar, Milford. Connecticut

HABITAT SUITABILITY ASCERTAINED BY GROWTH
AND SURVIVAL OF BAY SCALLOPS IN TIERED CAGES.
Joseph Choromanski, Sheila Stiles, Mark Dixon, and David
Veilleux USDOC, NCAA, National Marine Fisheries Service,
Northeast Fisheries Science Center, Milford Laboratory, Milford,
CT 06460; Christopher Cooper, Ocean Technology Foundation,
UCONN-Avery Point Campus, Groton, CT 06340.

A laboratory-spawned line of bay scallops (Argopecten iinuli-
ans irnidiuns) was used to evaluate the effectiveness of commer-
cial three-tier, rigid-mesh cages for growing scallops in two proxi-
mate hut dynamically different sites in eastern Long Island Sound.
With permission of the Gi-oton Shellfish Commission and the State
of Connecticut Aquaculture Division, we established comparative
field sites on the western side of Ram Island (low dynamic, gentle
tides), and the southern end of the island (high dynamic, tidal
current of up to 2 knots).

The scallops were spawned in March and held m temperature-
controlled tanks in the Milford Laboratory at 22"C until May when
ambient seawater temperature reached about IS'C. The scallops
were then acclimated to the lower temperature and distributed in
outdoor raceway tanks using densities determined as optimal — 5 L
biomass per tank. In late July, scallops with a mean size of 25 mm
were deployed at each site, with a starting biomass of 2 L (ap-
proximately 400 scallops) in the top tiers of a set of two cages at
each site. The cages were made of plastic-coated wire with a
7.5-cm mesh. Each cage measured 56 x 56 x 94 cm and was
divided horizontally into three sections or tiers. Two ballast areas
below the bottom tier provided an offset from the sea floor of
approximately 15 cm. Cage inserts of smaller mesh (10 and 17.5
mm) measuring 41 x 10 x 81 cm were used to hold the scallops.
After I month, the scallops were divided into three densities of 50.
100, and greater than 1 50 scallops per tier in each of the cages. The
cages were checked monthly to the end of the experiment to de-
termine survi\al and growth, with the added attention to shell
indentations that might indicate density problems, and to check
and remove fouling organisms.

Results from the field experiment indicated that satisfactory
growth of scallops occurred at both sites. The initial growth rate
was rapid, but then leveled off as the water temperature decreased.
Growth of scallops was slightly better at the western side of the
island (mean size of 51 mm) than at the southern end (mean size
of 48 mm). There was no difference in the mean size of scallops
in the top and middle tiers, with 50 and 100 scallops, respectively.
Bottom tiers of all cages, stocked with greater than 150 scallops,
did show slightly slower growth and higher mortality. Scallops in
the cages from the southern, high-dynamic area exhibited more
shell indentations: this could be attributable to the strong currents
pushing the scallops together for periods of time, thereby causing
a temporary decrease in available cage area that may mimic effects
of higher shelf densities. Further studies are waiTanted in similar
habitats to corroborate these results.

HORSESHOE CRAB AQUACULTURE: PRELIMINARY
RESULTS FROM HATCHING AND REARING STUDIES.

Carmela Cuomo, Yale University. New Haven, CT 06520 and
Uni\ersity of New Haven, West Haven, CT 06516; Paul R. Bar-
tholomew, SUPERB Technical & Environmental, Hamden, CT
06517; Leslie Angelini, Brian King, and Jeffrey Bytzko, The

Sound School, 60 South Water St., New Haven, CT 06519.

Previous studies undertaken by the two senior authors during
the summer of 2000 at the National .Marine Fisheries Laboratory in
Milford, CT resulted in the successful spawning of captive adult
horseshoe crabs, Liiiutlii.s polxplwiuns. The fertilized eggs were
allowed to develop under conditions approximating field condi-
tions, although predators were kept to a minimum. The eggs had a
hatching rate of 60% over an initial 2-mo period, followed by a
98'/r hatching rate over the course of 10 mo. Evaluation of the
long-term survival (greater than 1 y ) of Llnuilm was not completed
successfully because of a malfunction m the rearing tanks.

This study was undertaken in an effort to evaluate the role of
different food sources on the growth and survival of post-hatch
horseshoe crabs. Approximately 4.500 eggs were kept in small,
experimental bowls filled with Long Island Sound (LIS) seawater
and aerated. Initial egg densities varied per bowl, with a maximum
density of 1,500 and a minimum density of 50. All bowls were
checked daily for hatchlings. Upon hatching, all hatchlings from
an individual bowl were removed to a separate bowl, with no more
than 50 per bowl. Hatchlings were kept in aerated LIS seawater
and a series of behavioral observations were made on them.
Hatchlings in bowls were checked daily for tiiolting.

Molted individuals were removed frotn hatchling bowls and
placed in individual bowls of aerated seawater. The number of
post-hatch molted individuals per bowl ranged between 10 and 30.
Horseshoe crabs at this stage were separated into three groups for
preliminary experiments examining the role of diet on growth and
survival. They were fed one of three food items: newly hatched
brine shrimp, rotifers, or concentrated dried food flakes. The ju-
veniles were checked every day and any secondary molts or deaths
were noted. When an organism underwent a secondary molt, that
organism was transferred to a new bowl and fed one of the three
food choices. This same procedure was repeated for every new
molt stage reached. At the conclusion of the experiment, all sur-
viving post-hatch molts (second and upward) were removed and
placed in aerated, 10-gal aquaria containing artificial seawater and
a bottom covered with 3 cm of fine sand.

The results suggest that diet may play a supporting role in the
growth and sur\ ival of post-hatch Liniiilu.s juveniles, although this
factor needs further investigation. In general, all juveniles reached
their first post-hatch molt between 5 and 12 days. The time to
second post-hatch molt varied with diet. There was a slight dif-
ference in molt timing among the three test groups. Juveniles (first
post-hatch molt) rea)ed on a diet of rotifers underwent a second
post-hatch molt within 7-12 days, and those reared on a diet of
brine shrimp generally molted between 10 and 14 days. Juveniles

Milford Aquaculture Seminar. Milford. Connecticut

Abstracts. 2002 Annual Meetins. February 25-27. 2002

.Ul

reared on flaked food generally molted between 9 and 12 days. Of
these juveniles, those fed on a diet of brine shrimp experienced a
mortality rate of up to 5%, whereas those fed on rotifers or flaked
food experienced a mortality rate of <l'7'r. Time to third post-hatch
molt varied, with juveniles reared on flaked food molting sooner
than juveniles reared on either brine shrimp or rotifers. Those fed
flaked food were significantly more likely to molt oW'/r) and
survive (>85%) than those fed either rotifers or brine shrimp.

The results from these initial studies suggest that food quality
and type has only a slight effect on the two earliest post-hatch molt
stages of Limulus polyphenuis juveniles, but has a strong effect
upon the third post-hatch molt. It appears likely that this effect is
related not only to the food type, but also to the life habit of the
horseshoe crab, and that any rearing plan for this species must
include at least a two-phase food supply ciinsisting initially of
zooplankton. followed by food that is mixed in with the sediments.
Research on the effects of food quality on the growth of Limulus
juveniles continues at this time.

A COOPERATIVE STUDY ON THE AQUACULTURE OF
PORPHYRA LEUCOSTICTA (RHODOPHYTA) FOR AN IN-
TEGRATED FINFISH/SEAWEED RECIRCULATING
AQUACULTURE SYSTEM IN AN URBAN APPLICATION.
John J. Curtis. Sherry W. Lonergan, and Paul J. Trupp.
Bridgeport Regional Vocational Aquaculture .School. Bridgeport.
CT 06605; Peimin He, Shanghai Fisheries University. Shanghai,
People's Republic of China; Raquel Carniona and Charles
Yarish. University of Connecticut, Stamford. CT 06901; George
P. Kraemer, State University of New York. Purchase. NY 10577;
Christopher D. Neefus, University of New Hampshire, Durham,
NH 0,^824; Thierry Chopin, University of New Brunswick, Saint,
John, NB E2L 4L5; George Nardi, GreatBay Aquaculture LLC,
Portsmouth. NH 03801,

Aquaculture represents an excellent opportunity to help reju-
venate blighted coastal urban areas on the Northeast coast. On
land, aquaculture requires a relatively small amount of space;
building space can often be acquired at reduced cost in ungentri-
fied city areas and aquaculture can represent an attractive, envi-
ronmentally benign form of commerce. However, finfish and
shellfish aquaculture operations are a source of an effluent with
high concentrations of dissolved inorganic nutrients (N, P). To
prevent eutrophication, the EPA is developing stringent guidelines
for the release of N and P into coastal waters. An integrated re-
circulating aquaculture .system, coupling the growth of seaweed
and fish, can solve these problems for urban aquaculture facilities.
Not only is the effluent remediated (the seaweed requires for
growth the waste products produced by the fish), but an additional
multiproduct, high-value crop can be generated. Marine aquacul-
ture in the Northeast consists primarily of coastal pen-based
salmon production and shallow benthic production of shellfish
(mussels, clams, and oysters). There are very few tank-based (on
land) marine aquaculture operations in the Northeast. One opera-

tion is GreatBay Aquaculture LLC (Portsmouth. NH). a land-based
hatchery and grow-out facility for summer flounder and cod. high-
value fish that are being sold to the U.S. and Japanese sushi and
Sashimi markets and to "white tablecloth" restaurants.

Our work is part of a multi-university effort (University of
Connecticut. State University of New York at Purchase. University
of New Hampshire. University of New Brunswick, and Shanghai
Fisheries University! with GreatBay Aquaculture LLC to develop
an integrated finfish/seaweed recirculating aquaculture system
(RAS) suitable for urban aquaculture. The unique aspect of our
RAS system is that it will be integrating the culture of finfish (i.e..
summer flounder) and native species of seaweed (i.e.. Porphyra).
The red alga. "Pdiphyra". is the most valuable maricultured sea-
weed in the world, with an annual value of more than $1.8 billion.
Porphyra (nori) is primarily used for food as the wrapping around
sushi rolls; it is a major dietary source of taurine (controls blood
cholesterol levels) and is rich in proteins, vitamins, trace minerals,
and dietary fiber. On a dry weight basis. Porphyra can be worth
more as a source of biochemicals than as food. It is the preferred
source of the pigment, r-phycoerythrin, utilized as a fluorescent tag
for biotechnological applications. The United States is dependent
primarily upon foreign sources (i.e.. China. Japan, and Korea) for
nori via coastal net culture. BRVAS students are working along-
side undergraduate and graduate students in the construction and
operation of these systems.

The life history of Porphyra includes a microscopic filamen-
tous stage (the conchocelis stage) and the more conspicuous mac-
roscopic blade stage that grows attached to intertidal and shallow
subtidal substrate. There are at least seven recognized species of
Porphyra in the Northeast. We have begun evaluating one of these
native species {P. leucosticta) as a candidate for the integrated
RAS, because it may be one of the best sources for the sushi food
industry and for r-phycoerythrin. We are developing the mass
culture techniques (in both free culture or attached to nets) for this
native species of Porphyra. We will report on the mass seeding
technologies that we have developed and the specific growth rates
of P. leucosticta at the BRVAS culture facilities.

UPDATE ON SHELLFISH RESTORATION AND REVIEW
OF LOCAL HATCHERIES IN NEW JERSEY. Gef FHnilin.

Rutgers Cooperative Extension. Toms River. NJ 08755.

Cooperation among several sectors that work with water and
shellfishery issues has led to two new accomplishments that com-
bine an interest in improving both the ecology of the waterways
and the potential economy of the shellfish industry in New Jersey.

In the summer of 2001, 10,000 bushels of washed oyster shell
were deposited to construct a new reef in a near shore area in
Keyport Harbor in western Raritan Bay. The project was spear-
headed by the New York/New Jersey Baykeeper, with assistance
from the National Marine Fisheries Service, Rutgers Cooperative
Extension, local shelUishermen, and the NJ Department of Envi-
ronmental Protection's Bureau of Shellfisheries. In a community-

342 Abstracts. 2002 Annual Meeting. February 25-27, 2002

Miltord Aquaeulture Semuiar. Millord, Connecticut

supported effort. 10.000 oysters, which had been raised in Taylor
Floats by volunteers from the area, were deposited on the reef to
establish a community. This effort of growing oysters by the vol-
unteers continues.

Later that year in the fall, with the support from the South
Jersey Economic Development District, the last oyster boat on the
New Jersey Atlantic Coast moved 2.000 bushels of oyster seed to
an old bed at the mouth of the Mullica Riser. The Fitney Bit bed
will have been closed by the Bureau of Shellfisheries for a year when
it is opened for public harvest. Industi7 representatives and Rutgers
Cooperative Extension, who acquired the funds to do this work, are
presently looking for more funds to expand this work.

Shellfish hatcheries in New Jersey have been reevaluating their
production in light of challenges from Brown Tide blooms and
unexplained hatchery mortalities.

FLOW CYTOMETRY AS A TOOL TO QUANTIFY THE
OYSTER PHAGOCYTOSIS. RESPIRATORY BURST. AND
APOPTOSIS. Michael J. Goedken and Sylvain l)e Guise, De-
partment of Pathobiology. University of Connecticut. 61 N Ea-
gleville Road. U-89, Storrs. CT 06269.

Infectious diseases are a significant problem in oyster aquacul-
ture and cause immense production losses. The protozoan parasites
Perkinsus mariniis and Haplosporidium nclsam have generated
losses estimated in the hundreds of millions of dollars over the last
35^5 y in the Middle Atlantic states.

The relationship between parasites and oyster defense mecha-
nisms is unclear. A better understanding of the immunopathologic
association may reduce these economic losses. Defense mecha-
nisms of the eastern oyster {Crassostrea virt>iiuca) were quantified
at the single-cell level utilizing flow cytometry. Phagocytosis was
measured using fluorescent beads. Respiratory burst activity was
quantified as the increase in dichlorofluorescein-associated fluo-
rescence upon stimulation. Apoptosis was evaluated with TUNEL
assay. Three subpopulations of hemocytes (granulocytes, hyali-
nocytes, and intermediate cells) were identified with unique func-
tional characteristics. Granulocytes were most active at phagocy-
tosis and peroxide production, whereas hyalinocytes were rela-
tively inactive. TUNEL assay application allowed quantification of
hemocyte programmed cell death with temperature-dependant
changes. Flow cytometry can rapidly, accurately, and directly
quantify the morphology and function of a large number of indi-
vidual cells, and will lead to a better understanding of the bivalve
immune system.

COBIA CULTURE. Josh Goldman. Fins Technology. Inc.. l.'S
Industrial Rd., Turners Fall. MA 01376.

Cobia {Rcichycentron canadiim). is considered a prime candi-
date for culture, given its extremely fast growth rate and excelleni
flesh quality. Interest in cobia culture has grown substantialh in
the last 5 y. fueled by reported successes in extensive, net-pen
operations in Taiwan, and as U.S. researchers have gained some

familiarity with captive fish. Fins Technologs. with support from
NOAA"s National Sea Grant, has investigated a variety of aspects
of intensive cobia culture in an effort to develop protocols for
commercial production. We have evaluated growth and feed con-
version efficiency at 10. 20. and 30 ppl salinity in artificial sea-
water, and have instituted routine bacterial and histological moni-
toring of our populations to assess the risks and develop appropri-
ate health management strategies needed to support commercial
production. During the last 2 y. we and our collaborators hope to
demonstrate commercial feasibility of this promising species.

CREATING OPPORTUNITIES FOR STUDENT AQUACUL-
TURE PROJECTS. Eric Goodman, Colleen Cook, and Michael
Weiss. Friends Academy. Duck Pond Rd.. Locust Valley, NY 1 1560.
The goal of our ongoing project is to increase awareness and
participation in shellfish biology, habitat preservation, and aqua-
culture techniques among our peers. This group currently includes
seven students in Friends Academy, our high school, as well as
five students from Locust Valley and Oyster Bay, two nearby
public schools on the North Shore of Long Island. The project
began w ith a program designed as a basic introduction to shellfish
aquaculture for local educators to motivate them to start similar
projects in their schools. We held a series of seminars for students
who showed an interest in our work and who wished to design an
aquaculture project of their own. The topics of these seminars
included working in the lab with algae and constructing grow-out
equipment. Our next initiative was to construct a facility that was
open to the public to increase the number of educational and re-
search opportunities in the community. This facility, which is un-
der development, is a hands-on center that includes a classroom as
well as a fully functioning shellfish hatchery. We will be growing
the Atlantic bay scallop (Argopecten iinulwiis inadians) as well
as the eastern oyster iCrassostrea virgiiiica). Lessons in hatchery
operation and maintenance, shellfish life cycles and anatomy, and
the use of a FLUPSY are also taught in the hatchery. We hope that
through our work in the center, students will use the hatchery to
conduct their research relating to shellfish life cycles during the
school year.

AN EXPERIMENTAL INVESTIGATION OF DIETARY
FATTY ACIDS AND STEROLS AND THE IMMUNOLOGY
OF THE AMERICAN OYSTER, CRASSOSTREA VIR-
GINICA: A WELL-FED OYSTER IS A HEALTHY OYSTER.
N'EST-CE PAS? Helene Hegaret. Ecole Nationale Superieure
Agronomique de Rennes. France; Gary H. Wlkfors, NCAA Fish-
eries. NEFSC. Milford. CT 06460; Philippe Soudant, Universite
de Brest. France; Maryse Delaporte. Jeanne Moal, and Jean-
Fran(;ois Samain, Laboratoire de Physiologic des Invertebres.
IIREMER. Brest. France.

The lipid composition of the algae fed to oysters is very im-
portant because fatty acids and sterols in the membranes of oyster
cells to some extent have a dietary origin. The fatty acid and sterol

Milford AquacultLire Seminar. Miltord, Connecticut

Abstracts. 2002 Annual Meeting. February 25-27. 2002 343

composition of hemocyte membranes is thought to affect immune
function, thereby Hnking nutritional status with abiUty to respond
to environmental and health stresses. To investigate this linkage,
we designed an experiment in which two replicates of seven feed-
ing treatments were applied to the oyster. Crassostrea virginica,
and hemocyte function was evaluated before and after a subse-
quent high-temperature stress. Twelve oysters (ca. 50 mm) were
put into each of 14 computer-controlled feeding chambers. Algal
diets fed at 10% and 509f^ daily rations (dry wt/dry wt) were LB
1077/lB (Skeletonemu aistatuin). PLY429 {Tetmselinis chiii). and
a 50/50 (dry wt) mix; we also included two unfed controls. These
two algae have different lipids: LB 1077/1 B contains the fatty acids
C20:5(n-3) and C22:6(n-3) and cholesterol; whereas. PLY429 con-
tains C20:5(n-3). but no C22:6(n-3). and 24-methyl and 24-
niethylene cholesterol. After 5 wk of feeding at 20"C. we sampled
half of the oysters for hemocyte analyses and gill-membrane lipid
composition, subjected the remaining oysters to I wk of high-
temperature (28"C) stress, and sampled as previously. Gill mem-
brane lipids will be analyzed by GLC (work in progress).
Hemocyte function was evaluated by five methods employing the
FACSCAN flow cytometer: identification of hemocytes by SYBR
green fluorescence, viability by propidium iodide fluorescence,
adherence and aggregation by forward- and side-scatter, phagocy-
tosis using fluorescent plastic beads, and respiratory burst by a
newly modified method using Zymosan A as the activator. These
response variables were tested statistically, first using a nested
ANOVA model, with feeding ration and regime as independent
variables, and in second way using discriminant analysis.

Individual hemocyte functions were not affected significantly
by feeding ration or regime; however, a consistent trend relating
the higher ration with enhanced hemocyte function was apparent.
By contrast, differences between unfed and fed oysters were, in
many cases, highly significant and appreciable, with unfed oysters
showing decreased function in most subpopulations of hemocytes
(granulocytes, hyalinocytes. and intennediate cells). Discriminant
analysis, using data from multiple hemocyte measurements, was
able to differentiate between high- and low-quantity feeding treat-
ments, as well as between the different algal diets. These findings
indicate that nutrition does affect immune function in oysters,
thereby mediating their response to stress.

THE ROLE OF THREE BACTERIA IN SHELL DISEASE
OF THE AMERICAN LOBSTER {HOMARUS AMERICA-
NVS), Andrea Hsu. Erin Summers, and James Estrada, Boston
University Marine Program. Marine Biological Laboratory. 7
MBL St., Woods Hole. MA. 02543; Roxanna Smolowitz, Marine
Biological Laboratory, 7 MBL St., Woods Hole, MA 02543.

Although typically infecting impounded lobsters, lobster shell
disease is becoming increasingly more prevalent in wild popula-
tions throughout the New England region. This study utilizes scan-
ning electron microscopy (SEM) and histological analyses to de-

scribe the morphology and prevalence of bacterial cells present on
carapace samples taken from two wild-caught lobsters with shell
disease and one lacking any noticeable infection. SEM analysis
revealed and statistical tests verified three separate morphological
types of bacteria present on both carapace samples and cultures
taken from shell lesions. Results from bacterial identification sug-
gest two bacteria to be of the genus Vibrio, whereas the third is a
Pasteiirclla-Wke organism. Bacteria on infected carapace were
seen to concentrate on intact epicuticle. the edge of lesions, mi-
croscopic cracks and holes, and setal pores. Halo-like holes sur-
rounded all bacterial types, closely matching the shape of the
bacteria, suggesting that each bacterium is boring into the epicu-
ticle. Healthy carapace showed substantial bacterial concentrations
present only around carapace setae. Several of these bacteria were
seen with small-bore holes surrounding them, but active boring
and degradation of the epicuticle was minimal in contrast to the
infected carapace. Histological observations show that there is a
difference between the breakdown of wild and impoundment le-
sions. Shell lesions of wild lobsters show lattice-like cuticular
remnants attached to underlying less degraded cuticle in several
eroded foci. This study documents not only the presence of three
bacterial species in shell disease of wild-caught New England
lobsters, but also illustrates their role in the degradation of the
carapace. This is the first evidence of the mechanism that is used
to break down the epicuticle of the lobster shell, and suggests that
different organisms are involved in causing wild and impoundment
shell disease.

DERMO INVESTIGATIONS, RAZOR CLAM NURSERY
TRIALS, AND PRELIMINARY BAY SCALLOP ADHE-
SION CULTURE EFFORTS. Richard C. Karney and Aman-
dine Surier, Martha's Vineyard Shellfish Group. Inc.. Box 1552.
Oak Bluffs. MA 02557; David W. Grunden, Town of Oak Bluffs,
Box 1327, Oak Bluffs, MA 02557; Tliomas E. Berry. Martha's
Vineyard Shellfish, Box 1660, Edgartown, MA 02539.

To compare the infection patterns of dermo disease (Perkiiisiis
tnarimts) in Edgartown Great Pond and Tisbury Great Pond, cages
of wild and disease-free cultured oysters were deployed in both
ponds on June 20 and 21 and sampled monthly for mortality and
dermo infection progression. Man-made breaches of the salt
ponds' barrier beaches allowed for some manipulation of water
chemistry within the ponds. Edgartown Great Pond was managed
to maximize its exchange with the sea to reduce eutrophic condi-
tions believed to be stressing its oyster population. Management in
Tisbury Great Pond sought to limit exchange with the ocean in
hopes of achieving salinities unfavorable to the dermo parasite. By
October, infection rates in both ponds for all of the wild and two
of the three groups of cultured oysters were 100%. The infection
rate of one group of the cultured oysters in Tisbury Great Pond did
not rise above 76%. By November, cumulative mortality of the
wild oysters was 19.8% in Tisbury Great Pond and 29.5% in

344 Ahslnicts. 2002 Aniuuil Meeting. February 25-27, 2002

Miltord Aquaculture Seminar. Milford. Connecticut

Edgartown Great Pcind. Mortality of the cultured oysters was 4.4%
in Edgartown. The mortalities of cultured oysters at two sites in
Tisbury Great Pond were 5.5% and 3.4%. The variation in mor-
tality of the cultured oysters appeared to correlate with the densi-
ties of natural oyster beds at the sites.

In late August, seed razor clams {Eiisis direcliis) with an av-
erage length of 20. 1 mm were planted at two densities (54.5/ft" and
163.5/tr) in four different nursery systems — a tidal upweller.
mesh-covered bottom boxes, and mesh-covered and open floating
sand boxes. Final growth measurements taken in early November
were poorest for the high-density tidal upweller (28.8 mm) and
best for the low-density open floating sandbox (54.1 mm). In No-
vember, clam survival in the sandbox nurseries was as follows:
low density/no mesh 56%; high density /no mesh 50%; high den-
sity/mesh 48%: low density/mesh 39%.

.Several adhesives have been tested in efforts to develop a cul-
ture technology based on gluing bay scallops to plastic nets. A
suitable adhesive has yet to be located.

SOME CULTURE STRATEGIES FOR GROWING ROTI-
FERS (BRACHIONVS PUCATILIS) AS FEED FOR AQUA-
CULTURE APPLICATIONS. Robin Katersky. Barry Smith,
Dean Perry, and David Nelson, USDOC, NCAA, National Ma-
rine Fisheries Service, Northeast Fisheries Science Center. Milfnrd
Laboratory. Milford, CT ()64(i().

Rotifers are provided as first feed to larval finfish when they
are absorbing their yolk sacs and their digestive systems have
developed sufficiently to consume food. Marine finfish aquacul-
ture, therefore, requires them in large numbers at these times.
Rotifers can be fed marine microalgae for growth and reproduction
as well as for enrichment just before they are fed to the fish.

Rotifers typically are grown in large tanks with algal culture
added to the volume to replace the algal biomass previously con-
sumed by the rotifers. Culture water is usually changed by draining
the tank (and remaining algae), catching the rotifers on a screen,
and resuspending them in clean water. Rotifers can consume large
quantities of algae. Finfish studies at the Milford Laboratory re-
quire production of millions of rotifers. The finfish are fed daily at
a density of 10 rotifers/mL in 893-L tanks. With an algal supply of
1 X 10" cells/niL. how does one get enough algal biomass for
maximal rotifer growth in an economical space and time? We
investigated two culture methods to achieve our requirements.

The first culture method investigated was to concentrate the
rotifer tank using a hollow-fiber filter to remove 40 L/day of water
only. The rotifer tank then was refilled with algal culture. This
procedure was repeated daily, providing an average feeding of
200,000 algal cells/mL in 200- to 240-L rotifer tanks. Rotifer tanks
were started with 46 rotifers/mL and reached 233 rotifers/mL in
4.8 days.

The second culture method used a different strategy. Rotifers
were introduced into a full tank volume of algal culture (150 L) at

a density of -300.000 cells/mL. The rotifers then were left to grow
for 4 days with only experimental samples removed. Rotifer den-
sities consistently went from 59/mL to 232/mL in 3 days.

In all cases algae was consumed to below countable levels. The
second culture strategy was much less labor intensive than both the
traditional and the first method. The second, or all-algae method,
reduced labor by over 50% and required three culture vessels to
meet demand. For the remainder of the larval rearing period (sum-
mer 2001), the all-algae system was used for rotifer production
because of its reliability and ease of use. This system produced an
average of 30 x 10" rotifers/day.

These experiments, together with past work, indicate that there
is much more potential for optimizing the culture of rotifers.

ASSESSING HABITAT VALUE OF MODIFIED RACK AND
BAG AQUACULTURE GEAR IN COMPARISON WITH
SUBMERGED AQUATIC VEGETATION, IN PARTICU-
LAR, AN EELGRASS [ZOSTERA MARINA) BED. Brian Kil-
patrick, Joseph DeAlteris. and Robert Rheault, Department of
Fisheries. Animal, and Veterinary Science, Uni\ersity of Rhode
Island, Kingston, RI 02882.

Submerged aquatic vegetation (SAV) has attracted consider-
able attention in recent years because of its role as an essential fish
habitat necessary for sustainable fish production. Recent regula-
tions that protect SAV have been critical in conserving and restor-
ing this resource. Current policies that serve to protect SAV have
affected applications for construction, docks, dredging, and aqua-
culture. Opponents of shellfish aquaculture operations argue that
the gear used for the grow-out phase of the shellfish reduces the
potential for SAV restoration, and may negatively impact estuarine
ecosystems.

This study was conducted to compare and contrast habitat value
of modified rack and bag aquaculture gear (MRBAG), submerged
aquatic vegetation (SAV), and a nonvegetated shallow seabed
(NVSB). Habitat value is defined herein by descriptive species
diversity statistics and each habitat's ability to support an abun-
dance of organisms throughout the year. The study took place over
the course of 1 y, in which each of the three habitats was studied
at the end of each season (three replicate samples per habitat).
Sampling was performed in Pt. Judith Pond, an estuarine pond in
Rhode Island that discharges directly into Block Island Sound.
Specially constructed lift nets were used to sample the aquaculture
gear, whereas the remaining two habitats were sampled using a
drop-net of identical size and a venturi-driven suction sampler.
Organisms (>5 mm) in each sample were identified, enumerated,
and measured to the nearest millimeter. Environmental data were
collected at the time of each sampling period to discern similarities
and/or differences among the three habitats. Biofouling organisms
were measured in terms of percent cover, and an average surface
area (square centimeters per square meter of seabed) ot each bio-
fouling phyla and/or class was estimated for each habitat and sea-

Milford Aquaculture Seminar. Milford. Connecticut

Abstracts. 2002 Annual Meeting. February 23-27. 2002 345

son. Structural components, in particular, emergent surface area,
were measured to determine their role in providing habitat.

En\ ironmental parameters (temperature, salinity, and dissolved
o.xygen) were not significant between habitats but were signifi-
cantly different between seasons. Sediment type was primarily
sand and was not found to be significantly different (P > 0.05)
between habitats when the gravel size component (shell hash due
to the aquaculture operation) was removed from the MRBAG
habitat. The MRBAG habitat supports a significantly higher abun-
dance of organisms per unit area (m") of seabed throughout the
year. Total numbers of specimens collected are as follows: MR-
BAG. 6.777 individuals: SAV. 577 individuals; NVSB, 211 indi-
viduals. Species richness is consistently higher throughout the
sampling periods in the MRBAG habitat as compared to the SAV
and NVSB habitats. A two-way ANOVA using the richness esti-
mates generated by the jackknife procedure using quadrat samples
(Heltshe and Forrester 1983) indicated significant differences be-
tween all three habitats (P < 0.001 ). A Tukey HSD test indicated
that MRBAG > SAV > NVSB in terms of species richness values.
The species diversity of each habitat was computed using the
Shannon-Weiner Index and showed a significant difference (P <
0.001 ) using a two-way ANOVA between habitats. A Tukey HSD
test indicated that there were no differences between MRBAG and
SAV habitats, but there were differences between MRBAG and
NVSB habitats. The Smith and Wilson measure of evenness was
used to describe how each habitat was organized in terms of spe-
cies abundance proportions. MRBAG habitat had a consistently
lower evenness value because the MRBAG habitat was dominated
by a few species {Neopanopeiis sayi. Tautogolabnis adspersiis,
Mytitus edulisj. Habitat structural complexity was determined us-
ing emergent surface area of the structures found within each
habitat. A two-way ANOVA indicated a significant difference {P
< 0.001) between the MRBAG and SAV habitats in terms of
surface area of structure (MRBAG or SAV) per .square meter of
seabed. These findings suggest that shellfish aquaculture gear pro-
vides habitat to many organisms throughout the year and may be
ecologically beneficial to many native species of recreationally
and commercially important fish and invertebrates, especially in
the early life stages of these resources. This research is the first
step in determining habitat value for shellfish aquaculture gear.

THE CULTURE OF BLACK PEARL OYSTERS ON SUB-
SURFACE LONGLINES IN SAVU SAVU. FIJI. Gordon
King, Taylor Resources. Inc.. 130 SE Lynch Rd.. Shelton. WA

98584.

Continuing an informal company policy to diversify into new
species and geographical areas. Taylor United. Inc.. used family
knowledge and contacts to develop a Black Pearl Oyster Farm in Savu
Savu Bay on the Island of Vanua Levu in the island nation of Fiji.

The subsurface longline system as promoted by Dr. John
Bonardelli was adapted for grow-out of the black pearl oyster.

Pinctada inarguritifera. The installation and development of the
farm was complicated by political instability and an armed coup in
Fiji. Despite these difficulties, the farm now has more than 30
longlines and 30.000 oysters, with several hundred pearls already
harvested and several thousand due for harvest; many more are
being implanted in 2002. Taylor Resources is employing a three-
pronged approach for production of oysters for implantation: spat
collection, buying mature oysters, and a joint-venture hatchery. It
is believed that the farm will be a solid source of income for the
company and is already providing important employment and rev-
enue for the local Fijian population.

URBAN COMMUNITY MEETS AQUACULTURE: A CASE
STUDY IN THE NORTHWEST. Gordon King. Taylor Re-
sources. Inc.. 130 SE Lynch Rd.. Shelton. WA 98584.

Taylor United, Inc.. an established shellfish farming company
in Washington State, sought to expand their suspension mussel
farming operation beginning in 1995. This led to a series of events
including the formation of a local association to stop further aqua-
culture development; the production of a sophisticated anti-
aquaculture propaganda video; many hundreds of protest letters to
the local politicians, the press, and agencies; the requirement by
Thurston County for Taylor United to complete an EIS; and a
citizen law suit under the Clean Water Act claiming that the com-
pany should be required to get an NPDES permit.

Seven years and many tens of thousands of dollars later, the
expansion is still on hold. The presentation documents this series
of events and explores some of the various parties' attitudes, and
asks how much of the friction is due to bona fide environmental
concerns and how much is due to the gulf in knowledge and
understanding between urban Americans and the industries that
priiduce their food?

PROGRESS WITH CULTURING THE RAZOR CLAM (£,V-
SIS DIRECTUS). Dale Leavitt, William Burt, Diane Murphy,
and Rebecca Hanson, SouthEastem Massachusetts Aquaculture
Center. Massachusetts Maritime Academy, Buzzards Bay, MA
02532.

As reported in 200 1 . SEMAC has embarked on a study to
assess the feasibility of farming the razor clam {Ensis directus) in
the northeast. Operating on research funds from the Northeast
Regional Aquaculture Center (USDA-CSREES), we have con-
tracted with a commercial shellfish hatchery (.Aquaculture Re-
search Corporation. Dennis. MA) to produce 1-cm seed razor
clams for distribution to commercial shellfish growers throughout
the northeast. The growers' role is to test existing or new technol-
ogy for grow-out of a product for market. This presentation will
provide an update of the results of this study after year 1.

Approximately 4 million razor clam larvae were spawned in the
hatchery and reared through the nursery stage using conventional

346 Ahstmcr.s. 2002 Annual Meeting. February 25-27. 2002

Milford Aquaculture Seminar. Milfurd. Connecticut

hatchery technology similar to that used lor the surf clam (Spisula
solidissiina). Roughly 150.000 I -cm juveniles were delivered to
SEMAC in July lor distribution to the growers. Although the
hatchery process needs refinement to increase the larval and early
juvenile survival rate, we demonstrated that the hatchery stage of
razor clam production is feasible and can potentially produce
enough juveniles to sustain a commercial effort in razor clam
farming. Adjustments to the hatchery procedures are currently be-
ing made as we enter our second year of hatchery production under
this project.

The 2-cm juvenile razor clams were distributed to 10 commer-
cial shellfish growers, ranging froin New Jersey to Massachusetts,
who had been selected through a competitive proposal process.
Each grower received an allotment of seed w ith funds to construct
their proposed culture system. Technology being tested ranges
from conventional quahog culture techniques using netted race-
ways to floating culture trays and containment systems deployed
on the bottom. The seed were distributed late in the growing sea-
son this year, but it allowed the growers to field test their proposed
grow-out system for modification and improvement in anticipation
of next year's larger scale seed distribution.

The two parameters of interest during this preliminary stage of
the study were recovery of planted individuals and their growth as
measured by a change in length. Recovery of juvenile razor clams
using a variety of field grow-out technologies varied from very
poor, where intensive digging within the grow-out area uncovered
few individuals using a conventional quahog netted raceway, to
very high, where two 4-in. cores produced more than 50 living
individuals in a boarded and netted raceway. Growth rate also
varied from very little increase in seed size during the I l-wk test
period to significant growth where the 2-cm seed had grown to
more than 5.4 cm in length. Given these preliminary results, the
prospect of farming razor clams seems to be achievable and the
imprinement of hatchery and rearing technology will continue
thrt)Ui;h the next growina season.

A COMPARISON OF SURVIVAL IN JUVENILE AR-
GOPECTEN IRRADIANS IRRADIANS USING VARIOUS
CULTURE TECHNIQUES AT THE SOUND SCHOOL RE-
GIONAL AQUACULTURE CENTER. Kathryn R. Markey
and John J. Roy, Ttie Sound School. 60 South Water St.. New
Haven. CT 06519.

Shellfish aquaculture is expanding in the northeastern Linited
States. With the increased interest in commercial aquaculture has
come the advancement of the techniques employed by the growers.
As such. The Sound School Regional Aquaculture Center is rap-
idly expanding the portion of the school's curriculum that deals
with shellfish aquaculture. We have successfully sought assistance
from both commercial growers and scientists dedicated to the ad-
vancement of aquaculture. They have supported us in our endeav-
ors to provide our students with a state-of-the-art education when-

ever possible. We have been involved in the culture of both oysters
and hard clams for several years and are currently engaged in our
second year of working with Argnpectcn irnidimis irnuUuns. the
bay scallop, at the school.

This year, in August, the National Marine Fisheries Service in
Milford. Connecticut, donated approximately 3,000 Argopecten
inaditins irradians to the school. The Groton Shellfish Commis-
sion made arrangements with our students to over-winter 6.000
>2() mm and 26,000 10- to l5-mni bay scallops at the school. In
addition, the Noank Aquaculture Cooperative made 20.000 4- to
6-mm bay scallops available to our program for a nominal charge.
In our previous attempts to work with bay scallops, we concerned
ourselves with monitoring growth as well as survival. We have
learned, as have many in the industry, that when mortality in a
scallop crop reaches UJO^i. most concerns with growth are un-
founded. This year we have refocused our experimental regimes to
deal with the issues of survival of the scallops over winter.

A variety of culture techniques ha\e been employed to over-
winter the juvenile bay scallops. Cages and ADPI bags, holding
scallops at +25 mm in low densities, have been successful to date,
with a 91% survival rate. Three upwellers have been designed.
One is located on a fixed pier outside the school. The other two
were built inside a wet lab in the school. All three upwellers have
unit|ue design modifications. The outside upweller holds approxi-
mately 1.200 +25-mm scallops and mortalities have been <3% of
the total. More than 40.000 bay scallops of various sizes have been
successfully held in the inside upwellers. where we attempt to
maintain water temperature between 13 and 18"C and provide the
juvenile bay scallops with supplemental feedings of microalgae.
To date, mortalities in the.se systems have been <IO'7f. Experience
has shown that increased mortalities in the juvenile bay scallops
are to be expected during the winter months. Complicating matters
further are the issues that cold weather creates with the mechanical
equipment. Pump failure or frozen pipes will dramatically increase
mortality. However, there is no substitute for experience. With
each setback, we learn, and each time we learn, we advance our
techniques so that the next year's students have an increased like-
lihood of success.

SUSPENDED AQUACULTURE DEVELOPMENT IN CON-
NECTICUT. Paul D. Maugle, Mohegan Aquaculture LLC, 5
Crow Hill Road. Uncasville. CT 06382.

Aquaculture in Connecticut has for the last 150 y traditionally
harvested native set shellfish from the bottom. Connecticut's oys-
ters are the most valued oysters reared in the United States. Know-
ing that this approach is not inherently sustainable in eastern Con-
necticut waters, Mohegan Aquaculture LLC has chosen to have at
its core the production of oysters in suspended longlines and float-
ing cage systems (FADPI) culture systems.

It is not only necessary to treat the natural re.source as a re-
newable resource, it is also necessary for the business to set up

Milford Aquacultiire Seminar. Milford. Connecticut

Abstracts. 2002 Annual Meeting. Fehmary 25-27, 2002 347

systems that can provide a sustained income. Traditional ap-
proaches to nursery rearing seed stock in the Long Island Sound,
and in fact the approach taken along the entire east coast of the
United States, is small scale. East coast floating upvvelling systeins
(FLUPSY) development has been a bottleneck to furthering suc-
cessful aquaculture of culchless oysters. In a FLUPSY. shellfish
are nursery-reared from 1 .5 to 2^ mm. Current east coast FLLIPSY
technology produces about 800.000 to 1 million seed stock per
FLUPSY and uses the labor of two personnel. West coast tech-
nology produces 8 to 12 niillion stockable seed per FLUPSY with
two personnel.

The grow-out process plans will initially focus on producing
hard clams for shellfish habitat restoration and individual culchless
oysters. Hatchery-pioduced hard clams are reared in FLUPSY un-
til they reach 8-12 mm, and are then seeded into our nursery areas.
Oysters reared in the Stonington hatchery are transferred to the
FLUPSY at 2.5-3 mm and reared until they reach 20-25 mm. Seed
oysters are grown in floating cage systems or in trays along sub-
merged longlines. Once the shellfish reach >50 mm, they are
tumbled and transfened to suspended oyster rearing trays for rear-
ing to market size.

To accommodate sail boating and other recreational activities,
these tray units hang at regular intervals from a submerged long-
lute 10 ft below the surface in waters that range from 18 to 28 ft
at MLT.

Sustained rearing operation will enhance and sustain recre-
ational shellfish activities because each spawn will bring new
shellfish seed into the environment. These operations will increase
essential fish nursery habitat, remove bacterial and nitrogen from
coastal waters, mitigate coastal eutrophication, and serve as an
environmental sentinel.

IT TAKES A COMMUNITY TO GROW A SCALLOP. Mary

F. Morgan, Kathleen K. Becker, and Kim Tetrault, Cornell
Cooperative Extension of Suffolk County. Marine Environmental
Learning Center, Southold, NY 11791.

Cornell Cooperative Extension of Suffolk County. New York,
is in the second year of an expansion of its Marine Program to
include a community-based shellfish restoration model to foster
stewardship of the marine environment. The project, called SPAT
(Special Projects in Aquaculture Training) is based on the under-
standing that enhancement of shellfish beds contributes greatly to
the health of estuarine ecosystems, and that local communities can
play a significant role in stewardship and restoration. Bay scallops,
Argopecten irradians irradians, hard clams. Mercenaria merce-
naria notata. and eastern oysters, Crassostrea virginica. are com-
mercially, recreationally, ecologically, and historically important
species to the Peconic Estuary. However, cuirently the estuary
supports only 1% of its historic oyster stocks and less than 1% of
its historic scallop stocks, due in large part to the occurrence of

brown tide algal blooms and its effects on habitat and shellfish
health.

The SPAT project incorporates a community-based shellfish
enhancement effort with intensive aquaculture training and infor-
mation gathering. The active membership is currently 191 families
from 41 different communities throughout Suffolk County. The
families are currently in training to become Master Shellfish Gar-
deners, learning the process of growing local shellfish species in
containment and collecting data on growth rates and survival. Each
participant has been licensed by the NYS Department of Environ-
mental Conservation and local townships to possess shellfish un-
der a scientific collector's permit. In addition, more than 2001)
volunteer hours have been donated to date to assist in culturing and
planting millions of seed clams, oysters, and scallops in local
waterways and select test plots. Dozens of participants assisted in
a 250.000 bay scallop relay effort in March at Goose Creek.
Southold. A planting of millions of seed clams took place with
more than 30 community members at Cedar Beach Creek, in
Southold. in August. Thousand of eastern oysters are being grown
in containment to adult size in 48 local creeks and embayments.
From June to December, two dozen participants have lent their
time, effort, and expertise to build a community-operated shellfish
hatchery on location, which will concentrate on the culture of bay
scallops. The hands-on transfer of techniques that enable commu-
nity participants, be they high school students, local business own-
ers, or retired professionals, to restore locally important marine
resources goes a long way toward raising awareness and develop-
ing a stewardship ethic within communities.

BAY SCALLOP {ARGOPECTEN IRRADIANS IRRADIANS)
RESTORATION ON CAPE COD. Diane Murphy, Dale Lea-
vitt. Bill Burt, and Bill Clark, Cape Cod Cooperative Extension,
P.O. Box 367, Deeds & Probate Building, Barnstable, MA 02630.

The bay scallop {Argnpecieii irradians irradians) fishery has
long held a historic supplemental niche for Cape Cod and south-
eastern Massachusetts fishermen. However, within the last 15-20 y.
bay scallop populations have experienced a dramatic decline, with
some localized extirpations. Some suggested causes for this de-
cline include habitat degradation in the form of nutrient enrich-
ment and loss of eelgrass beds as well as increased predation from
introduced species such as green crabs (Carcinus maenus).

In 1999, in response to the diminished stocks of wild scallops
on Cape Cod, a collaborative restoration effort was initiated be-
tween Cape Cod Cooperative Extension, SouthEastern Massachu-
setts Aquaculture Center (SEMAC). and selected Barnstable
County municipalities. Why bay scallops? Rapid growth rate, high
market value, unstable supply, coupled with the evidence of de-
clining and/or absent natural populations from historically produc-
tive areas suggest that bay scallops would be viable candidates for
enhancement purposes. Pursuant to an exhaustive review of cur-
rent information available on the bay scallop, a regional workshop

348 Abstracts. 2002 Annual Meeting. February 25-27. 2002

Milford Aquaculture Seminar. Milf'ord. Connecticut

was held to design an effective restoration program. It was con-
cluded that spawning sanctuaries would provide the best means of
creating self-sustaining scallop stocks for commercial and recre-
ational fishing purposes.

Now in its third year, the Barnstable County restoration pro-
gram consists of purchasing small scallop seed derived from local
broodstock in late summer to over-winter in off-bottom cages.
These cages make optimal use of the water column for filter-
feeding, as well as reducing predation and slltation. The following
spring, scallop survival is assessed and scallops are redeployed
into floating spawning sanctuaries sited in historically productive
areas. Embayments are chosen with the greatest potential of larval
entrainment — avoiding sites with extreme tidal fluxes to ensure
localized settlement of seed. Optimal stocking densities also in-
crease the likelihood iif successful spawning. Spat bags are de-
ployed in close proximity to the spawning sanctuaries in order to
monitor recruitment. In addition, genetic fingerprinting (using
RAPD-PCR) is being used to evaluate the success of the bay
scallop restoration program.

EFFECTS OF CRASSOSTREA VIRGINICA POPILATIONS
ON SEDIMENTATION. PHVTOPLANKTON SPECIES
COMPOSITION, AND AMMONIA CYCLING IN EXPERI-
MENTAL MESOCOSMS. Jennifer Mugg-Pietros and Michael

A. Rice, Department of Fisheries, Animal and Veterinary Science.
University of Rhode Island. Kingston, RI 02881.

To determine the effects of oyster populations on water quality,
a mesocosm study was performed from June to October 2000.
Mesocosms with a volume of 1.^.000 L were used, in which there
were triplicate control tanks without oysters and triplicate experi-
mental tanks each with 200 oysters (=35 mm in valve height;
nominally filtering about 55 L day"' ind'' ). Experiments were run
sequentially in time for 3-wk periods, with water exchange rates
ranguig from 0% to 100% per day (0-13.000 L day''). Several
parameters were measured and compared between the control and
experimental tanks, including chlorophyll-a, particulate organic
and inorganic matter, sedimentation rates, nitrate, ammonia, phy-
toplankton species and numbers, and oyster growth rates. There
were no significant (P < 0.05) differences between tanks for most
parameters, with the exceptions of rates of sedimentation and spe-
cies composition of phytoplankton in the water column. Diatoms
of the genus Nitzsclun were predominant in mesocosms with oys-
ters; in control tanks. Skeletoiwma were dominant. Rates of atn-
monia excretion by oysters of various sizes were determined by the
sensitive salicylate-hypochlorite method, allowing for rapid deter-
mination of excretion rates to minimize biases introduced by vola-
tilization or transformation of the ammonia over time. The excre-
tion of ammonia by oysters can be described by the allometric
equation E = 50.65\r"'^'''''-, when E is the excretion rate in |jLg
NH3-N hr"' and w is the dry soft tissue weight in g. On the basis
of these data, it would be expected in the 3-wk experimental period

that an additional 470 p.g L"' ammonia above the average control
concentrations of about 40 |j.g L" would be present in each me-
socosm with oysters, but no significant difference was noted be-
tween the experimental and control tanks. This suggests that there
is rapid cycling of ammonia, perhaps by uptake by the rapidly
regenerating phytoplankton populations.

This is publication number 3910 of the College of the Envi-
ronment and Life Sciences at the University of Rhode Island, with
support from the RIAES under project number H-886.

A REVIEW OF DISEASES IN THE BAY SCALLOP [AR-
GOPECTEN IRRADIANS IRRADIANS) AND SOME OBSER-
VATIONS ON MORTALITIES AT THE MILFORD LABO-
RATORY. Steven Pitchford and Richard Robohm, USDOC.

NOAA. National Marine Fisheries Service. Northeast Fisheries
Science Center. Milford Laboratory. Milford. CT 06460.

Various diseases and other causes of mortality in the bay scal-
lop, Argopcctc'ii imulians irnuUans. will be reviewed, especially
those that have been encountered historically and reported during
hatchery and grow-out in the northeast Atlantic region. Unlike
some of the other cultured bivalve species such as the eastern
oyster. Cnissostrea virginica. which is affected by MSX and
dermo. the adult bay scallop does not appear to be susceptible to
specific pathogenic agents that can cause widespread epizootics.
Examples of infections in bay scallops to be discussed briefly
include those caused by prokaryotes (rickettsia. chlamydia. Vibrio
ssp.). protozoans, algae, and fungi.

As part of larger studies relating to immune system function
and disease resistance in bay scallops, a program to screen for
potential pathogenic targets was started in 1994. More than 70
bacterial isolates, the majority of which were Vibrio sp., were
recovered from dead and moribund larval and adult bay scallops.
High-concentration (approximately 10'' bacteria/larvae) screening
assays were conducted with 46 of the strains. Seven of these
caused >80Vf mortality. Next. 1.000 2-day-old larvae/L were ex-
posed in 48 h. using serial dilution challenges with the seven
bacteria. Only two isolates were considered to be pathogenic for
larvae, with median lethal concentrations (LC^d) of 8.65 x 10^ and
1 .98 X 10' colony forming units (cfu)/niL after 48 h. Follow ing the
crash of a bay scallop larval culture in 1998 at the Milford labo-
ratory, monitoring of the cultures, the ambient seawater, and algal
food sources for potential bacterial targets also was initiated.

Highlights of the monitoring program include consistent high
counts of Vibrio sp. for 1 wk following periodic, hot, freshwater
flushing of the seawater lines. These high bacterial loads may have
resulted from the large amount of dissolved and undissolved or-
ganic matter that remained in the piping system. In addition, newly
established mass algal cultures also would invariably show very
high numbers of Vibrio sp. OlO"^ cfu/mL) soon after being started;
however, the counts from these cultures would decrease to near 0
cfu/mL. usualh after 4-S w k.

Milford Aquaculture Seminar. Milford, Connecticut

Absrmcts. 2002 Annual Meeting. February 25-27. 2002 349

Other occurrences of high mortality at the Milford laboratory
also will be discussed; these include events with a possible infec-
tious disease etiology and those where increased mortality resulted
from biotic and abiotic causes, such as overcrowding and fouling.
Several episodes of gas bubble trauma have resulted in mass mor-
talities, killing VS^-lOO^c of adult scallops in very short periods.
Over-wintering mortalities routinely reach 409^ or even higher in
cold winters, when water temperatures approach 0"C.

SHELLFISH AQUACULTURES EFFECT ON TOTAL OR-
GANIC CARBON (TOO IN THE BENTHOS. Perry Raso and
Michael A. Rice, Department of Fisheries. Animal & Veterinary
Science. University of Rhode Island, Kingston. RI 02881.

Rhode Island waters provide a suitable habitat for shellfish
aquaculture. Rhode Island waters are also highly valued by both
local residents and tourists. If shellfish aquaculture is going to
expand as a successful industry in Rhode Island, it must progress
in a manner that does not negatively affect the estuary or bay in
which it is conducted. To examine the effects of shellfish aqua-
culture on the benthos, sediment samples were tested for total
organic carbon (TOC). Fifteen 0.5-L sediment sainples were taken
at each of 14 different shellfish aquaculture leases in Rhode Island
waters. Five samples were taken within the lease; five within 10 m
of the lease, and five no less than 100 m from the lease in a similar
water body (depth, flow, distance from shore). Results showed
normal variance between TOC levels of samples taken within,
around, and away from aquaculture leases. Mean TOC of all
samples away from the lease was 1 .48%, 1 .49% near the lease, and
1.21% inside the lease, with confidence intervals overlapping. This
study provides data showing that shellfish aquaculture. at the scale
that it is currently practiced in Rhode Island, does not significantly
affect TOC levels in the benthos.

This study is partially funded by RIAES project number H-886
and is publication number .^918 of CELS-URI.

INDUSTRIAL-SCALE SCALLOP CULTURE IN CHILE—
THE C.M.I. EXPERIENCE. Edwin Rhodes, Aquatecnics, LLC,
Milford. CT 06460.

Cultivos Marinos Internacionales, S.A. (CMl) is the largest
scallop aquaculture company in the world. CMI grows Argopecten
piirpiiniliis. native to Chile and Peru. Seed for the operation are
primarily hatchery-produced, but seed collection is also employed.
The original concept of producing 5-mm seed from a land-based
hatchery and nursery did not work reliably, but settling hatchery-
produced larvae on plastic mesh and putting them directly into the
sea suspended from longlines proved to be successful. The scallops
are subsequently grown in two pearl net stages, and three lantern
net stages, all on longlines. CMl leases more than 1 100 hectares of
growing area, split about evenly between two bays about .300 miles
apart, and uses a total of about 1 .500 longlines. each 200 m long.
Net changes to control fouling and to adjust scallop density are

done on land in one bay, and from barges in the other. Scallops
reach the market size of 80 mm shell height in 18 mo. and have an
adductor muscle plus roe weight yield of 20 g each. CMFs pro-
duction in the mid 1990s exceeded 5 million scallops/mo. Final
product form is usually individually quick frozen (IQF), and vir-
tually all of the scallops are e.xported from Chile to France.

THE DEVELOPMENT OF AN INSTRUCTIONAL SHELL-
FISH HATCHERY: A COLLABORATIVE EFFORT BE-
TWEEN AEROS CULTURED OYSTER CO. AND THE
SOUND SCHOOL REGIONAL AQUACULTURE CENTER.
Karen Rivera, Aeros Cultured Oyster Company. 100 Main St..
Noank, CT 06340; Amber L. Beitler and John J. Roy, The Sound
School. 60 South Water St.. New Haven, CT 06519.

The Sound School Regional Aquaculture Center, in coopera-
tion with Aeros Cultured Oyster Company, designed the Interdis-
trict Marine Educational Program's Instructional Shellfish Hatch-
ery (IMEP/ISH). The Instructional Shellfish Hatchery program is
an Interdistrict Cooperative Grant and is funded by the Office of
Urban and Priority School Districts of the Connecticut State De-
partment of Education. The IMEP/ISH project was formed to pro-
mote outcome-based learning between science classes from sev-
eral coastal Connecticut high schools. The IMEP/ISH incorporates
the vocational agriculture attitude of "learning through doing" as
the method of education that best enables young researchers to
conceptualize abstract points in their investigations as well as pro-
viding them with the techniques necessary to achieve the definable
outcomes being sought.

The IMEP/ISH was constructed in the Purity Processed Sea-
food building at the Noank Shipyard in Noank, Connecticut. The
program began on May 7 and finished on May 31, 2001. During
that time period, Karen Rivara of Aeros, in conjunction with staff
from The Sound School and instructors from the participating
schools successfully implemented 10 laboratory lessons that had
been designed for the IMEP/ISH cuiriculum. The laboratory les-
sons each considered an aspect of shellfish husbandry. The lessons
included the following topics: water quality, shellfish biology, bi-
valve reproduction and spawning techniques, microalgae culture,
hatchery technology, nursery systems, grow-out technology, shell-
fish health, harvest and handling methods, and depuration tech-
niques.

The Sound School acted as host school for the project. Ella T.
Gras.so/Southeastern Vocational Technical School, Groton, Con-
necticut, functioned as a partner school in the program, providing
the necessary benchtop space (in the school's BET lab facilities)
for the shellfish dissection and the shellfish health laboratories.
Students from Ledyard High School's aquaculture classes (VoAg
Department) participated in the program as well. When the pro-
gram was completed. 55 different students had participated in 199
student days of laboratory activities involving the major aspects of
shellfish culture as practiced by professional aquaculturalisis in
Long Island Sound.

350 Ahstracts. 2002 Annual Meeting. February 25-27. 2002

Miltbrd Aqiiacullure Seminar. Milford. Connecticut

THE EAST COAST SHELLFISH GROWERS ASSOCLA-
TION: A WORK IN PROGRESS. Karen Rivara, East End
Marine Farmers Association. Soiitlioid. NY I 1971.

During the last 25 y. shellfish aquaculture has grown on the
East Coast of the United States despite many obstacles. Newcom-
ers in the industry come from many sectors of the economy, from
retired schoolteachers and recent college graduates, to members of
the traditional fisheries who look to aquaculture to maintain a
sustainable living on the water. Established shellfish companies
continue to use and improve various culture methods to meet the
challenges of consistent production presented by losses due to
disea.se and the loss of good cultivation areas.

Other stakeholders in the marine environment have challenged
the growth of this industry. This impediment to the growth and
survival of shellfish aquaculture stems predominantly from a lack
of understanding regarding the actual impacts of the shellfish
aquaculture industry. Those who are opposed to the growth of this
industry overlook many of the benefits of shellfish aquaculture. In
other cases, shellfish aquaculture is viewed as an impediment to
the development of other industries in the marine environment.
Unfortunately, those who are against the growth of the industry
have in many cases taken their grievances to legislators and the
press, further fostering a lack of understanding and animosity to-
ward the industry. Too often aquaculturalists are put in the position
of having to take time from growing their businesses to defend
them against unreasonable public opposition.

The shellfish aquaculture industry clearly needs to become or-
ganized. We need to establish an East Coast Shellfish Growers
Association (ECSGA) that will identify socio-political obstacles
and work to remove them, will work in a proactive way with other
stakeholders, and will develop a public outreach/public awareness
protocol that will prevent the industry from constantly having to
defend itself. The ECSGA would also take the lead to implement
best management practices to guide individuals involved in shell-
fish cultivation as well as to inform legislators and other policy
makers.

It is important that this association be industry-driven, but in-
volve participation from related areas of the industry such as pub-
lic shellfish aquaculture facilities, state extension programs, aca-
demia. government, and regulatory agencies. This year presents a
unique opportunity to organize an East Coast Shellfish Growers
Association. A roundtable discussion at the 22"*^ Milford Aqua-
culture Seminar will be devoted to the formulation of this asso-
ciation. We will discuss organizational considerations such as geo-
graphic scope, types of membership, mission statement, and the
concerns of the industry that this association will address. In April,
the National Shellfisheries .'Association annual meeting will be
held m Mystic. Connecticut. This affords the shellfish aquaculttire
industry another opportunity to continue the process of forming an
association. The goal of the ECSGA meeting at NSA in Mystic
would be to continue discussion regarding the formation of the
association and establish a committee of interested individuals that

would also serve as the first Board of Directors. These individuals
would complete the legal process of forming an association, es-
tablishing an office, setting meeting dates, and establishing mem-
bership.

There are many reasons why the East Coast shellfish aquacul-
ture industry should begin to organize. We need to take advantage
of the opportunity to do so this year.

HEAVY METAL SURVEY OF FUCVS SPIRALIS COL-
LECTED FROM SOUTHWESTERN LONG ISLAND
SOIIND. Rene Sanz. Sherry Lonergan. Jennifer Sutorius. and
Dania Lieberthal. Bridgeport Regional Vocational Aquaculture
School, 60 St. Stephens Road. Bridgeport, CT 06605.

Finns spinilis. commonly know n as rockweed. is a brown alga
found in the upper intertidal and niidtidal /ones of the North At-
lantic. Similar to red algae. Fiiciis spinilis contains phycocolloids.
which are valuable emulsifiers used in the manufacture of food,
pharmaceutical, and healthcare products. In general, inacroalgae
take in heavy metals, some of which are beneficial for nutrition
and others that are possibly harmful to algae and humans in high
levels. These heavy metals accumulate in algae, which makes them
useful in biomonitoring. marine pollution assessment, and phytore-
mediation. Because algae are becoming more popular for use in
food, industry, and environmental applications, determining heavy
metal concentrations is important to a wide range of audiences.

Samples from five sites known as. sites A. B. C. D. and E,
located in southwestern Long Island Sound, were surveyed to de-
termine the concentration of heavy metal intake by Fiicus spiralis.
The heavy metals being tested include: cadmium (Cd), chromium
(Ca), copper (Cu), lead (Pb), and zinc (Zn). Samples were col-
lected within 1 h of low tide. After samples were collected, the
alga was washed, oven-dried, ground, and digested using a micro-
wave lab station. Samples were then analyzed using atomic ab-
sorption spectroscopy. Some samples were analyzed in replicate to
ensure quality control. Collection for test samples began on Oc-
tober 26, 2001. with the most current being December 19. 2001.
Results of the sampling were as follows:

• Cadmium (Cd) at site A ranged from below detectable limits
(n/d) to 0.04 |jig/g: site B value was 0.04 jxg/g: values at sites C
and D were all n/d: and site E value ranged from n/d to 0.04
M-g/g.

• Copper (Cu) at site A was 0.18 p.g/g; site B value was 0.2.^
jjig/g; site C values ranged from 0.036 to 0.0582 (Ag/g: site D
values ranged from 0.048 to 0.1104 (xg/g; and site E values
ranged from 0.048 to 0.26 |J.g/g.

• Chromium (Ca) at site A was 0.02 |xg/g: site B value was 0.03
pig/g; site E values ranged from n/d to 0.026 (j.g/g; site D values
ranged from n/d to 0.082 [xg/g; and site D values ranged from
n/d to 0.083 (jig/g.

• Lead (Pb) at site A was 0.48 jig/g: site B value was 0.5 |j.g/g:
site C values ranged from n/d to 0.134 |j.g/g: site D values
ranged from 0.022 to 0.14 |jLg/g; and site E values ranged from
0.02 to 0.51 |ig/g.

Milford Aquaciilture Seminar. Milford. Connecticut

Abstracts. 2002 Annual Meetina. February 25-27. 2002 351

• Zinc (Zn) at site A was 0.64 jjis/g: site B value was 0.7 (Jig/g;
site C values ranged from 0.1882 to 0.2332 |ji.g/g: site D ranged
from 0.06 to 0.214 [jLg/g; and site E ranged from 0.136 to 0.86
M-g/g.

Analysis of these metals will continue at additional sites in
Long Island Sound. Baseline data will be used in future compara-
tive research of this and other algal species.

in our recirculating aquaculture systems in order to study the in-
cidence of liver tumors and mortality in Atlantic tomcods.

Educational outreach programs: AREAC is developing cur-
ricula for pre-K-1 2'^ grade students, teachers, and their families to
enhance their knowledge and teaching ability on issues relating to
aquaculture and the environment.

Our programs have been funded by NPS, USAGE, DEC. Con
Edison, and CUNY.

AQUACULTURE ACTIVITIES IN BROOKLYN. NEW
YORK? Martin P. Schreibnian. Chester Zarnoch. John T.
Tanacredi, Lucia Magliulo-Cepriano, Jacob Raz, and Stefano
Diomede, Aquatic Research and Environmental Assessment Cen-
ter ( AREAC 1. Brooklyn College. 2900 Bedford Avenue, Brook-
lyn. NY 11210.

In Brooklyn College's Aquatic Research and Environmental
Assessment Center (AREAC), state-of-the-art recirculating aqua-
culture systems (RAS) have been used to conduct a number of
diverse aquaculture and enviriinmental assessment/restoration pro-
grams. These projects include the following topics.

Finfish aquaculture; Our program consists of induction of
spawning and/or grow-out of commercially important species. Our
achievements include production of market-si/e walleye and tila-
pia, as well as the culture of winter and summer flounder.

Bivalve aquaculture: Recirculating systems for downwelling
and broodstock conditioning were utilized in the culture of hard
clams (Merceiiaria moxenaria). Seawater used in the static algal
and larval cultures was biologically and chemically treated and
then reused. In its first season of operation, the hatchery produced
2 million hard clams; when 2.0 nmi in shell length, they were
moved out to a field site in Jamaica Bay, New York. An intensive
study is underway to measure the survival and physiological con-
dition of juvenile hard clams during the winter period. In addition,
oyster seed were cultured in Taylor float systems at two sites in
Jamaica Bay to monitor growth and water quality.

Ornamental and research aquaculture: AREAC has more than
600 aquaria dedicated to the culture of freshwater species for fish
hobbyists and scientific research. Soft coral is also being propa-
gated.

Captive breeding of horseshoe crabs: Adult horseshoe crabs
were spawned in AREAC in July 2001. Developing animals were
cultured to determine optimum temperature, medium, and nutri-
tional parameters in recirculating systems. Currently, we have
2,500 horseshoe crabs with carapace widths ranging between 2 and
35 mm.

Environmental assessment and restoration: The impact of en-
docrine-disrupting chemical pollutants on freshwater and saltwater
fishes has been studied for u number of years in AREAC. A
program of field and laboratory experiments has begun to examine
the effects of nonylphenols on winter flounder reproduction (in
collaboration with Dr. Anne McElroy, SUNYSB). In another proj-
ect, we have simulated the Hudson River by creating microcosms

SHELLFISH AQUACULTURE: GOOD EOR THE
ECONOMY, GOOD FOR THE ENVIRONMENT, GOOD
FOR YOU! Sandra E. Shuniway, Department of Marine Sci-
ences, University of Connecticut, 1080 Shennecossett Road, Gro-
ton, CT 06340.

The United States ranks 3"' worldwide in the consumption of
seafood, yet it ranks 1 I "' in aquaculture production, with just 1.1%
of global production by weight. World production of bivalve mol-
lusks has increased steadily over the past decade, and 80'/f of
world bivalve production is cultured product. Oysters comprise
35% of that total; clams and arkshells combined constitute about
33%. with China producing approximately 64% of the world total.
The United States ranks i"' among bi\alve-producing nations and
only 19"' in production via aquaculture. Possible reasons for these
discrepancies and the benefits of shellfish aquaculture will be de-
lineated and discussed.

HEALTH MANAGEMENT GUIDELINES FOR SHELL-
FISH CULTURE IN THE NORTHEASTERN UNITED
STATES. Roxanna Smolowitz, Marine Biological Laboratory,
Woods Hole, MA 02543; Susan Ford, Rutgers University, Port
Norris, NJ 08349; Lisa Ragone-Calvo, Virginia Institute of Ma-
rine Science, Gloucester Point, VA 23062.

Bivalve diseases can cause significant losses of both cultured
and wild stocks. To prevent the spread of disease among popula-
tions, most states generally do not allow the importation of bivalve
shellfish unless the animals are considered to be "disease free."" As
a result, examinations for disease have become a necessary pre-
requisite for determining health status. However, shellfish transfer
regulations vary by state, often appear capricious, and complicate
the sale and transport of commercially important seed. Further-
more, both regulators and producers have few guidelines that
would help them respond to and manage disease outbreaks, when
they occur. Lines of communication need to be developed between
diverse but knowledgeable groups to produce a set of standardized
monitoring/management guidelines for use by state regulators. We
describe a project intended to provide a set of uniform, scientifi-
cally based recommendations for the health management of com-
mercially important bivalve species in the northeastern L'nited
States.

The guidelines will be produced as a result of a series of work-
shops and meetings proposed for the next 2 y that will include

352 Abstracts, 2002 Annual Meeting, February 25-27, 2002

Milford Aquaculture Seminar. Milford. Connecticut

scientists, industry, regulators, and extension agents in the north-
eastern United .States. This interchange will first identity the
strengths and weaknesses of, as well as alternatives to. current
approaches to bivalve health management in the region, and in-
vestigate those in use or proposed by other states, by the federal
government, and by other countries. The guidelines that follow
from these discussions will be published in booklet form and will
be posted on the Web.

In addition to the guidelines, the document will also contain
sections pertaining to ( I ) concepts in disease spread, control, and
diagnosis; (2) descriptions of standard diagnostic methods; (3) an
examination of the potential application of novel molecular-based
diagnostics, including an assessment of their accuracy and sensi-
tivity, and hov\ their outcome should be interpreted; (4) a fact sheet
for each disease or disease agent; and (5) a glossary of general
terms. The fact sheet will include the common and scientific names
of the disease agent and its host or hosts, known geographic dis-
tribution, known environmental limitations, life cycle, method of
transmission, recommended diagnostic procedures, and treatment
or amelioration methods. Each disease agent will be classified
according \.o its potential danger to bivalve stocks. Although the
guidelines will have no force of law, we anticipate that the inclu-
sion of all interested parties in their development should lead to
their use by all northeastern states because the recommendations
are reasonable, scientifically based, and because they will benefit
the shellfish uidustries in the recion.

PREVALENCE AND MORTALITY ASSOCIATED WITH
SSO AND SSO-LIKE INFECTIONS OF CRASSOSTREA
VIRGINICA IN THE NORTHEAST. Roxanna Smolowitz, Ma

rine Biological Laboratory, 7 MBL St.. Woods Hole, MA 02543;
Inke Sunila. State of Connecticut. Dept. of Agriculture. Milford.
CT ()(i4f)(); Nancy Stokes and Lisa Ragone-Calvo, Virginia In-
stitute of Marine Science. College of William and Mary. Glouc-
ester Point, VA 23062.

Haplosporidium costale (seaside organism. SSO) was identi-
fied as a cause of mortality in the eastern oyster (Ciassostiea
virainwa) on the Atlantic coast of Maryland and Virginia in 1962
and is now endemic there. Early ( 1962) investigations in Virginia
showed mortality associated with SSO could reach 60% in some
years. SSO plasmodia are first identified in tissue sections in early
spring. Mortality resulting from synchronous sporulation in the
connective tissues occurs in May-June each year. Until recently,
post-sporulation SSO plasmodia have not been positively identi-
fied in oyster tissues until the following spring.

SSO-infected oysters have also been found along coastlines of
the more northern states, but historically. SSO morbidity and mor-
tality has not been considered significant. Northeast regulatory
agencies do not restrict shipments of oysters on the basis of posi-
tive SSO findings. In late spring 1998, oyster culturists in Katama
Bay, Martha's Vineyard. Massachusetts, observed IWc-lWr mor-
tality of cultured stocks. The cause was identified as SSO. thus

indicating that SSO can cause significant mortalities in some years
in the northeast. Notably, in I99S in both Connecticut and Mas-
sachusetts. SSO-like sporulating plasmodia were noted in sections
of oysters in the late fall of the year. Although no other cases have
since been identified in Massachusetts. SSO-like. fall sporulating
organisms continue to be identified in Connecticut.

Haplosporidiiiin nclsoni (MSX) also produces plasmodia in
oyster tissues and is a cause of significant mortality in mid-
Atlantic and northeast oyster populations. It is difficult to differ-
entiate H. nt'lsoni and //. costule plasmodia using traditional diag-
nostic methods, and it is possible that SSO and SSO-like plasinodia
have been misidentitied as MSX over the last several years, resulting
in the confusion between mortality associated with MSX and SSO.

In this study, species-specific DNA-based diagnostic PCR
methods and in situ hybridization (ISH) were employed in con-
junction with traditional histological examination to differentiate
H. nelsoni and H. castalc infections in cultured oyster populations
in Massachusetts and Connecticut, with the main objective of de-
termining to what extent mortality is attributable to SSO and/or
MSX. Additional objectives were to identify the SSO-like organ-
ism and to determine if fall and winter infection characteristics
(i.e.. prevalence, tissue forms, and locations) of SSO are of value
in predicting SSO-related disease severity in the following spring.

Results indicate that pathologists cannot reliably differentiate
MSX. SSO. and SSO-like plasmodia in traditionally stained tissue
sections. Using species-specific DNA primers and probes, PCR
and ISH methods can differentiate between these infections. In
addition. ISH can help quantity the relative proportion of SSO and
MSX Plasmodia within tissue sections. Some plasmodia identified
in oyster tissues collected in the fall, which might have been iden-
tified as MSX, are positive for SSO and SSO-like organisms. The
relationship of SSO-like organisms to SSO and MSX plasmodia
continues to be studied. Combining the history of oyster mortality
in a specific location with results from various diagnostic tests is
the best way to predict morbidity and mortality associated with
these oyster diseases m any population.

This work is sponsored by a NOAA/Sea Grant Oyster Disease
Research Award.

RHODE ISLAND'S SHELLFISH RESTORATION PRO-
GRAM IN RESPONSE TO THE NORTH CAPE OIL SPILL.
Karin A. Tanimi. Najih Lazar. and .Vrthur Ganz. Rhode Island
Department of Environmental Management. Coastal Fisheries
Laboratory. 1231 Succotash Road, Wakefield. RI 02879; James
(;. Turek, National Oceanic and Atmospheric Administration
Restoration Center, 28 Tarzwell Drive. Narragansett, RI 02882;
and John G. Catena, National Oceanic and Atmospheric Admin-
istration Restoration Center, One Blackburn Drive. Gloucester,
MA 01930.

On the evening of January 1 9. 1 996. the tank barge North Cape
struck ground off Point Judith. Rhode Island, and began leaking oil
in the vicinity of two National Wildlife Refuges, several salt

Milt'ord Aquaculture Seminar. Milford. Connecticut

Abstracts. 2002 Annual Meetinc, Februarv 23-27, 2002 353

ponds, and public and private beaclies. Wmd and wave action
dispersed the oil into the atmosphere, throughout the water col-
umn, and into the benthic sediment. Approximately 828,000 gal-
lons of heating oil were released into the surrounding offshore and
inshore environment, affecting large numbers of crustaceans, mol-
lusks. birds, amphipods, and fish. It was determined that the spill
was responsible for the loss of about 150 million surfclams,
Spisuki solidissiiiKi. with a total bioniass of 379,000 kg. for a value
of $1.5 million. The spill resulted in the formation of a natural
resource trustee group, composed of Rhode Island Department of
Environmental Management, the National Oceanic and Atmo-
spheric Administration, and the United States Fish and Wildlife
Service, to evaluate the injury to the natural resources and to plan
the resulting restoration activities. Because the surfclam popula-
tion should recover to natural baseline levels within 3-5 y. a com-
pensatory shellfish restoration program will be launched in Nar-
ragansett Bay and in the coastal salt ponds. Beginning in 2002. the
trustees will initiate a multifaceted and multispecies approach to
shellfish restoration with programs for the eastern oyster. Cnis-
sostrea virginica: Northern quahog. Mercciiaiici iiifireiuiriii. and
the bay scallop, Argopecten inaJiaiis iinnUans. The shellfish res-
toration strategy will utilize many techniques, which include a
remote setting program for C. virgincia and spawning sanctuaries
and spat collection for bay scallops. A. irradians irnidiuns. The
shellfish restoration initiatives in response to the North Cape oil
spill disaster offer tremendous opportunities for Rhode Island's
shellfish resources.

operated hatchery on location is expected to be operational for the
2002 growing season, which will concentrate on the culture of bay
scallops {Argopecten irradians irradians).

Various projects are planned for the 2002 season. These include
restoration work on two community spawner sanctuaries funded
by NOAA (oysters) and 5-Star (clams/scallop) grants. Two addi-
tional spawner sanctuaries, one for oysters and another for scal-
lops, have been designated by the town of Southold. .All sanctu-
aries will be stocked and monitored by SPAT trainees. The focus
will be on early field planting and predatiir control techniques for
select species of clams, oyster bed preparation and reef building,
and intensification of scallop deployment at multiple stages within
healthy eelgrass beds. The community hatchery is expected to
significantly increase the production of bay scallops with a target
goal of 5-10 million post-set for the 2002 season. A grant to
examine the sociological components of the SPAT initiative has
been awarded by an anonymous foundation. Two pilot educational
programs, one at the third-grade level and one at the teenage level,
are actively in progress. New internships in community-based
shellfish restoration activities are available.

It is the intention of the Cornell Cooperative Extension SPAT
initiative to establish a model for community-supported shellfish
restoration efforts thiough intensive training and active participa-
tion. The founding members of SPAT are excited and prepared to
meet the challenges in the upcoming year.

SPAT (SPECIAL PROGRAMS IN AQUACULTURE TRAIN-
ING) UPDATE. 2002. ESTABLISHING A MODEL FOR
COMMUNITY-BASED SHELLFISH CULTURE AND RES-
TORATION. Kim Tetrault, R. Michael Patricio, and Mary
Morgan. Cornell Cooperative Extension of Suffolk County, Ma-
rine Environmental Learning Center, Southold, NY 1 1971.

Cornell Cooperative Extension of Suffolk County, New York,
has expanded a component of their marine program division to
include a series of educational and training initiatives collectively
referred to as SPAT (Special Programs in Aquaculture Training).
The SPAT campaign is now 1 y old and has made tremendous
progress. The active membership of SPAT Master Shellfish Gar-
deners is currently 191 families strong, with attendance at the
monthly workshop series (II, 2-h lectures) consistently exceeding
KJO participants. More than 2,000 h have been logged into the
community hands-on training sessions held weekly throughout the
year. SPAT members assisted in culturing and planting millions of
seed clams, oysters, and scallops in local waterways and selected
test plots, as well as maintaining personal "garden" stocks. A new
web site (www.cce.cornell.edu/Suffolk/MARprograms/
Aquacultruemain.htm) now allows members to record data that
have been collected on a monthly basis for growth and survival of
cultured oysters {Crassostreu virginica). A community-built and

A NEW CLAM FOR CONNECTICUT. John Wadsworth,

Niantic Bay Shellfish. LLC. 15 First Street. Waterford, CT 06385;
Tessa Simlicli and Nancy Balcom. Connecticut Sea Grant, Uni-
versity of Connecticut. 1084 Shennecossett Road. Groton, CT
06340.

The razor clam Ensis directus. is one of the few untapped
resources in shellfish aquaculture in the northeastern United States.
The market supply of razor clams is low and inconsistent, particu-
larly because of the difficulty in harvesting these fast-digging
shellfish. If razor clam production were to become economically
feasible and less labor intensive, there would be great opportunity
to expand this underutilized species to niche inarkets. The North-
eastern Regional Aquaculture Center (NRAC) has provided fund-
ing for the aquaculture industry to test various clain grow-out
methods in four stales: Connecticut, Massachusetts, New York,
and Rhode Island. In Connecticut, a project was initiated to inves-
tigate razor clam grow-out in cages. Seed clams were obtained
from the Aquaculture Research Corporation (ARC) in Dennis,
Massachusetts, in September 2001. The clams were held initially
in upwellers and then transferred to cages for field experiments.
The project consisted of two experiments, the first of which was
designed to compare razor clam growth rates in cages lined with
felt set at two different heights. Replicate cages were lined with
felt to the top of the cages or. the high position (HI). The remain-
ing cages were lined up to the level of the sediment, or low posi-

354 Abstracts. 2002 Annual Meeting. February 25-27. 2002

Miltord Aquacullure .Seminar. Milford. Conneclieut

tion (H2). The second experiment was designed to compare razor
clam growth rates between two different growing areas, the Nian-
tic River (NR) and the Niantic Bay (NB) in Waterford. Connecti-
cut. The clams were divided among replicate cages and stocked at
a density of one clam/2.5 cm- (one clam/in.-). Monthly inventories
to determine clam density and growth rate (length and width to
±0.01 mm) were performed beginning in September 2001. As of
early February, preliminary results showed that there was no sig-
nificant difference in growth rates between felt heights (HI vs. H2)
or growing area (NR vs. NB ) ( ANOVA. P < 0.05 1. The clams grew
in length from 18.84 ± 2.22 mm to 34.71 ± 4.46 mm. High den-
sities were observed in the NR cages (>907f of original stocking
densities), however, the clams were present in very low densities
in the NB cages.

LIFE IN A TRAILER— DEVELOP.MENT OE A NEW
SHELLFISH HATCHERY AT THE MASSACHUSETTS
MARITIME ACADEMY. Bethany A. Walton, Aquaculture
Laboratory. Massachusetts Maritime Academy. 101 Academy
Drive. Buzzards Bay. MA 02532.

The Aquaculture Lab at the Massachusetts Maritime Academy
(MMA) in Buzzards Bay. Massachusetts, officially opened its
doors in April 2001. Located at the southern entrance to the Cape
Cod Canal on Taylor's Point, the hatchery is a cooperative venture
between MMA and Barnstable County; the SouthEastern Massa-
chusetts Aquaculture Center (SEMAC) also provides technical
support. This facility is not a production-scale shellfish hatchery,
but rather, a "teaching hatchery."" This teaching hatchery ser\es as
an integral component of an introductory aquaculture course
taught at MMA to provide "hands-on"" training for students in
shellfish biology, hatchery techniques, and intermediate culture.

The building (a refurbished trailer that measures appro.ximately
1100 ft") consists of two primary areas, one devoted to algae
production and the other to larval production. The greenhouse area
holds twelve 250-L Kalwall tubes and has space for several 3-L
carboys and 1-L jugs. Microalgae production consists primarily of
batch culture of more than 10 species. The larval production area
has a capacity to produce approximately 30-50 million 1-mm lar-
vae and holds four 937.5-L larval tanks; there is also a small area
devoted to setting animals and broodstock holding tanks. A limited
amount of upweller space is also on site. Two innovative features
of the hatchery are its seawater system and protected interior sur-
faces. The seawater system is a multiple pump system that feeds
into a common manifold consisting of four titanium submersible
pumps with a capacity of 50 gal/min. In addition, the interior
surfaces of the facility are protected by a commercial polyurethane
formulation (typically used for spray-on truck bed liners) applied
to the floor and walls to seal them from conosion and water
damage.

Not only will we continue to use the hatchery as a teaching tool.
we will also be coordinating our 2002 spawning activities with the
Eastham Aquaculture Technology and Training Center to maxi-

mize shellfish propagation and enhancement efforts in the Cape
Cod area. We plan to produce razor clams tEiisis directiis). bay
scallops iArgopecteii irradians irradians). and eastern oyster
iCrassostrea viri;iiuca) larvae for remote sets in various Cape Cod
towns. Currently, we are culturing a small number of razor clam
larvae.

THE NEW OYSTER WARS: POLICY PERSPECTIVES ON
THE INTRODUCTION OF CRASSOSTREA ARIAKENSIS IN
THE CHESAPEAKE BAY. Donald Webster. University of
Mar\land. Wye Research & Education Center. P.O. Box 169.
Queenstown. MD 21658.

Oyster harvests in the Chesapeake Bay have declined more
than 907f in the past 50 y. largely due to the inlluence of the oyster
diseases Haptosporidiim uelsoni and Perkinsus mariniis. Recent
studies regarding the potential of the species Cnissostrea ariak-
ensis have show n that this animal may pros ide beneficial attributes
desirable both for reconstruction of the oyster fishery as well as for
en\ ironmental management of the Bay. However, because it is a
nonindigenous species, there are factors to be considered prior to
an\ large-scale introduction. Variations in the current status of the
Cnissostrea virfiinica resource, as well as historical differences of
management techniques inherent in the two states, have led to
challenges regarding open-water introductions, as well as plans to
increase stocks of the nonnative oyster in the future. This paper
focuses on the policy differences between Maryland and Virginia
and the attitudes of \arious user groups that are interested parties
in the potential introduction of C. ariakcnsis in the Chesapeake
Bay.

DEVELOPMENTS IN SOFTSHELL CLAM HATCHERY
AND NURSERY PRODUCTION ON MASSACHUSETTS'
NORTH SHORE. Scott Weston, Mark Fregeau, and Joe Butt-
ner. Northeastern Massachusetts Aquaculture Center and Depart-
ment of Biology. Salem State College. Salem. MA 01970.

A major goal of the Northeastern Massachusetts Aquaculture
Center (NEMAC) focuses on nurturing a sustainable aquaculture
industry on Massachusetts North Shore amenable to existing so-
cial, economic, and en\ ironmental conditions. Several North Shore
communities are exploring aquaculture as a means to diversify and
supplement their capture fisheries while restoring and enhancing
endemic populations. Historically, the softshell clam {Mya
ari'iuiria) supported significant commercial and recreational har-
vests. Efforts initiated in the 1990s to augment natural populations
now in\ol\e a half dozen towns. Culture protocols adapted to local
conditions have demonstrated the efficacy of stocking 10- to 15-
mm spat in spring. Availability of suitable seed is limited and has
impeded stocking efforts. With the encouragement and participa-
tion of local communities. NEMAC has targeted reliable and ul-
timately large-scale production of spat as a priority objective.

NEMAC"s second year of operation (2001) started eariy as

Milford Aquaculture Seminar, Milford, Connecticut

Abslracts. 2002 Annual Meeting, February 25-27. 2002 355

200.000 juvenile clams were collected by local shellfishers from
the Rowley River in November 200(1 and successfully over-
wintered in the Cat Cove Marine Laboratory. Clam survival ap-
proached lOO'^'K though growth at 15-17°C averaged -0.5 inm/ino
between 3 January and 7 May. Clams were returned to the Rowley
community and released in early May 2001 at approxed sites.
Starting in early July 2001 and continuing to the present. NEMAC
personnel and North Shore shellfishers have monitored sites in the
Rowley River to ascertain water quality, clam abundance, and
optimal release sites.

NEMAC personnel induced four spawns of local clams be-
tween early May and mid July. Inadequate setups for settling lar-
\ae diminished survival of initial spawns and polyspermy ad-
versely impacted larval development during the fourth spawn.
NEMAC produced 75.000 post-set spat, supplemented by 500.000
clams imported from the Beals Island Regional Shellfish Hatchery
(BIRSH) in Maine. All clams were cultured in the laboratory on
Tetiaselmis clniii and T-ISO. As clams reached -3.5 mm in length,
they were transferred to screen-bottom trays at 10.000 clams/tray
(-3.600 cm"). Appro.ximately 30 periwinkles (Littorina sp.) were
added to each tray to crop macroalgae and other fouling organisms.
The first tray was stocked in mid July and stocking continued
throughout the summer. All trays were covered with white plastic,
floated in Smith Pool, and exposed to tidal flush. Initial growth
averaged 2.5-5 mm/mo. At 8-9 mm. growth of clams diminished,
which is indicative of overcrowding. Approximately 70% of the
clams stocked in upwellers survived. Clams are being over-
wintered. 250.000 (8-15 mm) in spat bags suspended in Smith
Pool and 150.000 (3-8 mm) are distributed in trays stacked in a
trough with a continuous flow of filtered seawater. Laboratory-
housed clams are fed 1-2 times daily with a Telrasetmis chuii and
T-ISO mix. The objective is to release 200.000 spat of suitable size
onto approved tidal fiats in spring 2002. It is hoped and anticipated
that on-going, collaborative restoration/enhancement efforts will
evolve into commercial enterprises conceived and pursued by local
shellfishers.

RAPID GROWTH OF BAY SCALLOPS. ARGOPECTEN IR-
RADIASS IRR^iDIAyS. IN LONG ISLAND SOUND. James
C. Widman. Jr. and David J. Veilleux, USDOC, NOAA, Na-
tional Marine Fisheries Service, Northeast Fisheries Science Cen-
ter, Milford Laboratory. Milford. CT 06460.

Bay scallops. Argopecten irradians irradiuns. were held in
pearl nets to evaluate the suitability of western Long Island Sound
as a culture site for scallops. Scallops were deployed at six loca-
tions off the coast of Darien. Connecticut. Pearl nets were an-
chored with a cement block and vertical lift was provided by an
attached subsurface buoy. Nets were deployed singularly or in
groups of three. Thirty scallops were measured to the nearest 0.1
mm. placed in each of nine pearl nets, and transported to the site
in coolers filled with ambient seawater. Initial deployment was on
July 25. 2001. and the experiment ended on February 6. 2002.

Survival was high, although there was some initial mortality.
Survival for the entire experiment averaged 71% and ranged from
53% to 83%. After the initial mortality, survival after September
1 1 averaged 93% and ranged from 79% to 100%. The increase in
survival after the September 11 sampling period indicates that
there may have been some handling/transport problems.

Scallops with initial mean shell heights ranging from 6.8 to 7.7
mm on July 24. 2001. grew to mean shell heights ranging from
45.4 to 50.6 mm by February 6. 2001. Growth rates were high
from July 24 until October 16. when water teinperatures began to
decline below 15"C. Less than 1 mm of growth occurred after
December 4. when water temperatures were low. Rapid growth
rates ranging from 0.36 to 0.49 mm/day were observed during the
period from September 1 1 to October 16. 2001. a period of de-
clining water temperatures.

For the experimental regime. Western Long Island Sound ap-
pears to be a suitable site for culturing bay scallops.

LIVESTOCK DOMESTICATION IN THE THIRD MILLEN-
NIUM: ALL WET? Gary H. Wikfors. USDOC, NOAA, Na-
tional Marine Fisheries Service, Northeast Fisheries Science Cen-
ter, Milford Laboratory, Milford. CT 06460.

The dictionary defines "domestic" animals as those "kept by
and for the use of man." The first domestication of land animals
(sheep in what is now Turkey) is thought to have occurred between
13,000 and 9.000 y ago during the Neolithic-to-Paleolithic transi-
tion in human history. Since that time, the trajectory of human
food acquisition has been away from the hunter-gatherer mode to
increasingly sophisticated husbandry of both plants and animals.
The one exception to this historical paradigm has been seafood. It
appears, however, that mankind now is entering a transition period
from harvest of wild populations in the sea to domestication for the
same reasons that drove land-animal domestication — rising de-
mand and limited supply of wildlife for harvest. We have the
benefit of about 10.000 y of human experience to guide decisions
about marine plant and animal domestication — both successes and
failures. There has, however, been limited effort to transfer the
broad principles of animal husbandry downstream; therefore, the
first objective of this presentation will be to advance this transfer.

At this early stage in the transition to aquaculture. the first few
global, industrial-scale aquaculture products — salmon and
shrimp — have reached a point of development to impact world
seafood markets, as well as generate controversy about the future
of aquaculture expansion on several fronts, chietly economic and
environmental. Unquestionably, mistakes have been made with
shrimp and salmon, as they have with agricultural livestock pro-
duction methods. What lessons can be learned from modem agri-
culture and our limited experience with industrial aquaculture and
how can these be related to broad principles identified from the
history of animal husbandry? These questions will be discussed
comparatively.

356 Ahsrmcls. 2002 Aniuuil Meeting. February 25-27. 2002

Mllford Aquaculture Seminar. Millord. Conneetieut

BLUE MUSSEL AQUACULTURE IN LONG ISLAND
SOUND. Lawrence Williams, Jessie D.. Inc.. 68 Anchorage
Drive. Milford, CT 06460: and Tessa Simlick. Connecticut Sea
Grant, University of Connecticut. 1084 Shennecossett Road. Gro-
ton, CT 06340.

A pilot-scale investigation into the longline culture of blue
mussels Mytilus ediilis in Long Island Sound has been initiated.
The project, in progress since the spring of 2001. includes collect-
ing and grading seed for grow-out on dynamic longlines at three
sites in Long Island Sound and comparing production rates. A
horizontal head rope (100 ft in length) was submerged 6 ft below
the surface (at MLW) and anchored with concrete blocks (150 lb
total weight). Vertical seed collecting lines (11 ft in length) were
attached to the head rope at 3-ft intervals. Seed collectors were
placed over submerged mussels beds in Branford. Milford, Fair-
field, and Stonington. Connecticut, in late April 2001. A large
seed-set appeared in Stonington in early June 2001 . however, mus-
sels were not visible on the collectors in western LIS until mid-
July 2001. In late September 2001. a second set of seed attached to
the seed collectors.

ARACHIDONIC ACID REQUIREMENTS IN LARVAL
SUMMER FLOUNDER, PARAUCHTHYS DENTATUS.
Stephen Willey and David A. Bengtson, Department of Fisheries.
Animal and Veterinary Science, University of Rhode Island,
Kingston. RI 02881; Moti Harel, University of Maryland Center
of Marine Biotechnology, 701 East Pratt Street, Baltimore. MD
21202.

Highly unsaturated fatty acids (HUFAs) have been identified as
essential fatty acids in marine fish and the dietaiy requirements for
the n-3 (omega-3) HUFAs eicosapentaenoic acid (2():5n-3. EPA)

and docosahexaenoic acid (22:6n-3. DHA) have been well docu-
mented. Only recently has attention been given to HUFAs of the
n-6 series, in paiticular arachidonic acid (20:4n-6. AA). The im-
petus for much of this attention is the fact that AA is highly
conserved during periods of starvation and also serves as a pre-
cursor in the biosynthesis of eicosanoids. physiologically active
compounds that aid in stress response, among other things.

The AA requirements of larval summer flounder were deter-
mined for the rotifer- and Artemia-feeding stages. Experimental
emulsions contained adequate n-3 HUFA ratios and emulsion lev-
els of AA were set at 0%, 3%. 6%. 9%. and 12% (AAO. AA3.
AA6. A9. and AA12). Examination of fatty acid levels in live
feeds and larval tissues confirmed the physiological incorporation
of fatty acids relative to dietary levels. In the first experiment.
survival, growth, and salinity tolerance (2 h in 70%c) were mea-
sured at 18 days after hatch (dah) after feeding the larvae the
various levels of AA. Larvae fed AA6-enriched rotifers were better
able to survive the salinity tolerance test. AA enrichment up to
12Vf had no effect on growth and survival. In the second experi-
ment, larvae fed AAO- or AA6-enriched rotifers until 23 dah.
followed by unenriched 24- and 48-h Artcnua nauplii until 32 dah.
These larvae were then subdivided and fed AA-enriched Anemia
from 33 to 45 dah. At the end of this experiment, larvae fed
AA6-enriched rotifers had higher survival, increased growth, and
survived better in the salinity tolerance test (2 h in 809ff ) than did
those fed AAO (unenriched) rotifers. The AA enrichment oi Ar-
temia did not have any significant effect on these variables. Thus,
the provision of AA6-enriched rotifers early in larval development
may serve to enhance larval stress tolerance at the rotifer stage,
while also increasing larval survival, growth, and stress tolerance
later in the Arlcniiii stage.

Joimuil of SlwUfish Research. Vol. 21. No. 21, 357^4U. 2UU2

ABSTRACTS OF TECHNICAL PAPERS

Presented at The 94th Annual Meeting

NATIONAL SHELLFISHERIES ASSOCIATION

Mystic, Connecticut
April 14-18, 2002

357

National Shellfisheries Association, Mystic, Connecticut Abstracts, April 2002 359

CONTENTS

I'ERKINSUS

George R. Abhe, Brian W. Albright. Carol B. McColloitgh. Christopher F. Diingan and Stephen J. Jordan

Environmental effects on Perkiusiis nuiriinis infection rates, growth and survival among Dermo-disease-free juvenile

oysters planted at three salinity regimes in the Patuxent River, Maryland 37 1

Bassem Allam and Susan E. Ford

Transport of particles across epithelia from oyster mantle cavity: a model for Perkinsiis inarimis invasion 371

Louis E. Burnett and Christopher S. Milardo

Effects of o\ ster emersion on the grow th and the metaboiisin of Perkinsiis iinirimis 371

David Bushek, John Scarpa and Susan E. Laramore

Susceptibility of the Caribbean oyster Crassoslrea rliizopliorac to Perkinsiis iiuinniis 37 1

Fu-Lin E. Chu, Eric Lund and Ellen Harvey

Does the unique fatty acid synthetic capability of Perkinsiis iiianniis have implication for virulence? 372

Lewis E. Deaton and Percy J. Jordan

Effect of Perkinsiis inanniis on phenoloxidase activity in bi\alve hemolymph 372

Christopher F. Dungan, Rosalee M. Hamilton, Carol B. McCollough, Kimberly S. Reece and Karen L. Hudson

Epizootic diseases in Chesapeake Bay clams 372

Vincent G. Encomio, Shawn Stickler and Fu-Lin Chu

E\ aluation of physiological condition in Dermo resistant oysters 373

Tarquin Dorrington, Maria Gomez-Chiarri and Lenore Martin

Evaluation of antimicrobial peptides for recombinant feed-based delivery in shellfish aquaculture 373

Julie D. Gauthier, Jerome F. La Peyre and Jill A, Jenkins

Flow cytometric analysis of lectin binding to in vitm cultured Perkinsiis nuiriniis surface carbohydrates 373

Eileen E. Hofmann, John M. Klinck, Eric N. Powell, Susan E. Ford, Stephen Jordan and Eugene Burreson

Climate variability and Dermo disease in Chesapeake Bay 374

Stephen L. Kaattari, E. Alanna Maclntyre and Christopher G. Earnhart

Modulation of Perkinsiis nuiriniis functions by host-derived products 374

Jerome F. La Peyre, Amy D. Nickens, Sandra M. Casas and Antonio Villalba

Viability and growth of Perkinsus marinus and Perkinsiis ailanticiis at three temperatures 374

Eric D. Lund, Fu-Lin E. Chu and Philippe Soudant

Is the temperature and salinity-dependent virulence of Perkinsiis nuiriniis associated with increased

lipid metabolism? 374

Carol B. McCollough, Christopher F. Dungan, Stephen J. Jordan, George R. Abbe and Brian W. Albright

Perkinsiis marinus infection rates in specific-pathogen-free juvenile oysters planted at three salinity regimes in the

Patuxent River. Maryland 375

Kennedy T. Paynter, Tim Koles and Don Meritt

Perkinsiis prevalence in oysters produced in the hatchery and planted in the field 375

Sammy M. Ray, Thomas M. Soniat, Enrique V. Kortright and Lance Robinson

Recent trends in levels of infection of Perkinsiis marinus in oysters from Galveston Bay. Texas: results of the

Dermowatch Monitoring Program 375

Kimberly S. Reece

Utilization of molecular genetic data for detecting, identifying and describing Perkinsiis species 376

Pilar Mui'ioz Ruiz and Maria Gomez-Chiarri

Protease activity in the eastern oyster Crassoslrea virginica after experimental infection with the protozoan parasite

Perkinsus marinus 376

S. M. Stickler, V. G. Encomio, S. K. Allen, Jr., F-L. E. Chu and J. F. Im Peyre

"Natural Dermo resistance"" in eastern oyster stocks: Chesapeake studies and defense-related activities 376

THE BIOLOGY AND CONSERVATION OF FRESHWATER MUSSELS
Alan D. Christian. David J. Berg and B. Crump

Spatial and temporal analysis of ecosystem processing of freshwater mussels in two headwater streams 377

Susan Rogers, Richard Biggins and Steven Ahlstedt

Conservation acti\ities tor freshwater mussels by the United States Fish and Wildlife Service 377

William F. Henley and Richard J. Neves

Diet evaluation for the freshwater mussel Elliptio comptanala ( Bivalvia: Unionidae) 377

360 Ahslimts April 2002 National Shelltisheries Association. Mystic, Connecticut

Variability in condition index and tissue biochemistry of Elliplio loniplanalti held in the field and laboratory 378

Curt L. Elderkin. David J. Berg. Janice L. Metcalfe-Smith. Caryii C. Vaughn. Alan D. Christian and
Sheldon I. Guttman

Hierarchical analysis of MtDNA variation in Amhlema plicata. a widespread mussel species 378

Jeffrey T. Garner and Thomas M. Haggerty

Gametoizenesis. spawning, brooding and glochidial discharge in Mci^aUmuuis iicrvd.sa (Bivlavia: Unionidae) from the

^ ^ ^ ^ 378
Tennessee River in Alabama '°

Catherine M. Gatenhy. Daniel A. Kreeger. Rohyn Reinmiller and Richard J. Neves

Clearance rate and filtration efficiencv of EUiptic compUmaw (Bivahia: Unionidae) exposed to different laboratory

,.,. ' 378

conditions

Daniel A. Kreeger, Catherine M. Gatenby and Deborah Raksany

lity
Richard J. Neves, Jess W. Jones and William F. Henley

Propagation of endangered freshwater mussels in recirculating culture systems 379

Daniel E. Spooner and Caryn C. Vaughn

Small-scale ecological function of freshwater mussels (Family: Unionidae) in the Kiamichi River. Oklahoma 379

Caryn C. Vaughn, Daniel E. Spooner, Melissa Moore and Keith R. Gido

Comparative and experimental evidence for the functional role of freshwater mussels in streams 379

W.-H. Wong, Jeffrey S. Levinton and Benjamin Twining

Assimilation of micro/.ooplankton by zebra mussels: the benthic zooplankton loop 380

GENETICS AND MOLECULAR BIOLOGY

Charles L. Brockhouse and Gillian Richard

Unscrambling the lobster genome: complex heterozygosity in the American lobster? 380

Bruno Ernande, Joel Haurc, Lionel Degremont, Edouard Bedier and Pierre Boudry

Genetical basis of the plasticity of the resource allocation in the Pacific oyster Crassostrea gigas 380

Ximing Guo, Jian Wang. Brenda J. Landau. Li Li. Gregory A. DeBrosse and Krista D. Buono

The successful production of tetraploid eastern oyster. Crassostrea virginica Gmelin 380

Matthew P. Hare and Stephen R. Palumbi

Genetic distinctiveness of inshore and offshore Spisula clams

Dennis Hedgecock, Sophie Hubert, Gang Li and Katharine Bucklin

A genetic linkage map of 100 microsatellite markers for the Pacific oyster Crassostrea gigas 381

Gregg G. Hoffman. Ami E. Wilbur, Martin H. Posey and Troy D. Alphin

A sin>'le-step multiplex PCR identification assav to distinguish megalopae of Calliiiecles sapuliis trom

Callinectes siinilis in plankton samples

Brenda J. luindau, Arnaud Tanguy and Ximing Guo

Searching for differentially expressed genes in diploid and Iriploid eastern oyster. Crassostrea virginica Gmelin 382

Ziniu Yu and Ximing Guo

A basic AFLP linkage map for the eastern oyster. Crassostrea virginica Gmelin 382

Mingjdng Zhou and Standish K. Allen, Jr.

Reversion revisited in Crassostrea ariakciisis: chromosome set instability in field trials across salinity regimes 382

EAST COAST BIVALVE INDUSTRY SESSION
Standish K. Allen, Jr.

An overview of the potential for Crassostrea ariakensis in the Chesapeake Bay

Gregory A. DeBrosse, David R. Jones and Eric N. Powell

Surf clam [Snisula solidissima) culture in southern New Jersey: hatchery culture proKK'ols and preliminary field grow

, ' 383

out results

M. Richard DeVoe and G. Ross Nelson

Coastal states permitting procedures for deploying a tidal powered upwelling nursery system 383

George E. Flimlin, Jr.

Relevant issues for the east coast shellfish aquaculture industry: finding commonality to foster unity 383

Nation;il Shellfisheries Association. Mystic. Connecticut Ahsrracts. April 2002 361

Aiiiaii Liithra and William Walton

Stakeholders" perceptions and challenges to participatory management: the case of the soft-shell clam. Mya arenaria

L.. industry in Maine 384

Sandra Macfarlane

Embracing aquaculture BMP's; a concept whose time has come as a good neighbor or just one more thing

to deal with? 384

Dana L. Morse and John Riley

Optimization of tidal upweller design: project wrap-up 384

Robert B. Rheault and Jeffrey T. Gardner

Growth and economics of shellfish nursery culture in a commercially available floating upweller 385

Leslie N. Stunner, John E. Supan and Charles M. Adams

Enhancing seed availability for the hard clam aquaculture industry through application of remote setting techniques ... 385
William C. Walton

Using shellfish seed as a public enhancement tool: a review of its use and success in the northeast US 385

Christopher G. Warfel, P.E.

Growth and economic advantages of distributed powered upwellers: creating a new aquaculture niche 385

Donald Webster

The new oyster wars: policy perspectives in the introduction of Crassostrea ariakensis in the Chesapeake Bay 386

BIVALVE DISEASE STATUS AND TRENDS

Bruce J. Barber and hatherine J. Boettcher

Recent expansion of juvenile oyster disease ( JOD) in Maine 386

David Bushek, Dwayne Porter, Loren D. Coen, M. Yvonne Bobo and Donnia L. Richardson

Status and trends of Dermo and MSX in South Carolina 386

Lisa M. Ragone Calvo and Eugene M. Burreson

A decade of disease: a report of oyster disease monitoring efforts in the lower Chesapeake Bay 1990-2000 387

Ryan B. Carnegie, Gary R. Meyer, Janice Blackbourn, Susan M. Bower and Nathalie Cochennec-Laureau

Development and application of a PCR for Mikrocytos macktni. the causative agent of Denmaii Island disease

in oysters 387

Susan E. Ford

Development of high disease resistance in a wild oyster population 387

Stephen J. Jordan and Jessica Vanisko

The long-term role of parasitic diseases in oyster population dynamics 387

Dale Leavitt, D. Murphy, W. Burt, W. Clark, M. Hickey, J. Moles and R. Smolowitz

Cape Cod bivalve diseases monitoring program. Year I 388

Karen L. Mareiro, Marta Gomez-Chiarri, Katherine Kerr, Emily Carrington and Arthur Ganz

Prevalence and intensity of parasitic diseases in bivalves from Rhode Island waters 388

Spencer Russell, Salvatore Frasca, Jr., Richard A. French and Inke Sunila

Modification and field trials of a multiplex PCR for the detection of three protozoan pathogens of the eastern oyster.

Crassostrea \iri;inica. Gmelin 1 87 1 388

Thomas M. Soniat, Enrique V. Kortright and Sammy M. Ray

Dermowatch: a web-based approach for monitoring the oyster parasite Perkinsus marinus

(Dennocystidiuin inarinuin} 389

Mary F. Stephenson, Sharon E. McGladdery and Nancy A. Stokes

Detection of a previously undescribed Haplosporidian-like infection of a blue mussel (Myfihis ediilis) in

Atlantic Canada 389

hike Sunila

Monitoring bivalve health in Long Island Sound 389

DISEASES OF CRUSTACEA

Donald C. Behringer, Jr., Mark J. Butler, IV and Jeffrey D. Shields

Ecological ramifications of disease in the Caribbean spiny lobster. Paniilirus argus 390

362 Abstracts. April 2002

Nation;il Shclirisheries Association. Mystic. Connecticut

Kathleen Castro and Thomas Angell

Field observations on the development and progress of a shell disease episode tor American lobster in Rhode Island:

1 993-200 1 ^^^

Terry Glover, Lauren Bergey and Judith S. Weis

Effects of paiasites on beha\ lor oi grass shrimp. Pcilaciiioiuics />iigio ?90

Barbara Homey, Andrea Battison and Allan Mackenzie

Cytocentrifu>'e preparations: an alternate method to examine the hemocytes of the American lobster

" . ^91

Himiartis amencainis

Gretchen A. Messick and Tom F. Nalepa

Parasites in Dtpi'icia spp. amphipods from Lakes Michigan and Huron ^^ 1

Jeffrey D. Shields

Pathological alterations in the eyes of the American lob.ster, Homanis cimcriinnii.s. infected with Panimocba sp 391

Jeffrey D. Shields, Donald C. Behringer, Jr. and Mark J. Butler, IV

A pathogenic herpes-like virus from the spiny lobster, Piundirus urgus 391

BLUE MUSSEL BIOLOGY AND CULTURE

John Brake, Jeffrey Davidson and Jonathan Davis

Observations on growth, gametogenesis, and sex ratio of Iriploid and diploid Mytilns eihilis 392

Jeff Davidson. Frank Boolhroyd, Neil McNair and Thomas Landry

The distribution and biology of an invasive tunicate in Prince Edward Island. Canada 392

Ellen L. R. henchington, Kenneth R. Freeman, Benedikte M. Vercaemer and Barry W. MacDonald

A lield study of settlement depths of A/\7(7i/,s cdidis and M. trossidus in Nova Scotia, Canada, 1998-2000 392

Angeline R. LeBlanc, Gilles Miron and Thomas Landry

Nutrient uptake and release from fouling organisms associated with cultured mussels in Tracadie Bay, PEI 393

Afton McGowen. Matthew Gordon and Paul D. Rawson

Species-specific settlement patterns of blue mussels in Cobscook Bay. Maine 393

Marcelo Miranda, David Innes and Raymond Thompson

Genetic aspects of the blue mussel (Mxriliis cdidis and M\tihis trossidus) hybrid zone in Atlantic Canada 393

Valerie Moreau, Edwin Bourget and Rejean Tremhiay

Small scale distribution of A/vr//;/,v edulis and M. irossiilu.s m the bale des Chalcurs and the Gaspe Peninsula 393

Randy W. Penney, M. J. Hart and N. Templeman

spei

Paul D. Rawson

Cladistic analysis of genetic differentiation between populations of the blue mussel, Mxtdus trossidus 394

Judith Senechal and Jon Grant

Behavior and growth of juvenile mussels (Myldiis spp.) in suspended culture socks 394

Benoit Thomas, Valerie Moreau and Rejean Tremblay

Update on the distribution of two mussels species {Mxtiliis cdidis and Mytiliis trossulus) in the Quebec maritime

394

regions

Rejean Tremblav, Valerie Moreau. Thomas Landry, Bruno Myrand and Cyr Couturier

Performance of Mxiiliis cdiilis and AA//7((,s irossidits in the Gulf of St. Lawrence: a laboratory experiment 395

J. Evan Ward, Sandra E. Shumway and Jeffrey S. Levinton

Blue mussels as model systems to investigate pallial cavity function in bivalves 395

HARMFUL ALGAL BLOOMS

Marie-Claude Archambauh, Jon Grant, Monica Bricelj and Don Anderson

Effects of clay, used to control harmful algal blooms, on juvenile hard clams, Mcrccnaria mercemuia 395

Rita A. Horner

History, some recent HAB events, and their impacts on shellfish and finfish m Washington State 396

Jay R. Leverone and Norman J. Blake

Effects of the toxic dinotlaaellate, Karciiiu hrcvis. on lar\al mortality and juvenile feeding behavior in the bay

„ ^ ■ r" 396

scallop, Argopeclcn irnuhans

Mussel culture in a mixed species (A/, cdidis and M. trossidus) /.one — some commercial implications 394

National Shellfisheries Association. Mystic, Connecticut Abstracts. April 2002 363

Gaiy H. Wikfors, Jennifer H. Alix, Roxanna M. Smolowitz, iMcey Wallace and Helene Hegaret

Detrimental effects of a recent Pryiiim-siiiiii isolate from Boothbay Harbor. Maine (USA) upon juvenile bay scallops
Argopecten irradians 397

SHELLFISH BIOLOGY

Richard R. Alexander and Robert M. Baron

BuiTowing-induced internal fractures and external abrasion in shells of the hard clam Mercenaria mercenaria from

Raritan Bay, New Jersey 397

Eleanor A. Bochenek, Eric N. Powell, John M. Klinck and Eileen E. Hofmann

Influence of environment and food supply on survival of Crassostrea gigas larvae: a modeling study 397

Andrew T. Cogswell and Sainia Sarkis

Growth characteristics of Argopecten gihhiis juveniles reared in tw o suspended culture systems 398

W. R. Congleton, Jr., B. R. Pearce and M. Parker

Growth and dispersal studies of Mya arenaria using a numerical flow model 398

Zaiil Garcia-Esquivel, Marco A. Gonzalez-Gomez and Francisco Ley-Lou

Estimation of ingestion and biodeposition rates of the Pacific oyster. Crassostrea gigas. in a coastal lagoon

of NW Mexico 398

iMiira E. Gomez, Ma. Teresa Viana, Zaiil Garcia-Esquivel, Armando Shimada and Louis R. D'Abramo

Effect of dietary protein/energy ratio on growth and metabolism of juvenile green abalone (Haliotis fulgens) 399

John Kraeuter, Eric N. Powell, Eileen E. Hofmann, John M. Klinck, Ray Grizzle, Monica Bricelj and Stuart Btickner

Modeling the growth of the hard clam. Mercenaria inerceiiana 399

Eric N. Powell, Eleanor A. Bochenek. John M. Klinck and Eileen Hofmann

Influence of short term variations in food supply and critical periods on survival of Crassostrea gigas larvae 399

Melissa J. Southworth, Juliana M. Harding and Roger Mann

Population structure of the hard clam. Mercenaria mercenaria. in Hampton Roads. Virginia 400

Helen Woods, Ken Moore and Carl Hershner

An examination of potential conflict between hard clam aquaculture and SAV in the lower Chesapeake Bay 400

SCALLOP BIOLOGY AND CULTURE

V. Monica Bricelj, Anne Veniot, Celine Bane and Peter Beninger

Postlarval development of the gills and implications for feeding in the sea scallop. Placopecten magellaniciis 400

Maureen Davidson

The effects of stocking density in pearl nets on survival, growth, and reproductive potential of the bay scallop.

Argopecten irradians irradians 40 1

Scott Feindel and Daniel Schick

Developing a coastal Maine sea scallop enhancement program 401

Brad Harris and Kevin Slokesbury

Growth and movement of sea scallops in the southern part of the Great South Channel on Georges Bank:

a tagging study 40 1

Daniel L. Jackson, Barry W. MacDonald, Shaka James, Benedikte Vercaemer, Ellen L. Kenchington and Andre Mallet

Investigations w ith triploid Atlantic sea scallops. Placopecten luagellanicus 401

Lisa M. Milke, V. Monica Bricelj and Christopher C. Parrish

A comparison of microalgal diets for enhanced production of Placopecten magellaniciis postlarvae 402

Shawn M. C. Robinson, Jim D. Martin and Eddy J. Kennedy

Impacts and control of the spionid polychaete. Polydora wchsteri on the sea scallop. Placopecten magellaniciis in

midwater culture 402

Gregg E. Rosenkranz and Douglas Pengilly

Development of a stock assessment program for weathervane scallops in Alaska 402

Fabrice Pernet. Rejean Tremblay and Edwin Bourget

Biochemical indicator of giant scallop PUuopecten magellaniciis quality: larval growth, competency and settlement ... 403
Kevin D. E. Stokesbury and Brad Harris

Examination of sea scallop. Placopecten magellaniciis. aggregations using a video survey in closed areas

of Georges Bank 403

364 Ahstracts. April 2002 National Shellfislieries Association. Mystic. Connecticut

PARASITE AND HOST DEFENSES

Steven M. Allen and Louis Burnett

The effect of pH on the kilhng activity of heniocytes m the Pacific oyster, Crassostrea gificis 403

Robert S. Anderson, Brenda S. Kraiis. Sharon McGladdery and Roxanna Smolowitz

Mucoid secretions protect QPX Ironi antimicrobial agents 404

Gwenaelle Choqnet, Philippe Soudant, Christophe Lambert, Christine Paillard and Jean-Louis Nicolas

Measurement of \'ihii<i lapctis cytotoxic activity on Riidihipcs philippiiitiruiii heniocytes by flow cytometry 404

Christie-Sue Decker and Robert S. Anderson

Chemotaxis of heniocytes of the hard clam. Mercenariu mcrceiuiriu. to Quahog parasite tmknown (QPX) and

other microorganisms 404

Maryse Delaporte, Jeanne Moal, Jean-franfois Samain, Philippe Soudant, Gwenaelle Choquet, Christophe Lambert and
Christine Paillard

Effect of dietary fatty acid composition on lipid profiles of haemocyte membranes in oysters and clams and its impact

on immune functions 404

William S. Fisher and Leah M. Oliver

Activation of oyster defenses by environmental contaminants 405

Christophe Lambert, Christine Paillard and Jean-Louis Nicolas

Improvement by probiotics of Pcctcn maximus larvae defense capacity, measured by chemiluminescence 405

Christophe Lambert. Philippe Soudant, Gwenaelle Choquet and Christine Paillard

Development of a flow cytometric measurement of oxidative metabolism product formation by Crassostrea gigas

heniocytes and application to evaluate pathogenic Vibrio inhibiting capacity 405

Ann C. Mountz and Robert S. Anderson

Purification of a novel antimicrobial peptide from the eastern oyster ( Crassostrea virgiiiica) 405

Christine Paillard, Gwena'ella Choquet, Christophe Lambert, Philippe Soudant, Helen Reid and Harry Birbeck

Temperature effect on immunocompetence of clam R. pliillppiiuiniiii and on V. nipelis cytotoxicity 406

Christine Paillard and Susan E. Ford

Repeated hemolyniph sampling of individual clams: what does it Icll us about sampling procedures? 406

Eric J. Schott, Jose A. F. Robledo, Wolf T. Pecher. Florence A. Okafor and Gerardo R. Vasta

The antioxidant svslcni of the protistan parasite. Pcrkiiisiis iiiiiriiuis 406

Philippe Soudant, Gwenaelle Choquet, Christophe iMmbert, Alain Marhic and Christine Paillard

Seasonal and culture site effects on the physiological, immunological and Brown Ring disease status of the manila

clam Riulilapts pliilippiiiiinini 406

P. Soudant, C. Uimbert. G. Choquet, S. Ford, C. Paillard, L. Degremont, M. Delaporte, J. Moal, P. Boudry,
P. Soletchnick, J. -P. Joly, M. Ropert. E. Bedier, A. Huvet and J.-F. Samain

Relationships between summer mortalities and defence mechanisms in families of Crassostrea gigas reared in

different environmental conditions 407

Qiitg-Gang Xiie, Jerome F. La Peyre, Aswani K. Volety and Fu-Lin E. Chu

Purification and characterization of lysozyme from plasma of eastern oysters ( Crassostrea virgiiiica) 407

SEA URCHIN BIOLOGY, PATHOLOGY AND CULTURE
Ralph Elston

Sea urchin mortality in Maine, initial case report and overview 407

Eddy J. Kennedy, Shawn M. C. Robinson, John Castell and G. Jay Parsons

Importance of dietary minerals and pigments for increasing somatic growth of juvenile green sea urchins

(Strongylocc'i}!roli(s drochachicnsis) 408

Gregory MacCallum, Shawn Robinson, Sharon McGladdery and Mary Stephenson

Sea urchin disease concerns in Atlantic Canada 408

Michelle Moody and Charles W. Walker

Photoperiod. urchin "eyes'" and gametogenesis 408

Esther C. Peters

Abiotic and biotic threats to sea urchin health; w hat price culture? 409

National Shellfisheries Association. Mystic, Connecticut Ahstnicts. April 2002 365

Michael P. Russell

Growth response and acclimation of green sea urchins to tluctuating salinity 409

Paul Waterstral and Ted Creaser

Urchin health issues in Maine 409

LOBSTER BIOLOGY AND FISHERIES

Kathleen M. Castro, J. Stanley Cobb, Richard Wahle and John Catena

The effects of habitat addition and stock enhancement for American lobsters, Honmnis nineiicauus on artificial reefs

in Rhode Island 410

Andrei Y. Chistoserdov, Feliza Mirasol and Roxanna Smolowitz

Characterization of microbial assemblagies involved in the development of shell disease in the American lobster,

Hdiimnis umericanus 410

J. S. Cobb and M. Clancy

Changes in lobster populations in Narragansett Bay. Rhode Island, 1959-2000 410

Cent Giray and Deborah A. Bouchard

The role of Vihiio fluvinUs and other bacterial species in lobster mortalities in Maine 410

Diane Kapareiko, Richard A. Robohm, John J. Ziskowski, George R. Sennefelder and Anthony Calabrese

Shell disease prevalence and severity in offshore American lobster populations 411

Wendy Norden and J. Stanley Cobb

Depth-related predation pressure on larval lobsters [Hcmniriis ciiiicriciiiius) prior to settlement 411

Susan A. Little, Win Watson and Bonnie Spinazzola

The effect of temperature on sexual maturity in the female lobster, Htnnarus iiiiic'ricanus 411

James Manning, Bonnie Spinazzola, Patrice Farrey, David Casoni and Clare Grindal

En\ ironmental monitors on lobster traps 412

Roxanna Smolowitz, Andrea Hsu, Erin Summers and Andrei Christoserdov

Lesions associated with recent epizootic shell disease in Hniininis ciiiicricainis on the northeast coast 412

Barbara A. Somers. Kathleen M. Castro, John Sorlien and Tom Angell

What can data from industry conducted tag-recapture programs tell us? A study of the American lobster

(Homanis umericanus) 412

OYSTER REEFS AND RESTORATION
Elizabeth M. Flynn and Kennedy T. Paynter, Jr.

A characterization of ideal habitat structure for the striped blenny Chasinodes hosiiuianus 413

Raymond Grizzle, Larry Ward, Jamie Adams, Semme Dijkstra and John Nelson

A comparison of acoustic techniques, videography. and quadrat sampling for characterizing subtidal oyster reefs 413

Kimberly A. Hamilton, D. LaDon Swann and William Burkhardt, HI

Prevalence of enteric microorganisms in the eastern oyster {Cnissostrea virginica) and their overlying waters at

representative sites of an oyster gardening program in Mobile Bay, Alabama 413

J. C. Issacs, W. R. Keithly and A. Diagne

Use and value of oyster reefs among recreational fishermen in Louisiana 414

Earl J. Melancon, Jr., Dale Diaz and Badiollah Asrabadi

Relaying as a method to remove hooked mussels from oysters prior to reharvest for sale 414

Roger I. E. Newell, Evamaria W. Koch, Melissa K. Wood, Ray E. Grizzle, and Raleigh R. Hood

Modeling the inllucnce of filtration by oyster stocks on turbidity and seagrass growth 414

Stewart Harris and Kennedy T. Paynter

The effect of stocking density on benthic community development and shell height in the eastern oyster,

Crassostrea virginica 415

Rachel E. Sackett, Russ Peterson, Ami E Wilbur and Jim Swartzenberg

Evaluating the contribution of commercial oyster aquaculture to recruitment 415

Lisa Wall, Linda Walters, Kevin Johnson, Neysa Martinez and Ray Grizzle

Recruitment of the oyster Crassostrea virf;iiiiia on intertidal reefs in areas with intense boating activity in the Indian

River Lagoon. Florida 415

366 Ahslracls. April 2002 National Sliellfisheries Association. Mystic. Connecticut

OFFSHORE FISHERIES

Todd Gedamke and William DiiPaiil

Integrating vessel tracking, catch data, and depletion models to estimate commercial scallop dredge efficiency 416

Kevin D. Goff, William I). DuPaul and David B. Rudders

The use of a 4-incli (101 mm I sea scallop {PUiKipech-n ma,iicllciiuciis) ring dredges m the context of an area

41(1
management strategy ^ '"

Deborah Hart and Paul Rago

Rebuilding sea scalkip iPlaaipectcn iiuif;ellaiiiciis) stocks using area closures and rotational fishing 416

Paul J. Rago and Deborah R. Hart

Fleet d>namics of the Atlantic sea scallop fisheiy 416

Farhad Rajahipour

Population structure of the Indian squid. Lolii^o iliivuKielii Orbigny. 1848. in Iranian waters of the Oman Sea 417

Anne Richards, Michael Fogarty. David Mountain and Mirta Teichberg

Recruitment dynamics of northern shrmip {PainUilii.s horealis) in the Gulf of Maine 417

James R. Weinberg and Charles Keith

Deep sea red crabs off southern New England: has there been a fishery impact on the population? 417

DISEASES OF SHELLFISH: CONTRIBUTED SESSION

B. K. Diggles, P. M. Hine, V. L. Webb, E. W. Maas, J. Nichol, S. Wakefield, R. Roberts. C. S. Friedman,
N. Cochennec-Laureau, K. S. Reece and N. A. Stokes

Mortalities of cultured abalone. Hiiliciis iris, infected by a novel haplosporidian 418

Ralph Elston, Dan Cheney, Brian MacDonald and Andrew Suhrbier

Low salinity tolerance in Manila clams Vciicnipis pliilippiiwntm 418

Sean E. Matson and Christopher Langdon

A specific pathogen free culture system for C. gigas larvae and spat 418

Gary R. Meyer and Susan M. Bower

Intramitochondrial crystals within the haemocytes of mussels [Mytihis cdiilis) experiencing unexplained mortalities.... 419
James D. Moore and Carolyn S. Friedman

Environmental detection of the rickettsiales-like prokaryote causing withering syndrome in abalone 419

J.-L. Nicolas, M. Gamier, M. Gay and F. Lerou.x

Vibrio associated with juvenile oyster during summer mortality in France 419

Wolf T. Pecher, Jose A. F. Robledo. Calhleen A. Coss and Gerardo R. Vasta

Further molecular characterization of Perkiiisiis aiulrewsi and related isolates 420

Jose A. F. Robledo, Gerardo R. Vasta. Patricia A. Nunes and M. Leonor Cancela

III villi) culture of Peikinsiis alhiiiucus trom clams Tapes deciissatiis: characterization of the rRNA gene 420

Jose A. F. Robledo, Eric. J. Schott. Adam G. Marsh and Gerardo R. Vasta

Gene discovery in Pcrkiiisiis iiniriiiiis using expression sequence tags (F.ST) 4-0

Jose A. F. Robledo and Gerardo R. Vasta

The Nramp gene and competition for available iron between Crassostrea virgiiiica and Perkiiisiis imirinus 420

Arnaud Tanguy, Susan E. Ford and Ximing Giio

Characterisation of gene expression in response to Perkiiisiis imiriniis and HuplosporiJiiim iiclsoiii infections in the

eastern and Pacific oysters

BIVALVE HABITAT SUITABILITY AND THE ROLE OF BIVALVES IN ECOSYSTEMS

Loren D. Coen, Richard K. Wallace and Nancy Hadley

How community-based oyster restoration can enhance research efforts: examples from South Carolina

and Alabama

William S. Fisher

Oyster habitat suitability as a component of resource management 4-1

Dana M. Frank and J. Evan Ward

Continuous monitoring of pumping pressure and valve gape in the oyster Ciassosiicci virginica in response to

changes m environmental parameters ^ —

Niitidiial Shellfisheries Association, Mystic. Connecticut Abstracts. April 2002 367

Raymond Grizzle, Jennifer Greene and Mark Luckenbach

A simplitied sestt>n uptake model for bivalves: preliminary field tests 422

Mark Luckenbach, Janet Nestlerode, Paige Ross, Jr. and Alan Birch

The influence of reef architecture and scale on success of oyster reef restoration 422

Jennifer Miigg Pielros and Michael A. Rice

Effects of Crassostrea rirglntca populations on sedimentation, phytoplankton species composition and ammonia

cycling in experimental mesocosms 423

Thomas M. Soniat

A modified habitat suitability index for the eastern oyster. Crassastrea virginicu 423

S. Gregory Tolley, Aswani K. Volety, Emily C. Lindland and James T. Winstead

Use of oyster habitat by reef-resident fishes and decapod crustaceans in the Caloosahatchee Estuary, Florida 423

Aswani K. Volety, S. Gregory Tolley and James T. Winstead

Effects of freshwater releases and season on oysters {Crassostrea virginica) in Caloosahatchee Estuary. Florida 424

James T. Winstead. Aswani K. Volety and S. Greg Tolley

Parasitic and symbiotic fauna inhabiting oysters (Crassostrea virginicci] and mud crabs {Panopeus hcrhstii) sampled

from Caloosahatchee Estuary. Florida 424

POSTERS

Patrick Baker and Amy Benson

Habitat and ecology of green mussels, Perna viridis. in Florida 424

Shirley Baker, Ed Phlips, David Heuberger, Clay Montague and Leslie Stunner

Introducing the CLAMMRS project: clam lease assessment, management and modeling using remote sensing 425

Andrea Battison, Barbara Horney. Richard Cawthorn and Allan Mackenzie

Hemocytes of Homarus americanus stained with a modified Wright-Giemsa stain: description and comparison to

current classification schemes 425

Karine Bouilly, Alexandra Leitao, Helen McCombie and Sylvie Lapegue

Impact of atrazine on aneuploidy in the Pacific oyster, Crassostrea gigas 425

Francois Bourque, Bruno Myrand and Marcel Roussy

Status of the Mytiliis edtdis stock which supplies high-quality spat to mussel growers in lles-de-Ia-Madeleine (Gulf of

St. Lawrence ) 425

Jason P. Breves and .Andrew E. Tate

Predator induced phenotypic response by the common periwinkle (Littoriiia littorea) 426

Sandra M. Casas, Antonio Villalba, Jerome F. La Peyre, Kimberly S. Reece and Carlos Azevedo

Continuous in viiro culture of Pcrkiiisiis atlanticus, parasite of the carpet shell clam Tapes decitssatiis 426

Lise Chevarie, Bruno Myrand, Francois Bourque, Michel Giguere, Lizon Provencher, Philippe Archambault and
Rejean Tremblay

A R&D program to develop Mya arenaria culture in Iles-de-la-Madeleine (Gulf of St. Lawrence) 426

Vania R. Coelho and Jeffrey D. Shields

Loss of eye pigmentation in two gammaridean amphipods from the biosphere 427

Tricia L. Cranmer, Daniela Zima and Richard R. Alexander

Shell repair rates in surgically damaged valves of the blue mussel {Mytiliis edulis) and the ribbed mussel (Geiikensia

demissa) from New Jersey 427

Alan J. Erskine and Standish K. Allen, Jr.

Over-w intering of Crassostrea ariakensis in land-based systems in Virginia 427

Chwan-Hong Foo and Jerome F. La Peyre

IiTadiation of oyster primary cell cultures with ultraviolet-light to eliminate bacterial and protozoal contaminants 428

B. Gagnaire, H. Thomas-Guyon and T. Renauh

In vitro effects of heavy metals and atrazine on Pacific oyster, Crassostrea gigas, haemocytes 428

Holly A. Gefroh, Matthew J. Jenny, Ryan B. Carnegie, Kevin L. Schey and Robert W. Chapman

Inducible antibacterial activity in oyster {Crasso.strea virginica) hemolymph 428

368 Ahsnvcls. April 2002 National Shellfisheries Association. Mystic. Connecticut

Jennifer Greene. Raymond Grizzle, David Burdick, Larry Ward and Ann Reid

Constructing shellfish reefs in a polluted, urban estuary: scientists join with the community to work for a

common goal -+29

Rashel V. Grindberg. Erin C. Rasnake, Michael Savarese and Aswani K. Volety

The eastei-n oyster as an indicator species to establish restoration targets in southwest Florida estuaries 429

Raymond Grizzle, Jamie Adams and Linda Wallers

Long-term changes in intertidal oyster reefs in a Florida lagoon potentially caused by boating activities: an analysis of

aenal photographs from 1 943-2(100 430

Raymond Grizzle, Jennifer Greene, Stephen Jones, Mark Luckenbach and Roger Mann

An oyster (Crassosirea \ir)>iiuca) reef restoration experiment m Nev\ Hampshire inxolving CROSBreed stock and

native transplants 430

William J. Hargis, Jr.. Helen E. Woods, Rebecca Arenson, Sharon Dewing, Arman Kaltayev, Elizabeth Mountz,
Marcia R. Berman and Dexter S. Haven

What certain 19"' and early 20"' century navigational and species purpose survey charts reveal about changes in the

oyster reef morphology of the lower Chesapeake Bay 430

William J. Hargis, Jr., Helen E. Woods, Rebecca Arenson, Elizabeth Mountz, Marcia R. Berman and Sharon Dewing

Morphology of a Chesapeake Bay oyster reef system in 1871-1S73 431

Cindi A. Hoover and Patrick M. Gaffney

Geographic variation in nuclear genes of the eastern oyster Cmssostrea viri^inicu 431

Nicola J. Kernaghan, Eileen Monck, Carlo Wieser and Timothy S. Gross

Characterization and manipulation of sex steroids and \ itellogenin in freshwater mussels 43 1

Edouard Kraffe. Philippe Soudant. Yanic Marty. Nelly Kervarec and Pierre Guenot

A specific association of docosahexaenoic fatty acid with cardiolipins of some marine bivalves 431

Brenda S. Kraus. Amy E. Beaven and Robert S. Anderson

Hemocyte-mediated defense responses of the lobster Hoiminis amciicaniis 432

Megan K. G. La Peyre. Amy D. Nickens and Jerome F. Im Peyre

Potential elimination of the protozoan pathogen Perkiusus luaiinus from eastern oysters by freshet events 432

Jean Lavallee and Donald J. Rainnie

A field investigation of the effects of V-notching on the health and susceptibility to infection of ovigerous female

American lobsters 432

Li Li and Ximing Gito

A preliminary linkage map for the Pacific oyster Cmssostrea aiU'^i^' consti-ucted with RAPD and AFLP markers 433

Karin B. Lohrmann. Elisabeth von Brand and Cristian Gallardo

Gonadal maturation of triploid \cMo\-\s Aiiiopccteii jniipitniuis Lamarck. 1819 433

Bortolini R. Jose Luis. Torres G. M. Pilar and Montoya R. Leobardo

Detection of white spot syndrome virus (WSSV) in carcinologic faun associated to shrimp culture of Sinaloa. Mexico.

using Polymerase Chain Reaction (PCR) and in situ hybridization 433

Gregory MacCallum, Jeffrey Davidson. Garth Arsenault. Sharon McGladdery, Michelle Maillet and Neil MacNair

Factors, risks and significance of emergent neoplasia diseases in cultured and wild soft-shell clams {Mya areiuiha) in

Atlantic Canada 434

Clyde L. MacKenzie. Jr.

Identity of United States mollusk production declines in the 1900s 434

Aaron P. Malay, Katherine J. Boettcher and Bruce J. Barber

Development of a PCR-based assay for detection of the JOD-associated Roseohacter 434

Nassrin Mashaii

Preliminary study about feeding ecology of the rock lobster. Paindinis hoinanis. Linnaeus. 1785, at Iranian seashores

of Oman Sea 434

Sean McDermott, David Burdick, Raymond Grizzle and Jennifer Greene

Evaluation of the structure and function of a created blue mussel (Mytihis ediilis ) reef 435

Coren A. Milbury and Patrick M. Gaffney

Genetic monitoring of oyster stock enhancement in the Choptank River. Chesapeake Bay 435

National Shellfisheries Association, Mystic. Connecticut Abstracts. April 2002 369

Daniel P. Molloy, Denise A. Mayer, Michael J. Gaylo, Kathleen T. Presti, Alexander Y. Karatayev and
Lyiibov E. Burlakova

Progress in the hiological control of zebra mussels with microbial toxin 435

Thomas E. Mullen, Jr. and Salvatore Frasca, Jr.

Characterization of a parasitic amoeba in the American lobster by molecular systematics 436

Kim-Lien T. Nguyen, Jerome F. La Peyre and Terrence R. Tiersch

Seasonal changes in cell proliferation of oyster tissues 436

Amy D. Nickens, Jerome F. La Peyre and Sandra M. Casas

Development of a medium to induce hypnospore formation and zoosporulation of Pcrkiiisiis maritius 436

Rodney Peters and David Raftos

The effects of Maitcilia sydneyi on the host defense responses of the Sydney rock oyster. Saccostrea gloinerata 437

Alan J. Power, Mary Sweeney-Reeves, Todd C. Recicar, Dodie M. Thompson and Randal L. Walker

Population biology of melongenid whelks in the intertidal /one in Wassaw Sound, Georgia 437

Deanna L. Prince, Robert Bayer. Christina Congleton, Shannon Colby, Danielle La Vine, Danielle Volmulh,
Katrina Brooks, Margaret Berry, William Congleton and John Vetelino

A rapid method for assessing stress in the American lobster using a hand held glucometer 437

Michael Sheppard, Florian Rambow, Marc E. Frischer and Richard F. Lee

Routes of Heiinitihliiuiiin sp. transmission into blue crabs 438

Shelly M. L. Tallack

The relationship between black spot disease and limb-loss in Cancer pugunis from the Shetland Islands. Scotland 438

Anne Thessen, Q. Dortch, T. M. Soniat and G. J. Doucette

Oyster grazing on toxic and non-toxic PscnJo-nilzschia and Thiilassisinra wcisfhiggii. and Diryhiiii lirighlwclUi 438

Jessica M. Vanisko, Mary Christman, Kennedy T. Paynter and Stephen J. Jordan

Historic eastern oyster (Crassosirea virginica) dredge and patent long surveys in the Maryland portion of the

Chesapeake Bay 439

Anna Walker. Michael Sheppard. Richard F. Lee and Marc Frischer

Heinatodiniuin infection in blue crabs, spider crabs and stone crabs 439

Linda Walters, Kevin Johnson, Lisa M. Wall, Neysa Martinez and Ray Grizzle

Shell movement and juvenile survival of the oyster Crassnstrea virginica on intertidal reefs adjacent to waters with

intense boating activity in the Indian River Lagoon. Florida 439

Win Watson, Steve Jury, Jennifer Wishinski, Dan O'Grady, Walter Golet, Darren Scopel, Heidi Pye and Chris Rillahan

Development and testing of a simple field system for monitoring mussel shell gape size 439

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18, 2002 371

PERKINSUS

ENVIRONMENTAL EFFECTS ON PERKINSUS MARINUS
INFECTION RATES. GROWTH AND SURVIVAL AMONG
DERMO-DISEASE-FREE JUVENILE OYSTERS PLANTED
AT THREE SALINITY REGIMES IN THE PATUXENT
RIVER, MARYLAND. George R. Abbe* and Brian W. Al-
bright, Academy of Natural Sciences Estuarine Research Center.
St. Leonard. MD 206S.'S; Carol B. McCollough. Christopher F.
Dungan. and Stephen J. Jordan. Sarbanes Cooperative Oxford
Laboratory, Oxford, MD 21654.

Specific pathogen-free {SPFl oysters were set on oyster shell
and transplanted to three sites in the Patuxent River, Maryland
along a salinity gradient to investigate environmental effects of
Perkinsus mariiuis on infection rates, growth and survival. Oysters
were held in trays on PVC structures 0. 1 m off bottom on natural
oyster bars. Several thousand spat were deployed at each site, and
100 oysters in a separate tray were followed for growth and mor-
tality. From September 2000 to September 2001 salinities at Hol-
land Point (HP, upper river). Gatton (GAT, mid) and Town Creek
(TC, lower) averaged 11.1, 1.3.0 and 14.4. respectively. Oysters
were examined monthly for growth and mortality and 30 were
collected from each site for assay of P. iiuiriniis infections by
v\hole body burden technique. This allowed determination of time
to initial infection and subsequent progression of disease. An ad-
ditional 30 from the natural population at each site were also
examined monthly by rectal tissue assay. Oysters (initially 25 mm)
at HP, GAT and TC grew 23, 34 and 27 mm, respectively, and
survival was 95, 98 and 949f during the first 12 months. Mean
intensity of dermo disease among feral populations (on a scale of
0-7) at HP, GAT and TC ranged from 1 . 1 to 4.2. 0.7 to 4.6 and 0.7
to 4.7, respectively, and averaged 2.51, 2.72 and 2.79. It appears
that salinity had little effect on growth, survival and infection
intensity during the first year; however, it is generally later that
damage occurs. Preliminary data suggest tlial a mid-river site
might be the best area to locate oysters, but additional data during
subsequent years may dictate otherwise.

TRANSPORT OF PARTICLES ACROSS EPITHELIA
FROM OYSTER MANTLE CAVITY: A MODEL FOR PER-
KINSUS MARINUS INVASION. Bassem Allam and Susan E.
Ford.* Haskin Shellfish Research Laboratory. Rutgers University.
Port Norris, NJ 08349.

The digestive tract has long been considered the major portal of
entry for the oyster parasite, Perkinsus marimis. but recent studies
suggest that the mantle and gill may also be import invasion sites.
We used 5-|a.ni latex beads, placed in the mantle cavity, to deter-
mine whether P. marinua could be actively transported across
mantle and gill epithelia. At intervals after incubation, oysters

were processed for histological and cytological analysis. After 2 h,
beads were observed in hemocytes at the epithelial surface and
within the epithelium of the gill and mantle. After 6 to 16 h. beads
were found within hemocytes in the underlying tissues. Sixteen
hours following contact, only about 1% of hemocytes withdrawn
from the adductor muscle contained beads, although most of those
that did contained multiple beads. Over the next 96 h, the fraction
of hemocytes with beads gradually declined. Meanwhile, tissue
sections showed that beads were progressively transferred toward
the digestive tract. These results emphasize the role of mantle and
gill epithelia as portals of entry for P. iminiuis and other micro-
organisms, and suggest a role for hemocytes in their transport.

EFFECTS OF OYSTER EMERSION ON THE GROWTH
AND THE METABOLISM OF PERKINSUS MARINUS.
Louis E. Burnett* and Christopher S. Milardo. Grice Marine
Laboratory, University of Charleston, SC, 205 Fort Johnson,
Charleston, SC 29412

We examined the effects of pH, oxygen, carbon dioxide, and
temperature on the metabolism of the oyster parasite Perkinsus
nuiriniis simulating conditions that occur within the tissues of the
Eastern oyster Crassoslrea virginica during air exposure in the
summer. P. inarimis was cultured in a modified JL-ODRP medium
in ambient air at high humidity. The culture medium was gassed
with appropriate CO,. N, and O, mixtures, and pH adjusted in
order to simulate the conditions present in oyster hemolymph.
Oxygen uptake of the parasite decreased proportionately with de-
creasing ambient oxygen, and decreasing pH increased P. muriiuis
respiration under low CO, conditions (p = 0.0006); under 15 torr
CO,, there was no relationship between pH and oxygen uptake.
Increased CO, levels (simulating conditions during oyster air ex-
posure) significantly increased parasite O, uptake (p < 0.0001 ). as
did increased temperature (p < 0.0001). Cycling temperature (at
pH 6.3, 7.1 and 7.6) in 6-hour intervals from 25°C-35°C did not
affect culture growth; however, when cycled from 25°C— 10°C,
cells showed a significant decrease in growth (p < O.OOOl ). Simi-
larly, cells grown at 35''C and 15 torr ambient CO, showed de-
creased growth. These results suggest that summer environmental
conditions in the intertidal zone and especially in the tissues of
oysters play a role in keeping P. muriniis infections of C. virginica
at sub-lethal levels (SC Sea Grant R/ER-14).

SUSCEPTIBILITY OF THE CARIBBEAN OYSTER CRAS-
SOSTREA RHIZOPHORAE TO PERKINSUS MARINUS.
David Bushek.* Baruch Marine Field Laboratory. PO Box 1630.
University of South Carolina, Georgetown. SC 29442; John
Scarpa and Susan E. Laramore. Harbor Branch Oceanographic
Institution. Ft. Pierce, FL 34946.

The oyster pathogen Perkinsus marinus is a formidable prob-
lem for the American oyster, Crassostrea virginica. The Caribbean
oyster C. rliizopliorae is a closely related species of C. virginica.

372 Ahsmicfs. 2002 Annual Meeting. April 1 4- IS. 2002

Nutidnal .Shellfisheries Association. Mvstic. Connecticut

but little is known of its resistance to P. marinus (Dermo). There-
fore. Dermo resistance was compared between the Caribbean and
American oyster.

Two Dernio-free families were produced and reared separately,
under quarantine, for each species. Oysters from each family were
challenged once via shell-cavity inoculations with either saline
(control) or one of two genetically distinct isolates of P. nniriinis:
ATCC 50762 from Fort Pierce. FL or ATCC 30783 from Cotuit
Bay. MA. Parasite dosages were adjusted to I x \(y P. imiriniis per
gram of wet tissue weight for each oyster. Oysters were fed phy-
toplankton twice a day and water was exchanged weekly for 16
wk. A natural infection study was also performed by placing oys-
ters from each family in concrete tanks that received 7-10 L/min
unfiltered seawater from the Indian River Lagoon and examining
the oysters for infection over the course of 214 days. Infections in
each experiment were determined using the whole-body burden
method.

Fifteen America oysters and one Caribbean oyster died during
the challenge experiment. Log 10 transformed P. mariiiiis burdens
in surviving oysters were significantly heavier for Caribbean oys-
ters (1.25 ± 0.08) compared to American oysters (0.72 ± 0.06).
Control oysters did not develop infections (0.09 ± 0.01). In the
natural infection study, parasites were detected in all groups after
103 days, but intensities were negligible and there was no differ-
ence in burdens between the two species. At the end of the study
(day 214), most oysters had died (survival: 0 to 277f ). Peikinsiis
prevalence was 100% in all surviving C. \iii<inica. but lO'^'r of the
C. rhiziiphonie had escaped infection. There was no significant
difference between species, however, in body burdens of survivors
at the end of the natural exposure experiment. Results from the two
studies indicate that Caribbean oysters are as susceptible to Noith
American isolates of Perkiiisiis. but may be somewhat more tol-
erant of heavier parasite loads.

DOES THE UNIQUE FATTY ACID SYNTHETIC CAPA-
BILITY OF PERKINSUS MARINUS HAVE IMPLICATION
FOR VIRULENCE? Fu-Lin E. Chu,* Eric Lund, and Ellen
Harvey, Virginia Institute of Marine Science, the College of Wil-
liam and Mary, Gloucester Point, VA 23062; Philippe Soudant,
Universite de Bretagne Occidentale, Place Nicolas Copernic.
29280 Plouz.ane, France.

The fatty acid synthetic capability in all the studied parasitic
protozoans are limited. Perkiiisus nuiriiuis distinguishes itself from
all of the other studied parasitic protozoans by its ability to syn-
thesize a wide range of saturated and unsaturated fatty acids. Most
interestingly. P. marinus utilizes a two carbon substrate ( "C-
acetate) to synthesize the essential fatty acid, arachidonic acid
(AA). The ability of P. inaiiniis to synthesize AA is novel. No
parasitic protozoan has been reported to be capable of synthesizing
A A de novo. There is building evidence in the mammalian system
that excess AA-derived eicosanoids (powerful intercellular signal-

ing molecules) may be harmful to host defenses. Synthesis of AA
increased dramatically from day I to day 3 in cultured P. nnuiniis
meronts. It has been suggested that AA metabolites may poten-
tially affect the cellular immune function and the intlammatory
response to infection. This research was funded by Metabolic Bio-
chemistry Program, National Science Foundati<in (MCBy728284).

EFFECT OF PERKINSUS MARINUS ON PHENOLOXI-
DASE ACTIVITY IN BIVALVE HEMOLYMPH. Lewis E.
Deaton* and Percy J. Jordan. Biology Department, University of
Louisiana at Lafayette, Lafayette, LA 70504.

Phenoloxidase has been implicated as a component of host
defense in a variety of invertebrates, including molluscs. The en-
zyme is found in the hemolymph and hemocytes of bivalves. Per-
kiiisus luariiuis is a protist that causes disease in oysters, but does
not infect G. cleinissci. We collected hemolymph from the mussel
Ceukeiisia ileiiiissa and the oyster Crcissihsirea rirginica and mea-
sured the phenoloxidase activity in the blood with a colorimetric
assay. Perkinsus marinus was added to aliquots of hemolymph and
the phenolo.xidase activity compared to that in hemolymph not
exposed to P. marinus at 1,2, 4. and 6 hrs after addition of the
protist. After 2 hours of incubation, the phenoloxidase acti\ity in
both G. demissa and C. virginica hemolymph mixed w ith P. mari-
nus was significantly lowered. These results suggest that P. mari-
nus may suppress some components of the host defense mecha-
nisms of bivalves.

EPIZOOTIC DISEASES IN CHESAPEAKE BAY CLAMS.
Christopher F. Dungan,* Rosalee M. Hamilton, and Carol B.
McCollough. Cooperative Oxford Laboratory. Oxford. MD
21654; Kiniherly S. Reece and Karen L. Hudson, Virginia In-
stitute of Marine Science, Gloucester Point. VA 23062.

Chesapeake Bay commercial clam landings have consistently
declined to less than 2'7r of peak harvests during the past ten years,
prompting Maryland DNR resource assessment surveys that in-
cluded disease diagnostics. During 2000, ten sampled commercial
clam populations (8 Mya arenaria softshell clams and 2 Tagelus
pleheiiis razor clams) all showed Perkinsus sp. infections at preva-
lences of 30-1009;^. Seven of eight sampled M. arenaria popula-
tions also showed disseminated neoplasia (DN) prevalences of
3-37%, but neither sympatric T. plelvius sample was affected by
DN disease. Depleted and diseased softshell clam populations,
especially Eastern Bay stocks affected by both diseases, were pro-
jected to suffer heavy disease mortalities. Although both mesoha-
line commercial clam species were reported as Perkinsus nuirinus
hosts in 1954. Perkinsus sp. infections in Maryland M. arenaria
populations were not detected histologically in routine survey
samples until 1990, the same year in which the current decline in
landings began. Axenic in vitro Perkinsus sp. isolates were readily
acquired from both clam hosts sampled during 2000. and from

National Shellfisheries Association. Mvstic, Connecticut

Abstracts. 2002 Annual Meeting. April 14-1 R. 2002 373

three additional clam species (Macoma halthica. Mulinia lateralis.
Rangia ciineata) sampled during 2001. In vitro cell cycle and
genetic characteristics of Perkinsiis sp. isolates from Chesapeake
Bay clams differ from those of P. luarhtus isolates from sympatric
oyster hosts.

EVALUATION OF PHYSIOLOGICAL CONDITION IN
DERMO RESISTANT OYSTERS. Vincent G. Enconiio,*
Shawn Stickler, and Fu-Lin Chu, Virginia Institute of Marine
Science School of Marine Sciences College of William and Mary
Gloucester Point. VA 23062.

Distinct oyster stocks (FI progeny) were grown in the field to
compare resistance to Dermo. After two years, significant differ-
ences in mortality and growth were observed. To compare the
effects of parasitic stress (Perkinsiis marinus) on physiological
condition and energy reserves between oyster stocks, energy re-
serves (glycogen, lipids, and total protein) were quantified and
evaluated for their contribution to overall physiological condition.
Biochemical indices were correlated with changes in shell height,
condition index, and P. marinus infection. Total glycogen and
protein contents increased with shell height. Results also indicate
seasonal variation in condition index, which was highest in winter
and decreased during the summer when infection levels were the
highest. Glycogen also exhibited a similar trend. These trends were
consistent between sites and over all oyster stocks, but variation
between stocks was not significant, despite differences in infection
intensities and mortalities. Although condition indices decreased
as infections increased, data were not strongly coiTelated. This
project was supported by ODRP. NOAA (Virginia Sea Grant #
VA-OD-99-3).

EVALUATION OF ANTIMICROBIAL PEPTIDES FOR RE-
COMBINANT FEED-BASED DELIVERY IN SHELLFISH
AQUACULTURE. Tarquin Dorrington,* Graduate School of
Oceanography, University of Rhode Island, Narragansett, RI
02882; Marta Gomez-Chiarri. Department of Fisheries, Animal
and Veterinary Sciences, University of Rhode Island, Kingston. RI
02881; Lenore Martin, Department of Cellular and Molecular
Biology, University of Rhode Island. Kingston. RI 02881.

The long-tenn goal of this research is to develop recombinant
strains of microalgae expressing antimicrobial peptides (AMPs)
that could be fed to bivalves for treatment of infectious diseases.
We screened for candidate AMPs that are toxic against marine
pathogens without inhibiting the growth of candidate hosts for
eukaryotic expression. Pleurocidin (from the winter flounder Plcii-
ronecles americaniis) and lachyplesin (from the horseshoe crab
Tachypleits tridentatiis) were active against Vibrio spp. in the
range of salinity. pH and protease concentrations found in the
digestive system of oysters (Minimum Inhibitory Concentration
MIC50 at 24 hours 6.25-25 (jlM). Tachyplesin and pleurocidin had

no effect on the growth of Perkinsiis marinus. The yeast strains
Pichia pastoris and Saccharomyces cerevisiae were resistant to
tachyplesin and pleurocidin activity (MIC;^,, > 200 ij,M), whereas
three algal strains (Skeletonema costatiim. Chlamydomoiuis rein-
hardtii and C. Pulsatilla] were sensitive (MIC,,, < 50 (jlM). While
proteases from P. marinus significantly reduced the antimicrobial
activity of pleurocidin. they did not affect the activity of tac-
hyplesin. These results are consistent with the finding that the
substrate specificity of Perkiiisus proteases is similar to that of
elastase. Oyster clearance rates of C. reinhardtii. C. Pulsatilla, and
P. pastoris were similar to those of Tetraselmis hicia. a commer-
cially available alga used in aquaculture.

FLOW CYTOMETRIC ANALYSIS OF LECTIN BINDING
TO IN VITRO CULTURED PERKINSUS MARINUS SUR-
FACE CARBOHYDRATES. Julie D. Gauthier,* Biology De
partment. Nicholls State University, Thibodaux, LA 70310; Jer-
ome F. La Peyre, Cooperative Aquatic Animal Health Research
Program, Department of Veterinary Science. Louisiana State Uni-
versity Agricultural Center, Baton Rouge, LA 70803; Jill A. Jen-
liins. National Wetlands Research Center, U.S. Geological Survey,
Lafayette, LA 70506.

Parasite surface glycoconjugates are frequently involved in cel-
lular recognition and colonization of host. The present study iden-
tifies surface carbohydrates by flow cytometric analysis of fluo-
rescein isothiocyanate-conjugated lectin binding. Lectin binding
specificity was confirmed by sugar inhibition and Kolmogorov-
Smirnov statistics. Clear, measurable separation between fluores-
cence peaks and no parasite autotluorescence were observed. Para-
sites (GTLA-5 & P-l strains) harvested at log phase growth from
a protein-free medium reacted strongly with wheat germ agglutinin
(WGA) and concanavalin A (Con A), reflecting presence of N-
acetyl-D-glucosamine and glucose/mannose moieties, respec-
tively. Both strains also bound, although with lower intensity.
Madura pomifera agglutinin (MPA) and Baiiliinia purpurea ag-
glutinin (BPA) (N-acetyl-D-galactosamine specific lectins), peanut
agglutinin (PNA) (terminal galactose specific), and Griffhnia sim-
plivifolia II (GSII) (N-acetyl-D-glucosamine specific). Back-
ground fluorescence levels were detected for Ulex europaeus ag-
glutinin I (L-fucose specific) and Limulus polyplwmus agglutinin
(sialic acid specific). The order of lectin binding intensity differed
between strains: GTLA-5 reacted with MPA>GSII>PNA>BPA,
whereas P-l reacted with PNA>MPA>BPA>GSII. which may re-
sult from differential expression of glycoconjugates throughout log
phase growth. Fluorescence microscopy revealed that PNA bound
with high intensity primarily to schizonts (dividing cells), whereas
the other lectins bound with approximately equal intensity among
parasite growth stages. Future efforts will determine if P. marinus
rapid recognition and internalization involves a specific lectin-
carbohydrate interaction.

374 Ahslnicl.s. 2002 Aiiiuuil Meeting. April 14-1 S. 2(102

National Shellfisheries Association. Mystic. Connecticut

CLIMATE VARIABILITY AND DERMO DISEASE IN
CHESAPEAKE BAY. Eiletn E. Hofniann* and John M.
Klinck. Center for Coastal Physical Oceanography. Old Dominion
Llniversity. Norfolk. VA 23529; Eric N. Powell and Susan E.

P'ord. Haskin Shellfish Research Laboratory. Rutgers University.
Port Norris. NJ 08349: Stephen Jordan, Sarbanes Cooperative
Oxford Laboratory, 904 South Morris Street. Oxford. MD 21654;
Eugene Burreson. Virginia Institute of Marine Science. P.O. Box
1346. College of William and Mary. Gloucester Point. VA 23062.
A mathematical model that includes biological and environ-
mental processes that contribute to the observed annual cycles of
intensity and prevalence of the disease. Dermo. has been used to
determine the effect of predicted climate variations on Eastern
oyster (Crassosin-ii virginica) populations in Chesapeake Bay.
Simulations that use the salinity change predicted for Chesapeake
Bay by the Hadley Center Climate model for conditions of doubled
CO, suggest that the present pattern of Dermo disease in the Bay
will be altered to the benefit of the oysters. The predicted salinity
change in Chesapeake Bay for conditions of doubled CO, pro-
duces an overall decrease in Bay salinity, which results in a de-
crease in the prevalence and intensity of Dermo disease. However,
for reduced salinity conditions, there is a trade-off between re-
duced disease levels and reduced reproductive capacity of the oys-
ters. Also, conditions of increased food can offset the effects of
either increased or decreased salinity on Dermo disease levels. The
simulated total oyster biomass changes suggest that for conditions
of doubled CO,, northern Chesapeake Bay oyster populations may
not be viable over the long term without external inputs of juve-
niles. However, oyster populations in the Rappahannock and York
Rivers increase or have stable biomass over time for most climate
change conditions. One implication of these results is that oyster
populations in southern Chesapeake Bay may sustain the Bay-wide
oyster fishery. These simulations provide a basis for suggesting
management plans for diseased oyster populations under condi-
tions of climate change.

MODULATION OF PERKINSUS MARINUS FUNCTIONS
BY HOST-DERIVED PRODUCTS. Stephen L. Kaattari,* E.
Alanna Maclntyre, and Christopher G. Earnhart, Department
of Environmental Sciences. Virginia Institute of Marine Science.
College of William and Mary. Gloucester Point, VA 23062.

In an attempt to induce physiological changes in vitro that
would be comparable to those elicited by natural infection, we
have co-incubated P. mariiuts cells with extracts of Crassostrea
spp tissues and plasma. Co-incubation with these host-derived ma-
terials gave rise to a variety of complex effects including altered
differentiation, protease expression, growth rates, infectivity. and
in vitro parasite mortality. Preliminary evidence is suggestive that
a suite of such functions could be employed as a prognostic tool
for the selection of resistant oyster stocks. However, variations in
the relative activity of these materials suggest that preparative

procedures and/or conditions of storage could confound such
analyses. Thus, while indicative of prognostic value, the precise
mechanisms of these biological effects must be resolved.

VIABILITY AND GROWTH OF PERKINSUS MARINUS
AND PERKINSUS ATI^NTICUS AT THREE TEMPERA-
TURES. Jerome F. La Peyre* and Amy D. Nickcns, Coopera-
tive Aquatic Animal Health Research Program. Department of
Veterinary Science. Louisiana State University Agricultural Cen-
ter. Baton Rouge. LA 70803; Sandra M. Casas and Antctnio
Villalba, Centro de Investigacions Marinas. Xunta de Galicia.
aptdo. 13. E-36620 Vilanova de Arousa. Spain.

The ability to culture several protozoan parasites of the genus
Perkinsus allows comparison of their growth and tolerance given
different environmental conditions. This information is important
to understand the distribution ofthe.se molluscan parasites and can
be used to predict zones for high risk of mortalities of host popu-
lations. The effects of temperature on P. marinus and P. atlanticus
were determined because temperature is a major factor controlling
the prevalence and intensity of infection in their respective host,
the eastern oyster. Crassostrea virginica, and the carpet shell clam.
Tapes decussatus. Three isolates of each species were added to
96-well plates at a seeding density of 10'^ cells/ml and incubated at
4°C, 15°C and 28°C for 15 days. The parasite density, viability.
size and metabolic activity were measured every other day starting
on day 2. Parasite density was measured with a hemacytometer.
Parasite viability was determined by neutral red uptake. Parasite
metabolic activity was assessed by measuring the cellular biore-
duction of the fluorescent dye Alamar Blue. The mo.st significant
result was that all three isolates of P. atlanticus multiplied at I5°C
whereas no increase in parasite number of any P. marinus isolates
was detected at LS'^C. The growth rate of P. marinus and P. at-
lanticus was comparable at 28"C and neither species grew at 4''C.
While perkinsosis is generally considered a warm water disease.
this study suggests there will be differences in the temperature at
which different Perkinsus species kill their respective hosts.

IS THE TEMPERATURE AND SALINITY-DEPENDENT
VIRULENCE OF PERKINSUS I^ARINUS ASSOCIATED
WITH INCREASED LIPID METABOLISM? Eric D. Lund*
and Fu-Lin E. Chu. Virginia Institute of Marine Science. College
of William and Mary. Gloucester Point. VA 23062; Philippe
Soudant, Universite de Bretagne Occidentals Place Nicolas Co-
pernic. 29280 Plouzane. France.

Teinperature and salinity significantly affect the rate of prolif-
eration and development of Perkinsus marinus and the progression
of the disease in its host, the eastern oyster. To better understand
how these environmental parameters affect the nutritional metabo-
lism of this parasite, the effects of temperature and salinity on the
lipid metabolism of P. marinus were investigated. Using axenic

Nation:il Shellt'isheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting, April 1-1-1 8. 2002 375

cultures grown at 3 temperatures and 2 salinities, the uptake, in-
corporation and nielabolisni of two fluorescent lipid analogues in
meronts and the lipolytic activities of nieronts and extracellular
proteins (ECPs) were determined. Uptake and bioconversion of the
lipid analogues were positively coirelated to temperature. Salinity
did not affect the uptake, but bioconversion of these two compo-
nents was lower at low salinity. Triacylglycerol lipase activity of
cell homogenates and ECPs were positively correlated to tempera-
ture. Phospholipase activity was not detectable in ECP. This re-
search was supported by NSF (MCB9728284).

PERKINSUS MARINUS INFECTION RATES IN SPECIFIC-
PATHOGEN-FREE JUVENILE OYSTERS PLANTED AT
THREE SALINITY REGIMES IN THE PATUXENT RIVER,
MARYLAND. Carol B. McCollough.* Christopher F. Dungan,
and Stephen J. Jordan, Sarbanes Cooperatix e Oxford Laboratory,
Oxford, MD 21654; George R. Abbe and Brian W. Albright.
Academy of Natural Sciences Estuarine Research Center. St.
Leonard, MD 20685.

Specific-pathogen-free (SPF) seed oysters were set and reared
in filtered and sterilized Patuxent River water. They were trans-
ferred to three natural oyster bars in the Patuxent River along a
salinity gradient. Deployment sites were located at Town Creek
(TC, down river), Gatton (GA, mid-river), and Holland Point (HP,
upriver). Samples of 30 oysters were assayed at 2, 4. and 8 weeks
post-deployment for infection by Perkinsus mariniis. using an en-
hanced RFTM whole body burden technique. Assays continued at
4-week intervals until three consecutive samples from each area
tested positive for presence of P. mariuiis. Three discrete spat sets
and deployments were made: September 2000, June 2001, and
August 2001. Animals at sites TC and GA September 2000 de-
ployments acquired P. marinus infections within 2 weeks, with
prevalences of 10% and 3% respectively. Positive results at low
prevalences and intensities continued for 8 weeks. Sites TC and
GA June 2001 deployment acquiied infections within 2 weeks,
with 10% and 13% prevalences respectively, and positive results
continued for 8 weeks with increasing prevalences. Site HP ac-
quired one infection (3%) between 2 and 4 weeks. Positive results
continued through two additional 4-week sampling intervals, with
increasing prevalences. All August 2001 deployments acquired
infections within 2 weeks, with prevalences of 7% at TC, 87% at
GA, and 3% at HP, and positive results continued for 8 weeks.
Infection intensities among these samples ranged from 1-23 hyp-
nospores per host animal. The GA deployment was 100% infected
by 8 weeks post-deployment. These results show that juvenile
oysters acquire P. iiuiriiiiis infections as early as 2 weeks after
placeinent in dermo disease endemic areas, and that these infec-
tions persist in the planted populations over time.

PERKINSUS PREVALENCE IN OYSTERS PRODUCED IN
THE HATCHERY AND PLANTED IN THE FIELD.
Kennedy T. Paynter* and Tim Koles, Department of Biology.
University of Maryland. College Park. MD 20742; Don Meritt,
Horn Point Laboratory, University of Maryland Center of Envi-
ronmental Science, Cambridge, MD 21613.

Since 1995, millions of oyster spat have been produced by the
Horn Point oyster hatchery. Most of those oysters have been
planted on oyster bars in various locations in the Maryland portion
of Chesapeake Bay in association with oyster restoration effoils.
Oysters planted in most of these areas were tested for P. marinus
prior to being planted and then regularly tested, in some cases for
as many as five years, after planting. Salinity, temperature and
dissolved oxygen data were also collected at most sites. Data were
analyzed from all monitored sites planted with Pcrkinsiis-ixec spat
over the past 5 years. Analysis showed that very few oysters were
infected with P. marinus during their first year. In fact, very few
oysters were infected during their second and even third years. Of
the nearly 2,000 oysters collected in 2001 that were planted on
restoration sites in 1998, 1999 or 2000, only 44 were infected. In
addition, infections appear to be more related to local infection foci
than salinity. Infection rates in hatchery-produced oysters are
lower than expected compared to the naturally set oysters trans-
planted by the State as part of the repletion program.

RECENT TRENDS IN LEVELS OF INFECTION OF PER-
KINSUS MARINUS IN OYSTERS FROM GALVESTON
BAY, TEXAS: RESULTS OF THE DERMOWATCH MONI-
TORING PROGRAM. Sammy M. Ray,* Department of Marine
Biology. Texas A&M University, Galveston. TX 77553; Thomas
M. Soniat, Department of Biology, Nicholls State University. Thi-
bodaux. LA 70310; Enrique V. Kortright. Kortright Corporation,
102 Allendale Dr.. Thibodaux, LA 70301; Lance Robinson,
Texas Parks & Wildlife Department, 1018 Todville Rd.. Seabrook.
TX 77586.

Since December 1998 oysters in Galveston Bay have been
monitored for levels of the parasite Perkinsus marinus as part of
the Dermo Watch Program. Monthly sampling of six reefs (Red-
fish, Hannah's, Frenchy's. Fisher's, Confederate and April Fool)
and three leases provides good spatial coverage and adequate tem-
poral resolution for management purposes. Each month oysters are
collected, their length (L) measured, and checked for weighted
incidence ( WI) of infection; water temperature and salinity are also
determined. Initial WI, T, S, and L are used by a model embedded
in the DermoWatch web site (www.blueblee.com/dermo) to cal-
culate a time to critical level of disease (t-crit). The t-crit is the
number of days that it would take to reach a critical WI of 1.5
(assuming no change in T and S). Values of t-crit are low when T
and S are high. The effects of drought conditions during 1999 and
2000 were reflected in higher WI and lower t-crit values through-
out the Bay. Oyster growers and managers can use estimates of

376 Abstracts. 2002 Annual Meeting. April 14-18. 2002

National Shellfisheries Association. Mystic. Connecticut

t-crit to manage disease. For example, heavily infected populations
of oysters with t-crit values equal to or approaching zero can be
moved to lower salinity sites or harvested before they die of dis-
ease.

UTILIZATION OF MOLECULAR GENETIC DATA FOR
DETECTING. IDENTIFYING AND DESCRIBING PERKIN-
SUS SPECIES. Kimberly S. Reece,* Virginia Institute of Marine
Science. The College of William and Mary. Gloucester Point. VA
23062.

Morphological characters are unreliable for distinguishing
among Perkinsiis species. In addition, traditional detection meth-
ods for Perkinsiis species such as histological analyses or the
Ray's fluid thioglycollate assay do not differentiate among the
species. Therefore, Perkinsiis species designations are based
largely on differences in hosts and/or geographic ranges. In recent
years several species-specific molecular detection assays have
been developed targeting DNA sequence differences among de-
scribed species. DNA sequence data is also being used to support
and validate descriptions of new Perkinsiis species. Results from
several molecular genetic studies indicate that caution must be
employed, however, to ensure that adequate molecular data is
available to appropriately develop molecular diagno,stics and to
discriminate among species. Intra-, as well as inter-specific se-
quence variation should be examined to confidently target a par-
ticular nucleic acid sequence in molecular diagnostic assays or to
use DNA sequence data from a locus in phylogenetic analyses or
for taxonomic discrimination. Data will be presented suggesting
that genetic variation that was previously thought to represent
sequence differences between two Perkinsiis species is observed
within the genome of a single Perkinsiis species isolated from two
different hosts. In another study, a comprehensive examination of
sequence variation among Perkinsiis species within the internal
transcribed spacer region of the ribosomal RNA gene complex
supports designation of a new Perkinsiis species that is clo.sely
related to P. atlanticus and P. olseni. Currently, P. iitlanticiis and
P. olseni cannot be distinguished based on available molecular
sequence data suggesting that synonymization of these two species
may be warranted pending results of more extensive and complete
analyses.

PROTEASE ACTIVITY IN THE EASTERN OYSTER CRAS-
SOSTREA VIRGINICA AFTER EXPERIMENTAL INFEC-
TION WITH THE PROTOZOAN PARASITE PERKINSIIS
MARINUS. Pilar Muiioz Ruiz and Marta Gomez-Chiarri,* De-
partment of Fisheries, Animal and Veterinary .Sciences, University
of Rhode Island, Kingston, RI 02881.

Perkinsns mariniis. an endoparasitic protozoan parasite, has
long been recognized as a serious oy.ster pathogen that is often
blamed for widespread mortality of the Eastern oyster Crassotrca

virginica. In order to investigate the molecular interactions be-
tween P. mariniis and oyster hemocytes during early infection.
Perkinsiis-free Eastern oysters (Taylor Shellfish. Washington.
USA), were inoculated either in the mantle cavity or the aductor
muscle with cultured cells of P. mariniis. Protease and antimicro-
bial activity were measured in filtered supernatants of hemolymph
collected 4 hours. 24 hours. 4 days and 2 months after experimen-
tal infection. No antimicrobial activity was detected in hemolymph
supernatants of parasitized or control oysters. Protease activity in
hemolymph supernatants collected 4 h after experimental infection
was significantly higher in parasitized oysters than in control oys-
ters. There were no differences in secreted protease activity be-
tween control and parasitized oysters sampled 24 hours, 4 days and
2 months after experimental infection. Gelatin-gel electrophoresis
(zymography) analysis showed the presence of 5 high inolecular
weight bands (60-220 KDa) with protease activity in both control
and parasitized oysters. Large variations in both the amount of
protease activity and the relative abundance of each protease were
observed between individual oysters. The presence of low molecu-
lar weight proteases (35-50 KDa). corresponding to P. mariniis
serine proteases, was observed in hemolymph supernatants of a
small proportion of experimentally infected oysters.

"NATURAL DERMO RESISTANCE" IN EASTERN OYS-
TER STOCKS: CHESAPEAKE STUDIES AND DEFENSE-
RELATF:D ACTIVITIES. S. M. Sticliler.* V. G. Encomio.
S. K. Allen, Jr. and F-L. E. Chu, Virginia Institute of Marine
Science, College of William and Mary, Gloucester Point, VA
23062; J. F. La Peyre, Louisiana State University. Baton Rouge,
LA 70803.

The restoration of Eastern Oyster populations can be acceler-
ated, in part, by using stocks of oysters resistant to the parasitic
disease Dermo, caused by Perkinsiis mariniis. Similarly, identify-
ing correlated defense activities will expedite the breeding process
for disease resistance. We used putatively Demio-resistant wild
stocks from both the Gulf of Mexico and the Chesapeake Bay and
a disease-resistant hatchery strain, and compared the resistance of
their progeny to Dermo disease in a two-year common garden
experiment at two Dermo-enzootic Chesapeake Bay sites. Using a
modified body burden assay, we determined Perkinsiis infection
levels and Dermo-related mortality were considerably higher in
Chesapeake stocks than in Louisiana stocks. Chesapeake stocks
experienced wide variation in both disease infection levels and
mortality. These results indicate a genetic basis for disease resis-
tance, even within a single region. Monthly hernocyte counts and
hemocyte and serum potential for killing P. mariniis. and plasma
protein, lysozyme, and protease inhibition levels were obtained (or
correlation with Perkinsiis infection levels. This project was sup-
ported by ODRP. NCAA (Virginia Sea Grant # VA-OD-99-3).

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-1 «. 2002 377

THE BIOLOGY AND CONSERVATION OF
FRESHWATER MUSSELS

SPATIAL AND TEMPORAL ANALYSIS OF ECOSYSTEM
PROCESSING OF FRESHWATER MUSSELS IN TWO
HEADWATER STREAMS. Alan D. Christian, Department of

Zoology. Miami University, Oxford, OH 45056; David J. Berg,

Department of Zoology. Miami University. 1601 Peck Blvd..
Hamilton. OH 4501 1 ; B. Crump, Caddo Ranger District. Ouachita
National Forest, 912 Smokey Bear Lane, Glenwood. AR 71943.
The objectives were to determine food resources, food resource
nutrient composition, tissue nutrient composition and N and P
excretion rates of two species of mussels from two Little Darby
Creek. OH and two Ouachita River, AR headwater sites. Seston
stable isotope analysis revealed no differences in 8'^N values be-
tween sites within a streams or seasons, however, summer b"C
values were significantly different than spring and fall S'^'C and
8''^N values. No differences in mussel 8'''N values were observed
between sites within a stream, but were significantly different
seasonally and different regionally. Seston nutrient concentrations
were similar between sites within a stream and among seasons. No
significant differences in nutrient tissue concentrations were ob-
served between species within a stream, sites, or seasons. Excre-
tion rates of N and P were lowest in spring and highest in fall for
all species. Average N:P ratios were below 16:1 for some species
and above 16:1 for other species. Nutrient limitation experiments
may have indicated N and P limitation in the fall. Isotope values
were regionally different, locally similar, temporally variable and
suggest mussels utilize a subset of the seston, possibly bacteria.
Mussels may be providing an important ecosystem role by pro-
viding limiting nutrients to primary producers.

CONSERVATION ACTIVITIES FOR FRESHWATER
MUSSELS BY THE UNITED STATES FISH AND WILD-
LIFE SERVICE. Susan Rogers,* US Fish and Wildlife Service.
Conway. AK 72032; Richard Biggins, US Fish and Wildlife Ser-
vice. Asheville. NC 28801; Steven Ahlstedt. US Geological Sur-
vey, Knoxville. TN 37828.

The Fish and Wildlife Service (FWS) is active in freshwater
mussel conservation through several approaches: habitat restora-
tion, propagation and reintroduction of juveniles, collaboration
with partners, and conservation research. The FWS is involved in
conserving both federally listed and unlisted species. Before any
recovery program can be successful, however, habitat must be
restored and protected. A program such as that enacted in partner-
ship with The Nature Conservancy on the Clinch River can be
particularly successful in reaching this goal. Partnerships are inte-
gral to the FWS's freshwater mussel recovery programs. For ex-

ample, the Tennessee Valley Authority and the Army Corps of
Engineers also have worked with the FWS on mussel conservation
in relation to their water resource projects. In addition to habitat
restoration, propagation efforts have been ongoing for approxi-
mately 10-15 y. and endangered mussel populations throughout
the country are being augmented with propagated juveniles. Fi-
nally research is being conducted to determine the causes of the
bottleneck in survival that juvenile mussels experience around 60
d of age. Further information needs that would aid the FWS's
conservation efforts inckide quantification of the impacts of sedi-
mentation from dredging and navigation projects on mussel popu-
lations, and the impacts of pesticides on glochidial transformation
success.

DIET EVALUATION FOR THE FRESHWATER MUSSEL
ELLIPTIO COMPLANATA (BIVALVIA: UNIONIDAE). Wil-
liam F. Henley* and Richard J. Neves, Virginia Cooperative Fish
and Wildlife Research Unit. Department of Fisheries and Wildlife
Sciences. Virginia Tech. Blacksburg. VA 24061.

To determine the effects of holding conditions and different
algal diets on freshwater mussels, the tissues of adult eastern el-
liptio freshwater mussels. EUiptio coinpkmnta (56-106 mm) were
tested for physiological and gametogenic condition over 8 sam-
pling events. Treatments included a reference group from the
source population, the Nottoway River. Virginia (NR), and 3 cap-
tive treatments. Captive treatments were mussels fed Scencdesmus
quMtricaiuta (S). Neocliloris nleouhiindiins (N). and a no feed
treatment (NF). Patterns in levels of soluble protein, glycogen, and
percentages of moisture differed over the course of the experiment
(p < 0.01 ). Production of ripe and developing gametes did not
differ in the treatments (p = 0.22). but the NF mussels ceased
gametogenesis in spring. Carbohydrate levels of the N and S ani-
mals did not differ from those of the NF mussels (p = 0.99).
Soluble protein levels of the N and S mussels remained relatively
high throughout the experiment, those of the NF animals declined,
and those of the NR varied. Percent moisture in tissues of the
treatments differed over time (p < 0.001 ). Levels of total lipids in
the mantle tissues of the treatment groups, as well as muscle fiber
diameters in the foot, provided useful complimentary data for as-
sessing body condition.

378 Ah.stmct.s. 2002 Annual Meeting. April 14-18. 2002

National Shcllfisheries Association. Mystic. Connecticut

HIERARCHICAL ANALYSIS OF MTDNA VARIATION IN
AMBLEMA PLICATA, A WIDESPREAD MUSSEL SPECIES.
Curt L. Elderkin,* Dept. of Zoology, Miami University, Oxford,
OH 4.'S036: David J. Berg, Dept. of Zoology, Miami University,
Hamilton. OH 4.S01 1; Janice L. Metcalfe-Smith. National Water
Research Institute, Burlington, ON, Canada L7R 4A(i; Caryn C.
Vaughn, Dept. of Zoology and Oklahoma Biological Survey. Uni-
versity of Oklahoma. Norman, OK 73019; Alan D. Christian,
Dept. of Zoology. Miami University, O.xford, OH 4-^056; Sheldon
I. Guttman, Dept. of Zoology, Miami University, Oxford. OH
45056.

Knowledge of genetic structure of target species is essential for
the development of effective conservation plans. Amhiema plicata
is a common, widespread freshwater mussel species. Six or seven
individuals from six populations of Aiuhlcma plicata within the
Lake Erie and Ohio River drainages were analyzed by sequencing
restriction fragments from a 652-base portion of the mitochondrial
cytochrome oxidase I (COI) gene and a 530-base portion of the
nuclear internally transcribed spacer (ITS- 1 1 gene. Percent se-
quence divergence was determined and the results were analyzed
by calculating Tamura-Nei distances between individuals and then
constructing neighbor-joining trees from the distance matrix. Two
haplotypes were identified for the COI gene and all populations
shared these haplotypes. Greater diversity was found for the ITS- 1
gene, with a total of six haplotypes, although each population had
only one or two haplotypes. Cluster analysis revealed no real geo-
graphic structuring for either gene. These preliminary results are in
striking contrast to those we have found using allozymes. The
latter group of loci showed significant differentiation between the
Ohio River and Lake Erie basins. Further investigation is required
in order to explain this apparent contradiction.

GAMETOGENESIS, SPAWNING, BROODING AND
GLOCHIDIAL DISCHARGE IN MEGALONAIAS NERVOSA
(BIVALVIA: UNIONIDAE) FROM THE TENNESSEE
RIVER IN ALABAMA. Jeffrey T. Garner,* Alabama Division
of Wildlife and Freshwater Fisheries, 350 Co. Rd. 275. Florence,
AL 35633: Thomas M. Haggerty, Department of Biology, Uni-
versity of North Alabama, Florence, AL 35632, Rebekah L. Rog-
ers. Bradshaw High School. Florence, AL 35630.

The annual gametogenic cycle of Megalonuias iwrvasa was
monitored over a two-year period by observing temporal changes
in histological preparations of gonadal tissues. In male specimens,
temporal changes in cell numbers and relative abundance were
evaluated by counting different germ cell types along transects
through gonadal acini. Temporal changes in female specimens
were quantified by measuring diameters of oocytes and counting
the numbers of oocytes per acinus. The cycles observed in both
male and female M. nervosa differed from those observed in other
members of the Ambleminae. Little activity was observed through
most of the year, culminating in a Hurry of activity in late simimer

and early autiniin. In male specimens, acini filled with spermato-
zoa in July and August and all were released in September. In
female specimens, oocytes developed and grew quickly during the
period in which spermatogenesis occurred in males and all oocytes
were released to the marsupia over a short period, .showing a high
degree of spawning synchronicity with the males. Glochidia
quickly matured and were present in both inner and outer gill
demibranchs until December.

CLEARANCE RATE AND FILTRATION EFFICIENCY OF
ELLIPTIO COMPIANATA (BIVALVIA: UNIONIDAE) EX-
POSED TO DIFFERENT LABORATORY CONDITIONS.
Catherine M. Gatenby.* Daniel .A. Kreeger and Robyn Rein-
miller, Patrick Center for Environmental Research. Academy of
Natural Sciences, Philadelphia, PA 19103; Richard J. Neves, Vir-
ginal Cooperative Fish And Wildlife Research Unit, Department of
Fisheries and Wildlife Sciences, Virginia Tech, Blacksburg, VA
24061-0321.

As part of an ongoing research program improve laboratory
culture protocols for native freshwater mussels, we quantified and
compared feeding rates and particle size preferences of mussels.
Elliptio complaiuihi. that were exposed to different holding con-
ditions in the laboratory. Two culture parameters were examined:
the importance of a burrowing substrate (sand) and the effect of
food quality (natural seston vs. lab-cultured niicroalgae). A Multi-
Sizer was used to measure and compare clearance rates for the
whole diet (2-63 (xm diameter), specific size fractions of the diet
(e.g.. 2-3 p-m), and for various sized carboxylate microspheres. By
comparing clearance rates for different sized particles, we calcu-
lated their relative filtration efficiency, which tended to be greatest
for moderately large sized particles averaging 7-10 (xm in diam-
eter. Both clearance rate and filtration efficiency varied consider-
ably between sediment/no sediment conditions, and among dietary
treatments of different quality. These parameters were also moni-
tored for periods of up to 60 d to determine whether £. complanata
adjust either feeding rates or particle size preferences as an adap-
tive response to differences in physical (e.g. sediment) or nutri-
tional conditions.

VARIABILITY IN CONDITION INDEX AND TISSUE BIO-
CHEMISTRY OF ELLIPTIO COMPlJiNATA HELD IN THE
FIELD AND LABORATORY. Daniel A. Kreeger,* Catherine
M. Gatenby and Deborah Raksany, Patrick Center for Environ-
mental Research. Academy o\ Natural Sciences. Philadelphia, PA
19103.

Like then marine counterparts, native freshwater mussels can
be sufficiently abundant to affect key ecological functions (e.g.,
energy, biogeochemical cycles) and perform important ecosystem
services (e.g., removal of suspended particulate matter, use as
bioindicators). The effectiveness of freshwater mussels depends in

National Shelltisheries Association. Mystic, Connecticut

Ahsrnuts. 2002 Annual Meeting, April 14-18, 2002 379

part on their physiological rate functions, which may vary with
their physiological condition. Our goal w as to determine the level
of variability in physiological condition of Elliptio conipUinata, a
common unionid in the Atlantic drainage. Adults were subjected to
a variety of laboratory and field conditions. In the lab mussels were
held in continuous flow chambers with or without sediment and
fed isocaloric rations of either natural seston or lab-cultured mi-
croalgae. Field populations were caged and transplanted to waters
of varying quality. Condition index and pro,\imate tissue biochem-
istry (protein, lipid, carbohydrate, ash) were monitored for 6 weeks
in the lab and 1 year in the field. Both condition and tissue bio-
chemistry of wild mussels varied seasonally, as expected for adults
undergoing reproductive cycling. However, irrespective of these
normal seasonal patterns, condition and tissue biochemistry also
varied among all experimental comparisons; food quality (seston >
algal diets), sediment conditions (sediment > no sediment) and
environmental conditions (field site differences). Although E.
cnmplanata is considered to be one of the more hardy unionids.
these results indicate that its physiological condition is sensitive to
nutritional and environmental cues. Therefore, physiological fit-
ness measures for E. complanata should prove useful as bioindi-
cators, and studies of ecological processing by these animals
should quantify physiological rate functions under a variety of
conditions.

SMALL-SCALE ECOLOGICAL FUNCTION OF FRESH-
WATER MUSSELS (FAMILY: UNIONIDAE) IN THE
KIAMICHI RIVER, OKLAHOMA. Daniel E. Spooner* and
Caryn C. Vaughn, Oklahoma Biological Survey and Department
of Zoology. University of Oklahoma. Norman, OK 73019.

Freshwater mussel (Family Unionidael biomass and species
diversity is declining as a result of recent and historic anthropo-
genic impacts. Despite this marked trend, very little is known
concerning the ecology of unionids with respect to stream ecosys-
tem function and local macroinvertebrate assemblages. We per-
formed a 12-month field enclosure experiment to examine the role
of unionids in streams and document their associated sediment and
epilithic communities. Treatments were two mussel species, Acti-
nonaias ligameiuina and Ainhlenia plicaiii. shell treatment, and a
non-mussel sediment treatment. We observed colonization of al-
gae, ash-free dry mass and invertebrates in the sediment and on the
shells over three time periods: 1 month. 3 months, and 12 months.
Chlorophyll a, AFDM and invertebrate abundance were higher in
treatments containing live mussels. This trend was significant for
months where stream flows were low, but non-significant during
periods of high flow. However, there were no significant differ-
ences between species despite observed trends. This study indi-
cates that freshwater mussels have the capacity to impact local
algae, invertebrate and organic matter dynamics. However this
relationship appears to be context dependent and may be overrid-
den by advective forces such as flow.

PROPAGATION OF ENDANGERED FRESHWATER MUS-
SELS IN RECIRCULATING CULTURE SYSTEMS. Richard
J. Neves,* Jess W. Jones and William F. Henley, Virginia Co-
operative Fish and Wildlife Research Unit. Department of Fisher-
ies and Wildlife Sciences. Virginia Tech. Blacksburg. VA 24061.
A propagation facility for the production, culture, and release
of endangered ju\enile mussels has been established at Virginia
Tech, to address the conservation needs of 70 federally listed
mus.sel species in the United States. After years of research on
species' life histories, substratum and food requirements, and ef-
ficacy of culture systems, the facility has been successful in cul-
turing juvenile mussels for release to natal rivers. Nine species
have been cultured thus far. and over 250,000 juveniles have been
released to 3 rivers in Tennessee and 2 streams in Virginia. These
culture systems are seemingly suitable for holding and condition-
ing adult mussels for spawning, and graduate student experiments
are ongoing to test the range of applications to mussel conserva-
tion. A recent grant from the National Fish and Wildlife Founda-
tion has allowed construction of a new facility to test the recircu-
lating culture systems on a larger production scale, to begin in
2002. The new facility, coupled with an adjacent pond, will pro-
vide additional research opportunities and conditions for long-term
rearing of juvenile and adult mussels.

COMPARATIVE AND EXPERIMENTAL p:VIDENCE FOR
THE FUNCTIONAL ROLE OF FRESHWATER MUSSELS
IN STREAMS. Caryn C. Vaughn,* Daniel E. Spooner, Melissa
Moore and Keith B. Gido. Oklahoma Biological Survey and De-
partment of Zoology, University of Oklahoma, Norman, OK
73019.

Both the overall abundance and species richness of freshwater
mussels are declining at an alarming rate. If mussels play critical
roles in the functioning of river ecosystems, this significant loss of
benthic biomass may result in alterations of ecosystem processes
and functions, and also may impact other stream organisms that
have co-evolved with mussel assemblages. We used a comparative
field study and laboratory experiments to investigate the functional
role of unionids in streams. From 1999-2001, we sampled benthic
macroinvertebrates, meiofauna and fishes living in and around
mussel beds in nine rivers of the Quachita Uplands of Oklahoma
and Arkansas. Data analyzed to date indicate that densities of some
benthic groups, notably oligochaetes and filtering caddisflies, are
significantly related to unionid density. Laboratory experiments
compared community respiration rates, water column nutrient con-
centrations, algal clearance rates, excretion rates, and biodeposi-
tion rates of Aclin(n)aius Ugaincuthia and Aiiihlcimi plicahi. Both
species exhibited a strong, linear relationship between biomass and
most ecosystem processes at small spatial scales. However, there

380 Abstracts. 2002 Annual Meeting. April 14-18, 2002

National Shellfisheries Association. Mystic. Connecticut

were only subtle differences between species in their effects. Our
results indicate that unionids can have strong effects on ecosystem
processes when bioniass is high, but that these effects are context
dependent and can be oveiridden by physical forces.

ASSIMILATION OF MICROZOOPLANKTON BY ZEBRA
MUSSELS: THE BENTHIC ZOOPLANKTON LOOP. W.-H.

Wong* and Jeffrey S. Levinton, Department of Ecology and
Evolution. State University of New York. Stony Brook. NY
1 1794-5245; Benjamin Twining, Marine Sciences Research Cen-
ter. State Uni\ersity of New York. Stony Brook. NY 1 1794-5000.
Bivahes living at high densities exert strong effects on the
water column. Following their invasion of North American rivers
and lakes, zebra mussels (Dreisseini polyiiiorpha) caused major
declines in both phytoplanklon and microzooplankton. We tested
the hypothesis that the microzooplankton are a potential food
source for zebra mussels, and by extension, other bivalves. We
labeled phytoplankton with '""C and fed them to two species of
rotifers found abundantly in the Hudson River, which had declined
following the zebra mussel invasion. The labeled rotifers were fed
to 7.ebra mussels and we estimated clearance rates and assimilation
of carbon. For both species of rotifers the assimilation efficiency
was 40-50%. Before zebra mussels dominated the Hudson River
in 1992. the assimilation From rotifers was about 2 to 3 times
higher than its riiutine metabolic rate and also contributed about
0.349 (J/h) to 0.662 (J/h) to mussels growth and reproduction,
conferring a positive scope for growth. Since the zebra mussels
became abundant, the assimilation is still sufficient to explain
about 16.4% to 23.1% of the mussels routine metabolic rate.
Therefore, rotifers play a conceivably large role in the zebra mus-
sels energy budget whether at high rotifer concentrations (before
the invasion) or at low rotifer concentrations {after the invasion).

GENETICS AND MOLECULAR BIOLOGY

UNSCRAMBLING THE LOBSTER GENOME: COMPLEX
HETEROZYGOSITY IN THE AMERICAN LOBSTER?
Charles L. Brockhouse* and Gillian Richard, Marine Aquatic
Genetics. Biological Sciences, U. South Alabama. Mobile. AL
36688.

The American Lobster. Hoiiiarus amcricaniis Edwards, is both
a highly important economic species and a model for invertebrate
physiology. In spite of this, remarkably little is known about any
aspect of the genetics of this species. In view of the long breeding
cycle, genomics approaches are clearly preferable to classical breed-
ing genetics. As a prelude to a genomics program, we have un-
dertaken genome size and meiotic studies. Using Feulgen staining
densitometry, we determined the H. aiiu'ruaiiiis genome size to be
approximately 2.7 x 10'' base pairs. Confocal microscopy was used

to study meiosis in male lobsters. The chromosome number was
found to be n = 108, in approximate agreement with previous
studies. Large numbers of univalents with chiasma to multiple
other univalents were observed at metaphase I. Most or all of the
chromosomes were observed in these conformations. This is
strongly indicative of heterozygous interchanges among the chro-
mosomes, a condition which has the genetic effect of linking many
chromosomes into a single linkage group.

GENETICAL BASIS OF THE PLASTICITY OF RE-
SOURCE ALLOCATION IN THE PACIFIC OYSTER

CRASSOSTREA GIGAS. Bruno Ernande, CNRS-CREMA.
17137 L'Houmeau. France; Joel Haure, IFREMER-LCPL 85230
Bouin. France; Lionel Degremont, Edouard Bedier and Pierre
Boudry* IFREMER-LGP. 1 7390 La Tremblade. France.

Phenotypic and genetic correlations between fitness-related
life-history traits — such as survival, growth and reproductive al-
location— have to be considered to better understand selective pro-
cesses, both in the wild and in breeding programs. Quantitative
genetic experiments, based on nested half-sib mating designs, were
performed in the Pacific oyster C. f;igas ( 1 ) to estimate genetic
variance and the response to selection and (2) to document phe-
notypic and genetic trade-offs between life history traits. Physi-
ological trade-offs. i.e. plastcity of resource allocation, were
shown to vary among genotypes. Genetic polymorphism was pri-
marily observed for the plasticity of reproductive effort. In addi-
tion, the degree of plasticity in reproductive effort appeared to be
genetically cortelated with mean survival. The signs of some sig-
nificant genetic correlations were found to reverse accross envi-
ronments. These results could explain the maintenance of genetic
polymorphism for the studied traits. These may help us to better
understand the causes of summer mortalities in C. gificis juveniles,
on which a national program recently started in France.

THE SUCCESSFUL PRODUCTION OF TETRAPLOID
EASTERN OYSTER, CRASSOSTREA VIRGINICA GMELIN.
Ximing Guo,* Jian Wang, Brenda J. Landau, Li Li, Gregory
A. DeBrosse and Krista D. Buono, Haskin Shellfish Research
Laboratory, Rutgers University, 6959 Mdler Avenue, Port NoiTis,
NJ 08349,

Triploid eastern oyster grow 30—40% faster than normal dip-
loids. Fast growth reduces not only culture duration and cost, but
also losses from diseases inflicted mortality, which represents a
serious problem in the eastern oyster. The ideal way to produce
triploids is through diploid x tetraploid mating. Triploid production
from tetraploid is as simple as producing normal diploids, 100%
effective and free from genetic defects caused by polar body in-
hibition. Tetraploid Pacific oysters have been successfully pro-
duced anil commercialized. A small number of tetraploid eastern
oysters were previously produced hut lost. Here we reported the

N;iti(iii;il Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 381

production of a large number of tetraploid eastern oysters. The
tetraploids were produced using the Guo-Allen method —
inhibiting polar body I in eggs from triploids fertilized with normal
sperm. Triploid females were individually confirmed by flow cy-
tometry before use. Fertilized eggs were treated with 0.5 mg/L
cytochalasin B from about 10 to 30 min post-fertilization (PF).
Thirteen replicates were made, and eight produced significant
numbers of spats OlOO). Ploidy analysis at two months PF found
tetraploid spat in all replicates, ranging from 10 to 100%. Over
4000 tetraploid spats were produced from the eight surviving rep-
licates. Tetraploids were larger than triploids and diploids within
groups. At five months PF. about 10% of the tetraploids changed
to primarily triploid/Ietraploid mosaics. The tetraploids were made
from a Rutgers disease-resistant stock, which demonstrated strong
resistance to MSX. a parasitic disease. The combination of supe-
rior growth and disease-resistance may make triploid eastern oys-
ters extremely valuable for aquaculture production.

published microsatellite DNA markers, we obtained 1 14 microsat-
ellite DNA markers for testing on three outbred families, using
1 1 -day-old larvae, to reduce segregation distortion caused by re-
cessive deleterious mutations (Launey & Hedgecock 2001 Genet-
ics 139:255). Of the 102 markers that are informative in at least
one family. 98 are placed on a consensus map comprising 10
linkage groups and 880 centimorgans (cM; cf. karyotype of 10
chromosome pairs with an estimated length of 600-1000 cM based
on chiasma frequency). Map distances and, occasionally, marker
order differ between parents and among families. Of the 79 new
microsatellites tested on four other Cnissostrca species. 76 may be
amplified from C. angidatii. 65. from C. sikuinea. 30. from C.
ariakensis, and 8. from C. virginica. Decline in ability to amplify
these markers across congeneric species, together with a high fre-
quency of null alleles within C. gigas (40% of the loci are segre-
gating for a null allele in at least one family), suggests that primer-
target DNA sequences are rapidly evolving in oysters.

GENETIC DISTINCTIVENESS OF INSHORE AND OFF-
SHORE SPISULA CLAMS. Matthew P. Hare.* Biology De-
partment. University of Maryland. College Park. MD 20742:
Stephen R. Palumbi, Center for Conservation and Evolutionary
Genetics. 16 Divinity Ave.. Cambridge. MA 02138

SpisiiUi solidissiiiiii is restricted to the western North Atlantic
where it is commercially harvested by dredge from shelf waters
between Georges Bank and Cape Hatteras. A closely related taxon.
S. solidissima similis. inhabits the Gulf of Mexico and shallow
coastal waters along the Eastern Seaboard. Although studies of the
pi)pulation biology and demography of Spisitia clams have been
productive because of the ability to age cohorts from annular
growth rings, few data have been published on range distributions
and patterns of genetic variation. The mitochondrial and nuclear
DNA sequences presented here represent a preliminary effort to
describe the phylogeny and biogeography of Spisida clams and
relatives in the North Atlantic. Mitochondrial cytochrome oxidase
I and nuclear calmodulin intron sequence data show the southern/
inshore subspecies is genetically very distinct from Spisiila solUlis-
siimi populations from the Gulf of St. Lawrence (Canada). Georges
Bank and offshore from Ocean City. Maryland. Despite the re-
stricted inshore habitat of S.i. similis. genetic variation within this
taxon is exceptionally high, suggesting a large effective population
size or population substructure among coastal populations.

A GENETIC LINKAGE MAP OF 100 MICROSATELLITE
MARKERS FOR THE PACIFIC OYSTER CRASSOSTREA
GIGAS. Dennis Hedgecock,* Sophie Hubert, Gang Li, and
Katharine Bucl<lin, University of California, Davis, Bodega Ma-
rine Laboratory, Bodega Bay, CA 94923-0247.

A genetic linkage map will be an important tool for improving
the Pacific oyster Crassostiea gigas. the most farmed aquatic spe-
cies in the world. Combining 91 newly cloned and 23 previously

A SINGLE-STEP MULTIPLEX PCR IDENTIFICATION
ASSAY TO DISTINGUISH MEGALOPAE OF CALLl-
NECTES SAPIDUS FROM CALLINECTES SIMILIS IN
PLANKTON SAMPLES. Gregg G. Hoffman,* Ami E. Wilbur.
Martin H. Posey, and Troy D. Alphin, Department of Biological
Sciences and the Center for Marine Science, 5600 Marvin K. Moss
Lane. University of North Carolina at Wilmington. Wilmington.
NC 28409.

Understanding recruitment variation in economically important
species such as the blue crab. Calliiicctes sapicliis. requires the
collection, processing, and identification of large numbers of larval
ammals. The identification process typically involves sorting of
specimens based on morphological characters under a microscope,
and subsequent enumeration of the species of interest. For some
species combinations, morphological identification is complicated
by a paucity of clear diagnostic features that reliably survive the
collection process. In southeastern estuaries, the study of the re-
cruitment dynamics of Callinectes sapidus is complicated by the
co-occunence of its congener. C. similis. We have developed a
multiplexed PCR technique that efficiently and accurately distin-
guish C. sapidus and C. similis larvae. This assay relies on the use
of species specific primers to amplify distinctly sized PCR (poly-
merase chain reaction) products and targets a portion of the mito-
chondrial cytochrome oxidase I gene. We have applied this tech-
nique to larval samples (megalopae) collected on settlement traps
deployed during the 2000 and 2001 season in the Cape Fear estu-
ary (North Carolina) to discern temporal and spatial patterns in
larval abundance.

382 Abslrcicls. 2002 Annual Meeting. April 14-IS. 2002

National Shellfisheries Association. Mystic. Connecticut

SEARCHING FOR DIFFERENTIALLY EXPRESSED
GENES IN DIPLOID AND TRIPLOID EASTERN OYSTER,
CRASSOSTREA VIRG/NICA GMELIN. Brenda J. Landau,*
Arnaud Tanguy, and Xiniing Guo, Haskin Shellfish Research
Laboratory. Institute of Coastal and Marine Sciences. Rutgers Uni-
versity. 6959 Miller Avenue. Port Norris. NJ 08349.

Triploid shellfish are useful for aquaculture because of their
sterility, superior growth and improved meat quality. Triploids
also provide a unique model system for analysis of certain bio-
logical processes. Triploids grow faster than diploids in most bi-
valves studied so far including oysters. This phenomenon has been
referred to as triploid gigantism. Although several hypotheses have
been proposed to explain triploid gigantism, we know little about
changes in gene expression in triploid oysters. The identification of
unique expression profiles in triploids may enhance our under-
standing of basic mechanisms of growth regulation. Because trip-
loids are sterile, comparison of expression profiles during game-
togenesis between diploid and triploid oysters may lead us to genes
involved in sexual maturation and meiosis. We have begun a study
.searching for differentially expressed genes in triploid eastern oys-
ter in the aim to better understand the physiological processes
involved in growth and reproductive differences between diploids
and triploids. Subtractive libraries were made to identify differen-
tially expressed genes in both diploid and triploid oy,sters. Selected
clones from these libraries are being sequenced. Results from the
sequence analysis will be presented at the meeting.

A BASIC AFLP LINKAGE MAP FOR THE EASTERN OYS-
TER, CRASSOSTREA VIRGINICA GMELIN. Ziniu Yu* and
Xiniing Guo. Haskin Shellfish Research Laboratory, Institute of
Coastal and Marine Sciences. Rutgers University. 69.59 Miller Av-
enue. Port Norris, NJ 08349.

Genetic markers and maps are needed to map and improve
economically important traits in oysters. Amplified fragment
length polymorphisms (AFLPs) are popular markers for linkage
mapping in many agricultural species because of the relative ease
of developing a large number of reliable markers at low cost. We
tested AFLP markers for linkage mapping in the eastern oyster.
Crassostrea virginica Gmelin. Selective amplification primers
were labeled with fluorescent dyes, and amplified fragments were
detected with the ABI 310 genetic analyzer and analyzed with the
GenScan software. Foity primer combinations were screened, and
ten of them were selected for linkage analysis of 8 1 progenies from
a reference family derived from a Rutgers disease-resistant stock.
The 10 primer pairs produced 212 polymorphic markers in the
reference family; and 1 30 of them were informative or segregating
in one of the parents. Chi-square analysis indicated that 136 (91%)
markers segregated in Mendelian ratio, while the other 14 (9%)
showed significant segregation distortion (p < 0.05). A basic link-
age map was constructed with 84 segregating markers from the
male parent. The markers formed 10 linkage groups, correspond-

ing to the haploid number of 10 chromosomes in this species. The
map covered a total of 439 cM. with average interval of 5.2 cM per
marker. Additional markers including microsatellites are being
added to this first linkage map of the eastern oyster.

REVERSION REVISITED IN CRASSOSTREA ARIAKEN-
SIS: CHROMOSOME SET INSTABILITY IN FIELD TRI-
ALS ACROSS SALINITY REGIMES. Mingfang Zhou* and
Standish K. .Allen, ,Jr., Aquaculture Genetics and Breeding Tech-
nology Center, Virginia Institute of Marine Science, Gloucester
Point. VA 23062.

VIMS has been systematically examining the potential of sev-
eral non-native species for their potential in allex iating the serious
decline of oyster stocks in the Virginia portion of Chesapeake Bay.
One that has emerged as quite promising is the Suminoe oyster.
Crassoslrca aiiukeiisis. We were interested in determining the
stability of triploids from the period of seed to harvest (about 18
months). About 150 individually marked triploid C. ariakeiisis and
100 triploid C. virainica were deployed at each of seven sites in
Virginia and Noith Carolina. Every three months, all triploid C.
ariakensis were biopsied for hemolymph to determine the occur-
rence and extent of reversion (development of diploid cells within
a triploid) by How cytometry. As in other studies, C. ariakensis
outgrew C. virginica at all sites. Overall. 26 mosaics were found
among 919 individuals tested. The highest incidence of reversion
was uncovered in medium salinity (average: -5%) with high and
low salinities 2% or less. Frequency of diploid cells within indi-
vidual mosaics was generally less than lOVr although we observed
several "streakers" that obtained 65%, 46% and 28% diploid cells
by study's end. These results demonstrate that the "risk of rever-
sion" in commercial aquaculture of triploid C. ariakensis will
likely be very low. but emphasizes that reversion is an inherent
quality of autopolyploid oysters in the Crassostrea genus and per-
sists through time. Therefore, the principal risk for recovery of
reproductive potential among triploids seems to reside in unhar-
vested and "lost" oysters that remain in the Bay for long periods of
time.

EAST COAST BIVALVE
INDUSTRY SESSION

AN OVERVIEW OF THE POTENTIAL FOR CRASSOS-
TREA ARIAKENSIS IN THE CHESAPEAKE BAY . Standish
K. Allen, Jr.. Aquaculture Genetics and Breeding Technology
Center, Virginia Institute of Marine Science, Gloucester Point. VA
23062.

VIMS has been systematically examining the potential of sev-
eral non-native species for their potential in alleviating the serious
decline of oyster stocks in the Virginia portion of Chesapeake Bay.

Natioiiiii Shellfisherics Association. Mvstic. Connecticut

Abstracts. 201)2 Annual Meeting. April 14-lS. 2002 .^8.^

One that has emerged as quite promising is the Suminoe oyster.
Crassostrea arkiakensis. Field trials with sterile triploids have
shown a general resistance to disease, rapid growth rate, and high
survival. Formal and informal test marketing of the product has
been similarly successful. As with any non-native species, serious
concerns exist over the long-term implications of introduction.
Some of these issues are associated with simultaneous introduction
of pests or pathogens and some with the ecological effects of
sustained population growth in the Bay. By culturing this species
in the hatchery, pest and pathogen issues are largely, but not
wholly, addressed. Aquaculture of triploid-only individuals miti-
gates, but does not eliminate, most of the ecological concerns.
Aquaculture of hatchery raised sterile triploid seed represents an
intcrmediute solution to assisting the industry between abandoning
non-native research and wholesale introduction of diploids. The
industry potential is enormous, but there are lingering questions
about how precisely this "revolution" will proceed.

SURF CLAM (SPISULA SOLIDISSIMA) CULTURE IN
SOUTHERN NEW JERSEY: HATCHERY CULTURE PRO-
TOCOLS AND PRELIMINARY FIELD GROW OUT RE-
SULTS. Gregory A. DeBrosse,* David R. Jones, and Eric N.

Powell, Haskin Shellfish Research Laboratory. Institute of Marine
and Coastal Sciences, Rutgers University. M59 Miller Avenue,
Port Norris. NJ 0SM9.

The state of New Jersey harvests over 90% of the worlds surf
clams. Spisula sniidissiimi (Dillwyn. 1817). with an annual value
exceeding $.^0 million. The commercial fishing industry targets
large clams, typically those larger than 80 mm shell length. These
clams are used primarily for clam strips and chowders. Clams
smaller than 70 mm cannot be fished economically, even though
their per bushel value may be high. The raw and steamed clam
trade uses primarily soft-shell clams [Mya aiviuiria) and hard
clams (Mercenaria nieiveiuiriii). In the case of Mercenaria. the
majority of clams utilized are produced by aquaculture. and this
industry in New Jersey alone produced approximately $6 million
worth of clams in 1998. The surf clam grows well in New Jersey,
at about twice the rate of hard clams. Cultured yearling surf clams
compare favorably with hard and soft-shell clams in a variety of
product forms; steamed, fried, and on the half-shell, and therefore
represent another potential species for these markets. A key to
Spisula aquaculture success is to grow clams to marketable size
(~50-55mm) in one year. To do this requires spawning and rearing
animals out of season (e.g.. during fall, winter, and early spiing).
This requires conditioning of brood stock in the hatchery, and
defining the parameters of successful larval and juvenile culture.
The specific parameters of brood stock conditioning, larval and
juvenile culture, and preliminary field giow out data will be de-
scribed in the presentation.

COASTAL STATES PERMITTING PROCEDURES FOR
DEPLOYING A TIDAL POWERED UPWELLING NURS-
ERY SYSTEM. M. Richard DeVoe,* South Carolina Sea Grant
Consortium. 287 Meeting Street, Charleston. SC 29401; G. Ross
Nelson, General Engineering Laboratories. 2040 Savage Road,
Charleston, SC 29407.

Shellfish at|uaculture is a growing segment of the U.S. aqua-
cultiue industry. Several nursery techniques have been developed
for nursery growout of oysters from seed pioduced by hatcheries,
including land-based runway systems and upwellers, and both bot-
tom and off-bottom nurseries. The Tidal-Powered Upwelling
Nursery System (TPU) was developed as a low cost alternative to
these traditional nursery methods. The TPU has the advantage of
being a floating structure using tidal power to upwell water into
bins that house oyster seed.

A questionnaire was sent to coastal state permitting contacts
during the year 2000 to determine if the TPU is a viable nursery
option for oyster growout for culturists in their states, to determine
the permits that would be necessary to deploy the TPU. and to
estimate the costs associated with these permits. From this initial
survey, four states were selected for a more complete analysis of
the permits necessary to operate the TPU — Massachusetts. South
Carolina. Washington, and Alaska. The permitting processes in
these states were analyzed to compare and contrast the regulatory
structures among these four states and with the results of the initial
survey.

RELEVANT ISSUES FOR THE EAST COAST SHELLFISH
AQUACULTURE INDUSTRY: FINDING COMMONALITY
TO FOSTER UNITY. George E. Fliinlin, Jr..* Rutgers Coop-
erative Extension. 162.'^ Whitesville Rd.. Toms River. NJ 087.'S,'S.
Hatchery supported shellfish aquaculture on the East Coast of
the United States has been growing since the 1970s. Once practi-
cally obscure, growers are now in every state bordering the At-
lantic with over 60 hatcheries or seed nurseries scattered among
the coastal states. Some of the newer states involved in hard clam.
Mercenaria mercenaria. or American oyster. Crassostrea vir-
ginica. culture have shown very quick industry growth while
places where shellfish aquaculture took hold over 25 years ago
have practically slowed to a stand-still. With growth however
comes scrutiny by governmental agencies, both state and federal,
increased regulations, competition for space, environmental issues,
disease concerns, and other challenges. Presently individual states
have associations which can address concerns as they arise but
there is no cohesive organization which can represent the common
problems, as well as. promote pro-active activities for the growers
throughout the region. This discussion will review the industry
priorities respective to the .social, political and scientific or tech-
nological problems that face the shellfish growers. A survey of
growers, associations, hatchery/nursery operators, and extension
personnel provides the input. A compilation of these issues will

384 Abslnicts. 2002 Annual Meeting. April 14-1 S. 2002

National Shelltisheries Association, Mystic. Connecticut

serve as a basis lor the lormation of a shelHish aquaculture indus-
try group on the East Coast.

STAKEHOLDERS' PERCEPTIONS AND CHALLENGES
TO PARTICIPATORY MANAGEMENT: THE CASE OF
THE SOFT-SHELL CLAM, MYA ARENARIA L., INDUSTRY
IN MAINE. Aman Luthra* and William Walton. University of
Maine at Machias. Machias. ME 04654.

Local management of the soft-shell clam (A/v</ aieiuiiia. L.)
fishery in Maine involves the interaction of several different com-
munities including commercial harvesters, shellfish committee
personnel, scientists and the state, who share a common interest of
increasing the productivity of their mudtlats. Efficient manage-
ment is hindered due to a lack of communication between these
different communities. In 2000-2001, we surveyed 26 municipal
shellfish committee chairs (7 in eastern. 8 in mid-coast and I I in
southern region) and interviewed 41 commercial harvesters (II in
eastern, 14 in mid-coast and 16 in southern region) to identify the
knowledge gaps that exist between these 2 groups and across the
3 regions. Responding towns differed significantly in the number
of commercial licenses issued and the price of recreational and
commercial licenses across the 3 regions. Interviewed harvesters
differed regionally in the number of active clamming months per
year and in the number of commercial licenses held by each har-
vester. Both harvesters and chairs identified pollution, predalion
and overfishing as the factors that most severely limited the num-
ber of harvestable clams on the flats. Both groups identified a
diverse array of predators with seagulls {Lunis spp.) and green
crabs (Cairinus maemis) topping their list. Harvesters identified
raccoons {Procyon totor) as the third most damaging predator
while only one chair mentioned it. Stock enhancement is being
pursued at different intensities and with different foci among the
responding towns in the three regions. While most towns in mid-
coast (.57%) and eastern region (71%) are transplanting wild seed
as a stock enhancement strategy, only responding towns from mid-
coast (50%) and southern Maine (33%) are planting hatchery-
reared seed. Both harvesters and chairs were opposed to reducing
the two-inch minimum as well as leasing of intertidal flats. The
survey and interviews identified areas of agreement and disagree-
ment among these communities involved in the management of
soft-shell clams, and these results can be used to guide both re-
search and improved management.

EMBRACING AQUACULTURE BMP'S: A CONCEPT
WHOSE TIME HAS COME AS A GOOD NEIGHBOR OR
JUST ONE MORE THING TO DEAL WITH? Sandra Mac-
farlane,* Coastal Resources Specialists. Orleans. MA 02653.

In a labor intensive, high risk and competitive industry Irought
with uncertainty and reliance on natural conditions for growing
food, initial efforts to foster an industry-wide or area-wide agree-

ment on the most environmentally sound and "giiod neighbor"
approach to the business have received a luke-warrn reception at
best from many growers. However, misinformation is rampant
outside the industry from stakeholders who also have a vested
interest in the manner in which the at|uaculture is conducted in
their back yard. In order for aquaculture to be sustainable, issues
such as water quality, disease prevention and control, and care and
maintenance of gear as well as animals is essential. Other stake-
holders must be convinced that the aquaculture operation is con-
ducting itself in the most prudent of manner on these issues and
adopting BMP's or a Code of Practice is an effective mechanism
to project a positive image. However, bringing all the voices to the
table and bridging gaps in understanding among stakeholders is a
crucial element in developing effective BMP's.

OPTIMIZATION OF TIDAL UPWELLER DESIGN: PROJ-
ECT WRAP-UP. Dana L. Morse* and John Riley. Maine Sea
Grant Program/UMaine Cooperative Extension. Darling Marine
Center. Walpole, ME 04573.

A fifth-scale model of a conimonh-used tidal upweller design
was built, and tested m the tow tank at the University of Maine, in
Orono. The goal was to better understand the effects of design
components on overall flow through the system, thus allowing
future construction to be maximally efficient, and therefore prof-
itable.

Three engineering considerations were tested at different
speeds in the laboratory: scoop size, silo outlet opening size, and
reduction of mooring line forces. Site visits were also made to
local shellfish hatcheries, to examine the effects of shellfish seed
size, bed depth, and shellfish species, on frictional flow loss in an
upweller silo.

Results indicate that it is critical that the upweller platform
maintains a le\el profile, as changes in horizontal aspect have
adverse effects on flow and growth. Silo outlets should be made as
non-restricti\e as possible, by increasing the diameter of the outlet
holes, submergence of the outlets, or by renun ing the upper por-
tion of the silo wall entirely, and protecting against shellfish loss
with plastic netting. Enlarging the scoop area over the initial de-
sign size has a limited effect on increasing flow through the up-
weller. Smaller shellfish seed tended to create stronger frictional
head losses than the larger seed. Lastly, placement of an escape
\cnt in the rear of the upweller can help to maintain a le\el profile
for the platform.

In general, the original Mook design was well thought out.
Though some iiKHlifications have resulted m performance im-
provements, the approach is sound and to the extent possible,
makes use of the potentials afforded by tidal action.

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 ,^85

GROWTH AND ECONOMICS OF SHELLFISH NURSERY
CULTURE IN A COMMERCIALLY AVAILABLE FLOAT-
ING UPWELLER. Robert B. Rheault,* Moonstone Oysters.
1121 Mooresfieid Rd.. Waketleld. RI 02879: Jeffrey T. Gardner,
227 Shore Rd.. Westerly. RI 02891.

A small, commercially-available, tloatinj; upweller that serves
as a dual-use dock and work platform has been deployed in several
sites on the east coast. Five seasons of testing and development
have led to production of a unit that is durable, functional, eco-
nomical to operate and easy to maintain. Several producers claim
that the device has resulted in substantial impro\ements in their
production economics by accelerating growth, reducing \ariation
in growth rates and cutting labor costs.

Growth and economic data from several Coastal Aquacultural
Supply floating upwellers are compared with other conventional
nursery culture methods such as a "Taylor tloat" and rack and bag
systems. Capitol costs, labor costs, maintenance and operational
expenses are described as well as siting and permitting consider-
ations.

ENHANCING SEED AVAILABILITY FOR THE HARD
CLAM AQUACULTURE INDUSTRY THROUGH APPLI-
CATION OF REMOTE SETTING TECHNIQUES. Leslie N.
Stunner.* Cooperative Extension Service. University of Florida.
Cedar Key, FL 32625; John E. Supan, Office of Sea Grant De-
velopment. Louisiana State University. Baton Rouge. LA 7080."?;
Charles M. Adams, Food and Resource Economics Department.
University of Florida. Gainesville, FL 3261 1.

Hard clam. Mercenaria mercenaria. aquaculture has developed
rapidly in the southeastern United States. Adequate seed a\ ailabil-
ity is a major industry concern and has recently faced critical
shortages. Technical procedures were developed and demonstrated
to determine the economic feasibility of transferring remote setting
technology from the Pacific Northwest molluscan shellfish indus-
try to the hard clam culture industry. Competent pediveliger larvae
obtained from commercial hatcheries were refrigerated, stored
overnight and delivered chilled to remote set locations for evalu-
ation of technique, site and season. Participating land-based nurs-
eries were modified to incorporate mechanical filtration of water
supply, remote setting tanks and downwellers. Management re-
gimes evaluated over 4 trials included: 1 ) supplemental feeding
with a commercial algal paste versus none, and 2) duration of
shipping. Biological features documented included survival and
time to reach a I mm seed, the minimum size presently obtained
by nurseries. Results will provide for operational procedures and
guidelines for remote setting of hard clam seed. The economic
characteristics associated with the remote setting system will be
described. Costs associated with producing the seed, including the
original larvae cost, will be compared with the current market price
for 1 mm seed. Remote setting of hard clam seed would allow

nursery operators to become less dependent upon traditional seed
sources and ensure a reliable supply of seed to growers.

USING SHELLFISH SEED AS A PUBLIC ENHANCEMENT
TOOL: A REVIEW OF ITS USE AND SUCCESS IN THE
NORTHEAST US. William C. Walton, Wellfleet Shellfish De-
panment. 300 Main Street. Welltleet. MA 02667.

In response to dwindling stocks of shellfish (hard clams. Mer-
cenaria mercenaria. oysters. Crassostrea virginica. soft shell
clams. Mya areiiaria. etc.) and increased fishing pressure, fisheries
managers are implementing a variety of restrictive and proactive
measures. One proactive strategy available to resource managers is
to supplement the existing wild stock w ith hatchery-reared juve-
nile 'seed" supplied by commercial and public shellfish hatcheries.
Planting seed on public grounds may enhance the fishery directly,
by introducing shellfish that survi\e and grow to a legally harvest-
able size, and indirectly by increasing larval supply and subsequent
year classes. This technique has been widely adopted in southern
New England for hard clams and in northern New England for soft
shell clams. Despite such implementation, quantitative assess-
ments are rare. Here I review 1 ) local managers' perceptions of the
success of such programs based on interviews and surveys. 2)
quantitative and experimental studies of the survival of shellfish
seed, and 3) known limiting factors. Although perceived and actual
survival rates span the spectrum, this variation can largely be
attributed to predation. which is a function of seed size at least in
hard clams. Unlike private aquaculture. however, the scale of pub-
lic seeding generally prohibits the use of nets to protect the seed.
To illustrate the choices faced by public managers. I will present
a case study of potential public seeding strategies of hard clams in
Welltleet, Massachusetts, highlighting the costs, risks and benefits.

GROWTH AND ECONOMIC ADVANTAGES OF DISTRIB-
UTED POWERED UPWELLERS: CREATING A NEW
AQUACULTURE NICHE. Christopher G, Warfel, P.E..*

ENTECH Engineering, PC Box 871. Block Island. RI 02807. with
support from the Rhode Island Slater Office of Technology.

Distributed powered upwellers. (also known as grid-isolated, or
renewable energy based upwellers) allow upweller technology to
be used in areas where traditional sources of electrical/mechanical
energy are not possible or desirable. The author presents the
growth and cost performance findings from two years of research
in the development of distributed generation upwellers utilizing
solar energy. The design and prototypes went through several
changes during the two years to facilitate its operability in a harsh
environment. The present configuration has proved to have high
reliability, provided for good growth, low mortality, and good
ergonomics. Growth of Crassostrea virginica averaged 320% over
base case for a relatively short four month growing seasons. The
major benefits of this technology is the use of upwelling technol-

386 Abstnicts. 2002 Annual Meeting, April 14-18. 2002

National Sliclltlsheries Association. Mystic. Connecticut

ogy in areas that are not conduci\e to traditional utility powered sears. JOD mortalities occuned in three additional locations, and

upwellers, allowing for the benefits of upwellers to be realized in in all cases, animals were heavily colonized by the same species of

locations traditionally thought not to be ammenable to such tech- Rosedhacter. The potential for utilizing genetic signatures of this

nology. bacterium for epidemiology will be discussed.

THE NEW OYSTER WARS: POLICY PERSPECTIVES ON
THE INTRODUCTION OF CRASSOSTREA ARIAKENSIS IN
THE CHESAPEAKE BAY. Donald Webster. University of
Maryland. Wye Research & Education Center. P.O. Box 169.
Queenstown. MD 216.^8.

Oyster harvests in the Chesapeake Bay have declined over
ninety percent in the past fifty years due largely to the influence of
Haplnsporidiuin nclsoni and Perkinsus marinus. Recent studies
regarding the potential of the species Crassostrea ariakensis have
shown that this animal may provide many features desirable in the
reconstruction of the oyster fishery as well as environmental man-
agement of the Bay. However, since it is a non-indigenous species,
there are many factors to be considered in its introduction. Varia-
tions in the cunent status of the C Virginia resource, as well as
historical differences of management techniques inherent in the
two states have led to challenges regarding open-water introduc-
tions as well as plans to increase stocks of the non-native oyster in
the future. This paper focuses on the policy differences between
Maryland and Virginia and the attitudes of various user groups that
are interested parties in the potential introduction of C. ariakensis
in the Chesapeake Bay.

BIVALVE DISEASE STATUS
AND TRENDS

RECENT EXPANSION OF JUVENILE OYSTER DISEASE
(JOD) IN MAINE. Bruce J. Barber.* School of Marine
Sciences. University of Maine. Orono. ME 04469: Katherine J.
Boettcher, Dept. of Biochemistry. Microbiology and Molecular
Biology, University of Maine, Orono. ME 04469.

Juvenile oyster syndrome has caused mortalities of cultured
Eastern oysters. Crassostrea virginica. in Maine since 1988. We
have identified a likely etiological agent, a novel taxa in the Roseo-
bacter clade of the marine alpha-proteobacteria. In oysters exhib-
iting signs of the disease {reduced growth and uneven valve
growth), this bacterium comprises 40-95^r of the total bacteria
colonizing gill and mantle surfaces. Until recently, all documented
outbreaks in Maine have occurred in the Damariscotta River. One
strategy employed by oyster growers to avoid JOD mortality has
been to use nursery sites outside the Damariscotta River. In the
summer of 2000, however, mortalities of 709f in the New Mead-
ows River and 20% in Maquoit Bay were reported by growers. In
2001, mortalities of 40% and 50% occurred in the New Meadows
River and Sheepscot River, respectively. Thus in the last two

STATUS AND TRENDS OF DERMO AND MSX IN SOUTH
CAROLINA. David Bushek* and Dwavne Porter. Baiuch In-
stitute, University of South Carolina. PC Box 16,^0. Georgetown.
SC 29442; Loren D. Coen. M. \vonne Bobo. and Donnia L.
Richardson, Marine Resources Research Institute. SCDNR. 217
Fort Johnson Rd.. Charleston. SC 29412.

South Carolina has a relatively abundant and extensive popu-
lation of oysters throughout the state. Dermo (Perkinsus marinus)
and MSX (Haplosporidium nelsoni) are both present, but they do
not cause widespread oyster mortality as reported in other regions.
Surprisingly, little attention has been paid to understand why this
difference exists. In South Carolina, virtually all oysters inhabit
high salinity (20-35 ppt) estuaries where they form extensixe
fringing and mounding reefs in the 1 .5 to 2 m intertidal zone. Their
intertidal existence exposes them to daily temperature fluctuations
that often exceed 30''C. with winter extremes below freezing and
summer extremes that can exceed 50°C.

Since 1972, SCDNR's Marine Resources Research Institute
(MRRI) has been documenting the occurrence of Dermo through-
out the state. MSX has been documented since at least 1986 and
monitored since 1994. In addition, the NOAA-funded Urbaniza-
tion of Southeastern Estuarine Systems (USES) project has moni-
tored these diseases in pristine and developed areas to identify
potential relationships with different land-use patterns and land-
scapes. The data indicate that P. marinus is ubiquitous and abun-
dant throughout the state. Prevalence and intensity peak from late
summer to early winter, but unlike populations in the northeast, the
parasite remains prevalent throughout the year. By comparison. H.
nelsoni is relatively rare or altogether absent with mean prevalence
generally below 20%. Haplosporidiwn nelsimi is known to be very
sensitive to low salinities ( lOppt or less). Low salinity is also often
invoked as a major control that reduces prevalence and intensity of
P. marinus. In South Carolina, most sites where oysters thrive
rarely encounter such low salinities for more than a few hours.
Furthermore, lowest salinities occur during low tide when the oys-
ters are exposed. Instead, elevated temperatures may exceed the
thermal tolerances of the parasites just long enough to shift the
host-parasite dynamic in favor of the host. In this talk, we sum-
marize recent findings and discuss implications for intertidal oys-
ters along the southeast Atlantic coast of the United States.

Natiiinul Shellfisheries Association. Mvstic. Connecticut

Ahstmcls. 2002 Annua] Meeting. April 14-18. 2002 387

A DECADE OE DISEASE: A REPORT OF OYSTER DIS-
EASE MONITORING EFFORTS IN THE LOWER CHESA-
PEAKE BAY I990-2()()0. Lisa M. Ragone Calvo* and Eugene
M. Burreson, Virginia Institute of Marine Science. School of
Marine Science, College of William and Mary. Gloucester Point.
VA 23062.

In the late 1980s oyster populations in the Virginia portion of
the Chesapeake Bay were significantly impacted by epizootics of
the oyster parasites Perkinsiis marinus and Haplospohdium nel-
soiii as a consequence of dry and warm climatic conditions. The
distributions of both parasites were widespread and the range of P.
marinus was extended into upper tributary areas that had histori-
cally been disease free. Since that time VIMS has been extensively
monitoring lower bay oyster populations for the presence of the
pathogens via annual fall surveys of 30-40 lower bay oyster popu-
lations, monthly surveys of four oyster reefs in upper James River,
and monthly summer surveys of naive sentinel oyster that are
transplanted to a site in the lower York River in May of each year.
Perkinus maiinus continues to persist in upper tributary areas,
despite the occurrence of several years that were relatively wet and
cold. Record high prevalences and intensities of P. maiinus were
observed in 1999 and 2000, and the parasite was found in all areas
where significant oyster stocks remain. Haplnsporidium nesloni
I pre\alences were generally low in 1990. 1993. and 1998; however,
record high levels were observed in 1999. In 1999, epizootics of
the parasite were extensive and caused severe oyster losses in
many areas. Clearly, both pathogens continue to be a significant
threat to lower Cheaspeake Bay oysters. The correlation of disease
patterns and environmental conditions will be discussed.

DEVELOPMENT AND APPLICATION OF A PCR FOR
MIKROCYTOS MACKINl, THE CAUSATIVE AGENT OF
DENMAN ISLAND DISEASE IN OYSTERS. Ryan B. Carne-
gie,* Gary R. Meyer, Janice Blackbourn, and Susan M. Bower,

Fisheries and Oceans. Science Branch. Pacific Biological Station.
Nanaimo. British Columbia. Canada V9R 5K6; Nathalie Cochen-
nec-Laureau, IFREMER, Laboratoraire de Genetique, Aquacul-
ture et Pathologic. BP 133. Ronce les Bains. F- 17390 La Trem-
blade. France.

Mikrocytos mackini has caused Denman Island disease of Cra.v-
sostrea gigas and Oslreci eclulis in British Columbia. Canada since
at least 1960. A small protistan parasite. M. mackini is commonly
observed with standard histopathological techniques around the
green pustules characteristic of Denman Island disease. In oysters
with subclinical infections, however. M. mackini is not easily de-
tected, and it is rarely observed before March and after June. Its
mode of transmission, life cycle, and taxononiic affinities are un-
known.

Our objectives were to design and validate molecular assays for
M. mackini and to use these tools to re-examine the sea.sonal
prevalence of M. mackini infections. We sequenced 1457 base

pairs of M. mackini SSU rDNA and developed a PCR specific for
this gene. We used this PCR to screen palp DNA samples from
cultured C. gigas from Henry Bay, Denman Island. BC monthly (N
= 100/month) from January to December 2001. PCR detected M
mackini more sensitively than did standard histopathology. PCR-
based M. mackini prevalence estimates met or exceeded histopa-
thology-based estimates in every month, and only two oysters M.
niackini-posil\\e by histopathology were misdiagnosed as negative
by PCR. PCR confirmed a trend in seasonal M. mackini prevalence
identified earlier by histopathology. Prevalence peaked in April
and particularly May, when 22% of lower intertidal C. gigas were
M. mackini-posilixe by PCR (12% by histopathology). However,
PCR also detected M. mackini in the palps of apparently healthy C.
gigas in every month of the year (through September 2001 ). a first
indication that M. mackini may persist subclinically in oyster
populations year round.

DEVELOPMENT OF HIGH DISEASE RESISTANCE IN A
WILD OYSTER POPULATION. Susan E. Ford, Haskin Shell-
fish Research Laboratory. Rutgers University. 6959 Miller Av-
enue. Port Norris. NJ 08349.

In 1957-1959. the introduced parasite. Haplosporidium ncl-
soni. killed 90-95% of the oysters (Crassostrea virginica) in lower
Delaware Bay and about half of those in the upper Bay. Shortly
thereafter. H. ;ie/5on/-caused mortalities in the native population
declined, equalling that of first-generation selectively bred oysters.
For two decades, no further change in the wild population oc-
curred, although steady improvement was achieved by continued
selective breeding. Survival of the wild population is though to
have plateaued because most oysters inhabited the upper bay and
were protected from H. nelsoni by low salinity. From 1957 through
1989. H. nelsoni prevalence was cyclic, but overall high (annual
maxima of 50 to 90%). After 1989. however, prevalence in wild
oysters rarely exceeded 30% even though unselected oysters con-
tinued to become heavily infected, and molecular detection indi-
cated that infective stages were present throughout the Bay. This
apparent "second step" in the development of resistance in the
native oysters occurred after the incursion of H. nelsoni into the
upper bay in the mid 1980s, with widespread and heavy oyster
mortalities c)ccurring for the first time since the 1957-59 epizootic.

THE LONG-TERM ROLE OF PARASITIC DISEASES IN
OYSTER POPULATION DYNAMICS. Stephen J. Jordan and
Jessica Vanisko. Sarbanes Cooperative Oxford Laboratory. 904 S.
Morris St., Oxford, MD 21654.

Populations of the Eastern oyster. Crassostiea virginica. suffer
high rates of mortality from Dermo. MSX. or both diseases
throughout most of the range of the species. The principal effect of
high mortality rates on population structure is truncation of the size
distribution, skewing the populations toward smaller oysters. Sec-

388 Abstracts, 2002 Annual Meeting, April 14-18, 2002

National Sliellfisheries Association. Mystic. Connecticut

ondary effects include reductions in population bioniass. harvests.
and spawning potential, although there does not appear to be a
strong effect on recruitment of juveniles. In Chesapeake Bay. the
diseases, in combination, have had their most negative effects
since the late 1980s, when Dermo spread throughout the popula-
tion. Over the past decade, disease intensity and oyster mortality
rates have been strongly dependent on salinity, intensifying in
drought years, moderating during wet periods, and affecting most
severely sub-populations in higher salinity regions. Time series of
oyster population data from Maryland have been used to construct
a model that simulates trends in the population at various rales of
disease mortality, fishing mortality and recruitment. Current rates
of total mortality appear not to be sustainable without significant
management intervention.

CAPE COD BIVALVE DISEASE MONITORING PRO-
GRAM, YEAR 1. Dale Leavitt,* D. Murphy. W. Burt, and W.
Clark, Cape Cod Cooperative Extension. Barnstable. MA 026.^0;
M. Hickey and J. Moles, Department of Marine Fisheries. Poc-
assett. MA 02559; R. Smolowitz, Marine Biological Laboratory,
Woods Hole. MA 0254.\

The effect of disease on bivalve populations is an important
factor in determining management methods tor the cultme. harvest
and movement of bivalves. In order to provide the information
needed by extension and regulatory agencies and the aquacullur-
ists, to effectively manage these populations on Cape Cod. MA, a
monitoring program has been established as a cooperative project
between Cape Cod Cooperative Extension Agency and the MA
Department of Marine Fisheries. Hard clams. Eastern oysters and
soft shell clams will be monitored over the course of each year. It
is anticipated that the monitoring program will become a routine
method for disease Information gathering in MA.

Oysters and hard clams were collected in the fall of 2001.
Twenty-five oysters from each site underwent testing for Peikin-
siis nuiriiuis using Thioglyocollate culture methods. Samples were
also evaluated histologically for M.SX and SSO. Sixty hard clams
from each selected site were examined grossly for QPX. Twenty-
five of these were examined histologically. In 2002. twenty five
soft shell clams from several sites will monitored. Results of the
first year of monitoring will be presented at this session.

PREVALENCE AND INTENSITY OF PARASITIC DIS-
EASES IN BIVALVES FROM RHODE ISLAND WATERS.
Karen L. Mareiro.* and Marta Gomez-Chiarri, Fisheries, Ani-
mal, and Veterinary Science, University of Rhode Island, Kings-
ton, RI 02881; Katherlne Kerr and Emily Carrinston. Depart-
ment of Biological Sciences, University of Rhode Island; Arthur
Ganz, Division of Fish and Wildlife, Department of Environmen-
tal Management, Wakefield, RI 02879.

The goal of this Shellfish Disease Survey is to assess disease
prevalence and intensity In Rhode Island bivalves. Eastern oysters
(Crassostrea virginica) were collected from several wild popula-

tions and local farms In August and November 1998-2001. Hard
clams (Mercoiaiia iiwrcfiuiiia} were collected from selected lo-
cations In 1998. 2000 and 2001. Dermo disease (caused by Perk-
insiis mariiiiis) is widespread in Rhode Island oysters, while preva-
lence of Multinucleated Sphere X {caused by HaplnsporidhiDi iiel-
soiii) and Seaside Organism (caused by HapUispdiiduim cusiale) is
low and restricted to a few locations. Prevalence of Dermo disease
ranged from 0-IOOVf . remaining low In oysters from aquaculture
leases and a few wild locations. Intensity of Dermo disease has
remained unchanged or increased since 1998. Several factors could
be responsible for differences in prevalence of Dermo disease,
including genetic factors, lack of Infective particles and differences
In environmental conditions. Trematode Infections were common
in Rhode Island oysters, but high prevalences of heavy infections
were found only in wild oysters from Point Judith Pond and Block
Island. Wild oysters from Pawcatuck River showed a high preva-
lence of the apicomplexan parasite Nematopsis ostreanim. No
Quahog Parasite Unknown (QPX) was detected in Rhode Island
hard clams.

MODIFICATION AND FIELD TRIALS OF A MULTIPLEX
PCR FOR THE DETECTION OF THREE PROTOZOAN
PATHOGENS OF THE EASTERN OYSTER, CRASSOS-
TREA VIRGINICA, GMELIN 1871. Spencer Russell,* Salva-
tore Frasca, Jr., and Richard A. French, Department of Patho-
biology and Veterinary Science. University of Connecticut, Storrs,
CT 06269; Inke Sunila, Bureau of Aquaculture, Department of
Agriculture, Department of Agriculture, State o'i CT. Mllford. CT
06460.

Populations of eastern oysters (Crassostrea virginica) along the
eastern coast of North America have repeatedly experienced out-
breaks of epizootic disease and mass mortality due to Infections by
protozoal parasites. As polymerase chain reaction (PCR) method-
ologies become routine laboratory procedures and diagnostic tools
of choice, it Is Imperative to incorporate PCR quality controls to
avoid false positive and negative results. In this study we describe
the modification of a previously developed multiplex PCR
( MPCR) for the detection of the eastern oyster parasites Hoplospo-
r'uUiim nelsoni, Haphisporidiwn costale and Perkinsus mariinis by
incorporation of a quality control extraction and amplification
PCR product from primers directed against an 805 base-pair (bp)
sequence of the 28S SSU rRNA sequence of Crassostrea vir-
ginica. The modified MPCR simultaneously amplified 805bp,
564bp. 30lbp and I51bp fragments of the SSU rRNA of C. vir-
ginica. H. nelsoni, P. marinus and H. costale, respectively. The C.
virginica gene product tests the quality of extracted DNA used to
support amplification in an optimized and appropriately performed
PCR. In addition, we examined the species specificity and sensi-
tivity of the newly modified MPCR for the detection of H. nelsoni,
H. costale and P. marinus and compared its performance to that of
the conventional diagnostic techniques of histopathological tissue

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 389

examination and the Ray/Mackin fluid thioglycollate medium as-
say. Five hundred thirty oysters were sampled from 12 sites along
the East Coast of the United States from the Gulf of Mexico to
New England throughout the entire range of these parasites. Our
results indicate that the newly modified MPCR is specific for the
detection of H. iiehoni. H. costale and P. nuiiiiuis. Through spike/
recovery experiments, the sensitivity of the MPCR for H. nelsoni.
H. costale and P. mariniis was 100 fg. 10 fg and 100 fg, respec-
tively, from 25 mg of oyster tissue. The modified MPCR detected
1 18 of 530 (22%) oysters with H. nelsoni. 12 (2%) oysters with H.
costale and 212 (40%) oysters with P. mahniis infections. The
RMFT assay detected P. marinus infection in 163 (31%) oysters.
Histopathological examination detected H. nelsoni and H. costale
infections in 30 (6%) and 4 (0.8%) oysters, respectively. The
MPCR is a more sensitive diagnostic assay for the detection of H.
nelsoni, H. costale and P. marinus. and the inclusion of an oyster
quality control product in the multiplex limits the number of false
negative results from extraction or PCR failures.

DERMOWATCH: A WEB-BASED APPROACH FOR
MONITORING THE OYSTER PARASITE PERKINSUS
MARINUS (DERMOCYSTIDWM MARINUM). Thomas M.
Soniat,* Department of Biology. Nicholls State University. Thi-
bodaux, LA 70310; Enrique V. Kortright, Koiiright Corporation.
102 Allendale Dr.. Thibodaux. LA 70301: Sammy M. Ray, De-
partment of Marine Biology. Texas A&M University, Galveston.
TX 77553.

A web site called DermoWatch (www.blueblee.com/dermo)
is being used to monitor Perkinsns marinus ( = Dennocystidiitm
nuiriiuim) in Galveston Bay. Texas. The main page provides the
most recent data from nine locations. Data include measured water
temperature (T) and salinity (S). calculated percent infection and
weighted incidence (Wl) of parasitism, and estimated time to a
critical level of disease (t-crit). (The t-crit is calculated as the time
in days to reach a critical WI if 1.5, assuming no change in T and
S.) By expressing the solution in days, oyster growers can make
informed decisions about when to move or harvest their oysters.
Furthermore, they can relate observed mortalities to estimates of
t-crit, and thus P. marinus ceases to be an "unseen" killer. With a
utility called the DermoCalculator the web site is useful, not just in
Galveston Bay. but wherever P. marinus is found. It allows anyone
with information on T and S, oyster length and initial WI to de-
termine a t-crit. A limitation of the approach in Galveston Bay is
that t-crit is calculated from a single (monthly) measurement of T
and S. A monitoring station is being constructed in Louisiana with
which real-time measures of T and S will be used to constantly
update calculations of t-crit. More frequent inputs of T and S
should increase the reliability of the t-crit estimates.

DETECTION OF A PREVIOUSLY UNDESCRIBED HAP-
LOSPORIDIAN-LIKE INFECTION OF A BLUE MUSSEL
(MYTILUS EDULIS) IN ATLANTIC CANADA. Mary F.

Stephenson* and Sharon E. McGladdery, Gulf Fisheries Centre,
Department of Fisheries and Oceans. P.O. Box 5030, Moncton,
New Brunswick, Canada EIC 9B6; Nancy A. Stokes, Department
of Fisheries Science, Virginia Institute of Marine Science, P.O.
Box 1346, Create Road, Gloucester Point, VA 23062.

Microscopic examination of a blue mussel, Mylilus edulis Lin-
naeus, with an unusual macroscopic appearance revealed the pres-
ence of a haplosporidian-like infection throughout the soft tissues.
This is the first documented occurrence of this group of parasites
in Atlantic Canada. Large numbers of blue mussels and American
oysters, Crassostrea virginica Gmelin, are sampled on an ongoing
basis to monitor their disease profiles within Atlantic Canada.
Twelve years of histological sampling has established a disease
profile including many protozoans and diseases of local concern
but no infectious agents resembling those listed by the Office
International des Epizooties (OIE). Samples were sent immedi-
ately to the Reference Laboratory for Haplosporidiosis at the Vir-
ginia Institute for Marine Science, where they were screened for
OIE-notifiable disease agents by in situ hybridization using Hap-
losporidium nelsoni and H. co.s/a/t'-specific DNA probes. These
did not hybridize with the parasite in the mussel. The spores of the
parasite are operculate and measure 3-5 microns by 6-8 microns.
They show a diversity of forms from roughly spherical to pyriform
with or without filamentous extensions. These resemble the hap-
losporidians from Mxtilns californianus and Mylilus edulis from
California {Haplosporidiuin lumefacientis). as well as a Haplospo-
riilium sp. described from mussels in Maine. As with these previ-
ous descriptions, the cuiTent infection, although massive, was not
associated with any haemocyte defense response indicative of an
acute pathogenic infection. No other infections have been found in
Atlantic Canada, and the significance for the lucrative Atlantic
Canadian mussel induslrv is unknown.

MONITORING BIVALVE HEALTH IN LONG ISLAND
SOUND. Inke Sunila, State of Connecticut. Department of Ag-
riculture. Bureau of Aquaculture. Milford, CT 06460.

Long Island Sound (LIS) has a viable, economically important
culture of eastern oysters (Crassostrea virginica) and quahogs
{Mcrcenaria mercenaria). Connecticut has 65.000 underwater
acres leased for bi\alve culture. New York 3,500 in LIS. Bivalve
health is constantly monitored in the field and hatchery operations,
and diagnostic service is available in the case of mortalities. Oys-
ters and clams arc diagnosed for inflammatory responses (acute or
chronic), degenerations (ceroidosis, atrophy), cell and tissue death
(necrosis vs. apoptosis). hemodynamic derangements (hemor-
rhage, edema), growth derangements (hyperplasia, metaplasia) and
tumors (benign or malignant). Possible pathological irritants, such
as infective agents or environmental factors are identified. An

390 Absli-ucls. 2002 Annual \4eeting. April 14- IS. 2002

National Shelllisheries Association. Mystic. Connecticut

epizootic caused by HupldspnriJuiiii iwlsdiii (MSX) in 19y7-199S
caused a 76% reduction in oyster production. At the present time
Haplosporidium costale (SSO) and possibly other hapiosporidiaii
species occur as coinfections with MSX contributing to an annual
loss of 1.^% in oyster stocks. Impact of the 1997-98 MSX epi-
zootic still affects the oyster industry since tho.se year classes
would be on the market now. Dermo disease (Perkinsiis marimis)
occurs with a high prevalence (80%) and a low intensity (1 on
Mackin Scale). It has not been associated with significant field
mortalities. On the contrary, hard clam harvest has seen a six-fold
increase during the last five years. Hard clams in LIS are healthy,
and the ecosystem does not appear to provide optimal conditions
for the clam parasite QPX to proliferate. We have recendy adapted
molecular methods to monitor bivalve diseases.

DISEASES OF CRUSTACEA

ECOLOGICAL RAMIFICATIONS OF DISEASE IN THE
CARIBBEAN SPINY LOBSTER. PANULIRUS ARGUS.
Donald C. Behringer. Jr.* and Mark J. Butler. IV, Old Domin-
ion University. Norfolk. VA 23529; Jeffrey D. Shields. Virginia
Institute of Marine Science. Gloucester Point. VA 23062.

Spiny lobsters have few reported diseases. The identification
and prevalence of pathogenic diseases in wild populations of spiny
lobsters are poorly known. We recently discovered the first patho-
genic viral disease (HLV-PAl known from a lobster, in this case
the Caribbean spiny lobster. Paniilirus argiis. Our findings suggest
that the disease alters the behavior and ecology of this species in
fundamental ways, in part via remarkable changes in the social
behavior of healthy individuals in response to diseased conspecif-
ics. Both field and laboratory data show significant avoidance of
infected lobsters by healthy conspecifics. Since 1999. we have
identified infected juvenile lobsters at 75% to 100% of the 14
nursery habitat sites that we have surveyed twice a year (summer
and winter) in the middle and lower Florida Keys, USA. The
disease is highly prevalent with prevalences of up to 40% in ju-
veniles (mean = 8%). Infected animals are often moribund, ex-
hibit lethargy, and have milky or chalky hemolymph. Prevalences
in initial challenge trials using infected hemolymph from infected
donors resulted in a prevalence of 90% (n = 20, control n = 10).
The virus appears to be highly pathogenic and moderately lethal
with deaths occurring after 60-90 days in inoculation trials. An
intriguing epidemiological twist is that commercial and recre-
ational fishing activities for this economically valuable species
may potentially contribute to the spread of the pathogen.

FIELD OBSERVATIONS ON THE DEVELOPMENT AND
PROGRESS OF A SHELL DISEASE EPISODE FOR
AMERICAN LOBSTER IN RHODE ISLAND: 1995-2001.
Kathleen Castro. University of Rhode Island Fisheries Center.
Kingston. Rl; Thomas Angell. Rl Department of Environmental
Management. Division of Fish and Wildlife, Wakefield. Rl 02880.
From 1995 to 2001. shell disease in the American lobster was
monitored in Rhode Island waters including Nairagansett Bay,
Rhode Island and Block Island Sound and the offshore areas of
Block and Hudson Canyons. A tag-recapture study conducted by
Rl lobstermen also included a shell disease category. In the inshore
population, a significant increase in frequency and severity of the
disease was documented beginning in 1996, reaching an 31% pro-
portion infected overall in 2001. Spatial information is available
and describes a rapid increase in the Upper East, Lower East and
West Passages from 1997-1998. Proportions in the Upper East
Passage and West Passage in Narragansett Bay continue to rise,
while the proportion infected in the Lower East Passage has de-
clined. Proportion observed with the disease is correlated with the
molting period, with rapid increases in September and October
after the major molt. In 2000 and 2001, there appears to be a shift
to higher infection rates in small animals and greater number of
males and immature females than previously observed. Tag-
recapture data is providing information on the progress of the
disease on individual lobsters and re-infection percentages after a
molt. Field data such as these may provide valuable information
concerning the causes and consequences of the disease on the
lobster populations.

EFFECTS OF PARASITES ON BEHAVIOR OF GRASS
SHRIMP. PALAEMONETES PVGIO. Terry Glover.* Social/
Behavioral Sciences, Bloomfield College, Bloomfield, NJ 07003;
Lauren Bergey and Judith S. Wets, Department of Biological

Sciences, Rutgers University — Newark, Newark. NJ 07102.

The effect of parasites on grass shrimp Palaemonetes piigio
was studied using shrimp from three sites which had varying levels
of contamination. There was an inverse relationship between level
of contamination and level of parasites. Parasitized shrimp had
either Microphallus .v/).. Microphalhts hyperparasitized by Uro-
spurklium sp.. or Prohopynis pamlalicoUi parasites. Microphallus
and its hyperparasite are endoparasites within muscle, while P.
paiiJaliciila. which lives in the gill chamber, is considered an
ectoparasite. Parasitized shrimp were compared to unparasitized
controls on latency to swimming in a novel environment, activity,
freezing to a startle stimulus, and latency to finding food. Shrimp
from the least contaminated site had the longest latencies and
lowest activity levels. Shrimp w ith P. paudalicola had the longest
freezing times. Shrimp with Urosporidiwu tended to have longer
swimming latencies than controls. Microphallus did not system-
atically affect behavior, even in shrimp in which they were nu-
merous. Although site differences have a greater effect on behavior

National Shellfisheries Association, Mystic, Connecticut

Abstracts. 2002 Annual Meeting, April 14-18. 2002 391

than parasite load, there is a complex relationship between con-
tamination, parasite levels and behavior.

CYTOCENTRIFUGE PREPARATIONS: AN ALTERNATE
METHOD TO EXAMINE THE HEMOCYTES OF THE
AMERICAN LOBSTER HOMARUS AMERICANUS. Barbara
Hornej and Andrea Batti.son,* Department of Pathology and
Microbiology: Allan Mackenzie, The AVC Lobster Science Cen-
tre, Atlantic Veterinary College, University of Prince Edward Is-
land, Charlottetown, PE. Canada, CIA 4P3.

Current techniques used for examination of crustacean
hemocytes can be labour intensive, costly, and have a slow turn-
around time as is the case with transmission electron microscopy.
Phase contrast microscopy, although inexpensive and rapidly pei-
formed. does not provide a permanent record of the results.

Cytocentrifugation of anticoagulated hemolymph samples, fol-
lowed by staining with a modified Wright" s-Giemsa stain, pro-
vided an excellent means to evaluate the hemocytes of Homarus
americanus. The technique is simple to perform, requires rela-
tively inexpensive laboratory equipment, and provides a perma-
nent record of results within one hour of sample collection. Opti-
mal results were obtained when slides were prepared within 6 to 8
h of sample collection.

Preliminary findings using hemolymph samples from the rock
crab. Cancer irroratiis suggest that this technique could be com-
patible for the examination of hemocytes of other crustacean spe-
cies.

PARASITES IN DIPOREIA SPP. AMPHIPODS FROM
LAKES MICHIGAN AND HURON. Gretchen A. Messick*

NCAA, National Ocean Service. Cooperative Oxford Laboratory,
Oxford. MD 21654: Tom F. Nalepa NOAA. Great Lakes Envi-
ronmental Laboratory, Ann Arbor, MI 48103.

Diporeia populations began to decline in Lake Michigan in
1992, just three years after the nonindigenous zebra mussel
Dreissena polymorpha was first reported. Diporeia are detriti-
vores, feeding upon organic material freshly settled from the water
column. In turn, they are fed upon by most fish species found in
Lake Michigan and are a major food-web link between pelagic
production and upper trophic levels. Although the decline in Di-
piircia populations was thought to be due to zebra mussels inter-
cepting food material (i.e., algae) before it settles to the bottom,
sampling efforts indicate sufficient food is still available to the
aniphipods. and Diporeia lipid content remain high, indicating the
population is not deprived of food. An alternative explanation for
the amphipod population decline may be pathogens.

Diporeia aniphipods were sampled to assay the prevalence of
disease and see whether prex alence of disease varied by time or
location. Surveys revealed numerous parasites in amphipod tissues
including virus, rickettsia-like microorganisms, fungus, a hap-

losporidan. microsporidans, external ciliates, gregarines, and
worms. Prevalence of nodules and parasites varied among surveys,
dates sampled, and locations sampled. No one etiologic agent has
been identified as causing the amphipod population decline but
several parasites identified during this investigation including mi-
crosporidans, rickettsia-like microorganisms, haplosporidan. and
fungus likely result in amphipod mortalities.

PATHOLOGICAL ALTERATIONS IN THE EYES OF THE
AMERICAN LOBSTER. HOMARUS AMERICANUS. IN-
FECTED WITH PARAMOEBA SP. Jeffrey D. Shields.* Vir

ginia Institute of Marine Science, Gloucester Point, VA 23062.

In September, 2001. 31 lobsters, Homarus americanus. from
Western Long Island Sound were examined for pathologies asso-
ciated with infection by Paramoeba sp. Only 1 animal (3.2%)
presented with gross morbidity and pathology indicative of a pre-
sumptive infection by Paramoeba sp. Histologically, light to mod-
erate infections were observed in 35.5% of the lobsters. Several
altered tissues were observed in the lamina ganglionaris, optic
nerve complex and ommatidia of the eyes. Eye pathologies ranged
from minor shifts or los.ses of optic pigments associated with the
optic nerves, to necrosis of the optic nerve with a complete shift of
pigments into the ommatidia. In more severe cases, the optic
nerves were obliterated, with a partial to complete disruption of the
basement membrane proximal to the ommatidia. Necrosis of the
retinular cells varied with the severity of the damage to the optic
nerves. There was marked infiltration of hemocytes into the space
formerly occupied by the optic nerves, and infiltration of
hemocytes into the ommatidial complex. In addition, infected ani-
mals showed relatively high intensities of non-specific granulomas
in many tissues. The high prevalence of non-specific granulomas
indicates that the lobsters in WLIS continue to be subjected to a
variety of disease-inducing stressors such as parasitic or microbial
infection, or exposure to metals or other toxicants. Interestingly,
the severity of the pathology in the eyes was not well correlated
with the intensity of infection of the amoebae in the optic nerves.
However, more lobsters should be examined to fully assess sever-
ity in relation to disease.

A PATHOGENIC HERPES-LIKE VIRUS FROM THE
SPINY LOBSTER. PANULIRUS ARGUS. Jeffrey D. Shields,*

Virginia Institute of Marine Science, Gloucester Point, VA 23062:
Donald C. Behringer. Jr.. Old Dominion University. Norfolk,
V.^ 23529: Mark J. Butler, IV, Old Dominion University, Nor-
folk, VA 23529.

A pathogenic herpes-like virus was diagnosed from juvenile
Caribbean spiny lobsters from the Florida Keys. Moribund lobsters
had characteristically milky hemolymph that lacked the ability to
clot. With light microscopy, the virus infected hyalinocytes and
semigranulocytes, but not granulocytes. Infected hemocytes had

392 Abstracts. 2002 Annual Meeting, April 14-18, 2002

National Shellfisheries Association, Mystic, Connecticut

emarginated, condensed chromatin, hypertrophied nuclei and faint
nuclear bodies resembling Cowdry-type-A inclusions. With elec-
tron microscopy, the large (187 nm ± 15 nm sd.) icosahedral.
capsid-enclosed nonoccluded virions were diffusely spread around
the border of the condensed chromatin with virogenic stroma
present in the cytoplasm and free in (he hemolymph. In some
ca.ses. virions were found in connecti\e tissue cells. Virion and
capsid assembly occurred w itinn the nucleus of infected cells, but
envelopes were not apparent. The virus ranged in prevalence over
time from 6% to 89^ with certain foci reaching up to 40'7f. The
virus was transmissible to uninfected lobsters using inoculations of
raw hemolymph from infected animals and through feeding trials.
Inoculated animals showed morbidity and began dying from the
virus after 60-80 d. Adult lobsters have not been observed with the
infection. Additional infection trials, impacts on the social behav-
ior of juveniles, and a TEM study to identify the agent are cur-
rently underway.

BLUE MUSSEL BIOLOGY AND CULTURE

OBSERVATIONS ON GROWTH, GAMET0GP:NESIS, AND
SEX RATIO OF TRIPLOID AND DIPLOID MYTILUS EDU-
LIS. John Brake,* Hatfield Marine Science Center. Newport. OR
97365: Jeffrey Davidson, Atlantic Veterinary College. Charlotte-
town. PEL Canada. CIA 4P3; Jonathan Davis Baywater Inc..
Bainbridge Island. WA 98110.

Concerns in the Prince Edward Island mussel aquaculture in-
dustry over product quality during, and immediately after the
spawning period prompted research on de\eloping triploid Mytihis
edidis. Triploid shellfish are sterile and retain quality during and
after the spawning period.

Field evaluations of diploid and triploid mussels demonstrated
that triploids had a greater growth rate than diploids. The growth
difference was evident in just nine months after deployment in
highly productive waters. This difference was not detectable in less
productive waters until the second year of growth, suggesting pos-
sible differential growth of triploids versus diploids, related to
environment. Mussels in the less productive waters were notably
less sexually mature in the first year, therefore the differential
performance of triploids may have been related to spawning. Trip-
loids had a mean shell length 1 .05% larger than diploids in the less
productive waters versus 8.09% larger in the highly productive
waters. Triploids examined after a spawning event showed no
histological evidence of spawning, while 7l'/r of diploids showed
some evidence of spawning. -Shell length, relative soft tissue
weight, and condition index were all higher in triploids. This re-
sulted in an increase in dry tissue weight of 62.82% and a mean
shell length increase of 10.95% when triploids were compared to
diploids at one site after the local spawning event. As well, a
highly skewed sex ratio confirmed a previous study on Mytihis

ifiiUiiproviinuilis showing a male dominant sex ratio in triploid
mussels.

THE DISTRIBUTION AND BIOLOGY OF AN INVASIVE
TUNICATE IN PRINCE EDWARD ISLAND, CANADA. Jeff

Davidson* and Frank Boothroyd, Atlantic Veterinary College,
University of Prince Edward Island, 550 University Avenue, Char-
lottetown. Prince Edward Island. Canada, CIA 4P3; Neil McNair,
PEI Department of Fisheries, Aquaculture and Environment, P.O.
Box 2000. Charlottetovvn. Prince Edward Island, Canada, CIA
7N8; Thomas Landry, Department of Fisheries and Oceans. Sci-
ence Branch. Maritimes Region. Gulf Fisheries Center, P.O. Box
5030. Moncton. New Brunswick. Canada. EIC 9B6.

The presence of the club tunicate Styeki cliiva was recently
noted in Eastern Prince Edward Island (PEI). Canada. This tuni-
cate presents a significant fouling problem for the blue mussel
{Mytihis ediilis) farms located in affected areas. The spread of this
new tunicate species in the waters of PEI is presently limited to a
few rivers and seems to be mainly from anthropological mode as
opposed to natural mode. ln\estigation on its reproductive biology
is being conducted through the monitoring of gonad development
and seasonal recruitment. Preliminary results are suggesting that
spawning may occur throughout the summer months, while re-
cruitment is limited to a relatively short period. The impact of this
new fouling organism is also being investigated by evaluating its
competition for food in relation to the blue mussels. Methods to
control this fouling problem are presently being developed and
tested. The eradication of this invasive tunicate from PEI waters is
considered impractical and therefore the development of farm
management strategies is considered as the only economically vi-
able solution.

A FIELD STUDY OF SETTLEMENT DEPTHS OF MYTI-
LUS EDULIS AND M. TROSSULUS IN NOVA SCOTIA,
CANADA, 1998-2000. Ellen L. R. Kenchington, Kenneth R.
Freeman,* Benedikte M. Vercaemer. and Barry W. Mac-
Donald, Department of Fisheries & Oceans. Bedford Institute of
Oceanography. PO Box 1006. Dartmouth. NS. Canada. B2Y 4A2.
Larvae of Mytihis ediilis and M. trossiiliis were shown previ-
ously to have different settlement preferences in the laboratory.
However, it was not known whether these results could be extrap-
olated to field conditions with the much greater degree of envi-
ronmental variability. Consequently, both temporal and spatial
variability in larval settlement between these species, with depth
were examined in the field. The relative proportion of M. edulis
spat was determined using genetic markers at each of 3 depths (1,
3, 5 m) at intervals over the spawning season (minimum 3 collec-
tions) for 3 consecutive years at one site in Parrang Cove, St.
Margaret's Bay. Nova Scotia. In the last year a second site in Ship
Harbour, Nova Scotia was studied to add a spatial dimension to the

National Shellfisheries Association, Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 393

project. There was no significant interaction between sites, years
and timing with respect to depth of settlement. M. edulis consis-
tently settled deeper than M. trossiiliis although there was both
temporal and spatial variability in the relative percentages of M.
cdtilis collected. In addition to opening up certain ecological ques-
tions, these results show promise for direct application to hus-
bandry practices at mussel farms having both species and where
aquaculturists wish to preferentially collect the more commercially
desirable M. edulis.

significant spatial and temporal variation in the number of settlers
observed on these collectors. Using molecular techniques, we have
also estimated the frequency of M. trossidus and M. edulis spat in
each sample. Appreciable frequencies of M. edulis spat were ob-
served at all locations throughout much of the experiment where-
a.se sizeable frequencies of M. twssuhis were more temporally
restricted. Our results suggest that species-specific differences in
post-settlement mortality must be invoked to e.xplain populations
containing exclusively M. twssulus adults within Cobscook Bay.

NUTRIENT UPTAKE AND RELEASE FROM FOULING
ORGANISMS ASSOCIATED WITH CULTURED MUSSELS
IN TRACADIE BAY, PEL Angeline R. LeBlanc* and Gilles
Miron, Universite de Moncton, Moncton, NB El A 9B6; Thomas
Landry, Department of Fisheries and Oceans, Science Branch.
Maritimes Region. Gulf Fisheries Center. P.O. Box 5030. Monc-
ton. New Brunswick. Canada. EIC 9B6.

Fouling organisms are causing concerns among mussel growers
in PEL Canada. Most of these foulers are sedentary filter feeders,
and are therefore a potential competitor with mussels for resources.
This could translate into a reduction in meat yield in mussels. We
carried an experiment to determine the relative impact of fouling
organisms on the uptake and release of nutrients. Chlorophyll a.
ammonium, suspended particulate matter and oxygen were inves-
tigated. This preliminary study was undertaken in December 2000.
before the ice cover, and showed that foulers had only a small
effect on nutrient use. Foulers accounted for about one tenth of
chlorophyll a consumption by mussels and foulers together. They
also contributed about one tenth of the ammonium released by
mussels and foulers. There was no significant use of suspended
particulate matter by the mussels or the foulers. The use of oxygen
was not significantly different between mussels and mussels with
foulers. A temporal and spatial investigation has been initiated in
2001 and preliminary results will be discussed.

GENETIC ASPECTS OF THE BLUE MUSSEL (MYTILUS
EDULIS AND MYTILUS TROSSULUS) HYBRID ZONE IN
ATLANTIC CANADA. Marcelo Miranda* and David Innes,

Dept. Biology. Memorial University or Newfoundland. St. Johns,
NF, AlC 3X9; Raymond Thompson, Ocean Science Center, Me-
morial University of Newfoundland, St. Johns. NF. AlC 3X9.

The blue inussel (Mytilus edulis and M. trossidus) hybrid zone
in Atlantic Canada provides an opportunity to study the process of
speciation. adaptation and species interaction. Species composition
in different size classes was determined at several aquaculture sites
and natural populations in Newfoundland using nuclear DNA
markers. M. irossulus and hybrids occur at a higher frequency in
the smaller size classes and M. edulis dominates in larger size
classes. This pattern suggests that M. tnissuius in these areas may
have a higher mortality rate or a shorter life span than M. edulis.
In addition, offspring from inter and intra-specific crosses were
reared in the laboratory to study the dynamics of hybridization and
species differences. Fertilization rate, larvae abnormality and
sperm competition were used to estimate barriers to hybridization
at the gamete stage. Growth rate and survival of the different
families were compared during the larval stage, spat (6 months)
and until maturity (18 months). These data were used to assess
hybrid fitness and also to compare the performance of both spe-
cies, which recently had been a major concern to the mussel in-
dustry.

SPECIES-SPECIFIC SETTLEMENT PATTERNS OF BLUE
MUSSELS IN COBSCOOK BAY, MAINE. Afton McGowen.
Matthew Gordon, and Paul D. Rawson,* School of Marine Sci-
ences. University of Maine. Orono, ME 04468-5751.

The blue mussels Mytilus edulis and M. trossulus are sympatric
throughout much of the Canadian Maritime Provinces, as well as
in easternmost Maine. We have observed marked variation in the
frequency of adult M. trossulus mussels among sites within Cobs-
cook Bay. Maine. Little is known regrading the degree to which
local variation in species-specific larval supply versus post-
settlement mortality determine the relative frequency of this spe-
cies in Gulf of Maine mussel populations. To examine the impor-
tance of larval supply, we deployed and sampled mussel spat col-
lectors (ropes) on a monthly basis at three separate sites within
Cobscook Bay during the summer and fall of 2000. There was

SMALL SCALE DISTRIBUTION OF MYTILUS EDULIS
AND M. TROSSULUS IN THE BAIL DES CHALEURS AND
THE GASPE PENINSULA. Valerie Moreau* and Edwin
Bourget, GIROQ. Departement de biologic. Universite Laval, Ste-
Foy. Quebec. Canada. GIK 7P4; Rejean Tremblay, Centre
Aquicole Marin-Universite du Quebec a Rimouski. Grande-
Riviere, Quebec, Canada, GOC IVO.

Mytilus edulis and M. trossulus are the two species found in the
Bale des Chaleurs and the Gaspe Peninsula. To date, few studies
bring out the effects of environmental factors (temperature, salin-
ity, tidal height, wave action) on the local or regional distribution
of these two species. Mussels were sampled on rocky shores ac-
cording to a factorial design including six locations, three degrees
of wave exposure (exposed, semi-exposed, sheltered) and two in-

394 Abstnuts. 2002 Aiiiuial Meeting, April 14- IS. 2002

National Sliellfisheries Association. Mystic. Connecticut

tertidal level (mid and low). PCR amplification using a diagnostic
DNA marker (Gtu-5) was used to distinguish species. Species
relative frequencies show no clear patterns of distribution with
wave exposure or tidal height. Although, there appears to he a
pattern of distribution at the regional scale, this pattern could not
be related to salinity or temperature gradients observed.

MUSSEL CULTURE IN A MIXED SPECIES (M. EDUUS
AND M. TROSSULUS) ZONE— SOME COMMERCIAL IM-
PLICATIONS. Randy VV. Penney,* M. J. Hart, and N. Temple-
man, Department of Fisheries and Oceans. Science. Oceans and
Environment Branch. P. O. Box 5667. St. John's. Ntld. Canada.
AlC 5X1.

The island of Newfoundland. Canada, is a zone where two blue
mussel species. M. ediilis and M. trossulus, overlap in distribution.
Typically, indigenous populations at most sites contain mixtures of
both species and hybrids. Sites in clo.se proximity (eg. <10 kilo-
meters) often vary as much in their relative species proportions as
sites hundreds of kilometers apart. Intra-site growth variability is
significantly affected by the mixed-species nature of these stocks.
In a 14 month commercial rearing trial of rope-cultured mussels
from sleeving to harvest at three farm sites, the M. edulis stock
component had higher growth rates in shell and total weight than
the sympatric M. trossulus at all three farms, while inter-specific
shell length growth rates were different at only one farm. Growth
rates of hybrids were typically intermediate between the two. Mor-
tality rates were similar between sympatric M. edulis and M. tros-
sulus at all three sites. We conclude that naturally-occurring stock
genetic variability may have a significant impact on commercial
production indices at farm sites within zones of species overlap.

differentiation or a reduction in genetic diversity among the At-
lantic coast populations. These results are contrary to what would
be expected if Gulf of Maine M. trossulus populations have only
recently been established.

BEHAVIOR AND GROWTH OF JUVENILE MUSSELS
mYTlLUS SPP.) IN SUSPENDED CULTURE SOCKS. Ju-
dith Scnechal* and Jon Grant, Oceanography Opt, Dalhousie
University. Halifax. NS B3H 4J1.

Suspended mussel culture is based on loading high densities of
juvenile mussels into mesh socks, and hanging Ihem from floats.
This leads to severe intraspecific crowding, and potentially reduces
growth and mussel yield. The highly mobile juveniles position
themselves according to size, shell gape, ambient current and food,
among other factors. Despite this critical stage in culture, there are
little data on behavioral mechanisms that lead to adult density and
growth rate. We conducted a series of field and laboratory experi-
ments with culture socks to examine the effects of stocking den-
sity, species (Mytilus edulis and Mytihis trossulus) and environ-
ment on early development of the culture population. //; situ pho-
tography was used to capture a time series of mussel images in
experimental socks at a commercial farm in Ship Harbor. Nova
Scotia. Moored CTD-current meters and water sampling were used
to characterize the sites. Image analysis on mussel size, gape, and
position was used to quantify temporal changes over several
months in the socks. In the laboratory, socks were hung in a tall
flow-through tank with variable temperature, current speed and
direction, allowing controlled experiments on mussel emergence
and growth. Initial results are discussed in the context of optimiz-
ing husbandry practices.

CLADISTIC ANALYSIS OF GENETIC DIFFERENTIA-
TION BETWEEN POPULATIONS OF THE BLUE MUS-
SEL, MYTILUS TROSSULUS. Paul D. Rawson, School of Ma-
rine Sciences. University of Maine. Orono. ME 04468-575 I .

Two species of blue mussel. Mytilus edulis and M. trossulus.
have overlapping distributions on the Atlantic coast of North
America. Populations containing a mixture of these species and
their hybrids are commonly observed throughout the Canadian
Maritime Provinces. Recent work, in my lab, has shown that the
range of M. tro.'^sulus extends well into the Gulf of Maine, much
further south than previously observed. In this study. I have em-
ployed a DNA sequence-based cladislic analysis to examine
whether the presence of M. trossulus in the Gulf of Maine is due
to a recent range expansion. DNA .sequences for a portion of the
female mitochondrial lineage D-loop region (-670 base pairs)
were obtained from 140 M. trossulus by PCR amplification and
direct sequencing. Analysis of these sequences indicates there is
significant genetic divergence between Atlantic and Pacific M.
trossulus populations. In contrast, there was noexidence of genetic

UPDATE ON THE DISTRIBUTION OF TWO MUSSELS
SPECIES {MYTILUS EDULIS AND MYTILUS TROSSULUS)
IN THE QUEBEC MARITIME REGIONS. Benoit Thomas,*

Centre aquacole marin, MAPAQ. Grande-Riviere. Quebec. GOC
IVO; Valerie Moreau and Rejean Tremblay, Centre aquacole
marin-Universite du Quebec a Rimouski. Grande-Riviere, Quebec,

GOC IVO.

This study attempted to determine the distribution of the two
species of mussels and hybrids along the main Quebec maritime
regions of the Gulf of St. Lawrence, as well as evaluate the bian-
nual pattern of distribution among different sites. Following 1996
near shore mussel sampling, we started a study based on mussel
spat fixed on artificial collectors immersed at 8 sites along the 460
km long Gaspe peninsula and at 5 sites along some 1000 km of the
North Shore of the Gulf of St. Lawrence. Biannual sampling was
conducted at these sites following initial results obtained in 1997.
At all sites, collectors and thermographs were immersed at 2 m in
mid-June (sea bottom depth: IS m) and saniplcil in mid-October of
the same year. Sampling the next June was also conducted along

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 395

sites located along the North Shore, where spat growth was found
to be low the first season and site accessibility was limited. In 1996
and 1 997. analyses were done by electrophoresis on MPI and by
diagnostic DNA marker (GLU-5) for other years. According to the
initial two year results, a predominance of M. irnssiilus occurs on
the northern side of the Gaspe peninsula as well as in the extreme
east of the North Shore. The best sites for presence of M. ediilis
was in the mid-region of the Gaspe peninsula as well as the west-
ern side of the North Shore. Results show that some sites are
variable, with yearly variations. This annual change or steadiness
in the proportion of the two species will permit mussel growers to
orient towards better M. editlis spat provision sites; accepting, for
now. the hypothesis that the commercial value of M. trossulus is
lower.

PERFORMANCE OF MYTILUS EPULIS AND MYTILUS
TROSSULUS IN THE GULF OF ST. LAWRENCE: A LABO-
RATORY EXPERIMENT. Rejean Tremblay* and Valerie
Moreau, Centre aquacole niarin-Universite du Quebec a
Riniouski, Grande-Riviere, Quebec. GOC IVO; Thomas Landry,
Gulf Fisheries Center. DFO. Moncton. New Brunswick. EIC 9B6;
Bruno Myrand, Station technologique Maricole. MAPAQ. Cap-
aux-Meules. Quebec. GOB 1 BO; Cyr Couturier, Marine Institute,
Memorial University of Newfoundland. St John's. Newfoundland.
AIB 3X5.

During the past 15 years, reciprocal transfer experiments with
mussels. Mxtiliis ediilis and A/, trossulus in Gulf of St. Lawrence
have shown that their performance, expressed in term of growth
and survival, is different between stocks. Moreover, the relative
performance level of a stock may vary between sites. These results
suggest that the performance of mussels is probably genetically
based. The genetic diversity of mussels in the Gulf of St. Lawrence
could be very important in relation to their ability to adapt to the
highly variable environmental conditions. The goal of this study, is
to characterise the performance of different mussel stocks and to
determine the relation between physiological parameters and ge-
netic traits, for both species of mussels. These analysis were con-
ducted on mussels spat sampled from artificial collectors in Que-
bec. Prince Edward Island, New-Brunswick and Newfoundland.
Bi nionthy growth and survival data was collected from twelve
different mussel stocks maintained in laboratory with non-treated
seawater. over a one year period. Temperature, seston and chlo-
rophyll-a were monitored. Physiological measurements, including
scope for growth and basal metabolism, as well as genetic analysis,
were conducted on each stock. Genetic analysis included species
determination by PCR technique and genetics variability deter-
mined by electrophoresis technique. Results will be discussed in
terms of mussel culture strategy for spat supply.

BLUE MUSSELS AS MODEL SYSTEMS TO INVESTI-
GATE PALLIAL CAVITY FUNCTION IN BIVALVES.
J. Evan Ward,* and Sandra E. Shuniway. Department of Marine
Sciences. L'niversity of Connecticut. Groton. CT 06340; Jeffrey S.
Levinton. Department of Ecology & Evolution. S.U.N.Y.. Stony
Brook. NY 1 1 794.

For over fifty years, mussels in the genus Mytiliis have been
used as model .systems to study aspects of bivalve behavior, physi-
ology, and genetics. Because of their relatively simple, non-
plicate, homorhabdic ctenidium, mussels are also ideal animals in
which to investigate general mechanisms of particle handling. In
this talk we will discuss our studies on particle capture, transport,
and selection in mussels and how they have furthered our under-
standing of pallial cavity function and its connection to ecosystem
processes. In particular, we will examine where gaps in our knowl-
edge exist and compare and contrast feeding processes in mussels
with those of other bivalves.

For our studies, mussels were delivered polystyrene tracer par-
ticles alone or in combination with defined diets consisting of
either ground, aged Spartiua sp. (3-IOp,m). similar sized phy-
toplankton (Rhodoinonas sp.), or a 50/50 mixture of both at three
concentrations (10\ 10"*, lO'' particles ml"'). Particle capture,
transport, and handling by the pallial organs were studied in vivo
by means of video-endoscopy and discrete samples of particulate
material were collected from various ciliated tracts. Image analysis
was used to track particle movement on the ctenidia and labial
palps, and flow cytometry was used to analyze samples for evi-
dence of particle selection. Particle depletion and handling time
experiments were also performed to measure residence times on
the ctenidia and labial palps. Results indicate the following: 1 .
ctenidal filaments are directly involved in particle capture. 2. diet
quality has little effect on particle handling mechanisms. 3. diet
quantity has significant effects on particle handling mechanisms,
and 4. particle selection is confined to the labial palps. Studies
such as these will lead to a better understanding of pallial organ
function in mussels, and allow us to better model the critical lim-
iting factors that mediate particle-feeding in bivalves and ulti-
mately affect the trophic dynamics of benthic ecosysteins.

HARMFUL ALGAL BLOOMS

EFFECTS OF CLAY, USED TO CONTROL HARMFUL AL-
GAL BLOOMS, ON JUVENILE HARD CLAMS, MERCE-
NARIA MERCENARIA. Marie-Claude Archambault* and Jon

Grant, Oceanography Dpt. Dalhousie University. Halifax, NS
B3H 4JI; Monica Bricelj, Institute for Marine Biosciences, Na-
tional Research Council of Canada. Halifax, NS B3H 3ZI; Don
Anderson, Biology Dpt.. Woods Hole Oceanographic Institution.
Woods Hole, MA 02543.

Increased interest in using ecologically inert clays to mitigate
harmful algal blooms at nearshore mariculture sites has prompted
studies on the effectiveness of this method on prolific U.S. blooms,

396 Abstracts. 2002 Annual Meeting. April I4-I,S. 2002

Nalional Shellfisheries Association. Mystic. Connecticut

such as Florida's neurotoxic blooms of Kaiciiia brevis. Potential
repercussions of this control strategy revolve around the increasing
flux of suspended particles to the benthos. Ju\enile suspension
feeding bivalves are potentially vulnerable as they could suffer
burial and a decrease in clearance rates and/or increase in pseu-
dofeces production in response to suspended clay, leading to re-
duced growth and delay in attaining size refuge from predators.
The main objective of the research was to assess lethal and sub-
lethal effects on juvenile hard clams. Mercenaria mercenaiui. in a
two-week flume application of phosphatic clay (by-product of
phosphate mining) to a simulated bloom of a non-toxic dinotlagel-
late (Heterocapsa triqiietra or Pinrocentnim mkaiis). Flow re-
gimes simulated two extreme conditions, representing end mem-
bers of a continuum expected in the field, a) where low flow
allowed complete settling and formation of a sediment layer, and
b) where high tlow maintained complete particle resuspension. No
clam mortalities occurred in either treatment. The sedimentation
treatment showed variable growth inhibition in shell and/or tissue,
but effects were not significant compared to controls (no sediment
layer), and clams rapidly resumed siphon contact with the overly-
ing water column. In contrast, a highly significant growth effect
(-90?^ reduction in shell and tissue growth) occurred in trials with
suspended clay compared to no-clay controls. Analysis of particle
size-spectra, using a sampling method designed to maintain the
integrity of floes, showed evidence of clay tlocculation, such that
clay particles were found above the 100% retention efficiency size
limit of the clam gill. These results suggest that repeated clay
applications in the field are likely more detrimental to clams under
flow conditions leading to prolonged /// siik resuspension of clay
than under conditions that promote rapid sedimentation.

HISTORY. SOME RFXENT HAB EVENTS, AND THEIR
IMPACTS ON SHELLFISH AND FINFISH IN WASHING-
TON STATE. Rita A. Horner,* School of Oceanography, Uni-
versity of Washington. Seattle. WA 98195-7940.

Harmful algal blooms and their effects, especially paralytic
shellfish poisoning (PSP). have a long history in the Pacific North-
west starting in June. 1793. when one of Captain George Vancou-
ver's crew members died and four others became ill after eating
mussels in central British Columbia. The next known occurrence
was in May, 1942, when three people died and eight became ill
from eating clams or mussels in Barkley Sound, British Columbia,
and three others died near Port Angeles, Washington, after eating
clams. Investigations suggested that only beaches on the Strait of
Juan de Fuca and the open Pacific coast were affected and the
Washington Department of Fisheries issued an annual closure of
these areas from April through October. Since then, PSP has
spread to all of western Washington's inland marine waters, except
Hood Canal, and is also present along the open Pacific coast and
in coastal estuaries. Beach closures are frequent and human ill-
nesses continue to occur, albeit infrequently, even with increased

monitoring. In 1991. domoic acid was found in razor clams and
Dungeness crabs on Washington's Pacific coast, where it contin-
ues to be found sporadically in razor clams with consequences for
their harvest. The causative organisms, several species of the dia-
tom genus Pseiido-iiitzscliia. are sometimes present in bloom con-
centrations in inland waters of Puget Sound and in coastal estuar-
ies, but if domoic acid occurs, levels are low and no closures have
been required. No confirmed cases of amnesic shellfish poisoning
(ASP) caused by domoic acid have been reported in the state.
Other potentially harmful phytoplankton species, including Het-
erosigina akashiwo and Chuetoceros spp. occur here, primarily
affecting finfish in net pens. Reactive oxygen species and possibly
an unknown toxin are associated with Heterosi^ina. while Cha-
etoceros effects are mechanical. Thus, western Washington waters
harbor a variety of potentially harmful marine phytoplankton spe-
cies that continue to plague shellfish and finfish growers in more
places and usually without warning.

EFFECTS OF THE TOXIC DINOFLAGELLATE, KAREMA
BREVIS, ON LARVAL MORTALITY AND JUVENILE
FEEDING BEHAVIOR IN THE BAY SCALLOP, AR-
GOPECrEN IRRADIANS. Jay R. Leverone* and Norman J.
Blake. College of Marine Science. University of South Florida. St.
Petersburg. FL 33701.

Florida populations of the bay scallop, Aiiiopccten irnuliaiis,
occur in areas prone to recurring blooms of the toxic dinofiagel-
late. Karenia brevis [=Cyinnocliiiiiiiii breve). These blooms can
have serious consequences for bay scallop recreational fisheries,
aquaculture and restoration activities. Surprisingly, there are few
published data regarding the effects of A', brevis on any aspect of
bay scallop biology or ecology in Florida. A series of laboratory
experiments was conducted to observe the direct effects ot expo-
sure to A.', brevis on early life stages of the bay scallop, A. irradi-
ans. For bay scallop larvae, an LC^,, of 900 cells/ml K. brevis was
calculated after a seven-day exposure. Larval development was
delayed and metamoi-phosis inhibited at concentrations above 500
cells/ml. Larvae responded similarly when exposed to either
"whole" or sonicated "lysed" cultures of K. brevis. Clearance rates
of juvenile scallops (5-15 mm shell height) were size dependent
and significantly (p = 0.05) reduced at A', brevis concentrations of

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 397

50 cells/ml and higher. At 100 cells/ml. reduced clearance rates
resulted in slower growth and reduced weight gain after one-week
exposure. At 500 cells/ml. clearance rates were irreversibly af-
fected even 20 hours after a spiked exposure. Findings will he
discussed in light of recent efforts to restore bay scallops along the
west coast of Florida.

DETRIMENTAL EFFECTS OF A RECENT PRYMNESWM
ISOLATE FROM BOOTHBAY HARBOR. MAINE (USA)
UPON JUVENILE BAY SCALLOPS. ARGOPECTEN IRRA-
DIANS. Gary H. Wikfors* and Jennifer H. Alix, NCAA Fish-
eries, NEFSC. Milford, CT 06460 USA; Roxanna M. Smolowitz.
Marine Biological Laboratory. Woods Hole. MA 02543; Lacey
Wallace. Southampton College of LIU. Southampton. NY 1 1968;
Helene Hegaret. Ecole Nationale Superieur Agronomique de
Rennes. Rennes. France.

The brackish-marine flagellate Piyiiiiu'siiini has been known to
be toxic to tmfish for decades. Symptoms in fish exposed to toxic
PnniiH'siiitii include copious mucuous production and bleeding
from the gills, and death by asphyxiation. Two strains of Piyin-
iifsium. isolated recently by Dr. R.R.L. Guillard were tested for
ichlhyotoxicity using cunner (Taiitogolabnis adspersus): we con-
finned toxicity and histopathology of one isolate, strain 97-20-1.
Further, we determined and documented effects of the toxic Ptym-
iwsiiiin strain (97-20-1) to bay scallops. Argopecten iiradians.

In one exposure experiment. 10-mm scallops were placed in
basins of ultrafiltered seawater. and cultured (bacteria-free) Piym-
iicsiiiiii 97-20-1 were added to achieve a cell density of 10' cells/
ml. Controls were scallops given an equivalent quantity of Iso-
chiysis sp. (strain T-ISO) or an equivalent volume of algal culture
medium. Scallops given T-ISO fed normally and produced fecal
pellets; whereas, scallops exposed to Prymnesium twitched vio-
lently, produced pseudofeces and abundant mucous, and eventu-
ally displayed valve gape that was not responsive to stimulation.
Within 24 hours, all scallops exposed to Prymnesium were mori-
bund or dead. Histopathological analysis revealed severe, acute,
total or near-total necrosis of the digestive gland and ducts, gill,
and other tissues in scallops exposed to Pryinnesiiim and normal
tissue histology in controls.

In a later experiment, larger scallops (50-mm) were exposed to
Prymnesium 97-20-1 (again with T-ISO as the control), and
hemolymph samples were removed after 2.5 hours for analysis of
hemocytes by flow-cytometry. Significantly more dead hemocytes
were seen in scallops exposed to Prymnesium than to T-ISO. but
no differences in aggregation and adherence or phagocytosis of
plastic microbeads were detected. We believe this to be the first
report of HAB effects upon the bivalve immune system. These
experiments revealed effects of an ichthyotoxic microalga upon
scallops ranging from behavioral to histopathological and immu-
nological, and may serve as a template for studies of additional
HAB-bivalve trophic interactions.

SHELLFISH BIOLOGY

BURROVMNG-INDUCED INTERNAL FRACTURES AND
EXTERNAL ABRASION IN SHELLS OF THE HARD
CLAM MERCENARIA MERCENARIA FROM RARITAN
BAY, NEW JERSEY. Richard R. Alexander.* Department of
Geological and Marine Sciences. Rider University, Lawrenceville.
NJ 08648; Robert M. Baron. Institute of Marine and Coastal
Studies. NOVA Southeastern University. Fort Lauderdale. FL
33004.

The hard clam Mercenaria mercenwiu from Raritan Bay. New
Jersey, reburrowed once a month into sediment in flow-through
tanks for a period of one year. Twelve specimens, between 34 and
38 mm dorsal-ventrally. reburrowed in each of five different tex-
tured sediments, namely 100% mud. lOO'-i- sand, admixture of
25% by volume ground shell hash and 75% mud. admixture of
257r shell hash and 75% sand, and admixture of 50% sand and
50% mud. Lacking tidal flushing through the tanks, mean shell
growth, dorsal-ventrally. was one mm/yr. Shell accretion was sta-
tistically significantly depressed in sand and shell-sand vs. mud.
and mud-shell substrata, a disparity attributed to repeated abrasion
of valve margin's during monthly reburrowing in coarser textured
sediments. Clams in sand and shell-sand had more concentric, fine
ornament or lamellae worn smooth relative to specimens in mud
and mud-shell mixtures. Upon sacrifice. 30 clams revealed "stuc-
coed." internal fractures initiated at the shell margin. Five linear
fractures radiate dorsally. Seven cracks curve or diagonal from the
shell margin. Three show right angle deflections, twice through
adductor muscle scars. Four merge or branch from the margin. In
1 2 specimens, fractures are in both valves. In ten specimens, frac-
tures were faintly visible on valve exterior. In four and two speci-
mens, respectively, fractures were initiated at the posterior or an-
terior margin. Repaired fractured shells are randomly distributed
among sediment textures. Nevertheless, fractures were probably
induced and/or exacerbated by the reburrowing process.

INFLUENCE OF ENVIRONMENT AND FOOD SUPPLY
ON SURVIVAL OF CRASSOSTREA GIGAS LARVAE: A
MODELING STUDY. Eleanor A. Bochenek* and Eric N. Pow-
ell, Rutgers University, Raskin Shellfish Research Laboratory,
6959 Miller Avenue, Port Norris, NJ 08349; John M. Klinck and
Eileen E. Hofmann, Center for Coastal Physical Oceanography,
Old Dominion University, Norfolk. VA 23529.

A biochemically-based model was developed to simulate the
growth, development, and metamoiphosis of larvae of the Pacific
oyster. Crassnstrea gigas. The model is unique in that it: 1 ) defines
larvae in terms of their protein, neutral lipid, polar lipid, carbohy-
drate, and ash content. 2) tracks weight separately from length to
follow larval condition index, and 3) includes genetic variation in

398 Ahsiructs. 2002 Aniuial Meeting. April 14-18. 2002

National Shellfisheries Association. Mystic. Connecticut

growth efficiency and egg quality to better simulate cohort popu-
lation dynamics. Simulations show that departure of temperature,
salinity or food content from optimum levels reduces larval cohort
survival, generally either because some larvae fail to metamor-
phose successfully or because an increase in larval life span in-
creases losses to predation. Also, different food compositions pro-
duce widely varying survivals at the same food concentration. The
simulations suggest that the ratio of the combined carbohydrate
and lipid pools to protein may best describe the overall quality of
the food. In simulations emphasizing genetic variability within the
cohort, larvae with high growth efficiency originating from large
eggs outperform other egg quality-growth efficiency combinations
for most environmental variables, including temperature, salinity,
and food content. In contrast, whereas the simulations suggest that
the influence of suboptimal temperature, salinity, or food content
is to compress genetic variation by uniformly favoring high growth
efficiency and large eggs, the simulations with food quality pro-
vide evidence of a mechanism that would expand genetic variation,
because variations in food quality favor a much broader range of
genetic types. The simulations support the supposition that food
quality is an important variable controlling larval cohort success.

GROWTH CHARACTERISTICS OF ARGOPECTEN GIB-
BUS JUVENILES REARED IN TWO SUSPENDED CUL-
TURE SYSTEMS. Andrew T. Cogswell and Samia Sarkis, Ber

niuda Biological Station for Research. Inc.. 17 Biological Lane.
Ferry's Reach. St. George's, Bermuda, GE 01.

Grow-out of hatchery reared calico scallops {Argopecten gih-
bus) is conducted in Bermuda's inshore waters using .square Irish
scallop trays and triangular Japanese pearl nets. Both enclosures
have advantages and disadvantages in both maintenance and han-
dling throughout the grow-out phase. To assess optimum technol-
ogy, yielding minimum labour demand and maximum scallop
growth and survival, a comparative study was performed.

A pool of juvenile scallops was distributed in triplicate in 6.0
mm pearl nets, and in scallop trays lined with 6.4 mm black poly-
ethylene "pouches". Scallop growth and survival were monitored
monthly in trays and pearl nets of comparable mesh size and
stocking densities ranging from 0.20 scallops-cm~- to 0.10 scal-
lops-cm"". At this time, sub-samples (n = 50) were collected from
the pool of triplicates and both shell and tissue growth of juscnile
scallops was recorded.

Preliminary results revealed that scallops cultured in pearl nets
achieved higher growth rates than scallops in trays from juvenile to
market size. Results also imply that the limiting effect of trays on
scallop growth is immediate above the initial height and weight
used in this experiment and increases until scallop growth nearly
ceases. Comparisons for scallops grown in pearl nets and trays are
made using Statview statistical package. Evaluation of trays and
pearl nets as grow-out enclosures is discussed in terms of optimal
grow-out strategy.

GROWTH AND DISPERSAL STUDIES OF MYA
ARENARIA USING A NUMERICAL FLOW MODEL. W. R.
Congleton, Jr.,* Marine BioResources; B. R. Pearce, Civil and
Environmental Engineering; M. Parker, Marine BioResources.
Univ. of Maine, Orono, ME 04469.

Growth: Current velocities averaged over the flood tide were
estimated by a numerical flow model and by clod cards for loca-
tions in an Eastern Maine bay and were compared to the annual
shell size increment of clams collected at the same locations. Sta-
tistical models including initial shell size, year of sample, high-low
current category estimated by clod cards or the numerical model
and interactions explained 57-58% of the variability in growth
increment after a difference transformation. High current simu-
lated growth, although the effect on growth increment was less
than that of sample year or initial size. The adjusted least squares
mean for the growth increment at the sites with low flow, as
identified by clod cards that averaged 4.35 ± .37 cm/s was 9.56 ±
.247 mm. and low flow that averaged 2.99 ± .43 cm/s using the
numerical model was 9.51 ± .274 mm. High flow sites averaging
5.86 ± .62 cm/s using clod cards had estimated growth increments
of 1 1 .90 ± .323 mm and high sites averaging 5.84 ± .46 cm/s using
the numerical model had estimated growth increments of 1 1 .70 ±
.33 mm.

Dispersal: Clam populations, particularly in eastern Maine, are
prone to recruitment failure of larvae onto intertidal flats due to the
large tidal amplitude and resulting high flushing rates. Larvae
move offshore during a larval development period that is extended
in cool waters in the summer along the eastern coast. High vari-
ability in ME landings, mudtlat sampling of settlement, spat bag
studies of larval distributions are consistent with this theory. Pres-
ently, studies using Eulerian and LaGrangian numerical flow mod-
els are being utilized to determine the effect of tidal magnitude,
length of dispersal period and variation in coastal geology on
larval dispersal.

ESTIMATION OF INGESTION AND BIODEPOSITION
RATES OF THE PACIFIC OYSTER. CRASSOSTREA Gl-
GAS. IN A COASTAL LAGOON OF NW MEXICO. Zaiil
CJarci'a-Esquivel,* Marco A. Gonzalez-Gomez, and Francisco

Ley-Lou, Universidad Autonoma de Baja California. Apdo. Postal
453. Ensenada, B.C. Mexico.

Four short-term (6-8 h) experiments were carried out at San
Quintin Bay (SQB) in 1999 (June, October) and 2000 (January,
April) in order to estimate "in situ" ingestion rates (IR) of the
Pacific oyster, Cmssosrn'a gigas. Experiments were carried out by
pumping seawater directly into experimental trays containing adult
oysters (94 ± 1 .7 cm shell height. 3.64 ± 0.45 g). Total and organic
particulate matter (TPM and POM, respectively) were measured
every 1 .5 h in the feces and pseudofeces produced by oysters and
in the input seawater. Clearance and ingestion rates were calcu-
lated by using the ash balance method. In situ POM/TPM ratio

National Shellfisheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting, April 14-18. 2002 399

(0.15 to 0.30) and oyster clearance rates (2 to 7.5 L h~') were
inversely related to TPM concentration (2-14 mg L"' ). while feces
(7.9-13.7 mg TPM oyster"' h"') and pseudofeces production (4.8-
13.2 mg TPM oyst"' h"' ) were directly proportional to TPM con-
centration. Net organic ingestion rates (3-4.2 mg OM h~') were
not dependent on total particle concentration. It is suggested that
particle resuspension plays an important role in regulating the
variability of food quality in SQB, but such variability does not
affect the net amount of organic matter ingested by C. gigos
throughout the year.

KFFFXT OF DIETARY PROTEIN/ENERGY RATIO ON
GROWTH AND METABOLISM OF JUVENILE GREEN
ABALONE {HALIOTIS FVLGENS). Laura E. Gomez,* FES

Cuautitlan UNAM. Mexico; Ma. Teresa Viana and Zaiil Garcia-
Esquivel, UABC. BC. Mexico: .Armando Shiniada, UNAM,
Mexico: Louis R. D'Abramo, MSU. USA.

Juvenile green abalone (initial size = 1 1.84 ± 1.003mm) were
fed different practical diets formulated to contain different P/E
ratios (mg/kcal) ranging from 62 to 108 mg/kcal. The dietary
protein level increased from 25.8 to 44.1% while the level of
energy remained constant (4056 to 4154 cal/g). After 61 days in a
How through system at 2 1 C. growth of abalone fed diets with a P/E
ratio of 100 and 108 mg/kcal (SGR = 2.42 ±0.11 and 2.51 ±0.10)
was significantly greater than that of abalone fed the other diets.
Food intake and therefore ingested energy per g of abalone were
similar for all dietary treatments. Protein and energy digestibility,
amonia excretion and oxygen consumption were determined after
the growth experiment was terminated. Intake of digestible energy
was different among dietary treatrnents, but intake of digestible
protein did not change. Abalones ingest food to satisfy their energy
requirements, and digestibility of protein and energy decreases as
the P/E ratio increases. The most efficient diet for growth probably
consists of a 100 P/E ratio with lower levels of dietary protein and
energy.

MODELING THE GROWTH OF THE HARD CLAM, MER-
CENARIA MERCENARIA. John Kraeuter* and Eric N. Pow-
ell, Rutgers University. Haskin Shellfish Research Laboratory,
6959 Miller Avenue, Port Norris, NJ 08349: Eileen E. Hofmann
and John M. Klinck, Center for Coastal Physical Oceanography,
Old Dominion University, Norfolk. VA 23529: Ray Grizzle,
Jackson Estuarine Laboratory. 85 Adams Point Road, Durham.
NH 03824: Monica Bricelj, Institute for Marine Biosciences.
National Research Council. 1411 Oxford Street. Halifax, NS, B3H
3Z1, Canada: Stuart Buckner, 23529 Town of Islip, Environmen-
tal Control, 401 Main Street. Islip, NY 1 1751.

A physiologically-based model that simulates the growth of the
hard clam. Mercenariu merceiuiria. in response to environmental
conditions of temperature, salinity, and food supply has been de-

veloped. The processes included in the clam model are those that
are responsible for changes in clam soft tissue weight, length, and
condition. Changes in clam soft tissue weight over time result from
the difference in assimilation and respiration. Changes in clam
condition are determined from a length-weight relationship that is
reflective of average clam growth. Changes in clam length
(growth) occur only when condition index is greater than zero,
which happens when the clam has attained excess weight for a
given length. No change in length occurs if condition index is zero
(mean case) or negative (less weight than expected at a given
length). Initial simulations with the clam model, that used allo-
metric relationships to relate weight and length, resulted in growth
rales that did not match those observed for hard clams over a range
of weights and lengths. However, when weight and length are
related using a third-order polynomial the simulated growth rates
match observed rates. The implication of this result is that small
clams are heavier for a given length and that large clams are longer
for a given weight. This approach represents a change in the way
that models are formulated for growth of clams and potentially
other bivalve species. Additional simulations illustrate the effect of
changing salinity and food environments and genetic variation in
growth efficiency and respiration on hard clam growth and sur-
vival.

INFLUENCE OF SHORT TERM VARIATIONS IN FOOD
SUPPLY AND CRITICAL PERIODS ON SURVIVAL OF

CRASSOSTREA GIGAS LARVAE. Eric N. Powell* and

Eleanor A. Bochenek, Rutgers University, Haskin Shellfish Re-
search Laboratory, 6959 Miller Avenue, Port Norris, NJ 08349:
John M. Klinck and Eileen Hofmann, Center for Coastal Physi-
cal Oceanography, Old Dominion University, Noii'olk. VA 23529.
A biochemically-based model was developed to simulate the
growth, development, and metamorphosis of larvae of the Pacific
oyster, Cmssostrea gigcis. The model defines the larvae in terms of
protein, lipid, carbohydrate, and ash content and includes genetic
variation in growth efficiency and egg quality. This model is used
to investigate the premise that certain periods of larval life are
more critical than others with respect to the availability of food and
that food quality is as important as food quantity. Simulation re-
sults indicate that critical periods in larval life do exist. However,
the critical portion of larval life depends on the structure of the
food that the larvae encounter. Overall, the most critical time is
late in larval life as the larvae approach metamorphosis. Increased
protein at this time always improves larval survival. Increased lipid
has the most effect about midway in larval life, but also exerts a
positive impact late in larval life. However, exposure to certain
types of food early in larval life can dramatically change cohort
survival. Additional simulations show that larvae with high growth
efficiency are more successful, as are larvae coming from large
eggs. Changes in food quantity influence larval survival primarily
by varying the length of larval life. Changes in food quality, how-

400 Abstracts. 2002 Annual Meeting, April 14-18, 2002

National Shellt'isheries Association, Mystic, Connecticut

ever, by restricting the range of genotypes in the cohort that sur- suggest that a management approach which annually detmes a 50

vive. as well as by varying larval life span, produce large changes meter buffer surrounding existing SAV beds would adequately

in survivorship. The simulations support the adaptive advantage of protect SAV while minimizing areas legally unavailable but oth-

larval cohorts with a relatively wide range of genotypes and sug- erwise suitable for clam aquaculture. thus minimizing conflict,
gest the important inHiicnce of variations in food quality in main-
taining aenetic variability.

POPULATION STRUCTURE OF THE HARD CLAM, MER-
CENARIA MERCENARIA. IN HAMPTON ROADS, VIR-
GINIA. Melissa J. Southworth,* Juliana M. Harding;, and
Roger Mann, Department of Fisheries Science. Virginia Institute
of Marine Science. Gloucester Point. VA 2.'?062.

The hard clam. Mcnenaiui iiHixenuna. supports one of the
most valuable fisheries in the Virginia Portion of the Chesapeake
Bay. In recent years with the displacement of oystermen moving to
clam harvesting as an alternative source of income, there has been
a gradual decline in catch per unit effort in the clam fishery.
Despite growing concern for the decreasing catch and the increas-
ing pressure of proper management there has only been one survey
in recent years that focused on stock assessment for management
purposes (Wesson. 1995). Preceding this the last substantial sur-
veys were prior to and just after Hurricane Agnes in the early
1970's. As such a survey of the Hampton Roads area was carried
out in the summer of 2001. The survey documented the distribu-
tion of the cunent demographics including size and age structure
of the hard clam stocks. The cuirent distribution of hard clams
exists over a cline of differing salinity and bottom types. From this
we compare site versus size. age. and the frequency of recruitment.
The frequency of recruitment, size, and age is then compared to the
environment. Comparisons of historic data and current survey data
shov\ changes over decadal time scales that are useful indicators of
long term environmental change in the Hampton Roads area.

AN EXAMINATION OF POTENTIAL CONFLICT BE-
TWEEN HARD CLAM AQUACULTURE AND SAV IN THE
LOWER CHESAPEAKE BAY. Helen Woods,* Ken Moore,
and Carl Hershner, Center for Coastal Resources Management.
Virginia Institute of Marine Science. Gloucester Point. VA 2.^062.
The Commonwealth of Virginia wishes to promote both hard
clam iMcneiiaria ttwixeiuiria) aquaculture and the growth of sub-
mersed aquatic vegetation (SAV) along its shallow subaqueous
bottom. Conflict can arise in areas where aquacullurists plant
clams in SAV habitat and/or when SAV coloni/es an area being
used for aquaculture. This project examined the issue of bottom
use conflict on the Eastern Shore of Virginia involving clam
aquacullurists and SAV interest groups. This issue was examined
by studying historic SAV and aquaculture trends, creating spatial
CIS models to predict areas suitable for the growth of SAV and the
development of clam aquaculture. and examining the current law s
in Virginia and neighboring states which affect this issue. Results

SCALLOP BIOLOGY AND CULTURE

POSTLARVAL DEVELOPMENT OF THE GILLS AND IM-
PLICATIONS FOR FEEDING IN THE SEA SCALLOP,
PLACOPECTEN MAGELLAN ICUS. V. Monica Bricelj,* Anne
Veniot and Celine Barre, Institute for Marine Biosciences, Na-
tional Research Council. 1411 Oxford St.. Halifax. NS B3H 3ZI.
Canada: Peter Beninger, Laboratoire de Biologic Marine, Faculte
des Sciences. Universite de Nantes. 44322 Nantes, France.

Hatchery-reared bivalves often experience poor growth and
survival during post-settlement stages, yet limited information is
available on postlarval moiphogenesis and feeding, especially in
scallops. Our research uses scanning electron microscopy to docu-
ment critical changes during the ontogeny of pallial feeding organs
(gills, mantle, foot, lips and labial palps) in sea scallops (and
recently in bay scallops, Argopeclen irnuUans) and their correla-
tion with size, between 0.3 and 14 mm in shell height (SH). We
present here the results concerning gill development in Pki-
copecten iiuigelUiniciis. Pronounced changes in morphology of the
filibranch gill produce a transition from a homorhabdic gill basket
to the heterorhabdic, W-shaped. plicate gill characteristic of adults.
Suspension-feeding is probably rather ineffective prior to reflec-
tion of the inner demibranch (occuiring at - 1 mm SH and followed
by accelerated proliferation of gill filaments), and formation of the
outer demibranch (completed at ~2 mm SHi. The onset of the
heterorhabdic condition via differentiation of principal filaments,
which allows bidirectional particle transport and thus the potential
for selection and ingestion volume regulation on the gill, occurs
fairly late in development, at -3.3 to 5.0 mm. Full plication of the
gill is only achieved at -7 mm SH. This protracted development in
P. nnif^elUinicKs contrasts with that in oysters, which undergo more
rapid gill differentiation and metamorphosis. We thus demonstrate
that the gills are relatively undifferentiated at 1-2 mm SH, when
sea scallops are commonly transferred from the hatchery to field
niuseries by commercial growers. We are cunentlv investigating
the relationship between these morphological changes and evolv-
ing mechanisms of food capture and ingestion, in order to better
define stage-specific nutritional requirements and growth perfor-
mance during nursery culture of this commercialh important spe-
cies.

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting, April 14-18. 2002

401

THE EFFECTS OF STOCKING DENSITY IN PEARL NETS
ON SURVIVAL, GROWTH, AND REPRODUCTIVE PO-
TENTIAL OF THE BAY SCALLOP, ARGOPECTEN IRRA-
DIANS IRRADIANS. Maureen Davidson. New York State De-
partment of Environmental Conservation. Bureau of Marine Re-
sources. 205 North Belle Mead Road. East Setauket. NY 11733.
In order to investigate the influence of slocking density on
scallop production, hatchery reared bay scallops were held in pearl
nets at three densities. 80/pearl net. 240/per net and 800/per net for
67 days during the summer. Surviving scallops were counted and
shell heights were measured to determine growth. The animals
were transferred to lantern nets and stocked at two different den-
sities. 50/tier and 200/tier. and overwintered, grouped by their
initial densities in the pearl nets. The following spring survival,
shell heights, and gonadal index (a measure of reproductive po-
tential) were detemiined. Survival and growth were inversely pro-
portional to stocking density in both pearl and lantern nets. Stock-
ing density in the lantern nets was found to have a greater effect on
overwintering survival than did the density in pearl nets. Gonadal
indices indicated that scallops initiated spawning at the same time,
regardless of density treatment. There was no significant pearl net
effect observed on gonadal index. Bay scallops cultured for direct
market should be held at low densities in pearl and lantern nets to
maximize growth and survival. Bay scallops raised for resources
restoration should be held at moderate densities in pearl nets and
low densities in lantern nets to maximize survival and reproductixe
potential.

DEVELOPING A COASTAL MAINE SEA SCALLOP EN-
HANCEMENT PROGRAM. Scott Feindel* and Daniel Schicli.

Maine Department of Marine Resources. W. Boothbay Harbor.
ME 04575.

Increasing the stock of wild scallops along the Maine coast by
capturing wild seed at spatfall. holding them captive through what
would be a time of high mortality in the wild and then releasing
them has captured the imagination of many Maine scallopers. This
concept builds upon a Japanese methodology that was exported
successfully to New Zealand several years ago. We are working
collaboratively with several groups of fishermen with assistance
from a New England Consortium grant. The project is currently
into its third season of spat collection and millions of scallop seed
were released this last fall in several locations along the coast from
last years spatfall.

Future work will involve continued collection of spat, defining
the best locations for spat collection, monitoring survival of re-
leased seed, looking at increasing survival through holding the
seed beyond the first year, addressing the issues of ownership and
adjusting harvest practices to maximize yield.

This paper will focus on current results and near future efforts.

GROWTH AND MOVEMENT OF SEA SCALLOPS IN THE
SOUTHERN PART OF THE GREAT SOUTH CHANNEL
ON GEORGES BANK: A TAGGING STUDY. Brad Harris*
and Kevin Stoltesbury, Intercampus Graduate School of Marine
Science and Technology. University of Massachusetts Dartmouth.
706 Rodney French Boulevard. New Bedford. MA 02744-1221.

A rotational fisheries management strategy is being considered
for the sea scallop. Placopecten maniieUanicns. fisheries of the
northeast United States. To implement a rotational management
strategy site-specific information on sea scallop population dy-
namics is required. For example, the sea scallop is the best swim-
mer of the 400 known species of scallops, and can move as much
as 1 5 km on Georges Bank. This could influence the size of the
rotational areas closed or open to harvesting. Further, sea scallop
growth rates show substantial variability over their geographic
range. This could influence the time period rotational areas are
open or closed. To begin the development of a site-specific data
set. a sea scallop growth and movement experiment was conducted
in the southern part of the Great South Channel of Georges Bank.
Approximately 13,000 scallops were tagged and released in May
2001. Presently. 677 tagged shells have been returned showing
movement of up to 7 km. and growth spanning more than 9
months. This preliminary data illustrates the need for further site-
specific movement and growth experiments over the sea scallops
ranae.

INVESTIGATIONS WITH TRIPLOID ATLANTIC SEA
SCALLOPS, PLACOPECTEN MAGELLANICUS. Daniel L.
Jackson,* Barry W. MacDonald, Shaka .James, Benedikte Ver-
caeiner. and Ellen L. Kenchington, Fisheries and Oceans
Canada. Bedford Institute of Oceanography. Halifax. Nova Scotia,
Canada B2Y 4A2: Andre Mallet. Mallet Research Services Ltd..
Dartmouth, Nova Scotia, Canada, B2X 3H3,

Bivalve aquaculture has benefited from the introduction of trip-
loids, but successful commercial production of triploid sea scal-
lops, Placopecten magellaniciis, remains elusive. Experiments
were conducted to determine the optimum combination of incu-
bation temperature (8^C and U^C) and Cytochalasin B (CB) con-
centration (0.25 mg/1 and 0.50 mg/1) for inducing triploidy in scal-
lops. Two replicate spawning lots of larvae were produced (Lot 1 ;
one female parent, one male; Lot 2: two females, two males), and
ploidy analyses were performed via fiow cytometry on Day 4 to
determine the initial levels of triploid induction. In the 8^C incu-
bation treatments, more triploids were found amongst the larvae
from Lot 1 (~24<7f in both CB treatments) than in Lot 2 ( 139f in the
0.25 mg/1 CB treatment, and 16% in the 0.50 mg/1 CB treatment),
numbers similar to those found in the untreated controls (16'7f in
Lot 1 and 20% in Lot 2). Among embryos incubated at 14°C. both
levels of CB produced -60% triploids in Lot 1. while in Lot 2 more
triploids were produced in the 0.50 mg/1 CB treatment (61%) than
in the 0.25mg/l CB treatment (39%). Many triploids were found in

402 Ahsrnicl.s. 2002 Annual Meeting. April 14-18. 2002

National Shclltisheries Association. Mystic. Connecticut

the 14 C controls (41'! in Lot I and 22'r in Lot 2l. Studies
comparing feeding rate.s between diploid and triploid sea scallop
larvae will also be discussed.

A COMPARISON OF MICROALGAL DIETS FOR EN-
HANCED PRODUCTION OF PLACOPECTEN MAGEL-
LANICUS POSTLARVAE. Lisa M. Milke* and V. Monica
Bricelj, Institute for Marine Biosciences. National Research Coun-
cil. Halifax. NS B.^H ,^ZI; Christopher C. Parrish, Ocean Sci-
ences Centre. Memorial University of Newfoundland. St. John's.
NF AlC 5S7. Canada.

Little is known concerning stage-specific diets that optimize
hatchery production of the sea scallop, Pkicopeclen inagellaniciis.
especially during vulnerable, post-settlement stages. Poor growth
and survival of these developmental stages may be related to nu-
tritional deficiencies. Therefore, our primary objectives were to
identify high-performance algal diets, involving a minimum num-
ber of species, for cost-effective implementation in commercial
hatcheries, and to determine algal properties (e.g., size, biochemi-
cal composition) which may contribute to differences in scallop
growth. We also compared the lipid composition (including essen-
tial fatty acids) of postlarvae and their diets. To this end. P. ma-
gellanicus postlarvae (initial mean shell height = 388 p.m) were
exposed for 28 days at I4°C to five mixed microalgal diets, each
consisting of one diatom and one tlagellute, (at a constant, volume-
equivalent concentration of 40 T-Iso cells p.r') in 400 L meso-
cosms. Diets consisted of one of three diatom species (Thalassio-
sira H'eissflogii. Chaetoceros imielleri. or Fntgilaiia familica). and
one of three flagellates [Pavlova lutheri. Pavlova sp. (CCMP 4.'i9l.
or Tetraselmis striata (PLAT-P)]. Shell growth trajectories, deter-
mined by video imaging, showed that F. familica. a previously
untested isolate from Mahone Bay, Nova Scotia, was the diatom
yielding the highest growth rate (22.6 |jim/day). Pavlova sp.
(CCMP 459). which is known to support excellent growth of sea
scallop larvae, yielded the highest growth rate (28.1 (ini/day) of
the three flagellates, perhaps due to its high levels of ARA (20:
4n6: arachidonic acid). Although T. striata is known to be an
excellent diet for oyster (Crassostrea virgiiiica) spat, it resulted in
the lowest scallop growth rate for both shell height (5.5 jxm/day)
and ash-free dry weight, providing a final mean size almost half
that of scallops fed the highest performing diet. Poor performance
of this diet may be partly related to the very low levels of DHA
(22:6n-3; docosahexaenoic acid; as a % of total fatty acids) in its
two algal constituents. Sea scallop growth rates on the two highest
performing diets were comparable to maximum literature values
for this developmental stage using 4 to 6-species diets.

IMPACTS AND CONTROL OF THE SPIONID POLV-
CHAETE. POLYDORA WEBSTERI ON THE SEA SCAL-
LOP, PLACOPECTEN MAGELLANICiS IN MIDVVATER
CULTURE. Shawn M. C. Robinson,* Jim D. Martin and Eddy
J. Kennedy, Dept. Fisheries and Oceans, St. Andrews Biological
Station. 531 Brandy Cove Rd.. St. Andrews. New Brunswick.
Canada, E5B 2L9.

Biofouling on culture structures and species is a normal phe-
nomenon to shellfish growers in all countries, however, some foul-
ing species are more damaging than others. In 1997, we initiated
an experiment to look at the full-cycle grow-out characteristics of
the sea scallop at a site in Passamaquoddy Bay that was judged to
be the best oceanographically for culture based on food and cir-
culation. Animals were grown at a depth of 5m and 20m from the
surface in either pearl nets or lantern nets depending on the size of
the scallops at the time. Nets were changed monthly during the
period of highest biofouling (late spring to mid fall) and as re-
quired outside of that window. The results of the 20m treatment
revealed that the shells were severely perforated by the polychaete
Polydora websteri causing death. We postulated that the growing
of the scallops in midwater effectively isolates them from their
normal ecosystem with regular interspecific interactions (such as
grazers) and allows commensal/parasitic organisms to grow out of
control. Therefore, in the summer of 2001. we initiated a second
phase to the project where we introduced juvenile green sea ur-
chins grazers (Strongylocentrotus droehachieiisis) to some experi-
mental cages. Initial results indicated that the sea urchins were
effective in reducing the density oi Polydora on the scallop shells.
Further results will be discussed during the talk as the study is still
in progress.

DEVELOPMENT OF A STOCK ASSESSMENT PROGRAM
FOR VVEATHERVANE SCALLOPS IN ALASKA. Gregg E.
Rosenlvranz* and Douglas Pengilly. .Alaska Department of Fish
and Game. 211 Mission Road, Kodiak, AK 99615.

Guideline harvest levels for Alaska's weathervane scallop iPa-
linopecten cauriniis) fishery are currently based on historic aver-
age catches. Although vessels participating in the fishery are sub-
ject to a mandatory observer program that monitors catch and
effort, research indicates that these statistics cannot reliably index
changes in scallop abundance. In 20(J0. the Alaska Department of
Fish and Game began work aimed at development of fishery-
independent stock assessment methods that could ultimately he
used to manage the fishery based on estimates of exploitable abun-
dance. To date, we have made four research cruises that surveyed
known scallop beds with a video drop camera, a lowed video sled,
and a New Bedford offshore survey dredge. Results indicate that
the drop camera does not survey enough area to provide good
density estimates. The video sled appears to be a more promising
tool, but reviewing tapes to obtain scallop counts is time consum-
ing. We are now experimenting with using the video sled to esti-

National Shellfisheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 40.^

mate dredge efficiency. A combination of video and dredge survey
methods may ultimately prove to be the best choice for assessing
weathervane scallop populations in Alaska.

BIOCHEMICAL INDICATOR OF GIANT SCALLOP PLA-
COPECTEN MAGELLAiMCUS QUALITY: LARVAL
GROWTH. COMPETENCY AND SETTLEMENT. Fabrice
Pernet.* GIROQ. Universite Laval. Cite universitaire. Quebec.
Canada, GIK 7P4; Rejean Tremblay. Centre Aquicole Marin. 6
rue du Pare C.P. 340. Grande-Riviere. Quebec. Canada. GOC 1 VO:
Edwin Bourget, Rectorat a la recherche. Universite de Sher-
brooke, Quebec, Canada, JIR 2R1.

The purpose of this study was to monitor the lipid class content
of larvae over the entire cycle, to verify the potential effect of
varying feeding regimes on larval lipid content, quality, growth
and survival, to evaluate the potential use of lipid class ratios to
forecast larval growth and survival, and tlnally. to examine the
effect of larval quality on .settlement behavior and success. When
larvae were able to feed from exogenous sources at day 4. three
diet treatments were applied. Larvae were periodically sampled for
lipid class analysis, growth measurement and survival assessment.
Behavior of pediveliger larvae for each diet treatment were re-
corded uith a videotape and endoscope set-up during settlement
period. During the course of experiment, lipid class composition of
the three diets were different in terms of triacylglycerol (TAG) rich
in saturated and monounsaturated fatty acids. Our study show that
TAG le\'el in larval food was positively correlated with growth
rate. TAG content and as a consequence, larval quality — as ex-
pressed with TAG-sterol (ST) or TAG-phospholipid (PL) ratios —
prior to settlement. A positive relation between number of com-
petent larvae produced and larval quality at day S has been found,
suggesting that survival at competency was partly explained by the
recovery efficiency of energetic reser\es after enibryogenesis. Lar-
val quality was correlated with settlement success in the way that
higher quality larvae has lead to poor settlement but explore the
same time whatever the age. whereas low quality larvae decrease
exploration time with age. As a consequence, the low settlement
success observed in our experiment with high physiological con-
dition larvae might be the effect of metamorphosis delay in re-
sponse to poor environmental conditions.

EXAMINATION OF SEA SCALLOP, PLACOPECTEN MA-
GELLANICUS, AGGREGATIONS USING A VIDEO SUR-
VEY IN CLOSED AREAS OF GEORGES BANK. Kevin D. E.

Stokesbury* and Brad Harris, School for Marine Science and
Technology. University of Massachusetts Dartmouth. 706 South
Rodney French Boulevard. New Bedford. MA 02744-1221.

Georges Bank is the world's largest natural scallop resource.
During the summer months of 1999/2000/2001 SMAST in asso-
ciation with the scallop industry developed and conducted 23

video surveys on Georges Bank. These surveys produced a series
of maps of the sea floor of Georges Banks containing high aggre-
gations of sea scallops. The video survey detailed the distribution
of substrate, depth, number of live and dead scallops, and macro-
invertebrates (sponges, starfish, filamentous fauna). The video
technique allow s a previously unattained precise, accurate measure
of these variables and allows correlation analy.ses between them.
Further, the closed areas of Georges Bank have scallop densities
higher than any previously observed. For example, the three areas
surveyed in 1999 (1940 km") contained approximately 652 million
scallops representing approximately 17 million kilograms (32 mil-
lion lbs worth approximately S161 million) of harvestable scallop
meats. This research addresses scallop stock assessment and the
critical regional and national issue of the effects of mobile fishing
gear on the marine benthic community. It has direct implications
for rotational fisheries management, on an appropriate spatial scale
(km), under consideration by the New England Fishery Manage-
ment Council.

PARASITE AND HOST DEFENSES

THE EFFECT OF PH ON THE KILLING ACTIVITY OF
HEMOCYTES IN THE PACIFIC OYSTER, CRASSOSTREA
CIGAS. Steven M. Allen* and Louis Burnett, Grice Marine
Laboratory. University of Charleston South Carolina. 205 Fort
Johnson Rd.. Charleston. SC 29412.

In recent years there has been an increase in the occurrence of
summer mortalities of the commercially important Pacific oyster.
Crassostrea gigus. These mortalities occur during the late summer
when water and air temperatures are at their highest. C. gigas are
grown intertidally and are. therefore, air exposed for hours at a
time. An oyster closed during air exposure depletes the oxygen
stores within the shell and builds up CO, acidifying the tissues.
The average pH of hemolymph from an oyster which is submerged
in well aerated water ( 18 C) and ventilating is 7.52 (0.04 SEM: N
= 35). The average pH of of hemolymph from an oyster which is
aerially exposed for 4h in 30X air is 6.83 (0.02 SEM; N = 26).
We hypothesize that stresses associated with air exposure inhibit
the immune system of the oyster and contribute to the summer
mortalities. The focus of the present study was to determine if the
innate immunity provided by hemocytes was decreased by low pH.
The ability of hemocytes to kill the bacterium Vibrio para-
luieinolyticus was assessed using an in viiro killing assay.
Hemocytes were treated with low pH and challenged in vitro with
V. parahaemolyticus. A tetrazolium dye reduction assay was used
to quantify the number of viable bacteria, from which a killing
index was calculated. No significant difference was found between
the two treatments pH 7.6 and pH 6.6 (p < 0.01; N = 14). ODRP
Grant No. NA96RG0488.

404 Ahslnicts, 2002 Annual Meeting, April 14-18. 2002

National Shellfisheries Association. Mystic. Connecticut

MUCOID SECRETIONS PROTECT QPX FROM ANTIMI-
CROBIAL AGENTS. Robert S. Anderson* and Brenda S.
Kraus, Chesapeake Biological Laboratory, University of Mary-
land Center lor Environmental .Science, P. O. Box 38, Solomons.
MD 20688; Sharon McGladdery. Oceans and Aquaculture Sci-
ence. 200 Kent Street. Ottawa. Ontario KIA OE6; Roxanna
Smolovvilz, Marine Biological Laboratory. 7 MBL Street. Woods
Hole, MA 02343.

Quahog parasite unknown (QPX) has recently caused signifi-
cant mortality in aquacultured Mercenaria menenaria in Massa-
chusetts. It secretes a viscous, mucoid substance which may pro-
tect the parasite from host defense mechanisms; it has been re-
ported that clams injected with QPX washed free of the mucus coat
did not develop infections or disease. In this study the antimicro-
bial activity of M. mercenaria serum was measured against QPX
with, or washed free of. its secreted coat. Massachusetts QPX
cultures were grown for 7 days in the presence or absence of clam
serum in the medium; the parasites were still in log stage growth.
When coat-free QPX were added to serum-containing medium,
dose-dependent growth mhihition was seen in cultures with 10-50
lig/ml serum protein; lower concentrations were often slightly
stimulatory, higher concentrations produced -100% inhibition.
Growth of cultured QPX, when fully enveloped by mucus, was not
inhibited by clam serum proteins at all concentrations tested (<60
(jLg/ml). If aliquots of coat-free QPX were incubated for various
time intervals (during which time they produced mucoid enve-
lopes) prior to the addition of clam serum to the cultures, there was
a time-dependent reduction of serum-mediated growth inhibition.

MEASUREMENT OF VIBRIO TAPETIS CYTOTOXIC AC-
TIVITY ON RUDITAPES PHILIPPINARVM HEMOCYTES
BY FLOW CYTOMETRY. Gwenaelle Choquet.* Philippe
Soudant. Christophe Lambert, and Christine Paillard. lUEM
UBO, LEMAR, Technopole Brest-Iroise, 29280 Plouzane,
France; .lean-Louis Nicolas. LPL IFREMER, Plouzane,
France.

Vihriii tapetis is the causative agent of Brown Ring Disease
which affects the clam. Ruditapes philippinanim. After incubation
with V. tapetis. hemocytes loose filopods and become rounded,
indicating production of a virulence factor by the bacteria. To
rapidly quantify this factor, a flow-cytometric test has been devel-
oped. This test is based on the capacity of V. tapetis to inhibit
adhesion of clam hemocytes to plastic. Several bacteria/hemocyte
ratios, other Vibrio spp. pathogenic to bivalves and various V.
tapetis isolates have been tested. Inhibition of adherence is detect-
able with as few as five bacteria per hemocyte. The greater cyto-
toxic activity of V. tapetis compared to V. splendidiis. Vibrio sp.
(strain S322). and V. pectenicida suggests a specific pathogenicity
of V. tapetis to R. philippinarum hemocytes. Though all V. tapetis
isolates possess the capacity to inhibit adhesion, significant varia-
tions of cytotoxicity among i.solates has been demonstrated. These

results are in agreement with //( vivo pathogenicity tests. The iden-
tification and characterisation of the genes involved in V. tapetis
cytotoxicity is in progress.

CHEMOTAXIS OF HEM0CYTE;S OF THE HARD CLAM,
MERCENARIA MERCENARIA, TO QUAHOG PARASITE
UNKNOWN (QPX) AND OTHER MICROORGANISMS.
Christie-Sue Decker* and Robert S. Anderson, University of
Maryland Center for Environmental Sciences. Chesapeake Bio-
logical Laboratory. PO Box 38. Solomons. MD 20688.

Quahog Parasite unknown (QPX) is a protist pathogen in the
phylum Labyrinthulomycota affecting the hard clam. Mercenaria
mercenaria. It causes a disease that has impacted hatchery and
broodstock clam populations in Canada and the United States.
Chemotaxis is the directed migration of cells in a chemical gradi-
ent, and is a well-documented immune response of hemocytes. It
is postulated that there is a con'elation between pathogenicity and
chemoattraction; invading organisms able to escape detection by
hemocytes would be better able to colonize their host. M. merce-
naria hemocytes are known to exhibit chemotaxis in response to
live Escherichia coli bacteria and to cell-free E. coli culture fluid.
This study is the first investigation into host chemotactic response
to QPX or other Labyrinthulomycota species. The chemotactic
effects of several pathogenic and non-pathogenic organisms were
compared. There was no chemotaxis by clam hemocytes toward
QPX cells devoid of their mucofilamentous secretions, but positive
chemotaxis for spent QPX media. There was positive chemotaxis
for Bacilhis megateriiim. a non-pathogenic bacterium. Other or-
ganisms investigated included a non-pathogenic Labyrinthulomy-
cota, a non-pathogenic Protist, and a pathogenic bacterium. Com-
parisons yielded a profile of M. mercenaria chemotactic response
for a broad range of parasitic challenges.

EFFECT OF DIETARY FATTY ACID COMPOSITION ON
LIPID PROFILES OF HAEMOCYTE MEMBRANES IN
OYSTERS AND CLAMS AND ITS IMPACT ON IMMUNE
FUNCTIONS. Maryse Delaporte,* Jeanne Moal, and Jean-
fran^ois Samain, LPI. Ifremer de Brest. 29280 Plouzane. France;
Philippe Soudant, Gwenaelle Choquet. Christophe Lambert,
and Christine Paillard, LEMAR. lUEM. 29280 Plouzane. France.

This study was designed to assess the influence of micro-algal
diets on the fatty acid profile of haemocyte membranes and on
immune functions. The oyster Crassostrea gigas and the clam
Tapes phUippinarum were fed three diets with varying PUFA com-
position.

The fatty acid composition of haemocyte and gill membranes
of both bivalves was greatly influenced by the diet. Nevertheless,
a selective retention of certain specific PUFA was observed in the
analysed tissues; 22:6(n-3) for clam and 20:.'i(n-3) for oyster. Im-
mune parameters were also affected. Indeed, a 20:5(n-3) and 20:

National Shellfisheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting. April 14-1 S

2002

405

4(n-6) enrichment appeared to increase the phagocytic rate and the
metabolic activity of clam haemocytes. A smaller positive effect of
20:5(n-3) on metabolic activity of oyster haemocytes was ob-
served. Interestingly, when oyster haemocytes were incubated one
hour at 18'C or 30"C. a positive correlation between the 22:6(n-3l
content of haemocytes and the phagocytic rate was noticed.

ACTIVATION OF OYSTER DEFENSES BY ENVIRON-
MENTAL CONTAMINANTS. William S. Fisher* and Leah

M. Oliver, U. S. Environmental Protection Agency. National
Health and Environmental Effects Research Laboratory. Gulf
Ecology Division. Gulf Breeze, PL 32561.

Four field studies performed on eastern oysters Crassostrea
virgmica support a hypothesis that Cu, Zn, and perhaps butyltins
and polycyclic aromatic hydrocarbons (PAH) can stimulate he-
mopoiesis, hemocyte locomotion and hemocyte bactericidal capac-
ity. The first study found circulating hemocyte numbers and loco-
motion were positively associated with contaminated sites in
Tampa Bay. particularly those where pooled oyster tissues con-
tained high concentrations of trace metal and PAH analytes. Sub-
sequently, the relationships between these particular contaminants
and hemocyte activities were extended to four additional bays (St.
Andrew, Choctawhatchee. Pensacola. and Biscayne). A third
study, which analyzed chemicals from individual oysters in Pensa-
cola Bay, verified that circulating hemocyte numbers and bacteri-
cidal activity were positively correlated with Cu, Sn, Zn. butyltin.
total metals, total polychlorinated biphenyls and total PAH. A
fourth study showed that circulating hemocyte number and bacte-
ricidal activity were significantly elevated when oysters were
moved from a relatively clean site to one with high concentrations
of Cu. Zn. butyltins and PAH. These data provide a weight of
evidence that certain chemical contaminants can stimulate de-
fense-related hemocyte activity in oysters. Although chemical con-
taminants are generally suspected to suppress defense functions of
oysters, these chemicals, for unknown reasons, appear to have the
opposite effect.

respectively 9.9 and 2.4 times the chemiluminescence activity. The
peak level was obtained about one hour after activation. This CL
test was used to compare CL activity of scallop larvae reared with
antibiotic (Chloramphenicol 4 ppml. probiotic bacteria (available
in our laboratory) or without treatment. The defense capacity of
larvae reared with probiotic was higher than those with antibiotic
or without treatment. Moreover, probiotic reared larvae were
shown to be less sensitive to in vitro CL inhibition of V. pecteni-
cida. This led to the conclusion that protiotics not only compete
with bacterial flora but were also able to impro\e scallop larvae
defense system (oxidative burst).

DEVELOPMENT OF A FLOW CYTOMETRIC MEASURE-
MENT OF OXIDATIVE METABOLISM PRODUCT FOR-
MATION BY CRASSOSTREA GIGAS HEMOCYTES AND
APPLICATION TO EVALUATE PATHOGENIC VIBRIO
INHIBITING CAPACITY. Christophe Lambert.* Philippe
Soudant, Gwenaelle Choquet, and Christine Paillard, LEMAR.
Laboratoire des Sciences de rEnvironnement Marin. UMR6539.
lUEM-UBO, place Nicolas Copemic, Plouzane, France.

A flow cytometric method to measure the production of oxi-
dative metabolism products was adapted to Crassostrea gigas
hemocytes. Measurement was based on the oxidation by hydrogen
peroxide (H^Gi) of intracellular 2',7'-dichlorotluorescin (DCFH)
in green fluorescent dichlorotluorescein (DCF). Activation by zy-
mosan particles of the respiratory burst metabolic chain was shown
to stimulate the DCFH oxidation in C gigas hemocytes and a rate
of 20 zymosan particles per hemocytes was found to be optimal. At
the opposite. DCFH oxidation using phorbol myristate acetate
(PMA) was not obtained. Anti-aggregant solution, used to avoid
hemocytes clamping after bleeding, was shown to inhibit the re-
spiratory burst measured by DCFH oxidation. Finally, the flow
cytometric method developed during this work was used to evalu-
ate and grade the DCFH oxidation inhibiting capacity of four
Vibrio species, known or suspected to be pathogenic for bivalves.

IMPROVEMENT BY PROBIOTICS OF PECTEN MAXI-
MUS LARVAE DEFENSE CAPACITY, MEASURED BY
CHEMILUMINESCENCE. Christophe Lambert* and Chris-
tine Paillard, LEMAR. lUEM-UBO. Place Copernic. Plouzane.
France; Jean-Louis Nicolas, LPI, IFREMER. Plouzane. France.

Literature concerning evaluation of defense system capacity of
bivalve larvae are scarce. It's why this work intended to adapt to
oy.ster and scallop larvae a chemiluminescence (CL) test to mea-
sure oxygen intermediate synthesis (mainly H,0,) during phago-
cytosis of zymosan particles and inhibiting effect of Vibrio
pectenicida. pathogenic for scallop larvae on this activity. As a
result, activation of whole alive Crassostrea gigas and Pecteii
ma.ximus larvae by zymosan particles have been shown to increase

PURIFICATION OF A NOVEL ANTIMICROBIAL PEP-
TIDE FROM THE EASTERN OYSTER {CRASSOSTREA
VIRGINICA). Ann C. Mountz* and Robert S. Anderson, Uni-
versity of Maryland Center for Environmental Science. Solomons,
MD 20688.

Oysters are routinely exposed to a variety of microbes: few of
these microbes are pathogenic, however oyster diseases, such as
Derino and MSX. have lead to drastic reductions in oyster popu-
lations along the Atlantic coast of the United States. Understanding
the oyster's immune system is an important element in determin-
ing how the remaining populations can best be protected or in-
creased. Many aspects of the eastern oyster immune system have
been characterized, including reactive oxygen species, phagocyto-
sis, and lytic enzymes. Antimicrobial peptides are a more recently

406 Ahslnuls. 2002 Annual Meeting. April 14-1,S. 2002

National Shellfisheries Association. Mystic. Connecticut

recognized component ot the bivahe defensive capabilities, which
have not yet been identified in the eastern oyster. A new antimi-
crobial peptide was purified from oyster serum via methanol pre-
cipitation, size-exclusion centrifugation. Sep-Pak filtration, and re-
verse-phase HPLC. Activity against the model Gram-positive bac-
terium was measured at all steps using a tetrazolium dye reduction
assay. The purified peptide is 4.3 kDa based on mass spectrometry
analysis and has preliminarily been named CVAP-I.

results, and .i) how much repeated sampling itself altered the mea-
surements. Most parameters were relatively consistent over time
(individuals had consistently high or low values), but more so in
the field than in the laboratory. There were significant day-to-day
variations when sampling occurred over a several-day period, but,
sampling time explained very little of the overall variation. Re-
peated sampling increased mortality and altered hemolymph con-
stituents in the laboratory, but not the field, study.

TEMPERATURE EFFECT ON IMMUNOCOMPETENCE
OF CLAM R. PHILIPPINARUM AND ON V. TAPETIS C\-
TOTOXICITY. Christine Paillard,* Gwenaelle Choquet,
Christophe Lambert, and Philippe Soudant, lUEM-UBO,
LEMAR Laboratoire de renvironnement marin. TechnopcMe
Brest-Iroise. 29280 Plouzane. France; Helen Reid and Harry Bir-
beck. University of Glasgow, Division of Infection and Immunity.
GLASGOW G12 8QQ. Scotland.

Adult clams. R. philippinarum. were conditioned for one month
in the laboratory at three temperatures 7. 1 3 and 2 1 ' C. and then
inoculated with V. tapetis. One month after V. tapctis challenge,
clam phagocytic activity and V. tapetis cytotoxicity were mea-
sured. An increase of phagocytic activity was demonstrated in
control clams maintained at the highest temperature. At 13°C.
higher phagocytic activity was measured in V. tapetis inoculated
clams compared to controls. Also, highest Brown Ring Disease
prevalence and intensity was obtained at 13°C. Cytotoxic assays
based on hemocyte-bacieria interactions were performed with bac-
teria grown at these three different temperatures. Generally, higher
cytotoxicity was found when V. tapetis was grown at 13°C. which
corresponds to the lower end of its optimal growth range. Further,
cell rounding percentage depends on whether the clams were ex-
perimentally challenged with the bacterium and temperatures at
which clam are maintained.

REPEATED HEMOLYMPH SAMPLING OF INDIVIDUAL
CLAMS: WHAT DOES IT TELL US ABOUT SAMPLING
PROCEDURES? Christine Paillard.* LEMAR. UMR 6539.
lUEM-UBO. Plouzane. France, Su.san E. Ford, Haskin Shellfish
Research Laboratory, Rutgers University, Port Norris, NJ.

Hemocyte concentrations, protein levels, and enzyme activities
are often used as measurements of the health status of marine
bivalves and are frequently considered an index to assess their
susceptibility to infectious agents. Yet these parameters are known
to vary considerably on a seasonal, regional, and individual basis.
and are likely to be affected by laboratory procedures. Sources of
variation such as these add uncertainty to the interpretation of
hemolymph assays. We repeatedly sampled the hemolymph of
clams, Ruditapes philippinarum, in both laboratory and field con-
ditions, to determine 1 ) how consistent measurements were over
time for the same individual, 2) whether time of sampling affected

THE ANTIOXIDANT SYSTEM OF THE PROTISTAN
PARASITE PERKINSVS MARIMS. Eric ,1. Schott.* Jose
A. F. Robledo. Wolf T. Pecher. Florence A. Okafor, and Ger-
ardo R. Vasta, COMB. UMBI. University of Maryland. Balti-
more. MD 21202.

Spread of the protistan parasite Perkiiisus iiiariiius within the
oyster Crassostreu virginica is believed to be via engulfment and
migration by phagocytic hemocytes. Because phagocytosis of
killed P. marimis trophozoites elicits production of reactive oxy-
gen intermediates (ROIs) by oyster hemocytes, but phagocytosis of
live trophozoites does not, P. marimis appears to have mechanisms
to prevent the accumulation or production of ROIs. We previously
described two Fe-type SOD genes {PmSODl. PmSODl) from P.
marimis and demonstrated the ability of their products to convert
O; • to H,0,. P. marimis is resistant to moderate levels of H^O,.
suggesting that it also possesses a mechanism for H^O, removal.
However, significant efforts to detect P. marinus catalase activity
and gene sequences have been unsuccessful. Instead, we have
found that P. marimis trophozoites possess abundant ascorbate
dependent peroxidase (APX) activity. We have partially purified
P. marimis APX. which co-migrates with a 35 kD band on non-
denaturing gels. Continuing genetic, biochemical, and cellular
studies of P. marimis FeSODs and APX will contribute to further
characterize the P. marimis antioxidant defense system. [Sup-
ported by Grant No NA06RG0101-5 from ODRP. NOAA. through
the Marvland Sea Grant, to GRV).

SEASONAL AND CULTURE SITE EFFECTS ON THE
PHYSIOLOGICAL, IMMUNOLOGICAL AND BROWN
RING DISEASE STATUS OF THE MANILA CLAM RUDI-
TAPES PHILIPPINARUM. Philippe Soudant,* (Jwenaelle
Choquet, Christophe Lambert, Alain Marhic, and Christine
Paillard LEMAR UMR 6539. lUEM-UBO. Place Nicolas Coper-
nic. Plouzane. France.

To assess the effects of environmental conditions on Brown
Ring Disease (BRD) expression, physiological and immunological
status, juveniles of clams were distributed in four rearing sites
.selected for their varied ecological characteristics. Clams were
sampled for analysis every three months for eighteen months in
each sites. Significant site and seasonal effects have been estab-
lished for the condition index, the growth rate, the haemocyte

National Shellfisheries Association, Mystic. Connecticut

Abstracts. 2002 Annual Meeting, April 14-18, 2002 407

concentration and cytomorphology. and the enzymatic activities
and protein content of the total hemolymph. Some significant re-
lationships between immunological and physiological parameters
were observed. The haemocyte concentration was correlated with
size and mortality rate of haemocytes while their complexity was
correlated with phenoloxidase activity. Also, correlation was
shown between the haemocyte concentration and the size of clams
while haemocyte size and hemolymph protein content were cor-
related to condition index. Surprisingly, the seeded clams showed
very low BRD prevalence in all sites and for all seasons. Mean-
time, high prevalence was observed in natural stock from one of
the site. This suggests hatchery seeded clams may be BRD resis-
tant and claimed further studies.

RELATIONSHIPS BETWEEN SUMMER MORTALITIES
AND DEFENCE MECHANISMS IN FAMILIES OF CRAS-
SOSTREA GIGAS REARED IN DIFFERENT ENVIRON-
MENTAL CONDITIONS. P. Soudant,* C. Lambert. G.
Choquet, S. Ford, and C. Paillard, LEMAR. lUEM-UBO. Place
Nicolas Copernic. Plouzane. France; L. Degreniont, M. De-
laporte, J. Moal, P. Boudry, P. Soletchnick. J.-P. Joly, M. Rop-
ert. E. Bedier, A. Huvet, and J.-F. Samain. LGP. LPI. LCs of
IFREMER. France.

High individual variability is often encountered when measur-
ing defence mechanisms in bivalves. Such variation is suspected to
result from both environmental and genetic factors. Determine
whether defence mechanisms of C. gigas are genetically based was
thus part of a national program conducted by IFREMER and ded-
icated to understand the causes of summer mortalities in C. gigas
juveniles. Fifteen bi-parental families, obtained from a nested half-
sib crossing design, were reared four months in three sites. Six
families were selected on their survival performance (three "good"
and three "bad" ones) to compare their immunological status. As
expected, most of immunological parameters tested were signifi-
cantly different according to the culture site. More interesting is
the significant differences measured between good and bad fami-
lies. For instance, higher total haemocyte counts and lower oxida-
tive metabolism were observed in good families. Moreover, incu-
bation with pathogenic Vibrio sp. S322 inhibited significantly
more the adhesion capabilities and oxidative metabolism of
haemocytes from bad families.

PURIFICATION AND CHARACTERIZATION OF
LYSOZYME FROM PLASMA OF EASTERN OYSTERS
{CRASSOSTREA VIRGINICA). Qing-Gang Xue* and Jerome

F. La Peyre. Cooperative Aquatic Animal Health Research Pro-
gram, Department of Veterinary Science, Louisiana State Univer-
sity Agricultural Center. Baton Rouge, LA 70803; Aswani K.
Volety. Division of Ecological Studies. Florida Gulf Coast Uni-
versity. Fort Myers. FL 33965; Fu-Lin E. Chu. Virginia Institute
of Marine Science, College of William and Mary, Gloucester
Point, VA 23062.

Lysozymes are antimicrobial proteins which are defined as 1,4-
3-N-acetylmuramidases cleaving a glycosidic bound between N-
acetylmuramic acid and N-acetylglucosamine of peptidoglycan.
Lysozyme activity has been detected in the body fluids and tissues
of many bivalve molluscs but lysozymes from only a few bivalve
species have been purified. The molecular weights of the purified
lysozymes range from 11 to 18 kDa. The analysis of N-terminal
amino acid sequences, when determined, indicated that they all
belong to a distinct type of lysozyme. the i-type. Lysozyme from
plasma of eastern oysters was recently purified by a combination
of ion-exchange chromatography on CM-Sepharose Fast Flow and
gel filtration chromatography on a Siiperdex G-7.'i column. The
final preparation showed a single band on SDS-PAGE gel with a
molecular weight of 18.4 kDa. About 1.5 mg of lysozyme was
purified from one liter of oyster plasma and the lysozyme specific
activity increased 150 fold. The enzymatic properties, stability and
antimicrobial activity of the purified plasma lysozyme are cur-
rently being determined.

SEA URCHIN BIOLOGY, PATHOLOGY
AND CULTURE

SEA URCHIN MORTALITY IN MAINE, INITIAL CASE
REPORT AND OVERVIEW. Ralph Elston, AquaTechnics/
Pacific Shellfish Institute, PC Box 687, Carlsborg, WA 98324.

Compared to some other species of marine invertebrates, health
parameters and disease conditions of sea urchins are less well
studied. The purpose of this case report is to present initial obser-
vations that may suggest causes or significant factors related to sea
urchin mortalities occurring in Maine in 1999 and 2000. These
observations may be useful in indicating what further investiga-
tions are needed to make a more definitive diagnosis regarding
cause of the losses.

Sea urchin (Strongylcentrotus droebachiensis) mortalities were
observed in two areas of Frenchman's Cove, Maine in late October
2000. Previously, urchin mortalities were reported in August 1999
by divers and during an urchin transfer in August 2000. The
samples from 1999 were examined bacteriologically and histologi-
cally by Dr. Paul Waterstraat of the Maine Department of Natural
Resources. The presence of a purple exudate from the teste was

408 Abstracts. 2002 Annual Meeting. April 14-18. 2002

National Shellt'isheries Association. Mystic. Connecticut

reported. The urchins were also examined for the presence of a
paramoeba. previously associated with urchin mortalities in Nova
Scotia, and none of these parasites were found.

Two groups of urchins were received live and chilled on No-
vember 29, 2000. One group consisted of four urchins collected
from Winter Harbor and the other group consisted of two urchins
collected from Schoodic Point. All urchins were examined on
receipt, and processed for necropsy and histological examination.
The teste diameter/height ranged from 53/34 mm to 58/33 mm.
Clinically, three of six urchins appeared healthy and exhibited
mobility of the spines. Two urchins showed loss of spines, one
from all except the oral surface and the second unilaterally in a
band froin the oral to aboral surface. Three were confirmed males
and three were females.

Of six animals examined histologically, no lesions were found
in two of the healthy appearing individuals. In both urchins with
loss of spines, there were multifocal areas of eosinophilic intra-
nuclear inclusion bodies in epithelium of the spines and their mus-
culature and associated necrosis of epithelium and myocytes.
These inclusion bodies were relatively common in one individual
but rare in another. This finding corresponds to the loss of urchin
spines noted by divers and other on site investigators. Such inclu-
sion bodies may represent a viral infection but there are other
non-infectious causes of inclusion bodies. Verification of a viral
infection would require examination by transmission electron mi-
croscopy.

IMPORTANCE OF DIETARY MINERALS AND PIG-
MENTS FOR INCREASING SOMATIC GROWTH OF JU-
VENILE GREEN SEA URCHINS {STRONGYLOCENTRO-
TUS DROEBACHIENSIS). Eddy J. Kennedy.* Shawn M. C.
Robinson, and John Ca.stell. Department of Fisheries and Oceans.
Biological Station, 531 Brandy Cove Rd., St. Andrews, NB,
Canada, E5B 2L9; G. Jay Parsons, Dept. of Fisheries and Oceans,
200 Kent Street. Ottawa. ON KIA 0E6.

Understanding the nutritional requirements of juvenile sea ur-
chins is necessary for diet formulation and optimizing somatic
growth. From Feb 2000 to July 2000, different mineral sources and
concentrations (modified Bernhart-Tomerelli salt mix at 0%. 1 .5'7r.
3%, 6%, and 15% and Shur-Gain at 3% and 6%) were incorporated
in pigmented (beta-carotene) diets to detemiine the effect on ju-
venile sea urchin (14 mm to 15 mm initial test diameter) somatic
growth. Non-pigmented diets were also used with 3% of each
mineral source incorporated in the diet. After the 154-day experi-
ment, the juveniles fed the non-pigmented diets were smaller (20
mm average TD) than those fed the 3% mineral pigmented diets
(23 mm average TD) (P<O.OOI). The juveniles fed the 15'^t min-
eral diet were larger (24.3 mm average TD) than those fed the 0%
and 1.5% mineral diets (22 mm average TD) (P<0.001 ). As well,
the juveniles fed the pigmented diet with y'i Shur-Gain mineral
(which lacked magnesium) were smaller (2 1 mm average TDj than

those fed the diet with 3% Bernhart-Tomerelli mineral (which
included magnesium) (23.5 mm average TD) (P<0.001). From
Nov 2()00 to April 2001, a pigmented (beta-carotene) prepared diet
with 159<- Bernhart-Tomerelli salt mix was compared to kelp (the
natural sea urchin diet) to determine differences in juvenile so-
matic growth production between diets. After the 159-day experi-
ment, the juveniles ( 1 mm to 2 mm initial average test diameter)
fed the prepared diet were larger (7.4 mm average TD) than the
kelp-fed juveniles (4.4 mm average TD) (P<0.001 ). Prepared diets
require pigments and high mineral content to optimize juvenile sea
urchin somatic growth.

SEA URCHIN DISEASE CONCERNS IN ATLANTIC
CANADA. Gregory MacCalluni,* Atlantic Veterinary College,
University of Prince Edward Island. 550 University Ave., Char-
lottetown, PE, COA 1 YO; Shawn Robinson, Biological Station,
Department of Fisheries and Oceans. 53 1 Brandy Co\ e Rd.. St.
Andrews, NB, E5B 2L9; Sharon McGIaddery and Mary
Stephenson, Gulf Fisheries Centre, Department of Fisheries and
Oceans, 343 University Ave.. PO Box 5030, Moncton. NB. EIC
9B6.

The green sea urchin (Strongylocentrdtus clroehachieusis) has
been fished in Atlantic Canada since the 1950's with a commer-
cially developed t~ishery emerging in the 1980"s in the Bay of
Fundy, New Brunswick and southwestern Nova Scotia (NS). In the
1990"s. Newfoundland, Quebec and to some extent Prince Edward
Island, also began fishing this species. The total Atlantic Canadian
sea urchin landings in 2000 were 3.050 MT (live weight) with a
value of $7.08 million.

The fishery, however, has not been without disease setbacks.
Between 1980 and 1983 sea urchin mortalities in NS were esti-
mated at 245,000 tons associated with a parasitic amoeba, Par-
ainoeha invadens. Since 1995. a die-off associated with paramoe-
bisasis of 50.000 to 100.000 tons has occurred representing an
estimated 10 to 100 times the weight of urchins taken by the NS
fishery. Currently, the Department of Fisheries and Oceans Canada
lists P. inviulciis and Bald-Sea-Urchin disease as "regional con-
cerns" with trematode metacercariae and turbellarian parasitism
listed as "negligible significance". The objective of this presenta-
tion is to give an overview and update on the current disease
concerns facing the green sea urchin fishing and aquaculture in-
dustry in Atlantic Canada.

PHOTOPERIOD. URCHIN "EYES" AND GAMETOGEN-
ESIS. Michelle Moody and Charles W. Walker, Department of
Zoology and Marine Biomedical Research Group. University of
New Hampshire. Durham. NH 03824.

Shortening daylength in the fall results in the initiation of
changes in two populations of cells within the gonads of both sexes
of the green sea urchin, Strongylocentrotus droebachiensis. These

National Shellfisherics Association. Mystic. Connecticut

Abstracts, 2002 Annual Meeting. April 14-18. 2002 409

cellular populations include spermatogonia in males and oogonia
in females and somatic cells called nutritive phagocytes (NP) in
both sexes. During the summer, gonial cells are amitotic and NP
store nutrients; during the photoperiod changes in the fall, gonial
cells begin mitosis and NP mobilize nutrients. Details of these
processes can be addressed on the web page http://zoology.unh.
edu/faculty/walker/urchin/gametogenesis.html The detectors for
these changes in day-length are unknown, but might include tube
feet, spines, the dermis or the gonads themselves. We are using
western blots and immunocytochemistry to detect PAX6 (the com-
plex eye master control switch gene) and rhodopsin proteins within
each of these tissues.

It is also unknown whether the response to photoperiod occurs
at the level of the NP, which then mobilize nutrients and thus cause
gonial cells to divide, or whether both populations of cells are
independently effectors for the photoperiod cue. In order to dis-
tinguish between these two possibilities, we are examining the
expression of the c-itnx protooncogene and of the SEAWI stem
cell controlling gene in both inales and females during the months
when the photoperiod que occurs (August — October).

Practical applications of this research for aquaculture might
result from preventing the photoperiod and thus suppressing ga-
metogenesis in the green sea urchin. Gonads in such sea urchins
should contain principally or exclusively nutritive phagocytes and
should be of superior size, taste, texture and firmness. Ovaries and
testes containing fewer gametes relative to NP are actually pre-
ferred in most cultures that each sea urchin gonads and such con-
ditions exist naturally in sea urchins during the summer.

Supported by USDA Competitive Grants Program, Hatch and
Sea Grant to CWW.

search on the immune system and impacts of environmental fac-
tors. Funds to support these studies must be factored into the
fishery equation.

GROWTH RESPONSE AND ACCLIMATION OF GREEN
SEA URCHINS TO FLUCTUATING SALINITY. Michael P.
Russell,* Biology Department, Villanovu University. Villanova.
PA 19085-1699.

Echinoderms are osmoconformers — external salinity deter-
mines internal osmotic concentrations. Green sea urchins are ex-
ceptional among echinoderms because they occur in brackish-
water environments. These habitats are among the most productive
fishing grounds and populations in these areas often have some of
the largest individuals. The states of Maine and New Hampshire
have granted sea urchin lease sites in these habitats and leasehold-
ers plan to seed these areas with hatchery-reared juveniles. How-
ever, previous workers have documented that larger sea urchins
can tolerate bouts of low salinity of both greater intensity and
duration than smaller individuals. I tested the growth response of
juvenile sea urchins to periodic bouts of sub-lethal hyposaline
conditions. I collected samples from an area not exposed to low
salinity and di\ided them into two groups: treatment (bouts of
hyposaline conditions) and control. After an initial acclimation
period, the treatment group displayed the same growth rate as the
control group despite repeated exposures to hyposaline conditions.
The acclimation period was equivalent to 10 days of growth. These
experiments demonstrate the importance and the feasibility of ac-
climating juveniles to hyposaline conditions before releasing them
into lease sites.

ABIOTIC AND BIOTIC THREATS TO SEA URCHIN
HEALTH: WHAT PRICE CULTURE? Esther C. Peters,*

Tetra Tech, Inc., 10306 Eaton Place, Suite 340, Fairfax, VA
22030.

Understanding how environmental factors affect urchin health
will be important to ensure a consistent supply of urchins for
human food resources; however, unlike other fisheries, our knowl-
edge of their susceptibility to toxicants and pathogens has been
limited and based on sporadic efforts. Sea urchin gamete fertiliza-
tion and embryo survival have been developed into a standardized
test to evaluate the toxicity of coastal sediments and used in re-
search on mechanisms of toxicity suggesting that toxicants enter-
ing nearshore waters or culture facilities might have adverse ef-
fects. Mass mortalities of adult urchin populations have occurred
because of bacterial, protozoal, or unknown pathogens, which
might be influenced by temperature, water movement, and popu-
lation densities, but the role of toxicants is unknown. No cases of
neoplasia in urchins have been submitted to the Registry of Tu-
mors in Lower Animals. Multidisciplinary approaches must be
consistently applied in monitoring urchin health, with more re-

URCHIN HEALTH ISSUES IN MAINE. Paul Waterstrat and
Ted Creaser. Maine Department of Marine Resources, Boothbay
Harbor Laboratory, PC Box 8, West Boothbay Harbor, ME 04575.
Maine urchin harvesters have reported mortalities of green sea
urchins, Strnngyloientrotus droebacliieiisis. over the last three
years. The urchin mortalities have been observed in at least 19
locations along the coast of Maine. Gross examination of urchins
taken from areas of mortality revealed a progression of lesions
ranging from the presence of an inflammatory exudates, loss of
tube feet and spines to complete denudement of the urchin test.
Examination of wet mount or squash preparations of coelomic
fluid, organ samples, and culture failed to indicate the presence of
Paramoeba mvadens, the agent responsible for recurring mortali-
ties of urchins in Nova Scotia. Elevated temperature, mortality
from draggers, and crashes of significant phytoplankton blooms
have each been implicated as a cause of mortality, but no consis-
tent etiology has yet been determined for the outbreaks. Attempts
at restoration by transplanting juvenile urchins or reseeding beds
have likewise met with limited success. Given the multifactorial

410 Ahslracts. 2002 Annual Meeting. April 14-18. 2002

National Shellfisheries Association. Mystic. Connecticut

nature of aquatic disease, the occurrence of disease outbreaks in a
fishery experiencing a significant declines in harvest landings,
does not bode well for the sustainability of the population. Rather
than attributing urchin mortalities to "natural causes'", there is clear
need to establish a mechanism for disease surveillance and control.
Unfortunately, there appears to be a surprising lack of information
about the pathobiology of sea urchins, despite the considerable use
of sea urchins as a laboratory model.

LOBSTER BIOLOGY AND FISHERIES

THE EFFECTS OF HABITAT ADDITION AND STOCK EN-
HANCEMENT FOR AMERICAN LOBSTERS, HOMARUS
AMERICANUS ON ARTIFICIAL REEFS IN RHODE IS-
LAND. Kathleen M. Castro* and J. Stanley Cobb. University of

Rhode Island, Kingston. RI 02881; Richard Wahle, Bigelow
Laboratory for Ocean Sciences. Boothbay Harbor. ME 04575;
John Catena. Restoration Center. National Marine Fisheries Ser-
vice, Gloucester, MA 01930-2298.

Six experimental artificial reefs were established in Narragan-
sett Bay, RI in February 1997. These have been monitored accord-
ing to a before-after-control-impact design by means of visual
surveys, traps, tag-recapture, photoquadrats and airlift sampling
for 6 years. Juvenile and adult lobster density at the reef site
increased from near zero to >1 lobster/m~. significantly higher than
before reef placement. Settlement of young-of-the-year lobsters
significantly increased. We used microwire tags to mark hatchery
reared lobsters and released them as 5"'-6'^ stage lobsters onto
three of the reefs at a density of 4.5/nr for three years. However,
subsequent density of young-of-the-year lobsters on the enhanced
treatment sites was not significantly different from the non-
enhanced sites. Despite intensive sampling, only three of these
tagged lobsters were recovered after placement. Placement meth-
ods may contribute to survival possibilities. Field and lab obser-
vations confirm behavioral differences between 4"' stage and 5''"
stage hatchery-reared lobsters as compared to field-caught lob-
sters. Predation rates in the lab were significantly higher for the 4"'
stage hatchery-reared lobsters raised in the URI facility compared
to the RI field-caught lobsters and hatchery-reared lobsters ob-
tained from Maine.

were collected in the Eastern Long Island Sound. NY (ELIS).
Buzzards Bay and Vineyard Sound, MA. Preliminary denaturing
gradient gel electrophoresis (DGGE) data indicate that up to five
independent phylotypes of bacteria are present in lobster lesions.
At least two of them were found in all lobsters from ELIS. This is
consistent with the five to six morphotypes of bacteria which were
cultured on a marine agar. All isolated bacteria were either aerobic
or facultatively anaerobic. No strictly anaerobic or microaerophilic
bacteria were isolated from lesions. Only two different types of
fastidious chitinolytic bacteria were isolated on a medium contain-
ing chitin. One of the two is a motile bacterium forming bright
yellow colonies, which was present in all analyzed lobsters (from
Buzzards Bay, Vineyard Sound and ELIS). The second is a bac-
terium forming white colonies. It was isolated from only two Buz-
zards Bay lobsters. No correlation has been found between the
presence or severity of shell disease and the presence of bacteria in
hemolymph.

CHANGES IN LOBSTER POPULATIONS IN NARRAGAN-
SETT BAY. RHODE ISLAND. 1959-2(»00. J. S. Cobb and M.
Clancy, Department of Biological Sciences, Unisersity of Rhode
Island, Kingston, RI 02881.

The past 35 years have seen an extraordinary increase in the
catch of lobster (Homanis americanus) along the northeast coast
of North America. Rhode Island landings ha\e tripled since the
late 1960s. We explored a fishery-independent data set of lobster
abundance developed from weekly research otter trawls made at
two locations in Narragansett Bay. RI by the University of Rhode
Island on a continuing basis since 1959. The patterns of abundance
over time differed substantially at the two stations; one of the
stations was highly correlated with the NMFS catch data set for the
same time period. Water temperature, which has increased steadily
since 1960, was significantly correlated with commercial landings
and with abundance at one of the stations. Lobster abundance at
both stations and the commercial landings were significantly cor-
related with the NAG winter index. At one of the stations the
timing of greatest abundance in the trawl samples shifted to earlier
in the summer. It seems likely that there have been effects of
climate on lobster abundance, however the effect appears to have
acted differentially at the two stations in Narragansett Bay.

CHARACTERIZATION OF MICROBIAL ASSEMBLA-
GIES INVOLVED IN THE DEVELOPMENT OF SHELL
DISEASE IN THE AMERICAN LOBSTER. HOMARUS
AMERICANUS. Andrei V. Chistoserdov* and Fellza Mirasol.

Marine Sciences Research Center, SUNY at Stony Brook, Stony
Brook, NY 1 1720; Roxanna Smolnwitz. Marine Biological Labo-
ratory, Woods Hole. MA 02543.

A combination of culture-based and molecular techniques was
used to study the consortia of bacteria isolated from shell lesions
and hemolymph of lobsters. Diseased lobsters used in this study

THE ROLE OF VIBRIO FLUVIALIS AND OTHER BACTE-
RIAL SPECIES IN LOBSTER MORTALITIES IN MAINE.
Ceni Giray* and Deborah .\. Bouchard. Micro Technologies,
Inc.. 41 Main Street, Richmond, ME 04357.

Weak lobsters {Httiiuinis aiiwrUaiuis) and mortalities have
been reported by pounds in Maine for several years with Vibrio
fliiviiilis implicated as the etiological agent responsible. Bacterial
screening of moribund lobsters was pertbrmed by Micro Tech-
nologies, Inc. through a contract with the Maine Department of

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 41 1

Marine Resources. The majority of isolates collected were puta-
tively identified as Hyplioiuicrobium iiuliciini through 16S rRNA
sequencing while V. fhivia/is was not isolated from any of the
lobsters. Further characterization demonstrated that H. iiidicuin
could grow at a temperature range from 3'C to a higher limit
between I6-20°C while V. fluvialis grew best between 16-30°C
with minimal growth at 8°C. The virulence of H. iiulicum and V.
fluvialis was tested by injection into lobster hemolymph at various
dosages. Both isolates resulted in 75-100% mortality withm 24
hours when injected at 10** colony-forming units (CFU). but only
H. incliciiiii resulted in mortalities (12.59!-) when injected at 10"
CFU/lobster. V. fischeri was used as a negative control while Li-
stonella ani^uillarum and Aerococcus viridans. both indicated as
lobster pathogens, were utilized as positive controls. The identifi-
cation of H. indiciiin as the primary isolate collected from mori-
bund lobsters and the observation of lobster mortalities mainly
during lowered water temperatures suggest that the isolate respon-
sible could be H. indiciini hut not \'. fhiviidis.

SHELL DISEASE PREVALENCE AND SEVERITY IN OFF-
SHORE AMERICAN LOBSTER POPULATIONS. Diane Ka-
pareiko, Richard A. Robohm. Joiin J. Ziskowski, George R.
Sennefelder, and Anthony Calabrese, National Marine Fisheries
Service. Milford Laboratory. 212 Rogers Avenue. Milford. CT
06460.

During the period 1990-1992, 15.004 lobster from 146 com-
mercial catches at nine offshore canyon sites surrounding the 106-
Mile Sewage-Sludge Disposal Site were examined for signs of
shell disease. Overall. 1.184 lobster (7.9%) had lesions. Females
were more affected by this condition than males. Shell-lesion oc-
currence was independent of carapace length (CL), but strongly
related to location (proximity to the 106-Mile Dumpsite as well as
to the 12-Mile Dumpsite). Data collection for the shell-disease
study included not only evaluation of presence or absence of dis-
ease, but also measurements of lesion size and carapace length.
This was done in anticipation of developing a method that would
determine the percentage of total surface area of each lobster af-
fected by shell disease: this percentage is the basis of a Disease
Severity Index (DSI). An estimate of lobster surface area could be
derived mathematically from carapace length, for both male and
female lobster in our database, using the formula y = 1.1034 +
1 .9677 * log(CL). The percentage of surface area covered by shell
lesions, multiplied by 10\ provides a DSI that may allow better
statistical correlations between mean disease severity and site of
lobster collection.

Regression Tree analysis of this multi-variate database indi-
cated that, unlike prevalence, the most important variable affecting
the DSI was carapace length. Overall. DSI's for smaller lobsters
(CL < 95). were significantly higher (p < 0.02) than larger lobsters,
regardless of sex or location. Small females had significantly
higher DSI's (p < 0.04) than large females. Males showed no

significant differences when similarly compared. A complete non-
parametric regression analysis of our DSI in relation to proximity
to the 106-Mile Site, may indicate whether sewage sludge dump-
ing had any effect on the severity of shell disease lesions in off-
shore American lobster populations.

DEPTH-RELATED PREDATION PRESSURE ON LARVAL
LOBSTERS {HOMARUS AMERICANVS) PRIOR TO
SETTLEMENT. Wendy Norden and J. Stanley Cobb, Univer-
sity of Rhode Island. Kingston. Rl 02880.

Prior to settlement, lobster postlarvae swim near the surface of
the water for several days. Settlement behavior begins between
two and six days after metamorphosis from the third larval stage.
During settlement, postlarvae dive from the surface to the benthos
to seek out suitable habitat. This potentially exposes the postlarvae
to a variety of predators not present at the surface. Understanding
where in the water column predation pressure is the greatest will
help in understanding behavioral strategies used by lobster post-
larvae when seeking appropriate habitat. We tethered postlarvae at
various depths to experimentally test the potential predation pres-
sure throughout the water column. Tether lines were deployed over
mud and cobble substrate types to look at the difference in preda-
tion pressure as it relates to habitat w ith four depths (0.5m. 1 .5m.
2.5m. 4m) above substrate. Higher predation occurred over cobble
substrate than over mud. Most of the predation occuired within
1.5m of the bottom and was probably primarily due to the cunner.
Tautogolabrus adspersiis. Laboratory observations with
epibenthic fish species confirmed their ability to consumer post-
larvae.

THE EFFECT OF TEMPERATURE ON SEXUAL MATU-
RITY IN THE FEMALE LOBSTER, HOMARUS AMERICA-
NVS. Susan A. Little* and Win Watson, University of New

Hampshire, Zoology Department, Durham, NH 03824; Bonnie
Splnazzola, Atlantic Offshore Lobstermen's Association, I 14 Ad-
ams Road. Candia. NH 03034.

The purpose of this study was to determine if female American
lobsters {Homarus americaniis) inhabiting areas with dissimilar
annual temperature profiles (measured in degree days >I0°C),
reached sexual maturity at different sizes. We compared the size at
maturity of lobsters captured in 3 different offshore locations (two
southern and one northern). 1 inshore site (Isles of Shoals. NH)
and one estuarine site (Great Bay Estuary. NH). Lobsters inhabit-
ing the Great Bay Estuary experienced the warmest temperatures
( 1320 degree-days), followed by the southern offshore sites (876:
906). the northern offshore site (669), and the Isles of Shoals (416).
Lobsters that migrate could gain a considerable thermal advantage.
For example, southern offshore lobsters would increase their de-
gree-days from 216 to 906. For this reason temperature exposure
for the offshore lobsters was calculated assuming a seasonal mi-

412 Abstracts. 2002 Annual Meeting. April 14-18. 2002

National Shelltisheries Association. Mvstic. Connecticut

gration. The maturity of female lobsters was determined using a
combination of abdominal width/carapace length ratios, cement
gland stages, and egg stages. As expected, lobsters that experi-
enced the most degree-days above 10°C reached sexual maturity at
smaller sizes. The size at which 50% of the lobsters were mature
was 84 mm CL for the Great Bay estuary (n = 7 1 ). <83 mm CL for
the two southern offshore sites (n = 80 lobsters), 91 mm CL for the
northern offshore site (n=40), and 89 mm CL for the Isle of
Shoals (n = 33). This difference in size at maturity was also mani-
fested in the average size, and range of sizes of berried females
captured in each location. These data suggest that regional differ-
ences in size at maturity should be taken into account when man-
aging the fishery. However, it is important to fully understand all
the behavioral, developmental and oceanographic processes that
influence size at maturity before implementing any new regula-
tions. This project was funded by the Northeast Consortium.

ENVIRONMENTAL MONITORS ON LOBSTER TRAPS.
James Manning, Northeast Fisheries Science Center, Woods
Hole. MA. Bonnie Spinazzola, Atlantic Offshore Lobster Asso-
ciation; Patrice Farrey. Maine Lobstermen Association; David
Casoni, Mass Lobstermen Association; Clare GrindaU Downcast
Lobstermen Association.

Beginning in early 2001, electronic temperature probes were
distributed to over 70 New England lobstermen by the four largest
associations. These units are recording hourly temperatures at
fixed locations throughout the Gulf of Maine including many off-
shore canyons sites. The objective is to occupy these same loca-
tions year-after-year for documenting interannual variability of the
deeper bottom water. Phase II of the project, just getting underway
in 2002, will deploy several salinity sensors around the region. The
primary objective is to characterize the water mass and to as.sess
the influence of remote source water. Participating lobsteren are
asked to record catch wherever probes are attached. Understanding
the biological significance of the physical variability is a second-
ary but a potentially worthwhile bi-product of the study. Details
are posted on the project website http://www.nefsc.nmfs.gov/
-jmanning/emolthtml.

LESIONS ASSOCIATED WITH RECENT EPIZOOTIC
SHELL DISEASE IN HOMARLS AMERICANLS ON THE
NORTHEAST COAST. Roxanna Smolowitz,* Andrea Hsu.
and Erin Summers, MBL. 7 MBL St.. Woods Hole. MA 02543;
Andrei Christoserdov, Marine Sciences Research Center, SUNY,
Stony Brook. NY 11794.

Shell disea.se is a commonly recognized problem in lobsters
held in impoundments during winter months. However, recently,
the economically devastating disease has reached high proportions
in free-living populations along the coast from Eastern Long Island
Sound, New York, to the Vineyard Sound, Massachusetts. The

disease is characterized by multifocal to confluent, shallow to deep
erosions, primarily of the dorsal carapace, often resulting in thin,
easily compressed shells and, rarely, ulcerations. Grossly, lesions
usually show marked melanization. but no obvious pathogen.

Histopathologically, carapace erosions are of variable depth;
but deeply extensive erosions are common. Carapace matrix in the
erosions is usually missing, but in some instances, unlike lesions
noted in impoundment disease, pillars of carapace matrix still re-
main attached in the eroded tissue. Intlammation is composed of
increased numbers of hemocytes in the underlying connective tis-
sues and accumulations of usually necrotic hemocytes between
layers of carapace. A second type of intlammation consists of
variable amounts of carapace proliferation produced by the intact
hyperplastic epithelium underlying the eroded site. Various organ-
isms are identified in the erosions, but the predominate organism
found at the interface of necrotic and li\e shell are bacteria. Work
is underway to identify the pathogenic bacteria at the lesion inter-
face.

WHAT CAN DATA FROM INDUSTRY CONDUCTED
TAG-RECAPTURE PROGRAMS TELL US? A STUDY OF
THE AMERICAN LOBSTER {HOMARUS AMERICANUS).
Barbara A. Soniers,* and Kathleen M. Castro, University of
Rhode Island. Fisheries Center. East Farm. Kingston. RI 02881;
John Sorlien, Rhode Island Lobstermen's Association. Box 421,
Wakefield, RI 02880; Tom Angell, Department of Environmental
Management, Coastal Fisheries Lab. Wakefield. RI 02880.

Tag-recapture data for the American lobster {Himuiriis ameri-
cciiuis) compiled from a two year experimental tag-recapture pro-
gram done by the Rhode Island Lobstennen's Association (R1L.\)
in cooperation with Rhode Island Sea Grant and the Rhode Island
DEM is being used to determine migration and life-history char-
acteristics for the Southern New England lobster stocks. Twenty
boats participated in the program; number of lobsters tagged and
recaptured varied among fishing areas, taggers and recapture re-
porters. A total of 1 1 .964 lobsters were tagged and released over a
16-month period. The majority of the lobsters were tagged in
August and September of 2000 and the months with the highest
recapture rates were Jul\, August and September of 2000 and
2001. 982 lobsters have been recaptured to date giving a recapture
rate of 8.29(^. Recaptures were received by phone and by mail with
a total of 91 fishermen returning tags. Biological data estimating
growth, egg frequency, shell disease and movement will be re-
ported as well as perceptions about cooperatise research from the
project participants.

National Shellfisheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 413

OYSTER REEFS AND RESTORATION

A CHARACTERIZATION OF IDEAL HABITAT STRUC-
TURE FOR THE STRIPED BLENNY CHASMODES BOS-
QUIASUS. Elizabeth M. Flynn* and Kennedy T. Paynter, Jr..

Department of Biology. University of Maryland. College Park.
MD 20742.

Historically, oyster reefs dominated the trophic interactions of
the Chesapeake Bay. Besides improving water quality and forming
an integral link in the food web. oysters provided the physical
structure that facilitated the development of a complex benthic
community. In the past 150 years, disease, overfishing, and pol-
lution have decimated the oyster population and destructive har-
\ esting methods have reduced complex, high relief reef structures
to flat "footprint" bars. The impact of this systemic loss of vital
habitat on the small demersal reef fish has gone relatively unstud-
ied. The objective of this study is to separate and identify the
structural components of shell aggregations that constitute desir-
able habitat for the striped blenny, Chasmodes hosqiiiainis.

Disarticulated oyster shells were arranged in a 30-gallon glass
aquariumin various vertical, horizontal and angled arrays to tested
for spatial preferences. Individuals were released into the tank and
remotely observed via overhead video for one hour. Mean perch
duration, perch frequency, minimum and maximum perch dura-
tions, and total duration of association with the shell arrays were
analyzed. The results indicate that C bosqidaniis prefer 1-2 cm
NN distances. C. bosquianus similarly preferred 60 degree arrays
as compared to 90 degreeswith regard to total association, maxi-
mum perch duration, and frequency. The short-term behavioral
patterns analyzed here suggest that purely structural elements of
oyster reefs are critical factors influencing demersal fish popula-
tion distributions.

A COMPARISON OF ACOUSTIC TECHNIQUES,
VIDEOGRAPHY, AND QUADRAT SAMPLING FOR
CHARACTERIZING SUBTIDAL OYSTER REEFS. Ray-
mond Grizzle,* Larry Ward, and Jamie Adams, Jackson Estua-
rine Laboratory, University of New Hampshire, Durham. NH
03824; Semme Dijkstra, Center for Coastal & Ocean Mapping.
University of New Hampshire; John Nelson, New Hampshire Fish
& Game Department. Marine Fisheries Division. Durham. NH
03824.

Acoustic techniques, videography. and quadrat sampling were
used to characterize several subtidal oyster reefs in the Great Bay
Estuary in New Hampshire and to compare their effectiveness,
with the long-term goal being a general protocol for reef mapping
and monitoring. The acoustic techniques consisted of single beam,
multibeam. and sidescan sonar. Preliminary analysis of the acous-
tics data indicated that reef boundaries could readily be mapped.

Videography was conducted by systematically imaging each of 40
sampling cells in a grid covering the approximate area of each reef.
A single drop was made in each cell and a 5 to 10-s recording
made of a 0.25 m" area. A still image was produced for each of the
40 cells and all were combined into a montage that revealed the
approximate boundaries of the reef. Five to ten cells on each reef
were randomly chosen and sampled by divers using a 0.25 m"
quadrat; all live oysters were measured (shell height) to nearest
millimeter using calipers. Oyster counts were also made directly
from each video image and compared to the quadrat data from the
same photo; preliminary analyses showed good correlations be-
tween these counts. Overall, our results to date indicate that acous-
tic techniques generally can delimit the boundaries of oyster reefs,
as has been demonstrated in other studies. Their potential for in-
ferring other reef characteristics (e.g., oyster densities), however,
is being assessed. Thus far, the major finding is that videography
may be a powerful and relatively inexpensive tool for detailed reef
mapping, including inferring oyster densities and perhaps other
characteristics.

PREVALENCE OF ENTERIC MICROORGANISMS IN
THE EASTERN OYSTER {CRASSOSTREA VIRGINICA)
AND THEIR OVERLYING WATERS AT REPRESENTA-
TIVE SITES OF AN OYSTER GARDENING PROGRAM IN
MOBILE BAY. ALABAMA. Kimberly A. Hamilton,* D.
LaDon Swann. and William Burkhardt. III. Department of Fish-
eries and Allied Aquacultures, Auburn Uni\ersity. Auburn. AL
36849 and U.S. Food and Drug Administration. 1 Iber\ille Dr.,
Dauphin Island. AL 36528-0158.

An oyster gardening program for the restoration of the eastern
oyster. Crassostrea virgiiiica. in Mobile Bay. AL began in May
2001. Thirty study locations were established in Mobile and Bald-
win counties along Mobile Bay. Of these sites three from Mobile
county and two from Baldwin county for a total of five sites were
purposefully selected in July 2001 to determine if a relationship
exists between enteric microorganisms in oysters and their over-
lying waters at different geographic locations along Mobile Bay.
The oysters were suspended under piers inside Eastfields floats
(surface area of 0.35 m") which positioned the oysters directly
below the surface of the water. Oyster and water samples were
taken from each site on a monthly basis and levels of fecal
coliforms. Escherichia coli. and male-specific bacteriophage (an
enteric viral simulant) were quantified using pre-established pro-
tocols.

The results have shown intermittent high levels of coliforms in
the shellfish, but levels in their overlying waters were low. The
bacteriophage levels have been consistently lower than the level of
detection; thus indicating that human wastewater treatment source
is not a likely cause of fecal coliforms. A high number of birds and
their droppings have been observed at some of the study sites from
which the Eastfields floats are suspended. These results may have

414 Absli-acts. 2002 Aiiiuial Meeting. April 14-IX. 2002

National Shelltisheries Assdciation, Mystic. Connecticut

implications tor future site selection of the gardening program.
Findings from these analyses will be used to inform local com-
munities and regulatory agencies of any impacts water quality may
have on shellfish quality.

USE AND VALUE OF OYSTER REEFS AMONG RECRE-
ATIONAL FISHERMEN IN LOUISIANA. J. C. Isaacs.* Loui
siana Department of Wildlife and Fisheries, Baton Rouge. LA
70898; W. R. Keithly, Coastal Fisheries Institute, Louisiana State
University. Baton Rouge. LA 7080,^; A. Diagne, University of
Arkansas at Pine Bluff. Department of Business Administration,
1200 North University - Mail Slot 4976. Pine Bluff. AR 71601,
Oyster reefs serve a wide variety of purposes. Until recently,
the majority of research has focused on the relationship between
the quantity and quality of reefs and commercial harvests derived
therefrom. Recreational fishermen in Louisiana, however, also
make extensive use of oyster reefs. This study serves two pur-
poses. First, it provides an examination of catch rates and species
diversity among inshore recreational fishermen fishing over oyster
reefs in comparison to inshore fishermen not fishing over oyster
reefs. Second, it provides an estimate of "willingness to pay"
among individual recreational fishermen for the privilege of fish-
ing over oyster reefs. This study employed a telephone survey of
Louisiana anglers to generate an estimate of the \alue of oyster
reefs in Louisiana to the recreational fishing sector. Overall, the
value is found to be significant, gives a justification for maintain-
ing oyster reefs in addition to commercial harvest.

RELAYING AS A METHOD TO REMOVE HOOKED MUS-
SELS FROM OYSTERS PRIOR TO REHARVEST FOR
SALE. Earl J. Melancon, Jr.,* Biology Department. Nicholls
State University, Thibodaux. LA 70.^10; Dale Diaz, Mississippi
Department of Marine Resources, Biloxi, MS 39530; Badiollah
Asrabadi, Math Department. Nicholls State University,

The hooked mussel. Ischadiuin recumtm, is considered a na-
tive species of the northern Gulf of Mexico, but introduced in the
Chesapeake, It is a small bivalve that can be found in great num-
bers attached to subtidal oysters located in low salinity environ-
ments. In the northern Gulf of Mexico, oystermen have tradition-
ally relayed mussel-laden oysters to higher salinity waters with the
expected results of killing them, and thereby removing them, be-
fore reharvest of the cleaned oysters for sale.

Results of laboratory, small-scale field and two commercial-
scale field experiments indicate that removal of mussels due to
transplanting to higher salinity waters is less a function of physi-
ological salinity stress and more a function of the transplanting
process itself. The hooked mussels were crushed, dislodged or
stressed to the point of permanent gaping during the commercial-

scale relaying operations. This resulted in an immediate 32-34%
mussel moilality, with a corresponding negligible oyster mortality.
The dead and dying mussels attracted predators, such as the blue
crab. CalliiH'ctes sapidus. and the southern oyster drill, Stranumita
hcu'imi.\iiniHi. which resulted in continued mussel losses in the
ensuing weeks.

In the more coastal location, where salinity is relatively high,
mussel-fouled oysters were cleaned by the third week after a sum-
mer transplant. The dilemma is that oysters in the higher salinity
waters may also become more vulnerable to predators and the
oysteriiian must consider reharvest in a short time. In the more
inland location, where salinity is more intermediate, and perhaps
with less abundant oyster predators present, mussel removal was a
longer process with 76% removed by the fifth week. Results of
these experiments strongly suggest that salinity is a key element in
the removal of mussels from oysters, but more in the role of a
habitat response and less as a physiological response to salinity.

MODELING THE INFLUENCE OF FILTRATION BY OYS-
TER STOCKS ON TURBIDITY AND SEAGRASS
GROWTH. Roger I. E. Newell,* Evamaria VV. Koch, Melissa
K. Wood, Ray E. Grizzle, and Raleigh R. Hood, Horn Point
Laboratory, University of Maryland Center for Environmental Sci-
ence, PC Box 775, Cambridge, MD 21613-0775,

Chesapeake Bay has undergone severe ecological changes.
Oysters are almost completely absent due to overharvest and dis-
eases and seagrass beds either are in decline or have disappeared
due to high water turbidity reducing light availability. Oyster reefs
and seagrass beds tended to impro\e water quality by actively
filtering particles out of the water, reducing wave energy, and
minimizing sediment resuspension. In part because of the loss of
these ecosystem functions water quality is poor and seagrass res-
toration not always successful. We hypothesized that the reestab-
lishment of oyster stocks, either via restoration of reefs or aqua-
culture, could benefit seagrass populations. Results from labora-
tory experiments and field studies were used to paramterize a
model predicting the extent to which oysters and seagrasses can
affect water quality and enhance the chances of survival and ex-
pansion of seagrasses. Oysters were capable o\ increasing light
penetration due to high summer filtration rates. The ability of
seagrass beds to minimize resuspension was a function ot water
depth and their capacity to attenuate waves. Large reproductive
seagrass plants that occupied the entire water column were more
effective at minimizing resuspension than small vegetative plants.
Our model results suggest that increasing oyster stocks can dra-
matically enhance light penetration and thereby increase the area
of Bay bottom where seagrasses can grow.

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 1-H8. 2002 415

THE EFFECT OF STOCKING DENSITY ON BENTHIC
COMMUNITY DEVELOPMENT AND SHELL HEIGHT IN
THE EASTERN OYSTER. CRASSOSTKEA VIRGINICA.
Stewart Harris* and Kennedy T. Paynter, University of Mary-
land. College Park. MD 20742.

The eastern oyster. Crassostrea virginica. is a gregarious, reet-
formins organism. Oyster populations that once dominated the
Chesapeake have declined significantly and interest has recently
arisen to restore the economic and ecological benefits of native
oyster populations. Understanding the ecological importance of
oysters and oyster reefs is critical to the restoration of the estuary's
ecosystem as a whole. Oyster densities on most Maryland reefs are
very low (about 3 oysters/m"). however, natural reefs formed in
other areas are comprised of high densities of oysters (>500/m^).
In order to maximize the effectiveness of oyster restoration, it is
important to determine how oyster density may affect oyster
growth, parasite prevalence and the formation of reef habitat uti-
lized by the benthic community.

In the fall of 1999, twelve 0.2-acre experimental plots were
constructed in the Patuxent River by placing fossil oyster shell on
a barren natural oyster bar. The plots were assigned one of four
treatments, zero. 124, 247. 494 oysters/m". in a randomized de-
sign. Oyster growth was 0.12 (± 0.004 SEM) mm/day for the 2000
season and slowed to 0.07 (± 0.006) mm/day in 2001. The data did
not show any effect in shell height due to density of oysters.
Colonization of the oyster reefs with fouling organisms was related
to density of oysters. In summer and fall of 2000. barnacle density
declined with increasing oyster density.

EVALUATING THE CONTRIBUTION OF COMMERCIAL
OYSTER AQUACULTURE TO RECRUITMENT. Rachel E.
Sackett,* Russ Peterson, and Ami E. Wilbur, Department of
Biological Sciences and the Center for Marine Science. .5600 Mar-
vin K. Moss Lane. University of North Carolina at Wilmington.
Wilmington, NC 28409; Jim Swartzenberg, J&B AquaFood.
Holly Ridge. NC 28445.

Commercial oyster {Crassostrea virginica) aquacullure opera-
tions concentrate large numbers of potentially reproductive ani-
mals. Such aggregations may function to produce large numbers of
larvae that subsequently recruit to natural beds. Anecdotal obser-
vations have suggested the enhancement of recruitment in the vi-
cinity of commercial aquaculture operations; however, confirma-
tion of such an effect has generally not been possible, as new
recruits derived from the cultured stock generally cannot be dis-
tinguished from those resulting from the spawning of wild oysters.
Recent stocking of commercial leases in North Carolina with oys-
ters produced in a hatchery in Louisiana has provided an oppor-
tunity to evaluate the potential for the enhancement of local re-
cruitment due to the spawning of cultured stock. Oysters from the

Gulf coast and Atlantic coast have been previously shown to ex-
hibit genetic differences in mitochondrial DNA (mtDNA) haplo-
type. PCR (polymerase chain reaction) amplification and DNA
sequencing of 409 base pairs of the 16s ribosomal gene are being
used to screen recent recruits collected from 7 natural beds sur-
rounding 1.5 acres of leased bottom (stocked with -70, 000 Gulf
oysters), as well as a sample of the culture stock. Sequence data
will be analyzed to determine what proportion of recent settlers
exhibited mtDNA haplotypes consistent with the hypothesis that
they were produced by the spawning activity of the culture stock.
Preliminary analyses revealed some recruits exhibit mtDNA hap-
lotypes identical to those exhibited by the culture stock, suggesting
that there is some local recruitment resulting from reproduction of
commercial aquaculture stock.

RECRUITMENT OF THE OYSTER CRASSOSTREA VIR-
GINCA ON INTERTIDAL REEFS IN AREAS WITH IN-
TENSE BOATING ACTIVITY IN THE INDIAN RIVER LA-
GOON, FLORIDA. Lisa WalL* Linda Walters, Kevin
Johnson, and Neysa Martinez, Department of Biology. Univer-
sity of Central Florida, 4000 Central Florida Blvd. Orlando, FL
32816; Ray Grizzle, Jackson Estuarine Laboratory. University of
New Hampshire, Durham. NH 03824.

Productivity, diversity and survival of estuaries are threatened
by explosive coastal population growth and associated recreational
activities. One major area of recreational growth has been the
number of people motoring in small pleasure craft at high rates of
speed. In counties bordering Mosquito Lagoon (northernmost sec-
tion of the Indian River Lagoon systein. east coast of central
Florida), there were 51.000 registered boaters in 1998. Numbers
have increased 10% annually since 1986 and continue to grow. In
areas of Mosquito Lagoon with intense boating activity, intertidal
reefs of Crassostrea virginica with dead margins commonly occur.
The dead margins consist of mounds of disarticulated shells. The
cause(s) of the reef die-offs is unclear. However, the disarticulated
shells may be reducing reef sustainability if these surfaces are
unavailable for oyster recruitment. Recruitment trials were run on
eight reefs (4 impacted. 4 healthy) in two eight-week trials in
Summer 2001 and Winter 2001-2002. Sediment loads, tempera-
ture and water motion at all sites were monitored. In the Summer
2001 trial, no significant differences were found between or within
sites for settlement or recruitment. However, temperatures reached
over 40°C on portions of the dead reef. Data of this type is needed
to identify causes of reef declines, habitat-specific management
protocols and appropriate restoration techniques.

416 Abstnicts, 2002 Annual Meeting. April 14-18, 2002

National Shelltisheries Association, Mystic, Connecticut

OFFSHORE FISHERIES

INTEGRATING VESSEL TRACKING, CATCH DATA.
AND DEPLETION MODELS TO ESTIMATE COMMER-
CIAL SCALLOP DREDGE EFFICIENCY. Todd Gedamke*
and William DuPaul. Virginia Institute of Marine Science, School
of Marine Science. College of William and Mary, Gloucester
Point, VA 23062.

In June of 1999, Georges Bank Closed Area II was opened to
the United States sea scallop fleet after a five-year multi-species
fishing closure. During the five-month opening, nearly six million
pounds of scallop meats were harvested, catch-per-unit effort data
were collected from o\er 1.000 commercial tows, and the tine-
.scale distribution of fishing effort was recorded hourly by manda-
tory vessel monitoring systems. A spatial analysis of both catch
and effort data was perfoimed to locate areas consistent with the
DeLury model assumptions. Gear efficiency was estimated to be
45'7c utilizing a combined maximum likelihood analysis of CPUE
declines in all suitable regions. An additional mdependent estimate
of efficiency was generated from survey stations that were
sampled before and after the opening. A kriging analysis was used
to determine mean catch rates and the index-removal method was
applied to compare the change in catch rates to the total landings
reported for the opening. Dredge efficiency was estimated to be
54Vc.

The results of this study suggest that the 25% efficiency esti-
mate used in calculations prior to the opening resulted in the over-
estimation of absolute biomass and the setting of a quota that
exceeded target exploitation levels. In addition, the results ot this
study suggest that information from the vessel monitoring systems.
now in use on many commercial fleets, can provide the fine scale
spatial details necessary to successfully apply depletion models to
open-ocean commercial fishing operations.

(<80 mm) scallops ha\e stimulated interest in developing an area
wide management strategy for sea scallops.

During commercial openings of these areas during 2000-2001,
the performance of a 4-inch (101 nnii) ring scallop dredge was
evaluated against the standard 3.5-inch (88 mm) ring dredge. De-
tailed catch data from 208 tows on eight commercial trips showed
a reduction in scallop harvest of up to 42.5% for scallops <90 mm
and increases in harvest efficiency of up to 12.9% for scallops
>l 15 mm. These results are closely related to the size frequency
distribution of the scallop resource. When the size frequency dis-
tribution of the scallop population has a modal shell height of
gi eater than I 1 0 mm, the use of a 4-inch ring dredge may play a
significant role in realizing the benefits of area-based management.

REBUILDING SEA SCALLOP (PLACOPECTEN MAGEL-
LANICUS) STOCKS USING AREA CLOSURES AND RO-
TATIONAL FISHING. Deborah Hart* and Paul Rago, NCAA
Fisheries, Northeast Fisheries Science Center, 166 Water St..
Woods Hole, MA 02543.

In the seven years since the closure of three large areas on
Georges Bank to trawling and dredging in December, 1994, sea
scallop biomass on Georges Bank has increased by more than a
factor of twenty. Limited fishing in the closed areas in 1999 and
2000 yielded about 1 1 million lbs of scallop meats while still
allowing for increases in biomass in these areas. Dramatic in-
creases in biomass have also been seen in the Mid-Atlantic Bight
after two areas there were closed to scallop fishing for three years
in 1998. These experiences indicate that area closures can be an
effective way to rebuild shellfish stocks and alleviate growth over-
fishing. Strong recruitment observed since the closures suggests
that the closed areas have also become important sources of scal-
lop larva. Both theory and practice indicate that rotational man-
agement, where areas are successively closed and then opened to
fishing, can increase both yield and spawning-stock biomass in the
sea scallop fishery.

THE USE OF A 4-INCH (101 MM) SEA SCALLOP {PLA-
COPECTEN MAGELLANICUS) RING DREDGES IN THE
CONTEXT OF AN AREA MANAGEMENT STRATEGY.
Kevin D. Goff, William I). DuPaul.* and David B. Rudders.

Virginia Institute of Marine Science. College of William and
Mary, Gloucester Point, VA 23062.

Early attempts to manage the sea scallop (Placopecten magel-
lanicus) fishery focused on establishing age-at-entry controls.
Since 1994, the primary management strategy shifted to an effort
control program and an increase in scallop dredge ring size to 3.5
inches (88 mm) from 3.0 inches (76 mm), with the intent of in-
creasing yield per recruit.

Dramatic increases in scallop biomass in three areas of Georges
Bank closed to mobile fishing gear in 1994 and two areas in the
mid-Atlantic closed in 1998 to protect concentrations of small

FLEET DYNAMICS OF THE ATLANTIC SEA SCALLOP
FISHERY. Paul J. Rago and Deborah R. Hart, NCAA Fisher-
ies, Northeast Fisheries Science Center. 166 Water St.. Woods
Hole, MA 02543.

The sea scallop (Placopecten magellanicus) supports the sec-
ond most valuable commercial fishery in the northeast USA. Clo-
sure of large areas of Georges Bank to scallopers in 1994 not only
resulted in rapid increases in scallop biomass but also altered the
spatial distribution of fishing effort. Detailed electronic informa-
tion on the hourly position of each vessel was used to study the
behavior of the fishing fleet for the period 1998 to 2000. Addi-
tional information from at-sea observers cortoborated the analyses

National Shellfisheries Association. Mystic. Connecticut

Ahstruit.s. 2002 Annual Meeting. April 14-18, 2002 417

of vessel monitoring data. Limited re-openings of the closure areas
in 1999 and 2000 induced marked shifts in fishing effort. Fine-
scale changes in fishing patterns can be related to the scallop
density, size composition and economic value as well as the pres-
ence of bycatch species. The scallop fishery is conducted over an
area of about 1 2.000 nni', but more than 75% of fishing activity is
concentrated within an area of about 3,000 nni". When scallop
density is low, however, fishing vessels disperse more widely.
Such variations in the concentration of fishing activity have im-
portant implications for impacts on habitat and finfish bycatch and
provides insights into management strategies for bivalve fisheries.

POPULATION STRUCTURE OF THE INDIAN SQUID, LO-
LIGO DUVAUCELII ORBIGNY, 1848. IN IRANIAN WA-
TERS OF THE OMAN SEA. Farhad Rajabipour,* Offshore
Fisheries Research Centre, Chabahar, Iran.

The population biology of the Indian squid Loligo duvauceli
Orbigny, 1848 was described from monthly sampling. December
1997 to January 1999. Samples were caught as by-catch of bottom
fishing trawlers from continental shelf area of Oman Sea at south-
east of Iran. 1281 samples including 694 males and 587 females
with the mean length of dorsal mantle and weight of 109.8 ± 34.95
mm and 48.32 ± 36.23 gr for males, and 1 10.6 ± 22.4 mm and
54.34 ± 28.35 gr for females, were collected. Maximum length
(ML) of dorsal mantle in males and females was observed in
January and June. Length-weight relationship was acquired. It is
reversed at 72 mm length of dorsal mantle in logarithmic model.
ML50 of males and females is 15.4 cm and 8.7 cm. There is no
significant difference between females and males frequencies at
the beginning of simimcr and ending of winter. NGI and GSl
indices detected that the pick of reproduction occurs at the begin-
ning of summer and winter. Males have three length classes, two
recruitment peaks at beginning of summer (17.4%) and winter
(82.6%), but females have only one length class and two recruit-
ment peaks at beginning of winter {58.979'f ) and summer (41.3%);
(tO = 1.4/year). Age and monality indices were measured. Catch
rate of L. diivcniceli in Iranian waters of Oman Sea was 0-100
kg/hour.

RECRUITMENT DYNAMICS OF NORTHERN SHRIMP
(PANDALUS BOREAUS) IN THE GULF OF MAINE. Anne
Richards,* Michael Fogarty, and David Mountain, NCAA
Northeast Fisheries Science Center. Woods Hole, MA 02543;
Mirta Teichberg, Boston University Marine Program, Woods
Hole, MA 02543.

The western Gulf of Maine is the southern limit of distribution
for northern shrimp Pandalus borealis. Previous studies showed

that recruitment of northern shrimp in the Gulf of Maine is sig-
nificantly affected by spring surface temperatures as well as
spawning stock biomass. The temperature effect corresponds to the
period of planktonic larval development. The purpose of this study
was to extend these analyses by examining the influence of addi-
tional environinental factors (freshwater runoff, wind patterns, and
climate indicators) on recruitment and to consider mechanisms for
the spring surface temperature effect. Runoff and wind patterns
strongly affect circulation within the western Gulf of Maine and
thus may affect recruitment through effects on retention and/or
transport of larvae. Results to date indicate no significant effect of
mean river discharge, timing of peak discharge, other temperature
effects, or the NAO winter index; however, the spring surface
temperature effect continues to be significant. The match-
mismatch hypothesis is a possible explanation for this effect, and
we are evaluating the hypothesis by developing estimates of timing
of onset of the spring phytoplankton bloom along with estimates of
timing of the shrimp hatch.

DEEP SEA RED CRABS OFF SOUTHERN NEW EN-
GLAND: HAS THERE BEEN A FISHERY IMPACT ON
THE POPULATION? James R. Weinberg* and Charles Keith,

NMFS. NEFSC. 166 Water St., Woods Hole, MA 02543.

A directed trap fishery for the deep-sea red crab, Chaceon
(formerly GeiyoiO i/iiiiit/Kcdeiis Smith, has taken place off the
coast of southern New England since the mid-1970s. These slow-
growing crabs occur from about 250-1000 m, and males grow
larger than females. The National Marine Fisheries Service con-
ducted a survey of population size-structure in 2001 and measured
the relationship between carapace width and body weight. This
inforination had not been updated since the late 1970s. Given that
the fishery targeted large males, we examined whether there was a
decline over tmie in the percentage of large males in the population
and in the individual weights of large males. Overall, the percent-
age of large males in 2001 was lower than in 1974, whereas the
fraction of large females either stayed the same or increased
slightly during the same time period. Data collected between 1973
and 2001 suggested that male body weight, for a given body size,
has declined over time. There was no trend over time in female
body weight. Thus, compared to the 1970s, the population appears
to have a lower percentage of large males in 2001, and these males
have lower biomass for their carapace size. The results might be
due to harvesting of large, heavy males over time; however, the
surveys are too widely spaced in time, and too few in number, to
draw strong conclusions about causality.

418 Abstracts. 2002 Aniiiwl Meeting, April 14-18. 2002

National Slicllfisheries Association. Mystic. Connecticut

DISEASES OF SHELLFISH:
CONTRIBUTED SESSION

MORTALITIES OF CULTURED ABALONE, HALIOTIS
IRIS, INFECTED BY A NOVEL HAPLOSPORIDIAN. B. K.
Diggles, P. M. Hine, V. L. Webb, and E. W. Maas, National
Institute of Water and Atmospheric Research, Kilhirnie. Welling-
ton. New Zealand; J. Nichol and S. Wakefield, School of Medi-
cine. University of Otago. Wellington South. New Zealand: R.
Roberts, Cawthron Institute. Nelson. New Zealand; C. S. Fried-
man. School of Aquatic and Fishery Sciences. University of
Washington. Seattle. WA; N. Cochennec-Laureau, Laboratoire
de Genetique et Pathologic. IFREMER. La Tremblade. France;
K. S. Reece and N. A. Stokes.* Virginia Institute of Marine Sci-
ence. Gloucester Point. VA.

Chronic mortalities of juvenile abalone, Haliotis iris, occurred
in a commercial culture facility in New Zealand during the Austral
summer and autumn of 2000 and 2001. Histopathology of mori-
bund abalone showed heavy systemic infections of a uni- to multi-
nucleate protozoan parasite associated with severe tissue damage.
Heavily infected abalone exhibited lethargy, loss of righting reflex,
and weak surface adherence. Mortality levels reached 90% in the
affected raceways. The parasite was identified as a haplosporidian
based on TEM and molecular analyses. Ultrastructural character-
istics of the parasite included the presence of multinucleate Plas-
modia, lipid droplets, an anastomosing endoplasmic reticulum and
the production of haplosporosome-like bodies from nuclear mem-
brane-bound golgi apparatus that matured into haplosporosomes.
Molecular confirmation of the TEM identification was accom-
plished by performing in situ hybridization (ISH) and by PCR-
amplifying and sequencing the parasite's small subunit ribosomal
RNA (SSU rRNA) gene. A DNA probe specific for several mem-
bers of the haplosporidia exhibited hybridization to the Plasmodia
in ISH of infected animals. The SSU rRNA gene sequence was
novel, but phylogenetic analyses strongly supported grouping this
parasite with the haplosporidians. The parasite was at the base of
the phylum Haplosporidia. ancestral to Urosporidiiim crescens and
the Hiiplosporidtiitn. Mincliiniu. and Bonamia species.

LOW SALINTY TOLERANCE IN MANILA CLAMS
VENERUPIS PHILIPPINARUM. Ralph Elston, AquaTechnics/
Pacific Shellfish Institute. PC Box 687. Carlsborg. WA 98324
USA; Dan Cheney. Brian MacDonald. and Andrew Suhrbier,

Pacific Shellfish Institute, Olympia. WA.

The manila clam. Venenipis philippiiuiniin. is the second most
important product of molluscan aquaculture on the west coast of
the United States and is grown on several other continents. We
evaluated the tolerance and the low salinity response in this clam

species using experimental exposures of 17 populations of manila
clams. Ambient seawater (30 psu) and freshwater were first tem-
perature conditioned, then mixed using aperture controlled flows
into incremental salinity head tanks that supplied the test cham-
bers. Water supplied to the test tanks was supplemented with algal
feeds. Low salinity tolerance of clams was determined by perform-
ing exposures of up to four weeks in duration at constant salinities,
followed by a recovery period. Clams were tested in salinity in-
crements of 2.5 psu ( =ppt).

Tolerance to low salinity events in clams is both a function of
survival response (duration of shell closure) and the physiological
adaptability of the clams to exist in low salinity environments. All
individual clams from all sources were tolerant to \5 psu but
populations showed variable proportions of individuals with tol-
erance to 12.5 psu and none were tolerant to long term exposure to
10 psu. Variable tolerance to 12.5 psu appears to be a function of
the effectiveness and duration of the shell closure response. All
clains could withstand 6 days at 5 psu. but complete group mor-
tality occuned by 12 days at 5 psu. All clams could withstand 8
days exposure to 10 psu but complete group mortality occurred by
14 days at 10 psu. Little effect of temperature was found on the
clams" tolerance to marginal low salinities. The low salinity ex-
posures resulted in alterations in the digestive gland epithelium
including loss of granulation, cellular swelling and cellular slough-
ing. We concluded that candidates to .select for low salinity toler-
ance are available in most populations but some populations are
enriched with individuals with tolerance to the marginal salinity
(12.5 psu).

Research supported by Saltonstall-Kennedy project
NA96FD0194. National Marine Fisheries Service. U.S. Depart-
ment of Commerce to Pacific Shellfish Institute. Olympia. Wash-
ini;ton.

A SPECIFIC PATHOGEN FREE CULTURE SYSTEM FOR
C. GIGAS LARVAE AND SPAT. Sean E. Matson* and Chris-
topher Langdon. Hatfield Marine Science Center. Oregon State
University, Newport, OR 97365.

The Molluscan Broodstock Program (MBP). a selective breed-
ing program for the Pacific oyster. Crasso.slrca .(•ificis. uses a Spe-
cific Pathogen Free culture system for all production and mainte-
nance of larvae, spat, broodstock and microalgae. This system is
necessary to exclude infectious agents of Haplosporidian costale
(Seaside Organism, SSO). which has been found in Pacific oysters
grown in Yaquina Bay, Oregon, where the program draws its
seawater. and for the safe outplanting of MBP spat in field test
sites along the West coast (USA). All seawater entering MBP
facilities is filtered through sand, diatomaceous earth, and a series
of 20. 5. and 1 jjLm cartridge filters. Seawater to mass algal cultures
and the nurserv is afso irradiated with UV-light at >30.000 micro-

National Shellfisheries Association. Mystic. Connecticut

Abslnicts. 2002 Annual Meeting. April 14-18. 2002

419

Watts-sec/cni" (MWS) as a back-up precaution. .Since the system's
inception, no MBP spat have been identified as being contami-
nated with SSO. or any other infectious agent. A series of labora-
tory experiments was performed to assess the effects of UV v\'ater
on larval growth and survival, spat growth and sur\i\al. and mi-
croalgal culture density. Experiments with oyster larvae indicated
that both the micro-filtration system and UV water treatment had
a significant negative effect on larval growth (p = O.O(H)l). A
significant reduction in growth was evident at UV intensities as
low as 10.000 MWS (p < 0.05). Methods that have significantly
improved larval growth, survival, speed to metamorphosis and spat
growth within the SPF culture system include substituting a 0.2p.m
filter and charcoal for a UV filter when rearing larvae, and the
addition of calcium bentonite (2mg/ml/day) or calcium montmo-
rillonite (3mg/ml/day) to larvae and spat cultures (p < 0.05).

INTRAMITOCHONDRIAL CRYSTALS WITHIN THE
HAEMOCYTES OF MUSSELS {MYTILUS EDULIS) EXPE-
RIENCING UNEXPLAINED MORTALITIES. Gary R.
Meyer,* and Susan M. Bovver, Department of Fisheries and
Oceans, Pacific Biological Station. Nanaimo. British Columbia.
V9R 5K6. Canada.

Persistent mortality (having a cumulative total of about 75%)
was observed between August 1999 and April 2000 amongst a
stock of cultured blond mussels {Mytilus editUs selectively bred for
their blond coloured shell trait) grown near Quadra Island. British
Columbia, Canada. During this period, 7 samples of mussels (n =
30 per sample) were examined. Up to 27% had macroscopic le-
sions (orange pustules) and up to 50% had histopathology consist-
ing of multifocal and or diffuse haemocyte infiltration that con-
tained abundant phagocytic cells and necrotic cellular debris usu-
ally in the mantle/gonad and digestive gland. However in some
instances, this pathology was also observed in the adductor
muscle, gills, kidney or heart tissue. No etiological agent was
evident. Intracellular mycobacteria-like organisms were observed
within a few haemocytes in up to 20% of the mussels examined
however their role in the disease remains unknown. During ultra-
structural examinations, intramitochondrial crystalline arrays were
commonly observed within necrotic or lysed haemocytes. These
crystals were not observed "free" within the cytoplasm of affected
cells and never observed in adjacent "healthy-looking" cells. The
chemical composition and significance of these crystals is un-
known. However, we speculate that they are likely composed of
condensed protein. Similar crystals have been reported from the
mitochondria of a wide variety of organisms and were usually
affiliated with an abnormal situation such as disease, pathological
process, or response to cell damage.

ENVIRONMENTAL DETECTION OF THE RICKETTSI-
ALES-LIKE PROKARYOTE CAUSING WITHERING SYN-
DROME IN ABALONE. James D. Moore.* California Depart-
ment of Fish and Game. UC Bodega Marine Laboratory. 2099
Westside Road. Bodega Bay. CA 94923: Carolyn S. Friedman,
School of Aquatic and Fishery Sciences. Box 355020. University
of Washington. Seattle. WA 98195.

A gastrointestinal Rickettsiales-like prokaryote (WS-RLP) was
recently identified as the causative agent of Withering Syndrome,
a chronic, fatal disease of wild and cultured California abalone.
Using a polymerase chain reaction (PCR) diagnostic method de-
veloped to detect the WS-RLP in abalone gut tissue samples, we
have amplified WS-RLP DNA from necrotic tissue held at room
temperature for up to 7 days, from samples fixed and embedded in
paraffin, and from seed abalone as small as 2mm. A non-
destructive WS-RLP sampling method is desired since both wild
and farmed abalone are highly valued. We developed a method
involving tangential flow filtration that concentrates particles > 0.1
|j.m from large volumes of seawater. allowing purification of DNA
for PCR from 15-(- liter samples. Although sample processing is
laborious, using this method we have detected the pathogen DNA
in effluent from abalone holding units in both experimental and
culture facilities. The WS-RLP is present in feces from infected
animals, and PCR using fecal material appears to be useful for
efficiently detecting the WS-RLP in various stages of farm pro-
duction. We are currently investigating an hypothesis that filter-
feeding organisms concentrate the WS-RLP from endemic waters
and can. thereby, be used to monitor WS-RLP presence in natural
populations. Initial attempts to amplify WS-RLP DNA from gill or
gut tissue of mussels (Mytilus spp.) being held with WS-RLP
positive abalone have been unsuccessful. Yet it is clear that mo-
lecular-based tools will significantly aid management of this dev-
astating disease. Supported, in part, by California Sea Grant Col-
lege and the Marine Region, California Department of Fish and
Game.

VIBRIO ASSOCIATED WITH JUVENILE OYSTER DUR-
ING SUMMER MORTALITY IN FRANCE. J.-L. Nicolas and
M. Garnier Laboratoire de Physiologie des Invertebres. Ifremer
BP 70. 29 280 Plouzane. France; M. Gay and F. Leroux. Labo-
ratoire de Genetique et de Pathologic. Ifremer. Ronces Les Bains,
BPI33. 17390 La Tremblade.

Ainong the hypotheses advanced to explain summer mortalities
of oyster in France (Morest program), the bacterial disease ap-
peared one of most relevant. Indeed, recent works of Lacoste
(2fJOI ) and Waechter (thesis 2001 ) showed that of Vibrio spleiuU-
dus (I and II) strains were able to kill the juvenile or adult oysters
by bath or injection. To verify this hypothesis, oysters were
sampled along the French coasts and analysed during mortality
period. Only the half of moribund oysters were invaded by dom-
inant bacteiia. Haemolyniph of other diseased oysters as well as

420 Ahstiacts. 2002 Annual Meeting. April 14-1 «, 2002

National Shellfisheries Associatum. Mystic, Connecticut

healthy oysters was often colonised by more or less abundant and
diversified bacterial flora. The isolation of a same dominant bac-
terial strain on several oysters of the same batch, occurred only in
hatchery and on only one oyster park. The taxonomic studies (by
ARDRA of gyrase B. and sequences of 16S DNAr) made it pos-
sible to classify in ? species these opportunist bacteria; V aestn-
ariaims (3 types). I', splciulidiis (II) and V cifii;iiilUiriim. Except V.
aestuarianus (type II). Phenotypical characters of these strains
were fickle and could not be used to identify the bacteria.

a P. alkmticiis and compare by alignnienl uilh those pailically
characterize rRNA loci from other PcrkinsKs species. Actively
growing cultures exhibited considerable si/e heterogeneity with a
large proportion of clusters constituted by small cells. Behavior of
P. iiilaiiticiis in culture resembles that of P. nuinnus. Optimization
of the culture conditions for P. atlanticus is underway. Established
clonal cultures will be useful for genetic characterization of this
species, and to increase our understanding of its pathogenicity.
[Supported by Grant NAO6RGOI0I-5 from ODRP. NOAA,
through the MD Sea Grant College, to GRV, and the Portuguese
Government PERKLAM Program to MLC).

FURTHER MOLECULAR CHARACTERIZATION OF
PERKINSUS ANDREWSI AND RELATED ISOLATES. Wolf
T. Pecher, Jose A. F. Robledo, Cathleen A. Coss, and Gerardo
R. Vasta, COMB. UMBI, University of Maryland, Baltimore. MD
21202.

We previously described ultrastructure. behaviour in culture,
and the rRNA locus of a Pfikiiisns species isolated from the baltic
clam Macoma halthica. The morphological characterization did
not reveal features remarkable enough to cleariy indicate that the
isolate was a distinct Perkinxus species (Coss et al. 2001a). How-
ever, the degree of difference of all rRNA genes and intergenic
regions examined was comparable to or greater than differences
between accepted Perkiii.siis species, which lead to its designation
as P. aiulieusi n. sp. (Coss et al 2001a. b). Here we report further
molecular characterization of P. aiulrewsi and the characterization
of an additional isolate from the hard clam Mcneiuiria merce-
luiriu. Based on the molecular characterization both isolates are
closely related. Their relationship with other recognized Perkinsus
species will be discussed. [Supported by Grant No.
NA06RG010I-5 from ODRP. NOAA. through the Maryland Sea
Grant Colleae. to GRV[.

IN VITRO CULTURE OF PERKINSUS ATLANTICUS FROM
CLAMS TAPES DECUSSATUS: CHARACTERIZATION OF
THE rRNA gene. Jose A. F. Robledo* and Gerardo R. Vasta.

COMB. UMBI. University of Maryland. Baltimore. MD 21202;
Patricia A. Nunes and M. Leonor Cancela. Molecular Biology
and Biotechnology. CMS-CCMar. University Algar\e-UCTRA.
Campus Gambelas. XOOO-SIO Faro. Portugal.

Perkinsus allaiiiicus cultures were derived from fresh
hemolymph and gills, and from tissues incubated in fluid thiogly-
collate medium from infected clams Tapes (Icciissanis from Al-
garve (South of Portugal). P. uilaiuiciis cultures were established
in the medium developed and optimized for P. mariniis. Prolifer-
ating cells were cloned by limiting dilution, and the species iden-
tity was established by applying two PCR-based P. uihmticiis-
specific diagnostic assays and by sequencing the RNA locus. We
provide for first time the complete sequence of the rRNA locus of

GENE DISCOVERY IN PERKINSUS MARINUS USING EX-
PRESSION SEQUENCE TAGS (EST). Jose A. F. Robledo,*
Eric J. Schott. Adam G. Marsh, and Gerardo R. Vasta, COMB.

UMBI. University of Maryland. Baltimore. MD 21202.

Since the late 1940s the protistan parasite Perkinsus marinus
has been associated with mass mortalities of the eastern oyster.
Crassostrea virginica. Based on decades of research, several ap-
proaches have been proposed to control ""Dernio" disease includ-
ing eariy disease detection, selective breeding of disease-resistant
oysters, and treatment with anti-parasitic agents. However, none of
these approaches has proven effective. In recent years virtually all
fields of biology have benefited from the information generated by
genomic approaches. We are building a P. marinus EST database
sequencing cDNAs from two P. marinus Lambda ZAP libraries
constructed using P. marinus propagated in standard culture me-
dium and in medium supplemented with C. virginica serum. From
the first 300 ESTs two findings are evident; there are many ex-
pressed genes whose products are known to be involved in \iru-
lence of other parasites and there are notable differences in the
classes of genes expressed in control versus serum-supplemented
cultures. As the number of ESTs increases, so will prospects for
identifying new targets for therapy and for understanding the bio-
logical basis of parasite virulence, infectivity. and pathology.

THE NRAMP GENE AND COMPETITION FOR AVAIL-
ABLE IRON BETWEEN CRASSOSTREA VIRGINICA AND
PERKINSUS MARINUS. Jose A. F. Robledo* and Gerardo R.
Vasta. COMB. UMBI. University of Maryland. Baltimore. MD
21202.

In the past decades Perkinsus marinus has produced extensive
damage to oyster bars with catastrophic consequences for shell-
fisheries and the health of coastal waters. Despite intense parasite
pressure, no Dermo-resistant oysters have been identified so far in
natural populations. The identification of genes that are directly
linked to disease-resistance and assessment of their value as ge-
netic markers, may facilitate the establishment of disease-resistant

National Shellfisheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 421

oyster strains. Most parasites have strong iron requirements and
have developed efficient mechanisms for iron acquisition from
their hosts. Reciprocally, most hosts have developed mechanisms
to prevent pathogens from acquiring iron while maintaining avail-
ability for their own cells. Iron sequestration from the pathogen is
also a non-specific host response to infection (nutritional immu-
nity). The mammalian natural resistance-associated macrophage
protein (Nramp) is a membrane transporter thought to be a deter-
minant of resistance/susceptibility to intracellular pathogens. We
have cloned Nramp from C. virgiuica and P. iiniri)iiis. used them
to screen genomic libraries, and examine their gene organization.
The characterization of these genes and their products in both host
and parasite will provide insight into their competition for iron,
and yield information on the mechanisms underlying disease sus-
ceptibility [Supported by Grant No NA06RG0101-5 from ODRP.
NOAA, through the Maryland Sea Grant College, to GRV].

CHARACTERISATION OF GENE EXPRESSION IN RE-
SPONSE TO PERKINSUS MARINUS AND HAPLOSPO-
RIDIUM NELSONl INFECTIONS IN THE EASTERN AND
PACIFIC OYSTERS. Arnaud Tanguy.* Susan E. Ford, and
Ximing Guo, Haskin Shellfish Research Laboratory, Institute of
Coastal and Marine Sciences, Rutgers University, 6959 Miller Av-
enue. Port Norris, NJ 08349.

The eastern oyster Crassostrea virginica has two major dis-
eases: Dermo caused by the parasite Perkinsiis mariniis and MSX
by Haplosporidiwn nelsoni. Effects of infection in C. virginica
range from reductions in condition index, hemolymph protein con-
centrations and lysozyme activity to decline in reproductive out-
put, retarded growth and death. MSX-resistant strains of C. vir-
ginica have been developed at Rutgers University, and the Pacific
oyster C, gigas appears to be resistant to both diseases. At present,
however, we know little about molecular mechanisms of infection
and host defense against the two parasites. We have begun a study
searching for genes involved in host response to Dermo and MSX
infections, using subtractive expression analyses. For Dermo, con-
trolled artificial infections are conducted in both C. virginica and
C. gigas. For MSX, infected wild oysters and uninfected resistant
oysters, which were naturally exposed to the parasite, were used to
construct the subtractive library. Nine subtractive libraries were
made: six for Dermo and three for MSX exposures. About 70
clones were selected for sequencing from each library. Among the
first batch of sequences analyzed, one-third matched with known
genes in GenBank. and the other represent unknown genes. A
detailed characterization of all sequences will be presented at the
meeting.

BIVALVE HABITAT SUITABILITY AND

THE ROLE OF BIVALVES

IN ECOSYSTEMS

HOW COMMUNITY-BASED OYSTER RESTORATION
CAN ENHANCE RESEARCH EFFORTS: EXAMPLES
FROM SOUTH CAROLINA AND ALABAMA. Loren D.

Coen,* Marine Resources Research Institute. SCDNR. 217 Fort
Johnson Rd.. Charleston, SC 29412; Richard K. Wallace, Auburn
University Marine Extension and Research Center. 4170 Com-
manders Dr.. Mobile. AL 36615; Nancy Hadley, Marine Re-
sources Research Institute, SCDNR. 217 Fort Johnson Rd..
Charleston. SC 29412.

In both South Carolina and Alabama, we have been utilizing
community-based programs to construct and/or enhance larger
scale oyster restoration efforts at selected sites using criteria based
on science and best management policies. We are trying to use
these sites as research platforms to test various restoration meth-
odologies such as type of shell, alternative substrates, reef con-
figurations, time/method of planting, and stabilizing meshes. In
South Carolina we are actually placing "habitat' (over 40 tons of
shell in 2001 ). rather than oysters into tidal creeks across the state.
Routine monitoring (chemical, physcial and biological parameters)
is being conducted by both trained volunteers (students and adults)
and researchers. In South Carolina, we have also meshed our reef
monitoring efforts with HAB efforts through the NOS's South
Carolina Phytoplankton Monitoring Network (SCPMN) and we
are using the reefs also as 'living classrooms' for a variety of
groups (e.g.. one built adjacent to the SC Aquarium). In Alabama,
oysters produced by volunteers using gardening techniques are
being planted at relic reef sites that were already enhanced for
sportfishing (as artificial reef fish habitat). This strategy creates
opportunities for restoration research which can be supported by
multiple interest groups. By constructing oyster reefs, we are im-
proving the suitability of sites for oyster reestablishment, thereby
improx ing habitat quality for reef residents and transient species.

OYSTER HABITAT SUITABILITY AS A COMPONENT OF
RESOURCE MANAGEMENT. William S. Fisher,* US En

vironmental Protection Agency, National Health and Ecological
Effects Research Laboratory, Gulf Ecology Division. Gulf Breeze.
FL 32561.

Economic and ecological issues have led resource managers to
examine depletion of eastern oyster reefs along the U. S. Gulf of
Mexico and Atlantic coasts. Crassostrea virginica is a lucrative
commercial species (over $60M in 2000) that also supports eco-
system integrity by providing enhanced vigor, organization and

422 Abstracls. 2002 Annual Meetnig, April 1 4- IS. 2002

National Shellfisheries Association. Mystic. Connecticut

resilience. These values are threatened by overfishing, habitat per-
turbation and land use changes that are also economically-driven,
but diminish the survival of both oysters and reefs. Relevant and
defensible scientific information is required to assess costs and
benefits of healthy and productive oyster reefs and to establish
management goals for their conservation, mitigation and restora-
tion. Characterization of environmental conditions that support
oyster survival and propagation is a requisite for this assessment.
In this regard, habitat suitability indices (HSI) have been devel-
oped that can be used to identify potential sites for initigation and
restoration. However, greater attention is needed on conditions for
spat settlement, a bottleneck life-stage for oyster populations. Al-
though spat may settle on many substrates, success is greatest on
bivalve shells and limited by even thin layers of sediment depo-
sition. Better understanding of eunents. turbidity, sediment depo-
sition and other conditions that influence larv al settling are needed
to improve HSI evaluations and the potential for successful oyster
reefs.

CONTINUOUS MONITORING OF PUMPING PRESSURE
AND VALVE GAPE IN THE O'i STER CRASSOSTREA VIR-
GINICA IN RESPONSE TO CHANGES IN ENVIRONMEN-
TAL PARAMETERS. Dana M. Frank* and J. Evan Ward.

University of Connecticut. Department of Marine Sciences, 1080
Shennecossett Road. Groton. CT (16340.

We have developed an optical biomonitor capable of continu-
ous short and long-term recording of pumping pressure and valve
gape in bivalve molluscs. The pressure sensor is situated in the
suprabranchial chamber of the oyster. The valve gape sensor is
attached to the right valve during experimental trials. With this
arrangement, we are able to examine the relationship between
valve gape and pumping pressure, in response to measured
changes in environmental parameters such as temperature, food
availability, dissolved oxygen concentrations and current velocity.
Laboratory trials are cuirently underway to establish responses to
some of these variables with the goal of deploying the systetn.
along with sensor arrays to measure these parameters, in the field.
Using this method, we hope to expand the scope of our under-
standing about the compensatory responses of bivalve molluscs to
changes in environinental parameters //; situ. Additionally, it al-
lows us to explore more thoroughly the mechanisms available to
shellfish for controlling pumping rates. Results of laboratory trials
have revealed multiple patterns of association between pumping
and valve gape and have established that the system is able to
record changes in these physiological paiameters in real lime.

A SIMPLIFIED SESTON UPTAKE MODEL FOR BI-
VALVES: PRELIMINARY FIELD TESTS. Raymond
Grizzle* and Jennifer Greene. University of New Hampshire,
Jackson Estuarine Laboratory. Durham. NH 03824: Mark Luck-
enbach, Virginia Institute of Marine Science. Gloucester Point,
VA 23062.

A spreadsheet-based model was developed to predict the per-
cent of the total water column cleared of seston by suspension
feeding bivalve molluscs on a daily basis:

9c Water Clearance = (A x B x C)/(D x E) x 100

where A = mean bivalve density (# ind/nr). B = mean individual
clearance rate (mVindividual/da). C = bottom area of reef (nr). D
= cross-sectional area of water column (ni"). and E = mean water
flow speed (m/da). It is designed for use in estimating the impacts
of shellfish reef restoration projects on water quality. The tnajor
simplifying assumption is a completely mixed water column, and
mean clearance rates are based on literature values. We tested the
model over a portion of one tidal cycle at two different sites, both
blue mussel (Myrilus edulis) reefs, one natural and one con-
structed. On both occasions, upstreain and downstream locations
were sampled concurrently at 30-min intervals for 2 to 5 hr for in
situ fluorometry and/or chlorophyll a from a fixed height -10 cm
above the bottom, and water depth and mid-depth water flow speed
were measured. Replicate 0.16 nr quadrats were sampled on each
reef, and mean density and size (shell length) of the live mussels
were determined. The model predicted average clearance/seston
uptake for the ambient water flow conditions, mussels densities,
and other conditions measured on the constructed reef to be <2%
of the total water flow. The field trial data cortoborated the inodel
predictions, showing no significant differences between mean up-
stream and downstream samples of chlorophv II a. For data from
the natural reef, the inodel predicted a mean of 1 1 .b'k total clear-
ance. During the field trial, measured chlorophyll concentrations
were significantly lower downstream with a mean difference (rep-
resenting seston uptake) of l6.3'/f . In situ fluorometry indicated an
overall uptake of 28%. In conclusion, both preliminary tests
showed good agreement with model predictions. Further testing is
underway to determine the model's general applicability.

THE INFLUENCE OF REEF ARCHITECTURE AND
SCALE ON SUCCESS OF OYSTER REEF RESTORATION.
Mark Luckenbach.* Janet Nestlerode, Paige Ross. Jr. and Alan
Birch, Virginia Institute of Marine Science. College of William
and Mary. Wachapreague, VA 23480.

Current efforts to restore oyster reefs in the Chesapeake Bay
entail the placement of substrate on the shallow seabed to promote
oyster settlement and reef development. Because oyster shell and
other alternative cultch are in short supply and/or costly, it is
important that we optimize our placement of this material to inaxi-
mize restoration success. In several field experiments we have

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 423

been investigating the role of three components of reef architec-
ture— vertical relief, interstitial space and spatial scale — on the
development of oyster populations and associated fauna on re-
stored reefs. Our results indicate that modest differences in vertical
relief and interstitial space have dramatic effects on early post-
settlement survival of oysters and the development of viable reefs.
To investigate the issue of scale and the role of reef size we have
initiated a large-scale restoration experiment in the Chesapeake
Bay. In a replicated block design, we have consti-ucted high relief
reef bases ranging in size from 400 ni" to 8000 nr and are char-
acterizing the development of oyster populations and associated
assemblages on the reefs. Oyster recruitment and survival patterns
from the first year of this study reveal significant effects of scale.

EFFECTS OF CRASSOSTREA VIRGINICA POPULATIONS
ON SEDIMENTATION. PHYTOPLANKTON SPECIES
COMPOSITION AND AMMONIA CYCLING IN EXPERI-
MENTAL MESOCOSMS. Jennifer Mugg Pietros and Michael
A. Rice. Department of Fisheries. Animal and Veterinary Science.
University of Rhode Island, Kingston, RI 0288 1 .

To determine the effects of oyster populations on water quality,
a mesocosm study was performed from June to October 2000.
Mesocosms with a volume of 13.000 L were used, in which there
was triplicate control tanks without oysters and triplicate experi-
mental tanks each with 200 oysters (=35 mm in valve height;
nominally filtering about 55L day"' ind"'). Experiments were run
sequentially in time for three week periods with water exchange
rates ranging from 0% to 100% per day (0 to 13,000 L day"').
Several parameters were measured and compared between the con-
trol and experimental tanks, including chlorophyll-a, particulate
organic and inorganic matter, sedimentation rates, nitrate, ammo-
nia, phytoplankton species and numbers, and oyster growth rates.
There were no significant (P < 0.05) differences between tanks for
most parameters, with the exceptions of rates of sedimentation and
species composition of phytoplankton in the water column. Dia-
toms of the genus Nitzschia were predominant in mesocosms with
oysters, and in control tanks, Skeletonema were dominant. Rates of
ammonia excretion by oysters of various sizes was determined by
the sensitive salicylate-hypochlorite method, allowing for rapid
determination of excretion rates to minimize biases introduced by
volatilization or tranformation of the ammonia over time. The
excretion of ammonia by oysters can be described by the allome-
tric equation E = 50.65w"'''^"-. when E is the excretion rate in |j.g
NH,-N hr"' and w is the dry soft tissue weight in g. Based upon
these data, it would be expected in the three-week experimental
period that an additional 470 |xg L"' ammonia above the average
control concentrations of about 40 jxg L"' would be present in
each mesocosm with oysters, but no significant difference was
noted between the experimental and control tanks. This suggests
that there is rapid cycling of ammonia, perhaps by uptake by the
rapidly regenerating phytoplankton populations. This is publica-

tion number 3910 of the College of the Environment an Life Sci-
ences at the University of Rhode Island, with support from the
RIALS under project number H-886.

A MODIFIED HABITAT SUITABILITY INDEX FOR THE
EASTERN OYSTER. CRASSOSTREA VIRGINICA. Thomas
M. Soniat.* Department of Biology. Nicholls State University.
Thibodaux. LA 703 10.

A series of habitat suitability index (HSI) models have been
developed for the U.S. Fish and Wildlife Service for use in envi-
ronmental impact and planning studies. HSI models assess habitat
quality for particular species; their output is a numerical index
from 0 to 1. where 1 represents optimal habitat and 0 represents
unsuitable habitat. An HSI for eastern oysters was developed by
Cake (1983) and modified by Soniat and Brody ( 1988). The Cake
model includes variables important to larval stages (presence of
cultch. mean summer salinity and a gregarious settling factor, mea-
surable as the mean abundance of living oysters) and post-
settlement stages (historic mean salinity, frequency of killing
floods, substrate firmness, oyster drill density and intensity of the
parasite Perkiiisiis murimis). The modification of Soniat and Brody
removes the gregarious settling factor from the model, simplifies
model structure, and better accounts for the negative effects of
high salinity, parasitism and drill predation on oysters. The modi-
fied HSI should be tested against a new, independently-collected
data set.

USE OF OYSTER HABITAT BY REEF-RESIDENT FISHES
AND DECAPOD CRUSTACEANS IN THE CALOOSA-
HATCHEE ESTUARY, FLORIDA. S. Gregory Tolley.*
Aswani K. Volety. and Emily C. Lindland. Florida Gulf Coast
University. Fort Myers. PL 33965; James T. Winstead. United
States Environmental Protection Agency, Gulf Breeze, FL 32561.
Habitat suitability of oyster reefs for fishes and decapod crus-
taceans was examined monthly at three sites in the lower Caloosa-
hatchee Estuary. At each site, I-m" lift nets containing approxi-
mately 5 liters (volume displacement) of oyster clumps were de-
ployed for a period of two weeks. Fishes and decapods sampled
averaged 94 individuals m"~ or 23 individuals 1"' oyster cluster.
Nine species of decapods and 18 species of fishes were identified:
reef-resident fishes included Gobiesox slntmosus. Chasmodes
saburrae. Gobiosoma robustum. and Opsanus beta: the xanthid
Eui-\panopeus depressus and the porcellanid Petrolisthes armatus
represented the dominant decapod crustaceans. Organism abun-
dance ranged from 30-197 individuals m"", density ranged from
5.2-63.3 individuals 1"' oysters, species diversity (H') ranged
from 0.05-1.75, species richness ranged from 4—11 species per
sample, and species dominance ranged from 24.8-97.7%. Species
diversity, species dominance, and organism abundance all differed
significantly among stations. Among-station differences in organ-

424 Ahstmcts. 2002 Annual Meeting. April 14-1 S. 2002

National Shellfisheries Association. Mystic. Connecticut

ism density (individuals 1"' oyster cluster) were found for the
fishes Chasmades sulntrrae, Gohiesox stnimosiis. and Opscmus
beta, and foi' the crabs Eiirypaiiopciis ili'pressiis. Menippe mene-
naria, Panopeiis sp.. and Perriil is/lies annaliis. Of these, only E.
depressus exhibited significant seasonal variation in density
(Kruskal-Wallis test; n = 79, p = 0.00007). being more abundant
durina the warmer months.

EFFECTS OF FRESHWATER RELEASES AND SEASON
ON OYSTERS {CRASSOSTREA VIRGINICA) IN CALOOSA-
HATCHEE ESTUARY, FLORIDA. Aswani K. Volety* and S.
Gregory Tolley, Division of Ecological Studies, Florida Gulf
Coast University, Fort Myers, PL 3396.^; James T. Winstead,
U.S. Environmental Protection Agency. Gulf Ecology Division.
Gulf Breeze. FL 32561.

The influence of freshwater releases and season on disease
prevalence and intensity of Perkiiisii.s luuriinis. condition index,
gonadal condition, recruitment potential, and growth of oysters
was examined monthly at five locations along the Caloosahatchee
estuary. Florida. Temperatures and salinities at the study sites
ranged from I6-33°C and 3-39 ppt respectively. Higher tempera-
tures and salinities favored P. mahniis. While prevalence of P.
mariniis ranged from 0 (after heavy rainfall and/or freshwater wa-
ter releases) -70"7f (during dry winter months), overall intensity
was light (0-1.3). Comparison of mortality among sites indicated
that juvenile oysters tolerated salinities of 5-38 ppt. Condition
index of oysters was influenced by reproductive cycle and spawn-
ing events. Spat recruitment (1-5 spat/shell) and growth of juve-
nile oysters was higher at sudtidal than at intertidal locations,
where sparse oyster distribution and swift currents appeared to
limit growth and .settlement success. Oysters were reproductively
active between March and October, with peak reproduction occur-
ring from June-October. Overall, results suggest that periodic
short-term freshwater releases may benefit oysters by lowering the
salinity and thus the intensity of Perkiiisus nniriiuis. Laboratory
studies suggest that adult oysters can tolerate low salinities (3ppt)
for 1-2 weeks. Given high salinities and infection intensities dur-
ing winter months, it is recommended that freshwater releases take
place during winter instead of current summer releases.

PARASITIC AND SYMBIOTIC FAUNA INHABITING
OYSTERS [CRASSOSTREA VIRGINICA) AND MUD CRABS
[PANOPEVS HERBSTII) SAMPLED FROM THE CA-
LOOSAHATCHEE ESTUARY, FLORIDA. James T. Win-
stead,* United States Environmental Protection Agency. Gulf
Breeze, FL 32563: Aswani K. Volety and S. Greg Tolley, Florida
Gulf Coast University, Fort Myers, FL 33965.

Oysters, Crassostrea virginica. inhabiting five sites in the Ca-
loosahatchee River estuary were studied over a 13 month period to
determine the suitability of oyster habitat in relation to their health

and condition. Histological examination of 650 oysters ( 10 animals
per station per month) revealed a varied parasitic and symbiotic
fauna inhabiting these molluscs at all stations. Organisms found
included protozoans (Nematopsis sp. and Perkinsus mariniiin). di-
genetic trematodes (Bucephalus cucuhis and the first report from
this area of what appears to be Echinosloma sp.), ceslode larvae
[Tylocephaliim sp.) and a hydrozoan inquiline symbiont [Eutima
sp.). In addition, a significant number of mud crabs, Panopeus
iicrhstii. inhabiting oyster habitat at two sites were found to be
parasitized by rhizocephalan barnacles, Loxothylacus sp. Preva-
lence and pathological consequences of these organisms and the
role oyster-parasite relationships may play as an indicator of eco-
system complexity (biodiversity) is discussed.

POSTERS

HABITAT AND ECOLOGY OF GREEN MUSSELS, PERNA
VIRIDIS, IN FLORIDA. Patrick Baker,* Fisheries and Aquatic
Sciences. University of Florida. Gainesville. FL 3261 1 ; Amy Ben-
son, Florida Caribbean Science Center. USGS. Gainesville. FL
32653.

The green mussel. Pcrnii \iridi.\. was introduced to Tampa Bay,
Florida, prior to 1999. It is abundant from Tampa Bay to Charlotte
Harbor and has appeared elsewhere in Florida. As with invasive
freshwater zebra mussels (Dreissena polymorpha). early green
mussel sightings were in industrial water intake systems, and green
mussels currently foul many industrial and municipal structures.
Green mussels show other parallels to invasive zebra mussels, such
as reaching densities of over 10.000 per m" in an ecosystem that
lacks a close native analog. It was with zebra mussels in mind that
we initiated a study to examine the habitat and ecological impacts
of green mussels. Based on preliminary results, however, predic-
tions of green mussel ecological impacts should not be modeled
upon zebra mussel impacts, for several reasons. First, green mus-
sels in Tampa Bay have been limited to artificial substrata, such as
pilings and buoys. Contrary to expectations, they rarely appear on
mangroves, nor do they overgrow benthic sediments. Second,
green mussels have invaded a habitat that already has a high di-
versity of potential competitors, such as barnacles and oysters, and
known molluscivores, including flatworms, crabs, stingrays, and
fish. Third, there is no early evidence of negative impacts on native
taxa, with the possible exception of oysters on artificial substrata.
Green mussels increase the structural diversity of the fouling com-
munity on pilings, and both the shells and the interstitial space are
occupied by numerous other taxa, both native and nonindigenous.
At least five native bivalves commonly occur in green mussel
aggregations, for example. Ecosystem impacts of this abundant
invader are almost certain to develop, but may differ considerably
from those of other mussel-like invaders.

Green mussels have survived several winters in Florida, and
appear to reproduce more than once annually. Their initial spread

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting, April 14-18. 2002 425

southward is consistent with dominant coastal currents, which
could eventually can'y larvae around to Florida's east coast. Ad-
ditionally, juveniles have become abundant in aquaculture systems
in Charlotte Harbor, and culturists are concerned about both their
impacts on seawater systems and their potential spread via human
vectors. The northern (thermal) limit of green mussels has not yet
been determined.

INTRODUCING THE CLAMMRS PROJECT: CLAM
LEASE ASSESSMENT. MANAGEMENT AND MODELING
USING REMOTE SENSING. Shirley Baker,* Ed Phlips, and
David Heuberger, Department of Fisheries and Aquatic Sciences,
Institute of Food and Agricultural Sciences. University of Florida,
Gainesville. FL 3265.^; Clay Montague. Department of Environ-
mental Engineering Sciences, University of Florida, Gainesville,
FL 32653; Leslie Sturnier. Cooperative Extension Service, Insti-
tute of Food and Agricultural Sciences, University of Florida, Ce-
dar Key, FL 32625.

In cooperation with the Florida Department of Agriculture and
Consumer Services, Division of Aquaculture, we are addressing
the needs of the emerging Florida hard clam (Mercenaria inerce-
riaria) aquaculture industry. The needs we are addressing include:
1) Risk of catastrophic crop loss; 2) Optimum farm management
practices; and 3 ) Selection of new aquaculture areas. Adoption of
remote sensing technologies in management practices will enhance
the sustainable development of open-water clam farming by in-
creasing production, farm efficiency, and profitability. Specific
objectives are to; 1 ) Install remote water-quality and weather sta-
tions in clam aquaculture areas to provide growers with timely
information important to their management decisions; 2) Create a
water-quality database to be used by the pilot Cultivated Clam
Crop Insurance program to document events associated with crop
loss; 3 1 Develop new techniques to monitor changes in natural
food abundance and quality, such as the use of satellite imaging; 4)
Develop a clam production model to examine optimum manage-
ment practices to increase production and profitability; and 4) Use
the production model and remote water-quality monitoring in se-
lection of new highly productive aquaculture areas in Florida.

HEMOCYTES OF HOMARVS AMERICANUS STAINED
WITH A MODIFIED WRIGHT-GIEMSA STAIN: DE-
SCRIPTION AND COMPARISON TO CURRENT CLASSI-
FICATION SCHEMES. Andrea Battison* and Barbara Hor-

ney, Department of Pathology and Microbiology; Richard Caw-
thorn and Allan Mackenzie, The AVC Lobster Science Centre.
Atlantic Veterinary College. Charlottetown. University of Prince
Edward Island. Charlottetown. PE, Canada. CIA 4P3.

Transmission electron microscopy and phase contrast light mi-
croscopy are common methods used to examine crustacean
hemocytes. Using these techniques, three types of hemocytes are
currently recognised in Hoinanis americanns and many other de-
capod crustaceans; Large Granule. Small Granule, and Hyaline

hemocytes. These procedures are. however, either time consum-
ing, costly or. do not provide a permanent record of the results.
Hemocytes of H. uiiu-iicaiuis were classified, based on cytoplas-
mic and nuclear morphology, after staining cytocentrifuged prepa-
rations with a modified Wright-Giemsa stain. Up to 1 1 different
hemocytes. Types 1 to 11, could be identified in some samples.
Morphologic similarities to the Large Granule and Small Granule
hemocytes were observed. Based on similarities in appearance and
alterations in number during Aerococcus viriclmis infections, the
Type 1 hemocyte is considered the morphologic and functional
equivalent of hemocytes in the Small Granule hemocyte category
with the Type 6 hemocyte being its immediate precursor. Types 2,
7, and 8 may be subsets of hemocytes included in the Large Gran-
ule hemocyte category. A counterpart of the hyaline cell has yet to
be conclusively identified. This technique should prove useful in a
research setting when alterations in number and types of
hemocytes are being evaluated for assessment of lobster health.

IMPACT OF ATRAZINE ON ANEUPLOIDY IN THE PA-
CIFIC OYSTER. CRASSOSTREA GIGAS. Karine Bouilly.
Alexandra Leitao, Helen McConibie, and Sylvie Lapegue.

IFREMER. Laboratoire de Genetique et Pathologic. B.P. 133.
17390 La Tremblade. France.

Aneuploidy has previously been reported in the Pacific oyster.
Crassostrea gigas, and has been shown to be negatively correlated
with growth. This is especially important since high variability of
growth rate is one of the major problems in the aquacultural pro-
duction of the species. The present study investigated the effect of
an environmental factor on the level of aneuploidy. Crassostrea
gigas animals at adult and spat stages were subjected to different
concentrations of atrazine representing peak values found in the
Marennes-Oleron Bay (A; 0.01 mg/1) and a value ten times higher
(B: 0.1 mg/1). Although atrazine did not show any effect on the
oyster mortality, significant differences in aneuploidy level were
observed between the different treatments (8% for control, 16% for
treatment A and 20% for treatment B). Moreover, the same level
of response was observed at adult and spat stages. Finally, the
offspring of oysters previously exposed did not show any differ-
ence in larval growth but the hatching rate differed (74% for con-
trol, 62% for treatment A and 55% for treatment B). This is the
first evidence for an environmental cause of aneuploidy in the
Pacific oyster. Crassostrea gigas.

STATUS OF THE MYTILUS EDULIS STOCK WHICH SUP-
PLIES HIGH-QUALITY SPAT TO MUSSEL GROWERS IN
ILES-DE-LA-MADELEINE (GULF OF ST. LAWRENCE).
Francois Bourque and Bruno Myrand, Station technologique
maricole des Iles-de-la-Madeleine. Cap-aux-Meules. Canada. GOB
IBO; Marcel Roussy, Centre aquacole marin. Grande-Riviere,
Canada. GOC 1 VO.

Mussel growers in Iles-de-la-Madeleine rely mostly on Bassin
du Havre-Aubert for spat supply. This small (-3 km^) and shallow

426 Abstmcrs. 2002 Annual Meeting. April 14-IS. 2002

National Shellfisheries Association. Mystic. Connecticut

(max. depth = 3.5 m) basin have only a restricted opening to the
sea and mussels spawn earlier than in any other local areas while
larval growth is rapid. Thus, spat reaches sleeving size in early
September. These mussels have a higher mean heterozygosity than
all other local stocks and they show rapid growth and higher re-
sistance to stress (including summer mortalites). This basin is ded-
icated only to spat collection and no grow-out is allowed. An
unknown but possibly important harvest of wild mussels by local
residents arose questions about the status of this stock which pro-
duces larvae for spat collection. In a 1997 survey, the low abun-
dance of young mussels added worry about the future of this
population. Surveys were repeated in 1999 and 2001. A very lim-
ited recruitment was observed in 1999 while large numbers of
adults were moved to a new area possibly by the action of currents.
However, the overall population remained stable at about 1 1 mil-
lion individuals. In 2001. the area covered with mussels expanded
considerably and recruitment was abundant. The near future of this
population, and thus spat collection, seems not at risk in terms of
potential for larvae production.

PREDATOR INDUCED PHENOTYPIC RESPONSE BY
THE COMMON PERIWINKLE [LITTORINA LITTOREA).
Jason P. Breves,* RWU Box 5327, Roger Williams University,
One Old Ferry Road. Bristol. RI 02809; Andrew E. Tate, Center
for Economic and Environmental Development. Roger Williams
University, Bristol, RI 02809.

An organism's phenotype depends upon both genetics and en-
vironmental influences. The relative importance of these factors is
of great importance to the field of evolutionary biology. Pheno-
typic plasticity, the ability to provide a single generational re-
sponse to an environmental pressure, has been used to describe this
relationship between genetics, the environment and phenotype.
Northern Yellow Periwinkles (Liuorina obtiisata) exhibit intraspe-
cific variance in shell thickness when exposed to predation by
Green crabs (Caniniis nniciuis). This response is believed to give
Periwinkles a competitive advantage compared to other prey not
exhibiting phenotypic plasticity. It is unknown if the Common
Periwinkle {Littorina liltcrca) can alter shell morphology when
exposed to predatory stress. This study examines the morphologi-
cal response of Common Periwinkles to the threat of Green crab
predation. Periwinkles (n = 96) were assigned in equal numbers
to one of three treatment means. Mass, operculum diameter, and
shell length, width and thickness were compared among Peri-
winkles exposed to ambient seawater (negative control), seawater
containing crab effluent, or seawater with crabs and their effluent.
Initial data analysis indicates differences in growth rates among
the treatments. Periwinkles exposed to crabs and their effluent
grew more rapidly than those in the other treatments, suggesting
that rapid growth may be a response to the threat of predation.

CONTINUOUS IN VITRO CULTURE OF PERKINSUS AT-
LANTICUS, PARASITE OF THE CARPET SHELL CLAM
TAPES DECUSSATUS. Sandra M. Casas* and Antonio Vil-
lalba, Centro de Investigacions Marinas. Xunta de Galicia. aptdo.
13, E-36620 Vilanova de Arousa, Spain; Jerome F. La Peyre,
Cooperative Aquatic Animal Health Research Program. Depart-
ment of Veterinary Science, Louisiana State University Agricul-
tural Center, Baton Rouge, LA 70803: Kimberly S. Reeee, De-
partment Virginia Institute of Marine Science, Gloucester Point.
VA 23062; Carlos Azevedo, Department of Cellular Biology, In-
stitute of Biomedical Sciences, University of Oporto. P-4050
Porto, Portugal.

Continuous //; vitro cultures of the clam Tapes deciissaliis para-
site Perkinsus cukmikits were established from infected clam gill
fragments, infected clam haemolymph and parasite hypnospores
isolated from infected clam gill fragments incubated in Ray's fluid
thioglycollate medium (RFTM). No continuous cultures could be
initiated from P. atUmticus zoospores. The highest success rate in
establishing continuous cultures was obtained with cultures initi-
ated from hypnospores ( 100%), followed by cultures initiated from
gill fragments (93%) and from haemolymph (30%). The source of
parasite also influenced the time taken to establish cultures and the
size of cultured cells. In viim proliferation of parasites was mostly
by vegetative multiplication. Zoosporulation yielding motile bi-
flagellated zoospores was observed in low proportion (<l% of
dividing cells) in every culture. Morphology of cultured parasites
corresponded to that of P. atkmticus found in clam tissues. Cul-
tured parasites enlarged in RFTM and stained blue-black with
Lugol's solution, which are characteristic of Perkinsus spp. DNA
sequences of the internal transcribed spacer region of the riboso-
mal RNA gene complex matched those of P. atlanticus. All cul-
tures were established in a medium designated JL-0DRP-2A
which was similar in composition to the culture medium JL-
ODRP-I originally used to propagate Perkinsus marinus in vitro.
The commercial culture medium ( 1:2 v/v) DME:Ham's F-12 with
feiuin supported the proliferation of P. ailcnnicus in vitro.

A R&D PROGRAM TO DEVELOP MYA ARENARIA CUL-
TURE IN ILES-DE-LA-MADELEINE (GULF OF ST.
LAWRENCE). Lise Chevarie, Societe de developpement de
I'industrie maricole, Capaux-Meules. Canada. GOB I BO: Bruno
Myrand and Fran(;ois Bourque, Station technologique maricole
des Iles-de-la-Madeleine. Cap-aux-Meules. Canada. GOB I BO;
Michel Giguere, Lizon Provencher, and Philippe Archambault,
Institut Mainice-Lamontagne. Mont-Joli. Canada. G5H 3Z4; Re-
Jean Treniblay, Universite du Quebec a Rimouski-Cenlre aqua-
cole marin de Grande-Riviere, Canada, GOC IVO.

A 5-yr R&D program started in summer 2(J00 to develop a
profitable soft-shell clam culture in the lagoons of lles-de-la-

National Shellfisheries Association. Mystic, Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 427

Madeleine. This program looks at a scenario based on the transfer
of undersized clams from an unexploited population to an aquu-
culture site for grow-out until legal size (>3I mm) is reached. We
have focused mainly on the potential of this population to provide
an annual harvest of .'> million clams. It should not be a problem
considering its >26.^ million individuals of L'i-4.'i mm and high
recruitment. Further, the low predator abundance and the absence
of pathologies (e.g. neoplasia) suggest it should not suffer heavy
losses. The small hydraulic device used to resuspend the top sur-
face layer and ease collection seems not to have negative impacts.
The large size of these clams should minimize predation and net
protection over seeded areas could possibly be avoided. Work has
been initiated to optimize storage of large quantities of clams
during the extended periods between harvesting and subsequent
seeding. Experiments were also initiated to define the optimal
seeding period and optimal density according to shell length.

LOSS OF EYE PIGMENTATION IN TWO GAMMA-
RIDEAN AMPHIPODS FROM THE BIOSPHERE. Vania R.

Coelho, Columbia University. Biosphere 2 Center. Oracle, AZ
K562,^; Jeffrey D. Shields,* Virginia Institute of Marine Science.
Gloucester Point, VA 23062.

During an ecological assessment of amphipod populations in
the Biosphere 2, several specimens of Ceradocus rubromaculatus
and Leiicothoe spinicarpa were observed with alterations in their
eye pigmentation. For C. rubromaculatus. 1768 specimens were
examined and 64 (%) presented with some degree of alteration. For
L spinicarpa. 150 specimens were examined and 91 (%) presented
with some degree of alteration. Grossly, the eyes appeared either
completely white with the individual retinas unpigmented, or the
eyes were partially pigmented with pigmentation loss surrounding
a pigmented core within the ommatidia. Histologically, the optic
nerves showed a range of pathologies from minor losses of pig-
mentation, especially in the centrally-located ommatidia, to com-
plete loss of pigmentation. In all cases, there was little to no
damage associated with the optic nerves or the ommatidia. There
was also no indication of increased infiltration of hemocytes into
the optic nerve complex or the ommatidia. Pigment cells can be
negatively affected by contaminants or by the lack of phytopig-
nients in the crustacean diet. For example, crustaceans lack the
ability to synthesize carotenoids and must obtain them from their
diets. We speculate that the loss of pigmentation in the eyes of
amphipods from the Biosphere may be caused by the lack of
certain dietary phytopigments.

SHELL REPAIR RATES IN SURGICALLY DAMAGED
VALVES OF THE BLUE MUSSEL {MYTILUS EDULIS)
AND THE RIBBED MUSSEL {GEVKENSIA DEMISSA)
FROM NEW JERSEY. Tricia L. Cranmer, Department of Ma-
rine and Environmental Studies. University of San Diego. San
Diego, CA 921 10; Daniela Zima,* Richard Stockton College of
New Jersey. Pomona. NJ 08240; Richard R. Alexander, Depart-
ment of Geological and Marine Sciences. Rider University,
Lawrenceville. NJ 08648.

The blue mussel Mytilus eduUs and the ribbed mussel Ceiiken-
sia cleiiiissa were collected (n = 120) from the salt mashes of
Tuckerton, New Jersey. Equal numbers of specimens (n = \5)
were notched at. or had holes drilled near, the posterior or ventral
margin of the valves of each species. These shell regions bear scars
from shell-crushing crabs and holes drilled by moonsnails. Repair
progresses through four stages: 1 ) tissue plugging the shell gap or
hole, 2) extension of the periostracum over exposed tissue, 3)
calcification beneath the periostracum, and 4) valve-thickening,
pigmentation, and expression of a ny ornamentation. Shell repair
was completed between two to seven weeks. Average repair rates
are statistically significantly faster for epibyssate M. edulis relative
to the endobyssate C demissa (Mann-Whitney U test: p < 0.05)
pooled for all types and location of shell damage. Correspond-
ingly, field surveys show that shell repair frequency is greater
among blue mussels (19% of all specimens) than ribbed mussels
(109f of all specimens). Mantle tissue repaired posterior shell
notches faster than the ventral margin in M. edulis. although shell
repair rate is statistically indistinguishable between ventrally and
posteriorly notched shells of G. demissa. Correspondingly, field
surveys reveal that 96"^ of all shell repairs in M. edulis are located
near the posterior margin. Mortality before completion of shell
repair was significantly greater for experimentally drilled vs.
notched shells of either species. Mantle tissue was damaged by
penetrative shell-drilling, but not inevitably in valve margin break-

OVER-WINTERING OF CRASSOSTREA ARIAKENSIS IN
LAND-BASED SYSTEMS IN VIRGINIA. Alan J. Erskine and
Standish K. Allen, Jr., Aquaculture Genetics and Breeding Tech-
nology Center, Virginia Institute of Marine Science, Gloucester
Point, VA 23062.

Decimation of the Crassostrea viri^inica industry in Virginia
has been well documented. Recent interest has turned to non-
native C. ariakeiisis culture for experimentation. We hypothesized
that it was beneficial, biosecure and cost effective for industry to
over-winter C ariakeiisis inside their hatcheries. Triploid C. ari-
akeiisis were deployed in raceway and upweller systems at four
sites in Chesapeake Bay from November 2000 to April 2001.
Three size classes, 4-6mm. 6-8mm and 8-1 2mm, were deployed
in replicate at a maximum can'ying capacity of 2000 grams total
wet weight. Twenty-five random oysters were sampled monthly

428 Ahstnicts. 2002 Annual Meetuig. April 14-18. 2002

National Shelltisheries Association, Mystic, Connecticut

and shucked wet weights and hinge to lip shell height measure-
ments were recorded. A low salinity site ( 10-I2ppt) on the bayside
of Chesapeake Bay and a mid salinity site (22-25ppt) on the sea-
side had the highest growth at 30 mm and 3 grams. This experi-
ment shows that over-wintering juvenile C. ariakensis could be
incorporated as a step in the technology of non-native oyster cul-
ture.

IRRADIATION OF OYSTER PRIMARY CELL CULTURES
WITH ULTRAVIOLET-LIGHT TO ELIMINATE BACTE-
RIAL AND PROTOZOAL CONTAMINANTS. Chwan-Hong
Foo* and Jerome F. La Peyre, Cooperative Aquatic Animal
Health Research Program. Department of Veterinary Science.
Louisiana Agricultural Experiment Station. Baton Rouge. LA
70803.

Microbial contamination of oyster primary cell cultures is an
obstacle to cell line development. Perkinsus uinnnus and Vihrii)
vulnificus are two prevalent contaminants of oyster cell cultures.
Studies have shown that ultraviolet-light (UV) effectively kills
certain aquatic protozoa and bacteria. Our objective was to develop
a procedure to eliminate P. marinus and V. vulnificus from primary
cell cultures by UV irradiation, while retaining the viability of
oyster cells. Oyster heart cells. P. marinus and V. vulnificus cul-
tures were irradiated with 50.000, 100.000, 201.000. and 402,000
(jLJoules/cm- of UV light. Two days post-irradiation, oyster cell
viability was determined by measuring their metabolic activity
using the MTS/PMS assay. Perkinsus marinus growth. 2, 4 and 6
days after irradiation, and V. vulnificus growth, 3, 6. and 24 hours
post-iiTadiation. were both assessed by measuring turbidity and
metabolic activity (MTS/PMS assay). Results indicated that oyster
cells viability decreased significantly (P < 0.0001 ) with increasing
UV energy levels. Peikinsus marinus growth on days 2 and 4 were
significantly inhibited by all UV levels, while growth on day 6 was
inhibited with UV levels above 100,000 p.joules/cm-. Vibrio
vulnificus growth. 3 and 6 hours post-irradiation, was inhibited (P
< 0.05) by all UV levels, but growth rebounded 24 hours after
irradiation. This study suggests that UV irradiation has a very
limited ability to eliminate P. marinus or V. vulnificus without
negatively affecting oyster cell primary cultures.

IN VITRO EFFECTS OF HEAVY METALS AND ATRA-
ZINE ON PACIFIC OYSTER. CRASSOSTREA GIGAS,
HAEMOCYTES. B. Gagnaire,'^ H. Thomas-Guyon,' and T.
Renault.- 'Universite de La Rochelle, Laboratoire de Biologie et
Environment Marin (LBEMl. avenue Michel Crepeau. 17042 La
Rochelle; -Ifremer La Tremblade. Laboratoire de Genetique el
Pathologic (LGP). Ronce-les-Bains. 17390 La Tremblade.

In the last decades shellfish culture has developed in a signifi-
cant way around the world. However, culture areas are often sub-

jected to recurring pollutions. The recrudescent use of herbicides
in agriculture, including atra/iiie. implies pollutant transfer to-
wards the aquatic environment in estuarine areas. Moreover, in-
dustrial wastes are sources of heavy metal contamination. It is
appropriate to consider the harmful effects of pollutants in marine
species, particularly in cultured molluscs. Bivalves, including mus-
sels and oysters, have been suggested as ideal indicator organisms
because of their way of life. They filter large volumes of seawater
and may therefore accumulate contaminants within their tissues.
The development of techniques allowing analysis of the effects of
such compounds on bivalve biology may lead to monitoring of
pollutant transfer in estuarine areas. In this context, the effects of
atra/ine and the effects of various heavy metals on defence mecha-
nisms were analysed in Pacific oysters. CrassDstrea gigas. Pollut-
ant effects were tested //; vilro on oyster haemocytes. Cell viability
and activities were monitored by flow cytometry. Enzymatic phe-
noloxidase-like activity was also evaluated by spectrophotometry.
Atrazine induced no effect on oyster haemocytes under the con-
ditions tested. On the contrary, mercury caused a significant mor-
tality of haemocytes maintained //; vilro. Aminopeptidase and phe-
noloxidase-like activities were also modified in the presence of
this pollutant.

KEY WORDS: Pacific oyster. Crassostrea gigas. haemocytes. tlow cy-
tometry, heavy metals, atrazine. phenoloxidase, cellular activities, toxicity

INDUCIBLE ANTIBACTERIAL ACTIVITY IN OYSTER
{CRASSOSTREA VIRGINICA) HEMOLYMPH. Holly A. Ge-
froh, Matthew J. Jenny, and Ryan B. Carnegie. Program in

Marine Biomedicine and Environmental Sciences; and Kevin L.
Sehey. Department of Pharmacology. Medical Universitv of South
Carolina. 171 Ashley Avenue. Charieston. SC 29425; Robert W.
Chapman. South Carolina Department of Natural Resources. 217
Ft. Johnson Rd., P O Box 12559, Charleston, SC 29422.

Oyster (Crassostrea virginica) tissues resist infection by ma-
rine bacteria, but the basis of this is unclear. Bacteria invading
internal spaces are quickly agglutinated and phagocytosed by
hemocytes; however, areas where hemocytes are not active (e.g.,
epithelial surfaces) also resist heavy colonization. Oyster lysozyme
kills bacteria, but is thought to be only weakly effective against the
most common marine species (Gram-negatives). Soluble bacteri-
olytic peptides found in many organisms, including the mussel
Mvlilus edulis. may supplement phagocytosis and the activity of
lysozyme in oysters as well.

Our objectives were to determine whether or not anti-Gram-
negative bacterial activity could be induced in the hemolymph of
C. virginica. and if so, to isolate and characterize its source. Li-
popolysaccharide (LPS), laminarin. or dH,0 control was injected
into the adductor muscles of wild C. virginica (N = 6 oysters/
treatment) collected locally. Hemolymph was collected from the

National Shellfisheries Association, Mvstic, Connecticut

Abstracls. 2002 Annual Meeting, April 14-18. 2002 -il^

adductor muscles at 12 and 24 h and pooled by time interval and
treatment type. Soluble protein from cell-free hemolymph plasma
was desalted using a C,^ column, eluted with 509<- acetonitrile.
dried down and reconstituted in dH,0. and spotted on a plate lawn
of Esclu'richid coli. Plasma protein samples from LPS-challenged
( 1 2 and 24 h post-challenge) and laminurin-challenged (24 h) oys-
ters inhibited E. coli growth, while plasma protein from unchal-
lenged oysters did not.

The induced antibacterial activity passed through a 1(1-
kilodalton (kDa) cutoff membrane. Further separation by re\ersed
phase high performance liquid chromatography (HPLC) and
analysis by matrix-assisted laser desorption ionization mass spec-
trometry (MALDI-MS) revealed several peptides around 3-7 and
9.5 kDa to be candidates for its source. These candidate peptides
are currently being sequenced.

(November), mussel densities had dropped to .50 to 1 15 m~" on the
six original reefs but size-frequency plots showed an average in-
crease in shell length of 5 mm in the largest size class of mussels
over the first 3 months, indicating good individual growth rates.
Recruitment into the smallest size classes also occurred on some
reefs. Using the same sampling protocol, initial mussel densities
on the three new reefs ranged from 430 to 1376 m". Future work
will include continued monitoring of all nine reefs, construction of
abutting saltmarsh. and environmental monitorinc.

CONSTRUCTING SHELLFISH REEFS IN A POLLUTED.
URBAN ESTUARY: SCIENTISTS JOIN WITH THE COM-
MUNITY TO WORK FOR A COMMON GOAL. Jennifer
Greene,* Raymond (irizzle. David Burdiclv. and Larry Ward.

Jackson Estuarine Laboratory. University of New Hampshire.
Durham, NH 03824: Ann Reid, Great Bay Coast Watch. Sea
Grant Cooperative Extension. Durham. NH 03824.

South Mill Pond, a 7-hectare estuarine embayment in the City
of Portsmouth. New Hampshire has been receiving combined
sewer overflows since the late 1800s. A tide gate controls water
flow into and out of the Pond and there is a road causeway with
box culverts that separates the Pond into inner and outer sections.
The City has a major sewer re-construction project underway that
will, when completed, reduce inputs of sanitary sewer wastes.
Scientists joined with the City, students from local schools, and
local volunteers to begin ecological restoration of the Pond in
2001. Six shellfish "mini-reefs" (each 2 x 5 m in surface area)
were constructed in May using mussels (Mytiiits ediilis) trans-
planted from a nearby natural reef. Mortalities as high as 80%
occurred on the three reefs in the inner pond within the first 2
months, likely as a result of elevated temperatures caused by ex-
tended closure of the broken tide gate during June and July. Hence,
three additional mussel reefs were constructed in the inner pond in
October 2001. Oysters iCnissostrea virginicci) remotely set at
Jackson Estuarine Laboratory were added as 3-month old spat to
the three new reefs in the inner pond and the three original reefs in
the outer pond in October, resulting in six mussel/oyster reefs.
Eighth grade students participated in construction of the mussel
reefs and third graders participated with the oysters. Community
volunteers also participated in reef construction and post-
construction monitoring. Three quadrat (0.16 nr) samples per reef
showed initial mean mussel densities on the six original reefs
constructed in May ranged from 150 to 930 m"-. After 6 months

THE EASTERN OYSTER AS AN INDICATOR SPECIES TO
ESTABLISH RESTORATION TARGETS IN SOUTHWEST
FLORIDA ESTUARIES. Rashel V. Grindberg.* Erin C.
Rasnake, Michael Savarese, and Aswani K. Volety. Florida Gulf
Coast University. College of Arts & Sciences. 10501 FGCU Blvd
South. Ft. Myers. FL 33965.

Water management practices within Southwest Florida have
drastically altered natural water quality conditions within estuaries.
The ecological and physiological responses of oysters. Crassostrea
virginica, were compared among pristine (Blackwater River),
semi-altered (Henderson Creek) and altered (Faka-Union canal)
estuaries with historically similar hydrologic conditions. In the
Faka-Union estuary, a system that receives excessive freshwater
during the rainy season, the distribution of reefs, the regions of
maximum living density, and the foci of maximum productivity
are displaced seaward relative to pristine estuaries. Henderson
Creek, an estuary receiving pulses of nutrients and freshwater due
to weir design, has oyster populations with higher mean produc-
tivities and higher living densities. While upstream locations in all
three estuaries have lower disease prevalence among adult oysters,
juveniles experience heavy mortality due to freshwater releases.
Overall growth rates are higher at upstream locations. Spat recruit-
ment occurred between June and October in all estuaries.

Faka-Union and Henderson Creek estuaries are scheduled for
restoration. The patterns of oyster distribution and physiology will
help establish target restoration conditions and provide a founda-
tion for the monitoring of restoration effectiveness in Southwest
Florida estuaries. Henderson Creek is presently undergoing resto-
ration through the retrofitting of its upstream weir to permit dy-
nainic control of freshwater release. Sheet flow should be restored
to Faka-Union in order to re-establish salinity levels favorable for
maximum reef development, living density, oyster growth, recruit-
ment, and productivity. Changes in oyster ecological and physi-
ological conditions will be monitored over subsequent years to

430 Ahslnicts. 2002 Annual Meeting. April 14-18. 2002

National Shellfisheries Association. Mvstic. Connecticut

LONG-TERM CHANGES IN INTERTIDAL OYSTER
REEFS IN A FLORIDA LAGOON POTENTIALLY
CAUSED BY BOATING ACTIVITIES: AN ANALYSIS OF
AERIAL PHOTOGRAPHS FROM 1943-2000. Raymond
Grizzle* and Jamie Adam.s, Jackson Estuarine Laboratory. Uni-
versity of New Hampshire. Durham, NH 03824; Linda Walters,
Department of Biology. Uni\ersity of Central Florida. Orlando. FL
32816.

Previous research in the late 1990s had shown that some inter-
tidal oyster (Crassostrea virgiiiica) reefs in that pt)rtion of the
Mosquito Lagoon within the Canaveral National Seashore. Florida
had dead margins consisting of mounded up, disarticulated shells.
It was hypothesized that boating activities were the cause of the
damage because all the reefs were adjacent to major boating chan-
nels. We characterized the history of the appearance of dead mar-
gins (and other reef changes) using aerial photographs taken be-
tween 1943 and 2000. Imagery analyzed included prints (black &
white, color, or color infrared) from 1943, 1951, 1963, 1975. 1988.
and 1995, and digital imagery from 2000 (USGS 1:12,000 digital
ortho-quarterquads). at scales from 1:6,000 to 1:24.000. Prints
were scanned at a resolution sufficient to yield 1-m pixels. After
scanning, each set of images was referenced to the year 2000
imagery using ArcView and Arclnfo CIS software. All reefs found
to have dead margins based on 1995 and 2000 aerials were visited
in November 2001 to confirm the presence and extent of dead
areas. This provided a general ground-truthing for the "signature"
(a highly reflective, light-colored area adjacent to darker-colored
live reef) to be used to detect the appearance of dead margins in the
historical aerials. The earliest appearance of dead margins was in
the 1975 aerials on reefs adjacent to the intracoastal waterway
(ICW), a major boating channel. The total number and areal extent
of dead margins increased with time. Our current preliminary es-
timate is that between 1 0 and 20% of the reefs in the Seashore have
been damaged. The most dramatic changes have occuired in reefs
along the ICW. .some apparently migrating away from the channel
as much as 50 m and with empty shells mounded up a meter above
the high water line. This historical analysis provides strong (al-
though only correlative) evidence that boating activity has had
dramatically detrimental effects on some oyster reefs in the study
area. Ongoing studies are aimed at further testing this hypothesis
and elucidating the actual mechanisms involved.

AN OYSTER (CRASSOSTREA VIRGINICA) REEF RESTO-
RATION EXPERIMENT IN NEW HAMPSHIRE INVOLV-
ING CROSBREED STOCK AND NATIVE TRANSPLANTS.
Raymond Grizzle, Jennifer Greene, and Stephen Jones. Jackson
Estuarine Laboratory. University of New Hampshire, Durham.
NH: Mark Luckenbach and Roger Mann, Virginia Institute of
Marine Science, Gloucester Point. VA.

The first documented MSX epizootic in New Hampshire oc-
cuiTed in 1995. resulting in dramatic declines in oyster abundances

on some reefs. The present project is the first experimental scale
effort in the State aimed at development of a long-term program to
mitigate the effects of disease. In 1999, studies were initiated in the
Salmon Falls River to: (1) characterize a disease-decimated reef,
(2) restore portions of the reef using spat from CROSBreed stock
(an MSX and dermo-resistant line) and transplanting of native
oysters, and (3) determine the effects of the restored reef on water
quality. Here we report on the first two objectives. Diver surveys,
tonging, and underwater videography indicated the overall bottom
area probably dominated by oysters before 1995 extended along
the main channel a total distance of 350 m with an average width
of 15 m. Quadrat sampling in 2000 and 2001 showed this area was
numerically dominated by the ribbed mussel {Geukensia demissa)
with a mean density of 40 m"; oysters were 20 m", and the blue
mussel {Mytilns edulis) 5 nr. Maximum, mean horizontal free-
stream tlow speed measured over portions of two tidal cycles with
an acoustic Doppler velocimeter was 32 cm s~'. In May 2000,
approximately 230 bushels of native oysters, dredged from the
Piscataqua River about 1 km south of the study site, were depos-
ited in an area measuring 10 x 30 m on the natural reef bottom.
Spat from CROSBreed stock were remotely set in June of 2000.
held in bags for 4 months, then transferred to a 10 x 20 m area on
the natural reef In October 2001. after I year of development, the
CROSBreed reef area had a mean density of 226 oysters m",
compared to 90 m~ on the native transplant reef, and 36 m" on the
natural reef The CROSBreed oysters have shown good growth,
increasing from a mean size of 26.8 mm shell height when put out
in October 2000 to 48.6 mm in October 2001. and no measurable
mortality. Natural spat set in 2000 was greatest on the CROSBreed
reef suggesting a "minimum threshold density" for successful
natural recruitment to restored reef areas.

WHAT CERTAIN I9TH AND EARLY 20TH CENTURY
NAVIGATIONAL AND SPECIAL PURPOSE SURVEY
CHARTS REVEAL ABOUT CHANGES IN THE OYSTER
REEF MORPHOLOGY OF THE LOWER CHESAPEAKE
BAY. William J. Hargis, Jr.. Helen E. Woods,* Rebecca Aren-
son, Sbaron De«ing. .Arman Kaltayev, Elizabeth .Mountz,
Marcia R. Bernian, and Dexter S. Haven, Center for Coastal
Resources Management. Virginia Institute of Marine Science.
Gloucester Point. VA 23062.

The decline of commercial oyster production in the lower
James River is reflected in the reduction of oyster-reef dimensions
over time. Employing navigational hydrographic survey charts
made by the U.S. Coast Survey in 1854-55. 1871-73. and 1940s
and oyster survey charts made in 1878 and 1909 by the USCGS
and the old U.S. Fish Commission, we examine changes in the
dimensions of the natural oyster reefs of the lower James River.
Chesapeake Bay. and discuss the likely cause of those changes.

National Shellfisheries Association. Mystic. Connecticut

Abstracrs. 2002 Annual Meeting. April 14-18. 2002 431

MORPHOLOGY OF A CHESAPEAKE BAY OYSTER
REEF SYSTEM IN 1871-1873. William J. Hargis. Jr., Helen
E. Woods,* Rebecca Arenson, Elizabeth Mountz, Marcia R.
Berman, and Sharon Dewing. Center for Coastal Resources Man-
agement, Virginia Instuute of Marine Science. Gloucester Point.
VA 23062.

During research on oyster reef evolution in the Chesapeake Bay
it became apparent that those once prominent benthic features
must ha\e intluenced their sunounding significantly. To investi-
gate the extent of that influence on the geomorphology. hydrody-
namics, and other ecologically important features, we developed a
3 dimensional presentation of the once highly productive oyster
reef system of the James River estuary. Charts of the study are
drawn from soundings made by the U.S. Coast Survey in 1871-73
were employed. Soundings were digitized on a Numonics 2200
digitizing tablet interfaced with Arclnfo running in a UNIX oper-
ating environment. The coordinate grids were projected to
NAD27. A 3-D TIN (Triangulated Irregular Network) model gen-
erated the picture of the bottom of the study area extant in 1871-
73. The 3-D presentation, center-piece of our presentation, clearly
depicts the oyster reef system of 1871-73. Extending into the
intertidal and acting as a inassive system of weirs and baffles, the
upthrusting reefs (Hargis. In Press) must have affected deposition,
scouring and erosion, not only near field but more distantly, even
into the shallows and adjacent shorelines. Undoubtedly, they in-
fluenced biological processes too.

GEOGRAPHIC VARIATION IN NUCLEAR GENES OF
THE EASTERN OYSTER CRASSOSTREA VIRGINICA.
Cindi A. Hoover* and Patrick M. Gaffney, Graduate College of
Marine Studies. University of Delaware. Lewes. DE 19958.

Studies of genomic DNA in the eastern oyster, Crassostreci
virginica, have uncovered genetic discontinuities in population
structure. Like most benthic marine invertebrates. C. virginica has
a planktonic larval stage, which is in principle capable of wide-
spread dispersal. If extensive dispersal occurs, one would expect to
observe genetic homogeneity across the geographic range of the
species. However, studies have found geographic genetic hetero-
geneity in both mitochondrial DNA (mtDNA) and nuclear DNA
(nDNA) of C. virginica. The break in mtDNA has prompted the
division of the C. virginica into Atlantic and Gulf coast subpopu-
lations. However, unlike mtDNA. the degree of nDNA differen-
tiation is still poorly known. This study examines nDNA from
Atlantic and Gulf coast oyster populations for genetic polymor-
phisms using restriction endonuclease fragment length (RFLP)
analysis, denaturing gradient gel electrophoresis (DGGE). and di-
rect sequencing. Nuclear primers are currently being screened for
polymorphisms in a panel of organisms representing the geo-
graphic range from Canada to Mexico. This work will improve on
previous research by examining additional nuclear markers and
will help clarify the question of large-scale variation between Gulf

and Atlantic coast oyster populations as well as shed light on
smaller-scale regional patterns of variation.

CHARACTERIZATION AND MANIPULATION OF SEX
STEROIDS AND VITELLOGENIN IN FRESHWATER
MUSSELS. Nicola J. Kernaghan'' and pjleen Monck, Univer-
sity of Florida. College of Veterinary Medicine. Gainesville. PL;
Carla Wieser and Timothy S. Gross, USGS/BRD/FIorida Carib-
bean Science Center. Gainesville. FL.

The characterization and manipulation of sex steroids and vi-
tellogenin in freshwater mussels, is critical for the development of
artificial culture procedures and the evaluation of reproductive
health of populations. The current study included an evaluation of
reproductive cycles and vitellogenin in Ellipiio Inickleyi. In addi-
tion, female Elliprio hnckleyi and Lanipsilis teres were exposed to
exogenous estradiol to induce spawning. Body tissues were col-
lected from adult mussels and standard RIA procedures were uti-
lized for androgen, and estrogen analyses. Vitellogenin, an egg
yolk protein produced under the influence of sex steroids, was
determined using an indirect method developed by Blaise et al.
( 1999). Sex steroid concentrations were found to be closely cor-
related to reproductive activities and spawning. Tissue concentra-
tions of both vitellogenin and estrogen were significantly elevated
following exposure to exogenous estradiol. Histological examina-
tion of gonad tissue also indicated changes in reproductive status.
The development of these procedures for use with freshwater mus-
sel species will be critical to the elucidation of potential habitat and
contaminant effects on reproductive function, as well as the culture
of endangered species.

A SPECIFIC ASSOCIATION OF DOCOSAHEXAENOIC
FATTY ACID WITH CARDIOLIPINS OF SOME MARINE
BIVALVES. Edouard Kraffe.* Philippe Soudant. Yanic
Marty, and Nelly Kervarec, Universite de Bretagne Occidentale.
UMR 6521 and UMR 6539, BP 809 29200 Brest. France: Pierre
Guenot CRMPO. Universite de Haute Bretagne. 35042 Rennes
cedex. France.

A cardiolipin (CL) class, also termed diphosphatidylglycerol.
was isolated by high performance liquid chromatography from
lipid extracts of Pecten maximus, Crassostrea gigas and Mytiliis
edidis, and characterized by analytical and spectroscopic methods.
The fatty acid (FA) composition of this CL represents a specific
association with docosahexaenoic acid [22:6(n-3). DHA). which
accounted for more than 90'7f w/w of the total FA of this class. The
structural determination by nuclear magnetic resonance spectrom-
etry and positive ion electrospray mass spectrometry verified a
configuration having four identical 22:6(n-3) molecules. This spe-
cific composition is different from those already reported in other
eukaryotes. Indeed. CL acyl chains reported in the literature are
mainly composed of monounsaturated or diunsaturated chains w ith

432 Abstracts. 2002 Annual Meeting. April 14-IS. 2002

National Shellt'isheries Association, Mystic, Connecticut

16 or 18 carbon atoms. The DHA-enriched CL may reflect a
specific adaptation ni bivalves that enhances the strtictural and
functional mechanisms of biomembranes in response to environ-
mental variations (temperature, salinity, emersion).

HEMOCYTE-MEDIATED DEFENSE RESPONSES OF THE
LOBSTER HOMARUS AMERICANUS. Brenda S. Kraus.*
Amy E. Beaven, and Robert S. Anderson, Chesapeake Biologi-
cal Laboratory, Uni\ersity of Maryland Center for Environmental
Science, P. O. Box 38. Solomons. MD 20688.

Lobster hemocytes held in primary culture avidly phagocytosed
untreated yeast cells labeled with tluorescein succinimidyl ester.
However, serum-treated yeast cells were less readily ingested by
the hemocytes. Serum agglutinins can serve to facilitate phagocy-
tosis of certain foreign particles (opsonization). Although anti-
yeast agglutinins were present in lobster serum they seemed to
inhibit phagocytosis, perhaps by blocking sites involved with rec-
ognition by hemocytes. Bacterial agglutinins have also been mea-
sured in lobster sera; their role as opsonins is under study. After
appropriate stimulation by phagocytosis or membrane perturba-
tion, hemocytes can produce cytoto.xic reactive o.xygen species
(ROS) which contribute to host defenses by destroying microor-
ganisms. Luminol-augmented chemiluminescence (CL) was used
to quantify ROS generation by lobster hemocytes. Phorbol
myristate acetate (PMA), a protein kinase C activator, was shown
to elicit ROS in hemocytes withdraw n from healthy lobsters, based
on hemolymph protein concentration. However, phagocytosis of
Listonella atiguillarum infrequently (-25%) produced a ROS ac-
tivity in PMA-responding animals. Opsonization of L anguiUuiuin
with serum did not enhance CL; this was not unexpected because
no anti-Z.. augudlaniiu agglutinin activity was detected in lobster
serum. Studies related to the role of agglutinins in recognition and
effector mechanisms of lobster hemocytes using other biotic and
abiotic test particles are in progress.

POTENTIAL ELIMINATION OF THE PROTOZOAN
PATHOGEN PERKINSl'S MARINVS FROM EASTERN
OYSTERS BY FRESHET EVENTS. Megan K. G. La Peyre,*

U.S.G.S. Louisiana Cooperative Research Unit, School of For-
estry, Wildlife and Fisheries. Louisiana State University. Baton
Rouge, LA 70803; Amy D. Nickens and Jerome F. La Peyre,
Cooperative Aquatic .Animal Health Research Program, Depart-
ment of Veterinary Science, Louisiana State University Agricul-
tural Center, Baton Rouge, LA 70803.

Environmental conditions have long been held to be critical
controls on host-parasite interactions but little attention has been
paid to the effects of short-term events on host-parasite interac-
tions. We tested the hypothesis that freshet events may be detri-
mental to P. marinus while ha\ina only minimal impacts on C.
virginica survival. Research based predominantly on environmen-

tal averages has led to the consensus that enxironmental conditions
such as salinity and temperature control P. iiuiriiius infection in C.
virginica. In contrast, salinity records from the Gulf coast indicate
that there is considerable variation in salinity, and several studies
have suggested that freshet events may be related to low P. mari-
nus infection intensities of oysters in certain areas. In order to
determine the effects of freshets on C. virginica and P. marinus.
we initiated a controlled laboratory experiment. In April, July and
December, 2001, oysters collected from Grand Isle, LA were di-
vided equally between a control system (maintained at 20 ppt) and
a treatment system (salinity lowered from 20 ppt to 0-1 ppt over 48
hours). Thirty oysters were sampled weekly from each tank and
infection intensities determined. Oyster mortality was monitored
daily. Treatment oysters did have reduced P. marinus body burden,
but actual success was highly dependent on the season. Crassos-
trea virginica was not adsersely affected by freshet events in cool
temperature, spring and w inter experiments, but experienced high
mortality during a summer freshet event. Since most freshet events
occur in winter-spring months, the use of controlled freshwater
events might present a potential management tool for reducing P.
marinus infection in oysters.

A FIELD INVESTIGATION OF THE EFFECTS OF V-
NOTCHING ON THE HEALTH AND SUSCEPTIBILITY
TO INFECTION OF OVIGEROUS FEMALE AMERICAN
LOBSTERS. .lean Lavallee,* AVC Lobster Science Centre. Uni-
versity of Prince Edward Island. 550 University Avenue. Char-
lottetown. PE, Canada, CIA 4P3; Donald J. Rainnie, AVC Inc.,
550 University Avenue, Charlottetown, PE, Canada, CI. A 4P3.

V-notching is a fishery management practice consisting of
marking ovigerous lobsters by punching a V shaped notch in the
tail before returning it to the seawater. This study investigated
some of the possible effects of V-notching on the health of oviger-
ous lobsters during the 2000 fall fishing season on Prince Edward
Island. One hundred and thirty ovigerous lobsters were physically
examined and assigned to two treatment groups. Hemolymph
samples were collected from 15 lobsters from each treatment
group and analysed for total protein (TP), total hemocyte counts
(THC) and for the presence oi Aerococcus viridans. Anopluyoides
liacmaplnia and Vibrio spp. Lobsters were housed individually in
cages containing 2, 3 or 4 compartments. Lobsters from the treated
group were V-notched immediately prior to returning the cages to
the sea bottom. Monitoring for mortality, and vigor status was
conducted at least weekly. Hemolymph was taken on 15 lobsters of
both treatment groups for TP and THC at the mid-point in the
study. At study termination (55 days), all lobsters were re-
examined and hemolymph was sampled for determination of TP,
THC and presence of A. viridans. .An. Iiaemapltda and Vibrio spp.
Additionally, 2 V-notched lobsters were sampled on Days 1, 3, 6,
13, 27 & 55 for presence of A. viridans. .An. hacmopliila and Viiirio
spp. and for histological assessment of the wound. Mortality dur-

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 433

ing the course of the study was limited to one lobster, and reflected
an act of cannibalism. There was no significant difference ^p >
0.05) between the two treatment groups for any parameter moni-
tored. Histological examination of the V-notch wound revealed
that the wounds appeared well sealed off to the environment by the
rapid infiltration of hemocytes by 24 hours.

A PRELIMINARY LINKAGE MAP FOR THE PACIFIC
OYSTER CRASSOSTREA GIGAS. CONSTRUCTED WITH
RAPD AND AFLP MARKERS. Li Li,* Institute of Oceanology.
Chinese Academy of Science. 7 Nanhai Road. Qingdao. Shandong
266071. China; Ximing Guo, Haskin Shellfish Research Labora-
tory. Institute of Coastal and Marine Sciences. Rutgers University.
6959 Miller Avenue. Port Norris. NJ 08349.

The development of genetic markers and linkage maps is an
important step toward the identification and potential improvement
of commercially important traits in oysters. The construction of a
linkage map requires a large number of molecular markers, which
have been a challenge in oysters. Microsatellites are probably the
best markers for linkage mapping, but they are expensive to de-
velop and not readily available to many labs. Therefore, we de-
veloped relatively inexpensive markers such as the random ampli-
fied polymorphic DNA (RAPD I and amplified fragment length
polymorphism (AFLP I markers for linkage mapping in the Pacific
oyster Crassostrea gigas Thunberg. Selected markers were used
for linkage mapping in a reference family with 75 progenies. The
reference family was a backcross of an interstrain hybrid to one of
the parental strain. ((Miyagi x Hiroshima) x Miyagi). In this study.
1 10 RAPD primers were screened with the parents and t"ive prog-
enies, and 25 primers with at least one reliable and segregating
band were selected for mapping analysis. The 25 primers gener-
ated 259 bands, of which 102 (39%) were polymorphic in the two
parents. The number of segregating RAPD markers was 41 in the
mother and 1 7 in the father. Using ten AFLP primer combinations.
802 peaks were obtained, of which 253 (32%) are polymorphic.
The number of segregating AFLP markers was 81 in the mother
and 50 in the father. A preliminary female linkage map was con-
structed with 122 RAPD and AFLP markers covering all ten chro-
mosomes, and details of the map will be presented at the meeting.

GONADAL MATURATION OF TRIPLOID SCALLOPS
ARGOPECTEN PURPURATUS LAMARCK, 1819. Karin B.
Lohrmann,* Elisabeth von Brand, and Cristian Gallardo, Uni-
versidad Catolica del Norte. Facultad de Ciencias del Mar. Cas.
117, Coquimbo. Chile.

It is expected that in triploid organisms the energy normally
used for reproduction would be allocated to growth. However, not
all triploid molluscs are completely sterile; and in some cases even
gametes are produced. The aim of this study was to assess the

gonadal development in the native scallop Argopecten purpiimtiis
induced to triploidy.

A. purpimiuis is a functional hermaphrodite, the male gonad
being creamy-white, located proximal to the foot, and the distal
female gonad is bright orange-red. They were induced to triploidy
with 6-dimethylaniinopurine (6-DMAP). Treated ( = induced) and
control scallops were processed for histology using routine meth-
ods. At the age of 1 1 months, when the control scallops were
mature, some treated scallops had a gonad which showed a uni-
form brown colour. These were true triploids as evaluated through
chromosomal counts. They showed the tendency of reducing the
female gonad, only few pre-vitelogenic oocytes were observed, in
otherwise empty acini. The male gonad was relatively more de-
veloped, but no gametes further than secondary spermatocytes or
early spermatids with no flagella were detected.

DETECTION OF THE WHITE SPOT SYNDROME VIRUS
(WSSV) IN CARCINOLOGIC FAUN ASSOCIATED TO
SHRIMP CULTURE OF SINALOA, MEXICO, USING
POLYMERASE CHAIN REACTION (PCR) AND /,V SITV
H\ BRIDIZATION. Bortolini R. Jose Luis and Torres G. M.
Pilar, Lab. Invertebrados. Fac. Ciencias, Universidad Nacional
Autonomade Mexico. A. P. 70-371. Mexico D. F. 04510. Mexico;
Montoya R. Leobardo, Lab. Virologfa. Centre de Investigacion
en Alimentos y Desarrollo. U. Mazatlan. A. P. 711. Mazatlan.
Sinaloa C. P. 82010. Mexico.

One of the most important sanity problems in penaeid shrimp
populations of trade important in the world, is originated by virus
infection. Recently in our country was detected the White Spot
Syndrome Virus (WSSV), that produce high mortality in the popu-
lation of cultured shrimps, mainly in the states of Nayarit, Sinaloa
and Sonora. For this research, we collected samples in summer of
2001. The samples were from different species associated to cul-
ture farms, (Litopenaeus vannamei. L. stylirostris. Calliiwctes
sapidus. Uca sp. and copepods). The copepods were homogenized
and proceced in integral form by Polymerase Chain Reaction
(PCR) probe, one hemolymph sample was taken from each of the
different species to which was done the PCR getting the result of
sequence in acrilamide, and having the corresponding marks. The
samples for processing by //) situ hybridization, were fixed in
Davidson's solution for 48 hours and then in OH 70. The histo-
logical cuts were done 5 fj.m of thickness and were put on posi-
tively slides. By in situ hybridization, infected tissues of samples
of Catlinectes sapidus and Litopenaeus vannamei were easily dis-
tinguished. The presence of the blue precipitations it was present
in branchial and connective tissues and gut subcuticular epithe-
lium. In the other species of carcinological faun was negative by in
situ hybridization probe.

434 Ahstnwis. 2002 Annual Meeting, April 14-1«, 2002

National Shellfisheries Association. Mystic. Connecticut

FACTORS, RISKS AND SIGNIFICANCE OF EMERGENT
NEOPLASIA DISEASES IN CULTURED AND WILD SOFT-
SHELL CLAMS (V/)4 ARESARIA) IN ATLANTIC
CANADA. Gregory MacCalluni,* Jeffery Davidson, and Garth
Arsenault. Atlantic Veterinary College. University of Prince Ed-
ward Island. .^^50 University Ave., Charlottetovvn, PEI, CIA 4P3;
Sharon McGladdery and Michelle Maillet, Department of Fish-
eries and Oceans Canada, Gulf Fisheries Centre, .U3 University
Ave.. PC Box 5030. Moncton. NB. EIC 9B6; Neil MacNair. PEI
Department of Fisheries. Aquaculture and Environment, 1 1 Kent
St.. Charlottetovvn. PEI. CIA 7N8.

In 1999. mortalities of soft-shell clams {Mya arenaria) caused
by or associated with haemic neoplasia occurred at several sites
around Prince Edward Island (PEI) and in Richibucto. New Bruns-
wick (NB). Haemic neoplasia has been well documented in bi-
valves (clams, mussels and oysters) worldwide since the early
1970"s. The cause(s) of haemic neoplasia is/are unknown. They
have been linked to infectious triggers (neoplastic cells per se or a
viral vector), anthropogenic carcinogens (e.g.. polychlorinated hi-
phenyls) and changing natural conditions (e.g.. abnormally high
water temperatures). The most urgent question, from an environ-
mental and clam production perspective, is whether the neoplasia
is infective or non-infective.

The objectives of this study are to: i) examine the transmissi-
bility of this disease; ii) determine the geographic and seasonal
distribution of haemic neoplasia in soft-shell clams from PEI (in-
cluding a study to relate disease prevalence to five PEI clam popu-
lations); iii) examine common environmental variables between
affected and unaffected sites on PEI. NB. and Nova Scotia (e.g..
temperature, bottom-type, terrestrial run-off. human activities/
input); iv) determine whether or not sediment exposure affects
emergence of haemic neoplasia; and v) determine whether or not
clams which have survived haemic neoplasia have developed a
resistance to the disease which can be passed onto their offspring.

IDENTITY OF UNITED STATES MOLLUSK PRODUC-
TION DECLINES IN THE I900S. Clyde L. MacKenzie, Jr.,*

James J. Howard Marine Sciences Laboratory. Northeast Fisheries
Science Center. 74 Magruder Road. Highlands. NJ 07732.

In the 1900s. the historical declines in landings of estuarine
mollusks along the east coast of the United States rarely were
caused by overfishing, that led to permanent sharp declines in
production of their larvae. Instead, habitat degration including ad-
verse algal blooms, domestic pollution which led to closure of
production beds, poor markets, and diseases have been the main
causes. The oyster. Crassostrea virginica. landings, mainly, suf-
fered from poor markets, and oyster habitats were despoiled by
siltation and by dredgers removing shells while oysters were being
harvested, and finally by diseases. Oysters companies sold their
shells to the poultry industry and for hardening roads rather than
spreading them on beds to produce more oysters. Northern quahog.

MerceiHiria mcnciiaiia. landings declined due to the narrowing of
bay openings, adverse algal blooms, and bed closures due to do-
mestic pollution. Soft clam, Mya arenaria. landings declined due
to destruction of beds, bed closures due to domestic pollution,
diseases, and high temperatures. Bay scallop. Argopecteii irradi-
aihs. landings declined due to adverse algal blooms, changes in bay
openings, and losses of eelgrass. Zostera marina. Attributing the
declines to overfishing leads resource managers away from the
actual causes and delays habitat restoration.

DEVELOPMENT OF A PCR-BASED ASSAY FOR DETEC-
TION OF THE .FOD-ASSOCIATED ROSEOBACTER. Aaron
P. Maloy* and Katherine J. Boettcher, Departinent of Biochem-
istry. Microbiology and Molecular Biology, University of Maine,
Orono. ME 04469; Bruce J. Barber, School of Marine Sciences,
University of Maine. Orono. ME 04469.

Juvenile Oyster Disease (JOD) has resulted in substantial losses
of cultured Eastern oysters. C/a.s.vo.sT/cd virginica. in the north-
eastern United States. Despite management strategies utilizing se-
lected lines and early deployment of hatchery-produced seed, JOD
continues to occur annually in Maine's Damariscotta River. Fur-
ther, during the past two years, mortalities have also been docu-
mented in three previously unaffected areas throughout Maine. In
all years and locations, affected animals were extensively colo-
nized by a novel species of marine a-proteobacteria (in the Roseo-
Ixicter clade). We are cuirently evaluating a PCR-based diagnostic
assay for detection of the JOD-associated Riiseobacter. This assay
uses specific primers to amplify the approximately 1300 base-pair
internal transcribed spacer (ITS) region between the Roseolnicler
I6S and 23S rRNA genes. Direct amplifications from suspended
cells are successful at concentrations as low as 1.000 cells per
reaction. From sequence data and restriction length fragment poly-
morphism analyses, two genetic signatures are distinguishable.
One is characteristic of the 1997-98 isolates, while the other is
characteristic of the 2000-01 isolates. Thus PCR amplification
followed by restriction enzyme digestion provides data regarding
both the presence of. and specific genotype of. the JOD-associated
Roseolnutcr. It is expected that this assay will be invaluable as a
screening and diagnostic tool, and for regional management efforts
to control the spread of JOD.

PRELIMINARY STUDY ABOUT FEEDING ECOLOGY OF
THE ROCK LOBSTER, PANVLIRUS HOMARUS, LIN-
NAEUS, 1785, AT IRANIAN SEASHORES OF OMAN SEA.
Nassrin Mashaii, Offshore Fisheries Research Centre. Chabahar.
Iran.

Preliminary aspects of feeding ecology of the rock lobster,
Pannlirus Iwmarus. specimens collected by diving from January
1 999 to November 2000 w as considered. Stomach contents were

National Shellfisheries Association. Mystic. Connecticut

Abstnicls. 2002 Annual Meeting. April 14-18. 2002 4.^3

recorded by using the point method. Fi and Pi of different prey
items were measured. Monthly and seasonally frequencies of
empty stomachs and different preys were compared using Chi-
square tests. Monthly changes of macrobenthic communities
sampled by diving were studied. Ivlev index used for comparing
the importance of items between stomach contents and mac-
robenthics. Pearson correlation coefficient of different prey items
against some hydrological factors was obtained. Spearman corre-
lation coefficient was used for correlation between different preys
against size groups, sexes and ovigerous females.

EVALUATION OF THE STRUCTURE AND FUNCTION OF
A CREATED BLUE MUSSEL {MYTILUS EDULIS) REEF.
Sean McDermott, National Marine Fisheries Service. 1 Black-
burn Dr.. Gloucester. MA 01930; David Burdick, Raymond
Grizzle, and Jennifer Greene. Jackson Estuarine Laboratory.
University of New Hampshire, 85 Adams Point Rd.. Durham. NH
03824.

In May 2001 . blue mussels {Myiilia eiliilis) were collected from
a donor site and transplanted into South Mill Pond, Portsmouth.
NH. as part of a broad scale project to restore a degraded tidal salt
pond. A total of six mussel reefs were established in two locations:
three at the inner South Mill Pond (ISMP) and three at the outer
South Mill Pond (OSMP). Two general parameters were used to
evaluate the structure and function of the created reefs: ( 1 1 reef
population dynamics (survivorship, size frequency distribution,
density, and movement) assessed mussel response to transplanting
and local pond conditions and (2) faunal utilization to evaluate the
establishment of ecological functions for living marine resources
(LMR). Mussel population dynamics were sampled once per
month (June. July. August) using a random sample protocol. Mus-
sels initially responded negatively to pond conditions (poor water
quality; minimal tidal exchange), resulting in high mortality.
Smaller mussels had a greater survival ratio as noted in a decreased
average shell length. Survival and average shell length increased
after water quality and tidal flow improved. Fish community dy-
namics were evaluated in reefs and reference areas through the
summer (June. July. August). Reef areas had greater species di-
versity per sampling effort than reference areas under slow water
conditions. Apletes qiuulracus and Meiiidiu meiiiclia were most
common in reef areas. Fundulus heierocHuis and M. menidia were
most common in reference areas. The constructed mussel reefs
functioned as habitat for LMR\s immediately, providing shelter
and forage for small fish. Further assessment of utilization by other
LMR's (invertebrates) is required to properly evaluate the func-
tions and value of the created mussel reef

GENETIC MONITORING OF OYSTER STOCK EN-
HANCEMENT IN THE CHOPTANK RIVER, CHESA-
PEAKE BAY. Coren A. Milbury and Patrick M. Gaffney,

Graduate College of Marine Studies. University of Delaware.
Lewes. DE 19958.

The spread of parasitic diseases (primarily MSX and Dermo).
in conjunction with overharvesting. has led to the rapid decline of
Eastern oyster (Crassostrea virginica) populations. Regional
variation in disease resistance may be u,seful in restoration efforts.
In collaboration with the University of Maryland Horn Point Labo-
ratory, we have assessed the success of recent enhancement efforts
within the Chesapeake Bay using genetic markers. C. virginica
exhibits regionally diagnostic 16s mitochondrial DNA haplotype
profiles (North Atlantic, South Adantic, and Gulf Coast). In 1997
oysters from Louisiana broodstock were planted in the Choptank
River. Maryland. The presence of newly settled spat with the Gulf
Coast haplotype in the Choptank River confirms the survival and
propagation of the Louisiana broodstock. An automated mini-
sequencing technique (Pyrosequencing Inc.) was used to determine
the mitochondrial haplotypes of oyster spat collected throughout
the Choptank River. This rapid mass screening method revealed
that 95'7f of spat collected were of the North Atlantic haplotype
and approximately 59c were South Atlantic. Of 4538 spat
screened, four (0. 1 '^ ) possessed the Gulf Coast haplotype. The use
of these genetic markers has enabled us to assess the survival,
propagation, and dispersal of the Louisiana oyster stock within the
Choptank Ri\er. Chesapeake Bay.

PROGRESS IN THE BIOLOGICAL CONTROL OF ZEBRA
MUSSELS WITH MICROBIAL TOXIN. Daniel P. Molloy,*
Denise A. Mayer, Michael J. Gaylo, Kathleen T. Presti, Alex-
ander Y. Karatayev, and Lyubov E. Burlakova, Division of
Research & Collections, New York State Museum, Albany, NY
1 2230.

Recent progress in the development of bacterial strain
CL0145A as a biocontrol agent of zebra mussels, Dreissena spp..
is reviewed. Strain CL0145A is a North American isolate of
Pseudomonas fliiorescens. a ubiquitous, soil-water. Gram-negative
bacterium, and a U.S. patent for its use for zebra mussel control
has recently been issued. Pseiidomoiias fliiorescens is not a para-
sitic species, and histological analysis indicates that mussels die
from a biotoxin associated with strain CL0145A cells, not from
infection. Therefore, future commercial products based on this
microbe could contain only dead cells, further reducing enxiron-
mental concerns. When zebra mussels ingest strain CL0145A
cells, the biotoxin specifically destroys their digestive gland tis-
sues. All zebra mussel sizes tested to date (ca. 1-30 mm long) are
susceptible to kill by strain CL0145A, and high mussel mortality
is achievable at all temperatures examined (range. 5-27°C). Static
cultures currently produce cells of the highest toxicity, but shaking
culture protocols are being developed in order to proceed to large-

436 Ahsimcts. 2(102 Annual Meeting. April 14-18. 2002

National Shelltisheries Association, Mystic, Connecticut

scale t'eirnentation production. Very small-scale trials, designed to
simulate tlow-through conditions, have been conducted in tempo-
rarily-installed pipes within a hydropower facility, and they have
confirmed that exposui-e to bacterial cells for 2 days at 23°C
achieves high mussel kill. Evidence of nontarget safety has been
demonstrated in laboratory and mesocosms trials. Current studies
are focusing on biotoxin identification, fermentation scale-up, and
identification of the key biotic and abiotic factors needed to maxi-
mize zebra mussel mortality.

CHARACTERIZATION OF A PARASITIC AMOEBA IN
THE AMERICAN LOBSTER BY MOLECULAR SYSTEM-
ATICS. Thomas E. Mullen, Jr. and Salvatore Frasca, Jr., De-
partment of Pathobiology and Veterinary Science. College of Ag-
riculture and Natural Resources. University of Connecticut, 61
North Eagleville Road. U-3089. Storrs, CT 06269-3089.

Past mass mortality events of Long Island Sound (LIS) lobster
(Homanis americamis) have been associated with a number of
potential etiologies, one of which is neurologic infection by a
parasitic amoeba. Histopathologic examinations of nerves and gan-
glia revealed tissue invasion by an amoeba, with and without at-
tendant hemocytic infiltrates. This amoeba possessed a small,
round, secondary nucleus differentially stained using the Feulgen
technique. Transmission electron microscopic examination of ner-
vous tissue confirmed the presence of this nucleus-like organelle,
or Nebenkoiper, a consistent feature of members belonging to the
genus Paramoeba Schaudinn, 1896. Previous efforts to culture
parasitic Paramoeba spp. and recent attempts to culture the lobster
amoeba in vitro have been unsuccessful. Molecular data for the
family Paramoebidae is ab.sent, and the lack of such information
contributes to the ambiguity associated with classification ot these
organisms. In this study we propose to characterize the rDNA of
this parasitic Paramoeba by determining the molecular systemat-
ics of potentially related lobose amoeba. We have sequenced the
small subunit (SSU) rRNA gene of twelve previously identified
organisms that have morphologic (light microscopic and ultra-
structural) similarity to the amoeba infecting lobster. This SSU
rDNA has provided the data necessary to describe the current
systematics of the Order Euamoebida using bioinformatic com-
puter methods. Based on this rDNA sequence data, species of
Neoparamoeba and Korotnovella occupy a separate clade between
Vannella spp and the clade classically representing the Class Lo-
bosa (Acanthamoeba spp and the leptomyxid amoebae). In addi-
tion, such rDNA nucleotide sequence data has allowed for identi-
fication of variable and conserved sequences that could be ex-
ploited for the purpose of amplifying SSU rDNA from the parasitic
amoeba in lobster tissue. The rDNA sequence of this amoeba is
expected to allow for molecular characterization using molecular
evolution methods and provides the critical sequence elements
necessary to develop primers and probes for future polymerase
chain reaction (PCR) and in situ hybridization-based (ISH) diag-

nostic tests to delect the parasite in lobster tissue. These tests will
be important to histopathologic and molecular diagnostics integral
to future health sinvcillance programs.

SEASONAL CHANGES IN CELL PROLIFERATION OF
OYSTER TISSUES. Kim-Lien T. Nguyen and Jerome F. La
Peyre,* Cooperative Aquatic Animal Health Research Program.
Department of Veterinary Science. Louisiana State University Ag-
riciiltural Center. Baton Rouge. LA 70803; Terrence R. Tiersch,
Aquaculture Research Station. Louisiana Agricultural Experiment
Station. Louisiana State University Agricultural Center. Baton
Rouge. LA 70820.

Identification of mitotically active tissues should assist devel-
opment of cell lines from oysters and other bivalve molluscs.
Except for evaluation of embryonic tissues, limited consideration
has been given to identification of optimal tissue types and time of
year to establish oyster cell lines. Thus, cell proliferation in tissues
of the eastern oyster, Crassoslrea virginica. was evaluated
monthly for one year by an immunohistochemical assay for pro-
liferating nuclear antigen (PCNA), an endogenous marker of cell
proliferation that is evolutionarily conserved and present in all
active phases of the cell cycle (Gl, S, G2, M). A commercial
monoclonal antibody to PCNA (PC 10) was used to calculate a
labeling index (percentage of labeled nuclei for 1000 nuclei
counted) at 400x magnification for each tissue. We found highest
proliferation in somatic tissues (labial palps > digestive diverticula
and stomach epithelium > gills > mantle) in late fall to early spring
(November-April in southern Louisiana). Cell proliferation ceased
in these tissues in late Spring while proliferation in maturing go-
nads persisted. After spawning, proliferation resumed in somatic
tissues for a month or two before gonad proliferation resumed in
late summer. This agrees with earlier observations that cell pro-
liferation in bivalves is highly variable throughout the year.

DEVELOPMENT OF A MEDIUM TO INDUCE HYPNO-
SPORE FORMATION AND ZOOSPORULATION OF PER-
klNSUS MARINUS. Amy D. Nickens and Jerome F. La
Peyre,* Cooperative Aquatic Animal Health Research Program.
Department of Veterinary Science. Louisiana State University Ag-
ricultural Center. Baton Rouge. LA 70803; Sandra M. Casas.
Centro de Investigacions Marifias. Xunta de Galicia. aptdo. 13.
E-36620 Vilanova de Arousa. Spam.

The difficulty in inducing zoosporulation of P. nuiriniis hinders
our ability to study zoospores. We recently developed a medium to
induce hypnospore formation and zoosporulation of P. marimis.
This medium was formulated in 3 steps by determining the effects
of various solutions. indi\ idually and in combination, on the size,
viability and zoosporulation of cultured parasites. The solutions
tested included fluid thioglycollate medium (FTM) components
(i.e., yeast extract, casein hydrolysate, dextrose, cystine, sodium

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 437

thioglycollate. agar) in step 1 ; solutions of amino acids, carbohy-
drates, lipids, vitamins or nucleotides in step 2; and media supple-
ments (lactalbumin hydrolysate. Hyprep 4601 . pancreatin. Ex-cyte
VLE. egg yolk, oyster lysate) in step 3. Solutions tested at each
step which had significant positive effects on the enlargement,
viability, and/or zoosporulation of cultured parasites were included
in a medium for testing all solutions of subsequent steps. The size
(35 + 13 (i-m. N = 100). viability (95%, N = 400) and zoosporu-
lation (35%, N = 400) of parasites incubated in our final medium
for 6 days were significantly greater than the enlargement ( 11 ± 2
p.m), viability (309}-) and zoosporulation (O'/r ) of parasites incu-
bated in the FTM for 1 week. The initial mean parasite size was 4
± 1 jxm (N = 100). Interestingly, enlargement and viability of
parasites incubated in our medium was similar to the enlargement
(35 ± 9 [xml and viability (95%) of parasites incubated in oyster
lysate. Zoosporulation (12%) of parasites incubated in oyster ly-
sate was however significantly lower. The availability of P. mari-
nus zoospores for study will enable investigations on the role of
this cell staee in P. marinus life cvcle.

THE EFFECTS OF MARTEILIA SYDNEY! ON THE HOST
DEFENSE RESPONSES OF THE SYDNEY ROCK OYS-
TER, SACCOSTREA GLOMERATA. Rodney Peters* and
David Raftos, Marine Biology Laboratory. Mucquarie University.
Sydney. Australia 2109.

Marteilia sydneyi (paramyxean parasite) is the causative agent
of QX disease in Saccostrea glomerata, which has mortality rates
of 90 to 98%. Outbreaks of QX disease have reduced oyster farm-
ing in some of the major oyster producing estuaries in New South
Wales, Australia to critical levels. Once, farmers had a productive
period of 2 years during which they could grow out, or "fatten,"
the oyster to a reasonable market size. Now this period lasts only
from April/May to December of the same year in the QX contami-
nated rivers.

Invertebrates, according to all available evidence, lack antibod-
ies and lymphocytes. However, they are still capable of mounting
highly efficient cellular and humoral immune responses. Our
premise is that these immune responses must be either evaded or
overpowered by M. sydneyi to initiate QX disease. Specifically.
our investigations have centered on proteolytic cascades, such as
the prophenoloxidase (proPO) system, that in other species are
closely associated with host defense. The prophenoloxidase cas-
cade catalyzes the hydroxylation of tyrosine to dopa and the oxi-
dation of dopa to dopaquinone. finally forming the pigment mela-
nin. A number of intermediates in this pathway are cytotoxic or
bacteriostatic, and melanin itself contributes to defense by encap-
sulating foreign material.

Data presented here describes an outbreak of QX disease on the
Georges River, NSW during 2000/2001. A strict congelation was
evident between infection intensity and prophenoloxidase activity.
ProPO activities declined rapidly at precisely the time that infec-

tion was established. The data supports the contention that proPO
activity is specifically suppressed by M. sydneyi in order to estab-
lish infection.

POPULATION BIOLOGY OF MELONGENID WHELKS IN
THE INTERTIDAL ZONE IN WASSAW SOUND, GEOR-
GIA. Alan J. Power,* Mary Sweeney-Reeves, Todd C. Recicar,
Dodie M, Thompson, and Randal L, Walker. Marine Extension
Service. Shellfish Research and Aquaculture Laboratory, Univer-
sity of Georgia. 20 Ocean Science Circle. Savannah, GA 3141 1-
1011.

Four species of whelk (family Melongenidae) are found within
the coastal waters of Georgia: the knobbed whelk. Busycon carica:
the channeled whelk, Busycotypus canalicukitus: the lightning
whelk, Busycon cantrarium; and the pear whelk, Busycotypus spi-
ratus. These whelks are commercially harvested using trawls and
are also taken intertidally by oystermen. clammers, and sport fish-
ermen. To date the whelk fishery remains one of the most eco-
nomically successful molluscan fisheries in the state. There is,
however, a lack of fundamental biological information to allow for
sound management decisions regarding the sustainability of the
fishery. Consequently, the present study was initiated to examine
the temporal variability in the abundance and population structure
of each species in the intertidal zone in Wassaw Sound. Georgia.
Whelks were collected at low tide from six locations, on a seasonal
basis, over an annual period. Prior to releasing, all were tagged
with an identifying label, measured (shell length, shell width, total
wet weight), and sexed. Size frequency distributions, sex ratios,
and sexua! dimorphism in terms of body size were determined.
Growth rates and seasonal migration patterns were examined by
recapturing previously tagged specimens. The implications of
these results for the whelk fishery in Georgia are discussed.

A RAPID METHOD FOR ASSESSING STRESS IN THE
AMERICAN LOBSTER USING A HAND HELD GLUCOM-
ETER. Deanna L. Prince, Robert Bayer.* Christina Congle-
ton. Shannon Colby. Danielle LaVine. Danielle Volmuth, Kat-
rina Brooks. Margaret Berry, and William Congleton. Depart-
ment of Animal Sciences, and School of Marine Studies,
University of Maine. Orono. ME 04469; John Vetelino, Depart-
ment of Electrical and Computer Engineering. University of
Maine. Orono. ME 04469.

A number of parameters were measured in lobsters exposed to
temperature stress and anoxia from air exposure. Measurements
included electrical resistance across the tail membrane, resistance
across the body from mouth to anus. pH. and hemolymph calcium,
magnesium, and glucose. Hemolymph glucose appeared to be the
best indicator of stress as a result of an increase in hyperglycemic
honiione. Based on these preliminary findings, a rapid technique
was developed to measure hemolymph glucose levels using a com-

438 Abstracts. 2002 Annual Meetmg, April 14-18, 2002

National Shellfishenes Association, Mystic, Connecticut

niercial hand held gluconieter available from any pharmacy. The
techniL|ue insolves centrilugation of the samples and placing the
serum on a disposable strip, which is read by the gluconieter. In a
field sampling using this technique lobsters from areas that tended
not to survive well in shipping and storage showed elevated blood
glucose.

ROUTES OF HEMATODINIUM SP. TRANSMISSION INTO
BLUE CRABS. Michael Sheppard.* Florian Rambow, Marc
E. Frischer, and Richard F. Lee. Skidaway Institute of Ocean-
ography. Savannah. GA 3141 1.

Heinatodiniiim sp. is a parasitic dinoflagellate which causes
mass mortality of blue crabs duruig infection peaks in late spring
and fall in salt marsh estuaries of coastal Georgia. The life cycle of
Heiiiatodiniitin sp. in blue crabs involves several different stages
including dinospores. prespores, trophonts and plasmodia [Shields.
J.D.. Ann. Rev. Fish. Dis. 4:241-271 (1994)]. A series of studies
were carried out to detemiine the source and possible routes of
transmission of Hfiiuitodinium sp. into blue crabs in the Wassaw
Sound estuary system. Both histological and a recently developed
molecular diagnostic techniques, including a quantitative real-time
PCR method, were used to quantify Heiiuitddiimiiii sp. in crabs and
in estuarine water samples. Three possible routes of transmission
were investigated in these studies; consumption of infected tissues,
injection of hemolymph containing Hcimandiiuiim sp.. and expo-
sure to estuarine water and sediments where infected crabs were
found. Hematodinium disease was transmitted by all of these
routes, but the most effective route was the transmission to healthy
uninfected crabs after feeding on infected tissues from the diseased
blue crabs. In several experiments there was a lack of transmission
of Hcimilddiiiiiiiii sp. into crabs from water found to contain Hc-
nuiiodiiiiiiin. which we speculate was due to lack of infectious
forms in the water, e.g. dinospores. Partially supported by Georgia
Sea Grant.

THE RELATIONSHIP BETWEEN BLACK SPOT DISEASE
AND LIMB-LOSS IN CANCER PAGURUS FROM THE
SHETLAND ISLANDS, SCOTLAND. Shelly M. L. Tallack,

North Atlantic Fisheries College. Scalloway. Shetland Islands.
ZEI OTS, UK.

The edible crab. Cancer pai;iirtis. is a heav ily exploited crus-
tacean resource throughout the UK. including the peripherally lo-
cated Shetland Islands. Black spot disease is the primary infection
recorded in the local population and is believed to be most preva-
lent in specimens with reduced immune systems, resulting from.
for example, injury. The confrontational, minority fishing activitv
of removing only claws from this species in offshore Shetland
waters has led to concerns regarding a possible increase in the
number of severely injured crabs. Shetland's infection rates were
compared with other UK studies.

Black spot disease rates were higher in Shetland than in studies
on populations in Norfolk (Ayres & Edwards, 19X2) and Wales
(Davies. 1999). but lower than findings from Ireland (Vogan et al.,
1999). A positive relationship v\'as shown between crab size and
black spot disease. Greater infection severity was associated with
later intermoult stages. Sex differences were evident with the pro-
portion of infected individuals being higher in males (25.61'^)
than in females (I2.78'y{-). Finally, limb-loss and crab iniury were
positively correlated with infection.

It cannot be determined whether black spot disease rates in
Shetland are linked to claw tlshing activity rates. However, as
vulnerability to black spot disease may be increased through in-
jury, findings from the cuirent study in addition to earlier research,
imply the need for handling techniques which minimize severe
limb-loss and injury in discarded crabs.

OYSTER GRAZING ON TOXIC AND NON-TOXIC

PSEUDO-NITZSCHIA AND THALASSISIORA WEISFLOG-
GII, AND DITYIAM BRIGHTWELUI. Anne Thessen,*' - Q.
Dortch,' T. M. Soniat," and G.J. Doucette,' 'Louisiana Univer-
sities Marine Consortium. 8124 Hwy 56 Chauvin. LA 70344; "Bi-
ology Department. Nicholls State University. Thibodaux. LA
70310; 'Marine Biotoxins Program. NOAA/NOS. Charleston
LAB. 219 Fort Johnson Rd, Charleston. SC 29412.

Pseudo-nit: schio spp. are chain-forming diatoms that some-
times produce donioic acid, a potent neurotoxin that causes Am-
nesic Shellfish Poisoning (ASP). Despite high abundances of
Pscndo-nitzschia over Louisiana oyster (Crasscislrea virginicu)
beds, there have been no documented cases of ASP. Two possible
explanations are that oysters cannot feed on long, pointed chains or
they discriiuinate against toxic cells while grazing. Short-term (<2
hr) grazing experiments were conducted with non-toxic P.
pseitdodelicatissima. toxic P. niuhisencs (22-87 |jim/cell. depend-
ing on species and clone; 4 cells/chain median chain length).
Tlialassisiora weisfloggii (15-23 pm). and Dityluiii hrii;htucllii
(70-140 |jLm). Oysters (73 to 85 mm) were collected in the field,
maintained on flowing ambient seawater. and then starved for 48
hours. Cultures were added at approxiniatelv 10'' cells/liter to con-
tainers with individual oysters and to controls with no oysters or
killed oysters. During experiments grazing was monitored by mea-
suring decreases in //( vivo fluorescence, but cell counts and vol-
umes were also measured. Oysters grazed rapidly on all diatoms
and cells appeared in feces within I hour. Grazing rates, based on
fluorescence, were lower on both types of Pseudo-nitzschui than
for the other diatoms, but those differences may not be sustained
when srazins; is based on cell volume/carbon.

National Shellfisheries Association. Mystic. Connecticut

Abstracts. 2002 Annual Meeting. April 14-18. 2002 4.^9

HISTORIC EASTERN OYSTER (CRASSOSTREA VIR-
GINICA) DREDGE AND PATENT TONG SURVEYS IN
THE MARYLAND PORTION OF THE CHESAPEAKE
BAY. Jessica M. Vanisko.* Man Chrisfnian, and Kennedy T.
Paynter. University of Maryland, College Park. MD 20742;
Stephen J. Jordan, Maryland Department of Natural Resources
(MD-DNR). Cooperative Oxford Laboratory. 904 S. Moiris Street.
Oxford. MD 21654.

The distribution of the Eastern oyster. Crassostrea virgiiiica. in
the Chesapeake Bay remains spatially variable. The densities often
range from 0 to 230 oysters per m". The conversion of historic
oyster surveys to spatial tiles (CIS) has provided the means to
examine the potential relationships between oyster biomass and
spat settlement, as well as past distributions of oysters in spatially
complex ways. Extensive patent tong sampling was conducted by
the Maryland Department of Natural Resources (MD-DNR) during
two surveys from 1975 to 1979. and again from 1989 to 1995. Data
were compared to determine if significant changes in the density of
spat, small, and market oysters occurred during the time period
between the two surveys. Changes in oyster density in Maryland
can be attributed to environmental disturbances and the impacts of
disease. Oyster biomass. as calculated from the MD-DNR dredge
survey, was paired with patent tong samples in an effort to test for.
among other things, a relationship between oyster biomass and
spat settlement on several spatial scales. A significant relationship
between biomass and spat settlement could not be established with
the data available, however data from current sampling regimes are
being analyzed for relationships between oyster density, disease
incidence, and/or spat settlement.

HEMATODINIUM INFECTION IN BLUE CRABS, SPIDER
CRABS AND STONE CRABS. Anna Walker,* Department of
Pathology. Mercer University School of Medine. Macon. GA
31207; Michael Sheppard, Richard F. Lee, and Marc Frischer.

Skidaway Institute of Oceanography, Savannah, GA 3141 1.

Hematodinium sp, is a histoinvasive parasitic dinoflagellate.
We report heavy infections of Hematodinium sp. in blue crabs
{Calliuectes sapidus) and spider crabs {Lihinia emarginata) col-
lected during late fall in a Georgia estuary. The prevalence of
Hematodinium sp. in blue crabs and spider crabs ranged from 20
to 80% at different sampling stations. Prevalence oi Hematodinium
sp. in stone crabs {Menippe mercenaria) from this estuary was
only 5% (n = 20). Both hemolymph and tissues were examined
microscopically and by molecular techniques. Most infected crabs
had a high intensity of infection (15 to 90% of the hemolymph
cells were parasite cells). A sequence analysis from the 18S rRNA
gene ( 1682 bp) of Hematodinium sp. from spider crabs and stone
crabs showed 99.6 and y9,5'/f sequence similarity, respectively, to
Hematodinium sp. from blue crabs. Infected crabs had interstitial
infiltrates of parasites in all tissues, but most notably in gills,
hepatopancreas, cardiac and striated muscle. Focal muscle necrosis

was present in heavy infections. Following injection of infected
hemolymph. tissues of healthy crabs demonstrated an influx of
granular hemocytes with encapsulations.

SHELL MOVEMENT AND JUVENILE SURVIVAL OF
THE OYSTER CRASSOSTREA VIRGINICA ON INTER-
TIDAL REEFS ADJACENT TO WATERS WITH INTENSE
BOATING ACTIVITY IN THE INDIAN RIVER LAGOON,
FLORIDA. Linda Walters,* Kevin Johnson, Lisa M. Wall, and
Neysa Martinez, Department of Biology, University of Central
Florida. 4000 Central Florida Blvd.. Orlando. FL 32816; Ray
Grizzle, Jackson Estuarine Laboratory. University of New Hamp-
shire. Durham. NH 03824.

Resulting from anthropogenic influences such as boat wakes or
natural disturbance events, reefs of the eastern oyster Crassostiea
virginica in the Indian River Lagoon have been declining in size in
recent years. Additionally, dead margins (i.e. mounds of tightly
packed, disarticulated shells extending above the high tide line) are
commonly found adjacent to boating channels. To determine the
impact of boating activity on: 1 ) shell movements, and 2) juvenile
oyster survival, experiments were run on eight oyster reefs in
Mosquito Lagoon, the northernmost region of the Indian River
Lagoon. Florida. Four reefs had large dead margins, while the
other four monitored reefs had no dead margins. To measure shell
movement, 25 pre-weighed, oyster shells were deployed within
0.25m" quadrants on the exposed, middle and protected regions of
each reef and dispersal of indiv iduals was assessed weekly for 8
weeks. Boat activity adjacent to the eight reefs was also recorded
during the eight-week trials. There was a positive correlation be-
tween shell dispersal in the exposed regions of reefs and the av-
erage number of boats per hour that passed by the study reefs.
Additionally, shells that weighed less than 50 grams and those
which exceeded 100 grams experienced the least amount of overall
movement, and the protected sides of the reefs experienced the
least amount of shell movement, even for those reefs with dead
zones. To determine the impact of shell movement on juvenile
oyster survival, 1-cm diameter clay mimics were attached to 20
shells at the exposed, middle and protected regions on all eight
reefs. At weekly intervals for the first 4 weeks of the 8- week trial,
damage to the clay was recorded and used as a proxy to estimate
damage to C. virginica at this vulnerable stage in its life-history.

DEVELOPMENT AND TESTING OF A SIMPLE FIELD
SYSTEM FOR MONITORING MUSSEL SHELL GAPE
SIZE. Win Watson, Steve Jury, Jennifer Wishinski, Dan
O'Grady, Walter Golet, Darren Scopel, Heidi Pye, and Chris
Rillahan, Zoology Department and Center for Marine Biology,
University of New Hampshire, Durham, NH 03824.

Mussels and other bivalves are known to vary their shell gape
size in response to changes in various environmental conditions
such as salinity, current or the presence of contaminants. In fact.

440 Ahstivcr\. 2002 Annual Meeting. April 1 4- IX. 2002

National Shellfisheries Association. Mvstic. Connecticut

several research groups have developed biomonitoring systems
based on measuring the shell status of freshwater bivalves. The
purpose of our project was to develop and test a simple and rela-
tively inexpensive system for measuring the shell gape size of
estuarine and sahwater mussels (Mytilus edulis) in their natural
habitat. Battery-powered Hall-Effect sensors were used to detect
the gape size of mussels and their output, in volts, was logged at
10 sec intervals onto a HOBO datalogger. The datalogger, batteries
and customized electronics were placed in a waterproof case inside
a buoy that communicated with mussels on the bottom via a wa-
terproof cable. Monitoring systems were deployed at 6 different

locations in the Great Bay Estuary. NH and along the N.H. coast-
line. Data obtained was examined to determine if changes in gape
size were correlated with changing tides, light levels and/or am-
bient concentrations of heavy metal contaminants. Laboratory
studies, under more controlled conditions, were used to determine
their response thresholds to these same stimuli. While the system
developed proved very sensitive and reliable, the complex factors
influencing the gape size of mussels made it difficult to defini-
tively explain all the variations in gape size recorded from mussels
in their natural habitat. This study was supported by CICEET and
Gulf Watch grants to W.H.W.

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JOURNAL OF SHELLFISH RESEARCH

Original articles dealing with all aspects of shellfish re-
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taceans, or relevant publications for other geographic regions.

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Watts, R. J., M. S. Johnson & R. Black. 1990. Effects of re-
cruitment on genetic patchiness in the urchin Echinometra
nuilluiei in Western Australia. Mar. Biol. \i)5:\45-\5\.
Book:

Claudi. R. & G. L. Mackie. 1994. Practical manual for Zebra
Mussel monitoring and control. Boca Raton, FL; CRC Press.
227 pp.

Chapter in Edited Book:

Davio, S. R., J. F. Hewetson & J. E. Beheler. 1985. Progress
toward the development of monoclonal antibodies to saxitoxin;
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son, A. W. White & D. G. Baden, editors. Toxic dinotlagel-
lates. Amsterdam; Elsevier, pp. 343-348.

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""99
Erratum

Abstracts of technical papers presented at the 5th Internatumal Cunt'erence on Shellfish Restoration. Nanaimo B.C.. Canada.

September 1 S-20. 2001 ^0'

Abstracts of technical papers presented at the 55th Annual Meeting of the Pacific Coast Oyster Growers Association &

National Shellfisheries Association. Silverdale. Washington. September 20-22, 2001 -^21

Abstracts of technical papers presented at the 22nd Annual Meeting of the Milford Aquaculture Seminar. Milford.

Connecticut. February 25-27. 2002 ^~^

Abstracts of technical papers presented at the y4th Annual Meeting of the National Shellfisheries Association. Mystic.

Connecticut. April 14-18. 2002 ^57

COVER PHOTO: Carpet shell clam. Tapes deaissatus (Linnaeus. 1758). The species is of Atlantic-Mediterranean
origin and is distributed along the Euro-African coast from England to Senegal and throughout the Mediterranean with
penctiation in the Red Sea. While the introduced short-necked clam or striped venus (Tapes philippinanim) has almost
superseded the carpet clam in Italian waters. T. decussalus is grown commercially in Sardinia. Photo courtesy of
Consorzio Promo/ione Prodotti Ittici. Italy.

The Journal of Shellfish Research is indexed in the following: Science Citation Index". Sci Search'". Research Alert"'. Current
Contents®/Agriculture. Biology and Environmental Sciences. Biological Abstracts. Chemical Abstracts. Nutrition Abstracts. Current
Advances in Ecological Sciences. Deep Sea Research and Oceanographic Literature Review. Environmental Periodicals Bibliography.
Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts.

Huayong Que and Standish K. Allen, Jr.

Hybridization of tetraploid and diploid Ciassostrea gigas (Thunberg) with diploid C. ariakensis (FujitaJ 137

Pedro Saucedo, Carmen Rodrigtiez-Jaramillo, and Mario Monteforte

Microscopic anatomy of gonadal tissue and specialized storage cells associated with oogenesis and spermatogenesis in

the calafia mother-of-pearl oyster. Pinctada mazatlanica (Bivalvia: Pteriidae) I'l-S

Alan J. Power and Randal L. Walker

Growth and gametogenic cycle of the blood ark, Anadara oralis ( Bruguiere, 1 789) in coastal Georgia 157

J. A. Rodriguez-Valencia, and F. Caballero-Alegria

Temporal tluctuations (1989-1999) in the populations oi Haliotis fulgens and H. cornigata (Gastropoda: Haliotidae),

at Islas San Benito (Baja California, Mexico) 163

M. Kruatrachue, P. Laimek, C. Wanichanon, V. Linthong, P. Sretarugsa, E. S. Upatham, and P. Sobhon

Development of the nerve ganglia of abalone, Haliotis asinina Linnaeus 173

Ingrid Bahamondes-Rojas and Marta Bretos

Induction of spawning and early development in Fissurella picia (Mollusca: Archaeogastropoda) from southern Chile. 185
Ludwig C. A. Naegel and Chris J. Cooksey

Tyrian purple from marine muricids. especially from Plicopurpura pansa (Gould. 1 S.^?) 193

Dominique Sud. Jean-Marc Poncet, Armelle Saihi, Jean-Marc Lebel, Dominique Doumenc, and Eve Boucaud-Camou

A cytological study of the mantle edge of Haliotis tuherculala L. (Mollusca. Gastropoda) in relation to shell structure. 201
Getian Wu, Kangsen Mai, Beiping Tan. and Wei Zhu

Dietary biotin requirement of juvenile abalone. Haliotis discus hamuli Ino 211

Nicholas G. Elliott, Jason Bartlett, Brad Evans, and Neville A. Sweijd

Identification of southern hemisphere abalone {Haliotis) species by PCR-RFLP analysis of mitochondrial DNA 219

Alberto de Jesus Navarrete

Distribution and abundance of Slrombtis costatiis (Gmelin. 1791) larvae and adults at the biosphere reserve; Banco

Chinchorro, Quintana Roo. Mexico 227

O. R. Chaparro, A. E. Soto, and C. E. Bertran

Velar characteristics and feeding capacity of encapsulated and pelagic larvae of Crepidida fecimda Gallardo, 1979

(Gastropods, Calyptraeidae) -^^

Oscar Efrain Holgitin Quiiiones and Jesiis Emilio Michel-Morfin

Distribution, density, and length-weight relationship of Chiton arliciilatus Sowerby. 1832 (Mollusca-Polyplacophora)

on Isla Socorro. Revillagigedo Archipelago. Mexico 239

Bealriz Novoa, Carolina Tafalla, Angel Giierra, and Antonio Figueras

Cellular immunological parameters of the octopus. Octopus vulgaris 243

H.-Jorg Urban and Jose M. Riascos V.

Estimating gonado-somatic indices in bivalves with fused gonads 249

Arnold G. Eversole and Yavuz Mazlum

Comparative fecundity of three Procamharus species -55

D. J. Macintosh, J. L. Overton, and H. V. T. Thu

Confirmation of two common mud crab species (genus Scylla) in the mangrove ecosystem of the Mekong Delta,

'*S9
Vietnam "

Biplob Das, Yusuf Sharif Ahmed Khan, Khan Towhid Osman, Pranab Das, and Md. Nurul Amin

Physico-chemical changes in acid sulfate soil during semi intensive culture of Penacus monodon fabricius. in cleared

mangrove areas of The Chakaria Sundarbans. Bangladesh 267

Craig S. Lawrence, Noel M. Morrissy, Philip E. Vercoe, Ian H. Williams, and Yuk W. Cheng

Physical, chemical, and biological variation among freshwater crayfish {Cherax albidus Clark. 1936) research ponds . . 273
Fernando Negrete-Soto, Enrique Lozano-Alvarez, and Patricia Briones-Fourzdn

Population dynamics of the spiny lobster Panulirus guttatus (Latreille) in a coral reef on the Mexican Caribbean 279

Roy Melville-Smith and Yuk Wing Cheng

Responses of migrating western rock lobsters Panulirus cvgniis (George, 1962) to two different tagging release

J 289

procedures

A. Battison, R. Cawthorn, B. Horney, and A. Mackenzie

Mushroom tyrosinase as a control material for phenoloxidase assays used in the assessment of crustacean "health" .... 295

The Journal of Shellfish Research is indexed in the following: Science Citation Index®, Sci Search®, Research Alert®, Current
Contents*/Agriculture, Biology and Environmental Sciences, Biological Abstracts, Chemical Abstracts. Nutrition Abstracts, Current
Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography,
Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts.

CONTENTS CONTINUED ON PREVIOUS PAGE

JOURNAL OF SHP:LLF1SH RESEARCH
Vol. 21, No. 1 Jt'NE 2002

CONTENTS

Kenneth Chew

Honored Life Member: John B. Glude 1

Craig L. Appleyard and Joseph T. DeAlteris

Growth of the northern quahog, Mercenciria inercenaria. in an experimental-scale upweller 3

Bassem Allam, Kathryn A. Ashton-Alcox, and Susan E. Ford

Flow eytometrie measurement of hemocyle viability and phagocytic activity in the clam, Riuiuapcs philippiiniium 13

M. J. Stewart and R. G. Creese

Transplants of intertidal shellfish for enhancement of depleted populations; prelinimary trials with the New Zealand

21
little neck clam

Edward Fahy, Maria Lyons Alcantara, Mark Norman. Ronan Browne. Vincent Roantree. and Nick Pfeiffer

Mortalities in Eiisis aniiatus (Jeffreys) (Solenacea) in western Ireland 29

Michael L. Zettler

Ecological and morphological features of the bivalve /\,v/<//7c horcalis (Schumacher. 1817) in the Baltic Sea near its

geographical range

R. W. Penney and M. J. Hart

Is survival genotype-dependent in North American populations of farmed blue mussels. Mytilus spp? 41

Qin-Feng Gao. Zheng-Lin Wang. Wai-Hing Wong, and Siu-Gin Cheung

Effects of food quality and quantity on feeding and absorption in black-ribbed mussels, Seplifer viii^anis (Wiegmann)

(Bivalvia: Mytilidae) dominating wave-exposed habitats in Hong Kong 51

K. R. Freeman, E. Kenchington, and S. P. MacQuarrie

Comparative seltlcmenl depths of Mvliliis editlis C. Linnaeus. 1738 and M. trossuhis Gould. 1850: 1. A. mesocosm

, . 59

study

E. Kenchington, K. R. Freeman, B. Vercaemer, and B. MacDonald

Comparative settlement depths of M\tili(s edulis C. Linnaeus. 1758 and M. in>ssiiliis Gould. 1850: II. Field

observations

Anthony S. Anderson. A. Lelania Bilodeau. Matthew R. Gilg, and Thomas J. Hilbish

Routes of introduction of the Mediterranean mussel (Myliliis galtoprovincialis) to Puget Sound and Hood Canal 75

Julie A. Maguire, Maurice O'Donoghue. Stuart Jenkins, Andrew Brand, and Gavin M. Buniell

Temporal and spatial variability in dredging induced stress in the great scallop Peclen iiuaiiniis (L.) 81

Marcel Frechette and Gaetan Daigle

Reduced growth of Iceland scallops Chlamxs islaiulica (O.F. Muller) cultured near the bottom: a modelling study of

87
alternative hypotheses

Rosalio Maldonado-Amparo and Ana M. Ibarra

Ultrastructural characteristics of spermatogenesis in diploid and triploid catarina scallop (Argopecten ventricosus

Sowerby II. 1 842 ) ^^

Marcial Villalejo-Fuerte, Marcial Arellano-Martinez. Bertha P. Ceballos-Vdzquez, and Federico Garcia-Dominguez

Reproductive cycle of Spondylus a(/c//e'r carpenter. 1857 (Bivalvia: Spondylidae) in the "Bahia de Loreto" National

Park. Gulf of California. Mexico '^-^

Young Jin Chang, Min-Do Huh, Myung-Joo Oh, and Yoshio Sugawara

Baculovirus-like particles in epithelial cell of digestive diverticula of the scallop. Patinopecten vessoensis 109

/. .S(//!i7a, N. A. Stokes, R. Smolowitz, R. C. Karney, and E. M. Burreson

Haplosporidimn coshde (seaside organism), a parasite of the eastern oyster, is present in Long Island Sound 113

Kwang-Sik Choi, Kyung-Il Park, Ki-Wan Lee, and Kazumi Matsnoka

Infection intensity, prevalence, and histopathology of Pcrkiiisiis sp. in the Manila clam. Rmlilapcs plulippmanim. in

, , n I ' "9

Isahaya Bay. Japan

Pedro Saucedo, Hie Racotta, Humberto Villarreal, and Mario Monteforte

Seasonal changes in the histological and biochemical profile of the gonad, digestive gland, and muscle of the calatia
mother-of-pearl oyster, Pinctada mazatkmica (Hanley, 1856) associated with gametogenesis 127

CONTENTS CONTINUED ON INSIDE BACK COVER

JOURNAL OF SHELLFISH RESEARCH

VOLUME 21, NUMBER 2

DECEMBER 2002

The Journal of Shellfish Research

(formerly Proceedings of the National Shellfisheries Association)

is the official publication of the National Shellfisheries Association

Editor

Sandra E. Shumway

Department of Marine Sciences

University of Connecticut

Groton, CT 06340

Standish K. Allen, Jr. (2002)

Aquaculture Genetics and Breeding

Technology Center

Virginia Institute of Marine Science

College of William and Mary

P.O. Box 1346

Gloucester Point. Virginia 23062

Shirley Baker (2004)

University of Florida

Department of Fisheries and Aquatic Sciences

7922 NW 71"' Street

Gainesville. Florida 32653-3071

Brian Beal (2004)
University of Maine
9 O'Brien Avenue
Machias. Maine 04634

Peter Beninger (2003)

Laboratoire de Biologic Marine

Faculte des Sciences

Universite de Nantes

BP 92208

44322 Nantes Cedex 3. France

Andrew Boghen (2003)
Department of Biology
University of Moncton
Moncton, New Brunswick
Canada El A 3E9

Neil Bourne (2003)
Fisheries and Oceans
Pacific Biological Station
Nanaimo, British Columbia
Canada V9T 6N7

Andrew R. Brand (2003)
University of Liverpool
Port Erin Marine Laboratory
Port Erin, Isle of Man IM9 6JA
United Kingdom

EDITORIAL BOARD

Eugene Buneson (2003)

Virginia Institute of Marine Science

P.O. Box 1346

Rt. 1208 Create Road

College of William and Mary

Gloucester Point, Virginia 23062

Peter Cook (2002)
Department of Zoology
University of Cape Town
Rondebosch 77(J0
Cape Town, South Africa

Simon Cragg (2002)
Institute of Marine Sciences
University of Portsmouth
Ferry Road
Portsmouth P04 9LY
United Kingdom

Leroy Creswell (2003)
University of Florida/Sea Grant
8400 Picos Road, Suite 101
Fort Pierce, Florida 34945-3045

Lou D'Abramo (2002)
Mississippi State University
Department of Wildlife and Fisheries
Box 9690
Mississippi State, Mississippi 39762

Christopher V. Davis (2004)
Pemaquid Oyster Company, Inc.
P.O. Box 302
1957 Friendship Road
Waldoboro, Maine 04572

Ralph Elston (2003)

Aqua Technics/Pacific Shellfish Institute

455 West Bell Street

Sequim, Washington 98382

Susan E. Ford (2002)

Rutgers University

Haskin Shellfish Research Laboratory

6959 Miller Avenue

Port Norris, New Jersey 08349

Journal of Shellfish Research

Volume 21, Number 2
ISSN: 0730-8000
December 2002

www.shellfish.org/pubs/jsr.htm

Raymond Grizzle (2003)
Jackson Estuarine Laboratory
Durham, New Hampshire 03824

Karolyn Mueller Hansen (2004)
1524 Barley Circle
Knoxville, TN 37922

Mark Luckenbach (2003)

Virginia Institute of Marine Science

Eastern Shore Lab

P.O. Box 350

Wachapreague, Virginia 23480

Bruce MacDonald (2002)
Department of Biology
University of New Brunswick
Saint John. New Brunswick
Canada E2L 4L5

Roger Mann (2002)

Virginia Institute of Marine Science

Gloucester Point, Virginia 23062

Islay D. Marsden (2002)
Department of Zoology
Canterbury University
Christchurch, New Zealand

Tom Soniat (2002)
Biology Department
NichoUs State University
Thibodaux, Louisiana 70310

J. Evan Ward (2002)
Department of Marine Sciences
University of Connecticut
1080 Shennecossett Road
Groton, Connecticut 06340-6097

Gary Wikfors (2002)

NOAA/NMFS

Rogers Avenue

Milford. Connecticut 06460

Juurnal oj Shellfish Research. Vol. 21. No. 2. 441^44. 20U2.

3=-

CD

IN MEMORIIIM

HAROLD HALEY HASKIN

1915-2002

Hal Haskin died on June 23. 2002 at the place he loved best — his Cape Shore cottage on Delaware Bay. Mo.st of his 87 years-since
he spent the summer after his junior year at Rutgers working on oyster drills — had been devoted to teaching and research on the marine
environment, and Delaware Bay was its focus. Hal's "Honored Life Member Biography" appeared in 1*^)99 in Volume 18(2) of the
Journal of Shellfish Research. Here we reflect more on his character and recount memories of the man.

Hal was bom in 1915 at Niagara Fall, NY to George and Laura Haley, the second of three children. Three years later, the children
were orphaned when both parents and a grandmother died within a week of each other in the 1918 flu pandemic. A family friend,
Frederick Haskin. adopted Hal — an unusual arrangement, particularly for the time, because Fred Haskin was a bachelor. Haskin was a
pipefitter, a job that forced him to travel around the country, so he lodged young Hal with a retired farm family living in southern New
Jersey, near the DuPont chemical plant where he sometimes worked. The daughter of the family, whom Hal knew as Aunt Jenny,
supervised the home and became his de facto mother. Later, Aunt Jenny and Fred Haskin married so that Jenny officially became the
mother that she had unofficially been throughout most of his childhood. It wasn't until many years later that Hal became reacquainted
with his many Haley relatives still living around Niagara Falls and for the first time was called "Uncle Hal".

Always a good student and with an unusually strong work ethic. Hal graduated from Rutgers College in 1936. the first student to do
so with a perfect grade score. It was at Rutgers that he came under the tutelage of Thurlow Nelson and began his life-long fascination
with oysters. During summers spent investigating the predatory oyster drills, Hal observed that the snails preferred young oysters to older
ones. Curious about mechanisms of chcnioattraction. he entered Harvard to work with John Welsh on this phenomenon, but he spent his
first summer as a graduate student at the Bermuda Biological Station studying lobster neurohormones. He grew a beard, which came in
red, was introduced to sailing, and acquired a taste for his research subjects.

Hal switched to algal physiology for his PhD dissertation, which he carried out under the supervision of the oceanographer Alfred
Redfield. His work included the development of a method for estimating chlorophyll concentrations using spectrophotometry — a
precursor to the present-day Strickland and Parson's method. At Harvard. Hal supported himself as a dorm proctor and a teaching
assistant. He was a natural teacher who excelled at hands-on instruction in both the laboratory and the field.

Upon receiving his PhD in 1941, Hal entered the US Army and spent the next 5 years training troops and supervising units guarding
the coast from Long Island to the Virginia Capes. He returned to Rutgers as an assistant profes.sor in the Department of Zoology in 1 946
and began developing a research program devoted to marine bivalves. His early work involved culturing and rearing of hard clams on
a grant from Campbell's Soup Company. In 1 950. however, he succeeded his mentor Thurlow Nelson as head of the Oyster Investigation
Laboratory in the New Jersey Agricultural Experiment Station, and for many years thereafter devoted most of his research efforts to
protecting and enhancing the oyster resource of Delaware Bay. Although he later became deeply involved in acquiring data on, and
developing a management plan for, offshore surf clams, his heart and mind never strayed far from oysters and Delaware Bay. From his
early efforts to institute harvest limits on the depleted natural seed oyster beds, through the devastation brought by the MSX parasite,
to providing management data when populations later rebounded, Hal considered the Bay's oysters to be his personal responsibility. He
brought to this and other tasks a unique combination of intelligence, scientific integrity, and the ability to work with people from all sides
of a problem. He never shirked from controversy, but he was always armed with reams of data, which he carried in whiskey boxes in
the back of his car, to backup his point of view. Hal took pains to make the data understandable to both the regulators and the fishermen.
He spoke his mind, albeit diplomatically, and he rarely lost a battle.

441

442 Ford et al.

One ongoing stuggle, which he inherited from Thurlow Nelson, was maintaining freshwater flow into the Delaware estuary. In this
battle against diversions and dams, he had the enthusiastic participation of his wife. Peg. whom he had met when she was a recent
graduate of Smith College and he was a graduate student at Harvard. The two of them eventually became a formidable team fighting
for rational water management in New Jersey, particularly concerning those policies affecting Delaware Bay and its oyster populations.

Although he ran a large research laboratory. Hal devoted much of his time to teaching. As a young assistant professor, he taught
mainly undergraduate courses in general biology, limnology, animal physiology, and invertebrate zoology. Somewhat later, he developed
graduate courses in coastal oceanography, estuarine ecology, and malacology. Most of the graduate students in the Rutgers Zoology
Department took at least one of these courses at some point in their academic careers — and never forgot them. Hal did not confine his
teaching to the classroom, nor did he limit it to official students. He delighted in showing anyone the anatomy of a shucked oyster or
talking about the intricacies of an oyster community. His audience might be a businessman or an oyster grower — it didn't matter — his
enthusiasm captivated them all. Anyone who was associated with Hal in any capacity always wound up considering himself or herself
a student. And Hal. himself, never stopped being a .student.

Hal retired in 1984 but he remained active long thereafter. During his nearly 50-year career, he touched many people — students,
colleagues, university and government officials, and shellfish producers. More than 200 of them gathered at the Haskin Shellfish
Laboratory on September 28, 2002 to remember him. People came from all along the eastern seaboard and from as far west as Colorado
to tell stories, share memories, renew acquaintances, look at old photographs, and eat good food — including freshly shucked oysters
farmed at the Cape Shore near the Haskin cottage.

His first and last graduate students were there, as were a host of students in between. They recalled how taking one of his courses
had been the stimulus to go into marine science or shellfish biology. Several remarked on how tough a questioner he was during thesis
defenses and how his criticisms were right on the mark, sometimes requiring a return to the bench to do a critical experiment. Although
Hal was remembered most for applied research that directly benefited the shellfish industry, his intellectual curiosity was profound. He
insisted that applied research conform to the standards of basic research. Further, how could one provide sound scientific advice for
management without understanding fundamental biological and ecological principals? Woe to any student or colleague who failed to
convince Hal that he or she knew the basic scientific principles underlying a concept.

Students remembered his Saturday courses and that he worked well into the night on Friday, and often into the early morning hours
of Saturday, preparing. The classes officially ran from 8 in the morning to 5 in the evening, but leaving at 5 was viewed with a highly
disapproving eye. Several students remarked on his indifference to physical discomfort on the field trips — and that he was equally
indifferent to discomfort of the students. The discomfort usually took the form of "cold and wet'" or "seasick", but on one occasion it
was much more senous. On an oceanography class field trip. Hal lacerated his hand severely on the pulley wheel of an outboard motor
that he was attempting to start. The deep cut ran along the base of his fingers and down the outside of his palm. Flapping fingers indicated
severe damage to his hand, but he merely wrapped it in a handkerchief and was fully prepared to continue the trip. At the overwhelming
insistence of his students, however, he reluctantly canceled the excursion in favor of a trip to the hospital and several hours of
microsurgery to rejoin the tendons leading into two fingers.

Neighbors, who grew up with the Haskin children at the Cape Shore, and whose fathers were businessmen and auto mechanics,
couldn't figure out what Hal did for a living. He was like a kid in an adult body who actually got paid for mucking about on the tide
flats playing with oysters. But he could answer all the questions they had about the organisms they found in the bay and along the shore.

Hal was an advocate of simplicity. He was not interested in "building an empire" because he felt that he would then have to spend
all his time supporting and defending it — certainly much less stimulating to him than the hands-on field and lab activities that he relished.
Colleagues and students remembered him as a quiet, low-keyed person who was a good listener. He was thoughtful and one knew that
he had truly reflected upon a question or an issue before he gave advice.

One of the fondest memories, shared by nearly all the students, was of the biannual "tray moves". These events took place each spring
when trays of selectively bred oysters had to be moved from winter quarters, where they were protected from ice damage, onto the tidal
flats in front of the Cape Shore Laboratory where they were exposed to disease pressure and could be easily tended. Each fall, the reverse
move took place. All able hands — students, faculty, technicians, friends — were marshaled for the event because the oysters in each
tray — and there were often more than 100 trays — had to be counted, samples removed, and the oysters had to be placed in freshly
prepared trays. The weather was usually cold and miserable, and ones fingers quickly became numb. Tying lines on the trays so that they
could be hung in the marina that served as winter quarters was especiall\ challenging under these conditions. Hal would check every
single knot (four per tray, half-hitches, not bowlines) and retie most, while being silently cursed by the small group of students standing
around on the dock — cold. wet. tired, and anticipating the reward that awaited them at the Haskin cottage where Peg was preparing the
traditional turkey dinner and a fire was blazing in the wood stove. Fifteen, twenty, occasionally up to thirty famished people would arrive.
The first order of business was the preparation and distribution of gin and tonics, and if Hal made them, they contained plenty of gin.
People pitched in to set the table, make the salad, and cook the peas, but certain tasks were Hal's own prt>vince: making the gravy,
mashing the potatoes, and carving the turkey. These jobs had to be performed in a very specific fashion, with Hal describing to anyone
within earshot his way to successful gravy or mashed potatoes. The evening ended with homemade (by Peg) pie — cherry, pumpkin,
blueberry, and pecan — along with ice cream, coffee and tea. and very muted memories of the earlier discomforts. The tray moves were
planned to end on Saturday night when the turkey dinner was held. Sunday morning one could sleep late, but not so late as to miss the
"flapper" breakfast — again at the Haskin cottage with Hal busy making blueberry pancakes. The tray moves eventually became very
popular events that attracted ex-students and often, their friends, back to the Cape Shore for a weekend of work and good fun.

Hal's publication record was modest and belies his research accomplishments. He was totally disinterested in maintaining a CV. In
fact, it was difficult to locate citations, among his own files, to list in this remembrance. His exacting standards applied to writing — both
style and content — and to his own as well as that of others. He had difficulty with the concept of publishing a paper that didn't tell a

In Memorium: Harold Haley Haskin 443

complete story, and he usually telt that he needed more data than he had. This was particularly true for field studies, even though his
data sets extended for years, and. in some cases, for decades. Although Hal always analyzed the results and used them in meetings or
to advise management agencies, formal reporting was less interesting than starting a new project or going on a field expedition. Students
recalled late night sessions in which everyone pitched in to help copy and assemble reports at the very last minute — typically well after
the deadline. Nevertheless, Hal had little difficulty obtaining funds to run his laboratory because grant managers knew the quality of the
data would be high — when they eventually received it.

When he wasn't working, which often seemed like never, Hal had three great recreational passions: woodcutting, sailing, and growing
lilacs. For years. Hal and Peg heated their house in southern New Jersey and their Cape Shore cottage with wood stoves. Their house
in Piscataway had multiple fireplaces. Hal always carried a chain saw and splitting wedges in the trunk of his car in case he had an hour
or two free to cut wood. Friends recalled that he cut down only dead or dying trees and knew exactly when they should be cut to
maximize dryness and minimize decay.

The principal Haskin sailboat was a 17 ft Thistle that was anchored on the tidal flats in front of the summer cottage, and sailed as
often as possible at high tide with Hal at the helm and a boat full of family, friends, and students from the Cape Shore Laboratory, just
down the beach. Annual beach parties were a chance for sailboat races between the Thistle and any other sailboat that could be pressed
into service.

More than 100 lilac bushes representing 70 different varieties, formed a fragrant hedge around the Piscataway house. Hal knew all
the varietal names and delighted in escorting visitors around the hedge, "introducing" them to each plant and pointing out its unique
characteristics. Each May when the lilacs bloomed, he would bring buckets full of blossoms into the lab to distribute among the staff
and faculty. He stored them in the cold room, which temporarily at least, smelled delightfully of lilacs rather than aged oysters.

The memorial service included tributes from representatives of numerous agencies, organizations, and institutions that Hal served,
including the National Shelltlsheries Association. He joined NSA in the late 1940s and rarely missed a meeting for almost 50 years. He
was Vice President in 1966-67. and President during 1967-69. He became an Honored Life Member in 1979 and. most precious to him.
was given the David Wallace Award in 1984.

In addition to Peg, Hal is survived by five children, Kathleen Haskin of New York City. Jean Haley of North Kingstown, RI, Elizabeth
Haskin of Cape May Court House, NJ, Frederick James Haskin of Piscataway, NJ, and Mary D. Haskin of Washington, DC: and four
grandchildren. Harold Haskin II. a student at Rowan University, Allegra and Elijah Penny, of Washington. DC. and William Schroer of
Cape May Court House. NJ.

Hal. alone and with Peg, received numerous awards over the years (see Kraeuter and Ford. 1999. J. Shellfish Res. 18: 337-339), but
one of the finest was presented at the memorial service: The New Jersey Nature Conservancy named a portion of its Delaware Bayshores
Tract near the Cape Shore cottage "The Harold and Margaret Haskin Nature Preserve." It was an entirely fitting honor for a lifetime of
dedicated service to environmental teaching and preservation.

Susan Ford

John Kraeuter

Walter Canzonier

Haskin Shellfish Research Laboratory

Port Norris. New Jersey

PUBLICATIONS

Welsh. J. H. and H. H. Haskin. 1939. Chemical mediation in crustaceans. IM. Acetylcholine and autotomv in Pcinilisrlws aniuilus (Gibbes). Biol. Bull.

76:405^15.
Haskin. H. H. 1942. A spectrophotometric method for the analysis of chloroplast pigments. J. Biol. Chem. 144:149-160.
H.iskin. H, H. 1949. Growth studies on the quahaug. Venus mercenaria. Proc. Nat'l. Shellfish. Assoc. 1949:67-75.
Haskin. H. H. 1950. Selection of food by the common oyster drill Urosalpiiu cinerea (Say). Proc. Nat'l. Shellfish. Assoc, 1950:62-68.
Huskin. H H. 1952 Further growth studies on the quahaug, Venus mercenaria. Proc. Nat'l. Shellfish. Assoc. 1952:181-187.
Haskin, H. H. 1954. Age determination in molluscs. Trans. N.Y. Acad. Sn. 16: 300-304.
Pomeroy, L. R. and H. H. Haskin. 1954. The uptake and utilization of phosphate ions from sea water by the American oyster. Crassostrea virginica

(Gmelin). Biol. Bull. 107:123-129.
Pomeroy, L. R.. H. H. Haskin and R. A. Ragotzkie. 1956. Observations on dinotlagellate blooms. Limol. Oceanog. 1:56-60.
Dean. D. and H. H. Haskin. 1964. Benthic repopulation of the Raritan River estuary following pollution abatement. Limnol. Oceanog. 9:551-563.
Haskin. H. H. 1964. The distribution of oyster larvae. Proc. Symposium on Exp. Marine Ecology. Occasional Pub. #2. Rhode Island Univ. Grad. School

of Oceanography, pp. 76-80.
Haskin. H. H., W. J. Canzonier & J. L. Myhre. 1965, The history of MSX on Delaware Bay oyster grounds, 1957-65. Amer. Malacol. Union Reports

/or 7965 32:20-21.
Haskin, H. H., L. A. Stauber& J. A. Mackin. 1966. Mincliinia nelsoni n. sp. (Haplosporida. Haplosporidiidae): causative agent of the Delaware Bay oyster

epizootic. Science 153:1414-1416.
Hamwi, A. and H. H. Haskin. 1969. Oxygen consumption and pumping rates in the hard clam Mercenaria mercenaria: a direct method. Science.

163:823-824.
Hidu. H. and H. H. Haskin. 1971. Setting of the Amencan oyster related to environmental factors and larval behavior. Proc. Nat'l. Shellfish. Assoc.

61:.V5-.50.
Van Winkle. W., S. Y. Feng, and H. H. Haskin. 1976. Effect of temperature and salinity on the extension of siphons by Mercenaria mercenaria. J. Fish.

Res. Brd. Can. 33:1540-1546.

444 Ford et al.

Douglass. W. R. & H. H. Haskin. 1976. Oyster-MSX interactions: alterations in hemolymph enzyme activities in Crassostrea virf>iiiicci during the course

o( Minchinia nelsoni disease development. J. Invertehi: Puthol. 27:317-323.
Hidu. H. and H.H. Haskin. 1978. Swimming speeds of oyster larvae Crassosuea virj^inica in different salinities and temperatures. Estuuries 1:252-255.
Haskin. H. H. & S. E. Ford. 1979. Development of resistance to Minchinia nehoni (MSX) mortality in laboratory-reared and native oyster stocks in

Delaware Bay. Mar. Fisheries Rev. 41:54-63.
Ford. S. E. & H. H. Haskin. 1982. History and epizooliology of Haph>spi>ruliiini nehnni (MSX). an oyster pathogen, in Delaware Bay. 1957-1980. J.

Inverlebr. Pathol. 40:118-141.
Haskin. H. H. & S. E. Ford. 1982. Haplnspnridiinn nelsoni (MSX) on Delaware Bay seed oyster beds: a host-parasite relationship along a salinity gradient.

/ Inveriehi. Pathol. 40:388-405.
Haskin. H. H.. R. A. Lut/ & C. E. Epifanio. 1983. Benthos (Shellfish). Chap. 13. In: J. H. Sharp, (ed). The Delaware Estuary: Research as Background

tor Estuarine Management and Development. University of Delaware College of Marine Studies and New Jersey Marine Sciences Consortium. Lewes.

Delaware, pp. 183-207.
Haskin, H. H. & S. E. Ford. 1983. Quantitative effects of MS.X disease {Haplnsporuhnni nelsoni) on production of the New Jersey oyster beds in Delaware

Bay. USA. Proceedings of ICES. CM I983/Gen:7/Mini-Syinp. Goteborg. Sweden. October. 1983. 20 p.
Haskin. H. H. & S. E. Ford. 1986. Breeding for disease resistance in molluscs. Proceedings of EIFAC/FAO Symposium on Selection. Hybridization and

Genetic Engineering in Aquaculture. EIFAC/86/Symp. R27. Bordeaux. France. May, 1986. 25 p.
Haskin, H. H. & S. E. Ford. 1987. Breeding for di.sease resistance in molluscs. In: K. Tiews, (ed). Proceedings World Symposium on Selection.

Hybridization, and Genetic Engineering in Aquaculture, Bordeaux 27-30 May, 1986. Vol. II, Heenemann Verlagsgesellschaft, Berlin, pp. 431-441.
Ford. S. E. & H. H. Haskin. 1987. Infection and mortality patterns in strains of oysters Crassostrea \irf;inica selected for resistance to the parasite

Haplosporiclinm nelsoni (MSX). / Parasilol. 73:368-376.
Ford. S. E. & H. H. Haskin. 1988. Comparison of in vitro salinity tolerance of the oyster parasite Haplosporidinm nelsoni (MSX) and hemocytes from

the host. Crassostrea virginica. Comp. Biochem. Physiol. 90A:I83-I87.
Ford. S. E. & H. H. Haskin. 1988. Management strategies for MSX iHaplospmidinm nelsoni) disea.se in eastern oysters. In: W. S. Fisher, (ed). Disease

Processes in Marine Bivalve Molluscs. 18. American Fisheries Society. Bethesda, MD. pp. 249-256.
Haskin, H. H. & J. D. Andrews. 1988. Uncertainties and speculations about the life cycle of the eastern oyster pathogen Haplosporuhiim nelsoni iMSX).

In: W. S. Fisher, (ed). Disease Processes in Marine Bivalve Molluscs. 18, American Fisheries Society, Bethesda, MD. pp. 5-22.
Barber. B. J.. S. E. Ford & H. H. Haskin. 1988. Effects of the parasite MSX [Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy

metabolism. I. Condition index and relative fecundity. J. Shellfish Res. 7:25-31.
Barber. B. J.. S. E. Ford & H. H. Haskin. 1988. Effects of the parasite MSX [Huplosporidinni nelsoni) on oyster [Crassostrea virginica) energy

metabolism. U. Tissue biochemical composition. Comp. Biochem. Physiol. 91A:603-608.
Vrijenhoek. R. C. S. E. Ford & H. H. Haskin. 1990. Maintenance of heterozygosity during selective breeding of oysters for resistance to MSX disease.

J. Hcrerf(0'8I:418-+23.
Ford, S. E.. A. J. Figueras & H. H. Haskin. 1990. Inlluence of selective breeding, geographic origin, and disease on gametogenesis and sex ratios of

oysters. Crassostrea virginica. exposed to the parasite Haplosporidium nelsoni (MSX). Aquaculture 87:285301.
Hillman, R. E., S. E. Ford & H. H. Haskin. 1990. Minchinia teredinis n. sp. (Balanosporida. Haplosporidiidae). a parasite of teredinid shipworms. J.

Protozoal. 37:364-368.
Kraeuter. J. N.. S. R. Fegley. S. E. Ford & H. H. Haskin. 1993. Delaware Bay oyster populations: effects of seed movement, harvesting, and diseases.

In: R. F. Dame. (ed). Bivalve Filter Feeders in Estuarine and Coastal Ecosystem Processes. G 33, Springer-Verlag. Berlin, pp. 531533.
Dittman. D. E.. S. E. Ford & H. H. Haskin. 1998. Growth patterns in oysters from different estuaries. Mar Biol. 132:461-469.

JoKnial of Shellfish Rc.winh. Vol. 21. No. 2. 44.'i-t46. 2002.

Jay Donald Andrews
Honored Life Member

Jay Andrews, affectionately known as -Andy" by his many friends and colleagues, is widely respected for his fLindamental research
on the ecology of the major oyster pathogens in the Chesapeake Bay, Haphsporidium iwl.soni and Perkinsus munnus. Less well known,
but equally important, are his 22-y dataset on oyster spatfal! patterns in the Chesapeake Bay and the disease management strategies he
developed for industry.

Andy was bom on September 9. 1916 in Bloom, Kansas. He grew up on a wheat and cattle farm in western Kansas south of Dodge
City during the Depression and drought of the 1930s. Andy attended high school in Bloom and was the top student in a class of 10.
Studies were apparently easy for him and he spent much time reading Zane Gray wild-west stories and following baseball scores and
players. When Andy graduated from high school in 1934, the nation was in full depression, and the western plains were in a severe
ten-year drought that was making farmmg very difficult, if not impossible. Lack of moisture prevented raising wheat or fodder for cattle.
Over time, Andy remembers, pastures of the family farm were filled with annual weeds, prickly pear cactus, and an explosion ot
jackrabbits. After graduation, Andy's father took him to a bank in Dodge City, borrowed $100 and sent him to Kansas State College in
Manhattan. College opened a new world for Andy, and he worked hard. In addition to classes, he worked 40 hours each month in the
state 4-H Club for 23 cents an hour and participated in the ROTC. which paid $15 a month. He was designated the top agricultural student
and obtained a degree in agriculture in 1 938, but farming in western Kansas was not an attractive proposition for Andy. Instead, he began
graduate study at the University of Wisconsin in Madison, where he obtained a teaching assistantship in biology. He earned a salary ot
$600 a year and had money for extracurricular activities for the first time. His love of opera developed during these years. Andy earned
an M.S. in 1940 and continued on for his Ph.D.

Andy's graduate studies were intenupted by World War II; he was called up in January 1941 and spent four years in the infantry.
He saw action in the South Pacific at the battle of Leyte Gulf and the battle of Bataan and survived multiple kamikaze attacks on his
troop ship. In 1946, he returned to Madison to finish his degree, which he earned in 1947. In the spring of 1946, Andy and fellow student.
Willard Van Engel, attended a fisheries meeting in St. Louis, where they met the Director of the Virginia Fisheries Laboratory in
Yorktown, VA (the precursor of VIMS), who offered them jobs that they both accepted. Van Engel was requested to work on blue crabs,
and Andy was asked to concentrate on oysters. A few months later, he was advised by a visiting professor from Yale not to work on
oysters, because there was already a large literature and "everything had been done already." Thankfully, Andy ignored the advice. When
Andy and "Van" arrived in Virginia in the fall of 1947, they learned that the three scientists who had been at the laboratory had left that
summer for positions at Texas A&M. They had been working with mussels on some compound important to the war effort. The three
scientists were John Mackin, Sewell Hopkins, and Winston Menzel.

44.5

446 BURRESON

Shortly after he an'ived in Virginia. Andy began monitoring oyster spatt'all patterns in the tributaries. After years of monitoring, it
became clear that setting patterns could be easily separated into two distinctive types. The large tributaries and the Chesapeake Bay
proper require very large stocks of brood oysters to acquire regular spatfall because of the large tidal exchange and flushing. Only the
upper James River estuary met this requirement and that was because of its special circulation patterns and the large beds of transplanted
oysters in the lower James being grown by private industry. Such other large rivers as the York, Rappahannock, and Potomac never had
enough broodstock to produce regular spatfall. but occasional large sets did occur. The small tributaries, with low runoff, exhibit a
completely different setting pattein than the large tributaries. These coastal plain subestuaries have moderate annual setting patterns, even
though oyster populations are low because of high larval retention caused by the winding channels and shallow flats. Uncharacteristi-
cally, Andy never published these data on spatfall patterns. Beginning in 1950. Andy tried to persuade managers in Virginia to use the
small estuaries to produce seed oysters for transplantation to larger tributaries, but this advice was ignored until long after he retired.
When the reef restoration strategy was initiated in Virginia in 1996. the first few reconstructed oyster reefs were placed in these small
tributaries because of the larval retention patterns that Andy had documented.

Andy's research on oyster diseases began when John Mackin visited VIMS from Texas to determine if PerkiiiMis nuiriiuis (Dermo
disease) was present in Chesapeake Bay oysters. The discovery of this pathogen in an area lacking oil drilling confirmed that oyster
mortality in the Gulf of Mexico was the result of P. marinus. not the oil indu.stry. Andy produced fundamental papers on the
epizootiology of Dermo disease during the 19.')0s, and his 1988 review on the pathogen is a classic paper still widely read and cited.
When Haplospondium nelsoni (MSX disease) appeared in the Chesapeake Bay in 1959. Andy immediately began conducting research
on this organism and produced many seminal papers on the ecology of this pathogen as well as on Haplosporidium coslale. One of
Andy's most important traits was that he published his results in a timely manner. In 1960, Andy initiated a monitoring program for H.
nelsoni at VIMS using imported susceptible oysters. This program continues to the present day and provides a 42-year database on H.
nelsoni prevalence and intensity that has proved invaluable for examining climate effects on pathogen abundance. Because of his broad
training and experience and his inquisitive nature, Andy was very insightful. For example, he was the first to hypothesize, in 1980, that
H. nelsoni was an introduced pathogen. It wasn't until 2000 that molecular data were obtained that support his supposition.

Andy worked hard and expected hard work of others. You arrived on time, and you didn't leave early. Andy loved the rigors of
fieldwork and scoffed at suggestions for an easier way to do things. He hauled oyster trays by hand and cleaned them by throwing
countless buckets of water that he dipped by hand. After such trips he returned to the campus at VIMS wet. co\ered \\ ith mud. shirtless,
in shorts, and barefoot. He surely startled more than one unsuspecting graduate student.

Andy is internationally recognized for his fundamental research on oyster diseases, but he was also a long-time educator. During his
career at VIMS he taught Ichthyology. Field Biology, Taxonomy and Systematics, Marine Ecology and Biologic Oceanography.

Andy was a capable and energetic volleyball player. In the "interesting" lunch time volleyball games at VFL/VIMS he played with
gusto, spiking near net-balls down the throats of opposing players with great vigor and glee. Road trips with Andy were always an
experience. Once, while driving north for a meeting, Andy spied a walnut tree along the road. He pulled over and took an old pair of
coveralls out of the trunk. He tied off the end of each leg, walked over to the tree and began filling the legs w ith w ainuts that had fallen
to the ground, commenting that he hoped we would not be shot by the owner.

Andy is an avid vegetable gardener and has a large plot near VIMS. He can still be seen hauling countless old milk jugs full of water
in his old truck during drought periods and undoubtedly still cursing the varmints that take half his crop.

Andy is a long-time member of the National Shellfisheries Association and served as President and Editor. He was elected Honored
Life Member in 198.^. In 1998, he received the David Wallace Award in recognition of his career-long efforts to use science for the
betterment of shellfish management and a sustainable industry.

Eugene Burreson
Gloucester Point, Virginia

JoiiriHil of Shellfish Research. Vol. 21, No. 2, 447-448, 2002,

Neil F. Bourne
Honored Life Member

Neil was bom August 1 1. 1929 in London. Ontario, Canada. He began his elementary school education there, and it was in London,
mainly through the influence of his father, that he became interested in natural history and particularly bird watching, a hobby and
passion that have continued to the present. It was this interest in natural history that eventually led him to become a biologist. The family
moved to Hamilton, Ontario in 19,^9, where he completed his elementary and high school education. By the time he moved to Hamilton,
he had over 150 bird species on his life list! He entered McMaster University in Hamilton in 1948 and graduated with a BSc in biology
in 1952, In 1953, he completed a MSc degree at McMaster under an Ontario Research Scholarship and in 195.^-54 spent a year in
Germany, where he undertook studies in limnology at the Freshwater Institute in Ploen and marine biology at the University of Kiel,
He returned to Canada in 1954, entered the University of Toronto, and graduated from there with a PhD in 1959, His thesis was entitled.
"Determination of carbon transfer from Chlorella vulgaris Beyerinck to Daphnia luagiui Strauss using radioactive carbon (C14) as a
tracer," Three years of his studies were supported by National Research Council of Canada scholarships.

On completion of his PhD in March 1959. Neil joined the staff of the Fisheries Research Board of Canada at the Biologic Station
in St. Andrews, New Brunswick working under the direction of the late Dr, J, C, Medcof. a former Honorary Member of NSA, At St,
Andrews, he was in charge of the sea scallop investigation that included investigations of the basic biology and ecology of sea scallops,
dynamics of sea scallop populations on Georges Bank and in the Bay of Fundy. scallop gear efficiency trials, and exploratory work to
locate sea scallop resources in other areas along Canada's Atlantic coast. While at St. Andrews, Neil spent two weeks at Dr. V.
Loosanoff s laboratory in Milford. Connecticut studying bivalve breeding technology. Thus began a life-long commitment to shellfish
aquaculture. He used knowledge gained at the Milford laboratory to begin initial attempts to culture sea scallops and was successful in
conditioning and spawning adults and raising larvae to the mature stage.

In 1965, Neil transferred to the Pacific Biologic Station in Nanaimo, British Columbia, first working for the Fisheries Research Board
of Canada and later with the Department of Fisheries and Oceans, Initially, he worked with the late Dr, Dan B, Quayle. another former
Honored Member of NSA. and remained at the Pacific Biologic Station until his retirement. During his career at the Pacific Biologic
Station, Neil undertook research studies on a wide range of molluscan subjects, including the basic biology of several Pacific coast
molluscan species, population studies of commercially important clams, harvesting gear efficiency, and studies to develop techniques to
culture several species. He developed techniques to predict Pacific oyster spatfall accurately for the industry, a service he continues to
provide. He undertook extensive surveys of bivalve resources in B,C, and. in particular, followed the dispersal of Manila clams
northward along the coast. As a result of these studies, a fishery for Manila clams became established in the central coast of B,C.

As a result of previous work and the influence of his stay at the Milford laboratory. Neil remained intensely interested in bivalve
culture and continued studies in this field. Initial studies were undertaken to investigate the potential of butter and littleneck clams and

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448 Chew

abalone. He undertook studies with Manila clam culture that were important in establishing the Manila clam culture industrv that exists

in B.C. today.

From 1981 to 1991 Neil led a program to investigate the feasibility of scallop culture in B.C. Several species were studied, but the
decision was made to work with the Japanese scallop. Mizuhopecteii yessoensis. and methods were developed to produce juveniles in
a hatchery and raise them to commercial size within a period of two years. Much of the information culminated in the publication. A
Maiuuil for Scallop Cidtnic in British Coliimhia. which continues to be used by many to the present day. Results of the work led to a
private company building a scallop hatchery and beginning commercial scallop culture in B.C. He continues to serve as an advisor to
this industry.

Neil has been active in foreign aid work, assisting at Universities and has been involved with several scientific societies. He worked
for the Canadian International Development Agency for 1 1/2 years in Fiji, where he served as Director of a Fisheries Training Program
at the University of the South Pacific in Suva. He undertook an assessment of the Cuban oyster industry for the Department of Fisheries
and Oceans and advised on a joint Canada-Cuba program to expand oyster culture in Cuba. He worked for the United Nations Food and
Agriculture Organization in the Peoples" Republic of China and continues to serve as a volunteer ad\ isor to the Canadian Executive
Service Organization. He served on the Aquatic Resources Subcommittee of the Science Council of British Columbia from 1987-2002
and was chairman from 1991 to 2002.

He is an affiliate professor at the University of Washington. School of Fisheries and has been a committee member for over 20
graduate students, mostly at the University of Washington, but also at the University of Victoria, the University of British Columbia,
and Simon Fraser University.

Neil served on the executive boards of the Canadian Society of Zoologists, the World Aquaculture Society, the Aquaculture
Association of Canada, serving as President of the latter Society from 1987-1988. He joined the National Shelltlsheries Association
(NSAl in 1961 and has been active in the affairs of the society since then. He served on the Executive Committee of the Association
from 1974-198.\ including tenure as President in 1981-1982; he was the first Canadian to serve as President of NSA. He has been a
member of the editorial board of the Joiiniul of Shellfish Research for many years. He was made an Honored Life Member of the
Association in 1990 for his exemplary service to NSA and the profession. In 1991 , he received the David H. Wallace award for promoting
understanding, knowledge, and cooperation among industry members, the academic community, and all levels of government and for
his outstanding success in bringing together shellfish scientists and industry officials for the benefit of shellfisheries.

Neil received an Award of Merit from the Department of Fisheries and Oceans in 1993 for an exceptional and distinguished
contribution to the effectiveness and efficiency of the Public Service and the Department, particularly for contributions to the aquaculture
industry of British Columbia. Neil retired from the Department in March 1994.

In May 2000, Neil was presented with an Honorary Lifetime Achievement Award from the Aquaculture Association of Canada. The
selection board cited long-time service to the Association, the research community, industry, and young scientists as exemplary examples
of the goals of that organization. He was the first recipient of this award.

Neil lives in Nanaimo and continues to work at the Pacific Biologic Station as a volunteer Scientist Emeritus, publishing results of
past and current work and encouraging young scientists in their careers. He still has time to enjoy his grandchildren, hike, work around
the house, and undertake other activities. He is keenly interested in golf and is a rabid bird watcher with the distinction of having birded
on all seven continents. One activity in his retirement is to compile his bird life list, he believes he has seen about 2,500 species of birds!
Those who have golfed with him have noted that he never lets golf interfere with birding although the opposite has occurred on occasion.

Kenneth Chew

University of Washington
Seattle, Washincton

Janimil of Shellfi.sh ReMccucli. Vol. 21. No. 2. 444_t.S0. 2002.

Victor G. Burrell
Honored Life Member

Dr. Victor (Vic) G. Burrell. Jr. has been a member of the National Shellfisheries Association (NSA) for over 30 years and served
as its president from 1982-1983. He was born in Wilmington, NC in 1925 and served in the U.S. Navy during WW II from 1943-1946.
He received a B.S. degree in English from the College of Charleston in 1949. In 196.5. after 15 years in his family's meat-packing
business, he went on to earn both Master's Degree and a Ph.D. Degree in Marine Science from the College of William and Mary in 1968
and 1972, respectively. In large part, this interest may have been kindled through his experiences operating a charter boat in South
Carolina and working part time in the commercial oyster industry. While a student at Virginia Institute of Marine Science (VIMS), he
served as the liaison to the fishing industry, especially with oystermen. At that time, he developed a method of denaturing the red
algal-derived seasonal color of oysters using the blower system.

" In 1972. he left the staff of the VIMS and returned to the Carolinas as an Associate Marine Scientist with the then just established
Division of Marine Resources in Charleston. He spent from 1972 to 1991 at the Marine Resources Research Institute (MRRI) of the
Division of Marine Resources, South Carolina Department of Natural Resources (then the Wildlife and Marine Resources Department).
He served as the Institute's Director from 1974 to 1991. "retiring" in 1991. Since then, he has continued to take an active role in South
Carolina's marine resources, including co-authoring numerous scientific and lay review publications for NOAA and the DNR as an
emeritus director and scientist at Fort^Johnson. In total, he has published over 65 scientific papers on zooplankton ecology, shellfish
biology, and oyster culture. These include a series for NOAA in 1996-1997 entitled "The History. Present Condition, and Future of the
Molluscan Fisheries of North and Central America and Europe, Volumes 1-3, with Clyde Mackenzie and others. In 2000, he authored
a DNR educational report entitled "The Recreational Fishery in South Carolina: the Little River Story," reviewing the first organized
offshore recreational fishing industry in South Carolina. He is also currently writing a history of South Carolina's oyster industry for
publication.

During his leadership at MRRI. Dr. Burrell saw the staff double to about fifty professional individuals, including eleven Ph.D-level
marine scientists. In addition to his active participation in the NSA, Dr. Burrell served also as president of the Southeastern Estuarine
Research Society (SEERS) and was named an honorary life member of SEERS. He served as president of the College of Charleston
Alumni Association and was honored as the college's Alumnus of the Year in 1963. He also saw the development and construction ot
the SC Aquarium serving on the executive board of the aquarium as a founding member.

He is listed in Community Leaders of America, Personalities of the South, American Men and Women of Science. International
Directory of Distinguished Leadership, Who's Who in the South and Southwest, Who's Who in America, and Men of Achievement. He
recently moved to new digs and we will miss him walking along the road tipping his hat and waving his hand as he traveled daily to
MRRI from his James Island house. However, we still expect to see him almost daily in his office (when he is not taking a vacation

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450 COEN AND BOBO

cruise) chatting witli everyone and being the gentleman that he is. He is an aficionado of big band swing and jazz, particularly that from
the Depression era through post-war periods. We remember his enjoyment of Ken Burn's "Jazz" and discussed with him the pros and
cons of Bum's choice of influential "characters." such as Louis Armstrong. He's knowledgeable enough about the music and musicians
of that era to do a creditable history. He's been an active member of St. James Episcopal on James Island for many years, serving as
Junior and Senior 'Warden on several occasions.

Vic and his wife Katherine have four children, eight grandchildren, and four great-grandchildren.

Loren D. Coen

M. Yvonne Bobo

MRRI

SCDNR

Charleston. SC 29412

Joiirmil of Shellfish Research. Vol. 21, No. 2. 451 -+52. 2002.

Original works of art surround the artist and NSA Honored Life
Member, Herb Hidu, at his home in Alna, Maine.

Herbert Hidu
Honored Life Memeber

Herb has traveled an unusual and convoluted path to distuielion in American shellfisheries science. He was born in 1931, the son of
a German immigrant and a 2nd generation Hungarian farmer and estate manager. The family never had the slightest inclination for
academia. He spent his youth hunting, fishing, and observing the pleasantries of living on a Connecticut rich man's estate as a cottage
inhabitant. He joined the army in 1951 and for 2 years was a paratrooper with the 82nd airborne division, which gave him his mindset
for later years. After his hitch, his drinking buddy made him aware of Korean G.I. Bill eligibility. Entering the University of Connecticut,
he majored in fisheries management and forestry (B.S., 1938). Along the way he had the opportunity to resume his parachuting paranoia,
working a summer as a smokejumper at the United States Forestry Service base in Winlhrop, Washington, during which time he made
four fire jumps.

At UConn, Dr. Slater of the Entomology Department convinced hint to pursue a graduate degree in the biological sciences. He
received a MS degree in Zoology in 1960 from Penn State University, working under the tutelage of Dr. Edwin L. Cooper on population
structure of largemouth bass in a farm fish pond.

Then, for some unexplained reason. Herb accepted a job in the alien science of shellfisheries biology, working for Dr. Victor
Loosanoff at the U.S. Bureau of Commercial Fisheries shellfish laboratory in Milford, Connecticut. His position entailed conducting
bioassays with shellfish larvae. The 3-year experience taught him the intricacies of shellfish hatchery culture and, perhaps more
importantly, perseverance. Indeed, having endured the extremely confining environment created by the very intense Russian scientist
Loosanoff, the world was now Herb's oyster and the oyster was now Herb's world.

The Mid- Atlantic MSX oyster mortalities of the mid-1960s provided Herb an excellent avenue for advancement. Rutgers University
and Dr. Harold Haskin were in great need of workers (students) who were versed in hatchery techniques. Efforts were concentrated on
the production of experimental disease-resistant stocks. This became Hidu's "bread and butter" as a graduate student, where for over 3
years he begat many of Rutgers' experimental oysters stocks at the Cape May laboratory. He used the abundant larval stocks to conduct
his study on laboratory behavior and field recruitment of Delaware Bay oysters. His 1967 Ph.D thesis is cited to this day as the definitive
study on Delaware Bay oyster recruitment.

After Rutgers. Hidu became a shellfisheries research biologist at the Chesapeake Biological Laboratory (CBL) in Solomons,
Maryland. It was hoped thai he would fill the shoes and carry on the traditions of the late Francis Beavin. the "dean" of Chesapeake Bay
oyster science. With four older veteran shellfisheries biologists, he pursued, for 3 years, field shellfish surveys of Chincoteague Bay and
the effects of power plant effluent on the early life history stages of Chesapeake Bay oysters. He developed the natural feeding method
for shellfish hatcheries in Maryland and stimulated an early commercial hatchery (Frank Wilde, Shadyside, MD). However, he was
miscast in the position at CBL, which he viewed as a mixture of research and personnel management. As an advocate of hatchery
production of shellfish to augment recruitment, he locked horns with the Maryland public oyster fishery. Furthennore. his lack of contact
with students at the University of Maryland's College Park campus, 100 miles distant inland, created for Herb an untenable situation and
he decided it was time to move on. His lifetime break came in 1970 when the University of Maine's Darling Marine Center entered the
Federal Sea Grant Program with a "Coherent Areas" grant entitled "Culture of Resources in a Cold Water Marine Environment". Hidu
was offered and accepted a position that required shellfish hatchery expertise and. more importantly to Herb, allowed access to excellent
students who would pursue M.S. and Ph.D degrees in Ocean<igraphy while accomplishing the Sea Grant aquaculture research.

The 15-H graduate students Hidu sponsored at the University of Maine won a record 5 NSA Thurlow Nelson Awards and contributed
significantly to the shellfisheries literature: blue mussel biology, R. Lutz, L. Incze. G. Podniesinski; triploid shellfish, S. Allen. M. Lee:
intertidal shellfish capacity adaptations. R. Gillmor; hatchery pathology and techniques, L. Plunket, M. Logue-Keller, R. Clime, and
others. In the 1970s Hidu developed the nation's first mariculture course. That field course and the associated aquaculture extension
effort helped stimulate a significant new Maine industry in oyster grow-out and hatchery production. For this, in 1990. he received the

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452 LuTZ

University of Maine's Presidentiul Public Service Award and, in tlie eyes of tliose of us who know Herb well, he took his place as
Maine's "father of aquacullure".

His own research resulted in over 73 papers, with topics ranging from hatchery techniques to field recruitment of oysters, gregarious
setting, biological fouling control, triploid shellfish, air winter storage of shellfish seed, blue mussel field recruitment, effects of
pollutants on shellfish larvae, and behavior of shellfish larvae. He ran the gamut of offices in the National Shelltlsheries Association
(NSA). becoming President in 1980. He won the NSA David Wallace award in 1991 and became an NSA Honored Life Meinher in 1994.
Herb retired in 1992 and is now Professor Emeritus of Animal and Veterinary Science at the University of Maine.

In retirement. Herb has gravitated toward his old roots with a move from aquaculture to horticulture, maintaining a small farm in Alna.
Maine at which he raises commercial Hosta plants. In fact, his farm bears a remarkable resemblance to a certain Connecticut rich man's
estate, with extensive trimmed pine hedges and espaliered trees. In 2001 he was a retired smokejumper volunteer in the Bob Marshall
Wilderness Area of Western Montana working trails and helping build a wilderness bridge. He is active in golf, watercolor art. and
woodlol management. He has been married 47 years to Judith and has .^ children and 4 grandchildren, all of whom live in Maine. He
is the man to whom the author of this biography owes his career — thanks. Herb.

Richard A. Lutz

Institute of Marine and Coastal Sciences

Rutgers University

New Brunswick. NJ 08901

Jdiirmil of Shellfish Research. Vol. 21. No. 2. 4.i.^-;54. 2002.

Sandra E. Shumway
Honored Life Member

Sandra "Sandy" Shumway was bom March 24. 1952 in Taunton. Massachusetts. She attended Mulcahey Grammar School before
going on to Taunton High School. v\here she graduated as Salutatorian in 1970. A tomboy as a youngster, she was more interested in
cowboys and Indians, football and baseball than dolls. She began piano in the second grade, but it was not her instrument. In fourth grade,
she took up the clarinet and later bass clarinet and played first chair in the All-State Band twice. While continuing to play throughout
college, music was for fun and science would win out. From the time that school let out until Labor Day, summers were spent at the
family summer home in Portsmouth. Rhode Island. She dug her first clam by the age of three, and as she got older she fished, sailed,
and tended a few lobster pots. She spent countless hours collecting marine life and cataloging it (unlike other normal children). She began
a small science project by the 5th grade and was involved in science fairs thereafter. She regularly won local and regional fairs and took
first place at the Massachusetts State Science Fair at MIT during her junior year. In her senior year, she was named a Ford Future
Scientist of America and to the NASA Youth Science Congress. She credits her parents and several tolerant and progressive teachers
for nurturing her scientific interests.

Thus, her love of the sea and its creatures has carried over into her adulthood, and it is not surprising that she majored in this field
in college. Sandy graduated Sumiiui Cum Laiiclc in 1974 with a B.S. in Marine Science/Biology from Southampton College. Long Island
University. From there, she went on to the University College of North Wales in Bangor. Gwynedd. Wales as a Marshall Scholar, where
she received her Ph.D. in 1976 and later a D.Sc. in 1992. As a post-graduate she continued at the Marine Science Laboratories in Menai
Bridge. Gwynedd. North Wales, then at the University of Otago in Dunedin. New Zealand and with the Department of Ecology and
Evolution. State University of New York at Stony Brook.

In addition to her investigations in Wales and New Zealand. Sandy has also conducted research in laboratories in Brazil and numerous
states including Georgia, Maine, Washington, and North Carolina. Her formal teaching experiences have been limited to several levels
at Southampton College, but she teaches constantly wherever she goes, and she has served on thesis committees at the University of
Maine, the College of William and Mary. University of Washington. College of Charleston. North Carolina State University. University
of South Florida. University of Maine. University of Connecticut, and Rutgers University. She has served as an External Examiner for
Ph.D. candidates at various universities in Australia. Canada. England. India. New Zealand, and South Africa.

Her travels have taken her far and wide. After returning from abroad, where she spent 1 974-1980 in Wales, New Zealand and Brazil.
Sandy spent two years at Stony Brook and then moved north to the Maine Department of Marine Resources as a Research Scientist from
19S.^ to 1993 and an adjunct scientist at the Bigelow Laboratory for Ocean Sciences. After this, she moved back to Southampton College
and spent the next seven years as a Professor of Marine Science and Biology. In 2001. she moved to the Department of Marine Science
at the University of Connecticut in Groton. where she is presently an Adjunct Professor in Residence.

Sandy's research covers more than 27 years in shellfish biology, filter feeding, and physiological ecology of marine invertebrates.
She pioneered the study of impacts of harmful algae on shellfish and introduced the use of flow cytometry to determine particle selection
in filter-feeding invertebrates. A primary focus has been on problems associated with the shellfish industry; for example, distribution of
toxins in shellfish tissues, detoxification rates, and timing and extent of toxicity between shellfish species with the goal of establishing
species-specific closures and helping the industry to prosper in the presence of harmful algal blooms. She has recently worked with
scientists at North Carolina State University to determine the impacts of Pfiesteria spp. on shellfish and public health. Two of these
collaborators. Drs. JoAnn Burkholder and Howard Glasgow, recently named a new species of dinofiagellate, Pfiesteria .slniiiiutnae, in
her honor. Sandy has a permanent illustration of it with her at all times.

Sandy has authored 120 publications, edited two books, and co-authored a shellfish cookbook. She has served as editor of the National
Shellfisheries Association's Journal of Shellfish Research since 1986 and also edits the Journal of Experimental Marine Biology and

453

434 Abbe

Ecology. In addition, she serves on the editorial boards of several other journals. Although this takes an incredible amount of time, she
finds it satisfying to see the results in print and says it also gives her a fantastic overview of the fields of shellfish biology and
experimental marine biology. The number of manuscript submissions to JSR from foreign countries has increased dramatically in recent
years, and Sandy enjoys helping scientists from underdeveloped countries get their work published. She recently launched a new journal
with Elsevier, Hcmnful Algae, co-edited by Ted Smayda.

Sandy has been responsible for much of the recent growth of the National Shellfisheries Association. When she became Editor of the
Journal of Shellfish Research, the publication was often behind schedule and barely 100 pages a year. Since then, the JSR has averaged
600-700 pages annually and reached a new level with the publication of five issues and more than 1.700 pages in 1998. She was the
first woman president of NSA, serving in 1991-1992, and was re-elected in 2002 to serve again in 2003-2004. She has worked to
increase membership in the association and has played an important role in the annual meetings of NSA with her organizational skills,
her management of students for registration, sales, and as AV operators, and for the student auctions, which she initiated in 1993. These
auctions raise money for the Student Endowment Fund, which helps defray the costs of students who attend the annual meetings. She
has good-humoredly dressed for these occasions in various costumes, including a court jester, a blue quahog, a geoduck, and a lobster.
On another occasion, she made her own bikini top, with the help of a hotel employee, from a pair of sea scallop shells, and has actually
sold the shirt{s) off her back. In her never-ending effort to nurture students, in 1992, Sandy began the Recruits, the organization of student
members of NSA. The Recruits now have their own forum on the NSA website, a nonvoting representative on the EXCOM and write
articles for the Newsletter.

Sandy has also held several offices in the World Aquaculture Society and was the push that NSA needed to become a sponsor of the
Aquaculture Triennial Meetings, first as an affiliate sponsor of Aquaculture '86 and then as a full sponsor of Aquaculture "89 in Los
Angeles. These meetings have allowed NSA members to meet with members of the World Aquaculture Society, the American Fisheries
Society Fish Culture Section, and many other smaller societies. Since that first meetmg, NSA has enjoyed the intellectual and financial
advantages that these larger meetings provide, and Sandy has been a major part of the organization and priiduction of each of these
including San Diego, Las Vegas, two in Orlando and the upcoming AQ "04 in Hawaii.

Despite her seemingly endless efforts with students, journals, scientific meetings, and the shellfish industry, Sandy still has time to
spend with her miniatures which she began building in 1982. She is a nationally recognized miniaturist, yet many of the people who know
her are unaware of her love of the small. Students are particularly surprised to find out that she devotes time to things other than science.
Her buildings have been featured in several magazines including Mhmiture Gazette. Nutshell News. International Dollhoiise. and
Miniature Collector. "Sandra Shumway"s Sea-Born Treasures"" by Mary Kaliski in the April 1998 issue of Miniature Collector is a
wonderful example. At one time, she ambitiously wanted to build an entire village in miniature ( 1 inch = 1 foot), but she has had to
settle for less because of lack of time and space. Thus far, however, she has created a general store, two fishing shacks, a bordello, a
bakery, a drug and candy store, a barber shop, a school house, and a funeral parlor (all complete with people).

Sandy is also an avid pool player. She began when she was in college, but became more serious about it when she moved to Maine.
She not only loves the game for what it is. but for the opportunity it provides to interact with fishermen. She organized local tournaments
for years and has a number of trophies to show for her efforts. Often after a day of paper sessions and a dinner at the annual NSA meeting,
she will find a local pool hall to unwind. Of all the honors and awards she has received over the years, one of her most prized recognitions
came from a well-seasoned, cynical fisherman in a local bar who, when asked "Who's the broad at the pool table?'" replied "She's a
scientist, but she's okay.""

Sandy was awarded NSA"s highest honorary citation, the Honored Life Member award at Aquaculture '01 in Orlando, Florida on
January 18, 2001 following the eloquent remarks of Dr. Melborne R. Carriker, who stated that "it is her warm outgoing personality,
genuine interest in people, and readiness to extend an unselfish helping hand to those in need that has endeared her most to so many
of us."

George R. Abbe

Academy of Natural Sciences

Estuarine Research Center

St. Leonard, Maryland

Joiinml of Shellfish Reseiirch. Vol. 21. No. 2. 455-460, 2002.

EVALUATION OF MIST ALERTt^' RAPID TEST KITS FOR THE DETECTION OF PARALYTIC
AND AMNESIC SHELLFISH POISONING TOXINS IN SHELLFISH

F. H. MACKINTOSH AND E. A. SMITH*

Fisheries Research Services. Marine Laboratory, P.O. Bo.x 101, Victoria Road,
Aberdeen. ABU 9DB. United Kingdom

ABSTRACT Rapid test kits (MIST Alert'") for the detection of paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning
(ASP) toxins in shellfish have recently been developed. In this study, these kits have been evaluated for their potential use in shellfish
toxin-monitoring programs and by the shellfish industry. These antibody-based tests were used to assess the presence of shellfish toxins
qualitatively in a variety of shellfish species (mussels, scallops, oysters, cockles, razor fish) while routine methods of detection were
simultaneously used to quantify any toxin present. All shellfish extracts found to contain PSP toxins at the regulatory limit of 80 p.g
saxitoxin equivalents (STX eq) 100 g"' shellfish flesh using the mouse bioassay (MBA) were confirmed as positive by MIST Alert
for PSP. Shellfish farmers and other professionals in the industry also used these test kits successfully, with all positive samples being
correctly identified, clearly demonstrating its potential application in shellfish harvest management and end product testing. MIST Alert
for ASP also detected toxin in all monitoring samples containing the regulatory limit for amnesic shellfish poisoning (ASP) toxins, 20
fig g"' shellfish fiesh. as determined by high-performance liquid chromatography (HPLC). In addition, among samples in which HPLC
did not detect toxin, the kit agreed in 99% of tests. Overall, these results suggest that MIST Alert'" for PSP and ASP could be used
as part of routine monitoring programs.

KEY WORDS: paralyUc shellfish poisoning toxins, amnesic shellfish poisoning toxins. MIST Alert, shellfish monitoring, mouse
bioassay, high-performance liquid chromatography

INTRODUCTION

Recently we have repotted on the use of a commercial anti-
body-based rapid test kit (MIST Aletl'" for PSP) for the qualita-
tive (yes/no) detection of paralytic shellfish poisoning (PSP) toxins
in shellfish (Mackintosh et al. 2002). These potent neurotoxins are
produced by certain algal species and can accumulate in filter-
feeding shellfish. Current European legislation requires that shell-
fish are monitored for these toxins, and if they are detected in
shellfish flesh above the regulatory limit of 80 |xg saxitoxin
equivalents (STX eq) 100 g"' shellfish flesh, restrictions on shell-
fish harvesting are imposed (Gallacher et al. 1998). The initial
study (Mackintosh et al. 2002) highlighted that the MIST Alert for
PSP kit could be used in monitoring programs as a prescreen for
toxin-negative samples and in end-product testing and thus war-
ranted further investigation.

Since our preliminary assessment of MIST Alert for PSP a
further antibody-based qualitative test for amnesic shellfish poi-
soning (ASP) toxins has also been developed (MIST Alert^" for
ASP). This kit utilizes sheep antibodies developed by Garthwaite
et al. (1998) for the detection of neurotoxic domoic acid (DA), the
principal compound of ASP toxins, produced by several species of
the diatom Pseiido-nitzschia. MIST Alert for ASP utilizes the same
working principals of immunoflow chromatography used in MIST
Alert for PSP (Fig. 1).

The first recorded outbreak of human ASP. following con-
sumption of contaminated mussels, was recorded in 1987 (Wright
et al. 1989). Subsequently, many countries implemented monitor-
ing programs for the detection of ASP toxins in shellfish with a
regulatory level of 20 ^.g DA g"' shellfish fiesh adopted. In the
UK, the Scottish shellfishery has been particularly adversely af-
fected by ASP toxin contamination of king scallops which was
responsible for widespread harvesting closures during 1999 and
2000 (Campbell et al. 2001, Gallacher et al. 2001 ).

♦Corresponding author: Telephone: 44 (0)1224 295522; Fax: 44 (0)1224
295511; E-mail: smitheCfSmarlab. ac.uk

To date, the main tools for PSP and ASP toxin-monitoring
programs have been the mouse bioassay (MBA) and the high-
performance liquid chromatography (HPLC) method of Quilliam
et al. ( 1995) with UV diode array detection, respectively. The use
of mice for the detection of PSP toxins is considered ethically
questionable, and the assay is known to be susceptible to matrix
interference (Schantzetal. 1958, Park et al. 1986). Chemical analy-
sis for ASP toxins is expensive and requires skilled operators.
Hence, the requirement for simple, cheap, quick, and accurate
detection methods for PSP and ASP toxins in shellfish have been
widely discussed (Gallacher et al. 1998, Garthwaite 2000, Llewel-
lyn et al. 2001).

The aim of this study was to evaluate further the use of MIST
Alert for PSP in regulatory monitoring, as a harvest management
tool, and end-product test by the shellfish growing and processing
indu.stries and to assess the potential of MIST Alert for ASP in
routine shellfish monitoring.

MATERIALS AND METHODS

Assessment of MIST Alert for PSP During Routine
Shellfish Monitoring

Throughout 2001, the MBA was used for the quantitative as-
sessment of PSP toxicity (p-g STX eq 100 g"' of shellfish flesh) in
acidic (0.1 M HCl) shellfish extracts (/; = 547) as part of the
Scottish shellfish-monitoring program. Simultaneously. MIST
Alert for PSP test kits (Jellett Biotek Ltd., Nova Scotia, Canada)
were used for the yes/no detection of PSP toxins and results com-
pared with those from the MBA in four categories: not detected,
<40; 40 - <80; and >80. |jig STX eq lOOg"'- as previously de-
scribed (Mackintosh et al. 2002).

Investigation into the Use of MIST Alert for PSP in Shellfish Harvest
Management and End-Product Testing

MIST Alert for PSP test kits were supplied from Fisheries
Research Services (FRS) to eight participants to evaluate its po-

455

456

Mackintosh and Smith

"0

w

"0
C/)

Figure 1. Tlu- MIST Altrt'^' (ts( kit. Test kit strips consist of an
absorption pad (Si, a nienihrant' striped with a mivtiire of toxin ana-
lof;s (the "T" line), and an antihody detection reayent (the "C" line), a
sample pad. and a conjugate pad containing the antibodies. A visible
"T" line indicates absence of toxin in the sample and no line indicates
the presence of toxin. The "C" line indicates that the sample fluid has
sufficiently resuspended and mobilized the antibody color complex. As
the level of toxin increases, the ""T" line progressively disappears.
Remnant "T" lines are scored as having 50, 25, or O'r of color intensity
relative to the "C" line. MIST Alert for PSP'^' — samples are consid-
ered positive if the 'T" line is 50% or less the intensity of the "C" line.
MIST Alert for ASP'^' — samples are considered positive if the "T" line
forms only a faint shadow (i.e., less than 25%) or is completely absent.

tenlial for field use. Participants comprised shellfish farmers, pro-
cessors, and environmental health officers (EHOs). Each partici-
pant was supplied with either 50 (farmers) or 100 (processors and
EHOs) test kits and the equipment/chemicals necessary to perform
acidic shellfish extraction as described by the manufacturer. Be-
fore the study, each participant received training in sample extrac-
tion and kit use. Subsequently, each participant assessed the pres-
ence/absence of PSP toxins in shellfish extracts using the test kits.
An aliquot of each shellfish extract analyzed, the test kit. and the
participant's interpretation of the result were returned to FR.S. The
kit result was reinterpreted by laboratory personnel and extracts
reanalyzed by MIST Alert for PSP by an experienced user within
the laboratory. Positive samples were further analyzed by the
MBA. If this was not possible (e.g.. if the sample volume sup-
plied from the participants was too small), samples were diluted
1:1 in a negative shellfish extract and retesled using MIST Alert
for PSP.

Assessment of MIST Alert for ASP Diirin/; Routine Monitoring

Shellfish samples (500) from the 2001 Scottish shellfish-
monitoring progratn were extracted in aqueous methanol. |50:50
(v/v) water: methanol] according to the extraction procedure of
Quilliam et al. ( 1995) and as detailed in Hess et al. (2001 ). Extracts
were concurrently analyzed by MIST Alert for ASP (Jellelt Biotek
Ltd.. Nova Scotia. Canada) and HPLC (Quilliam et al. 1995).
MIST Alert for ASP is peiformed as for MIST Alert for PSP. but
samples are only considered positive if the "T" line is completely
absent or appears only as a faint shadow (i.e., less than 25'7r) (Fig.
I). The qualitative presence of ASP toxins by the test kits was
compared with the quantitative analysis of these toxins (DA -i- epi
DA = total amnesic shellfish toxins; AST) by HPLC categorized
into five groups: not detected, the limit of detection (lod) that is
about 2.5 |j.g AST g"' shellfish flesh. >lod - <6. 6 - <20. and
>20 (xg AST g~' .shellfish tlesh.

Reproducibility of MIST Alert for ASP

Fifty aqueous methanol extiacts obtained fioni a variety of
different shellfish species (mus.sels. Mytilus echdis: cockles. Ceras-
idilenna ediile: queen scallops, Aqidpecten openidaris: Pacific
oyster. Crassoslrca fiificis: king scallop. Pecten iini.xiiuus — whole
animals, gonad, adductor, residual tissue — gut, hepatopancreas,
gills) were tested in triplicate using MIST Alert for ASP to inves-
tigate the reproducibility of the test kits. Extracts contained vary-
ing concentrations of AST, as determined by HPLC, and were
chosen to contain levels within the five categories above.

RESULTS

Use of MIST Alerl for PSP in Routine Shellfish Monitoring

Results from 547 shellfish extiacts tested by MIST Alert for
PSP were compaied with results obtained using the MBA (Table I .
Fig. 2). All extracts (/; = 77) deteiniined to contain toxin coiicen-
tiations above the legulatory limit (SO |jLg SIX eq 100 g"') by the
MBA were also found to be positive for PSP toxins by MIST Alert
for PSP. In addition, test kits gave a positive result for 91% (131/
135) of samples in which the MBA detected toxin below the regu-
latory limit (grouped as 40 - <80 and <4() p.g ST.X eq lOOg ' in
Fig. 2). The kit did not detect toxin in two mussel extracts (Mytilus
c'diilis). a king scallop gonad (Pecten maximiis). and a European
oyster (Ostrea edidi.s) exti'act, the mussel extracts both being in the
<40 (Xg STX eq 100 g"' category.

The MBA did not detect toxin in 335 shellfish extracts. Using
MIST Alert for PSP. 97 of these samples gave a positive result
(29',r) giving an overall "false-positive"" rate of I8'7r. Of these
samples, king scallop extracts (both gonad and whole animal) gave
a higher proportion of MIST Alert PSP positive results (35 and
53'/r, respectively) than mussel extracts (25%), the most com-
monly tested species in the PSP to\in monitoring program.

Field Trials of MIST Alert for PSP

Results from 259 shellfish extracts from eight field trial par-
ticipants are shown in Table 2. In general, participants and FRS
agreed in the positive or negative interpretation of the test kit
results. In one case. Participant 8 recorded a negative result, but
FRS considered it positive. A positive result was confirmed when
this extract was re-analyzed by MIST Alert for PSP at FRS. How-
ever, when further tested by the MBA. toxin was not detected. All
but one Participant recorded positive results when using the kit.

Detection of PSP and ASP Toxins by MIST Airrt™

457

TABLE 1.
Comparison of MIST Alert for the detection of PSP in shellfish extracts with the MBA.

No.

MBA

MIST

MBA

MIST

MBA

MIST

MBA

MIST

Tissue Type

Tested

gSO"

+

-

40 - <80

+

-

<4(l

+

-

ND''

+

-

Pecteit maximus (G)^

115

14

14

(1

->-)

21

1

1')

14

0

60

21

39

Peclen maximus {V^f

126

37

37

U

39

39

0

12

12

0

38

20

18

Mvtilus edulis

239

25

25

0

20

20

0

15

13

T

179

44

135

Ostrea edulis

6

0

1

0

1

0

5

0

5

Crassostrea gigas

27

0

0

0

27

1

26

Acjuipeclen operciikiris

16

0

1

1

0

1

T

0

13

6

7

Cenisknlerma edule

9

1

1

0

1

1

0

0

7

~t

5

Ensis. sp.

9

0

0

3

3

0

6

3

3

Total

547

77

77

0

84

82

2

51

49

-)

335

97

238

% compliance

lOO'J

9i.m

96. 1 %

71.0'*

■'MBA ((j,g STX cq 100 j
'' ND. not detected.
'"" G, gonad.
'' W. whole animal.

Only one sample was found to contain levels over the regulatory
limit when analyzed by the MBA.

Iiilerpretation of "T" Line Intensity

The majority of disagreement in results between participants
and FRS were attributable to differences in "T" line intensity
rather than whether a sample was considered positive or negative
(Table 2). Participant 2 seemed to misinterpret kit instructions and
in sotne cases, read the "C" line as a percentage of the "T"' line.
Despite this, positive and negative samples were correctly identi-
fied. Some trial participants attempted to read the kit too precisely;
for example, rather than scoring the "T" line as 0, 25, 50, or 100%
(see Fig. I ), results were reported as 5. 15, 30%. or so forth. Much
of the difference in interpretation between FRS and Participants 6
and 7 was attributable to this "overinterpretation." but results were
still correctly read as positive or negative.

Reanalysis of Shellfish Extracts

When shellfish e.xtracts were retested at FRS. there was over
90% agreement with the positive or negative result previously
obtained by Participants (Table 2). On four occasions when FRS
reanalyzed extracts (from Participants 3. 6, and 8) by MIST Alert
for PSP. the result changed from negative (on-site) to positive (at
FRSl. In one e.xtract from Participant 3, (a king scallop gonad) a
low level of toxicity (3l(xg STX eq 100 g"') was detected by the
MBA. In the extract from Participant 8, the MBA gave a negative
result, while insufficient extract volume was available for further
MBA testing from Participant 6, but when diluted and tested by
MIST Alert for PSP, the result was considered negative. Two
extracts from Participant 7 changed from a positive to negative
score when reanalyzed by the kit, but because the "T" line intensity
was close to 50% (see Fig. 1 ), the response was considered diffi-
cult to interpret.

Previous studies using HPLC showed that PSP toxin profiles of
stored Association of Official Analytical Chemists (AOAC) acidic
shellfish extracts do not change significantly over a five-day pe-
riod (data not shown). It was, therefore, considered that during

transportation of shellfish extracts from participants to FRS toxin
levels remained unchanged.

Use of MIST Alert for ASP in Routine Shellfish Monitoring

MIST Alert for ASP was used to test 500 shellfish extracts, and
the results were compared with the HPLC analysis routinely used
in the ASP toxin shellfish-monitoring program (Table 3 and Fig.
3). All samples over the regulatory limit (20 fjig AST g~'), as
determined by HPLC, were scored positive by MIST Alert for ASP
in = 162). In samples in = 1 14) that were below the regulatory
limit but above the HPLC lod (2.5 |jig AST g"' ), MIST Alert for
ASP detected toxin in 93 (81.5%). The kit also detected toxin in
10.6% (13/123) of samples at the HPLC lod and a further sample
(king scallop gonad) that was negative by HPLC. Of 39 samples
failing into the range >lod - <6 [x-g AST g"' 24 yielded a positive
result by the kit (61.5%). Of the commonly tested species, mussels
yielded the fewest positive results at the lod.

Reproducibility of MIST Alert for ASP

Overall, 90% of samples tested gave three identical "T" line
responses (Table 4). In samples wherein toxin was not detected, at

^^B Peclen mammus - Gonad
I 1 Peclen maximus ■ Whole animal
" ?! tr, Eu3MyMus edulis
c I i Other species

Toxicrty (pg STX eq 100 g ')

Figure 2, Percentage agreement between the MBA and MIST Alert
for PSP results for shellfish as determined by the MBA.

458

Mackintosh and Smith

TABLE 2.
Summary of results obtained using MIST Alert for the detection of PSP toxins in shellfish by field trial participants and FRS.

No.

Kits

Participant I sed

No. Samples

Sho«ini; Disagrt'cmt-nt

in +VC/-M' lntt'rpretatl(tn

Between FRS and

Participants

No. Samples

Shnwinji Disaureement

In "I" Line hiterpretation

BelHeen FRS and

Participants

I

13

15

74

4

45

0

5

20

0

6

34

0

50

14

0
1
9

16

No. Samples Siiowing

Disagreement in +ve/-ve

Interpretation Between

FRS and Participants

when Reanalyzed

by MIST at FRS

No. Samples Showinj;
l>isaj;reement in "T" Fine
Interpretation Bet'.seen
F R,S and Participants
Wlien Reana!>/.ed by
MIST at FRS

■' W. whole animal: G. gonad; R, residual tissue; A, adductor muscle.
" NA, Not apphcable.

0

No.

Positive

Tissue Type of

Samples

Positive Samples"

7

Pn un iiutAUims

IW, G. R)

3

Pecten maximus

(G, Wl

9

PfLlcn maximus

(W, G)

0

NA"

1

Pecten maximus (W)

10

Pecten maximus

(W. G. Ai

14

Mxtilus cdulis. Ensis

spp.. Aquipecten

opercularis.

s

.\f\nlus I'lhilis

the lod or >20 |xg AST g^' by HPLC. tlie kit gave three identical
responses in agreement with HPLC results. Eight extracts in the
range >l()d - <6 \xg AST g' ' gave three identical responses — two
negative and six positive. The remaining two samples each gave
two identical responses — one, two negatives, and a positive; and
the other two positives and a negative. Ten samples in the 6 - <20
jxg AST g"' category all gave three positive results, but only seven
samples gave three identical results (Table 4).

DISCUSSION

MIST Alert for PSP in Roiiline Monitoring

Using MIST Alert for PSP, a positive result was recorded for
all shellfish extracts, from a wide range of tissue types, known to
contain >80 |ji.g STX eq 100 g"' as determined by the MBA.
indicating that shellfish considered a threat to public health would
be detected by this method during routine PSP toxin monitoring in

Scotland (Table I. Fig. 2). In addition, approximately 97^^ of
samples containing toxins below the regulatory limit (by the
MBA) were also found to be positive by MIST Alert for PSP.
suggesting the kit has a similar sensitivity to the MBA (about 33
|jLg STX eq 100 g"'). an important factor when considering its
potential use. MIST Alert for PSP failed to detect toxin in four
samples in which the MBA detected toxin below the regulatory
limit (33. 38. 47. and 60 |xg STX eq lOOg-'). MIST Alert for PSP
detects the amount of toxin based on the ability of the antibody
mixture on the test kits to detect different STX analogs. It has been
reported that the test kit is less sensitive to neo-STX derivatives
and requires levels close to the regulatory limit to give a positive
result (Laycock et al. 2001).

Differing sensitivities of MIST Alert for PSP to different toxin
profiles may also account for the observed "false- positive" results
(i.e.. MIST Alert recorded a positive result; whereas, the MBA
indicated a negative result) observed in this study. For example.

TABLE 3.
Comparison of MIST Alert for the detection of ASP in shellfish extracts with HPLC.

MIST

MIST

MIST

MIST

MIST

No.
Tested

HPLC

20

HPLC

6-<20

HPLC
>lod - <6

HPLC

HPLC

Tissue Type

+

-

+

-

+

-

lod

+

-

ND"

+

-

Pecten maximus (G)*'

121

18

18

55

50

5

18

12

6

23

6

17

7

1

6

Pecten maximus (R)^

64

54

.'^4

5

5

0

T

1

0

3

0

3

0

Pecten maximus (W)''

KM

86

86

13

12

1

1

1

0

1

0

1

0

Pecten maximus (A)"

3(1

1

1

1

1

0

11

8

3

18

1

16

5

0

5

Mytilus edulis

12S

3

3

1

1

0

5

0

5

54

2

52

65

0

6.S

Aquipecten opercularis

17

0

0

1

1

0

12

3

9

4

0

4

Ostrea edulis

12

0

0

0

5

0

5

7

0

7

Crassostrea gigas

14

0

0

0

4

0

4

10

0

10

Cerastoilerma edide

7

0

0

1

(1

1

3

0

3

3

0

3

Total

500

162

162

0

75

69

6

39

24

15

123

13

110

101

1

100

% compliance

1009;

92^*

61.5%

10.6'7r

99.0<7c

' ND, Not detected; ''G, gonad; '•R, residual tis.sue; ""W, Whole animals; "A, adductor muscle.

Detection of PSP and ASP Toxins by MIST Alert^^'

459

TABLE 4.

Summary of the reproducibililv (>f triplicate analyses of shellfish
extracts using MIST for ASP.

Percentage of Number of Percentage of

HPIX' Samples Giving Samples Giving Samples Giving

(pg AST g"' Three identical Three Identical Two Identical
Shellfish Flesh) Responses Responses Responses

ND'

lUU

10/10

lod"

100

10/10

>Iog - <6

80

8/10 (6 positive.
2 negative)

20

6-<20

70

7/10

30

320

100

10/10

Total

90

45/50

10

' ND. Not detected.

'' led. limit of detection (about 2.5 |jLg AST g"').

extracts containing high levels of low-toxicity toxins (e.g.. some C
toxins) (Oshima 1995) may yield a negative result by the MBA but
a positive result by MIST Alert for PSP (Laycock et al. 2001 ). This
may be considered an advantage of the kit. because these toxins
can be readily converted to other more potent PSP toxins during
shellfish consumption. Shortcomings of the MBA must be taken
into account when considering inconsistent results. The MBA is
known to underestimate toxicity with an associated error of 207c
{b09c near the detection limit) (Park et al. 1986).

Use of MIST Alert for PSP in the Field

Results from data obtained by shellfish farmers, processors,
and EHOs show very little difference in the interpretation of posi-
tive or negative results obtained by these lay persons with less
experience of the test kits than staff at the routine monitoring
laboratory (FRS). Similarly, in-house (FRS) and on-site (Partici-
pants) results from shellfish extracts were, for the most part, com-
parable (Table 2). In this study, variability in the interpretation of
the "T" line response was observed. Previous trials (Mackintosh et
al. 2002) also demonstrated variation in interpreting the "T" line
response of MIST Alert for PSP by novice users. This trial sug-
gests that this may not be a serious problem, because positive and
negative samples were still correctly identified, and the manufac-

^^Mpecfen maxfmus - Gonad
i///lPeclen maumus - Residual

Pecten maximus - Whole animal

I 1 PprtRn wattmus - Adductor muscle

LLJJJ Myri'us edu/is

Other species

Toxicity (pg AST g^)

Figure 3. Percentage of samples found to be positive by MIST Alert
for ASP when compared with HPIX' results.

turer advises that if doubt exists because of "T" line intensity, a
positive result should be recorded. It is also considered that this
variable will be reduced with more extensive use of the kit.

Importantly, an extract determined to contain over 80 jxg STX
eq 100 g"' by the MBA was correctly identified by a participant.
A further positive extract that was not retested by the MBA (at-
tiibutable to insufficient sample volume) was diluted 1:1 in a nega-
tive shellfish extract and retested using MIST Alert at FRS. This
extract produced a negative response, suggesting toxicity in this
saiTiple was <80 jxg STX eq 100 g"'. although this would have
been dependent on the toxin profile of the extract.

Overall, even when considering inisinterpretation of instruc-
tions (e.g.. recording of the "T" line intensity), participants cor-
rectly identified positive and negative samples. Previously, we
have shown that novice users can make ertors in both use and
interpretation (Mackintosh et al. 2002). emphasizing that careful
attention to the instructions and interpretation of the results is
essential.

MIST Alert for ASP in Routine Monitoring

MIST Alert for ASP detected toxin in all samples containing
the regulatory limit of ASP toxin (20 jjig AST g"') and above, with
good reproducibility observed between test kits (Table 3, Table 4,
Fig. 3). Toxin was also detected in a high percentage (82%) of
extracts containing between the regulatory limit and the lod of the
HPLC method used. The manufacturer of MIST Alert for ASP
reports a detection limit of between 8 and 12 p,g AST g" (Jellett
et al. in press); our results show that the kit will detect toxin at
lower levels, particularly in king scallops. In Scotland, the con-
tamination of king scallops with ASP toxins has adversely affected
the offshore scallop fishery (Campbell et al. 2001, Gallacher et al.
2001); whereas, other shellfish species remain relatively unaf-
fected. Results from routine monitoring of Scottish king scallops
demonstrate that the proportion of scallops that currently do not
contain ASP toxin as determined by HPLC is about 30% (J. Petrie
pers. comm.). This implies that the majority of scallop extracts
tested by MIST Alert for ASP will give a positive result, regardless
of whether the scallops contain toxin below the regulatory limit,
and would still be marketable.

Overall, results from this study indicate that MIST Alert for
ASP is currently too sensitive to screen out low- toxicity king
scallop samples in routine monitoring programs, but it inay be
suitable for other shellfish species (e.g.. mussels). However, the
requirement for an alternative method to the current HPLC tech-
nique for ASP toxin detection in shellfish is not considered para-
mount, because the ethical concerns surrounding the MBA do not
need to be considered. It has been reported that MIST Alert for
ASP and PSP can be pertbrmed using the same extract; that is,
AGAC extract (sample homogenate extracted in O.IN HCl). Be-
fore this study, AOAC acidic extracts were examined by MIST
Alert for ASP and PSP (data not shown). It was found that, unless
these extracts were used immediately, they were unsuitable for
ASP testing because of the instability of DA in HCl, suggesting a
single extraction protocol would not be feasible. In addition, acidic
extracts are unsuitable for HPLC analysis if further investigation
were required.

CONCLUSION

In conclusion, the MIST Alert for PSP has been successfully
used dunng two monitoring seasons (Mackintosh et al. 2002) to

460

Mackintosh and Smith

determine the presence/absence of PSP toxins in Scottish shellfish.
The test Ivits confirmed the presence of PSP toxins in samples
considered to contain toxin at and above the regulatory limit (80
|a.g STX eq 100 g"'). MIST Alert for PSP could be used in moni-
toring programs as a screen to eliminate negative and low PSP
toxicity samples before use of the MBA for quantitative analysis of
positive samples. For example, if MIST Alert for PSP had been
used for this purpose during the study period in 2001. there would
have been a reduction of 439f of the MBAs. Funhermore. field
trial results indicate that the PSP kit has the potential to be used by
shellfish farmers as a shellfish harvest management tool and by
EHOs and processors in end product testing.

Results suggest that MIST Alert for ASP is too sensitive to use
as a screen in the Scottish ASP toxin-monitoring program and in

end-product testing for king scallops. A higher detection limit
would be desirable to reduce the high number of MIST Alert for
ASP positives observed at toxin levels <6 [Lg AST g"' by HPLC.
In contrast, it is possible that the kit may be used as a screen for
ASP toxins in other shellfish species and this should be studied
further.

ACKNOWLEDGMENTS

The authors thank Susan Gallacher for extensive advice. God-
frey Howard. John Turriff. Joyce Petrie. Margaret McCann. Nigel
Brown, and Alasdair Scott for the pro\ ision of samples and tech-
nical assistance. We also thank Joanne Jellett for advice and in-
formation. This work was funded by the Food Standards Agency,
UK. Grant No. B04006.

LITERATURE CITED

Campbell. D. A.. M. S. Kelly. M. Busman. C. J. Bulch. E. Wiggins. P. D.
R. Mcieller. L. Morton. P. Hess & S. E. Shumway 2001. Amnesic
shellfish poisoning in the king scallop. Peclen imiximiis. from the west
coast of Scotland. / Shellfish Res. 20:7,s-84.

Gallacher. S.. G. Howard. P. Hess. E. Macdonald. M. C. Kelly. L. A. Bates.
N. Brown. M. MacKen/ie. P. Gillibrand & W. R. Turrell. 2001. The
occurrence of amnesic shellfish poisons in shellfish from Scottish wa-
ters. In: G. M. Hallegraeff. S. I. Blackburn. C. J. Bolch & R. J. Lewis,
editors. Harmful algal blooms 2000. Inlergovemmental Oceanographic
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Journal of Shellfish Rcsennh. Vol. 21. No. 2. 461-463. 2002.

DIARRHETIC SHELLFISH POISONING ASSOCIATED WITH PROROCENTRUM LIMA
(DINOPHYCEAE) IN PATAGONIAN GULFS (ARGENTINA)

ANA MARIA GAYOSO,' * STACIE DOVER," STEVE MORTON,^ MARK BUSMAN.'
PETER MOELLER.- VANESA K. FULCO,' AND LUCIE MARANDA'

'Ceiitro Niicional Patagonico, Consejo Nacional de Investigaciones Cienti'ficas \ Tecnicas. B. Brown
s/n, 9120 Puerto Madryn. Chubut, Argentina: 'Marine Biotoxin Program USDOC/NOAA, 219 Fort
Johnson Road, Charleston. South Carolina 29412-91 10: Graduate School of Oceanography. University
of Rhode Island. Narragansett. Rhode Island 02882-1 1 97

ABSTRACT A serious diarrhetic shellfish poisoning (DSP) intoxication caused by the consumption of mussels harvested in the Gulfs
of San Jose and Nuevo. Patagonia, Argentina occurred in March (autumn) 1999. This was the first observation of DSP toxins along
the Argentine coast. Cells of the dinoflagellate Prorocemrum lima (Ehrenberg) Dodge were found in water samples, as were epiphytes
upon macroalgae and in the stomach contents of the mussels Aidacomya aim (Molina) and Mytilus ediilis platensis (d'Orbigny).
Extracts from both mussel species were positive for DSP-like activity using the nuorimetric phosphatase inhibition assay. When the
extracts were analyzed using liquid chromatography and tandem mass spectrometry, peaks of okadaic acid or dinophysisloxin I
(DTX-1 ) were not observed in the nonhydrolized samples. DTX-1 was detected in hydrolized extracts of Mytilus MdAulaLomya. The
Myiilus extract contained 21.2 ng DTX-1 per gram of the whole tissue: the Aulacomya extract contained 94.0 ng DTX-1 per gram.

KEY WORDS: Argentina; diarrhetic shellfish poisoning; okadaic acid: Prorocentrum lima, toxic dinoflagellate

INTRODUCTION

Harmful algal blooms have increased in geographic distribu-
tion, magnitude, and frequency during the last decades (Anderson
1989: Smayda 1990; Hallegraeff 1993). Diarrhetic shellfish poi-
soning (DSP), a severe gastrointestinal disturbance following the
consumption of shellfish, is also an increasing problem. DSP re-
sults from the accumulation, in shellfish tissues, of polyether tox-
ins produced by dinoflagellates belonging to the genera Dinophx-
sis and Prorocentrum (Wright & Cembella 1998 and references
therein). In his 1993 review, Hallegraeff recognized only the coast
of Chile as being affected by DSP in South America. Fenari et al.
(1993) and Mendez and Ferrari (1994) later reported DSP inci-
dents occurring in Uruguay. Proencj'a and Rorig (1995) and
Proeni;a et al. (1998) documented DSP occurrences in southern
Brazil. To date, only species of Dinopliysis have been the causative
agents of DSP cases reported from South America (Lembeye et al.
1993, Lembeye et al. 1996. Proen^a et al. 1998, Uribe et al. 2001 ).

Since 1995, oceanographic research along the North-
Patagonian coast of Argentina has included phytoplankton inves-
tigation, microscopical observations of stomach content of mus-
sels, and analysis of physical and meteorological data. Because of
recurrent blooms of another toxic dinoflagellate, Alexandrium
tamarense, in the area (Esteves et al. 1992, Caireto et al. 1998,
Gayoso 2001), monitoring programs are conducted only for para-
lytic shellfish poisoning toxins in shellfish (mouse bioassay ) by the
local (Chubut) government, who is responsible tor imposing bans
on shellfish harvest. Although the known DSP producers, Dino-
pliysis acuminata and Prorocentrum lima, are present (Esteves et
al. 1992, Santinelli et al. 1994, Gayoso 2001), monitoring pro-
grams for DSP are not currently conducted in Argentina.

On March 20. 1999. shellfish harvested in the Gulfs of San Jose
and Nuevo (Chubut) were served at a social event despite voiced
concerns from one of us (A.M.G.) that toxic dinoflagellates other
than Alexandrium tamarense were potentially present in the water
column and in the shellfish digestive glands. More than 40 people

*Corresponding author. E-mail: gayosolScenpat.edu.ar

suffered from gastrointestinal disorders after consuming the shell-
fish. Physicians from the Dr. Andres Isola Public Hospital reported
symptoms of diarrhea, nausea, and abdominal pain, consistent with
the DSP syndrome. After the incident, samples were collected
from the two north-patagonian gulfs for phytoplankton and epi-
phytic dinoflagellate identification, chemical analysis of mussel
tissues, and microscopic examination of the stomach content of
mussels was undertaken to document the causative toxin(s) and the
responsible organisms. This work reports on the first recognized
episode of diarrhetic shellfish poisoning on the Argentine coast.

MATERIALS AND METHODS

Water samples at two depths (surface and bottom) with a 2.5 L
Van Dom bottle and vertical and oblique hauls with a 25 (j.m mesh
plankton net were taken at two stations: Puerto Madryn. situated
on the west coast of Golfo Nuevo (42°46'S, 65°02'W) and San
Roman, located on the north coast of Golfo San jose (42M5'S,
64'15'W). Dominant macroalgae and mussels [Aulacomya aira
and Mytilus edulis platensis) were manually collected from the
bottom (ca. 18 m depth) at each station by scuba divers. The
samples were collected twice a month from March to June 1999.

The identification of species was made using the net samples,
a light microscope provided by Noniarsky illumination, and a
scanning electron microscope. For quantitative analysis, sub-
samples (from the bottle samples) were settled in a chamber from
a cylinder of 50 mL and counted with an inverted microscope. The
species of macroalgae were analyzed separately, subsamples (2-6
g wet weight) were placed in a tube containing a solution of
formaldehide-filtered seawater, the suspended epiphytic di-
noflagellate populations were examined and their abundance, ex-
pressed as cells per gram wet weight of macroalga, was estimated
from counts using a 1 niL Sedwick Rafter chamber. The mussel
stomach contents were analized within 4 h of collection, the stom-
ach content of at least six mussels was extracted with a Pasteur
pipette, the contents were pooled, diluted with filtered seawater,
and observed on a slide using a coinpound light microscope. Mi-
croalgae present were identified and their relative abundance was
estimated.

461

462

Gayoso et al.

Toxin Analysis

Toxin analysis was performed on the following two mussel
samples: (1) Aulacomya atra, which were collected in Golfo
Nuevo on March 18, 1999; and (2) Mytihis edulis platensis. which
were collected in Golfo San Jose on March 25. 1999. Both were
frozen whole until the time of analysis.

Sample Extraction

For each species, 5 g wet weight of whole mussels were ho-
mogenized in 80% methanol tor 2 min using a Polytron. The
resulting extract was filtered through a GHB glass fiber filter and
the filtrate brought to a volume of 5 mL. To convert all metabolites
to the parent okadaic acid (OA) or dinophysistoxin-1 (DTX-l ). 2.5
mL of each sample were hydroly/ed by heating at 76''C for 40 min
after addition of 250 jxL of 2.5 N NaOH (Mountfort et al. 1999).
Therefore, for each species two separate samples were analyzed
for DSP-toxins.

Protein Phosphatase Inhibition Assay

The prolciii phosphatase inhibition assay was performed in a 96
well format using the procedure of Vieytes et al. ( 1997). The assay
tests the ability of OA standard or unknown sample to inhibit
activity of purified protein phosphatase 2A against a niiorimetric
substrate, 6.8-difluro-4-methylum belliferyl phosphate (DiFMUP).
All samples were diluted in reaction buffer (50 iiiM Tris HCl pH
7.0, 0.1 mM CaCU) at least 4-fold to reduce the methanol con-
centration to s5% in the assay. This step was to eliminate inhi-
bition of the enzyme by methanol. For the assay, 10 p.L of a 1 : 1
dilution of 40 mM NiCl, and 1 mL/mL bovine serum albumin. 17
(J.L of reaction buffer. 33 p-L samples and standards (0.03-10 nM
OA; NRC, Ottawa Canada) and 200 (jlL of purified PP2A enzyme
(Upstate Biotechnology, Lake Placid, NY) were added to duplicate
wells of a 96 well plate (Costar, Corning, NY). To start the reac-
tion, 120 m DiFMUP (10 mM; Sigma, St. Louis, MO) was then
added to each well and the reaction allowed to proceed for 1.5 h at
37"C, followed by 30 min at 4°C. Protein phosphatase activity was
determined by fluorescence (360 nni excitation; 460 nm emission)
in the wells using a tluorimetric plate reader (Fluoslar. BMG Labo-
ratory Technologies. Durham, NC). OA-like activity in the sample
was quantified relative to the standard curve. The detection limit of
the fluorimetric phosphatase inhibition assay was approximately 1
X 10"'" M okadaic acid equivalents.

Liquid Chromatiinraphy and Tandem Mass Spectromelry (LC-MS/MS)

Samples that displayed protein phosphatase inhibition activity
were analyzed by LC-MS/MS using a Finnigan LCQ mass spec-
trometer. The methanolic extracts were injected on a C18 column
(Zorbax 2.1 x 150 mm) and eluted with a gradient of 50 to 95%
methanol/water containing 0. 1% TFA at a How rate of 0.2 niL/min.
A splitter device was used to direct 10% of the column effluent to
the electrospray source. The mass spectrometer was operated in
positive ion mode. Toxins were analyzed by trapping |M-t-Nal-i-
species for each toxin and conducting selected ion monitoring
experiments for distinctive fragment ions from the collisionally
activated dissociation of the trapped parent ions. Chromatographic
traces were acquired for the detection of the fragment ions and
undissociated parent ions. The limits of OA and DTX-l detection
were approximately I x 10"' and 1 x 10"'* M. respectively, as
measured by LC-MS/MS.

RESULTS

P. lima (Fig. 1) was unequivocally identified in net samples
from the gulfs of San Jose and Nuevo. The two main thecal plates
(valves) of the specimens found were obovate (length 33-38 jjim,
width 22-24.2 (j.m, n = 10) and cellular morphologic features
agreed with the descriptions given by Faust ( 1991 ) and McLachlan
et al. (1997, as Exuviaella lima). A row of conspicuous marginal
pores, scattered valve pores, and valve center free of pores were
observed. The species was registered in water samples (cell den-
sities less than 100 cells L"') on March 18. April 13 and June 29.
1999 ( 16.5-18 'C). Its occurrence coincided with the autiunn phy-
toplankton peak in which Rhizosolenia setigera Brightwell.
Chaetoceros citrvisetiis Cleve. C. diadema (Ehrenberg) Gran, C.
didymus Ehrenberg, and Pseudo-nilzschia pimgens (Grunow ex
Cleve) Hasle were most abundant, and A. tamarensc (Lebour)
Balech was also present. Dominant macroalgae in the gulfs San
Jose and Nuevo, Dictyota dichotoma (Hudson) Lamouroux
(Phaeophyceae),/4/iornc/H'i/m/i//r<»/«/»/)( (J. Agardh) Baldock. Ce-
ramiiim rubnim (Hudson) C. Agardh (Rhodophyceae). and Ulva
rigida (C. Agardh) Thuret (Chlorophyceae) showed an epiphytic
assemblage of diatoms and dinotlagellates that included P. lima.
Cell densities of P. lima varied from approximately 750 cells per
g wet weight of macroalga at station San Roman to 1 782 cells per
g wet weight at station Puerto Madryn.

Intact cells (with protoplasm) of P. lima were observed in the
stomach contents of the mussels on March 18, May 18, and June
29. 1999. During the study period, the diet of A. arra and M. edulis
platcii.si.s consisted mainly of diatoms, with benthic forms outnum-
bering pelagic species. Prorocentniin micans. benthic pennate dia-
toms. Puralia sulcata, and Thalassiosira spp. were the most abun-
dant organisms found in the mussel .stomachs.

Extracts of both mussel species were positive for DSP-like
activity using the tluorimetric phosphatase inhibition assay. The
Mylilus sample contained 33.5 ng of OA-equivalent activity per
gram of whole tissue, whereas the Aulacomya sample contained
150 ng/g. The difference between hydrolized and nonhydroli/ed
samples using this assay were not significant.

Extracts of the nonhydrolized samples analyzed using LC-MS/
MS did not exhibit peaks corresponding to okadaic acid or dino-

Figure 1.

DSP IN Argentina

463

physistoxin-1. However, dinophysistoxiii-1 was detected in the
Mxtiliis and Aidaaniiya extracts that hud been hydrolized. yielding
21.2 ng DTX-l/g whole tissue and 94.0 ng/g. respectively.

DISCUSSION

Dinoplivsis acuminata is a frequent component of the phy-
toplankton in the area; its abundance can reach up to 1 .9 x 10' cells
L"' (Gayoso 2001 ). However, no known gastrointestinal disorders
have accompanied its presence. The episode of diarrhea following
shellfish consumption observed in March 1999 appears to be re-
lated to the presence off. lima. It is an important component of the
epiphytic community associated with the dominant macroalgae in
the gulfs. The species was also present in the mussel stomach
contents and in the water samples.

Not only was P. lima present in mussel digestive glands, but
DSP toxin activity was also detected in M. edidis platensis and A.
alia, strengthening the causal relationship between P. lima and the
gastrointestinal disorders of March 1999. If toxin regulations such
as those implemented in the European Union had been in effect,
the shellfish harvest would have been halted; most EU countries
tolerate no DSP toxins above detection levels (Shumway et al. 1995).

LC-MS, a highly selective and sensitive method for detection

of DSP toxins (Quilliam & Wright 1995), showed a peak corre-
sponding to DTX- 1 in hydrolyzed samples of M. edulis platensis
and A. alia, whereas neither OA nor dinophysistoxin was observed
in nonhydrolyzed extracts. This finding is indicative of the esteri-
fied nature of DTX-1 within the mussels; esterified DSP toxins
(also called DTX-3 complex! are acyl derivatives that also possess
toxic activity (Quilliam & Wright 1995). Upon sample hydrolysis,
the esterified toxin converts to the active parent compound. Es-
terified DTX- 1 is found to be the main DSP toxin in two other
shellfish, the scallop Patinopecten yessoensis and the mussel Myti-
lus fialliiprovincialis (Suzuki & Mitsuya 2001 ). A significant dif-
ference was found in the toxin concentration of the two hydrolyzed
samples: 21.2 ng DTX-l/g of whole Mytiliis tissues versus 94.0
ng/g for Aulacomya. This finding may be explained by the time of
collection (1 week apart for the two species), different specific
feeding and digestion behavior, and location of the two populations.

ACKNOWLEDGMENTS

The authors thank M.L. Piriz for the taxonomical identification
of macroalgae. This study was funded by the Agenda de Pro-
mocion CientiTica y Tecnologica, Repiiblica Argentina, PICT/98
010.^709 and supported by funds from NOAA National Ocean
Service.

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Journal of Shellfish Research. Vol. 21. No. 2. 465-469. 2002.

REDUCING NEUROTOXIC SHELLFISH POISON (NSP) IN PACIFIC OYSTERS (CRASSOSTREA
GIGAS) TO LEVELS BELOW 20 MOUSE UNITS • 100 G"'

GRAHAM C. FLETCHER,' BRENDA E. HAY," AND MARGARET F. SCOTT'

'New Zealand Institute foi' Crop & Food Research Limited. Private Ba.ti 92169. Auckland. New
Zealand: 'AquaBio Consultants Ltd.. P.O. Box 560. Shortland St. Auckland. New Zealand: ^New
Zealand Institute for Crop & Food Research Limited. Private Bag 11030. Pahuerston North,
New Zealand

ABSTRACT Pacific oysters previously exposed to the algae Karenia hreve. which produces neurotoxic shellfish poisoning (NSP).
were held in experimental depuration tanks for up to 5 days prior to determmation of their NSP levels by mouse bioassay. Although
depuration reduced NSP levels in the oysters to around the regulatory limit (20 mouse units • 100 g"'). levels below the regulatory limit
could not be assured at temperatures of 1 5'C and 20=C and salinities of 24'tf and iA"c with the standard bioassay used. Detoxification
appeared less effective at 1 2 'C. The use of an in-line filter to remove free algae or ozone to eliminate algae and toxins did not enhance
detoxification. Use of a biological filter at I2°C in the tanks or feeding the oysters with Isochiysas galhana also did not assure NSP
levels below the regulatory limit. Exaniinafion of NSP distribution in the shellfish tissues revealed that during depuration, the
percentage of this lipid soluble toxin associated with the organs of the digestive system (gills and digestive viscera) decreased while
levels increased in high lipid tissues, such as the gonads. We postulate that NSP is difficult to eliminate from oysters because it is
incorporated into the lipid stores of the animal.

KEY WORDS: neuriitoxic shellfish poison. Pacific oyster. Cra.s.'io.sirea liii^as. detoxification

INTRODUCTION

Although the uptake of algal toxins by bivalve shellfish is a
problem worldwide for .shellfish producers, to date there have been
very few successful strategies to mitigate the effects of toxic algal
blooms. Current controls rely on growers monitoring toxin levels
in shellfish and ceasing harvesting when levels exceed prescribed
limits. This is in contrast to the situation when shellfish are ex-
posed to bacterial pathogens. In this case growers may have the
options of either relaying (moving shellfish to unaffected areas and
harvesting again when the shellfish have naturally purified them-
selves after a period of weeks) or processing the shelltlsh in land-
based depuration tanks where they will be cleared from bacterial
pathogens within a few days (Roderick 1994). Similar strategies
for the detoxification of shellfish from algal toxins are not com-
mercially available.

The uptake and elimination of three of the four main algal toxin
groups affecting shellfish have been investigated in laboratory
studies: paralytic shellfish poison (PSP) (Bricelj & Shumway
1998), diarrhetic shellfish poison (DSP) (Cembella et al. 1998) and
amnesic shellfish poison (ASP) (Wohlgeschaffen et al. 1992.
Whyte et al. 1995). These studies suggest that land-based detoxi-
fication only appears to be feasible for ASP. Relaying to eliminate
toxins could be developed but further research is required and
there would be risks of introducing toxic algae to unaffected areas.
Little work has been i^eported on detoxification of shellfish af-
fected by the fourth toxin group, neurotoxic shellfish poisoning
(NSP). Despite promising work that showed that NSP toxins were
susceptible to oxidation in ozonated seawater (Schneider & Rod-
rick 1995) little work on the toxins within shelltlsh has been pub-
lished (Fletcher et al. 1998). NSP occurs when people eat shellfish
that have been subjected to dinotlagellate blooms composed of
Karenia (syn. Gymnodinium) species. These blooms commonly
occur in the Gulf of Mexico where the causative alga is Karenia
breve (syn. Gymnndiiiiiiin breve. Prychodiseiis brevis). One out-
break, caused by a K. /);cr(--like species, has been recorded in New
Zealand (Chang 1995). We have previously reported the results of
six experiments designed to determine whether Pacific oysters
{Crassostrea i^igas) might be successfully detoxified of NSP tox-

ins in a depuration system (Fletcher et al. 1998). We found that,
although mean NSP levels could be reduced to levels below the
regulatory limit of 20 mouse units • 100 g"', levels below this limit
could not be assured in individual tests using the regulatory
method (APHA 1970). This was regardless of whether ozone was
u.sed in the seawater. We now report on four experiments designed
to confirm the previous results, investigate why levels below 20
mouse units were not consistently achieved and to evaluate other
detoxification methods that may result in additional decreases in
NSP levels.

MATERIALS AND METHODS

K. hreve cells were grown in a medium derived from the GP
medium of Loeblich and Smith (1968). The original GP medium
contained 85% seawater, but because a higher percentage of sea-
water gave better growth rates for K. breve (unpublished data) we
modified the GP medium in this study to the following composi-
tion: seawater (I L), KNO, (200 mg in 2 mL H,0), vitamin so-
lution ( 1 mL), trace element solution (5 mL), and soil solution (5
niL). This medium was autoclaved (12rC. 15 min). and then
K,HP04 (8.7 mg in 4 mL autoclaved) was added aseptically. The
vitamin solution contained thiamin HCl ( 100 mg), vitamin B,, (0.1
mg). biotin (0.2 mg) and H^O ( 100 mL). The trace element solu-
tion contained Na.EDTA (6 g), FeClj.eH^O (0.29 g), H^BO, (6.85
g), MnCK.4H,0 (0.86 g). ZnCK (0.06 g), CoCl_..6H,0 (0.026 g).
and H,0 (I L) adjusted to pH 7.9 with NaOH. The soil solution
was the supernatant after filtering (Whatman No. 2 filter) a mixture
of soil ( 1 kg) and H,0 (2 L) that had been autoclaved ( 12rC, 60
min).

Oysters were supplied with A', breve cells at rates of 10 to 25
X 10" cells.oyster"' (Table 1 ) and left for 24 h to ingest the cells
and take up the NSP toxin, as described previously (Fletcher et al.
1998). This feeding regime gave initial toxin levels from 29.6 to
70.7 mouse units • 100 g"' (Table 2).

Four detoxification experiments (designated Experiinents 1 to
4) were carried out in the experimental tanks described previously
(Fletcher et al. 1998). Briefly the tanks consisted of two closed
systems: one of four 50 L tanks plumbed in parallel to a tempera-

466

Fletcher et al.

TABLE 1.
Experimental conditions and NSP levels for uptake and detoxification of NSP from Pacific oysters.

Oysters

Detoxification

Treatment

Uptake
A. breve

Detoxification Conditions

Experiment

Weight'

Lipid'

Water

Number

(S.E.r

(S.E.I

Start Date

Number

Supplied

Treatment

Temp.

Salinity

Filter

1

12.14 (0.22)

1.79 (O.OS)

3/4/97

U

25

U.V.

15

24

lb

10

u.v.

15

24

Ic

10

U.V.

15

24

2

15.91 (0.56)

2.18 (0.19)

4/10/97

1

10

Ozone

20

34

5

3

16.29 (0.74)

3.94 (0.11)

8/27/97

3a

18

U.V.

20

34

5

3b

18

Biofilter

12

34

Bio

4

10.81 (0.14)

3.17 (0.09)

10/20/97

4a

23

U.V.

12

34

4b

23

U.V.-"

12

34

4c

23

Biofilter

12

34

Bio

4d

0

U.V.

12

34

' Mean drained wet weight of soft tissues for all oysters in the experiment,

- (S.E.) — Figures in parentheses are the standard errors of the means.

^ Mean lipid levels for all oysters in the experiment expressed as a percentage of the drained wet weight of soft tissue.

■* Fed hochrysiis during the experiment (days 2 and 3).

ture controlled sump and the other of a single 50 L tank niaintained
at the same temperature. Both systems had in-line 5 \i.m cartridge
filters (FilterPure 5PW10. Contamination Control. Auckland) and
ultraviolet lamps (Steriflo 369P, Contamination Control, Auck-
land) available. Ozone could be supplied to tanks 1-4 by a corona
discharge generator (CDIOOO. Novozone. Auckland) using oxygen
as the source gas. When applied, water was ozonated using an
electronic controller to provide a reading of 350 ± 20 mv (redox
electrode MC241Pt. Radiometer) in the shellfish tanks, which con-
verts to an Eh of 549 mv using the correction factor of -t- 1 99 mv
applicable to the Ag/AgCI. KCI reference electrode (Vogel 1961 ).
Additionally, in Experiments 3b and 4c, oysters were placed in a
1000-L biofilter tank ( 12 ± 0.5°C) where recirculating water was
purified by passing it through a sand and shell-based biologically
active filter.

Ozone and ammonia levels in the seawater were measured
using Palintest Photometer 5000 methods (Anon. 1994) and salin-
ity was determined using a reft-actometer (Atagol. NSP levels in

the oysters were measured using the APHA method ( APHA 1970).
Each NSP test was carried out on at least 100 g drained meats from
10-12 oysters. One or two such pooled samples were tested from
each treatment on each testing occasion with the two samples
being taken from separate tanks when the treatment was spread
over more than one tank (details of timing and numbers of samples
taken are recorded in Fig. 1 ). The NSP method can detect toxin
levels down to about 10 mouse units • 100 g"', depending on the
size of mouse used ( 1 8-22 g). Where toxicity was observed but the
relevant mice did not die, the toxin level was scored at half the
minimum detectable level. Crude lipid levels were determined
from the weight of lipid extracted in the ether extraction during the
NSP extraction procedure.

The experimental conditions under which the oysters were
made toxic and maintained during depuration are presented in
Table I. The depuration conditions in Experiments I and 2 were
designed to complement the half factorial design used in previous
work (Fletcher et al. 1998), increasing the replication for each

TABLE 2.
Fitted exponential curves.

NSP Levels (m.u.

• 100 g-')

Parameters (standard errors)'

Regression Coefficient

Treatment

Start (S.E.)-

End (S.E.)'

a

b

r

(R')

la

60.3(21.0)

15.0 (3,2)

lb

62.5(11.9)

23,7(7.1)

Ic

70.7 (3.5)

24.5 (3.9)

la.b.c

22.97 (3.49)

41.52(6.04)

0.116(0.226)

0.715

2

54.4(1,2)

12.8 (1.4)

13.36(1.20)

41.04(2.45)

0.065 (0.080)

0.976

3a

29.6 (5.5)

12.0(0.5)

11.77(4.89)

17.78(6.24)

0.329 (2.408)

0.859

3b

29.6(5.5)

15.2 (3,2)

0,00(157,14)

29.73(156.45)

0.880 (0.813)

0.710

4a

51.9(0.02)

24.7 (6,3)

26,13(6,73)

25.19(10.62)

0.343 (0.414)

0,426

4b

51.9(0.02)

23,2 (4,4)

0,00(58,22)

48.57(55,71)

0.823 (0.299)

0.640

4c

51.9(0.02)

31,6(2.0)

31.95(2.54)

19.95 (4.76)

0.000(0.312)

0.787

' Parameters fitted to equation I: Y = « + b.r^ with the standard error of the parameter in parentheses.
- Mean NSP level of the 1-2 samples taken at the start of the experiment.
•' Mean NSP levels of all samples taken from Days 3. 4, and 5,

Reducing Neurotoxic Shellhsh Poison

467

Experiment 1

H 1 1-

Expertment 3

■^il'

^

Experiment 2
Gill

r=^ Palps ^ )
^^ Gonad \i_y

' Digestive

Experiment 4

i5?:=_§—

Detoxification Time (days)

Detoxitication Time (days)

Figure 1. Detoxification progress. Kach point represents one NSP
analysis of a pooled sample of 1(> to 12 oysters. Curves represent the
best fit to Kq. 1: V = a + b.r^. Pic diagrams represent the distribution
of NSP in various tissues (as in Table 3) at the indicated times.

dotted line = regulatory limit (20 mouse units • g"').

Experiment I: Oysters in experimental detoxification tanks

with 15 C seawater at 24'7f salinity disinfected with U.V. light, (a) D
toxin level 1, (b) A toxin level 2. (cl • toxin level 3.

Experiment 2: • Oysters in experimental detoxification

tanks with 20 C seawater at 34% salinity disinfected by ozone.
Experiment 3: Detoxification with U.V. (20°C) or Biofilter (12°C).

• (al Oysters in experimental detoxification tanks with 20''C

seawater of 34'/, salinity disinfected with U.V. light. V (b)

Oysters in biofilter tanks with 12 C seawater at 34% salinity.
Experiment 4: Detoxification with U.V., (12 C), feeding with ho-
chnsus or not. or biofilter ( 12 C). • (a) Oysters in experimen-
tal detoxification tanks with 12°C seawater of 34% disinfected with

V.X. light. O (b) Oysters in experimental detoxification tanks

with 12 C seawater of 34% disinfected with U.V. light fed with Iso-

chrystis galbana. V (c) Oysters in biofilter tanks with 12 C

seawater at 34% salinity. ♦ (d) Oysters with no initial toxin.

factor (ozone or ultraviolet light sterilization, in-line t'lltration to 5
|xm or no filtration, water temperatures of 15°C or 20'-'C and sa-
linities of 247tr or 33%r). Also, in Experiment 1, three batches of
oysters were separately fed different levels of K. breve (Treatments
la, lb. and Ic). and then subjected to identical detoxification con-
ditions. Experiment 3 compared UV sterilization at 20°C (Treat-
ment 3a) with the use of the biofilter at 12°C (Treatment 3b).
Experiment 4 also compared UV sterilization (Treatment 4a) with
the use of the biofilter (Treatment 4c) but all oysters were held at
12°C. Additionally. (Treatment 4b) oysters were held in the same
conditions as Treatment 4a except that they were fed with 10
million cells Isnchrysiis gathana per oyster on days 2 and 3. Treat-
ment 4d was a control where oysters that were not toxic were
placed in the same tanks as Treatment 4a oysters and tested at the
end of the experiment (after 5 days) to see whether detectable
levels of toxin had been accumulated during the trial.

Nonlinear regression analysis was used to fit exponentially de-
clining detoxification curves of the form:

Y = fl -I- h.i

(1)

where Y was the mean NSP level for a treatment on a given day
and X was the number of days since the beginning of the experi-
ment. The parameter a is the asymptote of the curve (the level to

which NSP approaches after some large number of days); a ■¥ b is
the initial NSP level; and r is related to the speed of approach to
the asymptote. The model was constrained with r < 1 and a > 0.
Accumulated analysis of variance was used to compare the fitted
curves. Analysis of variance (ANOVA) was applied to the mean
NSP levels after day 2 (exclusive) and on parameter a in Equation
I to determine whether any of the experimental factors had statis-
tically significant {P < 0.05) effects on the outcome of detoxifi-
cation for 6 experiments providing a balanced set of factors (Runs
1 to 4 of previously published work (Fletcher et al. 1998). with
Experiments 1 and 2 of the current work). Residual Maximum
Likelihood (REML) statistics were similarly applied to compare
the effect of treatments in all experiments.

At the end of Experiment 2 (day 4) and at the beginning and
end (days 0 and 5) of Experiment 3a. the distribution of toxin in
various tissues was determined as well as the NSP levels in two
pooled samples of 10 oysters. To determine toxin distnbution. 100
oysters were dissected [see Quayle (1969) for a description of C.
gigas anatomy] into mantle (up to the junction between the mantle
and gill, adductor muscle, gill, labial palps, gonad (i.e.. the white
colored tissue surrounding the digestive organs) and digestive tis-
sues (remaining visceral mass). The tissues from the 100 oysters
were pooled, tested for NSP, and the results were related to the
total NSP level found in the pooled samples of the 10 whole
oysters.

RESULTS

The NSP results for the 4 detoxification experiments are pre-
sented in Figure 1 and Table 2. Initial toxin levels ranged from
29.6 (Experiment 3) to 70,7 (Experiment 1 Treatment c) mouse
units g"' with considerable variation in levels recorded from iden-
tical condiuons of uptake (Table 1). As observed previously
(Fletcher et al. 1998), there was a period of rapid detoxification
followed by a period of no significant changes in toxin levels
(from Days 3 to 5 inclusive). The results of fitting these data to Eq.
I are shown in Table 2. As separating the 3 batches of shellfish in
Experiment 1 did not improve the goodness of fit the NSP results
were analyzed as a single treatment. Statistical analyses of Experi-
ments 3 and 4 did not demonstrate significant differences in NSP
levels between treatments, with or without an in-line biofilter or
with or without feeding with /. galbana. Treatments with an in-line
biofilter did not result in increased ammonia levels in the water
with time while treatments in the smaller tanks did. as reported
previously (Fletcher et al. 1998). Adding the extra replication of
factors provided in Experiments 1 and 2 to the previously reported
(Fletcher et al. 1998) half factorial experiment (Runs 1^) effec-
tively reduced the confounding effect of shellfish weight and ini-
tial NSP level from the analysis and allowed valid ANOVA. How-
ever, this still showed no significant differences between the four
factors evaluated; temperature (1 5°C, 20°C), salinity (24%c. 34%r),
in-line filter (5 \xm. absent), disinfection agent (UV, ozone). Fur-
ther. REML analysis of all the experiments also showed no sig-
nificant effects (P > 0.05), although detoxification from treatments
at 12°C was possibly less effective than from those at 15°C (F <
0.10).

Results from the analyses of different tissues in Experiment 2
and Experiment 3 Treatment a are presented in Table 3. Immedi-
ately after toxification (Day 0. Experiment 3), the toxin was con-
centrated in organs associated with ingestion and digestion (gills,
palps, digestive tissues) while at the end of the detoxification

468

Fletcher et al.

TABLE 3.
Distribution of toxin in ovster tissues.

Tissue

Weight

Lipid

NSP

NSP

Experimem

3a.

Day 0

mantle

20.9

3.74

<10.0

II

aiiiiuclor

13.1

0.38

<10.0

0

gill

18.3

1.66

17.9

14

palps

11.2

5.27

14.6

7

gonad

22.8

4.98

< 1 ()-( 1

0

digestive

13.7

5

131

78

Experiment

3a.

Day 5

mantle

30.3

3.3

<10.0

tl

adductor

12.3

0.39

<1(1,0

0

gill

16.3

2.04

<I0.0

0

palps

10.3

5.55

13.4

22

gonad

17.8

5.6

11.9

33

digestive

13.1

5.57

22.1

45

Experiment

1

Day 4

mantle

30

2.75

<10.0

0

adductor

14

0.34

<10.0

0

gill

19.4

1.28

<10.0

0

palps

8.9

4.67

12.4

1 -)

gonad

17

3.88

12.4

->-)

digestive

10,7

4.3K

57.9

66

' Weighted contribution of the tissue to total amount of toxin for all tissues.

experiments (Day 4 of Experiment 2 and Day 5 of Experiment 3)
the remaining toxin was located in the palps, digestive tissues and
gonads, all tissues with high levels of lipid.

DISCUSSION

The fit of data to Eq. I (as indicated by the R" statistic) ranged
from a very good fit for Experiment 2 to poor for Experiment 4
Treattnent a. However, the poor fits can be explained by the large
variability between results from pooled samples of identically
treated oysters on a given day rather than the inappropriateness of
the exponential model. Similar results were found when fitting the
previously published data (Fletcher et al. 1998) from Runs I to 6
with the R- statistic ranging from 0.565 to 0.999 and the a param-
eter from 5.21 to 18.29. In New Zealand's only confirmed NSP
event, NSP stopped being recorded in shellfish (including Pacific
oysters) shortly after the causative organism disappeared from the
water column (Chang et al. 1995). In an open system where fresh
seawater was continually suppMed to the oysters it could be ex-
pected that parameter a in Eq. 1 would be /.cro (i.e., given suffi-
cient time the oysters would completely purify themselves of the
toxin as happened in the wild). In fitting the curves this only
proved true for two treatments: Experiment 3 Treattnent b where
the oysters were in large tanks filtered through a biofilter and
Experiment 4 Treatment b where oysters were fed with algae dur-
ing the detoxification experiment. The levels of NSP after 3-5
days for these two treatments were no lower than other experi-
ments, so detoxification was not more rapid in these treatments,
but the value of 0 for parameter a suggests that complete detoxi-
fication would occur with time. The overall results suggest that the
closed tank system is fundamcnlally limited in its ability to totally
detoxify oysters of NSP.

Practical requirements for bacterial depuration of shellfish al-
low (hat salinity can vary from ambient by up to 20"^^ without

affecting performance (e.g.. Interstate Shellfish Sanitation Confer-
ence 1997). In our work, reducing salinity from an ambient of 34
to 247cc (30% reduction) did not have a significant effect on toxin
loss, so a similar parameter could be applied for NSP detoxifica-
tion. Previous work showed that bacterial depuration of Pacific
oysters was more effective at 15°C-20°C than at temperatures
below I4°C (Fletcher et al. 1991 ). The current work confirms that
a similar pattern applies for NSP detoxification in this species with
some evidence of reduced detoxification at 12°C, but no differ-
ences in detoxification effectiveness at 15^'C and 20°C. Inclusion
of an in-line 5 |jim filter did not improve the effectiveness of
detoxification, suggesting that uptake of NSP associated with par-
ticles in the water (e.g., whole algal cells) is not important in this
system. As the products of ozonating seawater break down NSP
toxins (Schneider & Rodrick 1995) it was expected that NSP
detoxification would be more effective for oysters in ozonated
water. However, the results did not show any such effect, probably
due to the relatively low levels of ozone we were able to add to the
water, levels thai produced a change in redox potential in seawater
from the ambient of around 450 to 550 mv. This level was used
because, in preliminary trials. Pacific oysters were observed to
close their shells and cease pumping al redox potentials of over
600 mv. Thus, ozonation did not appear to be beneficial in reduc-
ing NSP toxin levels within oysters.

In the initial work, we observed that the levels of ammonia in
the water increased substantially during the detoxification experi-
ments. We postulated that these levels of ammonia and other meta-
bolic byproducts might have inhibited oyster function, thereby
preventing detoxification after the finst 2 days of the experiments.
Holding oysters in the larger tank where water was filtered by the
biological filter in Experiments 3 and 4 was designed to test this.
Although there was no build-up of ammonia in this tank, there was
still no significant detoxirication after the first 2 days (Fig. 1 1 and
rates and levels of detoxification were not significantly different to
those oysters in the experimental tanks without the biological fil-
ter. Similarly, feeding the oysters did not result in any marked
improvement in detoxification. Although these treatments may
have slightly improved detoxification, the differences were not
sufficient to assure the production of oysters below the regulatory
level within the 5-day period.

The location of toxins in the different oyster tissues goes some
way towards explaining the failure to achieve complete detoxifi-
cation. The locations of the NSP toxin at the start of Experiment 3
support the hypothesis that the toxin is associated with algal par-
ticles that are in the process of being ingested or digested. How-
ever, during the detoxification period the toxin was eliminaled
from the gills and appeared in the gonad tissues. After detoxifica-
tion, although the total levels of NSP were reduced, the remaining
toxin only appeared in high lipid tissues at the end of Experiments
2 and 3. This suggests that while some of the algal particles may
have been passed out in feces others have been digested and some
of the lipid-soluble toxin has been transferred to the animals" lipid
stores. Subsequent detoxification, therefore, will be a much slower
process, reliant on the oysters" need to metabolize their lipid
stores.

The considerable variability in toxin levels in oysters held un-
der identical conditions is of concern. The method we used is the
one accepted by regulatory authorities and the laboratory is ap-
proved under FDA protocols for this test. Although considerable
variability is expected when testing individual shellfish (e.g.,
Scotti et al. 1983, White et al. 1993), pooling 10 to 12 oysters

Reducing Neurotoxic Shellfish Poison

469

should limit the effects of oyster-to-oyster variability on the re-
sults. In some experiments (e.g.. Experiment 2) the results of rep-
licate samples were acceptably close, but in others using the same
test procedures (e.g.. Experiment 1 ) wide variances were observed.
The control in Experiment 4 Treatment d was designed to test
whether some of this variability might be due to the uptake of
toxins excreted by other oysters. However, this did not appear to
be the case, as the non-toxic oysters in this treatment did not
accumulate toxin to detectable levels. The observed variability is
critical in a regulatory environment where the test is usually car-
ried out on a single pooled sample of 10 to 12 oysters from a given
area. For example, in Experiment 1 Treatment c on Day 4 one
sample was below the regulatory liinit while another was well
above (Fig. la). Due to the variability in results, an argument could
be made that it would be better to base the regulatory limit on the
mean of a number of samples rather than a single pooled sample.
In this case many of our experiments would have met the regula-
tory limit. The extra testing required to obtain mean values would
place an extra cost on the industry when the current regulatory
limit has been effecti\e in preventing NSP illness. There is little
knowledge of the human dose-response curve for NSP but a 100-g

sample is a reasonable reflection of an average serving for a con-
sumer.

CONCLUSIONS

The levels of NSP in Pacific oysters were reduced to levels near
the regulatory limit of 20 mouse units • 100 g"' in 2-3 days when
held at temperatures between 15°C and 20°C and salinities be-
tween 24^;f and 349m. However, no treatment consistently pro-
duced levels below the regulatory limit within the 3-day trials. This
failure can be explained by the hypothesis that toxins migrate from
the readily accessible surfaces of digestive tissues and are incor-
porated into the animals" lipid stores. The level of variability en-
countered with the standard APHA test procedures suggests that
they need to be reviewed to confirm their suitability for assuring
safe levels of NSP toxins in shellfish.

ACKNOWLEDGMENTS

This work was funded by the New Zealand Foundation for
Research. Science and Technology. Contract CO2406. We are
grateful to G Summers for technical assistance and to J F Potter for
statistical analyses.

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Shellfish Safety. New York: Chapman & Hall. pp. 331-363.

Schneider. K. R. & G. E. Rodrick. 1995. The use of ozone to degrade
Gymnodinium breve toxins. In: R. Poggi. & J.-Y. Le Gall, editors.
Shellfish Depuration. Second International Conference on Shellfish
Depuration. Rennes 6-8 April. 1992. IFREMER. Plouzan pp. 277-289.

Scotti, P. D.. G. C. Fletcher. D. H. Buisson &. S. Fredencksen. 1983. Virus
depuration of the Pacific oyster [Crassostrea gigas) in New Zealand.
New Zealand J. Seience 26:9-13.

Vogel, A. I. 1961 . A Text-book of Quantitative Inorganic Analysis Includ-
ing Elementary Instrumental Analysis. 3rd edition. London: Longmans.

White, A. W.. S. E. Shumway. i. Nassif & D. K. Whittaker. 1993. Varia-
tion in levels of paralytic shellfish toxins among individual shellfish.
In: T. J. Smayda & Y. Shimizu. editors. Toxic Phytoplankton Blooms
in the Sea. Proceedings of the 5th International Conference on Toxic
Marine Phytoplankton, Newport, RI, 28 October-I November 1991.
Amsterdam: Elsevier Science Publishers, pp. 441^446.

Whyte, J. N. C, N. G, Ginther & L. D. Townsend. 1995. Accumulation and
depuration of domoic acid by the mussel. Myriliis califomianiis. In: P.
Lassus. G. Arzul. E. Erard-Le Denn, P. Gentien & C. Marcaillou-Le
Baut. editors. Harmful Marine Algal Blooms — Proceedings of the
Sixth International Conference on Toxic Marine Phytoplankton, Octo-
ber 1993. Nantes, France. Lavoiser, London, pp. 531-537.

Wohlgeschaffen G. D.. K. H. Mann. D. V. Subba Rao & R. PocklingUm.
1992. Dynamics of the phycotoxin domoic acid: accumulation and
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4:297-310.

Journal oj Slu'llfisli Rescaivh. Vol. 21. Nii. 2, 47I-+77, 2002.

RECIRCULATION OF DINOFLAGELLATE CYSTS BY THE MUSSEL, MYTILUS EDULIS L., AT
AN AQUACULTURE SITE CONTAMINATED BY ALEXANDRIUM FUNDYENSE

(LEBOUR) BALECH

F. M. HARPER,* E. A. HATFIELD, AND R. J. THOMPSON

Ocetin Sciences Centre, Memorial University of NewfounJUuul. St. Jolin's.
NewfoiindUnul. Canada AlC 5S7

ABSTRACT Holding suspension-feeding bivalves at an aquaculture site may facilitate the maintenance of toxic dinotlagellate
populations by concentrating transient vegetative cells or resuspended cysts. To examine the role of the mussel. Mytilus eiliilis. in
recirculating cysts within an aquaculture site contaminated with the dinotlagellate Alexandrium fundyense. sediment cores and fecal
samples were collected in September and October 1996. In the interim period, a bloom oi A. fundyense vegetative cells began. Mussels
egested similar concentrations of dinotlagellate cysts (Scrippsiella sp., A. fundyense. and an unknown Grey species) regardless of the
location of the mussel sock in the site, or the position of the mussel in the water column. In September, more putative A. ostcnfeldii
cysts were egested in feces collected from the bottom of two socks than in those from the top. One sock was located at greater depths
near a barrier island and the other in a shallow northeastern cove. Within each dinotlagellate species, there were no significant
differences between cyst concentrations in sediment throughout the site, the exception being the high concentrations in September of
putative A. oslenfeldii beneath the sock located near a barrier island (182 cysts-cm"^). Post-bloom, there were significantly fewer A.
fundyense cysts in the sediment underlying the sock near a barrier island. In contrast, there were significantly more putative A.
oslenfeldii cysts in the sediment in the shallow northeastern cove (580 cysts-cm"'). The daily replenishment rate of A. oslenfeldii cysts
in bottom sediments by mussel fecal deposition was estimated as 2 x lO'^ cysts m"" d"'. or about 8%. This may be a considerable
contribution to the maintenance of this dinotlagellate species in a mussel aquaculture site, but further studies are required to compare
other inputs and outputs of cysts to establish the relative importance of bivalve aquaculture.

KEY WORDS: dinofiagellate. Alexandiiuni. cysts, mussel, aquaculture. PSP

INTRODUCTION

Accumulations of resting cysts (or hypnozygotes) of benthic
dinotlage Hates have been observed in various marine environ-
ments: offshore trenches and depressions, fjords, and the shallow
coastal embayments often selected for shellfish aquaculture opera-
tions (Dale 1976. Dale et al. 1978. Anderson & Morel 1979, White
& Lewis 1982). Certain locations that accumulate cysts as a result
of selective deposition due to hydrographic and sedimentary pro-
cesses can act as "seed beds", producing motile cells that then
initiate blooms (Steidinger 1975, Anderson & Morel 1979, Tyler et
al. 1982). More than SO species of marine dinotlagellates are
known to produce cysts (Matsuoka et al. 1989). including species
of the toxic d\noi\digt\\aXt Alexandrium (Halim) Balech (formerly
classified as Gonyaidax spp. or the Protogonyaulax cateiwlla/
tamarensis species complex). In many parts of the world, A. taina-
rense (Lebour) Balech has been responsible for incidents of para-
lytic shellfish poisoning (PSP: see review by Hullegraeff 1993).

PSP is a .serious public health risk that threatens the commercial
harvest of both wild and cultured bivalve populations. Potent neu-
rotoxins can accumulate in bivalves through ingestion of vegeta-
tive cells of toxic dinotlagellates during suspension feeding
(Shumway et al. 1987, Shumway 1990). Cysts formed at the end
of Alexandrium blooms have also been implicated as vectors of
PSP toxin transfer to bivalves (Dale et al. 1978, White & Lewis
1982). There has been some debate concerning the toxicity of the
cysts relative to the motile vegetative cell (Dale et al. 1978.
Yentsch & Mague 1979, White & Lewis 1982, Cembella et al.
1990), although there is a consensus that A. tamarense cysts con-
tain PSP toxins. The abundance of A. fundyense (Lebour) Balech
cysts in sediments has been positively coiTelated with maximum-

*Correspondmg author. Present address: Department of Biology. Dalhou-
sie University Halifax. Nova Scotia. Canada. B3H 4J1

recorded levels of PSP toxins in the blue mussel, Mytilus edulis
(Schwinghamer et al. 1994). Mussels ingest A. tamarense cysts
and pass them in fecal pellets (Anderson 1984), but whether bi-
valves can extract the toxins from cysts and assimilate them re-
mains to be established.

Bivalves inhabiting areas with bottom sediments contaminated
with toxic cysts cannot avoid encountering these cells during re-
suspension events. Dense concentrations tif suspension-feeding bi-
valves in areas such as aquaculture sites may facilitate the main-
tenance of a seed bed of toxic dinotlagellate hypnozygotes by
concentrating transient vegetative cells or resuspended cysts and
depositing these cells onto the underlying sediment in the form of
pseudofeces or feces.

Mussel aquaculture is an expanding industry in Newfoundland:
production has increased from 320 tonnes in 1991 to over 1700
tonnes in 1999 (Statistics Canada 1999). Surveys of coastal sedi-
ments around the Province have revealed contamination by A.
fundyense cysts in many areas (McKenzie 1993, McKciizie 1994.
Schwinghamer et al. 1994). One former mussel farm, located in
Barred Island Cove, Notre Dame Bay, is permanently closed to
aquaculture due to persistent PSP contamination, and has been the
focus of ongoing research (Fig. 1 ). Sediment analysis at this site,
a cove with two barrier islands and a shallow sill, has revealed A.
fundyense cyst concentrations ranging from 20 to > 1,000
cysts-cm"^ (McKenzie 1993, McKenzie 1994, Schwinghamer et
al. 1994). This site experiences frequent sediment resuspension as
the result of strong winds.

The objective of this study was to examine the role of the
mussel, M. edulis. in recirculating dinotlagellate cysts and main-
taining Alexandrium populations within an aquaculture site. The
dinotlagellate cyst composition of feces collected from mussels
from the tops and bottoms of socks was determined to test the
hypothesis that mussels suspended further away from the contami-
nated sediment egest fewer cysts. To facilitate comparisons be-

471

472

Harpi-:r i-t al.

49°30'25" --

E

i 4.0.

5.6-

■+■

■+■

■+■

■+■

-+-

Sock 1 Sock 2 Sock 3 Sock 4 Sock 5

Figure 2. Location of mussel long-lines ( — I and socks ( ♦ ) at the study
site. Barred Island Cove, in Notre Dame Bay, Newloundland. Lengths
of s(Kks and sediment sampling depths are depicted for specified socks
(insert).

tween different sampling locations in the site, the concentrations of
cysts in the sediment beneath the mussel socks were determined.
The biodeposition rate of dinotlagellate cysts in fecal pellets was
then calculated to estimate the rate at which cultured mussels
returned resuspended cysts to the sediment surface.

MATERIALS AND METHODS

ColUctum and Examiiiatiiin nf Mussel Fecal pellets

Mussels were sampled in .September and October 1996 from
the tops and bottoms of mussel socks previously established at the
study site. Barred Island Cove, Notre Dame Bay, Newfoundland
(see Fig. 1 ). Wind speed and tide stage, factors that could induce
heavier sediment resuspension, were similar for the two sampling
periods. Sediment traps deployed in September and retrieved in
late October contained Alcxtintlrimii cells in various stages of en-
cystment, suggesting a mid- to late-October bloom (McKenzie et
al. 1998). Socks (4.6-ni length) were selected based on results of
earlier studies conducted at this site, which demonstrated that the
highest cyst concentrations occuired in the shallow sediment al the
northeastern edge of the cove (McKenzie 1994, Schwinghamer et
al. 1994). Socks 1 and 2 were located near the barrier islands;
.sock.s 3-5 were situated in the noitheastern cove (see Fig. 1 ).

On September 24, 1996. five mussels (Mytihts edulis. mean
shell length 6.^.5 mm ± 10.2 SD) were collected from the top and
five from the bottom of each of socks I, 2. and 4. Two species of
mussels. M. edulis and M. rrossidus. co-e.xist in Newfoundland;
those used in this study were identified as M. edulis ba.sed on size
and morphologic characteristics (Freeman et al. 1992). Further-
more, over 95% of mussels in this part of the coast of Newfound-
land are M. edulis according to genetic markers used by Innes and
Thompson (unpublished data).

Mussels were transported on ice (8 h) to the Ocean Sciences
Center. Logy Bay; no mortalities occuired. On arrival, mussels
were scrubbed tree of epibionts and placed in individual l-L con-
tainers with 700 niL UV-sterilized, filtered (1.0 |j.m) seawater
(FSW) at 15"'C. Mussels were removed after a 15 h incubation
period and the fecal pellets carefully collected and stored in the
dark at 4°C. No preservative was necessary, as low storage tem-
perature (4°C) and darkness do not stimulate germination of cysts
(Perez et al. 1998). Preliminary trials indicated that the gut reten-
tion time of dinotlagellate cysts was approximately 9 h. therefore
15 h was considered adequate time for complete digestion and/or
egestion; Scarratt et al. ( 199.^) proposed that 12 h would be suf-
ficient time to purge cysts from mussels. Since the mussels re-
mained closed during transport, there was no egestion of feces.
Prolonged retention of the material within the digestive tract dur-
ing transport did not appear to affect the egestion of intact di-
notlagellate resting cysts, although some cysts may have been
digested so that the values for cysts egested may be underesti-
mates.

On October 29. four mussels (mean shell length 4.^.6 mm ± 6.7
SD) were sampled from the top and bottom of each of socks 1 . 2.
.^. and 5. Fecal pellets were collected on-site: after being scrubbed
free of epibionts, individual mussels were placed in plexiglass
cylinders (diameter = 70 mm; height = 75 mm) filled with sur-
face water. Cylinders were sealed with a rubber stopper. lea\ ing an
air pocket, and submerged for 19 h at 7'-C. Fecal material was then
collected and transpoiled to the laboratory for processing. No
pseudofeces production was ever observed.

Fecal contents were disaggregated by repeated aspiration
through a micropipette tip, followed by gentle sonication for thirty
minutes in an ice-water ultrasonic bath (Cole Parmer 8851). In
preliminary testing of methods for disaggregating fecal pellets,
gentle sonication in the bath did not destroy any cysts and resulted
in a homogeneous suspension of fecal material (Harper 1997).
Sonication by microprobe and by ultrasonic bath has no significant
negative effects on germination of A. tunuirense cysts collected
from sediment (Perez et al. 1998). Cysts in each sample were
counted (triplicate samples) with an Utermiihl settling chamber.
Dinotlagellate cysts were identified and enumerated by direct cell
counting using phase and epitluorescence microscopy. Although
this approach is laborious and time-consuming, it yields the most
accurate information about the composition of the phytoplankton
and the condition of the cells (Sakshaug 1990). An electronic
particle counter could not be used for enumeration of the cells in
the fecal material as the cysts of each species were approximately
the same size (40 |j.iti diameter) and shape, and thus cysts of any
given species could not be distinguished from those of other spe-
cies.

To facilitate comparisons between mussels from different lo-
cations and of different sizes, cyst abundance values were ex-
pressed per mg total dry weight (DW) and per mg ash-free dry
weight (AFDW) of feces. Duplicate samples of 0.7-1.2 niL ho-

Recirculation oh Dinoflagellate Cysts by Mussels

473

moaeneous fecal suspension were retained on preweighed. pre-
combusted (450 "C) Whatman GF/C filters (25 mm) under gentle
vacuum, then rinsed with 10 mL 3% ammonium formate to re-
move salts. Filters were oven-dried to constant dry weight at 60°C,
then combusted at 450°C for 12 h. cooled in a desiccator and
icweighed to determine the AFDW of feces. Weights were deter-
mined within 1 |xg with a Mettler UM3 microbalance.

Collection, Conceittration, and Exaoiinotion of Cysts jroni Sediment

On each of the sampling dates, four sediment cores were col-
lected by hand by SCUBA divers within a 1-m" area beneath
mussel socks I— f in September and socks 1-5 in October. The
sediment was composed of small rocks, fine gravel and silt. The
top 6-8 cm of sediment and 2-3 cm of overlying water was col-
lected using a 50-mL polycarbonate centrifuge tube (cross-
sectional area 5.92 cm") with the conical tip removed (McKenzie
1993. McKenzie 1994; McKenzie & Schwinghamer 1994). Cores
were transported on ice in the dark to the laboratory where the top
3 cm of sediment and the overlying water were transfen'ed to clean
50-mL tubes, then stored in the dark, unpreserved. at 4°C. Di-
noflagellate cysts were enumerated in the top 3 cm of sediment
rather than just in the tlocculent surface layer (Anderson & Wall
1978. Dale et ul. 1978. Lewis et al. 1979). As many Alexcmdiium
tamarense (Conyaulax excavata) cysts can be present in the top
3^ cm of sediment as are found at the surface (White & Lewis
1982).

To ensure adequate dispersal of detritus, sediment samples
were sonicated with two bursts (each 45 sec, power level 4) of a
Branson 250 Probe Sonifier. Samples were then fractionated to
separate and concentrate dinoflagellate cysts following the den-
sity-gradient technique of Schwinghamer et al. (1991). In brief,
after sonication the sediment was sieved through 80- and 20-p.m
Nite.\ nylon sieves, retaining the material on the 20-jji,m sieve. A
step gradient was then formed by slowly injecting "light" and
"dense" solutions of a non-toxic, aqueous colloidal silica suspen-
sion (Nalco 1060), made isosmotic with seawater (salinity 329(0
using sucrose, beneath the sieved cyst suspension in a 50-mL
centrifuge tube. The tubes were then centrifuged at 1,500 g for 30
niin at 4°C and the concentrated layer of dinoflagellate cysts at the
interface between the light and dense layers removed for exami-
nation.

The dinoflagellate cyst composition of each sediment core was
determined in triplicate by examining the concentrated cyst layer
with phase and epifluorescence microscopy. Each count was per-
formed on 100-200 |j.L of the homogenous cyst slurry, the volume
settled for each core being adjusted to yield a total of 100-3(J0
cysts per slide (Venrick 1978. with reference to Lund et al. 1958).
Results were expressed as the number of cysts-cm"'' sediment.

Dinoflagellate Cysts in Feces and Sediment

The cysts of four dinotlagellate species were enumerated in
fecal and sediment samples: Scrippsielta sp. (Stein) Loeblich. Al-
i:\anclriiiiii fuinlyciise. and two unknown species. Cysts were iden-
tified using morphologic characteristics: the shape of the cyst body
and its ornamentation, cyst size and diameter, wall structure, and
the presence of food reserves and pigmented bodies (Matsuoka &
Fukuyo 1995). The first unidentified form resembled the resting
stage of A. ostenfcldii (Paulsen) Balech et Tangen. a toxic di-
notlagellate that has been observed in the Gulf of St. Lawrence
(Levasseur et al. 1996). and in Nova Scotian coastal enibayments

(Cembella et al. 1998. Cembella et al. 2000). These cysts matched
the description of the A. nsiciifcUlii cyst provided by MacKenzie et
al. (1996); a spherical cell (-40 [j.m diameter) containing one or
two red-brown pigmented granules and areas of condensed,
golden-brown pigmentation radiating from the center of the cell
towards the periphery. Identification of the vegetative cells that
emerged from these cysts was not possible, as repeated attempts at
establishing cultures were unsuccessful, but these vegetative cells
had the typical gonyaulax form (Hallegraeff 1995), although they
did not survive more than 48 h. These cysts are referred to here as
putative A. ostenfcldii pending further germination studies.

The second unidentified cyst is referred to as the Grey cyst
because of its appearance in phase microscopy. It is possible that
these cysts were partially digested A. fimdyense cysts as they were
approximately the same dimensions and clearly contained a red
pigmented body, but all were tapered at one end, in contrast with
the two rounded ends of cylindrical A. fundyciisc cysts. Grey cysts
did not germinate under cullurc conditions and currently remain
unklentitled.

Statistical Analyses

The concentrations of the dinoflagellate cysts in feces from
mussels collected from the top and bottom of each sock were
analyzed by two-way analysis of variance with the sock and the
position of mussel on the sock as fixed factors and the individual
mussel as the unit of analysis. Separate analyses were performed
for the abundance of each dinotlagellate species present in mussel
feces, expressed per mg DW and per mg AFDW feces. September
and October data were treated separately as only socks I and 2
were sampled in both months.

The concentrations of the four species of dinoflagellate cysts in
the sediment beneath the socks were analyzed by one-way analysis
of variance with the sock as the fixed factor. Analyses were per-
formed separately for September and October data, and foi' each
cyst type, expressed as the total number present-cm"'' sediment.

A two-way analysis of variance was performed to compare pre-
and post-bloom concentrations of the cysts in the sediment beneath
socks 1—1. Month and sock were fixed factors in the analysis; the
individual cores were the experimental units.

Where the F value exceeded the critical value at P = 0.05.
planned comparisons were made using Fisher's LSD method (Mil-
liken & Johnson 1984). The criterion for statistical significance in
all analyses was P < 0.05.

RESULTS

Concentrations of Dinoflagellate Cysts in Mussel Feces

The concentrations of dinoflagellate cysts egested were highly
variable between individual mussels (Fig. 2, Fig. 3). In September
(see Fig. 2), the concentrations of Sciippsiella cysts egested were
low. ranging from 0-142 cysts-mg"' DW and 0-98 cystsmg"'
AFDW feces. Few Alexandrium fimdyense cysts were egested,
from 0-28 cystsmg"' DW and 0-846 cystsmg"' AFDW feces.
Concentratit)ns of the unknown grey morphotype were also low,
ranging from 0-71 cysts-nig"' DW and 0—174 cysts-mg"' AFDW
feces. There were no significant differences in the concentrations
of Scrippsielta sp., Alexandrium fimdyense. and Grey cysts be-
tween mussel feces collected from the tops and bottoms of socks.

More putative Alexandrium ostenfeldii cysts were found in the
mussel feces collected in September (see Fig. 2) than any of the

474

Harper et al.

800

'01I600

1 1400
1 1200 -
§"1000 -
^ 800

o

1-T 1-B 2-T 2-B 4-T 4-B

Location of mussels (Sock - Position)

Figure 2. Concentrations ot dinotlagellate cysts egested in mussel feces
collected from the tops (T) and bottoms (B) of socks in September.
Values are standardized per mg dry weight of feces and per mg ash-
free dry weight. Each bar is the mean ± standard error Ih = 5). The
asterisk (*) indicates signillcant differences (/' 9 0.1)5) between the
concentrations of a dinollagcllate species in feces collected from the top
and bottom of a specific sock.

other dinoflagellate cyst types quantified. Putative A. ostenfehUi
concentrations varied from 10 to 846 cystsmg"' DW and 18 to
2,190 cystsmg"' AFDW feces. Significantly more of these cysts
were found in mussel feces collected from the bottom of sock 4
than from the top (P = 0.0001 for values expressed in terms of
DW; P = 0.004 for AFDW) and from the bottom of sock 2 than
from the top {P = 0.039 for DW). Sock 4 was located in the
northeastern cove; the bottom of the sock was only 0.6 m above the
sediment. Socks 1 and 2 were located near the bairier islands and
were further from the bottom. 1.9 and 2.4 m from the sediment
respectively (see Fig. 1).

In October (see Fig. .^), there were no significant differences in
the concentrations of any of the dinoflagellate species between
fecal samples from mussels collected from the tops and bottoms of
the socks. The concentrations of Scrippsiella sp. egested were
similar to those found in September, ranging from 0-90 cystsmg"'
DW and 0-216 cystsmg"' AFDW feces (see Fig. 3). Alexandniim
fundyense cysts concentrations in the mussel feces remained low.
varying from 0-170 cystsmg"' DW and 0 to 181 cysts-mg"'
AFDW feces. Concentrations of putative A. ostenfeldii cysts ege-
sted ranged from 0-130 cysts-mg"' DW and 0 to 325 cysts-mg"'
AFDW feces. Grey cyst concentrations were also low; consistent
with amounts egested in September, ranging from 0 to 130
cystsmg"' DW and 0 to 325 cystsmg"' AFDW feces.

The error in counting cysts in the fecal suspension was esti-
mated from five replicate counts of a single sample containing 494
cysts (putative A. ostenfeldii). The coefficient of variation (CV)
was 18% mg"' DW and mg"' AFDW feces. The error in this

method is comparable to the 20% CV reported by Ishikawa and
Taniguchi (1994) for enumeration of cysts of Scrippsiella sp. in
sediment. The counting error (CV) for the sediment cores was
14%, estimated by five replicate counts of putative A. ostenfeldii
cysts in one sediment core that contained 156 cystscm"''.

Concenlratiuns of Dinuflagellate Cysts in Sediment Beneath
Mussel Socks

On both sampling dates, concentrations of Scrippsiella sp.. Al-
e.mndriiiin fundyense and Grey cysts did not differ significantly
within each species between sediment samples collected through-
out the site {P > 0.05, Fig. 4). In September. Scrippsiella sp. cysts
numerically dominated the sediment with a mean concentration of
596 cystscm"' (±335.6 SD) compared with the mean A. fundyense
concentration of 89 cysts-cm""* (±88.7 SD). Grey cysts were scarce
(mean 4 cystscm"' ±6.8 SD). In October, mean Scrippsiella sp.
concentrations were 306 cystscm"' (±144.2 SD), A. fundyense
mean concentrations were 142 cystscm"'' (±137.3 SD). and very
few Grey cysts were found (mean 0.6 cysts-cm""" ±1.3 SD).

In September, there were significantly more putative A. osten-
feldii cysts in the sediment beneath sock 1 than elsewhere in the
site (P = 0.008). In October, however, there were no significant
differences in putative A. ostenfeldii cyst concentrations in the
sediment beneath the mussel socks (P > 0.05).

Sediment trap data collected in October as part of a concunenl
study (McKenzie et al. 1998) suggested that an A. fundyense
bloom began during mid- to late-October. Post-bloom, there were
significantly fewer A. fundyense cysts-cm"' beneath sock 1 than

1-B 2-T 2-B 3-T 3-B 5-T
Location of mussels (Sock - Position)

Figure i. Concentrations of dinoflagellate cysts egested in mussel feces
collected from the tops (Tl and bottoms (B) of socks in October, Values
arc standardized per mg dry weight of feces and per mg ash-free dry
weight. Each bar is the mean ± standard error (/( = 4|. Legend as in
Figure 2.

Recirculation of Dinoflagellate Cysts by Mussels

475

Figure 4. Concentrations of dinoflagellate cysts per cm' in the sedi-
ment beneath mussel socks sampled in September and October. Sock
numbers refer to designations on Figure 1. Each bar is the mean ±
standard error (/; = 3|. The asterisk (*) indicates a significant differ-
ence between the concentrations of putative A.oslenfeldii cysts beneath
sock 1 and concentrations beneath socks 2-4 in September (P = 0.008).
Legend as in Figure 2.

elsewhere (P = 0.001). In contrast, there was significantly more
putative A. ostenfeldii cysts beneath socks 3 and 4 in October than
there were in September {P = 0.006 and P = 0.02, respectively).

DISCUSSION

Significance of Mussel Position in the Water Column with Respect to
Cyst Contamination

Vertical repositioning of mussels in the water column may limit
their exposure to toxic dinoflagellate cells (Desbiens et al. 1990).
To reduce PSP contamination, Desbiens and Cembella (1993)
placed mussels near the bottom of the water column (13-15 m
deep) during a bloom of vegetative Alexandrium excavatum cells
and raised the mussels close to the surface when A. excavatum was
concentrated near the bottom of the water column. Mussels placed
near the bottom were the least toxic for most of the exposure
period.

In this study, we hypothesized that mussels suspended close to
the sediment would egest more dinoflagellate cysts than mussels
suspended near the top of the water column. We found that this
depended on the location of the mussel sock in the site and the
sampling time. In September, mussels collected from the bottom of
socks near a barrier island and in the northeastern cove of a former
aquaculture site at Barred Island Cove. Newfoundland, egested
more putative A. ostenfeldii cysts than mussels collected from the
top of the socks. In October, however, similar numbers of A.
ostenfeldii cysts were egested regardless of the proximity of the
mussels to the underlying sediment and their location in the site.

No differences were found in either sampling date within the con-
centrations of Scrippsiella spp., A. fundyense or Grey cysts in feces
from mussels collected from the tops and bottoms of the socks.

It is not possible to establish a direct relationship between the
concentration of cysts beneath a mussel sock and the concentra-
tions in feces egested by the overlying mussels. Mussels may have
been ingesting cysts from other sources (e.g.. horizontal advection.
newly formed cysts in the water column resulting from encysting
vegetative cells), or cells may have encysted within the digestive
tract. For example. Grey cysts were numerous in mussel feces
collected in October, but few were present in the underlying sedi-
ment. Samples from the water column are needed to determine the
source of the egested cysts, but it is clear that suspending cultures
near the top of the water column does not reduce the exposure of
mussels to cysts at this site.

The depth of sediment erosion may be a factor in the resus-
pension of dinoflagellate cysts, depending on the depth distribution
of the cysts of each species. White and Lewis (1982) demonstrated
that there are as many A. tamarense (Gonyaulax excavata) cysts in
the top 3 cm of sediment as in the flocculent surface layer, but it
is not known whether there are vertical gradients in the abundance
of other types of cysts within the sediment. There is no clear
relationship between the density of dinoflagellate cysts directly
beneath the mussel socks and the number of cysts passing through
the overlying mussels.

Distribution of Dinoflagellate Cysts in Bottom Sediments

Concentrations oi Scrippsiella sp.. Alexandrium fundyense and
Grey cysts did not differ significantly within each species through-
out Barred Island Cove. Sediment core samples collected in No-
vember and December of 1992 showed the highest concentration
oi A. fundyense (1,130 cysts-cm""*) in the shallow sediment on the
eastern edge of the site (McKenzie & Schwinghamer 1994). In
October 1993, the highest concentration of A. fundyense (118
cysts-cm"^) was detected near the barrier islands (McKenzie
1994). In September 1996. the highest concentration oi A. fundy-
ense cysts was found beneath sock 1, near the barrier islands (371
cysts-cm"''); in October, the highest concentration was found in the
northeastern edge of the cove beneath sock 3 (214 cysts-cm""*).
Similarly, the highest concentrations of putative A. ostenfeldii
were found in the sediment beneath sock 1 in September (182
cysts-cm""*) and beneath sock 3 in October (580 cysts-cm"'). The
distribution and concentrations of dinoflagellate cysts in the sedi-
ment are cleariy variable both temporally and spatially within this
site.

Between September and October 1996, concentrations of A.
fimdyense cysts either did not change or significantly decreased in
sediments beneath the mussel socks samples. In contrast, there
were significantly more putative A. ostenfeldii cysts beneath socks
in the northeastern cove in October than there were in September.
In the interim, a bloom of Alexandrium vegetative cells began
(McKenzie et al. 1998). Although this bloom was attributed to a
proliferation of A. fundyense cells, it is possible that it may have
been caused by both A. fundyense and A. ostenfeldii blooming
concurrently, triggered by the same environmental conditions; A.
ostenfeldii does not tend to form monospecific blooms (Cembella
et al. 1998). Vegetative cells of A. fundyense and A. ostenfeldii arc
difficult to distinguish under the light microscope, the method used
by McKenzie et al. (1998). In material fixed in Lugofs iodine,
Hansen et al. (1992) were unable to distinguish between the two
species.

476

Harper et al.

Blooms of motile, cyst-forming dinoflagellate species can in-
crease cyst deposition in the underlying sediments. For cysts to
accumulate, the deposition rate must exceed the rates of loss
through regeneration, excystment and transport (Cembella et al.
1988). In this study, we did not observe a post-bloom increase in
cyst concentrations of A. fuiidyense in the surface layer of the
sediment, but there was an increase in putative A. ostenfeklii that
may have been the result of transport of cysts within the site or
encysting of vegetative cells. Vegetative cells were still in the
process of encysting in the water column, as various encystment
stages of A. fiindyense were observed in sediment trap samples
(McKen/ie et al. 1998).

Previous studies at this site enumerated only those cysts that
appeared exactly like A. fiDidxi'iise (McKen/ie 1993. McKenzie
1994. McKenzie & Schwinghamer 1994). In 1996, putative A.
ostenfeldii cysts were as abundant as A. fiindyense in the sediment.
The vegetative cells of A. ostenfeldii have been documented in the
St. Lawrence Estuary and Gulf (Levasseur et al. 1996) and in
coastal Nova Scotia (Cembella et al. 1998). This species has not
been recorded in Newfoundland, possibly because it is difficult to
identify. Maximum concentrations of ,4. fiindyense (371
cy.sts-cm"'') and putative A. ostenfeldii cysts (580 cysts-cm"'') in
the sediment from this site are comparable with values from other
identified cyst beds in eastern Canada. Sediments in the lower
St. Lawrence estuary can contain 400-1., "it )()+ A. e.xcavaliim
cysts-cnr\ depending on the time of the year (Cembella et al.
1988, Cembella 1990). The southern Bay of Fundy. particularly to
the east and northeast of Grand Manan Island, is rich in A. fiindy-
ense (G. excavata) cysts, ranging from 2,000-8.000 cystscm"'
sediment (White & Lewis 1982). In Newfoundland, sediments at
Harbour Grace, Conception Bay, contain between 30-150 A.
fiiiuhense (G. excavata) cysts-cm"' (White & While 1985).

Estimated Biodepositioii Rate of Cysts from Suspended
Mussel Cultures

The replenishment rate of bottom sediments by putative A.
ostenfeldii cysts egested by mussels can be estimated from the rate
of biodeposition, the process by which feces and pseudofeces settle
to the bottom (Haven & Morales-Alamo 1966). Since this study
was not designed to investigate the biodeposition rates of di-
noflagellate cysts, mussels were not continuously exposed to the
cells of interest. Values from the following calculations later may.
therefore, underestimate the actual rates of biodeposition since the
mussels collected in this study were isolated from the seston and
were not permitted to feed continuously.

Over the 15 h incubation period in September, mussels (n =
30) egested an average of 333 putative A. ostenfeldii cysts-mg~'
DW feces. In October, mussels (n = 32) egested an average of 2.4
cysts-mg"' DW feces over 19 h. Navarro (1983, as cited in Na-

varro & Thompson 1997) reported that biodeposition rates of -53
mm shell length Mytihis cliileiisis ranged from 10-38 mg
DW-musseT' d"'. Using an intermediate value of 20 mg
DW-musseP' d"'. the biodeposition rate of putative A. ostenfeldii
cysts by an individual mussel was 6,660 cysts-mussel"' d"' in
September and 47 cysts-mussel"' d"' in October. Comparison of
these values with the concentrations of cysts in the bottom sedi-
ments requires information on the stocking density of mussels. In
Newfoundland, the average mussel farm has an estimated 30.9
mussels-m"" bottom (C. Couturier, pers. comm.), therefore in Sep-
tember approximately 2.1 x 10^ putative A. ostenfeldii cystsm""
d"' were transfened to the sediment in the form of biodeposits. In
October, approximately 1 .5 x 10"" cysts-m"" d ' were deposited on
the bottom.

Mean concentrations of putati\e A. ostenfeldii cysts in the sedi-
ment, expressed as numbers-cm"" integrated through the upper 3
cm. were 2.55 x 10'' cysts-m"" in September and 4.26 x 10''
cysts-m"" in October. Approximately 8% of the putative A. osten-
feldii cysts in the sediment were being replenished daily in Sep-
tember by fecal pellet deposition from the overlying mussel stocks.
The replenishment rate in October was considerably lower, less
than 1% of the cysts in the sediment being deposited in the form
of mussel feces.

The egestion of putative A. ostenfeldii hypnozygotes by M.
ediilis in feces provides a mechanism for the recycling of di-
noflagellate cysts to the sediment. An approximate daily replen-
ishment rate of 8'/(- may be a considerable contribution to the
maintenance of cyst populations, and could alter the composition
of the sediment over time. Before the contribution of mussel aqua-
culture to dinoflagellate cyst recycling within a site can be deter-
mined, the number of cysts in biodeposits must be compared with
other inputs and outputs of cysts, such as horizontal ad\ection or
natural sedimentation during blooins. Future research should in-
clude the determination of concentrations of cysts and vegetative
cells in the water column of aquaculture sites and comparison with
appropriate reference areas. The hydrographic characteristics of
each site must also be considered as these can clearly play an
important role in distribution of cysts.

ACKNOWLEDGMENTS

The authors thank the O.S.C. field services unit and D. Mc-
Kenzie for sample collection and transport, C.H. McKenzie and M.
Riehl for technical support, and H. Chen, D. Schneider and W.
Blanchard for statistical advice. The manuscript was improved by
comments from S. Shumway and two anonymous reviewers. This
work was supported by a research grant to RJT by the Natural
Sciences and Engineering Research Council of Canada and by a
graduate student fellowship from Memorial University of New-
foundland to FMH,

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Joiinwl of Shellfish Re.mirch. Vol. 21. No. 2. 47y-;88, 2002.

REPRODUCTIVE CYCLE OF THE CHOLGA PALETA, ATRINA SEMINVDA LAMARCK, 1819
(BIVALVIA: PINNIDAE) FROM NORTHERN PATAGONIA, ARGENTINA

RODRIGO CASPAR SORIA,* MARCELA SUSANA PASCUAL, AND
VICTOR HUGO FERNANDEZ CARTES

Laboratorio y Criadero de Mohiscos Bivuhos. Instltiito de Biologia Marina y Pesquera Alte. Slonii. CC
104. (8520) San Antonio Oesle. Rio Nei^ro. Argi'mina

ABSTRACT Alrina seminuda (Bivalvia:Pinnidae) is distributed naturally along the Atlantic coast of America from North Carolina
to the Valdes Peninsula. At the San Mati'as gulf (North Patagonia), its southern distributional limit, it inhabits subtidal areas of fine
and coarse sandy bottoms, on depths ranging from 4 to 30 m. The reproductive cycle of a population of A. seminuda was studied from
October 1999 to October 2000. Histologic samples of gonad tissue were obtained from adult individuals collected at Piedras Coloradas
(40 '53'S. eS'WWl. Six stages of gonad development were described on the basis of histo-morphologic characteristics: indifferent,
early active, developing, mature, spawning, and spent. This study indicates that A. seminuda exhibits an annual reproductive cycle.
Gametogenesis starts at the begmnmg of autumn and is prolonged during the winter. A high synchronism in maturation and spawning
occurs at November and December. The spent stage begins at mid-summer and concludes at the end of that season. Muscle index (Ml)
fluctuates inversely to the gonad index (Gl) along the reproductive season, suggesting that this species uses reserves stored in the
adductor muscle for gonadic maturation. A. senumida is a gonochonc stable species that exhibits, on a 1.05% of Ihe individuals, two
types of functional hermaphroditism

KFA' WORDS: bivalves. Pinnidae. Atrina seminuda. reproducti\'e cycle

INTRODUCTION

Species belonging to the family Pinnidae are commonly known
as pen-shell, hacha, rompechinchorro, wing-shell, razor-shell, and
locally as cholga paleta. They are distributed along sheltered sites
of warm and temperate seas, at depths up to thirty meters.

The cholga paleta, Atrina seminuda Lamarck 1819, is a mem-
ber of the Pinnidae family and is the only Pinnidae present at the
Patagonian coast. This species is distributed from North Carolina.
United States, to the San Jose Gulf. Argentina (Scarabino 1977,
Lodeiros et al. 1999). A. .'teminuda has been cited by the synonyms
Pinna seminuda. Pinna listeri. and Atrina (Sen-alrinat seminuda
(Scarabino 1977).

At the San Mati'as Gulf (40°40' S, 63°30' W) (Fig. I) this
species inhabits sandy bottoms at depths ranging from five to thirty
meters. The cholga paleta lives in a vertical position with the
anterior portion of its body deeply buried on the substrata, to which
it is attached by large and strong byssal threads.

The biology of A. seminuda had not been studied until present.
However, several studies have dealt with other members of the
family Pinnidae, focusing reproductive biology (Noguera &
Gomez-Aguirre 1972, Coronel 1981, Yongqiang & Xiang 1988,
de Gaulejac et al. 1995a, de Gaulejac et al. 1995b, Maeda-
Martinez el al. (unpublished manuscript), Rodriguez-Jaramillo et
al. 2001. Ceballos-Vazquez et al. 2000). ecology and population
structure (Butler & Brewster 1979, Keough 1984, Butler 1987,
Warwick et al. 1997. Richardson et al. 1999), fisheries, and aqua-
culture (Sung & Myong 1984. Cendejas et al. 1985, Bishui et al.
1987, Yoo & Yoo 1984, Yoo et al. 1988. Maeda-Martinez et al.
1996, Reynoso-Granados et al. 1996, Guo et al. 1999).

Studies on reproductive biology and gonadic cycle are impor-
tant tools in fisheries and aquaculture management of valuable
commercial species. Several methods can be used to estimate the
reproductive cycle in bivalves (gonadal macroscopic appearance

♦Corresponding author. E-mail: rgskua@yahoo.com. ar

or frottis, dynamics of embryos and larvae in the plankton, timing
of larval recruitment, physiologic indexes behavior, etc) being the
histologic study of gonadal tissues the most reliable method.

In this study we describe the reproductive cycle of Atrina sem-
inuda from Northwest San Mati'as Gulf (Patagonia, Argentina)
through histologic analyses and the role of the adductor muscle as
a storage organ of reserves used in gonad maturation.

MATERIALS AND METHODS

Samples were collected at Piedras Coloradas (40°53' S. 65°04'
W), a site located on open waters of the NW coast of the San
Mati'as gulf (see Fig. I ). The sampling site has bare bottom, com-
posed of coarse sand and gravel, covered with sparse bivalve shell
fragments. Tidal current speed is 0.3-0.5 m.s^'. and tides are
semidiurnal (average height: 6.31 m) (Servicio de Hidrografia Na-
val 1994). Monthly average sea surface temperature fluctuates
between 10.4°C in August, and 19.8°C in February, and salinity
varies between 34.19ff and 35.1%r (Fernandez 1989).

Sampling was carried out from October 1999 to October 2000.
on a monthly basis with the exception of the period October to
February when samples were collected fotlnightly. Individuals
were randomly collected by diving along a straight transect, at
depths ranging from 8-12 m. Surface seawater temperature was
recorded at each sampling date.

Seventy individuals of sizes ranging from 150-250 mm total
length were selected from the sampled lot for histologic treatment
and to estimate muscle index (Ml).

Histologic Treatment

The visceral mass of each individual was separated from the
adductor muscle. A section of gonadal tissue (approximately 1
cm') from the posterior-dorsal area was excised from each indi-
vidual. Each tissue section was fixed in Davidson's solution,
stored in 70% alcohol and dehydrated in consecutive baths of
ethanol and 1:1 mix of 1007c. ethanol and xylene. Tissue samples
were embedded in paraffin (56/58°C). Five to seven |j,m sections

479

480

SORIA ET AL.

San Antonio Bay

41° S-

San Matias Gulf

65'^' W

[ South
V Amenca

Las Gnitas
Beach

Piedras

Coloradas

Beach

San Matias Gulf- - - -la

Study Area

Figure 1. Location of the study area at the Northwest coast of the San Matias (lulf. (Patagonia) Argentina.

were made with rotary mierotonie and stained with hematoxiliii
and eosin.

Analysis of the Repriidiiilire Cycle

Histologic sections were examined under light luicroscope. Sex
was determined for each animal.

A scale of gonadal maturity was developed using as a basis a
scale of maturity stages described for other Pinnidae, namely At-
riiui ntaura (Maeda-Marti'nez et al. unpublished manuscript) and
Fiwui nigosa (Noguera & Gomez-Aguirre 1972, Coronel 1981,
Ceballos-Vazquez et al. 2000). Relative frequencies of gonadal
developmental stages were obtained throughout the annual cycle.
Sex ratio was estimated during the period over which mature and
spawning individuals appeared. A Chi-sqiuuc lest was used to
assess differences from the 1:1 sex ratio.

Frequencies Distribuliun anil Mean Size of Oocytes

Oocyte size was determined from histologic sections using an
eyepiece graticule calibrated with a stage micrometer (x400). Five
to eight females were randomly selected from the lot sampled
during each collection date, and at least 100 oocytes (among those
with a clearly visible nucleoli) per female were measured through
the longest axis, transverse to the nucleus, hidividuals with scarce
oocytes and extensive phagocytosis (spent and indifferent indi-
viduals) were not analyzed, following the criteria recommended by
Grant and Tyler (1983a. 1983b).

Gonad Index

A gonad index (GI) that represents the reproductive activity
was estimated using a numerical grading system based on the
maturity stages de.scribed from each monthly collection data (Hef-
fernan et al. 1989). Three categories were established on the basis
of gonad development: I = Indifferent (I) + Spent (VI), 2 = Early
Active (II) + Developing (III), and 3 = Mature (IV) + Spawning
(V). The GI was estimated by multiplying the number of individu-
als belonging to each category by the category score (1 to 3 ). then
summing the values and dividing the resulting value by the total
number of animals in the sample.

Muscle Index

Dry weight of both posterior adductor muscle and remaining
soft tissues from 40 individuals on each collection date was ob-
tained using a drying chamber (85^C) until constant weight was
reached (-72 h). Muscle index was estimated as MI = (adductor
muscle weight/soft tissue weight) x 100. Mean values were cal-
culated for each collection date.

RESULTS

Sex Ratio

A total of 378 individuals were studied (mean shell length =
194.37 mm; SD = 21.49 mm) of which. 175 were females
(46.3%). 124 were males (32.87r). 4 were hermaphrodites ( 1 .Q5''r )
and 75 (19.8%) could not be sexed (Fig. 2). Sex ratio during the
spawning season (1.15 females: 1 male, n = 90) did not differ
significantly (P s 0.05) from the expected 1:1 ratio.

Histologic Analyses

Macroscopic and histologic analysis of gonadal tissues, al-
lowed the authors to establish the following stages according to the
characteristics exhibited during development.

Indifferent

No evidence of gametic development, acini small with total
absence of gametes. Connective tissue is abundant. In this stage it
is not possible to determine sex (Fig. 3).

Externally the gonad is brownish, watery, and flaccid.

Females

Early Active: Acini irregular in shape containing oogonias,
giving place to pievitcllogenic pear shaped oocytes (average = 7.6
\xm SD = 3.2 |xm) attached to the walls of acini. Inteifollicle
connective tissue decreasing (Fig. 4A).

Macroscopically. gametogenic activity is evidenced due to
color and swelling changes. Gonad acquires a pale pink color in
the region next to the disiestive eland.

Reproductive Cycle of Atrina seminuda

481

100%

0%

I S^ 8^

B"" r5 ^

^ jH <N

O 1-^ 1— I

0\

<s

ON

o
o

o
o

I

o

o
o

o
o

<S tH

o

s

o
o

o

o
o

o

o
o

o

o

o

o

i

©

o
o

o

o

O iH

FEMALES

MALES

^

HERMAPHRODITES

INDIFFERENT

Figure 2. Frequency distribution of male, female, hermaphrodites and undifferentiate individuals of .4. seminuda in the period October 1999 to
October 2000.

Developing: Acini increase in size as a result of oocyte ac-
cumulation and growth. Previteliogenic oocytes are found attached
to the wall of acini, also several vitellogenic oocytes can be found
dispersed in the lumen (average = 31.43 p.m, SD = 12.4 jjim).
Acini walls are well defined and interfollicle connective tissue has
decreased (see Fig. 4B).

As gonadic tissue develops, it invades the digestive gland, ex-

• r ^M^ .:

*:~...7''

'.' .^S^ .*X».' -.-.

>:'s,i*-:, .•i.-^;*^-» .y.-i^j^ifv- ' •••■ ,.•• > J

^ .-V ■•>•■.--. • ■■ "■ • - ".:

Figure 3. Photomicrograph of gonadal stages: indifferent stage; scale
bar = 50 urn.

tending towards the posterior region. Gonadal tissue becomes or-
ange in color.

Mature: Acini are distended and fulfilled with ripe polygonal
shaped oocytes (average = 45.6 p.m; sd = 23.7 |j.m); some oo-
cytes remain attached to the walls of acini. Intrafollicle area is
reduced to a minimum. The boundary of acini is not distinguish-
able. Connective tissue is reduced or it is absent (see Fig. 4C).

Gonad reaches its largest area, gonadal tissue is swollen and is
red colored.

Spawning: Acini partially spawned containing few oocytes
that expand and lose the polygonal shape. Phagocytes are present.
Little connective tissue is present. A new proliferation of oocytes
attached to the walls of acini can occur (see Fig. 4D).

Externally the gonad exhibits red color, begins to decrease and
loses swelling.

Spent: Acini are collapsed as a result of oocyte release. Re-
sidual oocytes are irregular in shape; are free in the lumen and
begin to be absorbed. Phagocytes proliferate during this st;ige.
Connective tissue abundant. A new proliferation of oocytes can
occur attached to acini walls (see Fig. 4E).

Macroscopically. the gonadal tissue shows a pink-red color and
watery aspect.

482

SORIA ET AL.

- «^ ^••. ^ :';i ■ O <» -> '■ S

if

-f^.-';^!

Figure 4. Photomicrographs of gonadul stages ol .1. seminiida. Females: ( \l earl> acti\e, (B) de\eloping, (C) mature, (1)1 spawning (E) spent:
and males: iFl earl> active, ((i) developing, (H) mature, ll) spawning and (J) spent: scale bar = 50 jim.

Reproductive Cycle of Atrina seminuda

483

Males

Early Stage: Several primary germinal cells are attached to
the wall of the acini. A variable quantity of spermatogonia is
located along the internal wall of the acini arranged in bands of
several cells. Connective tissue is decreasing (see Fig. 4F).

Externally, this stage is evident due to color change and swell-
ing of gonadal tissue. It acquires a grizzly white color.

Developing: Spermatogenesis exhibits a centripetal evolution
from the internal wall towards the lumen. Spermatocytes and sper-
matids are free and tilling the lumen. Connective tissue continues
decreasing as gamete accumulation proceeds (see Fig. 4G).

Gonadal tissue grows invading the digestive gland, and extends
towards the anterior area. Gonadal tissue acquires a whitish color.

Mature: Acini are distended, filled with spermatozoa with
their tails pointing towards the acini lumen. Spermatocytes and
spermatides conform a thick layer against the follicular walls.
Connective tissue is absent. Boundaries between acini are not dis-
tinguished (see Fig. 4H).

Gonadal tissue is swelling and has a white color.

Spawning: Acini are partially empty, showing a marked de-
crease in the number of spermatozoa. Connective tissue is increas-
ing (see Fig. 41).

Externally, gonadal tissue begins to loose size and swelling,
and has a grizzly color.

Spent: Acini collapsed with few residual spermatozoa, con-
nective tissue is abundant. No evidence of active gametogenesis.
Phagocytes proliferate during this stage (see Fig. 4J).

Gonadal tissue shows a watery aspect and a whitish-grizzly
color.

Hermaphrodites

A. seminudci exhibits two types of functional hermaphrodites,
both equally represented.

Type 1; Gonads exhibit separated female and male acini (Fig.
5A). Externally, it is possible to distinguish male gonadal tissue
and clear reddish female "patches".

Type 2: Female and male gametes arranged inside the same
acinus. Oocytes area located at the periphery of the acinus and
spermatozoa in the center of the acinus (see Fig. 5B). This type
was not detected macroscopically during sampling. The individu-
als exhibit the appearance of a typical female gonad.

Seasonal Change of Reproductive Cycle

During the period of study, superficial water temperature ex-
hibited its highest value (21.2°C) in January 2000 and the lowest
value (9.7°C) on September 2000 (Fig. 6).

The annual reproductive cycle of the total population of A.
seminuda is summarized in Fig. 7a. In October 1999 only early
active (12.5%) and developing individuals (87.5%) are repre-
sented. The highest proportion of mature individuals is found in
late November (82%). sharply decreasing by middle December.
Spawning peaks by the end of December (84%) while, in the
following months, individuals at spent and indifferent stages
gradually increase. From the end of February to March, the ma-
jority of the population is in the undifferentiated stage (72%).
Gonad differentiation begins at middle March involving a small

Fifiure 5. Photoniicrodraphs of hermaphroditic individuals of .4. sem-
inuda. i.X) type 1: separate male and female acini, (Bl type 2: male and
female gametes in the same acini bar = 5(1 nm.

portion of the population. Intense gametogenic activity is evi-
denced through the winter with individuals showing early active
and, in lower proportion, developing gonads. By October 2000, as
opposed to the former year, the majority of the population has
developing gonads (60%) (see Fig. 7a).

Females

In October 1999 all females show developing gonads. The
highest proportion of mature individuals is found at the end of
November (86%), time at which spawning begins. Spawning peaks
in December (86%) and extends through the summer involving
low numbers of individuals (10% in January). Gonad proliferation
begins in March, with gonads in early active and developing stages
represented until October (see Fig. 7b).

Males

In October male gonads are mainly at the developing stage
(75%). during November and December gonads at the stages de-
veloping, mature and spawning coexist in the population. Evacu-
ation begins in November and. at the end of this month. 78% of
mature individuals are detected. Spawning mainly occurs in De-
cember, even when spawning extends along the summer until
May. Spent individuals are found from January to April. Gonads
development begins on April and proceeds throughout the winter
and spring months (see Fig. 7c).

Frequencies Distribution and Mean Size of Oocytes

In October 1999 mean oocyte diameter (MOD) was 27.07 (xm

(s

11.63 |jim). During November and December oocytes grow.

484

SORIA ET AL.

a
H

Figure 6. Surface seawater temperature recorded at each sampling date at Piedras Coloradas.

showing the maximal MOD on November 26 {45.63 |jim; s =
23.74).

In early December oocytes ranging 35-65 ^im decrease in niiiii-
bers, while oocytes of sizes ranging 5-35 jxm, increase. MOD
decreases at this time, reaching 29.47 jjim (s = 15.84). By the end
of December the pattern inverts and the MOD again increases
reaching 41.52 fxm (s = 12 \xm) on December 24. In January and
February 2000 the mode represented by 35-65 |jim oocytes have
diminished markedly and simultaneously a new mode of prolifer-
ating oocytes arises in individuals showing spawning and spent
gonads. This new proliferation is reflected by an abrupt decline in
MOD values that reach 16.11 fxm (s = 12.65) in January, and 14.4
p.m (s = 12.55) in February. This new summer proliferation does
not seem to culminate in a spawning event due to the fact that these
oocytes do not reach the typical mature oocyte size in the season
(Fig. 8). By the end of February there is a total absence of oocytes
in the gonad.

At the beginning of March, MOD shows the minimal values of the
annual cycle (mean = 8.08 |xm: s = 3.98), and the low dispersion of
the data shows the debut of gametogenic activity (see Fig. 8).

Gonad Index

Gonad index exhibits a marked seasonal pattern (Fig. 9). GI
increases at mid spring reaching its highest values by the end of
November and December as a result of the higher numbers of
mature and spawning individuals. Minimal is found by the end of
February when most individuals have undifferentiated gonads. Gl
increases again in March indicating the debut of gametogenesis
(see Fig. 9).

Muscle Index

MI also exhibits a seasonal pattern, showing an increase of
73.6'/f from the end of November, when it has its lower value
(20.64%), to August when it reaches its highest value (35.8%) (see
Fig. 9). The MI shows, during the reproductive season (October to
February), the opposite pattern to the GI. decreasing whilst this
increases. This behavior is held until March when both indexes
increase and remain stable during the winter months and the debut
of spring.

DISCUSSION

A. semiiuula exhibits an annual pattern of reproduction at the
Northwest of San Mati'as Gulf. Gametogenesis initiates at the be-
ginning of autumn, when suiface seawater temperature ( = 19 "O
begins to decrease. A high synchronism in maturity and spawning
is recorded. Spawning takes place during the end of spring (No-
vember to December) when seawater temperature is around 20°C.
By the end of December, most of the population was spawning or
had already evacuated their gonads. Females exhibit a higher syn-
chronism in maturation and spawning than males. Oogenesis be-
gins earlier in autumn and ends earlier in summer than spermato-
genesis. This fact suggests that the small amount of undifferenti-
ated individuals found from June to October can be mainly
attributed to males whose spent stage is displaced on time com-
pared with females.

The analysis of oocyte size supported and facilitated the inter-
pretation of the reproductive cycle of A. seminuda. Oocyte prolif-
eration and growth could be easily identified, suggesting that the
first cohort (December oocytes) is released; meanwhile the new-
cohort of oocytes that proliferates during spawning or in spent
individuals (February oocytes) does not reach maturity and is
probably not released. These summer oocytes are most likely re-
absorbed.

Gametogenesis is defined by exogenous (mainly temperature
and food) and endogenous factors and the relation among them
shapes the pattern of each reproductive cycle (Sastry 1979). A.
iiiciiira from La Paz. Mexico, behaves as many tropical and sub-
tropical species, maturing during the warmer months and spawning
when temperature decreases (Maeda-Marti'nez et al. unpublished
manuscript). On the other hand. P. iiii;i>sa. from Bahi'a Con-
cepcion. Mexico show the inverse pattern, with reproductive ac-
tivity occurrmg during the warmer months (March to November)
and reproductive inactivity in winter (November to February) (Ce-
ballos-Vazquez et al. 2000). Both species show a protracted re-
productive period. A. seminuda shows a similar reproductive pat-
tern to that ai A. pevtiiuihi japonicci from Yoja Bay, Japan (Yoo &
Yoo 1984). which shows a protracted developing period and a
shorter spawning season that coincides with the warmer months.

Reproductive Cycle oe Atr/na seminvda

485

100%

^ 80% H

> o

U ^ 60%

u O

g °" 40%

(a)

20% -

0% -I"— ^'

■■■■

S

^

!S

S5

lii^

T T T T T

T T T T

100% 1

□

indifferent

|»Ml mature

early active mSM developing

spawning ^ spent

Figure 7. Reproductive cycle of A. seminuda at Piedras Coloradas. Relative frequencies of gonadal stages from October 1999 to October 2000.
(a) Total population, (b) females, and (c) males.

486

SORIA ET AL.

^

80

60

40

20

>. 60

S 40

3

u

80
60
40

I

I

ll.

80 -

(a)
7-Oct-99
N=4
n=400

x=27.07
s(l=11.6

■ I ■ ^*^'

80
60 H
40
20
0

L"M

25 45 65

(d)
lO-Dec-99
N=5
n=503
\-=29.47

stl=15.8

(e)
24-Dec-99
N=8
n=751
x=41.52
sd=12.01

80
60
40
20
0

X

(c)
26-Nov-OO
N=5
n=500

x=45.6 40

.d=23.7 ^^,

80
60

0

80
60
40
20
0

80
60
40
20
0

1

(g)
3-Feb-OO
N=7
n=300
\-14.44
stl=12.5

(h)
14-Mar-()0
N=5
n=135
x=8.08
s(J=3.9

(i)
16-Apr-OO
N=5
n=607
x=10.63
s (1=7.03

0)

26-May-OO

N=6

n=606

x=15.04

s(l=13.3

25 45 65

Oocyte diameter

80

60
40

20

80
60
40
20

I

l.ll.

0)
3-Aug-OO
N=8
n=802
x=23.16
s(l=16.9

il.ii

(m)
19-Sept-OO
N=6
n=597
x=15.55
sd=15.5

xl 1 1 1«

(n)
20-Oct-OO
N=8
n=800
s=27.86
sd=15.6

25 45 65

Figure 8. Frequency (%) distribution of oocyte diameter ((jm). N = number of females: n = number of oocytes; x = mean oocyte diameter.

This pattern is also shared by other Northern Patagonian bivalve
species, as Amiantis purpurata (Morsan 2000). Aequipecten tehu-
elcluis (Narvarte 2001). Mytiliis ediilis platcnsis (Trancart 1978)
and Ostiea ptwkluma (Morriconi & Calvo 1979).

The adductor muscle of ,4. seminudu (localK called "callo")

shows marked seasonal changes becoming light in weight and
watery after spawning. The Ml sharply decreases during the start
of the spawning season, suggesting that this specie uses for repro-
duction the reserves stored in the adductor muscle. As the propor-
tion ot non-spawned individuals in the population decreases, the

Reproductive Cycle of Atrina seminuda

487

-Q- MI O- GI

40

35

30

MI

25

20

15

1

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•Q

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■ \

,©■

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1

1

1

1 1 1 1 1 1

1

2 GI

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ON

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o

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o
o

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o

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©

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©

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o

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^

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2

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o

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o

o

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o

Figure 9. Variation of O = mean gonad index (GI), and D = muscle index (MI) from October 1999 to October 2000.

MI initiates a gradual increase. This relation among the MI and the
reproductive cycle has not been described for other members of the
Pinnidae. Maeda-Marti'nez et al. (unpublished manuscript) found
no relation between the MI and the reproductive cycle for Atrina
Duiiirii. a warm temperate sea species that has a different repro-
ductive behavior than 4. seminuda.

Pelecypods exhibit a wide variation in the expression of their
sexuality ranging from strictly gonochoric species to those that are
invariably functional hermaphrodites (Sastry 1979). Hermaphro-
dite individuals are often found in normal gonochoric species. The
cholga paleta/1. seminuda is a stable gonochorist (Giese & Pearse
1974) that shows a low frequency of hermaphrodites (1.05%).

The most common form of hermaphroditism in pelecypods is
"type 1" where separate female acini and male acini occur in the
same individual (Sastry 1979). although the occurrence of both
sexes in the same acini can also exist. The cholga paleta shows
both types of functional hermaphroditism. In the case of "type 2"
hermaphroditism, the male products are arranged in the lumen and
the oocytes are arranged on the walls of acini, suggesting a protan-
dric pattern of gonad development.

Ceballos-Vazquez et al. (20(J0) documented 20.99f of her-
maphroditism in P. lugosa. and Butler (1987) reported Q.V/c of
hermaphrodites in P. bicolor from Australia. Coronel ( 1981 ) de-
scribed for P. rugosa a similar "type 2" hermaphroditism as the
described in our study. Hermaphroditism "type 1" was reported in
Aulacomya aler (Tortorelli 1987). Mytitus califomianus (Young
1941. Young 1945) and Mytilus edulis (Lubet 1959).

Hermaphrodites have not been reported for cogeneric species
A. maura (Maeda-Marti'nez et al. unpublished manuscript), and A.
pccunaui japanica (Yongqiang & Xiang 1987).

ACKNOWLEDGMENTS

We are grateful to Inslituto de Biologi'a Marina y Pesquera
Alte. Storni and the staff of the Laboratorio y Criadero de Molus-
cos Bivalvos (I.B.M.P. Alte. Storni), particularly to Cecilia
Castanos and Myriam Elvira for support during this work. We also
thank Norma Santinelli. Natalia Zais and Sebastian Leal for labo-
ratory and field assistance, and one anonymous reviewer for sug-
gestions made on the manuscript.

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Yongqiang, F. & Q. Xiang. 1988. Studies on ultrastructure of oocyte in
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Yoo, S. K., H. S. Lim, H. Y. Ryu & K. H. Kang. 1988. Improvement of the
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Young. R. T. 1941. The distribution of the mussel (Mytilus californianus)
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Joiinuil oj Shellfish Research. Vol. 21. No. 2, 489-49?, 2002.

THE PHYSIOLOGICAL ECOLOGY OF BLACK-RIBBED MUSSELS, SEPTIFER VIRGATUS
(WIEGMANN) (BIVALVIA: MYTILIDAE) ON A SUB-TROPICAL WAVE-EXPOSED SHORE IN

HONG KONG

QIN-FENG GAO, KA-KEI MAK, AND SIU-GIN CHEUNG*

Department of Biology and Chemistry. City Universit}- of Hong Kong. Tat Chee Avenue.
Hong Kong SAR. China

ABSTRACT Oxygen consumption and ammonium excretion by Septifer virgahis were measured in situ on an exposed rocky shore
montlily from January to December 1999. Condition index and reproductive cycle were determined simultaneously. Spawning was
diphasic, the first phase being recorded in spring (March to April). The second spawning was more prolonged (July to October), but
less intense than the first. Oxygen consumption was temperature-dependent and demonstrated a seasonal pattern with a peak being
recorded from summer to early autumn and with lowest values in winter. Seasonal variation in excretion rate was small with a major
peak in summer, possibly associated with post-reproductive stress and high temperatures. 0;N ratio reflected energy metabolism and
utilization with a peak in spring and early summer (>30) when growth and gametogenesis were resumed. In late summer, spawning
was initiated and temperatures rose high and became lethal. 0:N decreased to minimum values.

KEY WORDS: Septifer. oxygen consumption, excretion. 0:N ratio, mussels

INTRODUCTION

Numerous studies have been reported on the relationship be-
tween environmental variables and physiological processes of bi-
valves and corresponding mechanisms whereby the organisms are
functionally acclimated to their environments (Bayne 1976. Gos-
ling 1992, Dame 1993). Among various factors, exogenous ones
such as food, temperature, and salinity, and endogenous ones such
as size, age, and reproductive activity are imponant in controlling
an individual's physiological processes (Bayne 1976, Bayne &
Widdows 1978, Bayne & Newell 1983, Huang & Newell 2002).

Representatives of the Mytiiidae are highly successful coloniz-
ers of intertidal habitats, playing a significant role in energy flow
and nutrient cycling due to their dense populations and filter-
feeding patterns (Smaal & Prins 1993. Gili & Coma 1998). Among
various mytilids. northern temperate/boreal species of Mytiliis
have received most attention (Seed & Suchanek 1992). Black-
ribbed tiiussels, Septifer virgatiis (Wiegmann) are distributed
throughout the Indo West-Pacific and dominate the mid-littoral
zone, forming a continuous band on exposed rocky shores includ-
ing Hong Kong (Iwasaki 1995. Morton 1995). As the ecological
equivalent of Mvtihis ediilis and M. galloprovincialis on northern
Atlantic shores and M. califuritiamis on northwestern Pacific
shores (Morton 1995), it is surprising that very little is known
about this species. In Hong Kong, 5. virgatiis matures at a shell
length of -15 mm and lives for 4-5 y, although older individuals,
possibly up to 1 2 y of age. occur as solitary organisms lower down
the shore. Two groups of narrowly spaced growth bands are de-
posited annually, one during winter (December to March) and
another in summer from July to October (Morton 1995, Richard-
son et al. 1995). Most adult individuals remain mature year round.
Spawning is divided, however, into two phases, one in spring
(February to March) and another in autumn (September to Decem-
ber). Such a diphasic pattern is suggested to be the result of intense
stress in summer when high rock temperatures at mid-day coincide
with low spring tides (Liu & Morton 1994. Morton 1995). Such
conditions also cause mass mortalities of mussels, barnacles and
limpets (Williams & Morritt 1995). In spite of the above research

♦Corresponding author. E-mail; bhsgche@cityu.edu. hk

on S. virgatiis in Hong Kong and elsewhere (Tsuchiya 1983,
Iwasaki 1995), no systetnatic data have been reported upon sea-
sonal variations in physiological responses and their relationships
with e.xogenous and endogenous factors. The objectives of this
study are to determine the seasonal changes in physiological re-
sponses (oxygen consumption, ammonium excretion) under ambi-
ent teinperature and food conditions as well as body condition and
reproductive cycle. The results provide interesting comparisons
with data for other bivalves, particularly the ecologically equiva-
lent mytilids from temperate/boreal regions.

MATERIALS AND METHODS

Sample Collection and Determination of Hydrographic Conditions

S. virgatiis occurs as a continuous band in the higher intertidal
zone of a wave-exposed shore at Cape D'Aguilar on the south-
eastern extremity of Hong Kong Island. Hong Kong. About 50
individuals of S. virgatiis of shell lengths 14—66 mm were col-
lected monthly from the shore from January to December 1999
(except in Novetnber when sampling was prevented by strong
waves). Epibionts on the shells were removed and individuals were
kept for three days in an outdoor fiberglass tank supplied with
seawater pumped continuously from the study site.

Food conditions of the seawater. i.e., total particulate matter
(TPM: mg P' ). particulate organic matter (POM: tug P' ), particu-
late inorganic matter (PIM: mg 1"') and organic content (f), were
determined using the filter and ash method. Six samples of sea-
water (200 ml each) were filtered through ashed and pre-weighed
25 mm glass fiber filter papers (Whatman GF/C), rinsed with
isotonic ammonium acetate, dried at 90' C for 24 h, weighed to the
nearest 0. 1 mg, then ashed in a muffle furnace at 450"C for 6 h and
reweighed. The concentrations of TPM and PIM were thus mea-
sured directly. POM was calculated by subtracting PIM from TPM
and f was estimated as f = POM/TPM. Temperature (°C). dis-
solved oxygen (mg I"') and salinity (7cr) were also measured dur-
ing each visit.

Oxygen Consumption and Nitrogen Excretion

To determine oxygen consumption rate (Vo,: p-g h"' ). each S.
virgatiis was placed in a separate sealed perspex chamber (300-

489

490

Gao et al.

550 ml, depending on the size of the animal). For individuals
smaller than 20 mm, four to six were put in a container and the
calculated oxygen consumption was divided by their number. Two
empty chambers were used as controls. The sealed chambers were
bathed in a fiberglass tank supplied with seawater pumped con-
tinuously from the sea to reduce the effect of temperature on the
respiratory activity of the contained animals. Each chamber was
sealed for about 60 min, the time chosen according to a prelimi-
nary study such that the oxygen concentration declined neither too
excessively to affect the normal respiration, nor too slightly to
accurately determine variations in oxygen concentration (Wong &
Cheung 2001 ). Initial and final dissolved oxygen values were de-
termined with a YSI DO meter. The oxygen consumption rate was
ineasured as |xg h~' after correction with the control.

Ammonium excretion rate (V^: fxg NH^-N h"') was deter-
mined using the phenolhypochlorite method (Parsons et al. 1984).
Individual S. virgatiis were maintained in separate glass beakers
filled with 200 ml filtered seawater (GF/C) for 1 h and the initial
and final concentration of ammonium measured. Similar to the
oxygen consumption rate measurement, 4-6 individuals smaller
than 20 mm were used and the calculated ammonium excretion
rate divided by the number in each beaker. Two beakers without
animals served as controls.

Oxygen consumption and ammonium excretion rates were
transformed to atomic equivalents and the ratio of oxygen to ni-
trogen (0:N ratio) computed.

Condition Index and Size-Slandardization

After determining respiration and excretion rates, the shell
length of each individual was measured with vernier calipers to the
nearest 0.01 mm. Body tissues were then dissected out and soma
and gonad dried separately at 80°C to constant weight. Tissue dry
weight was measured by an electronic balance to the nearest 0. 1
mg. Body condition was represented by the condition index (CI)
calculated from tissue (TDW: mg) and shell (SHOW: g) dry
weights according to the equation CI = TDW/SHDW (Brown &
Hartwick 1988). Dry gonosomalic index (DGSI), representing the
reproductive cycle, was calculated as the ratio of gonad dry weight
(GDW: mg) to total tissue dry weight (TDW: mg), i.e., DGSI =
GDW/TDW (Lee 1985).

Respiration and nitrogen excretion rates were plotted against
shell length and body weight following the allometric equation, Y
= aX , where Y is the physiological parameters, X is body size,
and a and b are coefficients. To facilitate comparisons of physi-
ological rates in different seasons, physiological rates were size-
standardized according to the following equation:

Ys = (Ss/Sq)'' X Y„ (Strychar & MacDonald 1999)

Where, Y and S were the physiological parameters and bivalve
body size, respectively and standard and observed measurements
refer to the subscripts 's" and "o", respectively. Analysis of covari-
ance (ANCOVA) indicated that the slopes of the monthly allo-
metric equations were unequal, (i.e., no all-year pooled slope
might be regressed) b, therefore, was the coefficient in the above
allometric model derived from monthly data (Packard & Board-
man 1987).

Physiological parameters were frequently standardized with
body weight. One defect of body weight, however, was its seasonal
flexibility compared with shell length, especially when large varia-
tions in the condition index occur (Navarro et al. 1996), to which
the current study conformed. Moreover, it is widely accepted that

ventilation rates, one of the factors regulating respiration, is de-
pendent on ctenidial area, which is highly correlated with shell
length (Jones et al. 1992. Hughes 1969; Bayne et al. 1976). We,
therefore, used shell length instead of tissue weight to standardize
the physiological parameters (Iglesias et al. 1996, Labarta et al.
1997). Physiological rates were standardized to a 40 mm indi-
vidual of S. virgaliis. the dominant size at the study site.

Statistical Procedures

To obtain functional relationships between physiological re-
sponses and environmental conditions, a set of regression equa-
tions was fitted to experimental data, following standard least-
squares procedures. Regression analyses were performed by
simple linear and non-linear procedures, depending on the most
appropriate function to be fitted in each case (Zar 1999). Multiple
regression analysis was conducted when physiological parameters
were correlated with more than one environmental condition, and
the collinearity between independents was tested with coUinearity
statistics of SPSS measured with tolerance value and condition
index (Belsley et al. 1980. SPSS Inc. 1999a, SPSS Inc. 1999b).
Independent(s) highly correlated with others was (were) elimi-
nated from the independent list and the regressive model was
reconstructed until all the intercorrelations between the indepen-
dent variables were removed. Residuals were also analyzed to
check normality, constant variance of predicted dependents and
other necessary assumptions of the regression model. Data were
transformed if necessary to meet the regressive requisites, (i.e.,
normality of datum distribution and homogeneity of variances).

Analysis of variance (ANOVA) or analysis of covariance
(ANCOVA) was used, depending on whether covariance existed
or not, for comparisons among the groups of data. Prior to analysis,
raw data were diagnosed for normality of distribution and homo-
geneity of variances using the Kolmogorov-Smirnov test and Lev-
ene test, respectively. All statistical procedures were performed
with software SPSS, release 9.0 (SPSS Inc. 1999a. SPSS Inc.
1999b, SPSS Inc. 1999c).

RESULTS

Hydrography

Seasonal variations in hydrographic parameters including sea-
water temperature, dissolved oxygen, salinity and seston charac-
teristics are presented in Table 1. Temperature underwent seasonal
variations with high values (~29°C) being obtained in summer,
decreasing gradually to low ones (-17'C) in winter. Dissolved
oxygen level was quite constant throughout the year (except in
December 2000) and was centered around 6.5 mg 1"'. Salinity was
fully marine throughout the year except in summer (June to Au-
gust! when rainfall was highest and value fell to 269J(. Monthly
changes in food concentration (TPM) were small but an excep-
tionally high value was obtained in January, which was six times
that o'l the lowest value (5.1 I mg P') obtained in June and was
probably caused by strong winter monsoon winds and consequent
strong waves (Morton & Morton 1983). The value of f varied from
0.20 in April to 0.50 in October with higher values being obtained
in summer to autumn (June to December).

DGSI and CI

Temporal variations in total tissue, soma, and gonad dry weight
of a 40 mm mussel are shown in Figure 1 and DGSI and CI in

Physiological Ecology of Septifer virgatus

491

TABLE 1.

Seasonal variations in seston characteristics (±SD) including TPM (mg T'), POM (nig T'), PIM (mg I"'), and f. and temperature, dissolved

oxygen (DO I and salinity from January to December 1999.

TPM

POM

PIM

Temperature

DO

.Salinity

Month

(mgl"')

(mg r')

(mgr')

f

("C)

(mg r')

(%o)

Jan

33.82 ±1.33

7.74 ±0.14

26.08 ± 1.42

0.23 ±0.01

17.1

5.90

33.6

Feb

9.07 ±1.58

2.05 ± 0.40

7.02 ± 1.29

0.26 ± 0.04

18.2

7.60

33.8

Mar

5.74 ± 0.65

1.13±0.14

4.61 ±0.56

0.23 ± 0.03

22.1

7.07

34,0

Apr

7.67 ± 1.79

1.48 + 0.29

6.19 ± 1.51

0.20 ± 0.02

24.4

6.99

33.8

May

9.47 ± 3.23

1.96±0.18

7.51 ±3.08

0.23 ± 0.06

25.5

6.13

33.7

Jun

5.11 ±0.39

1.85 ±0.17

3.26 ± 0.39

0.37 ± 0.04

28.9

6.47

24.5

Jul

5.67 ±0.91

2.12 + 0.46

3.55 ± 0.42

0.38 ± 0.05

29.6

6.30

27.4

Aug

6.75+0.91

2.99 ±0.79

3.76 ±0.91

0.44 + 0.09

28.6

6.87

27.0

Sep

6.47 ± 1.10

2.84 ±0.97

3.63 ± 0.57

0.44 ± 0.07

28.0

5.61

32.5

Oct

6.89 ±1.32

3.36 ±0.75

3.52 ± 0,79

0.50 ± 0.05

25,7

6.25

32.0

Dec

7.2 1 ± 0.73

2.59 ± 0.29

4.62 ± 0.57

0.35 + 0.05

17.6

9.90

33.0

Annual mean

9.47 ± 8.05

2.76 ± L79

6.71 ±6.50

0.33 ±0,11

24,3 ± 4.5

6,81 ±1,13

31.3 ±3.3

Figure 2. Soma dry weight remained constant throughout the year
with a single peak in February. Two peaks, however, were iden-
tified for the gonad with a major one occurring from February to
March and a smaller but extended one from May to September.
Seasonal changes in the reproductive cycle were represented by
DGSI for which two peaks were identified, one in winter and the
other in summer (Fig. 2). DGSI was positively correlated with CI
(Pearson correlation coefficient r = 0.442. /; = 402, P < 0.001).
CI was also correlated with both temperature (Pearson correlation
coefficient, r = -0.265, n = 402. P < O.OOI ) and TPM (r = 0.054,
„ = 402, P = 0.276) but not f (r = 0.039, /; = 402. P = 0.436).

Oxygen Consumption, Ammonium Excretion, and Oxygen: Nitrogen
(0:N) Ratio

Allometric equations relating oxygen consumption (Vo,) and
shell length are presented in Table 2. Vo^ was significantly cor-
related with shell length for all months. The slopes of the regres-
sions were significantly different (ANCOVA, F,|,3k() = 5.82, P<
0.001 ) and varied from 1 .30 in September to 2.68 in February. Vo,
of a standard-sized (40 mm) 5. virgaliis varied significantly with
time (ANOVA, DF = 10, 391, F = 34.51. P < 0.001 ) with high
values in summer (May to September) and lowest one in January
(Fig. 3). The regression analysis showed that Vo, was significantly

Jan Feb Mar Apr May Jun Jul
month

Aug Sep Oct Nov Dec

Figure 1. Septifer virgatus. Monthly variations (±SD) of the total tissue
(TDW: mg). soma (SDW: mg), and gonad (GDW: mg) dry weight from
January to December 1999.

affected by temperature and the relationship between respiration
rate, temperature and shell length can be described by the follow-
ing equation:

Vo, = 0.0177 X SL'

= 529.54, P < 0.001)

0.726. F,

With temperature as the covariate. salinity did not significantly

affect Vo, (ANCOVA, F,

1.098. P = 0.295). There was a

negative correlation between Vo, and body condition with a Pear-
son correlation coefficient of -0.14 (;; = 402. P < 0.001 ).

Allometric relationships relating ammonium excretion (V^)
and shell length are presented in Table 3. V^, was significantly
correlated with shell length for all months with the slopes of the
regressions varying significantly with months (ANCOVA, F,u j^,,
= 10.09, P < 0.001). The regression coefficient ranged from 1.13
in September to 2,89 in January. V^, of a standard 40 mm S.
virf^aliis varied significantly with time (ANOVA, DF = 10. 391,
F = 33.13, P < 0.001) with the lowest value being obtained in
April and a maximum one in September (Fig. 3). As compared
with Vo,, the correlation between V,^ and temperature was weak
(P < 0.001) with a Pearson correlation coefficient (r) of 0.21.
Using teinperature as covariate, salinity did not significantly affect

Vn (ANCOVA, F,

.407, P = 0.237). V^. however, was

positively correlated with Vo, (r = 0.138, n = 402, P < O.OI ) and
negatively with CI (r = -0.176, n = 402, P < 0.001).

0:N of a standard 40 mm S. virt^iitiis varied significantly with

160
140
120
100 Q
80
■ 60

u.b -
0 5 -

■

h \

-•-DGSI
-»-CI

0,4 :

O 0,3 ,
a

k'

r

02:

\

K^-i^'^^^^

0,1 ■

1- -

L J

L

Np-— — ■

0-

■

^ , t -^A

40

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

month

Figure 2. Monthly variations (±SD) of condition index (Cll and the

dry gonosomatic index (DGSI) of a standard 40 mm Septifer virgatus

from January to December 1999.

492

Gao et al.

TABLE 2.

Allometric relationships between respiration rate (\o,: fig h') and shell length (SL: mm) as well as monthly variation of V02 of a standard

40 mm SL Septifer rirgatiis.

Regression

Residual sum

Standard Vo,

Month

Equation

sum of square

of square

F value

P value

r square

(mean ± SD)

Jan

Vo,

= 0.0063 X SL--'"'

11.149

3.871

F..,.,

=

95.05

<0.001

0.74

83.2 ± 25.2

Feb

Vo,

= 0.0053 X SL-*"

8.274

2.098

F,.24

=

94.66

<0.001

0.80

107.3 ±34.2

Mar

Vo,

= 0.087 X SL' "-

9.928

1.929

F|.36

=

185.32

<0.00l

0.84

107.5 ±26.1

Apr

Vo,

= 0.739 X SL'-"

4.748

4.407

F,28

=

30.17

<0.001

0.52

162.7 + 65.2

May

Vo,

= 1.346 xSL"»

5.488

2.402

F,,.

=

82.25

<0.001

0.70

228.8 ±65.3

Jun

Vo,

= 0.605 + SL'"'

9.453

2.089

F, ,s

=

158.40

<0.001

0.82

233.0 ± 49.2

Jul

Vo,

= 0.062 X SL--'

20.458

8.779

F,,7

=

86.22

<0.001

0.70

240.4 ± 116.8

Aus;

Vo,

= 0.075 X SL- '^

27.245

4.189

F,..u

=

260.149

<0.001

0.87

251.0 ±77.5

Sep

Vo,

= L895 X SL""

7.339

4.578

F,..,6

=

57.71

<0.001

0.62

243.1 ±80.2

Oct

Vo,

= 0.477 X SL' "

7.568

8.261

•^I.SK

=

34.81

<0.001

0.48

142.8 ±62.3

Dec

Vo,

= 0.01 1 X SL-"

19.168

4.031

F,..,7

=

175.96

<0.001

0.83

142.5 + 46.9

time (ANOVA, DF = 10. 391. F = 9.15. P < 0.0(11 ) with hiyli
values being obtained from April to August and low ones from
September to March (Table 3. Fig. 4). The annual mean value of
0:N was 24.0 with highest and lowest values being 36.1 and 10.5,
respectively. 0:N was significantly correlated with temperature
(Pearson correlation coefficient r = 0.236. n = 402. P < 0.001)
but not with salinity and food conditions.

DISCUSSION

Morton (1995) showed through histological studies that indi-
viduals of S. virgatiis from the same site as the present study
remained mature year round. Spawning, however, was limited to
two periods in spring (February to March) and autumn (September
to December). This matches with our data on DGSI for which the
values decreased in March to April and July to October, showing
that they reflect reproductive cyclicity satisfactorily. The increase
in somatic growth in winter to spring was probably an artefact as
the gonad in S. viifiatiis, like in other mussels, was not a discrete
organ with gonoglands ramifying throughout the body. Complete
separation of gonadal and somatic tissue, therefore, is impossible.
This is further confirmed by the suspension of growth from De-
cember to March with a winter growth ring formed (Morton 1995).
Lee (1988) also found the coupling of gametogenic event and
DGSI for another local mytlid, the green mussel Pcnni viridis. The
bimodal pattern of spawning and recruitment demonslrated in this

400 -,
350-
300-
250-
200 •
150 '

100 ;

50-

-^SV02

1

}

k

-•-SVN

t

\ ^

r T

\

' 1

T 1

ky

^

^ — '

/

^

\,

^

x^--r 1

L • r

I

Y' 1 -L i

18
16
14

<

10 ^

c

8 "O

IT

■■ 6 — "
■ 4
• 2

0

Jan Feb l^ar Apr May Jun Jul Aug Sep Oct Nov Dec

month

Figure 3. Monthly variations (±SD) of oxygen consumption (SV,,,: ng

h"') and ammonium excretion (SV^: pg h"') of a standard 40 mm

Septifer rirj>alus from January to December 1999.

study is common to other local mytilids (Morton 1991) and is
correlated with seasonal changes in hydrography in Hong Kong of
which high summer temperatures (possibly in conjunction with
lowered salinities as a result of enhanced summer rain) dividing an
otherwise single pattern of summer breeding into pre- and post-
summer phases (Morton 1991. Morton 1995). Although DGSI
showed two major peaks with similar amplitude, only one was
identified for CI in February with a less obvious decrease being
obtained from August to December. As growth was suspended in
winter to spring (Morton 1995). a higher CI value indicated that
reproductive output in spring was much higher than in summer. An
extended spawning in summer may help alleviate the problem of
post-reproductive stress and increase survival when high rock tem-
peratures at mid-day coincide with low spring tides (Morton 1995).
Similar observations were also documented for another locally
dominant mussel, Penui viridis, inhabiting a sheltered harbor
(Cheung 1993a).

Food availability is a major factor determining gonadal growth
and reproductive cycle (Bayne 1976, Newell el al. 1982, Kang et
al. 2000). Food availability in this study was the lowest when the
gonad was developing from April to July (Table 1). It was, how-
ever, also the time for body growth that was diphasic, with spring
growth occuiTing from March/April to June and autumn growth
from September/October to December/January (Morton 1995). A
trade-off for limited resources between growth and reproduction
may help explain a lower reproductive output in summer than
autumn/winter when food availability was highest (October to
February).

The oxygen consumption rate of S. virgiitus demonstrated a
seasonal pattern with peaks recorded from summer to early autumn
and low values in winter. A similar .seasonal pattern has been
documented for a number of bivalves (Bayne & Newell 1983,
Cheung 1993b, Hummel et al. 2000, Huang & Newell 2002) with
temperature, food availability, and reproductive condition being
the major determining factors (Babarro et al. 2000). This study
indicated that oxygen consumption in 5. vigaliis was most signifi-
cantly affected by temperature. The effect of food availability was
insignificant, although numerous reports have demonstrated that
oxygen consumption was reduced under low values of food quality
(Babarro et al. 2000) as the result of a lowering of digestive ac-
tivity and erowth (Bavne & Widdows 1978, Bavne et al. 1989).

Physiological Ecology of Septifek virgatvs

493

TABLE 3.

Allometric relationships between NHj-N excretion rate (\\,: pg h ') and shell length (SL: mm) as Htll as monthly variation of excretion rate

(S\\) and 0:N ratio of a standard 411 mm Septifer virgatus.

Regression

Residual

SV^

Month

Equation

sum of square

sum of square

F

V

jlue

P value

r square

(mean ± SD)

0:N

Jan

Vm

= 0.000155 X SL-'*^

14.215

11.624

F,„

=

40.36

<0.001

0.55

7.39 ± 3.08

14.38 ±20. 11

Feb

Vn

= 0.000209 X SL- ^"

8.990

9.511

F,,24

=

22.68

<0.001

0.49

7.24 ± 3.70

17.82+ 13.43

Mar

Vn

= 0.000456 X SL- "

17.532

20.885

F,,.

=

30.26

<0.001

0.46

7.05 ± 4.32

22.61+21.76

Apr

V^,

= 0.0259 X SL' '"

4.390

5.488

F,.2B

=

22.40

<0.001

0.44

4.73 ± 2.05

36.10 ±19.09

May

Vn

= 0.0270 X SL' ■•'

6.008

2.402

F,.,6

=

90.23

<0.001

0.71

5.80+ 1.66

34.50 ±15.63

Jun

Vn

= O.OllSxSL'"

10.847

4.359

F,„

=

87.10

<0.001

0.71

7.13 ±2.42

32.03 ± 13.73

Jul

Vn

= 0.00307 X SL-""

18.248

5.531

F,,7

=

122.07

<0.001

0.76

7.29+2.91

34.88 + 32.92

Aug

Vn

= 0.00391 X SL-"

25.270

12.492

Fl,40

=

80.92

<0.001

0.67

10.53 ±4.96

29.92 ± 30.37

Sep

v>,

= 0.245 X SL'"

5.520

5.946

F,,,,

=

33.42

<0.001

0.48

16.93 ±6.26

14.90 ±9.50

Oct

Vn

= 0.00365 X SL- -"

15.892

4.290

F,,,s

=

140.76

<0.001

0.79

12.96 ±4.51

10.52 ±5.04

Dec

Vn

= 0.0295 X SL' "

6.710

1.791

F,,7

=

138.62

<0.001

0.79

7.91 ± 1.68

16.04 ±4.58

Such a discrepancy may be due to small niictuations In food avail-
ability in this study when particulate organic matter was show n to
vary from 1.13 to 3.36 mg P' (except in January). Significant
correlation between reproductive activity and oxygen consumption
have been reported by a number of authors. Bayne and Widdows
(1978) obtained a significant correlation between oxygen con-
sumption rate and gametogenic index and not with temperature tor
Mvtilus edulis. Similar results were reported for the cockles Cenis-
todemm edule by Newell and Bayne ( 1 980), whereas other studies
have reported significant correlation between oxygen consumption
and temperature and reproductive period (De Vooys 1976. Iglesias
& Navarro 1991, Smaal et al. 1997). As the gametogenesis of S.
virgatus covaried with temperature and growth was resumed in
spring, it is difficult to distinguish the relative importance of tem-
perature, growth and reproductive activity in determining the oxy-
gen consumption rate in spring. Oxygen consumption rate, how-
ever, was lower in winter when the gonads were developing.

Unlike oxygen consumption, the correlation between excretion
rate and temperature was weak. A number of workers have docu-
mented a close relationship between excretion rate and reproduc-
tive cycle (Bayne & Scullard 1977. Bayne & Widdows 1978.
Smaal et al. 1997, Babarro et al. 2000). Such correlations were
suggested to be the result of a heavy reliance on protein catabolism
for energy when mussels are reproductively ripe and have low

■ 80
■70
•60

■ 50
.40°

30

' 20

10

0

Jan Feb Mar Apr t^ay Jun Jul Aug Sep Oct Nov Dec
month
Figure 4. Relationships between temporal variations (±SD| in DGSI
and 0:N ratio of a standard 40 mm Septifer virgatus from .lanuary to
December 1999.

0.4-,

-♦-DGSI

"

0.35-

r^^

y

-•-0:N

0,3 ■

\ , /

N

k

■

0.25-

L\

r/

\ ^

i -

; 1

" \

A-

'

<

\

^\X

0.15]

■■

/ ^ ^ ^

k

1

0,1 -

\^ ^ X"

■ ,

0.05-
0 -

^^•"^"^

glycogen content (Bayne & Scullard 1977). Spawning started in
August when excretion rate increased abruptly. As glycogen stores
are low during that time, energy for maintenance was derived from
substantial protein and ammonium production increases. This is
further confirmed by the coupled oscillation between DGSI and
0:N ratio from June to December (Fig. 4, see later). The problem
of post-reproductive stress is aggravated by high rock temperatures
at midday coinciding with low spring tides (Morton 1993). Such
stresses also resulted in high mortality of liinpets (Williams &
Morritt 1995), barnacles and 5. \irv,atus (Liu & Morton 1994).
Temperature started to decrease in October and there was a sus-
pension of spawning and a resumption of growth; excretion rate
also started to decrease and 0:N ratio increased.

A low 0:N ratio (<20) has been used as an indicator of nutri-
tional stress for marine bivalves (Bayne et al. 1985) as it shows an
increased reliance on protein as a catabolic substrate rather than
carbohydrates and lipids (Huang & Newell 2002). It may also be
related to the greater demand for dietary carbon than nitrogen so as
to preserve carbon based energy for utilization in seasons when
food resources are low (Kreeger 1993, Huang & Newell 2002).
High 0:N ratios (>30) coupled with gametogenesis and body
growth in spring (April to June), resource demands for gametoge-
nesis, therefore, were mostly derived from temporal nutrition ac-
quisition instead of body reserves. As stress became intense in
summer, growth was suspended and spawning was initiated and
there was a heavy reliance on protein catabolism for energy re-
sulting in a low 0:N ratio (Fig. 4). When growth and gametoge-
nesis resumed in autumn as temperatures decreased, the 0:N ratio
increased gradually and peaked in spring.

A number of studies have demonstrated the significance of
reproductive activity to oxygen consumption, ammonia excretion
and 0:N ratio for species with a well-defined reproductive cycle
such as Mytilus edulis (Bayne & Widdows 1978. Widdows et al.
1984. Smaal et al. 1997) and Mytilus galloprovincialis (Navarro et
al. 1991, Babarro et al. 2000). Being a subtropical species with a
diphasic spawning pattern, seasonal variations in energy metabo-
lism and utilization by S. virgatus are satisfactorily reflected in
0:N ratio and are closely related to reproductive cycle, in addition
to environmental influences such as temperature and food avail-
ability. Although oxygen consumption is most significantly af-
fected by temperature, excretion rate is mainly influenced by

494

Gao et al.

growth and nutritional stress associated with intense heat and re-
productive activity.

ACKNOWLEDGMENTS

The worl< described in this study was substanlially supported
by a grant t'roni the Research Grants Council of the Hong Kong

Special Administrative Region. China (CityU Project No.
9040279). The authors thank Professor Brian Morton for improv-
ing an earlier draft of this manuscript and the staff and graduate
students of the Swire Institute of Marine Science, The University
of Hong Kong for their assistance in facilitating the experiments
conducted in their laboratory.

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Jouniul of Shellfish Research. Vol. 21. No. 2. 497-501. 2002.

MICROALGAL FOOD OF THE RIBBED MUSSEL AULACOMYA ATRA (MOLINA, 1782) IN

GOLFO NLEVO (PATAGONIA, ARGENTINA)

NESTOR FERNANDO CTOCCO* AND ANA MARIA GAYOSO

Centra Nacional Patagonia). CONICET. Boulevard G. Brown s/n. 9120 Puerto Mudryn.
Chuhut. Argentina

ABSTRACT StoniaLli contents ol the ribbed mussel Auhuomya alia and phytoplanklon samples from the west coast of Golfo Nuevo
(Chubut Province. Argentina) were analyzed monthly between August 1997 and December 1998. Twenty-five taxa of microalgae
ranging from 9 to IM (xm in size were found in stomach contents along with other miscellaneous items. A marked .seasonal variation
of microalgal food was not observed in Aiilacomya atra. Diatoms such as Paralia siikata and Thalassiimra sp. were observed year
round. Variation in the benthic to total species ratio revealed that during most of the year benthic and pelagic microalgae contributed
equally to the diet of the ribbed mussel. In contrast, during dinotlagellate blooms, as occurred during late spring and summer,
dinotlagellate cells dominated the ribbed mussel stomach contents. This findmg is of special importance in relation to the recurrent
blooms of the PSP causative agent, Alexamlniim lamarense. in the Golfo Nuevo.

KEY WORDS:

ribbed mussel. Aiilaconmi aim. food resources, phytoplankton. Southwest Atlantic. Patagonia

INTRODUCTION

Mussels are suspension feeders. Quality and abundance of food
affects growth rate, gonad development and survival of bivalve
mollusks (Bayne & Widdows 1978, Newell et al. 1982, Berg &
Newell 1986). It is generally assumed that suspension-feeding spe-
cies rely on phytoplankton as their main source of energy. Yet it
has been demonstrated that benthic species can also play an im-
portant role in the feeding ecology of various bivalves (Mikulich &
Tsikhon-Lukanina 1981. Tsikohn-Lukanina 1982). In fact, it is
possible to find benthic and pelagic food species equally repre-
sented in the guts of bivalves from shallow waters (Shumway et al.
1987). However, several authors have pointed out that food selec-
tion occurs within in bivalves (Shumway et al. 1985, Sidari et al.
1998).

The ribbed mussel. Aiilacomya atra (often incorrectly cited as
Aiilacoima aier according to Cazzaniga. 1994) (Molina. 1782).
locally named cholga, supports shellfisheries in the northern Pat-
agonian gulfs of Southwest Atlantic (Ciocco et al. 1998). It is one
of the most common bivalve species around the coasts of southern
South America (Fig. I), ranging northwards to San Mati'as Gulf
(41°S) on the Atlantic and to Peril along the Pacific (I5°S).

The quantity and quality of food available is a major limiting
resource for suspension feeding organisms (MacDonald & Thomp-
son 1985. MacDonald & Thompson 1986, Chauvaud et al. 1998).
Until now information on the specific food items used by AiiUi-
comva atra was only available for the Chilean coasts (Guzman &
Campodonico 1975, Osorio et al. 1982). This is the first report on
feeding in Aiilacomya atra along the Argentina coast.

Golfo Nuevo, situated at 43°S on the Patagonian coast of Ar-
gentina (Fig. 1 ), is a rough elliptical, semi-enclosed body of water
in contact with the Southwestern Atlantic Ocean through a 17-km
wide strait. Phytoplankton is dominated by diatoms and dinotlagel-
lates (Gayoso 2001 ). Recurrent blooms of the toxic dinotTagellate,
Ale.xandrium tamarense (Lebour) Balech. have been documented,
resulting in human health hazard (Esteves et al. 1992. Ciocco
1995, Gayoso 2001 ). Other harmful dinofiagellate species includ-
ing Proroceiilnim lima (Fhrenberg) Dodge (Gayoso & Ciocco

2001) and Diiioplivsis acuminata Claparede & Lachmann have
also been found in the area adding potential risks for human health
and marine resources. In this study we describe the stomach con-
tents of ribbed mussels in order to: (1) characterize the nature of
the food items: (2) assess seasonal variation of food items; and (3)
provide preliminary information about the relationships between
microalgal species in water samples and algal cells in the stomachs
of the ribbed mussel.

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*Corresponding author. E-mail: cioccofe'cenpat.edu.ar

70 60

Figure 1, Map of South America showinj; dislriljution «X Aulacomya
ultra (gray circltsl and location of sampling area (arrow).

497

498

ClOCCO AND GaYOSO

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MiCROALGAL FOOD OF AULACOMYA ATRA

499

MATERIALS AND METHODS

Samples of phytoplankton and ribbed mussels (Aulacomya
atra) were collected monthly from August 1997 to December 1998
at a station located in the west coast of the Golfo Nuevo (42°46'S;
65°02'W). On each sampling date, several adult ribbed mussels
(length range: 55.2-1 16.4 |a.m) were collected by divers from the
bottom (approximately 18 m depth). The mussels were transported
to the laboratory and the digestive gland of six randomly chosen
individuals were dissected. For microscopic analysis, stomach
contents were diluted with filtered seawater and observed in a
Sedgwick-Rafter chamber using a compound light microscope.
Microalgae present in the stomach contents were identified and
counted. Water samples (200 mL) were collected with a Van Dorn
bottle at 0 and 15 m depth and immediately fixed in LugoFs iodine
solution. For quantitative phytoplankton analyses. 50 mL sub
samples, using mixed 0 ni and 15 m depths, were settled in a
chamber and counted using an inverted microscope.

RESULTS

Twenty-three taxa of microalgae. ranging in size from 9-231
IJim, were found in the stomach contents of ribbed mussels (Table
1 ). The diatoms PaniUa sulcala (Ehrenberg) Cleve and a Thalas-
siosira species characterized by linear areolae arrays and a diam-
eter ranging from 26.4 to 32 |jLm. were the most frequent species
(77% of all samples). A group of unidentified benthic pennate
diatoms that included more than one species also showed high
frequency. The dinotlagellate Piorocentnim mkaiu Ehrenberg
was the most abundant organism found in the ribbed mussel stom-
achs. During the summer (January to February, 1998), mean den-
sities up to 225 cells per individual mussel were observed. On
November 17 and December 2. 1998, cells of the toxic dinoflagel-
late. AleMindniim tainarense. were found in great number (mean
values up to 105 cells per individual mussel). Both dinotlagellate
species were observed mostly as intact cells. Additional items were
observed including macroalgal fragments, foraminifera and empty
tinlinnid loricae (mainly StenosemeUa avellana Meunier, Heticos-
tomella siihidatu Ehrenberg and Tiiuinopsis gracilis Kofoid &
Campbell). Broken cells of the diatom Dityhim brightweUii. (T.
West) Grunow ex Van Heurck. and fragments of pennale diatoms
were also frequently found.

Phytoplankton seasonal variation was characterized by two dia-
tom peaks, spring and autumn blooms (Fig. 2A), dominated by
Chaetoceros socialis Lauder. C.curvisetus Cleve, C.diadema
(Ehrenberg)Gran and C. didymus Ehrenberg, Skeletonema cos-
tatiim (Greville) Cleve, Pseudonitzchia piingens (Grunow ex
Cleve) Hasle and Gniiuirdia dclicatiila (Cleve) Hasle. Dinoflagel-
lates tended to be abundant during spring and summer (Fig. 2B),
when Pidiocciilnim inicans was the most abundant species. Other
dinotlagellates included Pyrophaciis horologiwn Stein and Dino-
physis acuminata. Ceratiwn horridmn (Cleve) Gran, C. fusus
(Ehrenberg) Dujardin and C tripos (O. F. Miiller) Nitzsch were
also present during the autumn. The toxic dinotlagellate A. taina-
rense, responsible for PSP toxicity in the area, was found in phy-
toplankton samples taken in November to December, 1998 (up to
1.5 X 10' cells L"'). Phytoplankton was dominated during late
spring (late October to December 1997) by small forms of
phytoflagellates, less than 10 |xm in diameter, including Phaeo-
cystis spp., coccolithophorids and small-unidentified monads.

All the microalgal taxa identified from the guts of ribbed mus-
sels were also found in water samples. Many of them, however,
were true benthic species and only occasional component of the

O

o
o

X

CD

5?

r,.|,^r.h~aooooooocooooo
= OOou-S5->^tnz

Figure 2. Cell number variation of diatoms (A) and dinoflagellales (B)
in tiie water samples of Golfo Nuevo and ratio of benthic species over
total species of Aulocomya altra guts in each sampling dale (C),

phytoplankton community. The average ratio of benthic species to
total species numbers from 6 stomachs per sampling date (Fig. 2C)
widely varied throughout the year; it varied from less than 0. 1 in
summer (February 1997) and late spring (November to December
1998), during Prorocentrum micans and Alexandriwn tamarense
blooms, to a ratio of almost 1 in May 1998. Percentages between
0.4 to 0.5 were commonly registered (Fig. 2C). Moreover, some of
the more representative phytoplankton taxa and algal groups, such
as Chaetoceros spp., Skeletonema costatum and phytoflagellates,
were never found in the stomach contents.

DISCUSSION

The size range of the food items detected in the stomach con-
tents in this study (9-23 1 ixm) is similar to that reported for A. atra

500

ClOCCO AND GaYOSO

from Arica, Chile (21-270 |j,m, exceptionally up to 725 ixm; Oso-
rio et al. 1982) and other species such as Myrilus eiliilis Linnaeus
(10-250 (j.m; Newell & Shuniway 1993) and inshore Placopecten
mageUanicus (GmelinK8-240 \i.m: Shumway et al. 1987) from the
Gulf of Maine. A wider size range of food items were reported in
offshore Placopecten iinigclUinicus (10-350 (jlui; .Shumway et al.
1987) and Patinopecten yessoensis (Jay)(9-950 |jLni; Mikulich &
Tsikhon-Lukanina 1981). Silicotlagellates. foraminifera, and spe-
cially tintinnids were other items observed in ribbed mussel stom-
achs in this study. The three groups are also miscellaneous items
reported in A. arm from Arica. Chile (together with abundant
detritus and other items; Osorio et al. 1982) and gut contents of
other bivalves such as Clihiinxs varia (Linnaeus) (foraminifera.
Hunt 1925). Placopecten ntagellanicus (silicotlagellates, Shum-
way et al. 1987), Aequipecten tehuelclms (d"Orbigny)(silico-
tlagellates, Vernet de Hall 1977), Mytilus echilis (silicoflagellates.
Newell et al. 1989) and Mytilus galloprovincialis Lamarck (sili-
coflagellates, Sidari et al. 1998).

Two diatom peaks are observed as a recurrent feature of the
annual phytoplankton cycle in Golfo Nuevo (Gayoso 2(MI ). This
common characteristic was not reflected in the annual variation of
microalgal food of the ribbed mussel. Among the most represen-
tative species of the diatom bloom. Skeletonema costatum. Clia-
etoceros socialis. C.curvisetus. C.diademu, C. didynuis and
Pseiido-nhzchia piingens. only the latest, was observed in the
ribbed mussel gut (Table I ). Absence of dominant pianktonic mi-
croalgae has been reported for other bivalve species such as the
absence of Chaetoceros spp. in Placopecten mageUanicus (Shum-
way et al. 1987) and S. costatum in Aequipecten tehuelclms from
Golfo San Jose, Argentina (Vernet de Hall 1977). These cases
suggest a probable selective feeding capacity as indicated for oth-
ers bivalves (Field 1922. Vernet de Hall 1977, Shumway et al.
1985. Shumway et al. 1987. Sidari et al. 1998), The absence of
phytoflagellates in A. atra stomach contents may be explained by
quick digestion of some small (mainly <10 (xni in diameter) algal

species ("Shumway et al. 1987). reduced retention efficiency of
particles less than 7 |jim (MLihlenberg & Riisgard 1978) and/or
difficulties in identification of micro-flagellates in bivalve stomach
contents due to the fragility of these small cells (Vernet de Hall
1977).

A marked seasonal variation was not observed in the microal-
gal food of Aulac(miya atra. Diatoms such as Paialia sulcata and
Thalassiosira sp. were observed year round. Variation in the ratio
of benthic over total species demonstrated that during most of the
year benthic and pelagic microalgae contributed equally to the diet
of the ribbed mussel in the Golfo Nuevo. In contrast, during di-
noflagellate blooms, as occurred during late spring and summer,
the percentage of benthic species dropped to less than 10% (Fig.
2C). The fact that P. micans and A. tamarense were found mostly
as intact cells in the stomachs suggests, however, that they are not
assimilated by the ribbed mussels. The capacity of concentrate
large amounts of dinoflagellates was also observed in Mytilus gal-
loprovincialis during a bloom of Dinophysis (Sidari et al. 1998). In
A. atra from Magellanic Region (Southern Chile) Guzman and
Campodonico (1975) idenlified Alexandrium catenella (Whedon
& Kofoid) Balech (formerly Gonyaulax catenella) in A. atra from
Punta Arenas, Chile. Lembeye (1981) reported the toxic di-
noflagellates Alexandrium catenella and Diiu>pliysis acuta Ehren-
berg (DSP causative agent) in stomach contents of A. atra from
Chilean Magellan waters. Great densities of Alexandrium tama-
rense can explain the high toxicity of the ribbed mussels when the
species is blooming in the Golfo Nuevo (Esteves et al. 1992,
Andrinolo et al. 1999). Toxicity values up to 1,750 |jig STXeq/100
g were reported in A. atra from Golfo Nuevo by Andrinolo et al.
(1999).

ACKNOWLEDGMENTS

The authors thank the anonymous reviewer for the constructive
criticisms that helped to improve our work. This study was funded
by CONICET, Republica Argentina, PIP 4269-96.

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Esteves. J. L., N. Santinelli, V. Sastre, R. Diaz & O. Rivas. 1992. A to.xic
dinoflagellate bloom and P.SP production associated with upwelling in
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Field. 1. A. 1922. Biology and economic value of the sea mussel Mytilus

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from North-Patagonian coastal waters (Argentina) a.ssociated with a

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copeclen mageUanicus. 1. Growth rates of shell and somatic tissue.

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food availability on the ecological energetic of the giant scallop Pla-
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Muhlenberg. F. & H. U. Riisgard. 1978. Efficiency of particle retention in
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Journal of Shellfish Research. Vol. 21, No. 2. 503-5US. 2U02.

TRIPLOID INDUCTION OF MYTILUS EDULIS USING 6-DIMETHYLAMINOPURINE

JOHN BRAKE,' JEFFREY DAVIDSON,^* AND JONATHAN DAVIS^

' Coastal Oregon Marine Exp. Station. Hatfield Marine Science Center. 2030 S. Marine Science Dr.,
Newport. Oregon 97365; -Department of Health Management. Atlantic Veterinary College, University of
Prince Edward Island. 550 University Avenue. Cluirlottetown. PEL Canada. CIA 4P3: ^Baywater Inc..
15425 Smoland Lane. Bainhridge Island. Washington 981 10

ABSTRACT The induction of tnploidy in Myiilus eduli.s. using 6-dimeth\iaminopurine (6-DMAP). was investigated as a potential
method of providing high quality sterile product in Prince Edward Island (PEl), Canada. Initial results indicated induction by blocking
the second polar body would require a treatment starting time of 21 min post-fertilization at lOX. Poor yields in subsequent spawns
caused a re-examination of these factors by investigating the meiotic events post-fertilization. The resulting description was used to
develop a series of suitable treatment time windows for testing inducing techniques. Triploids were successfully produced with the use
of 6-DMAP at various concentrations and at various times post-fertilization. The optimal investigated procedure for induction of
tnploidy in Mytihis ediilis was the treatment of eggs at 20°C with 400 ^JLmol/L of 6-DMAP starting at 24 min post-fertilization for a
treatment time of 10 min. This treatment yielded 83.1% triploids in induced samples with a survivorship of 1.39% to the D- vehger
stage. The investigated methods for producing triploid Mytilus edidis are not yet optimized to commercial levels. This work forms a
basis for further work in the optimization of this technique.

KEY WORDS: 6-dimethylaminopurine. meiosis. mussel, Mytilus edulis. triploid

INTRODUCTION

The Prince Edward Island (PEI) mussel aquaculttire industry is
a large contiibutor to Canada's aquaculture sector, accounting for
greater than 80% of all Canadian mussels over the past two de-
cades. One area of concern to the PEI mussel aquaculture industry
is the harvesting and marketing of mussels during, and just after
the spawning season. Ripe mussels (near spawning) can spawn due
to the stress of shipping. Also, mussels that have recently spawned
have significantly reduced meat yield making them less appealing
to the consuming public. In PEI. the marketing of non-spawning
triploid mussels during this period would alleviate these problems
and allow the industry to market a high quality product year round.

Triploids are organisms with three sets of chromosomes, dif-
fering from most other sexually reproducing organisms that have
two sets (diploids). Triploids also do not normally undergo meio-
sis, as the three sets of chromosomes cannot property synapse.
Therefore triploids have poorly developed gonads and produce far
fewer gametes than diploids (Allen 1988). Tnploidy is an aberrant
genetic state in molluscs, and most organisins, and therefore must
be produced artificially. The main objective of this study is to
develop and optimize a triploid induction strategy. The important
considerations for developing a triploid-inducing technique for
commercial use are effectiveness and safety. 6-dimethylaminopu-
rine (6-DMAP) is far less dangerous than cytochalasin B (CB) and
equally effective in most cases (Desrosiers et al. 1993). therefore
it was selected as the chemical induction agent for testing in this
study.

In molluscs, triploid induction occurs through the use of an
external treatment on a fertilized egg. The goal of coinmercial
triploid induction of shellfish is to produce a high percentage trip-
loid cohort with high survivorship throughout the hatchery stage
(Allen et al. 1986). Imperative to the induction of both a high
percentage of triploids and a high survival rate in a cohort of
triploid mussels is the determination of the proper time at which to

*Corresponding author. Tel.
E-mail: davidsonfeupei.ca

-f 1-902-566-0666; Fax; -f 1-902-566-0823;

begin triploidy induction, and the treatment duration. In the past,
the stage of development when 50% of the eggs showed polar
body formation has been used as a common cue to start triploid
induction treatment in oysters (Allen et al. 1989). Although the
duration of the treatment varies between hatcheries, this treatment
has a finite duration, the optimal time of which is determined by
the timing of meiotic events. The time, from which a triploid
induction treatment may be initiated until it is no longer effective.
may be referred to as the induction window. One of the objectives
of this study is to determine the optimal time for triploid induction
initiation in Mylihis edidis embryos. Another objective of this
study is to define the induction window for Mylihis edidis using an
effective concentration of 6-DMAP and a standardized set of pa-
rameters. This work serves as the foundation for the future opti-
iTiization of the triploid-inducing technique (using 6-DMAP) in
Mytilus edidis.

MATERIALS AND METHODS

All experiments were conducted at the Ellerslie Shellfish
Hatchery (ESH). in Ellerslie, PEI. Flow cytometry, to define
ploidy level in samples, was performed at the University of Wash-
ington in Seattle. Washington, and at the Whiskey Creek Shellfish
Hatchery in Tillamook. Oregon. Broodstock were obtained from a
mussel lease located in Lennox channel, near Lennox Island. PEI.
These animals were conditioned for 6 weeks, during which time
they were batch fed a mix of cultured microalgae at water changes
every 2 days. Spawning was induced by a -i-IO°C thermal shock.
Fertilization occurred within 2 h of gamete collection so as not to
compromise gamete quality.

Description of Polar Body Extrusion Timing

Temperatures of 15°C and 20°C were investigated. 20°C was
selected as it is an easy achievable temperature in most shellfish
hatcheries, and 1 3°C was selected, as it is closer to the temperature
during the natural local spawning in this species, and also common
in many shellfish hatcheries. Approximately 3 million eggs were
pooled from 3 females for use in each temperature treatment.
Sperm was also pooled from 4 individuals for egg fertilization.

503

504

Brake et al.

Figure I illustrates the sampling procedure used for ttiis experi-
ment. Eggs were placed on a 20 |xm sieve within a 20 L bucket of
I |jLm filtered sea water (FSW). This bucket was placed in a water
bath to keep the eggs and surrounding sea water at 20°C. Eggs
were sampled by raising the screen out of the water and aspirating
eggs from the screen (without an excess of sea water) and imme-
diately returning the screen to the bucket (Fig. 1). The aspirated
eggs were immediately placed into a pre-loaded 2-ml microcen-
trifuge tube containing 2*^ buffered formalin fixative. The sample
was mixed well so as to fix the eggs rapidly. The eggs were
sampled prior to fertilization, at fertilization, and at subsequent
3-min intervals up to 2 h post-fertilization. Simultaneous to this
sampling, the same procedure was being followed using the same
egg and sperm pools in a water bath of 15°C. Under light micros-
copy, polar bodies in the fixed egg samples were visible using a
0.5% aceto-orcein stain (Guo 1991). For each sampled time, the
number of eggs (out of the first 50 visualized on the slide) showing
visible polar bodies was recorded. When this number reached 25
(50% of eggs), the target had been reached.

Effect of 6-DMAP Concentration on Percent Triplvid Induction

The sperm from 6 males was pooled for this experiment. The
sperm was used to fertilize the pooled eggs from 4 females. The

Eggs developing on a
20nm sieve in FSW.

B

Sieve is removed,
and eggs are
aspirated without
FSW.

c

Eggs are placed in a
pre-loaded
microcentrifuge tube
and mixed well for
rapid fixation.

D

Sieve is placed back in
FSW to allow for more
egg development prior
to next sampling. ^'

Figure 1. Schematic of the experimental sampling procedure used in
the description of pnlar body extrusion timing and the description of
meiotic events.

pooled eggs were counted and loaded into IL plastic beakers at a
density of 100,000 eggs per beaker (100 eggs per ml of FSW).
Treatments were replicated three times in this experiment and
consisted of eight concentrations of 6-DMAP, at each of the two
investigated temperatures. The tested 6-DMAP concentrations
were similar to those used by Desrosiers et al. ( 1993) who ranged
concentrations from 0 to 600 jjimol/L. The same range was se-
lected for experimentation in this case as the resolution in their
results gave clear indications of the effect of chemical concentra-
tion on percent triploid induction. For each treatment the appro-
priate amount of 6-DMAP was weighed and placed in a labeled
aluminum foil packet. When a treatment was initiated the chemical
was emptied into the beaker and rapidly mixed into the water until
it was dissolved. Egg suspensions were mixed every few minutes
throughout the treatment duration to ensure adequate chemical
contact with all eggs. Water baths of 1 5 C and 20°C were used to
keep egg suspensions at the proper temperature. Eggs were al-
lowed to acclimate to the proper temperature for 30 min just prior
to fertilization for optimal induction (Allen et al. 1989). All eggs
were treated at 21 min post-fertilization for 20 min duration. At the
conclusion of the treatment duration the eggs were thoroughly
rinsed, placed onto a 20-|xm sieve, and resuspended into their
respective treatment beakers with new FSW. The egg suspensions
were allowed to incubate for 24 h, then samples were taken. Em-
bryos that had grown to the D-veliger stage were collected by
rinsing them onto a 64-|jim sieve. Sampled embryos were trans-
ferred into a 15 ml centrifuge tube filled with FSW. These samples
were used for ploidy determination.

Description of Meiotic Events

Eggs from 4 females were pooled for use, and sperm from 7
males was pooled for fertilization. This experiment was only per-
formed on one sample of pooled fertilized eggs held at 20°C. A
water temperature of 20°C was used as this was shown in the
results of the description of polar body extrusion timing (the first
experiment) to have more synchronous meiosis, (therefore it
should be more effective than I5°C for triploid induction).
Samples of eggs were taken every minute from fertilization until
one hour post-fertilization. Each of the first 50 eggs observed for
every sample were categorized as being in a particular stage of
meiosis. The first 50 readily scored comprised the sample for each
time investigated.

Evaluation of Several Treatment Windows

Sperm from 5 males was pooled to Icrlili/e the eggs pooled
from 12 females. All treatments were standardized using a tem-
perature of 20°C and a 6-DMAP concentration of 400 p.mol/L and
100 eggs/ml of FSW. The treatments investigated included differ-
ent treatment starting times based on the observed meiotic events
in the previous experiment. Four treatment durations of 5, 10, 15,
and 20 min were investigated along with four treatment starting
times of 24, 27, 30, and 33 min post-fertilization. Survivorship was
assessed by taking 1 5 ml samples of each culture at day 3 and
fixing them with Lugol's iodine. A survivor was later scored as a
fixed D-veliger that showed food in the gut, under light micros-
copy. Samples were taken for both ploidy and sur\'ivorship.

RESULTS

Description of Polar Body Extrusion Timing

Table 1 summarizes the results. In the 20'^C group polar bodies
appeared as early as 15 min post-fertilization and showed the mean

Triploid Induction of Mytilus epulis

505

TABLE 1.
Percent of polar body I extension in the 15 C and 20 C groups.

Time
(Min)

15 C

ICC

(1

3

6

9

12

15

18

21

24

27

2 ±2.0
30+ 10.58
38.67 ± 14.05
63 ±15.56

2.67 ± 1.15
23.33 ± 3.05
50.67 ±4.16

desired score of 50"^ polar body display at 21 min ( 50.67 Vr ±4.16)
post-fertilization. Samples taken from the 15°C group showed a
slower development of polar bodies, with none appearing until 1 S
min post-fertilization. All samples from the 15"C group showed
50% polar body display by 27 min post-fertilization (63% ±
15.56). The I5°C group was also less synchronous, with replicates
showing a standard deviation of as much as 15.56% (of eggs
.showing polar bodies) between them at 21 min post-fertilization.
The largest standard deviation in the 20°C group at any given time
interval was 4.16%.

Effect of 6-DMAP CnnceiUralioii on I'erceiil TripUiid Iiidiutioii

Figure 2 is a summary of the results. In all treatments the
number of live D-larvae was too low to run each replicate sepa-
rately for ploidy analysis. Therefore, all three replicates for each
treatment were combined for ploidy determination by flow cytom-
etry. There were no triploids in the controls, however with the
addition of 6-DMAP. all of the tested concentrations except the 50
p,mol/L treatment at 15°C produced triploids. In all cases except
the 500-|xmol/L concentration (that had an outlier percent triploid
of 9.8% and was therefore excluded) the I5''C treatments gave a
lower percent triploidy than did the 20''C treatments. For both
temperatures, the rate of increase in the percent of triploids in-
duced decreased at concentrations higher than 300 |xmol/L. There
is little increase in the percent triploids with an increase in
6-DMAP concentration beyond 500 |j.mol/L.

Description of Meiotic Events

Figure 3 is a summary of the results. Telophase I (the stage
when polar body 1 is extruded) was visible as early as 12 min

% Triploid
Induction

.100
90
80
70
60
50
40
30
20
10
0

/

15deg. C
20 deg. C

100 200 300 400
6-DMAP Concentration (pmol/L)

500 600

Figure 2. Percent triploid induction of Mytilus ediilis using 6-DMAP.

0 14 21 25 29 33 37 41 45 49 53 57
Time Post Fertilization (min) - - - Telophase 1
I Telophase 2
I — —Anaphase 2
Cell Cleavage

Figure 3. Summarized time-related meiotic events in Mytilus edulis at
20 C.

post-fertilization. Anaphase 2 (the stage that is a precursor to polar
body 2 being extruded) began at 22 min post-fertilization. Telo-
phase 2 (during which polar body 2 is extruded) began at a time of
27 min post-fertilization. Egg cleavage (the first mitotic embryonic
cleavage) began at 44 min post-fertilization. Approximately 50%
of the scored eggs were going through Anaphase 2. and 50%
through Telophase 2 at 32 min post-fertilization.

Evaluation of Several Treatment Windows

The investigated treatment windows varied greatly in both the
percentage of survivors to the D-stage (survivorship) and also in
the percent triploid induced. Table 2 is a suminary of the obtained
results. The control samples tested showed a 37.6% survivorship to
the D-stage. with no triploids present. The highest mean treatment
survivorship (9.337ri) was found in the treatment begun 24 min
post-fertilization for a duration of 5 min. The lowest mean survi-
vorship (0%) was found in the treatment begun at 27 min post-
fertilization for duration of 20 min. The highest mean percent
triploid induction (83.1%) was found in the treatment begun 24
min post-fertilization for duration of 10 min. The lowest mean
percent triploid induction (0%) was found in the treatment begun
at 33 min post-fertilization for duration of 15 min. Tetraploid
peaks were evident in a number of these treatments from the re-
sulting printout of the flow cytometry (Table 2).

DISCUSSION

Evaluating the Induction Window Using Meiotic Events

The determination of the proper induction window is crucial to
triploid induction for a number of reasons. First, the induction
treatment is potentially lethal to fertilized eggs when exposure
times are too long. Desrosiers et al. ( 1993) found that longer ex-
posures to 6-DMAP interfered with first cleavage and resulted in
developmental abnormalities, especially in Mytilus edulis. In cases
where the induction comes early, although a cohort with a high
percentage of triploids might be produced, the survivorship would
be low, making the cohort less useful from a commercial perspec-
tive. Secondly, poor timing might not only lower survivorship, but
also affect the percentage of triploids that would be produced in a
given cohort. If the induction window (although the proper length
of time) is initiated too late, meiosis II will not be blocked and the
production of mostly diploids will result. This would result in a
cohort with a high percentage of diploids, and thus a lower per-

506

Braki: i-:t al.

TABLK 2.
Results of investigation treatment windows.

Initiation Time

Duration (min)

(min)

U

5

10

15

2(1

0

M,

= 37.6 ±
= 0

lo.y

24

M,

=

9.33 ±5.7

M.

= 1.39 ± 1.6

Ms

=

0.98 ±0.8

M.,

=

0.28 ± 0.6

M,

=

56.1 ± 17.9

M,

= 83.1 ±8.8

M,

=

59.3 ± 18.3

M,

=

68.6 ±5.4

27

M,

=

3.90 ± 2.8

M,

= 0.84 ± 0.9

Ms

=

0.97 + 1 .0

M.,

=

0

M,

=

4H.0± 14.2

M,

= 65.8 ± 12.2

M,

=

76.5 ± 13.0

M,

=

49.2 + 19.0

30

M.

=

6.96 ± 2.9

M..

= 0.98 ± 1.5

M,

=

3.2 ± 2.5

M,

_

0.42 ± 0.6

M,

=

51.8 ±5.9

M,

= 49.5 ± 6.6

M,

=

11.6 ±4.6

M,

=

17.2 ±5.8

33

M,

=

5.58 + 2.1

M,

= 2.23 ±1.6

Ms

=

5.29 ± 3.0

Ms

=

3.07 + 2.9

M,

=

27.6 ±9.3

M,

= 14.9 ±13.7

M,

=

0

M,

=

2.20 ±3.8

M., = mean % survivorship to the D-veliger stage (3 replicates/treatment)
M, = mean % triploidy of the sampled D-veligers.
* tetraploids were found in the ploidy sample.

centage of triploid.s. It is important to note that not all eggs are in
the same meiotic stage at any time. If the induction window begins
too early, the result would be a blocking of nieiosis 1 for most of
the eggs, resulting in pentaploids (5N) being produced. Pentap-
loids do not survive to the D-veliger stage. This was shown to
occur in Mytihis galloprovincialis by Scarpa et al. (1993). The
authors exposed eggs to Img/L of cytochalasin B from 7 to 35 min
after fertilization, blocking meiosis I. The resulting developing
pentaploid eggs showed a subsequent inhibition of first cleavage
and died. Therefore the resulting cohort would have a large per-
centage of triploids at the D-veliger stage, as whatever wasn't a
pentaploid would likely be a triploid (having meiosis 1 completed
and being in meiosis II during treatment). This cohort would likely
have a lower survivorship however, as the pentaploids would all
die.

The first experiment (the description of polar body extrusion
timing) was the first attempt to find a starting tinie for triploidy
induction with reference to the time of fertilization. It was assumed
at that time that it was more critical to find the initiation time of
induction, as the exact duration of the treatment could be refined
in further experiments. It was also decided that both temperatures
of 15''C and 20' C would be investigated as they are both common.
and easily achievable working temperatures in shellfish hatcheries,
and they would give some insight as to a temperature effect in the
induction window. The niain objective of this experiment was to
determine the time at which 50'/r of fertilized eggs developed polar
bodies. Fifty percent polar body formation has been used as a
common cue to start triploid induction treatment for shellfish, with
treatments ending after various periods depending on the species
(Baker 1996. Allen et al. 1989). It is important that most of the
eggs are highly synchronous in terms of meiotic events. If most
eggs are in the same stage during induction, and it is the effective
stage, the result would be a higher percent triploidy. Lu (1986)
found that meiotic events of the eggs are more synchronous at
higher temperatures (not exceeding the physiologic limilsl. Given
this finding, a higher temperature within the physiologic limit
would be expected to produce more .synchronous meiotic events.
This was supported by the finding that more eggs were in the same

meiotic stage on average at a given sampling time in the 20°C
sample compared with the 15 °C sample in the first experiment.

One observation in related work was that during the prt)duction
of both triploid and diploid animals for a simultaneous growth trial
there was an unexpected high mortality level during repeated
spawns. This initiated a reevaluation of the treatment window,
particularly the induction initiation tiniing as detennined to be
between 21 and 24 min post-fertilization in the description of polar
body extrusion timing. One possible explanation for repeated low
survivorship (less than 0.01% to the D-veliger stage) may have
been that initiation of treatment to induce triploidy might have
been too early. If this were the case, polar body I extrusion would
be blocked in most eggs, forming pentaploids. Pentaploids do not
survive to the D-veliger stage. However, any eggs that were past
this stage would likely have polar body 2 extrusion blocked and
thus become a triploid. The observed trend in the related growth
trial spawns was in concordance with this phenomenon as the
survivors of these spawns.although few were mostly triploids (all
over 80% triploidy, with many over 90%).

Therefore, the description of meiotic events involved a more
in-depth study of the meiotic events in Mytihis editlis at 20°C. As
opposed to attempting to treat at the time just after polar body I
was extruded, a tinie-related description of the meiotic events was
used. This strategy was shown to be successful in Geoduck clam
triploid induction (Vadapolas 1999). Desrosiers et al. ( 1993) found
that triploid induction treatment using 6-DMAP was effective at
the metaphase I stage. In Geoduck clam induction however. Va-
dapolas (1999) found that extrusion blocking with 6-DMAP just at
the point where 50% of the eggs were in Anaphase 2 and 50% of
the eggs were in Telophase 2 produced optimum results.

The description of polar body extrusion timing and the descrip-
tion of meiotic events showed similar results in terms of polar
body 1 extrusion liines. The description of polar body extrusion
timing illustrated that most eggs had extruded, or were in the
process of extruding polar body 1 by 21-24 min post-fertilization.
The description of meiotic events similarly showed that most eggs
were undergoing polar body I extrusion, scored as Telophase 1, at
a time of 24 min post-fertilization. The fact that both experiments

Triploid Induction of Mytilus edulis

507

showed a similar time of polar body 1 extrusion infers that if the
induction initialization used in the growth trial spawns was in fact
early, causing mortality, it was not because the target of first polar
body extrusion was improperly identified.

Triploid Induction

The effect of 6-DMAP concentration on percent triploid induc-
tion demonstrated that 6-DMAP could be used at a number of
different concentrations to induce triploidy in Mytilus edulis. Al-
though 15°C was shown to be less synchronous (and thus less
optimum for triploid induction) in the determination of polar body
extrusion times, these results were not yet obtained at the time of
the first induction experiment. In all cases the 20"C samples had a
higher percentage of triploids than did the 15°C sample (Fig. 2l.
This is likely due to the fact that eggs at 20°C are more synchro-
nous in terms of their meiotic stage, resulting in a higher percent-
age of eggs in the effective treatment stage, and a higher percent
of triploids. This is in concordance with the results from the de-
scription of polar body extrusion timing that showed a higher
degree of meiotic synchrony in eggs at 20°C compared with 15°C.
This is also supported in the literature as Lu (19861 found that
meiotic events of eggs are more synchronous, and thus should give
a higher percent triploid induction, at higher temperatures (not
exceeding the physiologic limits). In past studies, treatments with
a higher degree of meiotic synchrony allowed for more effective
induction treatments as more eggs were in the target stage during
treatment, resulting in a higher percent triploid and a higher sur-
vivorship (Downing & Allen 1987, Allen et al.. 1989. Gerard et al.
1994). In light of these past studies, and given that the 20°C gave
both a higher percent triploid induction and a higher degree of
meiotic synchrony, the 20°C treatment is considered more optimal
than the 15'C treatment.

The evaluation of several treatment windows tested various
induction windows (initiation and duration times), which could be
compared with the meiotic events described previously. Survivor-
ship was low in all cases. This was likely because a relatively high
concentration of 6-DMAP (400 |j,mol/L) was used. A decrease in
the number of normally developed D-larvae (presumed to not be
capable of survival) with an increase in 6-DMAP concentration
was reported by Gerard et al. (1994). In the case of the current
experiment, the relatively high 6-DMAP concentration was re-
quired to ensure that a suitably high percentage of triploids was
produced, and was based on the results from the effect of 6-DMAP
concentration on percent triploid induction (Fig. 2). Treatments
initiated at 24 min post-fertilization produced both the highest
percent triploid (83.1 at a duration of 10 min) and the highest
survivorship (9.33 at a duration of 5 min). Taylor Resources Inc.,
a hatchery that commercially produces triploid Mytilus gallopro-
vincialis. typically obtains 95-100'7f triploidy with lO-lS'/r sur-
vivorship. It is difficult to compare these refined procedures with
the results from these preliminary experiments, as much more
optimization is required to reach commercial relevance. As these
were preliminary experiments, a triploid percentage of greater than
50% with as high a survivorship as possible was the objective. This
was based on the caveat that further optimization would improve
both the percent triploid induction and survivorship.

Past attempts at inducing triploidy in Mytilus edulis have given
a wide range of optimal treatments, resulting in a wide range of
percent triploid induction and survivorship. Beaumont and Kelly
(1989) produced, at best, 25% triploid-producing eggs using heat

shock alone (from 15°C to 25°C at 10 min post-fertilization for 10
min). The authors measured the percentage of eggs that were de-
veloping, and the percentage of those that were developing nor-
mally. They found that, at best, approximately 10% of treated eggs
developed. Of those that developed, approximately 60% developed
normally and would likely go on to develop as triploid embryos.
One treatment had less than 5% development and less than 7% of
those showed normal development. However, in the same study, it
was found that CB treatinents could produce up to 60% triploid
larvae in the optimal treatment group (using Img/L of CB al 5 min
post-fertilization for 15 min). Again there was a significant reduc-
tion in the percentage that developed, and of those, which devel-
oped normally. Approximately 8% of treated eggs developed, and
approximately 63% of those developed normally. The current re-
sults (Table 2) illustrate some treatments that showed better in-
duction performance when compared with the aforementioned
study, with up to 83.1% triploid induction (the 24-10 treatment)
and up to 9.33% survivorship (the 24-5 treatment) to the D-veliger
stage. Yamamoto and Sugawara ( 1988) were able to produce up to
97.4% triploidy by the use of heat shock alone (from 20°C to 32°C
at 20 min post-fertilization for 10 min). and found apparently no
negative effect upon survival rates. However, these results are
considerably higher than those produced by Beaumont and Kelly
(1989). As well. Yamamoto and Sugawara (1988) report yields
much higher than those found in these experiments that showed
results slightly higher than those of Beaumont and Kelly (1989).
Yamamoto and Sugawara ( 1988) gave high yields with heat shock
alone despite the fact that 6-DMAP is generally accepted as being
a more effective triploid inducer than temperature alone (Desro-
siers et al. 1993). Yamamoto and Sugawara (1988) also found
2-22% triploids in their controls that they attribute to handling
stress. No other known triploid shellfish work has shown triploids
in their controls. In the Chilean blue mussel. Mytilus chilensis.
Toro and Sastre (1995) found an optimum induction treatment
using heat alone (from 18°C to 32°C at 10 min post-fertilization
for 10 min) to induce 51%^ triploid with 29%: survival to an age of
15 days. The optimum percent triploid induction in their study was
lower than in these experiments, the survival rate was much higher
however. Scarpa et al. (1994) compared six methods to induce
triploidy directly (not including 6-DMAP) and found that cytocha-
lasin B (CB) was the most effective at inducing triploidy in Mytilus
gatloprovinciidis. obtaining 86%^ triploidy to the D-veliger stage.
These authors also found that, averaged between their two trials.
71.6%r survived the CB treatment. These reported results are
higher than the results of this study, however, as mentioned pre-
viously, we excluded CB from our trials due its carcinogenic ef-
fects.

Although percent triploid induction in this study is comparably
high (over 50%f ) in some treatments when compared with many
shellfish studied (Beaumont & Fairbrother. 1991 ), survivorship is
lower than expected. Future optimization of the triploid induction
method should focus on increasing survivorship while not com-
promising percent triploid induction. Improved hatchery tech-
niques and husbandry may also increase survivorship, but cannot
help to increase the percent triploidy. When considering a com-
mercial technique, a high percentage of triploids in a cohort are not
the only consideration in an induction technique. It has to be
balanced with finding a method that produces a high survivorship
of the triploids to remain economically feasible.

One further noteworthy result is the incidental production of
tetraploid mussel larvae during some of the treatments in the

508

Brake et al.

evaluation of several treatment windows (Table 2). Six different
treatment combinations produced some measurable number of tet-
raploids. There may be commercial significance in these findings.
Triploids can be produced at a rate of lOO'^r with a high survivor-
ship, and without using chemicals by mating a tetraploid to a
diploid (Guo et al. 1996). Therefore, if tetraploids could success-
fully be produced and reared, they might become commercially
important animals in the production of triploid mussels. Scarpa et
al. ( 199.^) found that tetraploidy could be induced in MytUiis gal-
loprovincUdis by suppression of both polar body 1 and 2. This may
have been the mechanism for the production of the tetraploids
found in these results. However, when these tetraploid-inducing
treatments are compared with Figure 3. a pattern that suggests
another mechanism is evident. All tetraploid producing treatments
but the 24-15 treatment overlap cell cleavage. Theoretically, if an
inhibition treatment overlaps cell cleavage, the chromosomes
could divide into two sets but the cell would not cleave. The result
will be one cell with double the diploid number of chromosomes,
and subsequent mitotic divisions will result in tetraploid larvae
(Beaumont & Fairbrother 1991 ). This has been shown as a viable
method of tetraploid induction. Guo et al. (1994) attempted to
produce tetraploids by inhibiting mitotic cell cleavage with heat
shocks, producing up to 45% in one case, however no tetraploids
in their study survived past the D-stage of development. Future
work on triploid production of Mytilus edidis should focus on the

production of tetraploid broodstock used to produce 1007f triploid
larvae without the use of chemicals. Many commercial triploid
Pacific oysters are now produced using this method (Chew 2000).
It has been demonstrated that using the puromycin analogue
6-DMAP at various concentrations can produce triploid mussels.
At the conclusion of this research the optimum investigated pro-
cedure for induction of triploidy in Mytilus edulis was to treat eggs
at 20°C with 400 (jimol/L of 6-DMAP starting at 24 min post-
fertilization for a treatment time of 10 min. This treatment yielded
83.1% triploids in induced samples with a survivorship of 1.39%
to the D-veliger stage. The current investigated methods for pro-
ducing triploid Mytilus edulis are not yet optimized to commercial
levels. These findings are a basis for further work in the optimi-
zation of this technique.

ACKNOWLEDGMENTS

The authors thank Garth Arsenault for technical support
throughout this study. We also wish to thank Benoit Eudeline for
assistance with How cytometry throughout the project. Funding for
this project was provided by a grant from the National Research
Council (NRC) through its Industrial Research Assistance Pro-
gram (IRAP). the PEI Aquaculture and Fisheries Research Initia-
tive, the Prince Edward Island Aquaculture Alliance (PEIAA). and
the PEI Department of Fisheries. Aquaculture and Environment.

Allen. S. K. 1988. Triploid oysters ensure year-round supply
31:58-63.

Allen, S. K., S. L. Downmg. J. Chailoti & J. H. Beattie. 1986. Chemically
and pressure-induced triploidy in the Pacific oyster Cicissostrea gigas.
Aquaculture 57:359-379.

Allen. S. K., S. L. Downing & K. K. Chew. 1989. Hatchery Manual for
Producing Triploid Oysters. Seattle: University of Washington Press,
6 pp.

Baker, B. 1996, Building a better oyster. Bioscience 46:240-244.

Beaumont. A. R. & K. S. Kelly. 1989. Production and growth of triploid
MmiIus cilulis larvae. ,/. Exp. Mar. Biol. Ecol. 132:69-84.

Beaumont. A. R. & J. E. Fairbrother. 1991. Ploidy manipulation in mol-
luscan shellfish: a review. / Shellfish Res. 10:1-18.

Chew, K. K. 2000. Update on Triploid Pacific Oysters. Aquaculture 26:
87-89.

Desrosiers. R. R.. A. Gerard. J. Peignon, Y. Naciri. L. Defresne. J. Mo-
rasse, C Ledu, P. Phelipot. P. Guerrier & P. Dube. 1993. A novel
method to produce triploids in bivalve molluscs by the use of 6-Dim-
ethylaminopurine. J. E.\p. Mar. Biol. Ecol. 170:29^3.

Downing. S. L. & S. K. Allen. 1987. Induced triploidy in the Pacific oyster,
Crassostreu giga.s: optimal treatments with cytochalasin B depend on
temperature. Aquaculture 61:1-15.

Gerard, A.. Y. Naciri. J.-M. Peignon. C. Ledu & P. Phelipot. 1994. Opti-
mization of triploid induction by the use of 6-DMAP for the oyster
Crassostrea gigas (Thunberg). Aquae. Fish. Manage. 25:709-719.

LITERATURE CITED

Ocearuis

Guo. X. 1991. Studies on tetraploid induction in the Pacific oyster, Cras-
sostrea gigas (Thunberg). Ph.D Thesis. Seattle: University of Wash-
ington. 167 pp.

Guo. X., W. K. Hershberger, K. Cooper & K. K. Chew. 1994. Tetraploid
induction with meiosis 1 inhibition and cell fusion in the Pacific oyster
iCras.w.ftrea gigas. Thunberg). J. Shellfish Res. 13:193-198.

Guo. X.. G. A. DeBrosse & S. K. Allen. 1996. All-triploid Pacific oysters
(.Crassostrea gigas Thunberg) produced by mating tetraploids and dip-
loids. Aquaculture 142:149-161.

Lu, J.-K. 1986. The Effects of Temperature and Salinity on Meiosis, Fer-
tilization and First Mitosis of The Pacific Oyster (Crassostrea gigas)
eggs. Masters Thesis. Seattle: University of Washington. 122 pp.

Scarpa, J., K. T. Wada & A. Komaru. 1993. Induction of tetraploidy in
mussels by suppression ot polar hody formation. Nippon Suisan Gak-
kai.ihi 59:20n-202i.

Scarpa. J.. J. E. Toro & K. T. Wada. 1994. Direct comparison of six
methods to induce triploidy in bivalves. Aquaculture 1 19:1 19-133.

Toro, J. E. & H. D. Sastre. 1995. Induced triploidy in the Chilean blue
mussel, Mytilus chilensis (Hupe, 1854) and performance of triploid
larvae. J. Shellfish Res. 14:161-164.

Vadopalas, B. 1999. Development and optimization of triploid induction
techniques in the geoduck clam, Panopea ahrupia. Masters Thesis.
Seattle: University of Washington. 81 pp.

Yamamoto, S. & Y. Sugawara. 1988. Induced triploidy ui the mussel,
Mytilus edulis. by temperature shock. Aqucuultioe 72:21-29.

Jotimal ol Shellfish Rf.saurh. Vol. 21, No. 2. 5(W-.'^I7. 2U02.

MUSSEL CULTURE AND COCKLE FISHERIES IN THE NETHERLANDS:
FINDING A BALANCE BETWEEN ECONOMY AND ECOLOGY

PAULINE KAMERMANS* AND AAD C. SMAAL,

Netherhmds Institute for Fisheries Research. Centre for Shellfish Research. P.O. Box 77. 4400 AB
Yerseke. The Netherlands

ABSTR.ACT 111 the Netherlands, wild stocks of mussel seed are fished and mussels are cultured on bottom plots. In addition, wild
stocks of the edible cockle are dredged for harvest. Two of the areas where these activities are carried out are nature reserves. In 1993.
the government implemented a policy in these reserves to ensure the conservation, protection and development of natural values and
processes in which human activities should fit in. Fishing for shellfish is considered a traditional activity in these waters. Therefore,
it is allowed, but under the restriction that no negative effects are caused. As a result of this policy, fishing for mussel seed and cockles
is not allowed in areas with a high potential for the development of mussel beds and seagrass fields. A number of bird species are
dependent on shellfish for their food requirements. Therefore, the policy makes use of a reservation system. This means that, in years
when mussel and cockle stocks are low, an amount is reserved for the birds and cannot be fished. The government and shellfish industry
agreed on co-management, (i.e., the fishermen are responsible for implementing the measures). This task is carried out by Producers'
Oroanizations. An overview of the viewpoints of the interest groups and the role of policy makers and scientists is given.

KEY WORDS:

Mvtihi.s echilis. Ceruslodcnmi edule. co-management, culture, fisheries, government regulations

INTRODUCTION

Shellfish are an important component of the ecosystem as they
filter the water and are a food source for organisms such as birds
and humans (Dame 1996). The protection and restoration of shell-
fish beds can contribute both to preservation of estuarine and near
coastal ecosystems and to sustainable economic development of
the coastal zone. In the Netherlands, six shellfish species are com-
mercially e.xploiled (Smaal & Lucas 2000). Different species are
harvested in different areas (Fig. 1). In the coastal zone fishing is
directed at wild stocks of the trough clam Spisula siibtnmcaia. and
the American razor clam Ensis directus. In the Oosterschelde es-
tuary and the saline lake Grevelingen the European oyster Ostrea
ediilis and the Pacific oyster Cnr^sostreci !>igii.s are cultured. Fish-
ing for seed of the blue mussel Myliliis edulis takes place in the
Wadden Sea and on rare occasions in the Oosterschelde estuary.
The mussels are cultured on bottom plots located in the Wadden
Sea in the North and the Oosterschelde in the South. Fishing on
wild stocks of the edible cockle Cerastudenna ediile takes place in
the Voordelta, Western Scheldt, Oosterschelde and Wadden Sea.

This article describes an attempt to combine the exploitation of
shellfish stocks and the protection of estuarine habitats in the Neth-
erlands. To reach such a goal agreement among stakeholders is
essential. A description of the mussel culture and fishing for mus-
sel seed and cockles is presented. Regulations concerning the man-
agement of shellfish stocks and other habitats are summed up.
Furthermore, an overview of the viewpoints of the interest groups
and the role of policy makers and scientists is given.

METHODS

Mussel Culture in the Netherlands

In the Netherlands, the culture of the blue mussel Mytilus edulis
depends on seed that is fished twice a year in the most western part
of the Wadden Sea (see Fig. 1). Seed mussels are approximately
10- ."^0 mm in length. These mussels are transported to the bottom

♦Corresponding author. Tel.: -1-31-113-672302: Fax: -1-31-113-573477:
E-mail: p.kamermansCs'rivo.wag-ur.nl

culture plots, which are leased from the government. The plots are
located in the Wadden Sea and the Oosterschelde (see Fig. 1 ). The
mussels are left to grow to market size (>45 mm). Depending on
the environmental conditions this size is reached in 1-3 y
(Dijkema 1997). The mussel seed catches show large annual fiuc-
tuations that reflect the variability in spat fall (Fig. 2a). Experience
of the mussel growers shows that a catch of 65 x 10" kg seed
mussels is needed to sustain a yearly production of 100 x 10'' kg
mussels (see Fig. 2b). This relatively low conversion is caused by
heavy mortality as a result of predation by birds and starfish, and
losses due to storms. The mussel seed target is not always reached.
In fact, during the last ten years the target was reached only once.
Thus, mussels are imported from other areas such as Germany
(Dijkema 1997). The Netherlands is the fourth largest blue mussel
producer in the world, after China, Spain, and Denmark (FAO
20G0a, FAO 2000b). In 1999 the total landing value of mussels
was Dfl 125 million = 55 million USD (Productschap Vis 2000).
Almost 40'7f of the mussels are exported to Belgium.

Cockle Fisheries in the Netherlands

Dredging for cockles takes place in the Wadden Sea, the
Voordelta, the Oosterschelde and Western Scheldt (see Fig. I).
Wild stocks are fished mechanically with suction dredges. In ad-
dition, a small group of fishermen makes use of rakes with nets
that are deployed by hand. The majority of the cockles is not sold
in their shell, but cooked and canned for transportation to Spain.
Therefore, landings are expressed in meat instead of fresh weight
(Fig. 3). The catches show considerable variability again caused by
fluctuations in spat fall. The main harvest area is the Wadden Sea.
In 1998 a large cockle stock was available, and the total value of
the catch was Dfl 60 million = 27 million USD (Steins 1999). The
Netherlands is the number one cockle producer in the world (FAO
2000a).

Producers ' Organizations

All groups involved in the mussel and cockle industry (i.e.,
fishermen, vessel owners and shellfish sellers) are organized in the
Mussel Producers' Organization (PO Mussels) and the Cockle Pro-
ducers' Organization (PO Cockles). The POs depend on voluntary

509

510

Kamermans and Smaal

Figure 1. Shelirish fishing and culture areas in The Netherlands.

membership. POs offer attractive facilities to its members and they
have legal powers to enforce binding agreements. The POs for-
mulate Shellfish Fisheries Management Plans {e.g.. Productschap
Vis 2(X)0). These include yearly fish plans, which are based on
results of yearly surveys that determine the amount and distribu-
tion of mussel seed and cockles. In some cases (e.g.. when mussel
seed is present on a sheltered location), seed is not fished in fall,
but left for next spring.

The Role of the Netherlands Institute for Fisheries Research

Every March, since 1992. the Netherlands Institute for Fisher-
ies Research (RIVO) carries out a subtidal mussel seed survey in
the western part of the Wadden .Sea. This survey is conducted by
order of the PO Mussels. The sampling set-up is stratified (i.e.. the
sampling grid is intensified at locations where subtidal mussel
beds are present). These locations are known from the previous
year and qualitative assessments carried out in early spring by
fishery inspectors of the Ministry of Agriculture, Nature Manage-
ment and Fisheries. At every sampling point a 20-cm-wide cockle
dredge is used for 150 m. The mesh size is 5 mm. For profitable
seed fishery a biomass of 0.1 kg mussels per m~ is necessary (Fig.
4). The RIVO akso monitors the distribution of seedbeds and the
development of mature mussel beds in the intertidal with aerial
photography surveys in combination with ground truthing.

Each May. since 1990. RIVO carries out a basin-wide cockle

survey in the Wadden Sea. the Oosterschelde and the Western
Scheldt. The survey is under contract by the Ministry of Agricul-
ture. Nature Management and Fisheries and the PO Cockles. The
sampling grid is stratified with a denser grid at cockle beds (Fig.
5). The location of these beds is derived from a qualitative survey
that the fishemien carry out just prior to the annual RIVO survey.
From the survey data the total cockle stock is determined.

Dutch Regulations

The Wadden Sea and the Oosterschelde do not only function as
areas where shellfish fishing and culture takes place, but al.so as
nature reserves. The birds, wetlands and other habitats in these
areas are protected under several national and international laws
such as the Ramsar Convention, the European Directive on the
Conservation of Wild Birds, and the UNESCO Man and Biosphere
Program. The areas used to have extensive seagrass meadows
and intertidal mussel beds. Renewed development of these habi-
tats is desirable. For migrating birds, the Wadden Sea and the
Oosterschelde are important wintering areas or stopover sites for
refueling on the flyway between the North Pole and Africa. In
addition, a number of resident species, such as oystercatchers
and eider ducks, are present (Rosner et al. 1993).

In these protected areas human activities are possible only
when they do not cause negative effects. In the early 199()s, low
stocks of both cockles and mussels were present in the intertidal

Mussel Culture and Cockle Fisheries in the Netherlands

1UU -
80-

-1 1 — 1

target

o) 60 -

1=

r 40-

20 -
0-

u

L_JjXI^

D spring
. ■ autumn

1

91/92 92/93 93/94 94/95 95/96 96/97 97/98 98/99 99/00
season

■ Import

D Wadden Sea

■ Oosterschelde

180 j
160

140

120

100

80

60

40

20

0

Seasons
Figure 2. Mussel seed catches and mussel landings in the Netherlands in million kg fresh weight. Targets are indicated with dotted lines.

Wadden Sea (Beukema 1993). This was a result of a number of
factors including reduced natural spat fall, storms and unrestricted
fishing activities (Dankers 1993). At the same time, high mortality
of a shellfish eating bird species was ob.served (Smit et al. 1998).
Thus, the possibility of a link between bird mortality and shellfish
fisheries appeared. The public awareness and social commotion
caused by these events triggered the formulation of regulations to
better protect the area.

In 1993. the government implemented a policy for the period
1993 to 2003 to ensure the conservation, protection and develop-
ment of natural values and processes in the Wadden Sea and Oost-
erschelde estuary (LNV. 1993). Human activities must fit into this
policy. Targets are restoring bird populations at levels of the
1980s, and promoting development of seagrass beds and 2.000-
4.000 ha of stable intertidal musselbeds (LNV 1999). Fishing for
shellfish is considered a traditional activity in these waters. There-
fore, it is allowed, but under the restriction that no negative effects
are caused. The government makes use of co-management (i.e.. the
fishermen are responsible for implementing the measures). The PO
Cockles and the PO Mussels carry out this task. Consumption-
sized cockles and mussels are also the preferred prey of oyster-
catchers and eider ducks (Zwarts et al. 1996. Nehls & Ruth 1994).
Therefore, the policy makes use of a reservation system in the
shellfish fisheries. In years when mussel and cockle stocks are low.

the aim of the policy is to ensure that 60% of the food requirement
of birds is reserved. However, the calculations underlying the
policy have been questioned by (Ens 2000). Figure 6 shows large
annual fluctuations in cockle stock as a result of variability in spat
fall. Since the implementation of the food reservation policy in
1993. fishing for cockles was not allowed in the Wadden Sea in
1996 and restricted to the subtidal areas in 1997. In the Ooster-
schelde fishing was not allowed in 1997. 1998, 1999. and 2000.
Another regulation is that fishing for mussel seed and cockles is
not allowed in areas with a high potential for the development of
mussel beds and seagrass fields. In 1993. 14% of the intertidal flats
in the Oosterschelde and 26% of the intertidal Wadden Sea were
permanently closed to shellfishing. In 1999. an additional 5% of
intertidal flats in the Wadden Sea were closed (Fig. 7). The loca-
tion of these areas is based on habitat maps for seagrass meadows
and mussel beds. The maps are produced with CIS models that
include parameters such as exposure time, wave action, sediment
characteristics (Brinkman et al. 2002. de Jonge & de Jong 1999).
To monitor that shellfish fishing does not take place in the closed
areas all vessels are equipped with a black box. This system reg-
isters the ship's position at 1-min intervals when the ships are
fishing. The data are retrieved from the boxes by an independent
agency and the POs give penalties to offenders. In addition to the
government measures, the Shellfish Fisheries Management Plan of

512

Kamermans and Smaal

16
14
12

^ Voordelta
D Westerschelde
■ Oosterschelde
D Wadden Sea

I

I

I

g

•^10

O

0) b
4
2
0

77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01

Years

Figure i. Cockle landings in the Netherlands in niilliun kg meat.

the POs for the period 1993 to 2003 includes a reduction in the Evaluation Projects

number of cockle vessels, a more spread out distribution of the

cockle fleet, restricted fishing periods for both cockles and mussel In 1998. the effectiveness of the governmental measures was

seed, and yearly fishing plans on amounts fished and fish locations evaluated for the period 1993 to 1998 in the so-called EVA I

based on survey data (Produclschap Vis. 2000). project (LNV 1998. Smit et al. 1998). Due to a series of cold

mussel biomass in g/m^

. -100

. 100-500

• 500 - 1000

• 1000 - 2000

• 2000 - 3500
no mussel

I mussel culture plots

10 km

6^

Figure 4. Subtidal mussel seed biomass in the western Dutch Wadden Sea. Results of RIVO sur\e) in 2000.

Mussel Culture and Cockle Fisheries in the Netherlands

513

cockle density in n/m^

• 0

. 1-500

• 500-1000

• 1000 - 5000

20 km

Figure 5. Cockle den.sit> in the Dutch Wadden Sea. Results of RIVO cockle survey in 2000.

winters with liigh shellfish mortality in this period, clear conclu-
sions on effects of shellfisheries could not be drawn from the
available dataset. The rate at which intertidal mussel beds devel-
oped was lower than expected. Intertidal mussel spat fall occurred
in 1994 only (van Stralen & Kesteloo-Hendrikse 1998). and the
intertidal mussel area in the Wadden Sea is approximately 1000 ha
in 2000 (Kater & den Os 2001 ) Therefore, an additional 59^ of the
intertidal flats was closed based on the newly developed mussel
habitat map (Brinkman et al. 2002). In 2000, the second phase of
the evaluation started. In this EVA II project (Ens et al. 2000) the
effects of shellfish harvests on nature in the Wadden Sea and
Oosterschelde is e\alualed for the entire 10-year period from 1993
to 2003 (LNV & VW 1999). The project is based on the following
policy questions (Ens et al. 2000).

• What are allowable effects of shellfish harvests on mussel beds,
seagrass meadows and shellfish consuming birds?

• Have the measures taken so far had the expected effect?

• Do we comply with the international regulations on bird pro-
tection?

• When effects are negative, what can we do to reduce these effects?

These policy questions were translated into a number of
research sub-projects. Effects of mussel fishery and culture on bird
populations are studied. The effects of cockle dredging on cockle
stocks; stable intertidal mussel beds, other zoobenthos, seagrass
meadows, and sediment composition are studied by comparing
their development in open (fished) and closed (unfished) ureas. In
addition, availability of black box data gives the opportunity to
further specify the fishing intensity within the open areas. Linking
survey information with black box data and catch data gives in-
formation on the relationship between density of shellfish, fishing
intensity and yields. Another subproject checks the assumptions on

which the calculation of the amount of food reserved for birds is
based. What is the profitable shellfish density for birds'^ Does it
matter where the food is located (both in the tidal zone and in the
horizontal plane)? Which fraction of the total stock is usable by
birds? And finally, the slow recovery of both seagrass meadows
and mussel beds warrants refinement of the habitat maps. In this
way a better selection of closed areas is possible. The results of the
evaluation project will play a role in the final decision on the
continuation of shellfish harvesting, which the government will
take in 2003.

Finding a Balance Between Economy and Ecology

Cockle fishermen and mussel farmers make a living with their
activities and represent the commercial economy, while nature
conservationists protect birds and habitats, such as seagrass beds
and mussel beds, and are concerned with ecology. Both groups
agree that the Wadden Sea and Oosterschelde should not be com-
pletely closed for human activities (e.g., recreation and fishing)
provided that these activities do not have negative impacts. The
discussions center on these impacts. Despite the policy to reserve
food for shellfish eating birds, a further decline in oystercatcher
population is taking place (Fig. 8). In addition, the development of
intertidal mussel beds and seagrass meadows is slow. These ob-
servations cause tension between the different stakeholders. The
different forms of shellfish fishing and farming show different
degrees of opposition between the stakeholders. Traditional fishing
for cockles by hand does not cause much tension. Fishing for
mussel seed in the subtidal area is starting to become an issue, as
these stocks also provide food for eider ducks. The main disagree-
ments are on fishing for intertidal mussel seed and mechanical
cockle fishing (Piersma et al. 2001. Smaal et al. 2001. Camphuy-
sen el al. 2002).

514

Kamermans and Smaal

250

200

150

<1>

E

c
o

E 100

50

0

a

I

ByBH

food reservation

25

sublit, from 1990

riRczica^EjRc:

<— og o ^ UD ti> r^ o:- -x- -i^ -— oj o f IT- -j^ r-- ■» -T^ o '- oj •■•'• ^ u"' •^' r-- o:- ix- -^
r^ f- 1-- r-- r-- r-- r-- r-- r^ o:' o:- CO o:- 05 CO CO CO co co 'n> cr- co ov m tx- ov 'X- -r^ x» o
CT)CT>CT>oo^o>o>o:'a:'"X>'7>oo^cr>0"^'7^<7^<7>cr>oo:>x^coocn'7:'Cri'T>a:>o

Figure 6. Cockle stock in the Dutch Wadden Sea as determined by extrapolation from various sampling campaigns in the VVaddcn Sea and the
basin wide RIVO surveys from 1990 onwards. It is assumed that both birds and fishermen need a cockle density of more than 50 per m* for
profitable use of the stock (grey pari of the bars). Part of the grey bar is black, which indicates the catch. From 1993 onwards 12.6 million kg
meat is reserved as food for birds. This is indicated with a horizontal line.

The cockle and mussel seed fishermen see an increase in re-
strictions, without the expected restoration of the ecosystem. As
they have already put considerable effort in a changed manage-
ment (e.g., the use of fishing plans and black box systems), they
are reluctant to agree to more. Shellfishermen point out that, al-
though bird populations are smaller than in the eighties, they have
increased since the seventies. Nature conservation groups see the
continued decline of bird populations as a sign to stress the pre-
cautionary principle. The definition of the precautionary principle
is under debate. The con.servation groups define it as: when un-
certainty on the effects of a human activity exists, do not allow it.
The European Commission have extended this with a balancing of
interests: measures taken on the basis of the precautionary prin-
ciple need to be in relation to the chosen protection level and need
to be based on research on possible costs and benefits of carrying
out that measure (COM 2000). Both fishermen and environmen-
talists question the calculated aniounts of food needed by the birds.
Fishermen ague that the lower level of bird populations in the
seventies did not coincide with lower shellfish stocks. Nature
groups and ornithologists (Ens 20(K), Camphuysen 1996, Cam-
phuysen et al. 2002) point out thai not the total shellfish stock, but
the stock that is available for birds is of importance. This may lead

to higher food reservation values. Furthermore, there is debate
about the closure of intertidal areas to proniote development of
mussel beds. Fishermen have the impression that trtoderate fishing
improves the sediment and enhances survival of niussel seedbeds,
while environmentalists are of the opinion that natural processes
should not be interfered with. The images that both groups have of
each other strongly influence the discussions that are part of the
decision-making process (Steins 1999). Thus, securing ongoing
communication between interests groups is of great importance.
Identification of common interests should play a key role.

In addition to the tension between fishernien and conservation-
ists, there is tension between science and politics. The government
wants to keep the shellfish industry and protect nature at the same
time. To do this properly they want to make decisions on objective
scientific grounds. The scientists are unable to reach consensus on
scientific grounds for the presence or absence of a link between
shellfishery activities and the recent ecosystem developments (de-
cline in bird populations, and slow recovery of intertidal mussel
beds and seagrass meadows). Scientists generally work on a micro
scale and can be fairly certain about their conclusions, while poli-
ticians need answers on a macro scale. The advice of the scientists
needs to be objective and exact. However, when findings from

Mussel Culture and Cockle Fisheries in the Netherlands

515

Figure 7. Location of areas that are closed for shelirish fisheries in the Dutch VVadden Sea.

250000

200000 '

150000

100000

50000

1984

1986 1988 1990 1992 1994

1996

1998 2000

1974 1976 1978 1980 1982

Figure 8. Development of total oyster catcher population in the Dutch Wadden Sea from 1975 to 1999. Counted numbers in fall (open squares)
and following winter (closed squares) are indicated. Running mean (black line) is calculated from these counts. Counts in severe winters (closed
squares with arrows) are not included in mean (Ens 2002).

516

Kamermans and Smaal

small scales are upgraded to larger scales the results will always
have a certain degree of uncertainty. To avoid scientific conflicts.
and remain credible to society, it would be better to present results
in the form of ranges instead of single values (Hauge 2000). How-
ever, this causes problems for the managers, as they need single
values when drawing up permits. Thus, alternative ways to deal
with scientific uncertainties concerning natural resources are needed.

CONCLUSIONS

The balance between economy and ecology is very delicate. In
fact, balancing between economy and ecology is a more appropri-
ate way to describe the current situation. Co-management has not
brought the solutions the government hoped for. At present, two
options dominate the discussion on the future of shellfisheries in
the Wadden Sea and Oosterschelde. These are:

• A continuation of the fisheries as before, which seems unac-
ceptable to the nature conservation groups.

• A buy out of the mechanical cockle fishery, which seems un-
acceptable to the fishermen.

The shellfish-fishermen, -farmers, and -sellers have set up a
foundation called ODUS (Development Sustainable Shellfish-
eries). This foundation proposes the following methods for cockle
fishing and mussel farming that aim for effects that are within the
restoration capacity of the system and could be more sustainable
(ODUS 2001).

• Selecting optimal areas for harvest may enlarge the efficiency of
cockle dredges. Newly developed fishing gear may reduce the
effects of the dredges on the bottom.

• Management of closed areas can be dynamic instead of static.
The location of closed areas could be based on annual variations
according to the location of the cockle stock

• Relaying of cockles may reduce mortality and enhance growth.
This can be the beginning of a development from fishery to
culture.

• 1-ishing of unstable mussel seed beds will use seed before it is
lost in storms.

• Methods should be developed to avoid large seed losses on
culture plots. This will enhance the yield and thus reduce the
amount of mussel seed needed.

• Optimization of the mussel stock on culture plots to the exploi-
tation capacity of the system w ill enhance the yield.

In our opinion, these promising options should receive more
scientific attention. Conservation of the natural system is a com-
mon interest of all stakeholders. Degradation of the system will
lead to reduced biodiversity and lower resilience. Both birds and
fishermen need a well functioning ecosystem that guarantees the
continued presence of shellfish stocks.

ACKNOWLEDGMENTS

We thank Bruno Ens and Mariette Kip for critically reading an
earlier version of the manuscript.

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Picrsma. T., ,^. Koolhaas. A. Dekinga, J. J. Beukema. R. Dekker & K.
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Journal of Shellfish Ri".ccinh. Vol. 21. No. 2. 519-527, 2002.

SETTLEMENT OF POMATOCEROS TRIQUETER (L.) IN TWO SCOTTISH LOCHS, AND
FACTORS DETERMINING ITS ABUNDANCE ON MUSSELS GROWN IN

SUSPENDED CULTURE

DIRK A. CAMPBELL AND MAEVE S. KELLY*

Scottish Association for Marine Science. Oban. Argyll. PA37 IQA. Scotland. United Kmi^dom

ABSTRACT In Scotland, some 2.000 tons of mussels {Mylilus ediilis ) are produced each year from suspended rope cultures. Mussels
can act as a settlement substrate for tubeworm, Pomatoceros spp. and badly fouled mussels are devalued and may be discarded. The
estimated cost to the Scottish rope-grown mussel industry is between £300,000 and £500,000 per annum. Established growers believe
the problem is worsening. Collector plates were deployed at two sites in two lochs on the West Coast of Scotland to monitor
Poimitoceros spp. settlement. In conjunction, in sint trials at a mussel farm site assessed tubeworm settlement on rope-grown mussels.
The tubeworm P. irujiieter was found to be the prevalent species settling on the collector plates and on the mussels. The timing of peak
tubeworm settlement differed between lochs hut was synchronous between sites and different depths within the same loch. Peak
settlement in both lochs occurred after the highest seawater temperatures were recorded. Settlement intensity differed between lochs,
sites, and depths, indicating the scale of variation in settlement withm lochs. Mussel shell size was distinguished as a significant factor
influencing Pomatoceros spp. settlement as higher numbers of tubeworm settled on larger mussels. In large mussels (mean shell length
60.8 mm ± 0.70 SE) tubeworm settlement was greatest in treatments where conspecific adults were already present and higher levels
of settlement were found in mussels stocked at lower densities. In small mussels (mean shell length 33.5 mm ± 1.03 SE) the initial
stocking density and tubing system had no effect on tubeworm settlement. Differences in the fouling intensity between mussel stocks
were attnbuted to variation m the abundance of tubeworm larvae in the water column and the size of the mussels at the time of retubing.
A loot"* mortality could be expected in adult P. uiqiieter after 24.1 h and 35.4 h when exposed to air at 7°C and I3°C, respectively.
In areas where tubeworm is a persistent problem consistent annual monitoring of the Pomaloceros spp. settlement is recommended as
part of a management strategy to avoid heavy fouling on mussel stock. Grow-out strategies to alleviate tubeworm fouling on
rope-grown mussels are discussed.

KEY WORDS: Ponmtoieros. Mxlilic ciliili.s. settlement, tubeworm fouling, mussel culli\ation

INTRODUCTION

Calcareous tube-dwelling polychaeles from the genus Pimiato-
ceros have long been considered to be primary fouling organisms
(Ciisp 1965. OECD 1967). In Scottish waters, mussels are likely to
be colonized by two species, Pomatoceros lamarcki (Quatrefages)
and P. triqiieter (L.). P. lamarcki is an intertidal to shallow sub-
tidal species, whereas P. triqiieter is generally considered subtidal.
particularly adjoining deep water (Hayward & Ryland 1995). Ma-
ture adults are capable of breeding all year round; however, re-
productive studies from other countries suggest episodes of peak
settlement over relatively short periods ( 1-3 wk) (Klockner 1976.
Castric-Fey 1983). The larval phase is temperature dependent and
lasts for approximately three weeks (Seagrove 1941); however,
monitoring larval populations from plankton tows has proved un-
satisfactory in the past because the soft-bodied larvae do not pre-
serve well (Senz-Braconnot 1968). Newly settled worms can reach
full tube length (20-60 mm) and sexual maturity in four months
(Castric-Fey 1983) and are therefore capable of rapid colonization.
However, the species distribution and settlement of P. lamarcki
and P. triqiieter in Scottish lochs has not been described.

The cultivation of Mytiliis ediilis L. in suspended culture in
Scotland increased to approximately 2.000 tons in 2000. The Scot-
tish rope-grown mussel industry maintains its competitive edge
over the Irish rope-grown mussel and UK dredge industries by
trading in premium-quality mussels only. Farmed mussels consti-
tute an excellent substrate for the settlement of many other organ-
isms, which from the perspective of the mussel farmer are collec-
tively termed fouling. Suspended long line cultures are particularly

*Correspondmg author. E-mail.' mke@dml.ac.uk

vulnerable to fouling because they are continually submerged and
considerable effort is required to clean crop for marketing (Hick-
man 1992). Both barnacles and tubeworms frequently settle on
mussel shells. Barnacles are not as problematic and are usually
removed by the brtishes used to strip mussels from culture lines,
however, tubeworm (Pomatoceros spp.) longer than 5 mm are
resistant to brushing and result in badly fouled mussels being
devalued or discarded. Mussels with more than 7% of the shell
fouled are not considered Grade A quality. Tubeworm-fouled mus-
sels are not suitable for sale as ••live-in-shell" product to retailers
as the fouled shells are considered visually unattractive, the tube-
worm can die before the mussel causing an offensive smell and the
tubeworm can be released from the tube on cooking and appear
unappetizing. In Scotland, it is considered uneconomical to pro-
duce Grade B rope-grown mussels because of the higher economic
investment involved compared with dredged mussel operations,
and the competition for the export market from the larger Irish
cultivation industry which produces primarily Grade B stock. The
estimated cost of tubeworm fouling to this industry is between
£300,000 and £500.000 per annum (P. Marshall SSMG) and es-
tablished growers believe the problem is worsening.

Previous work has focused on the competition for food re-
sources between fouling suspension feeders and rope grown mus-
sels (Lesser et al. 1992). However, few data exist with regard to
reducing the impact of fouling organisms in cultured mussel sys-
tems. A survey of Irish rope-grown mussel growers indicated the
most important factors in managing tube-worm fouling on their
farms were the timing of handling (delaying grading and thinning
of stock until after the main tubeworm settlement), the density of
mussels on the lines (outer mussels forming a barrier to settle-
ment), the influence of weather and the depth of culture line (Pur-

519

520

Campbell and Kelly

cell & Cross 1994). All of these factors have been identified by
Scottish mussel growers as potentially being of major importance
to fouling levels on their sites.

The objectives of this study were to first describe tubeworm
species colonizing Scottish rope-grown mussels, monitor settle-
ment, and determine the timing of peak settlement in two contrast-
ing lochs and at different depths, thereby ascertaining inter and
intra-loch variation in settlement abundance. Second, field trials
were conducted to assess the effect of mussel density, depth, re-
tubing material, and the presence of adult tubeworm conspecifics
on tubeworm settlement intensity in two size classes of mussels.
Third, to evaluate the use of aerial immersion as a method to
control tubeworm growth on mussels. The data collected provide
basic information for the development of husbandry practices to
alleviate tubeworm fouling.

MATERIALS AND METHODS

Sample Sites

Two lochs. Loch Stn\en (N55"59'.W5'07') and Loch Beag
(N56°53',W3°44'| with established mussel fanns, were selected as
the rope-grown stock here was known to be prone to annual settle-
ments of tubeworm species (Fig. 1). Loch Striven is a narrow
Fjordic loch, almost 13 km in length with the third longest flushing
time (12 days) of any sea loch (Edwards & Sharpies 1986). In
contrast. Loch Beag is not a true fjordic loch but an elongated
embayment with greater mixing and fresh water run-off (Howson
et al. 1994).

Timiiif; of SellkmeiU

The collector strings consisted of settlement plates (a roofing
slate, 40 x 23 mm) attached by cable ties to a length of 6 mm
polypropylene rope, at depths of 2. 5, and S m. Before deployment,
the plates were conditioned for two weeks in aquaria of sand-
filtered seawater and kept in darkness to promote a predominately
bacterial biofilm (Hammer et al. 2001 ) because lack of an estab-
lished biofilm on slates has been shown to limit settlement (Chan
& Walker 1998). Fortnightly, from mid-April to mid-December
2001, at each of two sites within Loch Striven and Loch Beag (Fig.
2), four replicate collector strings were deployed and two replicate
collector strings, which had been in place for a fortnight, were
recovered for tubeworm enumeration. Thus, on every sampling
visit, two replicate collector strings were added to those in the
water, and they remained there until completion of the monitoring
program. A Tinytalk' ' II temperature logger was deployed at a
depth of 5 m in each loch to record sea temperatines throughout
the sampling period. All tubeworm ob,served on both sides of the
12 slates recovered from each loch, every fortnight, were enumer-
ated with the aid of transparent counting grid.

Pomatoceros Species Identification

Tubeworms were identified from slates retrieved at the end of
the experiment, which had been placed in the water during the
peak settlement period. Individuals (>20 mm tube length) were
randomly selected from replicate slates at each depth, site, and
loch: removed from their tubes; and identified under a dissection
microscope on the basis of opercular structure (Zibrowius 1968),
which has been confirmed by previous genetic studies as suitable
to distinguish the two species (Ekaratne et al. 1982).

West Coast of
Scotland

Figure I. Map of the West Coast of Scotland and sampling locations
al Lochs Beag and Striven. Rectangles represent areas of long line
mussel cultivation. Small black squares show subsites at which collec-
tor plates were deployed (mld-.\pril to mid-December 20(11).

Kffecl of Mussel Size, Density, Retuhing System, and Conspecifics on
Tubeworm Settlement

At Loch Striven in mid-August 2001, before peak tubeworm
settlement, rope-grown mussels, with <2'> of individuals fouled
with tubewomi (termed "clean"), were graded to create two dis-
tinct size classes with 1) large clean mussels (mean shell length
39.9 mm ± 1.03 SE, n = 50) and 2) small clean mussels (mean
shell length 33.5 mm ± 1.03 SE, n = 50). Mussel cultures are
allowed to establish on 8-m lengths of polypropylene rope hanging
vertically. Before attachment by the byssal threads, the mussels are
supported by a mesh tube. Mussels may migrate through different
tubing systems at different rates and/or establish different feeding
structures on the culture line, which could influence tubeworm
abundance on mussels. For this experiment the clean large and
small mussels were retubed using two common tubing systems:
cotton tubing and plastic "pergolari" mesh, at a high and a low
stocking density forming eight different treatments (Tables 1 and
2). A further group 3), large mussels (mean shell length 61.7 mm

Mussels Grown in Suspended Culture

521

12000 -

10000

2.1-Stnven
site 1

200 -

0 -

2.3-Beag
site 1

1/5 1/6 1/7 1/8 1/9 1/10 1/11 1/12

400

200

0 -

Time (months)

Time (months)

Figure 2. Mean
Striven (Apr to

and standard deviation for Pomatmeros spp. settlement counts per slate at 2, 5. and 8 m at two sites 4 km apart within Loch
Dec 2001), 500 m apart in l,och Beag (note difference in scale on the Y-axis I. and seawater temperature ( C) at 5 m.

± 0.915 SE. n = 501 with >98% of individuals fouled with tube-
worm, were graded and retubed using plastic '"pergolari" mesh
only at a high and a low stocking density, forming a further two
treatments (Tables I and 2). The mean shell length was signifi-
cantly smaller in the small clean mussel treatment than in the two
large mussel treatments, which were not significantly different in
terms of shell length, thus allowing the inclusion of mussel shell
length as a treatment effect (F = 250.2. P < 0.001, df = 2,Tukeys
P < 0.05; Table 2).

Each of the 10 treatments had 5 replicates that were suspended
vertically and at random, approximately 40 cm apart, along the
horizontal main line. The treatments were removed from the water
after 16 wk (mid-Deceinber) during which peak tubeworm settle-
ment had occurred and then declined to a base level. The num-
ber of Pomatoceros spp. that had settled on individual mussel
shells sampled from a depth of 4-5 m from each replicate was
counted.

TABLE L
Treatment characteristics.

Treatments

Replicates

Tubing

Density

(A) Large clean mussels
1

2

3
4

(B) Small clean mussels
5

6
7
8
(Cl Large fouled mussels
9
10

Cotton

High

Cotton

Low

Pergolari

High

Pergolari

Low

Cotton

High

Cotton

Low

Pegolari

High

Pergolari

Low

Pergolari

High

Pergolari

Low

522

Campbell and Kelly

TABLE 2.

Shell length (mm) and stundard error (SEl and slocklnj; densities of
the mussel treatments.

TABLE 3.

Mean and SD Pninatmeros spp. counts at peak settlement for each
depth at sites in Loch Striven and Loch Beaj;.

Treatment

Length I mm I

(/I = 50 (±S.E.)

Stocking Density kg/m)

High

Low

Large clean

yLjty

(±1.05)

3

Large fouled

61. 7-'

(±0.92)

5

Small clean

33.5*'

(±1.03)

1.75

0.875

Means with different superscripts in the same column are significantly
different iP > 0.05. ANOVA and Tukeys).

Mussel Stock Fouling Comparisons

In early August 2001. the fouling intensity of tubeworm on
rope-grown mussel stocks with varying treatment histories in Loch
Striven was assessed. In Loch Striven the mussel spat generally
settles from the plankton in spring (late- April to early-May), by the
following spring the nius.sels are termed 1-y-olds. At intervals over
the growing cycle the mussel stocks may be stripped from the
culture rope, graded according to siz.e, and resuspended at a lower
density. The different groups and depths examined were deter-
mined by the stocks (settled in spring 1999 or 2000). which were
available at the site. Tubeworm fouling intensity was compared on
1-y-old ungraded mussels ( 1-y ungraded), 1-y-old mussels that had
been thinned in November 2000 (1 y Nov), and in small 2-y-old
mussels that had been graded in July 2001 (2 y July). Mussels from
2. 4, 6, and 8 m depth on culture line from each stock were
compared. A 1-m length of mussel culture line was removed from
five replicate culture ropes at the four different depths and indi-
viduals separated and washed. As a measure of mussel density the
total weight of individuals per m was recorded to the nearest 0. 1
kg. Finally, the percentage of mussels with tubeworm and the
number of tubeworm per mussel for a kg sample per replicate were
ascertained.

Aerial Immersion Time and Tubeworm Survivorship

In December 2001, rope-grown mussels heavily fouled with
tubeworm were collected from Loch Striven and transported in
cool boxes to the laboratory aquaria. In each of two temperature-
controlled rooms at 13°C and 7°C (both at 95% humidity), groups

80
70

20

10 -

■■ I

Gorton
Cotton

- high

- low

^H 3

1 — 1 4

Perg -
Perg -

high
low

Jl

2 3 4 5

No. of Settled Tube worm

Depth (m)

n

\lean No. Pomatoceros

spp

per slate (±SD)

Site

Loch Striven

Loch Beag

1

-)

1

2029-'(±552)

124" (±18.4)

5

2

4019" (±423)

389=" (±236)

8

2

4825" (+202)

222"" (±120)

T

2

T

5159" (±127)

194" (±53.0)

3

-)

9124" (±10.^9)

628"" (±5.0)

S

-)

11711'' (±65.0)

97 r (±94.0)

Figure 3, Pomatoceros spp. frequency on small mussels (%).

Means with different superscripts in the same column are significantly
different (P > 0.05. ANOVA and Tukeys).

of five mussels with three or more adult tubeworm per mussel shell
were divided among raised trays randomly placed within a con-
tinuous flow seawater tank. The mussels with tubeworm were left
for five days to allow recovery and acclimatization to laboratory
conditions before the start of the experiment. All tubeworms were
identified as P. triqueter. At time 0, all trays were removed from
the seawater, excluding six replicate control trays, which remained
immersed for the duration of the experiment. Six replicate trays of
mussels were returned to their respective tanks after 6. 12. 18. 24.
30. and 36 h of aerial exposure at either 13T and 7'C. Forty-eight
hours after each tray was returned to the seawater, the percentage
of tubeworms alive on each mussel was recorded. Individuals were
assumed to be dead when they did not retreat into their tubes when
touched by a pin.

Statistical Analysis

ANOVA was used to test for significant differences among
treatments and depths, the data having met the assumptions of the
test. Test of association (x") was used to compare tubeworm fre-
quency distributions among the treatments. The linear correlation
between variables was assessed using Pearson's correlation coef-
ficient (/■). Regression analysis was performed to determine the
functional relationship between tubeworm survivorship {9c) and
immersion time (hi. ANCOVA and Tukeys pairwise comparison
was used to compare regression lines between the different treat-
ments. The statistics package Minitab* version 13.1 was used for
all analyses.

RESULTS

Throughout the experimental period, juvenile Pomatoceros
spp. were the most abundant species observed on collector slates in
both lochs. The distribution of settled juvenile tubeworm on the
slates tended to be uneven, composed of aggregated groups with a
greater proportion of individuals on the lower half of the slate.
After two weeks of immersion at each site, the tube length of
individuals on the slates was <3 mm.

Pomatoceros spp. Species Identification

All individual tubeworms examined on slates from the peak
settlement period at Loch Striven and Loch Beag were identified
as P. triqueter. Therefore, P. triqueter was assumed to be the most
abundant of the Pomatoceros species present and, furthermore,
responsible for colonization and fouling of rope-grown mussels in
the lochs.

Mussels Grown in Suspended Culture

523

Pomatoceros spp. Settlement

The timing of peak settlement differed between lochs but was
found to be synchronous between sites and over depth within the
same loch. Significant differences in settlement intensity were ob-
served between lochs, sites, and depths. At the Loch Beag sites two
periods of peak settlement were observed (26.6.01-10.7.01 and
4.9.01-18.9.01) with the highest mean number of P. triqueter per
slate (971 ± 94 SD, site 2) recorded in the later peak (Table 3 and
Fig. 2). It is not known whether settlement at Loch Beag was in
fact two discrete periods or one potentially more intense period
disrupted by environmental conditions. A single period of peak
settlement was recorded at Loch Striven (26.9.01-15.10.01); with
the highest mean number of individual P. triqucler per slate
(11711 ±65 SD, site 2), an order of magnitude higher than in Loch
Beag (Table 3 and Fig. 2). The Loch Striven and Loch Beag later
peak events occurred as summer seawater temperatures declined.
In both Loch Striven and Loch Beag at peak settlement, site 2 had
significantly greater settlement than site I (Loch Striven. F =
280.0, P < 0.001. df = 1; Loch Beag, F = 27.2. P < 0.02, df =
1; Tukeys, P < 0.05), indicting the scale on which P. triqueter
settlement intensity can vary within lochs at sites 500 m to 4 km
apart (Loch Beag and Loch Striven, respectively).

In Loch Beag. no effect of depth on settlement intensity was
observed at site 1. whereas at site 2. significantly greater settle-
ment occurred at 5 m and 8 m than at 2 m. The effect of depth on
P. triqueter settlement was more pronounced at Loch Striven. Site
1 had significantly greater settlement at 5 m and 8 m than at 2 m
and at site 2 settlement increased significantly with increasing
depth.

Effect of Mussel Size, Density. Retubing System, and Conspecifics on
Tubeworm Settlement

The number oi Pomatocervs spp. settling on small mussels was
found to be significantly lower than that on the large mussel treat-
ments. Among the small size class treatments, the percentage of
mussels with si tubeworm on the shell ranged between 21.5%
and 28.4%, whereas in the large size class treatments, the percent-
age of mussels with >1 tubeworm on the shell ranged between
91% and 100% (Tables 4 and 5. Fig. 3 and 4). Thus, analyses on
small mussel treatments were performed separately to the large
mussel treatments.

Small Mussel Treatments

The small mussels migrated successfully from the two tubing
systems. Pomatoceros spp. were observed to have settled predomi-
nately along growth checks on the mussel shell exterior with a

smaller proportion having settled on the umbo and shell ligament
regions.

No significant difference in the mean percentage of individuals
fouled was found (ANOVA. F = 1.24. P = 0.329, df = 3) and no
significant differences were ob-served among frequency distribu-
tion of Pomatocero.s spp. fouling between the small mussel treat-
ments (four classes. 0. 1.2, 3-I-, x"' = 10.29, P = 0.328. df = 9;
Table 4. Fig. 3).

Large Mussel Treatments

Significant differences in number of tubeworm per individual
were identified between the large mussel treatments (ANOVA. F
= 17.1, P < 0.001. i// = 5; Table 5). However, in all large mussel
pergolari treatments, the mussels did not establish outside the tube
because the mesh size proved too small to allow mussels to mi-
grate through the apertures. Thus, the mussels were confined
within the tube for the duration of the field trial, affecting the
structure of the feeding colony and preventing comparison be-
tween pergolari and cotton tubing systems with respect to tube-
worm fouling.

Clean cotton-tubed mussels at low density had a significantly
greater number of tubeworm per mussel than clean cotton-tubed
mussels stocked at high density. No significant differences were
observed between clean cotton-tubed mussels at low density and
clean pergolari-tubed mussels at high and low densities. No sig-
nificant differences were observed between clean cotton-tubed
mussels at low density and fouled pergolari mussels at high and
low densities. Tubeworm numbers per individual were signifi-
cantly greater in fouled pergolari-tubed mussels at high and low
densities compared with clean pergolari mussels at high and low
densities (Table 5).

From the test of association analysis (x~), four significantly
different distributions among frequency classes of tubeworm on
large mussels were identified; 1 ) clean cotton-tubed and clean
pergolari-tubed at high density had the greatest proportion of mus-
sels with low frequencies of tubeworm and the proportion of mus-
sels decreased steeply in higher tubeworm fouling classes (Fig. 4.
4.1 and 4.3); 2) clean pergolari tubed at low density had a large
proportion of mussels with low frequencies of tubeworm fouling,
which did not decrease as steeply over higher tubeworm fouling
classes compared with distribution 1 (Fig. 4, 4.4); 3) clean cotton
tubed at low density comprised of an approximately even fre-
quency distribution of mussels throughout the tubeworm fouling
categories (Fig. 4. 4.2); 4) fouled pergolari-tubed mussels at high
and low density also consisted of an approximately even frequency
distribution of tubeworm frequency classes among mussels yet had
the lowest proportion of mussels in low tubeworm fouling catego-
ries (Fig. 4. 4.5 and 4.6).

TABLE 4.

Mean (%) of mussels with Pomatoceros spp. and SE in the small mussel treatments.

Treatment

Tubing

Density

Replicates

Mean % Mussels With
Pomatoceros spp (±SEl

CV %

Cotton
Cotton
Pergolari
Pergolari

High
Low
High
Low

22.3'' (±2.65)
28.4° (±4.82)
21.3° (±2.02)
21.5° (±1.81)

26.7
38.0
21.2
18.8

Coefficient of variation is given (CV%). Means with different superscripts in the same column are significantly different (P > 0.05. ANOVA and Tukeys).

524

Campbell and Kelly

M 40

0-) 5-9 10-14 15-

Pomatocews freq. classes

2 30

Z 20

0-4 5-9 10-14 15-

Pomatoceros freq. classes

0-4 5-9 10-14 15-

0^4 5-9 10-14 15-

Pomatoceros freq. classes

0^ 5-9 10-14 15-

Pomatoceros freq. classes

3 30

Z 20 -

0^ 5-9 10-14 15-

Pomatoceros freq. classes Pomatoceros freq. classes

Figure 4. Pomatoceros spp. frequency classes on large mussels (100 mussels per treatment).

Mussel .Slock Fouling Inlcnsily Coinparisniis

The results demonstrate the wide \ariation in the percentage of
mussels fouled that exists among different mussel stocks within
the farm site, with the mean percentage mussels fouled among the
different mussel stocks ranging between 0.7% (l-y ungraded
stock) and 5.9% (l-y Nov-graded stock; Table 6). The stocks — l-y
Nov, l-y ungraded, and 2-y July mussels were all significantly
different with respect to levels of tubeworm fouling, with a greater
percentage of mussels fouled in the l-y Nov stock and least in the

l-y ungraded stock (F =55.6. P < 0.001. elf =2; Tukeys P <
0.05).

Variation in fouling intensity was observed to increase as depth
increased. However, no correlation between the percentage of
fouled mussels and the variables depth (m) and density (mussels
kg/m) among individual stocks or stocks combined was found. A
negative correlation was found between depth (m) and mussels
density when all treatments were combined (;■ = -0.532. P <
O.OOl. n = 60). indicatmg that as depth increases mussel density
alom; the culture line decreases.

Mussels Grown in Suspended Cultlire

525

Treatment

TABLE 5.
Mean number of Pnmaloceros spp. per mussel and SE in the large mussel treatments.

Fouling

Clean

Clean

Clean

Clean

Fouled

Fouled

Tubing

Cotton

Cotton

Pergolari

Pergolari

Pergolari

Pergolari

Density

High
Low
High
Low
High
Low

lUO
100
100
100
100
100

Mean No. Pomaloceros
spp. Per Mussel (±S.E.)

5.S1' (±0.501
10.55" (±0.84)
5.07^ (±0.38)
7.24" (±0.58)
10.10^+0.65)
10.45N+0.66)

CV%

87.1
79.6
75.1
80.7
64.4
63.3

Coefficient of variation is given (CV%). Means with different superscripts in the same column are significantly different (.P < 0.05. ANOVA and Tukeys).

Aerial Immersion Time and Tubewonn Surviviirship

Although increasing P. irii/iieter mortality was clearly evitjent.
as aerial immersion time (h) increased, no mortalities amongst the
mussels occurred during the trials. No P. tnqiictcr mortalities were
observed in the control treatment, which was never immersed dur-
ing the experiment (Fig. 5)

The interaction term (time x treatment) was significant
(ANCOVA, F = 5.90. P = 0.02). indicating that the slopes. iP.
triqiieier % survival rates over time) were significantly different at
7°C and 13°C, at the 95% confidence level. Adult P. irkjuerer
were predicted to achieve a mean 100% inortality after 24.1 h and
35.4 h at an air temperature of 7°C and I3°C. i-espectively (Table 7).

DISCUSSION

P. iriqueter was the most abundant of any species settling on
collector plates and the only species of tubeworm found on the
mussels. This is in concordance with the observation that P. kumi-
rcki occurs more frequently in intertidal and shallow turbid sub-
tidal waters (Hayward & Ryland 1995) and that P. iriqueter can
out-compete P. lamarcki for settlement space at depths of 13 m.
although the dominance of one species over the other is thought to
be dependent on climatic conditions (Castric-fey 1983).

The pattern of settlement in the two Scottish lochs was consis-
tent with that in other temperate populations of P. triqiieier
(Klockner 1976. Castric-fey 1983), with peak settlement occurring
later in the year, possibly the result of the more northerly latitude
(Klockner 1976). The timing of peak settlement differed between
lochs, presumably as a result of specific biologic and physical
differences (Crisp 1974. Dirnberger 1990) but was synchronous
within each loch and over depth. Levels of settlement differed
between lochs, sites, and over depth within the same loch, giving
indication of the scale on which intra-loch variation in environ-
mental factors influence settlement abundance. Such monitoring

should be repeated to ascertain year-to-year variation in occur-
rence of peak settlement within Scottish lochs.

The higher settlement levels observed at Loch Striven may
result from a long flushing time combined with comparatively
little fresh water input and mixing (Tett et al. 1986): consequen-
tially, the larvae are retained within the loch for longer periods of
time and rarely e,\perience lowered salinity, which is reported to
reduce settlement (Castric-fey 1983). In addition Loch Striven has
had an established mussel site (8 y). whereas the site in loch Beag
was recently established (3 y) and is approaching its second har-
vest. The scales of the operations are also significantly different
(standing mussel stock at Loch Striven is 200 tons and Loch Beag
is 30 ton). A longer period of mussel production at Loch Striven
may have increased the standing population of tubeworm through
the accumulation of live mussels and shell material beneath the
farm, reported to increase site availability for settlement of calcar-
eous polychaetes (Tenore et al. 1982. Kaspar et al. 1985).

Increasing or peak seawater temperature may act as a direct or
indirect cue for optimum gamete production or spawning or create
optimal conditions for larval settlement and survival, resulting in
discrete and intense periods of tubeworm settlement. At higher
temperatures bacterial loading would also be maximal and may
also generate a cue for settlement. A high bacterial density has
been identified as a major cue for the settleinent response of P.
lamarM (Hammer et al. 2001). and mussel farms have been
shown to possess increa.sed microbial assemblages resulting from
elevated levels of organic enrichment (La Rosa et al. 2001).

Speculatively, the exaggerated metabolic output from mussels
intensively farmed within a restricted loch environment may en-
hance or promote a tubeworm larval settlement response. Natural
concentrations of ammonia (NH,). a principle component of ex-
cretion in bivalves, released by adult oysters can be sufficient to
induce settlement of oyster larvae, particularly at highest tempera-
tures when peak values for total NH,-NH4* occur (Fitt & Coon
1992). Mussels may therefore be more susceptible to tubeworm

TABLE 6.

Mean length of mussels (mm) with SE mean density of mussels (kg/m) with SE, mean ( -7, ) of mussels with Pomaloceros spp. and SE for each

mussel stock.

Treatment

Length (mm)

(H =50) (±.S.E.)

CV^f

Mean Density
kg/m (±S.E.)

1 y Ungraded

1 y Nov

2 y July

37.8 (±1.45)
40.4 (±0.66)
41.7 (±0.70)

27.1
11.5
11.9

6.0 (±0.80)
4,4 (±0.20)
4.0 (±0.20)

Means with different superscripts in the same column are significantly different (P < 0.05. ANOVA and Tukeys).

Mean % Mussels
Fouled (n = 100) (±S.E.)

0.66-'{±0.17)
5.89" (±0.51)
2.64" (±(J.,34)

526

Campbell and Kelly

100 .

\ \
\ \

• 13 °C

O 7"C

13 °C

80-

7°C

>

E

60
40-
20 -

0 -

i \

i

\

\ ^
\

\

0 5 10 15 20 25 30 35 40 45

Emersion Time (hrs)

Figure 5. Mean /'. triqiwler survival (%) and standard error against
aerial immersion time Ih) at 13 C and 7 C after a 48-li recovery period
for the linear section of the relationship. Regression lines are fitted.

fouling immediately upon re-immersion after grading, for in ad-
dition to offering an unprotected surface for settleinent. a pulse of
excreted nitrogenous waste, accumulated during immersion and
released on re-immersion, could further induce P. liic/iierer larvae
to settle. Tubeworm settlement cues with respect to environmental
conditions on a mussel farm warrant further research.

The field trial highlighted mussel size (shell length) as an im-
portant factor influencing P. thqueter abundance on farmed mus-
sels because small mussels had consistently fewer tubeworms.
This may be a result of the fast growth rates of smaller mussels,
preventing successful tubeworm attachment. Tubeworms were not
found on areas of fastest growth such as the posterior shell edge
but predominately on shell growth checks and on the umbo indi-
cating settlement to have occurred on regions of relatively slower
growth and/or at periods of disturbance and slow growth. The
density of smaller mussels established on culture lines is greater
than large mussels, having smaller interstices between individuals
and less of a surface area for settlement of fouling organisms. As
mussels age. a change in the nature of the inussel shell texture or
its biofilm may make them more attractive for tubeworm settle-
ment. No effects of initial stocking density or tubing system on
tubeworm settlement on small mussels were distinguished, indi-
cating that size has a greater influence on settleinent than either of
these two factors. Further work should determine the relationship
between mussel size and susceptibility to tubeworm settlement.

P. triqueier settlement was greatest in treatments with conspe-
cific adults on the mussels. However, there was no difference in
tubeworm settlement levels between high and low densities in the
fouled mussel treatments. This suggests that the cue to settle pro-

vided by conspecifics was sufficient to outweigh the effects of
stocking density. Settlement on or near conspecitlc adults has ben-
efits; adults derive reproductive benefits from being within aggre-
gations and larvae that settle near adults benefit from choosing a
habitat likely to support post larval growth (Pawlik 1992). P. lam-
arcki has been shown to settle in response to chemical substances
originating from the body of conspecific adults or juveniles (Chan
& Walker 1998). As a result, whenever practical, individuals with
fouling should be removed from mussels that are to be retubed for
on growing.

Tubeworm numbers were more abundant on the clean large
mussels stocked at low stocking densities; this may be attributed to
greater shell surface areas exposed for settlement and or a decrease
in overall feeding activity in mussels at lower densities on the
culture line. In areas of high tubeworm settlement, mussel lines
should be stocked at optimum densities for maximal rapid growth
and distributed evenly throughout the tubing.

A wide variation in fouling intensity was observed among mus-
sels stocks with different treatment histories at the farm site.
Differences in the fouling intensity between the stocks are most
likely to be attributed to the settlement intensity of tubewonii and
the size of mussels at the time of retubing. The influence of depth
on settlement was not as distinct along mussel culture lines when
compared with the collector slates. Consequently, adjusting cul-
ture line depth to control fouling may prove ineffectual. No cor-
relation could be found between inussel density and degree of
fouling. However, counting the number of mussels on a meter of
culture rope may have been too insensitive as a measurement
oi density as mussel density was not always homogenous within a
meter of culture line and settlement site selection by tubeworm
is influenced over small scales (Pawlik 1992). Further studies
of increased precision would determine the true effect of these
variables.

As a result of the discrete nature of P. triqiieter settlement,
mussel growers could routinely monitor tubeworm settlement and
to avoid practices such as grading and returning mussels to the
water in periods of intense tubeworm settlement. Monitoring
settlement should be conducted specifically for each loch. Growers
should monitor settlement in conjunction with physical variables
such as seawater temperature and salinity specific to individual
sites to reveal areas and conditions that consistently yield lower
levels of tubeworm fouling, and criteria for establishing new mus-
sel farm sites should include hydrographic conditions that mini-
mize tubeworm fouling. Furthermore, mussel-management strate-
gies should adhere to minimizing the time mussel stocks are in the
water, particularly in the second growing season when indi\ iduals
reach a critical size and become vulnerable to excessive tubeworm
fouling. In contrast, an alternative approach to limiting tubeworm
settlement might be to avoid grading in the second year, thereby

TABLE 7.

The regression coefficients a and p and respective .SE. Pearson's correlation coefficient (r), probahility and predicted aerial emersion times
Itprid ll""ll with upper and lower 959;: confidence intervals (C.I.). to achieve l(M)'7f mortalitj for /'. triqueier at 7 C and 13 C after a 48-h

recovery period.

Temperature

Coefficient a

(±SE)

Coefficient p

(±SE)

(h)

Lower •JS'/r C.L

Upper 95% C.L

7'C
13 C

24
24

116.8 (±6..'i2)
126.2 (±9.381

-4.S6 (±0.402)
-3..S7 (±0.323)

-0.919 {.P < 0.001)
-0.940 (P < 0.001)

3.S.4
24.1

33.0
22.3

38.6
26.5

Mussels Grown in Suspended Culture

527

leaving mussels undisturbed and allowing a mixed size-class feed-
ing colony to develop.

Adult P. triqueter were comparatively resilient with respect to
aerial immersion, with a predicted mean 100% mortality after 24.1
h and 35.4 h at an air temperature of 7°C and 13X. respectively.
However, as a result of the lengths of immersion time needed to
achieve 100% P. triqueter mortality, it may not be practical for
growers to use aerial exposure as a method to control tubeworm
growth on mussels to be retubed and harvested at a later date.
Although no mussels died within the experimental period, aerial
immersion over such periods of time may cause undue stress in

rope-grown mussels unaccustomed to exposure, resulting in higher
mortalities after grading and retubing and delayed growth.

ACKNOWLEDGMENTS

The authors thank David Scott of Loch Striven Mussels and Ian
MacKinnon of Loch Beag Mussels for their invaluable contribu-
tion towards the project. Sincerest thanks are also due to Simon
Howard and Galium Cameron of Loch Striven Mussels for their
support throughout. This study was funded by Highlands and Is-
lands Enterprise, The Highland Council and The Crown Estate.

Castric-fey, A. iy83. Recruitment and growth and longevity of Pomaioc-
eros triqueter and Pomatoceros lamarckii on experimental panels in
the Concareau area. Soiilli Brittany. Aim. Inst. Oceanngr Paris 59:69-
91.

Chan, A. L. C. & G. Walker. 1998. The settlement of Pomatoceros lam-
arckii (Polychaeta: Sabellida: Serpulidae); a laboratory study. Biofoitl-
ing 12:71-80.

Crisp. D. J. 1974. Factors innuencing the settlement of manne invertebrate
larvae. In: P. T. Grant & A. M. Mackie, editors. Chemoreception m
marine organisms. New York: Academic Press, pp. 177-265.

Crisp, D.J. 1965. The ecology of marine fouling. In: G. T. Goodman.
R. W. Edwards & J. M. Lambert, editors. Ecology and the industrial
society, 5"' symposium of the British Ecological Society. London:
Blackwell Science Publications, pp. 99-177.

Dimberger, J. M. 1990. Benthic determinants of settlement for planklonic
larvae: Availability of settlement sites for the tube building polychaete
Spirobis spirillum (Linnaeus! settling onto seagrass blades. J. Exp.
Mar Biol. Ecol. 140:89-105.

Edwards, A. & F. Sharpies. 1986. Scottish sea lochs: a catalogue. Scottish
Marine Biological Association and the Nature Conservancy Council.
Dunstaffnage Marine Laboratory Library, Oban, Argyll. PA37 IQA.

Ekaratne, K., A. H. Burfitt, M. W. Flowerdew & D. J. Crisp. 1982. Sepa-
ration of the two Atlantic species of Pomatoceros. P. lamarckii and P.
triqueter (Annelida: Serpulidae) by means of biochemical genetics.
Mar Biol. 71:257-264.

Fin. W. K. & S. L. Coon. 1992. Evidence for ammonia as a natural cue for
recruitment of oyster larvae to oyster beds in a Georgia salt marsh. Biol.
Bull 182:401-108.

Hammer, J. P., G. Walker & J. W. Latchford. 2001 . Settlement of Poma-
toceros lamarckii (Serpulidae) larvae on biofilmed surfaces and the
effect of aerial drying. J. E.xp. Mar. Biol. Ecol 260:1 13-131.
Hay ward. P. J. & J. S. Ryland. 1995. Handbook of the marine fauna of

Northwest Europe. Oxford: Oxford Univ. Press, pp. 258-260.
Hickman. R. W. 1992. Mussel cultivation. In: E. Gosling, editor. The mus-
sel Mytilus: Ecology, physiology, genetics and culture. Developments
in aquaculture and fisheries science. Vol. 25. Amsterdam; Elsevier, pp.
465-510.
Howson, C. M., D. W. Conner & R. H. F. Holt. 1994. The Scottish Seal-
ochs. J.N.C.C. Report, 164. Dunstaffnage Marine Laboratory Library.
Oban, Argyll. PA37 IQA.
Kaspar, H. F.. P. A. Gillespie. I. C. Boyer & A. L. Mackenzie. 1985. Ef-

LITERATURE CITED

fects of mussel aquaculture on the nitrogen cycle and benthic commu-
nities in Kenepuru Sound. Marlborough Sounds. New Zealand. Mar.
Biol. 85:127-1.36.

Klockner, K. 1976. On the ecology of Pomatoceros triqueter (Serpulidae
Polychaeta). Breeding season, choice of substratum, growth and mor-
tality. Helgolander wiss. Meeresuntez 28:352—400.

La Rosa. T., S. Mirto. A. Marino. V. Alon/o. T. L. Maugen & A. Ma^/ola.
2001. Heterotrophic bacteria community and pollution indicators of
mussel — farm impact in the Gulf of Gaeta (Tyrrhenian Sea). Mar.
Environ. Res. 52:301-321.

Lesser. M. P., S. E. Shumway, T. Cucci & J. Smith. 1992. Impact of foul-
ing organisms on mussel rope culture — interspecific competition for
food among suspension-feeding invertebrates. J. Exp. Mar. Biol. Ecol.
165:91-102.

OECD. 1967. Catalogue of main marine fouling organisms. Vol. 3: Ser-
pulids. Paris: Organisation for Economic Co-operation and Develop-
ment, pp. 79.

Pawlik. J. R. 1992. Chemical ecology of the settlement of benthic marine
invertebrates. Oceanogr. Mar Biol. Rev 30:273-335.

Purcell, R. & M. Cross. 1994. Technical Report on Tubeworm Fouling.
Cork: Aquaculture Development Centre. University College Cork. Ire-
land.

Seagrove, F. 1941 . The development of the ^erpulid Pomatoceros triqueter
L. Quart. J. Micros. Sac. 82:467-540.

Senz-Braconnot, E. 1968. Donnees ecologiques et biologiques sur la fixa-
tion des Serpulidae dans la Rade de Villefrance-sur-mer. Vie Milieu
198:109-132,

Tenore, K. F., L. F. Boyer. R. M. Cal, J. Corral. C. Garcia-Fernandez.
N. Gonzalez. E. Gonzalez-Gurriaran. R. B. Hanson, J. Iglesias. M.
Krom. E. Lopez-Jamar, J. Mcclain. M. M. Pamatmat. A. Perez.
D. C. Rhoads, G. de Santiago. J. Tietjen. J. Westnch & H. L. Windom.
1982. Coastal upwelling in the Rias Bajas. NW Spain — contrasting the
benthic regimes of the Rias de Arosa and de Muros. J. Mar. Res
40:701-772.

Tett. P.. R. Gowan, B. Grantham, K. Jones & B. S. Millar. 1986. The
phytoplankton ecology of the Firth of Clyde sea-lochs Striven and
Fyne. Proc. R. Sac. Ed. 90B:223-238.

Zibrowius, H. 1968. Etude morphologique systematique et ecologique des
serpulidae (Annelidae. Polychaeta) de la region de Marseille. Reel
Trav. Sm Mar Endoume 43:81-252.

Journal of Shellfish Research. Vol. 21, No. 2, 529-537. 20U2.

MUSSEL DREDGING: IMPACT ON EPIFAUNA IN LIMFJORDEN. DENMARK

PER DOLMER*

Danish Insliiiik' for Fisheries Research. Charhilleiiliiiul Castle. 2920 Charloltenlwul. Denmark

ABSTRACT Species composition and population density ol epibenthos are described in two areas in Limfjorden. Denmark. Both
areas covered both a mussel fishing ground and an area that has been permanently closed tor mussel dredging since 14SS. Furthermore,
mussels were dredged in a part of the mussel fishing grounds in both areas four months before the investigations. The rest of the fishing
grounds had not been exploited for at least four years. This study describes the short-term impact (4 mo) and long-term impact (>4
y) of mussel dredging using the permanently closed areas as controls. The data were analyzed by multivariate statistics. In both
short-temi study areas significant effects of dredging were observed. A number of taxa (sponges, echinoderms, anthozoans, molluscs,
crustaceans, and ascidians) had a reduced density or were not observed in fished areas four months after the fishing was ended. In one
of the two long-term study areas, significant differences in species composition and density were observed between fished and closed
areas, indicating that the fishery may have a long-term impact on the epibenthic community, whereas in the other long-term area no
difference was observed between fished and control areas. Significant reductions in the amount of shell debris and gravel were observed
in the dredged areas. The impact of the loss of these benthic stitictural compiments on ecosystem processes and functions is discu.ssed.

A"£)' WORDS: long-term impact, short-term impact, fishery impact, benthic epifauna, mussel dredging, .seabed structure

INTRODUCTION

The use of dredges, beam-, and otter-trawls in demersal fish-
eries and the development of heavier gear types with increased
fishery effort have raised a growing concern on the impact at
benthic ecosystems (see review by Jennings & Kaiser 1998. Hall
1999, Collie et al. 2000). Recent studies have investigated the
direct impact of towed-bottom fishing gear on benthic organisms
(Bergman & Hup 1992. Eleftheriou & Robertson 1992, Brylinsky
& Gibson 1994. Thrush et al. 1995. Cunic & Pany 1996. Collie et
al. 1997, Tuck et al. 1998. Hall-Spencer et al. 1999. Hoffmann &
Dolmer 2000, Dolmer et al. 2001), the impact on seabed topogra-
phy and seabed composition (Hall et al. 1990, Pranovi & Giova-
nardi 1994. Kaiser & Spencer 1996. Schwinghamer et al. 1998).
and the effect of suspension of bottom sediment and release of
oxygen-consuming substances and nutrients (Riemann & Hoff-
mann 1991, Dyekja;r et al. 1995, Pilskaln et al. 1998). Indirect
effects of the fisheries include changes in the trophic structure
(Babcock et al. 1999) as the reported increase in density of scav-
engers feeding on injured or discarded species (Gislason 1994,
Jennings & Kaiser 1998) or as changes in species behavior and
species interactions (Ramsay & Kaiser 1998).

Because the blue mussel is an economically and ecologically
important species in many coastal areas, mussel dredging inay
significantly affect the form and the function of benthic ecosys-
tems. Blue mussels form tridimensional matrices of byssus-thread
interattached mussels. These stabilized structures are important for
assemblages of associated organisms (Tsuchiya & Nishihira 1985,
1986, Svane & Setyobudiandi 1996, Ragnarsson & Raffaelli
1999). In soft-bottom habitats, solid components such as gravel
and biogenic structures such as mussel beds and shell debris sub-
stantially increase substratum heterogeneity and complexity. These
structures are reported to be important for invertebrates as spatial
refuges from predators (Heck & Crowder 1991. Dumas & Witman
1993, Lee & Kneib 1994, Thiel & Dernedde 1994) or as a sub-
stratum for settling invertebrates and sessile organisms (Witman &
Suchanek 1984. Baker 1997, Lapointe & Bourget 1999). Mussel
dredging may destroy mussel bed structures and remove important

"E-mail: pdo(s'dfu. min.dk

seabed structuring components such as shell debris and gravel. The
fishery thus exerts a potential diiect impact on the ecosystem by
changing benthic habitat structures and indirectly interfering with
invertebrate settling processes and prey-predator interactions.

In Limfjorden, Denmark, an extensive fishery for blue mussels,
Mxtilus edulis. exists. In a previous study in Limfjorden. Dolmer
et al. (2001 ) demonstrated that mussel dredging reduced the den-
sity of invertebrates living in the bottom. In particular, the poly-
chaetes were significantly reduced in number. Another investiga-
tion in 1997 of the long-term impact of mussel dredging on the
epifauna in an area that has been closed for mussel dredging since
1988, however, failed to show any effects of the mussel fishery
(Hoffmann & Dolmer 2000). The aim of the present study was to
compare the spatial cotnposition of asseinblages of epibenthic
fauna on mussel fishing grounds four months after a fishery was
finished, in areas that have not been fished for more than four
years, and in areas that have been permanently closed since 1988
to test the short-term and long-term impacts of mussel dredging on
the epifauna. The second objective of the study was to measure the
impact of mussel dredging on the amount of shell debris and gravel
on the seabed.

MATERIAL AND METHODS

The study was conducted in Limfjorden (Fig. I ). This area is a
micro-tidal, eutrophic water system of interconnected enclosures
in northern Denmark, supporting a high production of blue mus-
sels. The mean mussel biomass in the part of the area open to
mussel dredging (-750 km") is about 600.000 tons with large
temporal variations (Dolmer et al. 1999), This biomass supports
the largest fishery in Europe exploiting natural mussel populations.
The annual mussel landing is approximately 100,000 tons or 15%
of the mussel stock.

The short-term and long-term impact of mussel fishing on
benthic epifauna was investigated in two different areas in Limt-
jorden; in Lugstor Broad, in the central part of the fjord, and in the
Ager0 area, in the western part of the fjord (Fig. 1). Part of both
areas has been permanently closed to mussel fishing since 1988. In
L0gst0r Broad, the northern part of the area is closed and in the
Agero area, the central part is closed. During April-May 1999 (4
months before the investigation) mussels were commercially
dredged in the area just southwest of the closed area in L0gst0r

529

530

DOLMHR

Figure 1. Map of the two study areas and their stations. The double
hues indicate the separation between fished areas and areas that have
been closed to mussel nshin;; since 1988. In Logstor Broad (eastern
area), the area north of the double line is closed and the area south of
the line is fishing ground. In the Agero area (western area), the area
between the two double lines is closed to mussel fishing. The stations used
in the analyses of long- and short-term impact are separated with dotted
lines. (O) control stations and (•) stations with mussel dredging.

Broad and in the area just north of the clcsed area in the Ager0
area. Furthermore, data from the mussel stock assessment pro-
gram, where the inussel biomass at 250-,^,'>0 stations in the Lim-
fjorden is described annually (Dolmer et al. 1999). indicate that
that the area southeast of the permanently closed area in L0gst0r
Broad and the area south of the permanently closed Ager0 area has
not been fished at least since 1996. Epifauna in the Logstor Broad
and the Agero areas was described in September 1999 to test
whether the species composition and density differed between per-
manently closed areas, areas that were fished four months before
the study, and areas that had not been fished for niore than four
years. Replicated stations in the area closed to fishing were con-
trasted with replicated stations located in adjacent areas that were
subject to harvesting practices.

Shorl-Tcnn Impact (4 Mo)

Stations 7-8 in Logstor Broad and stations 2 1 and 23 in the
Agero area were fished by mussel dredge during April-May 1999
and significant impact on the seabed was observed when diving in
September. To test the impact of dredging on the epifauna species.
co]iipc)sition on the two stations in each area was contrasted with
two control stations in the nearby closed areas. Stations 7 and 8
were contrasted with stations 9 and 10 in LogstOr Broad and sta-
tions 21 and 25 were contrasted with stations 15 and 26 in the
Agero area (Fig. 1). No signs of dredging activity were observed
on the seabed when diving at the control stations.

Liiiiji-Term Impact (>4 >J

In the eastern part of L0gst0r Broad, the stations in the closed
area (12, 13, 14) were contrasted with the stations in the area open
for mussel dredging { 15, 18, 19. 20). In the Agero area, the stations
in the closed area (1 . 5. 7. 10. 11) were contrasted with the stations
in the area open for mussel dredging (6. 8. 9. 24).

At each station, the epifauna (>1 ) cm was identified and quan-
tified. Colonial species such as hydro/oans and bryo/oans were
omitted from the study because of difficulties in quantifying these
taxa. The sponges were included in the investigation, and here
each distinct colony was classified as one individual. At each
replicated station, the epifauna in 30 circles of 0.24 m" were quan-
tified by use of SCUBA diving. The circles were marked with an
iion ring I'andomly placed on the sea floor. The material in the last
1 0 circles was collected and the population density of blue mussels
and the weights of shell debris and pebbles were measured in the
laboratoi'y.

The epifauna species composition and density data was tested
with the PRIMER-5-package (Plymouth Routines in Multivariate
Ecological Research). Before the analysis, the ring samples with-
out epibenthic organisms were excluded from the datamatrix.
Bray-Curtis similarity indices were calculated on 4"' root trans-
formed species density data according to Clarke and Warwick
(1994). The stations were plotted in a Multi-Dimensinal Scaling
plot (MDS) to identify separate clusters of stations. Differences
between contrasted stations were tested in two steps by ANOSIM
analyses. It was tested if there were differences among replicated
stations within each treatment area (fished contra closed) in each
area and if differences (P < 0.01 ) were obtained then an ANOSIM
analysis tested for differences in species composition between con-
trasted stations on average ranked data. If no differences were
observed aniong replicated stations then an ANOSIM analysis
tested for differences between contrasted stations on the set of data
treating each ring observation observations as separate data. The
SIMPER procedure identified which taxa contributed most to the
dissiiiiilaritv among contrasted stations. The significances of the
differences in density of these taxa were tested with / tests.

Observed differences between contrasted stations can be due to
short-term and long-term impacts of mussel dredging or may be
caused by environmental gradients in the study area. It was as-
sumed that if an environmental gradient affected the similarity
between stations there would be a positive relationship between
spatial distance and similarity between stations. On a MDS plot.
the stations would then form a unidirectional track with the spatial
and similar close stations close to each other and most spatial
distant and dissimilar station at the longest distance. To test wheth-
er the similarity among stations was related to their spatial relation.

Mussel Dredging

531

the MDS plots of the stations, including an indication of the sta-
tions spatial relationships, was analyzed.

Impact on Substratum

The data on weights of shell debris and gravel at each of the
short-term impact study stations were tested separately for the
L0gst0r and the Ager0 Area in nested two-way ANOVAs with
fishery/closed status as the first factor and the stations as the sec-
ond factor — nested to the fishery/closed factor. Before the tests.
the data were ///-transformed and tested for homoscedasticity
(F,„,^-test). To test the relationship between diversity and substra-
tum composition, a Shannon-Wiener index was calculated for
each sample from the short-temi study stations. The relationships
between this index and the amounts of shell debris and pebbles
were analyzed by linear regression. To test the role of mussels
forming a biogenic substratum important to the associated fauna,
the relation between the Shannon-Wiener index and the density of
mussels in each sample was analyzed by linear regressions at
station 7 and 8 and 9 and 10 in L0gst0r Broad.

RESULTS

During the investigation, a total of 1 1 and 20 different epifauna
species were recorded in L0gst0r Broad and the AgerO area, re-
spectively. MDS ordinations of the stations in L0gst0r Broad and
the Ager0 area showed that the stations in the short-term study
areas were separated into two clusters, including stations from the
fished and the closed part i)f the study areas, respectively (Fig. 2).
The separation of the stations from the long-term study areas was
not that clear-cut. Low oxygen concentrations (<2 mg 0-, P' ) were
recorded during two weeks in August 1999 in parts of Logstor
Broad. Some mortality of blue mussels was observed after this
oxygen deficiency, but also other species may have been affected.
Because the distribution of areas with oxygen deficiency did not
overlap with the distribution of the tlshed areas, the low oxygen
concentration did not interfere with the results of this study.

Short- Term Impact

In the two short-term study areas, no differences were observed
among stations within each treatment area and the difference be-
tween dredged and closed stations was tested on complete sets of
data. The ANOSIM analysis showed that there were significant
differences between the two dredged stations and the two control
stations in both L0gst0r Broad (P = 0.001 ) and the Ager0 area (P
= 0.001 ). The SIMPER procedure and ; tests indicated that a large
number of species disappeared or had a reduced density in the two
dredged areas, including poriferans. echinoderms. anthozoans,
gastropods, crustaceans, and ascidians (Tables 1 and 2). Splitting
the data into two functional groups, mobile and sessile species,
indicated that the sessile species contributed to a larger part of the
dissimilarity between the control and the dredged stations (51-
61%) than the mobile species, although these species still contrib-
uted significantly to the dissimilarity (.30-42*). ANOSIM analy-
ses on data split into functional groups showed a significant dredg-
ing impact on sessile fauna both in L0gst0r Broad and in the Ager0
area (Table 3).

Long-Term Impact

In both L0gst0r Broad and the Agero area, significant differ-
ences among stations within each treatment area (fished contra
closed) were observed (Table 3). Consequently, the analysis of the

(a)

/

■"•"■

~7\

5

~ 11^

/

/

7

k* '"■

■^

/

10

/

, 1

( 25 )i

8 ;

9J

(...

y

/

6:

.^■'6

~^\ \

Agere

21

stress:

=0.06

(b)

/"io

\ ■'9

\ X

^

(f

•.. 18 N

•*

-^10 )

Logstor Br.
stress=0.07

J

7)

Figure 2. MDS plot of station means in the .Ajjero area (a) and l.ogstar
Broad (bl. The control area stations are delineated with a solid line and
stations from fished areas with a dashed line. The gray background
Indicates long-term study stations and the white background short-
term stations.

long-term fishery impact was conducted on average rankings and
re-ranked data. The ANOSIM analyses testing the species compo-
sition at the stations in the fished area in the southern part of
L0gst0r Broad and in the closed northern part showed that there
was no difference {P = 0.771) between stations indicating that no
long-term effects could be observed. In the Agero area, a signifi-
cant difference (P = 0.024) between the species composition in
the central closed area and the southern area where mussel dredg-
ing is legal may indicate a long-term impact. The SIMPER pro-
cedure and f tests on data from the Agero area indicated that a
number of species disappeared or had a reduced density in the area
open to mussel dredging (Table 4).

The MDS of the similarity between stations and their spatial
relationships (Fig. 3a and b) show no relationship between simi-
larity and spatial distance in the two short-term study areas. This
indicates that differences in species composition between stations
are not due to an environmental gradient in the area. In the long-
term study areas, some of the stations with the longest distance
in-between showed a relationship between similarity and distance

532

DOLMER

(stations 11-10-7 in the Agero area and stations 18- 19-20- 15 in
L0gst0r Broad). The spatial distribution of the dredged part of the
long-term study area in Ager0. with a central station (station 6) and
three stations in a semicircle, make an analysis of environmental
gradients impossible because no relation between similarity and
distance can be established because the distances from station 6 to
all three stations in the semicircle are equivalent.

Impact on Suhslratum

Amounts iif shell debris and gravel differed significantly be-
tween dredged and control stations in both areas (Fig. 4a and b). In
Logstor Broad, the weights of shell debris ranged from 0.5 kg m""
at station 7 and 8 in the fished area to approximately 2 kg m"" at
station 9 and 10 in the control area. In the Agero area, the weights
of shell debris ranged from 0 kg m"" at station 21 and 25 in the
fished area to I kg m~" at station 15 in the control station. Gravel
was only found at the stations in Logstor Broad ranging from 0 kg
m"" at station 7 in the fished area to 0.7 kg m"" at station 9 in the
closed area. The nested ANOVA analyses for both areas showed a
significant effect of dredging on the weights of shell debris (P =
0.000) whereas no significant differences were observed among
stations when nested to the fished/control areas (Logstor B: P =
0.467. AgerO: P = 0.215). The weights of gravel were not only
significantly affected by the dredging (P = 0.000). but also by the
stations {P = 0.001 ). Plotting the Shannon-Wiener diversity (H')
index in the samples from Logstor Broad as a function of the
amount of shell debris a significant (P < 0.01) positively correla-
tion was obtained for the data from the fished area. The similar
relationship hold constant in the permanently closed area (Fig. 5).
As the amount of shell debris in the Agero and the weight of
pebbles in Logstor broad are low at the fished stations, similar
relations cannot be plotted for these data. The relationship between
H' and the mussel density (Fig. 6) showed a similar pattern. A
significant positively correlation (f < 0.01) was obser\ed in the
area open to mussel dredging whereas in the pennanently closed
area only a trend can be detected (P = 0.09)

DISCUSSION

To achieve a holistic sustainable management of fisheries,
knowledge of the fishery impacts on the target populations and the

ecosystem is required. In respect to fishery impact on ecosystems,
such management has to consider both the short-term and long-
term impact and the recovery time for the ecosystem to re-establish
a habitat. This study describes significant differences in the species
composition among stations in areas tlshed four months before
sampling and control stations. A number of taxa (poriferans, echi-
noderms. anthozoans. molluscs, crustaceans, and ascidians) had a
reduced density or were not observed in fished areas four months
after the fishing was ended. These differences included reductions
in both mobile and sessile species. Differences in the species com-
position were also observed between an area that had not been
fished for more than four years and a closed control area. A sig-
nificant reduction in shell debris and gravel was observed in
dredged areas.

The observed differences between stations in fished and closed
areas may be due to mussel dredging but could also be caused by
environmental gradients in the study area (e.g., wind exposure,
water current, sediment composition, primary production). A MDS
plot relating similarity and spatial distance between stations, how-
ever, did not indicate any relation in the short-term areas, suggest-
ing that the observed differences in species compositions are due
to dredging activity and not due to environmental gradients. As the
distance between the closest dredged and control stations in the
short-term in Agero and Logstor Broad is 750 and 200 m, respec-
tively and that the demarcation line between fished and closed area
was drawn without any reference to physical conditions in the area,
it is reasonable to conclude that no environmental gradients were
involved. The stations used in the long-term impact analysis had a
more extensive distribution, with <10 km between the most distant
stations. In Logstor Broad, the dissimilarity was related to the
distance between stations, indicating that a gradient probably in-
fluenced the area. This environmental gradient may increase the
variability in species composition, making it more difficult to de-
tect long-term impact of the fishery. In the Agero area, the way the
stations were distributed made the detection of an environmental
gradient difficult. Consequently, it is difficult to judge whether the
observed differences in species compositions were due to long-
term impact of mussel dredging or to a gradient.

The design of fishery impact studies is a trade-off between a
robust experimental design and the amount of effort that can be
invested in the study. An often-used statistical design is a BACI

TABLK 1.

Short-ttrm impact: species contributing to the dissimilarity {%) between fished stations 7-8 and control stations 9-10 in a closed area in

Legster Broad.

Mobility

Dissimilarity ( % )

St 7-

-8 Dredged

St 9-

-10 Control

Mean

2SE

Mean

2 SE

1 test P

Corella parallelogrammu

S

37

O.Ot)

(1 III!

16.00

8.25

0.00**

Macropodia rostrota

M

14

0.00

u.oo

0.33

0.29

0.02**

Cran^on crangon

M

10

0.07

0. 1 3

0.60

0.46

0.03*

Mvliliis cthilis

S

9

15.40

9.85

1 5.00

7.70

—

Sagania irogladxtes

s

8

0.73

0.52

3.40

0.93

0.00**

Metridiitni senile

s

7

0.13

0.19

0.07

0.L3

0.56

Carcinus maena.s

M

6

0.00

0.00

0.13

0.19

0.16

Mobile species

.W

0.07

—

1.06

—

Sessile species

61

16.26

—

34.47

—

The species are separated into mobile species (M) and sessile species (S). The pooled mean density (individuals m'-) and 2 SE are given and the
differences in densities between dredged and undredged areas are compared with I tests (**P < 0.025. *P < 0.05). Pooled dissimilarity contribution and
mean-densities are given for two functional groups. Only species contributing more than 4% to the dissimilanty are included in the table.

Mussel Dredging

533

TABLE 2.

Short-term impact: species contributing to tlie dissimilarity (Vr) between fished stations 21-25 and control stations 15-26 in a closed area in

the Ajjero area.

St 21-25 Dredged

St 15-26 Control

Mobility

Halichondria panicea S

Asterias rubens M

Sagarlia troglodytes S

Teuita felina S

Metridium senile S

Carcinus maenas M

Macropodia rostnita M

Mytilus edulis S

Buccinum imdatuni M

Hinia reticulata M

Crangon crangon M
Mobile species
Sessile species

Dissimilarity %

lb
12
11
10

6
6
6

5

42
51

Mean

u.ou

0.27
0.27
0.07
0.93
0.60
0.07
0.00
0.07
3.87
1.67
6.55
1.27

2SE

O.UO
0.26
0.32
0.13
0.98
0.37
0.13
0.00
0.13
0.95
0.72

Mean

0.40
1.00
2.27
0.40
2.33
1.80
0.00
0.07
0.00
7.20
1.27
11.27
5.47

2 SE

0..^7
0.44
1 .06
0.31
1.89
0.67
0.00
0.13
0.00
1.72
0.62

( test P

0.03*

0.01*'

0.00*''

0.05*

0.19

0.00*<

0.32

0.32

0.32

0.00*'

0.40

The species are separated into mobile species (M) and sessile species (S). The pooled mean density (individuals m"-) and 2 SE are
differences in densities are tested with / tests (**P < 0.025. *P < 0.05). Pooled dissimilarity contnbution and mean-densities are given for
groups. Only species contributing more than 4% to the dissimilarity are included in the table.

given and the
two functional

Test area

Short-term impact Logstor Br.

Short-term impact Agero

Long-term impact Logstor Br.

Long-term impact Agero
Test on functional groups

Mobile species Logstor Br.

Sessile species Logstor Br.

Mobile species Agero

Sessile species Agero

TABLE 3.
ANOSIM analyses of epifauna species composition and density.

Similarity of Treatment
Stations ( % )

Data for .Analysis of
Fishery Impact

Fishery Impact
Global R

0.1-2

11^1

0.1

0.1

4-38
19-25
0.1-2
0.1-91

Complete set of data

0.224

Complete set of data

0.066

Average ranking

-0.093

Average ranking

0.363

Complete set of data

-0.082

Complete set of data

0.3

Complete set of data

0.016

Complete set of data

0.039

Fishery Impact

Pi 9c)

0.1

0.2

77.1

2.4

94

0

36

4

The data in each area was analysed in two steps. First it was tested if there was difference among stations within each treatment area (fished contra closed),
and if there were differences (P < 1%) then the analysis of the fishery impact was conducted on average rankings. If no difference was observed, then
the impact analysis was conducted on complete sets of data.

TABLE 4.

Long-term impact: species contributing to the dissimilarity ("7f ) between fished stations (6, 8, 9, 24) and control stations (1. 5, 7, 10, II)

closed area in the Agero area.

Hinia reticulata
Crepidula fornicata
Crangon crangon
Ascidiella aspersa
Metridium senile
Carcinus maenas

Dissimilarity (%)

28

22

11

7

6

5

Dredged

Control

Mean

2SE

Mean

2SE

/ test P

3.90

0.66

13.20

1.71

0.00**

0.00

0.00

62.40

20.98

1.53

0.41

3.79

0.79

0.00**

0.00

0.00

5.28

2.50

0.01**

0.10

0.11

2.32

0.70

0.00**

0.67

0.27

2.51

0.5S

0.29

The mean density (individuals m"') and 2 SE are given and the differences in densities are tested with ; tests {'•
contributing more than 4% to the dissimilanty are included in the table.

*P < 0.025. *P < 0.05). Only species

534

DOLMER

(a) 5,..

, 7

24

:

••••1

10

■■' ■■■ y

25 \
■■•\6 -•'■••

„. 8
... 9

11

2/-''

\

Agero

21

stress=0.06

(b)

.15

14.:-iV;

20

>/'..'.^V""''3

19 12

■^ \.

10

18/

Logstor Br.
stress=0.07

8

Figure 3. MDS plot ol station means in the Agero area (a) and Logstor
Broad (b). The spatial relationship between stations is indicated with
a dashed line between the nearest stations in the long-term study areas,
and with a solid line in the short-term study areas.

design (Bet'ore-After-Control-lmpact) that also tests for temporal
and spatial variability in a study area. It is important for the results
of experimental impact studies of trawling and dredging that im-
pacted areas are large (Thrush et al. 1995). A meta-analysis of the
impact of demersal fishing gear on benthos (Collie et al. 2000)
demonstrated that the recovery time is shorter in small impacted
patches because of larger edge effects. The results of experimental
studies may then be strongly dependent on the experimental de-
sign. This study investigated the impact of an unplanned mussel
fishery, in which the scale of the study area is very large compared
with the experimental studies. This ensures that the results are not
affected by the experimental design. Contrary, the character of the
study implies that a causal relation between the observed varia-
tions in species and substrate compositions and fishery impact not
can be established, although the study represents strong indices of
the impact of dredging.

Fishery impacts may significantly affect ecosystem function.
Seme of the species that were partly or fully eliminated from
fished areas were sessile filter feeders (poriferans. anthozoans,
molluscs, and ascidians). In shallow-water bodies such as Limf-
jorden. these filter feeders exert an important control mechanism
over the pelagic primary producers (Cloem 1982, 1991, Petersen

(a)

fished control ^
3000 ^ 4 ^ 4 ^

station

(b)

1500 n

fished

control

1000

E
2

SI

55 500 -

0 -^=

station

Figure 4. The weights of shell debris (solid linel and gravel (dashed
line) at two fished stations and two stations in a closed area in Logstar
Broad (a) and the Agero area (b). The weights are given as mean ± SE.

& Riisgiird 1992, Dolmer 2000) and reduction of a benthic filtra-
tion capacity may weaken bentho-pelagic coupling. Apart from the
filtration capacity, the coupling between the pelagia and benthos
depends on the mixing rates of the water column and the transport
rates of material and energy from the water column to
the benthos. Seabed roughness is a determinant of near-bed mix-
ing (Herman et al. 1999), so smoothing of the seabed by remov-
ing mussel beds and larger particles reduces the transport of par-
ticles to the seabed and reduces the population filtration rates.
Consequently, the fishery interferes with benthic filtration both
by removing filter feeders and by changing near-bed hydrodynamics.
The distribution of blue mussels was patchy, and in L0gst0r
Broad the density of mussels in the fished area was slightly higher
than in the permanently closed area. This suggests that the density

1.5

0.5

o

o oo %

HUH Fn , , 1

o

1 1

Mussel Dredging

2

535

1000 2000 3000 4000
shell debris (g m'^)

5000 6000

Figure 5. Relationship between the amount of shell debris and Shan-
non-Wiener indices for each sample at stations 7-10 in Logstor Broad.
(D) indicates samples from the area open to mussel dredging and ( I
indicates closed areas. Data were analyzed with linear regression. The
dotted line shows the regression on data from the fished area (R' =
fl..^85; P < 0.01 ), and the solid line that on data for the closed area {R-
= 0.017; P > 0.05).

I 1

0.5

O D

IB O ° D °

0 mBBD — Q — B-

10

15

20

25

Mytilus edulis {indviduals m )

Figure 6. Relationship between density of blue mussels and Shannon-
Wiener indices for each sample at stations 7-10 in Logstor Broad. (D)
indicates samples from the area open to mussel dredging and (O)
indicates closed areas. Data were analyzed with linear regression. The
dotted line shows the regression on data from the fished area {«' =
0.470; P < 0.01), and the solid line that on data for the closed area
(R- = 0.152; P = 0.09).

of mussels was much higher m the fished area than in the closed
area before the fishery was initiated. As the mussels fonn a bio-
genic substratum important for associated fauna, mussel density
may influence the distribution of other species. A higher initial
mussel density in the area open to mussel dredging, as in the
short-term study area in L0gst0r Broad, would result in an under-
estimate of the impact of mussel dredging, as a more diverse
associated fauna probably characterized the fished area before the
fishery was initiated.

In L0gst0r Broad, no long-term impact of mussel dredging was
apparent. A previous study also failed to show long-term effects
(Hoffmann & Dolmer 2000). In the present study, the multivariate
analyses of long-term impacts was conducted on average ranked
data resulting from differences among stations within each treat-
ment area. The power of this analysis is much lower than an
analysis of the complete data set. which can be used when no
difference is observed among stations. To detect an impact ot
mussel dredging fishery impact must be separated from the noise
of other factors affecting the ecosystem (Jennings & Kaiser 1998).
Limfjorden is almost bi-annually perturbed by extensive oxygen
depletion lasting for several weeks resulting in mass mortality of
mussels and other benthic invertebrates. In 1994 and 1997 oxygen
depletion caused mortalities of 25-50% of the mussel populations.
The scale of these events has to be considered when evaluating the
impact of the mussel fishery.

Seabed heterogeneity and complexity is an important feature
when discussing the character of benthic habitats. This study has
shown that mussel dredging removes larger sessile animals, shell
debris, and gravel from fished areas. Furthermore, dredging re-
moves mussels forming an important biogenic substratum for as-
sociated fauna. The investigations measured the largest impact on
sessile species, but the fishei^ also affected mobile species after 4
mo. Mussel and shell beds are reported to reduce predation by
creation of spatial refuges (Thiel & Dernedde 1994. Lee & Kneib
1994) and Kraeuter and Castagna ( 1977) reported that Mercenaria
mercenaria had a 75% better survival when reseeded on shell and
gravel substrata than on sand. The impact on epibenthic species is
2-fold: a direct impact of mussel dredging by killing and injuring

individuals by direct contact with the mussel dredge and an effect
from the changed habitat heterogeneity and complexity. The re-
covery of the fished habitats is a function of immigration of mobile
species and recruitment of sessile and mobile species. Recruitment
may also be affected by the changed seabed structure. In many
invertebrates larval settlement depends on solid substrata (Young
1983. 1985. Witman & Suchanek 1984. Baker 1997. Lapointe &
Bourget 1999) but also changed near-bed hydrodynamic forces
may modify larval distribution (Butman 1987, Jonson et al. 1991 ).

Changing the handling of benthic animals and materials in the
fishery by-catch can accelerate the recovery process of ecosystems
following mussel extraction. Today mussels below the legal mini-
mum size (<4.5 cm shell length) and waste material (other animals
and shell debris) are relayed in certain areas (Kristensen & Lassen
1997). Mussel shells from the cooking process must not be re-
cycled to the seabed and are deposited or used on land. As a
consequence of this procedure, important materials are transported
away from the fishing grounds. These important materials should
be brought back to fished areas. Habitat restoration by relaying
small inussels and other invertebrates, shell, pebbles, and larger
stones after mussel dredging would reduce the recovery-time both
in respect of the form and the function of the ecosystem and would
improve the sustainability of the mussel fishery.

The conclusion of this study is that the mussel dredging exerts
a significant short-term impact on the benthic fauna. No unequivo-
cal long-term impact could be demonstrated. The fishery changes
the seabed structure by removing solid particles and biogenic par-
ticles. This impact may be long lasting or even irreversible and
may significantly change the function of the ecosystem.

ACKNOWLEDGMENTS

The authors thank Prof J Collie and Dr. S. A Ragnarsson and
three anonymous referees for thoughtful comments and linguistic
corrections on the manuscript, and to A. Hansen for practical help
in the field. The study was part of the EU-project ESSENSE (Con-
tract FAIR CT98-420I ) and was also financially supported by the
Limfjords Counties.

536

DOIMER

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Jintrmil of Slwllfixh Ri:u'uivh. Vol. 21. No. 2. 539-547, 2002.

FOULING IN SCALLOP CULTIVATION: HELP OR HINDRANCE?

KATHERINE A. ROSS, JOHN P. THORPE, TREVOR A. NORTON, AND ANDREW R. BRAND*

Port Erin Marine Laboratory, University of Liverpool, Port Erin. Isle of Man. British Isles. IM9 6JA

AB.STR.ACT We examined the effects of fouling on physiocheniical and food conditions inside nets used for suspended great scallop
iPecten imixitiitis) cultivation. Conditions in clean nets and sites with no nets were similar for the parameters studied and differed fiom
those in fouled nets. Fouling of nets reduced water movement and. contrary to cominon assuinptions, was associated with high levels
of plankton and detritus. Obvious negative effects of fouling (e.g., accumulation of inorganic matter or nitrate and ammonia) were
absent. It was concluded that in some areas negative effects of fouling may be caused by foulers parasitising or mechanically interfering
with scallops rather than creating an unfavorable environment. Our results have important consequences for scallop growers, research-
ers modelling cultivation in an ecosystem context, and those investigating relationships between the growth of cultivated scallops and
environmental conditions.

KEY WORDS: scallop, aquaculture. biofouling. Pecien nui.xiimis. suspended culture, pearl nets, environmental conditions, particulate
matter, Irish Sea

INTRODUCTION

Immersion in plankton-rich water generally enables scallops in
suspended cultivation to grow faster than on the seabed, under
natural conditions (MacDonald & Thompson 1985. Wallace &
Reinsnes 1985. Hardy 1991 1. Unfortunately, the conditions that
promote scallop growth also encourage fouling of cultivation nets
and scallop shells. This is costly because it increases the weight
and drag of cultivation equipment and is difficult to remove
(Hardy 1991 ). Fouling can also affect scallop growth and appear-
ance.

Fouling of scallop shells and cultivation nets has been shown to
reduce the growth of immature scallops (Claereboudt et al. 1994a,
Lodeiros & Himmelman 1996. 2000). It is assumed that fouling
organisms reduce scallop growth by competing for food and space
or by reducing water flow through nets, and hence the supply of
food and o.xygen and removal of waste products (Duggan 1973,
Leighton 1979, Huguenin & Huguenin 1982. Cote et al. 1993.
Enright 1993, Claereboudt et al. 1994b. Lodeiros & Himmelman
1996. Lu & Blake 1997), Fouling of scallop shells increases the
weight of upper valves and can bind upper and lower valves to-
gether. This increases mortality or reduces growth, probably be-
cause scallop feeding and respiration is inhibited (Paul & Davies
1986. Minchin & Duggan 1989, Roman 1991, Lu & Blake 1997,
Lodeiros c& Hiinmelman 2000). Other potentially negative effects
of fouling include irritation of the scallop mantle (Getchell 1991,
Mortensen et al. 2000) and parasitism by species, including poly-
chaete worms and amphipods (Leibovitz et al. 1984. Mortensen et
al. 2000). However, the influence of fouling varies. Some re-
searchers have concluded that fouling does not affect bivalve
growth (Wallace & Reinsnes 1985, Widman & Rhodes 1991.
Lesser et al. 1992, Lodeiros et al. 1993, Lodeiros et al. 1999).
whereas other work suggests that fouling can have beneficial ef-
fects (Ross 2002). Potentially beneficial effects include a positive
influence on plankton abundance (inveilebrate assemblages re-
lease nutrients, promoting primary production. Dame & Dankers
1988, Asmus & Asmus 1991, Peterson & Heck 1999. Arzul et al.
2001, Mazouni et al. 2001) and. in areas of high flow, reduction of
fast water currents that might otherwise inhibit scallop feeding
(Cote et al. 1993. Devaraj & Parsons 1997).

The present study aimed to determine how fouling on pearl nets
alters the environment for great scallops [Pecien ma.ximiis (L.)).
Divers collected water samples from clean and fouled nets (both
containing scallops) and from sites with no nets (i.e., the water
column nearby), enabling us to isolate the influence of fouling on
physiochetnical and food conditions. This is the first investigation
to examine environmental conditions inside bivalve cultivation
nets and thus provides a unique description of how fouling could
affect scallop growth. Fouling is typically countered by frequent
net cleaning with high-pressure water hoses or regular net changes
(Hardy 1991. Laing & Spencer 1997). Both methods are labor
intensive, increase equipment requirements, and are thought to
stress scallops, reducing their growth rates (Wildish & Kristman-
son 1988, Parsons & Dadswell 1992. Enright 1993. McDonough
1998, Ross 2002). The results of this experiment should help
growers to tackle fouling efficiently, thereby saving time and
money. The study was conducted in an exposed Irish Sea location
off the Isle of Man. However, the major foulers (hydroids and
amphipods) are ubiquitous (see e.g., Hidu et al. 1981. Arakawa
1990. Enright 1993. Claereboudt et al. 1994a) and thus the results
are likely to be relevant where scallop cultivation is perfonned in
other areas with hich water flow.

MATERIALS AND METHODS

Field Work

♦Corresponding author. E-mail: arbrand@liv.ac.uk

Two longline systems (subsequently referred to as the north
and south systems) were positioned off the Southwest coast of the
Isle of Man in approximately 23 m of water (Fig. 1). Longline
head-ropes were approximately 10 m below the sea surface. The
tidal range in this area is 6 m and peak flows are approximately 1
m/s. Experiments were performed in June 2000 during spring
tides. Gross tidal flow runs parallel to the systems, but nearby
rocky outcrops cause erratic local flow patterns that sometimes
extended to the south system. Salinity is 34 ppt. and water tem-
peratures reach a summer maximum of about 15°C and winter
minimum of 6°C (T. Shammon. personal communication 2(;)01).
To the north east of the longlines untreated sewage from Port Erin
(population ca. 2.800) is discharged in the lower intertidal (Fig. 1 ).

Pearl nets were hung in strings of three, with a 2-kg weight
attached below the lowest net. The nets had a plastic covered
square wire frame base with sides of 34 cm and black, monofila-
tnent mesh with 16 mnr spacing. The tnesh is the same as that

539

540

Ross ET AL.

Figure 1. Location of longline systems off the Isle of Man. Irish Sea.

commonly used for lantern nets, and thus the results of these
investigations are probably relevant for on-growth in both pearl
and lantern nets. For logistical reasons only the top nets, hung
about 0.15 m below the head-rope, were sampled. The nets used
for nutrient samples contained ten 2-3 yr old P. nui.xinnis with a
shell length of 65-85 mm. For water flow experiments, nets con-
tained 10 flat pebbles whose combined weight equalled that of 10
scallops. Pebbles were used instead of scallops because scallop
movement may have abraded the plaster balls, leading to inaccu-
rate estimates of water motion. Scallop-sized pebbles were chosen
so that water flow and net movements matched those of nets con-
taining scallops. Nets for flow measurements had loops in the
central, supporting rope, and a door so that plaster balls could be
inserted and fixed centrally. Door fastenings and support were on
net seams so that they did not alter water flow.

Ammonia, nitrate, particulate matter, plankton, and water mo-
tion were measured in clean nets, fouled nets, and open water sites.
Fouled nets had been immersed for 16 weeks before the sampling
whereas "clean" nets had been deployed for only 2 wk. Open-
water sites were positions under the head-rope of the longline. at
the same depth as experimental nets. Treatments were arranged
randomly, at 1-min intervals, along the two systems. On each
system, treatments were replicated five times for nutrient experi-
ments and four times for water flow experiments. Nutrient and
water motion experiments were perfoimed side by side on the
longlines. Water motion was measured for 48 h. during which time
water samples were collected.

Diving was performed from the R,V. Sula. Syringes with 120-
mm Teflon tubing tips (.3-mm diameter) were used to collect
samples in preference to permanent sampling tubes (which would
have become fouled) or electronic probes, which are difficult to
use accurately in situ. Divers collected a complete set of 15 or 20
nutrient samples (for example, all of the ammonia samples from
one longline) using labeled syringes. To prevent disturbance, nets
were not touched or moved either before or during sampling. To
remove any trapped debris from the syringe tip, 5 niL of water was
taken up outside the nets and expelled once the tip was in position.
Water samples were then collected slowly to minimize disturbance
and to avoid sampling water from outside the net. Dives lasted a

maximum of 20 min, after which samples were returned to the
boat. Samples were collected from nets by two pairs of divers
deployed at lO-min intervals. Sampling was alternated between
longlines so that no more than two samples, totalling 160 mL of
v\ater ( 1 % of the net volume), were taken from a net in 4 h. Before
sampling, all bottles and syringes were washed in dilute acid and
rinsed in distilled water.

I'hysiochemical Conditions

Ammonia and nitrate were measured in lOO-niL water samples,
which were kept in the dark on ice during the short boat journey
back to the laboratory. In the laboratory ammonia and nitrate
samples were filtered, through GF/F papers, into bottles and fro-
zen. An Alpkem autoanalyser (RFA 2) was later used to determine
nutrient concentrations.

Plaster of Paris spheres can be used to accurately measure time
integrated water motion (Thompson et al. 1994); in these experi-
ments, they proved to be a reliable alternative to expensive micro-
flow meters. The spheres were made by combining 100 g of Plaster
o\ Paris (CaSOj) with 90 mL of distilled water. The plaster was
mixed to a smooth paste, tapped to remove air bubbles, and then
poured into moulds. Moulds were plastic spheres (70-mm diam-
eter) with a central wire. Filled moulds were vibrated for 10 min
to remove trapped air. Plaster spheres were removed from their
moulds after approximately 12 h and placed in a well-ventilated
area to dry. After at least 4 wk. spheres were dried at .30°C to a
constant mass (accelerated drying at high temperatures can affect
the crystalline structure of CaSOj (Muus 1968).

Before immersion, plaster spheres were wrapped in soft cloths
to prevent chipping and to minimize dissolution. Divers opened
nets and fixed spheres centrally so that they were not abraded by
contact with fouling organisms or nets. Al open water sites, wire
was used to suspend spheres below the head-rope, at the same level
as spheres inside nets. Once in position, the cloths were removed
and the nets were resealed. After 48 h, divers retrieved spheres and
wrapped them in soft cloths before returning to the boat. In the
laboratory, the spheres were dried to a constant mass and their final
surface area was calculated from volume measurements obtained
by fluid displacement.

Plaster dissolution rates (V,) provide an indication of relative
water motion;

K/ =

(W| -wo

Where VV, and W, are the weight of the sphere at the beginning
and end of the experiment respectively, A is the mean surface areas
of the sphere, calculated from start and end values, and T is the
time over which spheres were immersed (Thompson et al. 1994).

Food Conditions

Water samples ( 100 niL) for particulate matter analysis were
kept in the dark on ice during the boat journey back to the labo-
ratory. Particulate matter was filtered onto preashed papers imme-
diately on return to the laboratory. The papers were rinsed with
isotonic ammonium formate and then dried at 40°C to a constant
weight and ashed overnight at 450°C. The GF/F filter paper used
to collect particulate matter had a pore size of 0.7 p-m. Particulate
organic matter (POM), particulate inorganic matter (PIM), and
total particulate matter (TPM) were calculated as follows:

POM= dry weight of filter paper and sample

- ashed weight of filter paper and sample

Fouling in Scallop Cultivation

541

TPM = dry weight of filter paper and sample

- filter paper ashed weight
PIM = ashed weight of filter paper and sample

- filter paper ashed weight

As soon as the water samples were taken up to the boat. 60-mL
samples were transferred to bottles with 1 .2 niL of neutral LugoFs
iodine. The bottles were stored in the dark until plankton were
counted, measured, and categorized using inverted microscopy and
the computer programme SCION image analysis for Windows.
Samples were settled in a counting chamber following the methods
of Utermohl (Hasle 1978). Dense samples were diluted with fil-
tered seawater so that all of the plankton in the chamber could be
counted. To ensure that the precision of plankton counts was
greater than 20% of the total count, the volume of sample enu-
merated always contained more than 130 individuals of the most
abundant organisms (Postel et al. 2000). Organisms were recorded
according to type (small plankton, centric diatoms, pennate dia-
toms, diatom chains, solitary chain-forming diatoms, pelagic cili-
ates, benthic ciliates, dinotlagellates, flagellates, crustaceans, nem-
atodes, invertebrate larvae, and eggs and spores) and maximum .^

o

W
X

tion was used to slightly reduce the contributions to similarity of
the most abundant species). The similarity matrices were ordinated
and clustered using non-metric multi-dimensional scaling (MDS)
and hierarchical agglomerative clustering (on group-average link-
age), respectively (Clarke & Warwick 1994). The two-dimensional
MDS plot had a low stress value and hence the dendrogram from
CLUSTER analysis is not presented here. Instead, levels of simi-
larity from cluster analysis are indicated on the MDS plot (Fig. 4).
A priori tests of the differences between locations and treatments
were performed using a two-way, crossed ANOSIM (analysis of
similarity), and the plankton groups contributing most to any dif-
ferences found between the groups were determined using

length (5-10, 11-20, 21-50, 51-100, and >100 |jim). Small plank-
ton were all organisms of 50-10 pirn; generally these were flagel-
lates and diatoms.

Statistical Analyses

The experimental design was balanced; location was a random
factor with two levels, north and south systems, and treatment was
a fixed factor with three levels, open-water sites, clean nets, and
fouled nets (Underwood 1997). Physiochemical conditions were
measured in five replicates per treatment for each system, but time
constraints meant that plankton data were obtained only for three
replicates per treatment-longline combination. Concentrations of
plankton and nutrients and rates of plaster erosion for each treat-
ment were examined using two-way analysis of variance
(ANOVA). Heterogeneity of variance was tested for using Co-
chran's test (Winer 1971) and where necessary data were trans-
formed. Some data were heterogeneous even after transformation,
but ANOVA was still applied because the experimental design was
balanced and large (Underwood 1997). However, such analyses
increase the probability of a type I error, and therefore significant
results should be interpreted with caution. When ANOVA showed
that the probability of a treatment effect exceeded 0.05 and there
was no interaction between location and treatment [P > 0.25). data
for the two systems were pooled, thus increasing the power of
ANOVA to detect treatment effects (Underwood 1997). When
ANOVA indicated significant factors or interactions between fac-
tors, post-hoc Student-Newman-Keuls tests were performed to
determine which means differed. All analyses were performed
using GMAV5 (Underwood et al. 1998). Ammonia measurements
were analyzed by ANOVA and concentrations below the limit of
detection were included as 5 (Jig/L, the highest undetectable value.
This conservative approach increased the probability of type II
error.

As described above, plankton were classified as one of 45
groups according to their size and type. The data set was then
analyzed using nonparametric, multivariate techniques included in
the PRIMER (Plymouth Routines in Multivariate Research) soft-
ware package (Clarke & Warwick 1994). Bray-Curtis similarity
indices (Bray & Curtis 1957) were calculated between all pairs of
samples to produce a data matrix (after a square-root transforma-

E
o
bj

c
o

o

CO

CO

zi.

0

CO

_i

CD

'c
o
E
E
<

0.4
0.3
0.2
0.1

0.0

12
10
8
6
4
2
0

20
15
10

-

■

r-

— 1

T

p

E

1

open-
water

clean

fouleid

North system

open-
water

clean

fouled

South system

Figure 2. Physiochemical conditions. A, Water motion; B. nitrate con-
centrations: C. ammonia concentrations (mean ± SE) in clean and
fouled pearl nets and open-water sites.

542

Ross ET AL.

SIMPER (similarity percentages analysis. Clarke & Warwick
1994).

RESULTS

Macrofouling Communities

Fouled nets were almost entirely covered (>909f) in the hy-
droids Tubidaria indivisa L. and T. kuynx Ellis & Solander: the
amphipod Jassa falcata (Montagu) and its silt tubes were common
as were the nudibranchs Dendnmotus frondosiis ( Ascanius). Cory-
phelki lineata (Loven). and Facelina bostnniensis (Couthouy).
Small hydroids (e.g.. Obelia sp. and Clyiia hemispherica (L.))
were present but occupied little space compared with the large
TubuUuia spp. "Clean" nets were sparsely colonized (<59(') by
small hydroids.

Physiochemical Cimditiuns

The plaster spheres used to measure water motion remained
spherical throughout their deployment. Water motion was similar
on both longline systems (Fig. 2) and was only significantly re-
duced by fouled nets (Table 1 ). Nitrate and ammonia concentra-
tions were not affected by treatment (Fig. 2). However, only four
ammonia measurements exceeded the minimum detection level of
the autoanalyser (5 |jig/L): these were all on the north system,
which therefore had significantly higher rates of dissolution than
the south system (Table 1 ).

Food Conditions

Total particulate matter was most abundant on the south system
and here there was significantly more in fouled nets compared with
clean nets and open-water sites (Table 2). Although this trend was
apparent on the north system (Fig. 3), differences were smaller and
not significant. The ratio of PIM to POM was lowest in fouled

nets, for both noilh and south sy.stems (Fig. 3. Table 2). Micros-
copy revealed that particulate matter included detritus and inver-
tebrate faeces, in addition to plankton.

Plankton larger than 21 |xm were rare and thus numbers in the
three largest size classes were pooled for univariate analysis. Total
plankton and plankton in size classes 5-10 |xm and 11-20 |i.ni
were most abundant in fouled nets (Table 2). Multivariate analysis
also distinguished fouled nets from clean nets and open water sites
(which clustered together on the MDS. Fig. 4). Interestingly fouled
nets from the two systems were also distinct. ANOSIM revealed
significant differences between both locations and treatments (/?
= 0.42, P < 0.01 and R = 0.57, P < 0.01, respectively). Pairwise
comparisons found significant differences between fouled nets,
open-water sites [R = 0.78. P = 0.01 ). and clean nets (R = 0.89,
P = 0.01), but, as indicated by the MDS plot, open- water sites,
and clean nets, contained similar communities {R = 0.278. P =
0.08). SIMPER analysis showed that high abundances of centric
diatoms, flagellates, pennate diatoins (all 5-10 )a.m). and small
plankton primarily distinguished fouled nets from clean nets and
open water sites. High abundances of these organisms also distin-
guished samples from north and south systems. Although less
common, eggs and spores, nematodes, pennate diatoms, and in-
vertebrate larvae also appeared inost frequently or uniquely in
fouled nets. Consistently low plankton abundance explains the
atypical position of one sample from fouled nets on the MDS plot
(Fig. 4): despite its low content, this replicate contained most of
the plankton that characterized other samples from fouled nets.

DISCUSSION

Fouling of cultivation nets created a unique environment for the
scallops inside: physiochemical and food conditions differed from
those both in clean nets with scallops and those in the water

TABLE 1.

Two-way ANOVA and Student-Newman-Keuls multiple comparisons testing for the effect of location and treatment (clean nets, fouled nets,

and open-water sites) on pliysiocliemical conditions.

Source of Variation

df

MS

F

P

F Ratio \ ersus

Water motion

C = 0.4799. P > 0.05

Location

1

0.0012

3.18

0.091

Residual

Treatment

2

0.0425

196

0.005

Location x treatment

Location x treatment

2

0.0002

0.57

0.575

Residual

Residual

18

0.0004

Total

23

SNK multiple comparison

of treatment results;

North system:

open-uater = c

ean > fouled

Nitrate

C = 0..^539. P > 0.05

Location

1

0.1242

2.22

0.149

Residual

Treatment

2

0.1141

2.91

0.256

Location x treatment

Location x treatment

2

0.0392

0,70

0.506

Residual

Residual

24

0.0558

Total

29

Ammonia

C = 5574. P < 0.05

Location

1

160.5453

4,41

0.046

Residual

Treatment

2

2 1 .8963

1.00

0.500

Location x treatment

Location x treatment

2

21.8963

0.60

0.556

Residual

Residual

24

36.3827

Total

29

Cochran's test results are given. (Bold type mdicates a significant result. P < 0.05).

Fouling in Scallop Cultivation

543

TABLE 2.

Two-way ANOVA and Student-Newnian-Keuls multiple comparisons effect of location and treatment (clean nets, fouled nets, and

open-water sitesi on particulate matter and plankton.

Source of Variation

df

MS

F

P F Ratio Versus

TPM

C = 0.3013. P>0.05

Location

1

1040

7.08

0,014 Residual

Treatment

2

2378

4.35

0.187 Location X treatment

Location x treatment

2

547

3.73

0.039 Residual

Residual

24

147

Total

29

SNK multiple comparibon of interaction:

Open-

water:

North system = South system

North system: open-water = clean = fouled

Clean:

North

system = South system

South system: open-water = clean < fouled

Fouled: North system < South system

PIM:POM

Transformation = Ln (X + 1 ). C = 0.4331, P > 0.05

Location

I

0.0003

0.00

{).444 Pooled data

Treatment

2

0,3369

5.61

0,001 Pooled data

Location x treatment

2

0.0293

0.49

0,620 Pooled data

Residual

24

0.0627

Total

29

Pooled data

26

0.0601

SNK multiple comparison of treatment results:

Open-

water

= clean > fouled

Plankton 5-10 |xm

Transformation = Ln (Xl, C = 0.7020. P < 0,05

Location

1

0.15

0.17

0,686 Residual

Treatment

2

11.44

22..56

0,042 Location x treatment

Location x treatment

2

0.51

0,58

0,576 Residual

Residual

12

0.88

Total

17

SNK multiple comparison of treatment results:

Open-

water

= clean < fouled

Plankton 1 1-20 |xm

Transformation = Ln (X + 1), C = 0.7042, P < 0.05

Location

1

0.15

0,18

0,678 Pooled data

Treatment

2

11. .34

13,88

0,001 Pooled data

Location x treatment

2

0.50

0,61

0,559 Pooled data

Residual

12

0.87

Total

17

Pooled data

14

0.82

SNK multiple comparison of treatment results:

Open-

water

= clean < fouled

Plankton 21-H jjim

Transformation = Ln (X + 1), C = 0.5244. P > 0.05

Location

1

0.97

7,26

0,020 Residual

Treatment

2

1.85

5,24

0,160 Location x treatment

Location x treatment

2

0.35

2,66

0,111 Residual

Residual

12

0.13

Total

17

Total plankton (>5 ixm)

Transformation = Ln (X + 1). C = 0.4092, P > 0.05

Location

1

279000

1,25

0,283 Pooled data

Treatment

-)

16300000

7.29

0,007 Pooled data

Location x treatment

2

2890000

1.29

0.305 Pooled data

Residual

12

2130000

Total

17

SNK multiple comparison of treatment results:

Open

water

= clean < fouled

The results of Cochran's test are given, (Bold type indicates a significant result. P < 0,05,)

nearby. Other work has indicated that sometimes scallops in fouled
nets grow faster than scallops in clean nets (Ross 2002). High
water flow can inhibit scallop feeding (Wildish & Saulnier 1993,
Claereboudt et al, 1994b. Skjaeggeslad 1997) and it could be that,
in a high current area, heavy fouling aids scallop feeding by
roughly halving water movement. Similarly. Skjaeggeslad (1997)

found that water-motion inside plastic cages was reduced by up to
68% by fouling. In addition, fouling might prevent seston deple-
tion around scallops by creating turbulent flow and actively mixing
the water (Frechette et al. 1989. Larsen & Riisgard 1997),

Ammonia and nitrate concentrations were similar for all treat-
ments, indicating that neither clean nor fouled nets caused a build

544

Ross ET AL.

O)

E

O

Q.

E
c

CD

cn

O

c
o

c
ro

Q.

70
60
50

40
30
20
10
0

3.5
3.0
2.5
2.0

1.5
1.0
0.5
0.0

7000
6000
5000
4000
3000
2000
1000
0

open-
water

clean

Z^ '^rfifA

fouled

North system

open-
water

clean

fouled

South system

Fijjurt 3. Food conditions. A, Concentrations of total particulate mat-
ter (TI'M): B. ratios of inorganic to organic particles (PIM:POM); C,
concentrations of 5-1(1 pni (hatched areasi, 11-20 (im (white areas),
and >21 jim (grey areasi plankton (mean ± SF.), in clean and fouled
pearl nets and open-water sites.

up of decay or excretory products. A few apparently high ammonia
measurements may have been caused by the capture of detritus in
water samples (kelp detritus was abundant and the northern
longline was close to a sewage outfall pipe). Inoiganic nitrogen is
absorbed by phytoplankton and it is possible that high numbers of
phytoplankton reduced levels of nitrate and ammonia in fouled
nets. In finfish cages, fouling can reduce water exchange, leading
to oxygen depletion, fish mortality, or reduced growth (Cronin et
al. 1999). Similarly, reduced oxygen levels have been invoked to
explain reductions in scallop growth when fouling is heavy (Hu-
guenin & Huguenin 1982, Enright 1993. Lu & Blake 1997). How-

,.--"

"•sV

\ .SF

•spy

.sc~\_

.,--'

/' .SC ""^ \

SC

' • JJC 1

NO. so *'"';

NO, *S0

SO"NC

•■. NC

'',Nf . ,'

Stress = 0.04

Figure 4, MDS ordination of Bray-Curtis similarity matrix for
square-root transformed plankton-abundance-data. Samples are la-
belled with their location: north system (N), south system (S), and
treatment: clean nets (C), fouled nets (F), and open-water sites (O).
Samples are grouped at a 70% level of similarity from CLUSTER
analysis.

ever, oxygen concentrations have been found to track the abun-
dance of autotrophic plankton, peaking in fouled nets (Ross 2002).
Thus, it seems unlikely that oxygen depletion is a consequence of
fouling in scallop net culture in temperate locations with high
water flow.

Contrary to previous suggestions (Duggan 1973. Leighton
1979. Huguenin and Huguenin 1982, Cote et al. 1993. Enright
1993. Claereboudt et al. 1994. Lodeiros and Himmelman 1996, Lu
and Blake 1997). fouling did not reduce the (quantity or quality of
food particles available for scallops. Instead, net fouling was as-
sociated with abundant plankton and detritus and a favorable PIM/
POM ratio. Trends in plankton abundance were strong enough to
be identified, despite the low number of replicates and the noto-
riously patchy distribution of plankton (Hasle 1978).

Proximity to the shore and to a sewage outfall may explain why
the study area was characterized by high seston loadings (ca. 20
mg/L); similar loadings were found by Cranford et al. (1998) and
Lodeiros et al. (1998) in the sea off Canada and Venezuela, re-
spectively. High levels of organic matter in fouled nets suggest that
fouling may trap, produce, and perhaps support the production of
organic matter — a potential energy source for scallops. Even at
high seston concentrations ( \5 mg/L) scallop scope for growth can
be reduced by dilution of POM by PIM (MacDonald et al. 1998).
When seston concentrations are not limiting a low PIM/POM ratio
(critical values are between 3.5 and 6: the exact number varies
between authors) is required by scallops to maintain a positive
energy balance and maximum scope for growth (Vahl 1980. Wal-
lace & Reinsnes 1985, Cranford 1995, MacDonald et al. 1998). In
this study, open-water sites had PIM/POM ratios of around three,
close to the critical value, and significantly higher than in fouled
nets. Enhanced le\els of POM in fouled nets could thus prevent high
ambient PIM concentrations from depressing scallop growth rates.

Plankton communities were dominated by autotrophs of 5-20
|xm. reflecting Graziano's conclusion that 65% of primary produc-
tion in the northeast Irish Sea is from phytoplankton of 5-20 |xm
(Gra/iano 1988). Fouling may have encouraged primary produc-
tion by releasing nutrients, by retaining plankton in a favorable
light environment or by providing a substrate for benthic auto-

Fouling in Scallop Cultivation

345

trophs. Increased primary production seems, in turn, to have sup-
ported lieterotrophic and mixotrophic organisms such as di-
notlagellates and cihates. Although this finding contradicts com-
mon assumptions of those interested in shellfish cultivation, ecolo-
gists recognize that beds of suspension feeders have the potential
to induce the growth of more phytoplankton than they consume
(e.g., Asmus & Asinus 1991). This is because suspension feeders
increase local inorganic and organic phosphate and nitrate concen-
trations directly through excretion and indirectly via bacterial de-
cay of their faeces (e.g.. Dame & Dankers 1988, Asmus & Asmus
1991, Peterson & Heck 1999. Arzul et al. 2001, Mazouni et al.
2001). A possible mechanism by which suspension-feeding foulers
and scallops could produce and retain nutrients is described in
Figure 5. This is important because in most marine systems, in-
cluding the Irish Sea. phytoplankton are likely to be nitrate limited
at certain times of year (Allen et al. 1998. Kennington et al. 1999).
Mazouni et al. (2001), for example, suggest that during summer
months nutrient recycling by oyster culture units may drive pri-
mary production in a French lagoon.

Benthic plankton are often suspended by coastal turbulence and
generally survive well in the water column (Newell & Newell
1979); thus, the prevalence of benthic ciliates and pennate diatoms
in our open-water samples. However, inany benthic species were
most common in fouled nets where they may have proliferated
because of conditions described above or because of the presence
of a solid surface onto which they could attach or settle. Benthic
organisms might have been suspended by the passage of the sam-
pling syringe, but movement of scallops and water currents are
also likely to make them readily available as food for scallops.
Increased plankton abundances in fouled nets could promote scal-
lop growth because ambient levels (<600 cells/niL in this study)
are unlikely ever to exceed maximum concentrations for scallop
uptake or assimilation (ca. 15.000 cells/mL. Cahalan et al. 1989,
Skjaeggestad 1997).

Proliferation of phytoplankton in fouled nets indicates that light
levels were not reduced below their compensation point, even by
thick Tubulaha fouling. Perhaps strong sunlight in June penetrated
the translucent stalks of this hydroid. Fouled nets may also have
encouraged plankton growth and reproduction by preventing cells
from sinking below the euphotic zone. Future studies might try to
measure light attenuation by different fouling communities. Inver-
tebrate larvae (including decapods and echinoderms) and nema-
todes were only found in fouled nets; although relatively rare,
these potential predators and parasites could have deleterious ef-
fects on scallop growth and survival (O'Connor et al. 1999. Freites
et al. 2000). Net fouling could also be problematic if it promoted
the growth of plankton responsible for shellfish poisoning.

This is the first description of how fouling influences the en-
vironment inside nets used for shellfish cultivation. The data con-

Phytoplankton

Nutnents
via excretion

Heterotrophs
(Scallops, zooplankton. foulers)

Nutnents
via bactenal
activity

Detritus, faeces and pseudofaeces

Figure 5. Mechanism by which nutrients might be retained and re-
cycled in fouled pearl nets.

tradict the common assumption that fouling reduces food levels.
Instead, fouling can be associated with increased food availability
and does not necessarily encourage a build up of decay products or
inorganic matter, even when scallop densities are high. These find-
ings may help to explain why in high current areas scallops inside
pearl nets grow faster than scallops outside (Claereboudt et al.
1994b). They also support anecdotal evidence that a degree of
fouling proinotes the growth of cultivated oysters (Arakawa 1990,
Mazouni et al. 2001). It might be inferred that in some areas
negative effects of fouling on scallop growth are caused by fouling
organisms mechanically interfering with scallops (e.g.. binding
them in unfavorable positions, inhibiting shell opening or disrupt-
ing feeding behavior), or by harboring predators and parasites,
rather than altering the environment. Growers of scallops in high
current or oligotrophic offshore locations should perhaps strive to
reduce the mechanical inteiference of foulers rather than trying to
prevent fouling altogether. Strategies might include biological con-
trol, which can keep bivalves clean and free to move, but does not
completely remove biofouling (Hidu et al. 1981, Enright et al.
1983, Cigarria et al. 1998. Ross 2002). This approach could benefit
scallop growth by reducing mechanical interference whilst retain-
ing the potential food enhancing properties of fouling.

That the en\ ironment inside scallop cultivation nets can differ
significantly from the water-column also has important conse-
quences for two areas of research. First, studies often relate pat-
terns of scallop growth in suspended culture to environmental
conditions (e.g., Wallace & Reinsnes 1985, Cote et al. 1993,
Claereboudt et al. 1994a, Emerson et al. 1994. Lodeiros & Him-
melman 1994. Thorarinsdottir 1994, Velez et al. 1995, Kleinman
et al. 1996. Lodeiros et al. 1998, Lodeiros & Himmelman, 2000).
This relationship may be better understood if future studies con-
sider the influence of net fouling, or measure conditions inside
nets. On a wider scale, water column data have recently been used
by researchers assessing the affects of bivalve cultivation on nu-
trient and seston dynamics of bays, or to predict the capacities of
areas for shellfish cultivation (e.g., Penney et al. 2001, Pilditch et
al. 2001). Because of its potential to uncouple scallop processes
from water-column seston conditions, the influence of net fouling
should also be included in such models.

Here we have provided a snapshot examination of how fouling
can alter environmental conditions. There is evidence that effects
vary with season and the age or composition of the fouling com-
munity (Ross 2002). Though difficult, simultaneous assessment of
fouling communities, the environment inside nets and scallop
growth would enable the influence of fouling to be better under-
stood. We used wide-mesh peari nets containing intermediate sized
scallops in a fast current area. Future work might examine the
effects of fouling in low current areas or with the fine mesh nets
used for growing spat. Studies to determine how common fouling
assemblages (e.g., hydroid, tunicate and bivalve dominated com-
munities) affect the net environment could help growers to choose
cultivation sites, depths or methods of fouling control.

ACKNOWLEDGMENTS

We are very grateful to divers and boat crew for help with
sampling in cold Manx seas. Thanks to T. Shammon, I. Allen, 1.
Laing and J. Berges for advice on nutrient and plankton sampling
and J. Ludgate for help with maps. KAR was supported by a
NERC research studentship GT4/97/148/MAS and the work by the
Isle of Man Department of Agriculture, Fisheries and Forestry.

546

Ross ET AL.

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Jourtwl of Shellfish Rcseunh. Vol. 21. No. 2. 549-555, 2002.

ANNUAL FEEDING CYCLE OF THE PATAGONIAN SCALLOP ZYGOCHLAMYS PATAGONICA
(KING AND BRODERIP, 1832) IN RECLUTAS BED (3rS-55°W), ARGENTINE SEA

LAURA SCHEJTER,' - CLAUDIA S. BREMEC,' " * RUT AKSELMAN,"
DANIEL HERNANDEZ,- AND EDUARDO D. SPIVAK'

^Comejo Nacioiuil tie lnvestlt;aci<>nes Cientificas y Tecnicas (CONICET). Buenos Aires, Repi'iblica
Argentina: -Institiita Nacional de Investigacion y Desarrollo Pesquero. Paseo Victoria Ocampo 1,
(7600) Mar del Plata. Repiildica Argentina: and ^Universidad Nacional de Mar del Plata (UNMdP),
Mar del Plata. Repiihlica Argentina

ABSTRACT This article deals with the diet ol the Patagonian scallop. Zygochluiiiy.s p,ikis;i>niiu. during a yearly period at Recliitas
bed (39°S-55"W). Argentine Sea, and relate.s the results with the oceanographic conditions and the growth pattern known tor this
species. Scallops (n = 180) from six samples were dissected and the gut contents were identified and counted. Results showed a
predominance of diatoms in the diet and maximum food ingestion in spring. Maximum somatic (muscle) growth for this species at the
same study site was also found during spring. It is suggested that after the spring phytoplanktonic bloom, oceanographic conditions
(thermocline in development) allow the sedimentation of food particles to the bottom; thts input of energy could produce the somatic
growth documented for this filter-feeding species.

KEY WORDS: diet, annual cycle. Patagonian scallop gut contents, Zxgochlamxs patagniiica. Argentine Sea

INTRODUCTION

Scallops are suspension-feeding organisms, feeding on detritus
and phytoplankton (Bricelj & Shumway 1991). Particles aie
mainly captured by cilia and mucus on the dorsal infrabranchial
surface of the gill and the gill arch thus provides the major capture
site (Beninger & Le Pennec 1991 ). Ciocco ( 1995) documented the
importance of the labial palps in food particle transport to the
mouth and also their contribution in preventing reflux and favoring
agglutination and particle selection in Aequipeclen tehiiekhus
(d'Orbigny). It is known that particle selection may occur at the
labial palps and/or gills (Jorgensen 1990; Ward et al. 1997) and
also that selection is not only based on particle size but on other
important characteristics (Shumway 1985. Shumway et al. 1997).
The effective lower limit of particle retention in studied pectinids
ranges between 5 and 7 |jim. and therefore bacterioplankton (typi-
cally between 0.3 and 1 jxm; Bricelj & Shumway 1991) and pi-
coplankton are not available as food. Some gut content studies in
pectinids showed the importance of benthic and/or tychopelagic
algae in the diet of scallops (Vemet de Hall 1977. Shumway et al.
1987. Bricelj & Shumway 1991 ).

Seasonal growth in bivalves, including scallops, is influenced
by environmental changes, especially food supply (Bricelj &
Shumway 1991). It is known that food availability is coirelated
with growth in scallops (Griffiths & Griffiths 1987. Barber &
Blake 1991 ), and that it is possible to find abnormalities in growth
during diatom blooms (Lorrain et al. 2000). In addition. Ciocco
(1992) concluded that differences in growth between populations
of Aequipeclen lelniehluis were related with environmental fac-
tors, such as temperature, depth, and food availability, and not with
genefic factors. Valero et al. (2000) studied growth in Zy-
gochlamys patagonica (King and Broderip) using satnples taken
from the Reclutas bed of the Argentine Sea (Fig. 1 ). They found
that maximum growth in shell, muscle and gonad occurred during
different months of the year.

The Patagonian scallop, Zygochlamys patagonica, is distrib-
uted in the Magellanic Biogeographic Province, in the Atlantic

^Corresponding author. E-mail: cbremecCs'inidep.edu.ar

Ocean, from 35°S to Tierra del Fuego, and in the Pacific Ocean, up
to 42°S, between 40 and 200 m depth (Waloszek & Waloszek
1986, Ciocco et al. 1998). Zygochlanns pataganica and
Aequipeclen lelmelchiis are the two commercial pectinid species in
the Argentine Sea. There are nine Patagonian scallop beds in the
Argentine Sea; Two are located in the intermediate shelf between
60 and 70 m depth and seven in the shelf break front area along the
100 m depth isobath (Lasta & Bremec 1995, 1998, 1999: Fig. 1),
which is characterized by high productivity (Brandhorst & Cas-
tello 1971, Carreto et al. 1981, 1995, Podesta & Esaias 1988). The
Reclutas bed is located in the northern zone of this area, where
oceanographic seasonal changes occur. During summer, the water
column is stratified, and the surface and the bottom layers are
separated by a pronounced pycno and thermocline between 30 and
40 m depth. During autuinn and winter, the water column is ver-
tically mixed by convective circulation patterns, which result in
breakdown of stratification (Guerrero & Piola 1997). Baldoni and
Guerrero (2000) provided a more detailed study of the evolution of
the temperature vertical structure of the water column in Reclutas
bed area during the year.

The objectives of this work were to study the diet of the Pat-
agonian scallop. Zygochlamys patagonica, from samples taken at
Reclutas bed during an annual period, to detect possible seasonal
changes in diet, and to relate these results with the oceanographic
conditions in the study area and with the growth pattern of the
species,

MATERIALS AND METHODS

Gut contents of 180 scallops (35-50 mm shell height) were
analyzed from samples (;? = 30) preserved in formaldehyde
(10%). taken in June, August, October, and November 1996 and
February and March 1997 in Reclutas bed (39°24'S-55°56'W,
lOO-m depth). The protocol used involved the dissection of gut +
digestive gland complex in all sampled scallops. The gut was cut
open and the contents were washed in 2 mL of fresh water. A 1 -niL
subsample of the gut content washed suspension was analyzed in
a Sedgwick-Rafter counting chamber that was divided into 7 rows
and 17 columns. All panicles within six columns were counted and
idemified under the microscope ( 100-250x). This quantity repre-

549

550

SCHEJTER ET AL.

-3500

■^0 00

-5500

ARGENTINA

-5500

-70.00 -65,00 -6C00

LONGITUDE
Figure 1. Zygochlamys patagonica beds in the Argentine Sea.

sents approximately 1/6 of the total gut content. The number of
samples and columns studied were statistically tested and the es-
timated error of the procedure was <16% (Schejter 2000).

Multivariate MANOVA (Morrison 1976), univariate ANOVA,
and the Tukey test (Sokal & Rohlf 1979) were used to establish the
significance in the observed differences in food groups or species
found in gut contents during the study. The significance level was
P = 0.05 in all cases, but a Bonferroni correction (Morrison 1976)
was used when necessary. Data were normalized using the square
root transformation; the variance was found to be heterogenous.
However, as MANOVA and ANOVA are robust tests when there
is a balanced design (Ito & .Schull 1964, Ito 1988). it seemed
correct to use them in this case in which sample sizes were the
same in all months.

The IR index (an index of relative importance and abundance;
Bucher & Herrera 1981 ) was calculated for the main groups and
species in gut contents. This index [IR = (Ni/Nt) x (Mi/Mt) x
100] considers not only the total abundance of an item (or group)
per month in all guts (Ni/Nt), but also whether the item (group)
was present in all guts or just in a few (Mi/Mt).

RESULTS

Twelve species and resting stages of diatoms, five species of
dinotlagellates and cysts, two species of silicotlagellates, prasino-
phytes, and a number of other organisms and remains grouped as
miscellanea (tintinnids, foraminifers, nematodes, etc.) were found
in gut contents of the Patagonian scallop (Table 1 ). Particle sizes
varied between 19 and 170 (xm (mainly 25 to 60 jim), although
some crustacean remains (-250 \x.m) and some small diatoms (<19
(jLm) were found (Table 2). The majority of items found in gut
contents had a planktonic origin. This is the case of dinotlagellates.
silicoflagellates, prasinophytes and tintinnids, and most diatom
species. Nevertheless, some of them, namely Paralia sulcata, are
tychopelagic. Dinoflagellate cysts and resting diatom stages are
typically benthic.

TABLE \.

Food items found in Zygnchlamys patagonica gut contents during the
stud> period at Reclutas bed, Argentine .Sea.

Diatoms

Pill alia siilciiki (Ehrenberg) Cleve

Nitzschia ctuinuira Grunow

Nilzschia sp.

Thalassiosira spp.

Pleurosigma nonnanii Ralfs in Pritchard

Tluilassionema nilzscliioides (Grunow) Meereschkowsky

Siepliaiiopyxis nirris (Amott in Greville) Ralfs in Pritchard

Biililiilpliia sp.

Aclinoptychus sp.

Pennate 1

Centric 1

Centric 2

Resting stages
Dinotlagellates

Dinophysii inawsoni (Wood) Balech

Dinopliysis rolumiala (Claparede et Lachmann)

Dinophysis conlracia (Kofoid et Skogsherg) Balech

Dinophysis acuminata (Claparede et Lachmann)

Protoperidinium metananwn (Balech) Balech

Gonyaiilax digitalis (Pouchet) Kofoid (cysts)

Resting cysts
Silicoflagellates

Dictyocha speculum Ehrenberg

Dictyocha fibula Ehrenberg
Prasinophytes

Ph'rospfrma spp.
Tintinnids

Dicryocysta elegans var. lepida (Ehrenberg) Kofoid el Campbell

Dictyocysta elegans var. speciosa Jorgensen

Acanlhostomella sp.

Others
Miscellanea

Foraminifera

Rotifers

Nematodes

Nauplii larval stages

Invertebrate eggs

Copepod spermatophores

Radiolarian remains

Unidentified remains

Polychaete chaetae

Sponge spicules

Detritus

Pollen

The total number of ingested particles varied between 300 and
8,000 per gut and their mean number differed significantly among
months (F,,; ,-,4] = 55.859: P < 0.001). Maximum food ingestion
was registered in November, and minimum food ingestion periods
were October and March (Fig. 2). Total abundance of diatoms,
dinotlagellates, silicotlagellates, prasinophytes, foraminifera, and
tintinnids differed significantly between months (MANOVA;
F|3o.67si =-^6.86597; P < 0.001; Fig. 3).

Diatoms were always the most abundant food item (1R= 45-
90%, with mean values between 320 and 1450 diatoms per gut)
and were present in all guts during the entire sampled period (Fig.
4). The most abundant species was Paralia sulcata (maximum
values in November: IR = 66%; Fig. 5). This species was present
throughout all months and in all guts and was the dominant spe-

Feeding of the Patagonian scallop

551

TABLE 2.

Size ranges of main food ittms found in Zyguchlamys palagoiiica gut
contents from Reclutas bed.

Food Item

Size ((im)

Paralia sukalci
Thalassiosini spp.
Nitzschia spp.
Pleurosignm nonnanii
Pennate 1
Dlnophysis spp.
Dictyocha fibula
Dictyocha speculum
Plerospenna spp.
Dictyocysui elegans
Acanthostomella sp.
Foraminifera
Rotifers

Invertebrate eggs
Sponge spicules
Pollen

19-180-'

30-55

34-64

100-121
30-42
36-62
50-55
38-50
19-75
64-70
31-37
30-170
59-66

146-162
39-190
19-23

' Minimum individual frustule diameter and maximum chain length,
respectively.

cies, except in February. In this month, the typical planktonic
diatom genus Thalassiosira was almost the only food item found
in gut contents (IR = 80%). In March, this species was still an
important component of the diet (IR = 46%) but decreased in
importance during the remaining months (IR = 7-16%; Fig. 6).
Nitzschia spp. and Pleurosigina nonnanii made a minor contribu-
tion to total gut content (IR < 19% and IR < 4.5%, respectively;
mean = up to 140 items per gut and up to 23 items per gut,
respectively).

Dinoflagellates followed diatoms in importance (up to 1,000
items per gut, maximum IR = 25%, in November) in four of the
six sampled months (Fig. 4). Dinophysis rotundata was present
most of the time. Dinophysis mawsonii was present only in the
spring, Gonyaulax digitalis cysts were found by the end of summer
(February and March) and only a few in June. Unidentified di-
noflagellates cysts were found in February, March, June, and Au-
gust.

Silicoflagellates (two species of Dictyocha) were always ob-
served, and they were the second most abundant item in August

^ MeantSO
CH MeaniSE
a Mean

Figure 2. Mean abundance of particles per gut of Zygachlamys patag-
onica in the Reclutas bed during the study period. Different letters (a,
b, c) indicate significant differences iP < 0.05 1.

(IR = 0.02-12%; mean between 1 and 156 items per gut). Prasi-
nophytes were relatively important in gut contents in June and
March (IR = 9.6 and 5%. respectively, mean = 1 10 and 32 items
per gut, respectively). Foraminifers were present in all months and
in most guts (except in March) but with low IR and abundance
values (IR = 0.7-6.2, mean between 8 and 79 items per gut).
Tintinnids were frequent in spring (October and November) but
even in these months had low IR and abundance values (IR < 1.7;
mean <20 items per gut). Invertebrate eggs and copepod spennato-
phores were abundant in February and March.

DISCUSSION

The diet of Zygachlamys patagonica is mainly composed of
diatoms. These results agree with similar studies on bivalve gut
contents, which also showed a predominance of diatoms (Vernet
de Hall 1977. Pollovero 1984, Newell et al. 1989, Leonard! et al.
1996). In addition, it is remarkable that most of the food items
found had a planktonic origin. Paralia sulcata, one of the more
important diatoms recorded in the diet of Zygachlamys patagonica
and known as a tychopelagic species, is frequent in the shelf and
the shelf break area during most of the year(Lange 1985). Reclutas
bed, approximately 1 10 nautical miles offshore and 100-m deep, is
undoubtedly located in the pelagic zone.

It must be pointed out that among the food items found in
Zygochalnivs patagonica gut contents, there were two potentially
harmful dinoflagellate species present, Dinophysis acuminata and
Dinophysis rotundata. which could produce diairhetic shellfish
toxins (Lee et al. 1989).

The maximum food contents were recorded in November (up to
8,000 food items per gut; mean = 2,800 items per gut), being
Paralia sulcata the main food item. Paralia sulcata was also dom-
inant in gut contents during the year, except in February when
Thalassiosira spp. predominated and represented about 80% of
total gut content. It should be mentii>ned that samples were pre-
served in formaldehyde, some food particles could have been de-
stroyed because of the preservation, and also that highly digestible
particles or naked cells were not recorded by this method.

The vertical movement of particles in the seawater column is
very important for those animals that live far away from the photic
zone (Valiela 1995). Algal cells settling during spring and fall
blooms are one of the main inputs of particulate organic matter
from the pelagic to the benthic system. Downward mixing of
plankton during certain times of the year undoubtedly plays a large
role in making food organisms available to deep-water scallops
(Smetacek 1982, Shumway et al. 1987). The northern Argentine
shelf shows an annual phytoplankton growth cycle with two peaks
of which the most important one occurs in spring (Carreto et al.
1995, Akselman 1998). This peak was reported to occur by the end
of September-October at the boundary of the coastal system/
intermediate shelf (Akselman 1998), and maximum recorded chlo-
rophyll a levels increase from the intermediate shelf to the shelf
break (Can-eto et al. 1981, 1995, Bertolotti et al. 1996). Ther-
mocline formation begins in spring at the intermediate shelf and
extends all along the shelf (Carreto et al. 1995). In October and
November, the thermocline is already in development in the Re-
clutas bed area (Baldoni & Guerrero 2000), and oceanographic
data for October and November 1996 were similar to mean esti-
mated values for the study area by Baldoni and Guerrero (2000,
Baldoni, personal communication).

Smetacek (1982) indicated that nutrients accumulated in a

552

SCHEJTER ET AL.

DIATOMS

DINOFLAGELLATES

c

-

c

b

1 0 \"

c

m

S
1

S 600

[

3

■B (00

_

_

<

a

a

4 4

JUN AUG OCT NOV FEB

PRASINOPHYTES

SILICOFLAGELLATES

200

a

5

a

1 160

o

5.

1 ™

[

I

I 80

b

b

f

b

i 40
<

-

L r

b

b c
1 a 1

c

+

m

JUN AUG OCT NOV FEB MAR

FORAMINIFERS

TINTINNIDS

AUG OCT

OCT NOV

I Mean ± SD; Mean ± SE; I Mean

Figure 3. Mean abundance of principal food items per gut of Aygochlamys piitiifidiiica in the Reciutas bed during the stud\ period. Different
letters (a, b, c, d, e( indicate siyniflcanl differences (/' < 0.05).

Feeding of the Patagonian scallop

553

■ DIATOMS

El PRASINOPHYTES

Figure 4. Percent composition of different food items groups in gut contents of ZygocUlamys patagonica during the study period in the Reclutas
bed. The group Miscellanea (see text) also includes tintinnids.

17-m water column at Kiel Bight over the winter were depleted
within two weeks after phytoplankton bloom initiation. Sedimen-
tation of phytoplankton took about one week and attained maxi-
mum values three or four days after bloom initiation. Sedimenta-
tion rates of diatoms and other phytoplankton components are
related to their physiology, cell weight, and volume (Denman &
Gargett 1983). As the organic matter sedimented was formed by a
large number of living cells (and hence, was high in nutritional
quality), benthic metabolic response to this input was rapid. For
other deeper benthic systems a similar pattern is expected (Smeta-
cek 1982).

It is thus possible that, given the oceanographic conditions
described during spring at the Reclutas bed (thermocline in devel-
opinent). sedimentation processes or phytoplankton sinking down-

wards to the bottom have facilitated food availability to benthic
organisms after the phytoplankton bloom started at the surface.
This would explain the high abundance of cells observed in gut
contents from scallops collected in November. It is probable that at
the beginning of October (when samples were taken) increased
primary production started in surface waters, as previously docu-
mented (Carreto et al. 198 1 ). but availability of food at the bottom
remained low because of the time requirement for sedimentation
processes in a lOO-m water column. During summer, the stratifi-
cation of the water column is so pronounced that transport of
phytoplankton cells to the bottom would probably be limited; un-
der these circumstances we cannot explain the relatively higher
abundance of Thalassiosira spp. during February. It is known that
species of this genus are capable of producing blooms in other

Figure 5. Mean abundance of Paralia sulcata per gut of Zygochlaiiiys
patagonica in the Reclutas bed during the study period. Different let-
ters (a, b. c) indicate significant differences (/' < ().(I5|.

too

c

200

000

d

b

800

-

600

400

b

T

b

200

a

rm

b

a

i

i

nZ Mean t SD
CH Mean i SE
□ Mean

JUN AUG OCT NOV FEB MftR

Figure 6. Mean abundance of Thalassiosira spp. per gut of Zy-
gochlamys patagonica in the Reclutas bed during the study period.
Different letters (a. b, c) indicate significant differences iP < 0.05).

554

SCHEJTER ET AL.

ureas of the Argentine Sea (Carreto et al. 1981). but oceanographic
conditions at the Reclutas bed resulting in a strongly stratified
water column would prevent cell sinking. However, occasional
climatic events are able to disturb water column stratification,
increasing sedimentation rates (Nielsen & Kiorbe 1991). On the
other hand. Bode et al. ( 1998) conducted studies on the export of
organic matter to the bottom, and found that most of it was pelagic
in origin. Moreover, they determined that phytoplankton species
found in sedimentation traps were not the same as those found at
the surface, indicating that sedimentation traps were providing
records of past production events. In summaiy, simultaneous sea-
sonal information about gut contents and phytoplankton species
composition in the study area is needed to establish the degree of
particle selectivity in the feeding process, and whether the occur-
rence and abundance of food items are due to higher availability of
potential food after phytoplankton growth.

The results of this study agree closely with recent findings of
studies on biologic aspects of this species. Valero et al. (2000)
studied the grow th pattern of Zyf><>clilamys patagDiika at Reclutas
bed. As was already found for several species of scallops from
shelf and coastal areas (Barber & Blake 1991. Ciocco et al. un-

publ.). growth of different body components of the Patagonian
scallop are not simultaneous: ma.ximum shell growth occurred in
July, maximum gonadal growth was obtained in June, and maxi-
mum somatic (muscle) growth occurred in November. In view of
this pattern, it is possible to link the period of maximum muscle
growth with that in which scallops showed higher cell abundance
in gut contents, both of which occur in November. Consequently,
we hypothesize that somatic (muscle) growth of Zyiiochlumys pu-
uiiionica located in the Reclutas bed occurs during the period of
higher food availability in the bottom, which follows the period of
spring phytoplankton growth in the upper layer of the water col-
umn and its subsequent sinking to the bottom before the develop-
ment of the seasonal thermocline.

ACKNOWLEDGMENTS

The authors thank to Lie. R. Pinero, Lie. S. Incorvaia, Dr. H.
Mianzan, and Dr, N. Ciocco for useful suggestions and bibliogra-
phy. We are particularly grateful to Dr. S. Shumway and Lie. M.
Lasta for their encouragement during our investigation. We also
would like to thank the suggestions of the anonymous reviewers.

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Journal of Shellfish Resecinh. Vol. 21, No. 2, 557-561. 2002.

COMPARISON OF THE PARASITES AND PATHOGENS PRESENT IN A CULTIVATED AND IN
A WILD POPULATION OF SCALLOPS (ARGOPECTEN PURPURATUS LAMARCK, 1819) IN

TONGOY BAY, CHILE

KARIN B. LOHRMANN,'* ANDREW R. BRAND," AND STEPHEN W. FEIST'

'Universidad Catolica del Norte. Facidtad de Ciencias del Mar. Cas. 117. Coquimbo. Chile; 'Port Erin
Marine Lahoratoiy. University of Liverpool. Port Erin. Isle of Man. IM9 6JA. United Kiniidoni: 'CEFAS
Weymouth Laboratoiy. Barrack Road. The Nothe. Weymouth, Dorset DT4 SUB. United Kingdom

ABSTRACT Cultivation of the "ostion del norte". ArgopecWn purpuratus. is an important economic activity in tlie 3rd and 4th
Regions of Chile. Studies of disease v\'ere undenaken on wild scallops to gather baseline mformation on healthy populations. However,
as cultivated scallops are kept at higher densities than the wild populations, the occurrence and prevalence of parasites and tissue
pathology differed as indicated in this study that compares the types of parasites and their prevalence in wild and cultivated scallops
from Tongoy Bay, a major center for scallop cultivation. In January 1999, 151 cultivated and 154 wild scallops were e.\amined
histologically and via scanning electron microscopy (SEM). The same parasite taxa. as well as granulomas, were found in both
populations, but their prevalence differed. The granulomas were small, with no apparent etiology, but the cultivated population
harbored significantly more lesions than the wild population. The only parasites found were a prokaryote in the dige,stive gland and
the ciliate protozoan Tricliodina sp. on the gills of the scallops. Trichodina did not seem to elicit a host response and its prevalence
was significantly higher in the cultivated scallops. The prokaryote. a rickettsiales-like organism (RLO) was observed as basophilic
inclusions in digestive gland tubule epithelial cells. The intensity of infection was low. but significantly higher in the wild stock. No
host reaction was seen, and the prevalence showed no significant difference between the two populations. The infection with RLOs
was therefore independent of the source of the scallops, unlike the Trichodina or the granulomas, which were significantly more
prevalent in the cultivated population.

KEY WORDS: scallop, parasite, long-line culture, nckettsiales. Trichodina

INTRODUCTION

Argopecten piirpurariis. the ""ostion del norte"" {northern scal-
lop) is distributed on the Eastern Pacific coast from Sechura. Peril
(6°S), to Tongoy Bay, Chile (31°S) (von Brand et al. 2002), and is
harvested and cultivated in both countries. In Chile, the most abun-
dant beds are in the 4th Region, in the bays of Tongoy, Guanaque-
ros, and Coquimbo. These populations almost disappeared through
overfishing, prompting a total harvesting ban for this species in
1986 and this ban is still in force for wild populations. In the early
1980s, A. purpuratus culture became established (DiSalvo et al.
1984, lUanes-Biicher 1987) and currently the 3rd and 4th Regions
produce 97% of the cultivated scallop production of Chile ( Aqua-
noticias 2000).

Tongoy Bay is the single most important bay for production of
cultivated scallops. A serious disease outbreak here could have
significant consequences for this industry, so a survey was under-
taken to gather baseline information on parasites and tissue pa-
thology present in healthy members of the cultivated population.
There is no information available on any diseases of A. purpuratus
from Tongoy Bay, but some metazoan parasites have been re-
ported for this species in other bays in Chile and Peru. Mateo et al.
(1975) described germ sacs and cercaria of a hemiuroidean in the
gonad of A. purpuratus in Perii, causing castration of heavily in-
fected scallops. In Chile. Oliva et al. (1986) described two larval
cestodes located in the gonads of A. purpuratus in Antofagasta.
One of the cestodes belonged to the family Phyllobothriidae. and
the other possibly belonged to the family Oncobothriidae. In Co-
quimbo. Lohrmann et al. (1991) described a metacercaria of a
fellodistomid trematode in the labial palps of A. purpuratus and an
unidentified larval cestode in the intestine of a few animals of this
species (Lohrmann & .Smith 1993).

'Corresponding author. E-mail; klohrmanCaucn.cl

As there is still a small natural bed of A. purpuratus at the
southern tip of Tongoy Bay. both wild and cultivated scallops are
available for examination in this bay. The wild scallops from this
natural bed live on the sea floor in depths of up to 15 m. at a
density of 1 to 2 scallops m"- (Stotz & Gonzalez 1997). Cultivated
scallops are kept in pearl nets at densities of about 166 individuals
m"- for scallops at a size below 50 mm, and 19 individuals m"" in
lantern nets at sizes above 50 mm (lUanes 1986). Since the culti-
vated scallops are kept at greater densities than the natural popu-
lations, it might be assumed that the occurrence and prevalence of
parasites and other pathogens may differ. This investigation was
carried out on wild and cultivated scallops, to test for differences
in the prevalence of parasites and tissue pathology between the two
populations of A. purpuratus.

MATERIALS AND METHODS

In January 1999, 151 cultivated, and 154 wild scallops were
obtained from Tongoy Bay (Fig. 1). The 12-month-old cultivated
scallops had been hatchery-produced, and transferred to the sea at
age one month. They had been kept at a density of 19 scallops m^-
in lantern nets hanging from long lines at a depth of about 10 m
from the suil'ace. The natural scallops were obtained from a small
bed located near the fishing community of Puerto Aldea, which is
located at the southern tip of Tongoy Bay (Stotz & Gonzalez
1997). These scallops were adults, but their ages were not known.
They were collected by "hooka" diving (this consists in delivering
air to the diver through a hose from a compressor in the boat), from
a depth of 8 to 10 m. Each sample of scallops was transported to
the central aquaculture laboratory of the Universidad Catolica del
Norte in Coquimbo. where they were placed in tanks with running
seawater at ambient temperature. They were processed m batches
of 50 scallops daily, starting on the day after their arrival.

The soft tissues of the scallops were taken out of the shells, and

557

558

LOHRMANN ET AL.

CHILE

Pacific
Ocean

i

<■" Coquimbo

,i j 25"S

30° S

4 Guanaqueros
Bay

..^^ 4 Tongoy Bay

!71O20-W

45°S

I

/\ Tongoy Bay ^

lengua:

DE VACA
POINT

Puerto^
Aldea *■..-.

i^M

Scallop farms

0 1 2

/

Km

\ Si

« /
V

Antarctica

Figure I. Map of Tongoy Bay showing the area dedicated to scallop
farming (F) and the small natural bed (N) near Puerto Aldea.

were fixed for histology in Davidson's fluid (Sliaw & Battle 1957).
including the gills, digestive gland, gonad, kidney, mantle, and the
adductor muscle. They were prepared for histology using standard
methods and stained with haematoxylin and eosin fH & E).
Giemsa and Ziehl-Neelson methods were used as appropriate to
detect parasites and ceroid-like pigment respectively. Slides were
analyzed and photographed using a Nikon E600 Eclipse photomi-
croscope. For assessing the intensity of infection of rickettsiales-
like organisms (RLOs) and trichodinids. the most heavily infected
area of the histologic section was selected, and the number of
parasites present was counted at a magnification of 400 times, with
a field diameter of 1.240 |xm. Three categories of intensity were
defined: grade I (very light) one parasite present, grade II (light),
two to four parasites present, grade III (moderate), five or more
parasites present.

For scanning electron microscopy (SEM), sections from
selected wax blocks were cut at 12 p.m, and mounted on cover-
slips. Sections were de-waxed in three changes of xylene,
passed through three changes of H)09f ethanol (modified from
Toner et al. 1992). and critical point dried using CO,. Samples
were mounted on double-sided adhesive tape and ion sputtered
with gold. The sections were viewed and photographed using a
JEOL TS 300 microscope. Measurements of individual rickettsi-
ales-like organisms and Trichodina were taken from SEM photo-
graphs.

For analyzing the prevalence of each parasite, a 2 x 2 contin-
gency table was constructed with the frequency counts of each
parasite in the wild and the cultivated scallops, and a x" test for
goodness of fit was applied. The intensities of infection were coin-
pared using the Mann Whitney rank sum test (Zar 1999).

RESULTS

From the total of .305 scallops analyzed, surprisingly few patho-
gens were detected. These included rickettsiales-like organisms
(RLOs) and ciliutes [Trichodina sp.). Small, granuloma-like tissue
lesions were also detected. These are described in more detail later.

Rickettsiales-Like Organisms (RLOs)

Rickettsiales-like organisms (RLOs) were detected in digestive
gland tubule epithelial cells in the form of spherical basophilic
inclusions, with a diameter ranging between 7 and 14 fjLm. Occa-
sionally, the inclusions were also seen in the lumina of the diges-
tive tubules (Fig. 2A). In the interior of each inclusion there were
darker staining bodies, which at the scanning electron microscope
(SEM) level could be discerned as rod-shaped, and of fairly uni-
form size (Figs. 2B & C), ranging from 0.8 to 1.3 |xm in length,
and 0.38 to 0.46 ixm in width. The inclusions seemed to be en-
closed by a thin membrane, separating the RLOs from the cyto-
plasm of the host cell (Fig. 2B). The prevalence of these RLOs was
41'7f in hatchery-reared scallops and 37% for wild scallops (Table
I ) but this difference was not statistically significant (P =
0.6247). The intensity of infection in both populations is shown in
Table 1 . A significant difference [P = 0.001 ) was detected in RLO
intensity between populations, the natural population showing a
higher proportion of scallops with degree II and III of infection.

Trichodina sp.

Trichodina sp.. a ciliate protozoan, was found associated with
the gills of the .scallops (Fig. 3A). It was dome-shaped, with a
horseshoe-shaped macronucleus (Figs. 3 A & B). It measured 19 to
23 jxm in height, and the basal disc was 14 to 18 p.m in diameter.
The basal disc was surrounded by a ciliary girdle (Figs. 3A & B).
This trichodinid was always closely associated with the gill fila-
ments, but no pathologic changes to the gill were detected. The
prevalence was 56% for fanned scallops, but only 5.1% for wild
scallops (Table 2). and this difference was highly significant (P <
0.001). The intensity of infection is shown in Table 2 for both
populations of scallops. The difference in intensity between the
cultivated and the wild population was not statistically different.

Craiiiilomas

In the base of the gills, small tissue lesions were found embed-
ded in the connective tissue. These consisted of a central focus of
pigmented material resembling ceroid that appeared to be con-
tained within host cells and was surrounded by a thin capsule of
fibroblast-like cells (Fig. 4). These lesions are hereafter referred to
as granulomas. No evidence of infectious agents was detected in
association with these granulomas. The prevalence was 12% in
cultivated scallops, and 2.6% in the natural stock (Table 3) and this
difference was highly significant (P = 0.0034).

DISCUSSION

In scallops. RLOs have been described as basophilic inclusion
bodies in the gills of Placopeclen magellanicus (Gulka et al.
1983). Argopecten irradians (Leibovitz et al. 1984. Elston 1986,
Karlsson 1991 ), and Pcclen maxinuis (Le Gall et al. 1988. Le Gall
et al. 1991). They have also been found in the kidney of Ar-
gopecten irradians (Morrison & Shum 1983. Karlsson 1991,
McGladdery et al. 1993). as well as in the digestive gland
(McGladdery et al. 1993). The basophilic inclusions oi A. pitrpii-

Parasites from Wild and Cultivated Scallops

559

TABLE 1.

Prevalence and intensity of infection with a rickettsiales-like
organism in scallops from Toiihov Bay.

No. +/No.
Examined

Prevalence

Intensity of Infection {%)

Scallop
Group

Grade

Grade
II

Grade
III

Cullivated
Natural

61/151
57/154

41

37

78
22

20

32

46

was found that indicates they are most likely rickettsiales and not
chlamydiales-like organisms. Using SEM. the morphology and
surface characteristics were discerned. However, as no other SEM
images of RLOs were found in the literature, a comparison with
other RLOs was not possible.

Since the prevalence of this organism showed no significant
difference between the cultivated and the wild populations, host
density does not seem to be an important factor in its transmission.

Figure 2. Rickettsiales-like organisms (RLOs) in the digestive gland of
A. purpuratiis. A: Light micrograph showing several basophilic inclu-
sions (arrows) with RLOs in the cells of one tubule. One inclusion can
be observed in the lumen (L) of another tubule (short arrow). Stain: H
& E. Bar: 5(1 (im. B: Scanning electron microscopy (SEM) image of a
histologic section of one basophilic inclusion. IK': digestive tubule cell.
RLOs (*). Arrow: membrane separating the inclusion from the diges-
tive cell. Bar: 5 \im. C: RLOs at higher magnification. One RLO (*).
Bar: 1 (im.

rains were similar in size and structure to the rickettsial inclusions
described by Morrison and Shum (1983) in the kidney of A. irm-
dians. or in digestive gland tubules of clams by Elston and Peacock
(1984). For the RLOs from A. piirpunitus only one life cycle stage

Figure .^. Tricbodina sp on the gills of A. piirpuralus. A: Light micro-
graph of a few individuals in different orientations. The horseshoe-
shaped nucleus (short arrow) can be clearly seen, as well as the ciliary
girdle (arrow). Stain: H & E. Bar: 5(1 pm. B: SEM image of a histologic
section showing one complete, and part of another Trichodina sp. The
whole individual appears longitudinally sectioned, showing the horse-
shoe-shaped nucleus (n), and the ciliary girdle (eg). The incomplete
individual shows the basal disc (arrow) surrounded by the ciliary
girdle (eg). Bar: It) pm.

560

LOHRMANN ET AL.

TABLE 2.

Prevalence and intensity of infection with Trichodinu in scallops
from I onuoy Bay.

Intensity of Infection (%(

TABLE 3.
Prevalence of granulomas in scallops from Tongoy Bay.

Scallop No. +/No. Prevalence Grade Grade Grade
Group Examined i'7i\ I II III

Cultivated 84/151

Natural 8/154

56.0

5.1

64.0

37.0

29.0
37.0

7.0
26.0

The RLOs observed in A. purpuniuts do not .seem to cause any
harm, because there is no host response. Infections with RLOs are
common in bivalves, where they usually cause only mild effects, if
any (Lauckner 1983. Comps & Tige 1999). However, RLOs were
reported to have caused a few serious diseases but the relationship
between the presence of RLOs and the mortalities was not experi-
mentally demonstrated. In 1983. a mass mortality of Placopecten
magellaniciis occurred in Rhode Island, USA and a rickettsia-like
organism was found in the gills and other tissues of these scallops
(Gulka et al. 1983). Mass mortality due to branchial RLOs was
also reported for Pecten inaxiimis in Brittany. France by Le Gall et
al. (1988), for the giant clam Hippupiis hippopus (Norton et al.
1993). and for the clam Vi-iicnipis rlunnhoides from Spain by
Villalba et al. ( 1999). With such limited knowledge it is important
to undertake further studies on RLOs transmission and their effect
on scallops of different ages and culture conditions such as density,
temperature and depth.

Tiicluidina sp. ciliates are very common in bivalves (Lauckner
1983. Bower et al. 1994). They have been described from the
following scallop species: Mizuhopeclen yessoensis (Stein 1974, in
Lauckner 1983), Chlamys farreri (Kuidong et al. 1995) and Pla-
copecten magellaniciis (McGladdery et al. 1993). In invertebrates,
trichodinids are considered to be harmless commensals, feeding on
bacteria (Lauckner 1983). However, they are present in large num-
bers in weakened animals (Bower et al. 1994), and also in organ-
isms from areas polluted with chemicals and bacteria (Boussaid et
al. 1999). Boussaid et al. (1999) found that Ciassostrea gigas
heavily infected with Trichodina exhibited an intlamniatory re-
sponse of the gill, and numerous desquamated epithelial cells,
haemocytes, and tissue debris of host origin was observed sur-
rounding the parasites. They also stated that an excessive mucus
production covered the gill lamellae. This could interfere with the
respiratory function of the gill, and may result in the death of the

Figure 4. light micrograph of one granuloma located in the base of
the gills. Some degrading material and ceroid (c) can be observed,
encircled by flbroblast-like cells (arrows) encapsulating it. Stain: H &
E. Bar: 50 fim.

Scallop Group

No. +/No. Examined

Prevalence ( % )

Cultivated
Natural

18/151
4/1.54

12
2.6

oyster (Boussaid et al. 1999). A significant difference in preva-
lence of Trichodina was found between farmed (56%) and wild A.
piirpiiialiis (5.2%). This may be a consequence of the crowding of
the scallops, their proximity facilitating the transfer of this com-
mensal.

The granuloma-like tissue lesions found at the base of the gills
are similar to lesions (that they called ""swirl" encapsulation) ob-
served by McGladdery et al. (1991) in bay scallops Argnpecten
inadians infected by what was thought to be a Perkinsus species.
Goggin et al. (1996) made an assessment of these lesions, and
concluded that they were not produced by a Perkinsus species, but
were a general response to a foreign agent. In this study, the very
low prevalence and intensity of the lesions precluded ultrastruc-
tural investigations that are needed to determine the etiology of the
granulomas. However, Gonzalez et al. ( 1999) identified apparently
identical lesions in A. piirpuratiis from Valparaiso, which were
heavily infected with a protistan. Macroscopically, infected ani-
mals presented small dark brown pustules in the mantle, which was
also retracted. Based on one electron microscopy image of the
protistan they suggested that it could be an apicomplexan. How-
ever, details were indistinct and this finding needs to be confirmed.
Since pathogen involvement cannot be ruled out as a cause for
these granulomas and there was a significantly higher prevalence
in cultivated stocks, further investigations are needed to identify
their cause.

From the results of this study it can be concluded that both
cultivated and natural A. piirpitratus from Tongoy Bay harbored
very few putative pathogens, and those that were present, were the
same for the two scallop groups. The main difference between the
two groups of scallops was the density at which they lived: I to 2
scallops m'- for natural scallops in Puerto Aldea. and 19 scallops
per m"~ for the cultivated scallops. They also differed in the lo-
cation, with natural scallops living on the seabed and cultivated
scallops in cages suspended in mid-water. The increased density
can favor transmission of pathogens in two ways, either providing
hosts that are in close vicinity, and/or increasing stress. Over-
crowding can reduce food availability or increase the levels of
toxic waste products, all of which contribute to stress (Newell &
Barber 1988). Stressed organisms have less energy available to
defend themselves from disease and this is recognized as an im-
portant factor that can trigger disease in otherwise healthy animals
(Lauckner 1983. Newell & Barber 1988. Sindermann 1990).

It is surprising how few potential pathogens were harbored by
these two populations of scallops, since some metazoan parasites
have been previously found in A. piirpitratus in other northern
Chilean bays. Although the scallops examined during this study
were from healthy populations, the threat of disease is always
present. Any pathogens that are new to A. purpiiratus could have
a devastating effect on both cultivated and natural stocks. Patho-
gens may be involuntarily carried by fouling organisms on boats,
in the ballast water of big ships, or by transpoiling scallops or other
bivalves from other regions to Tongoy Bay. Newly introduced

Parasites from Wild and Cultivated Scallops

561

parasites can cause epidemics if the host's innate defense mecha-
nisms are not able to destroy it. or if the host is not able to defend
against a novel parasite strategy (Figueras & Fisher 1988). It is
therefore recommended that A. purpuratHS stocl<s be regularly as-
sessed for disease agents, so as to identify any different pathogen
from those known to be present in these populations.

ACKNOWLEDGMENTS

The authors thank Alejandro Abarca for providing the culti-
vated scallops, and Sergio Gonzalez and the fishermen from Puerto
Aldea for providing the natural scallops. Thanks to Wolfgang Stotz
for the map of Tongoy Bay.

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Journal of Shellfish Re.ieorch. Vol. 21, No. 2. 563-569. 2002.

BYSSAL ATTACHMENT OF AMUSIUM BALLOTI (BERNARDI, 1861) (BIVALVIA:

PECTINIDAE) SPAT

SIZHONG WANG,' ' PETER F. DUNCAN,'* WAYNE KNIBB," AND BERNARD M. DEGNAN'

^Department of Zoology ami Entomology University of Queensland Brisbane. Queensland 4072
Australia: -Bribie Island Aquaculture Research Centre Department of Primary Industries, Queensland
PO Box 2066 Bribie Island. Queensland 4507 Australia: ^Faculty of Science University of the Sunshine
Coast Maroochydore DC. Queensland 4558 Australia

ABSTRACT It has been previously reported that the saucer scallop. Amusium balloti. either lacked the ability to produce byssal
threads or could do so only bnefly. This present study reports our main conclusion that in the early spat stages, A. balloti does indeed
secrete byssus, albeit at a time different from most scallops. We found that .4. ballon first attaches by a byssus only after metamorphosis
(indicated by the presence of dissoconch shell), and we found no evidence of the byssal attachment before or during early metamor-
phosis. By the time spat reach a shell height of 1-2 mm they secrete two or three fine byssal filaments. Byssal attachment is maintained
until the spat reaches -4-5 mm. around the stage where they develop the ability to swim. This pattern is unlike that found m most other
scallops that initiate byssal production and attachment before metamorphosis. We also descnbe post-settlement behavior of A. ballon.
The newly settled postlarvae (-200 (jim shell height) crawl along the substratum by using their foot. During attachment, spat change
their positions daily, moving an average of 17 mm per day. In culture, a gentle water jet and hypersaline bath (40'7f() effectively
detached spat, with the majority rapidly reattaching.

KEY WORDS: Amusium balloti. byssal attachment, aquaculture. scallop, captive breeding

INTRODUCTION

The broad stages whereby free swimming planktonic mollus-
can larvae undergo transition into spat is similar in most scallops
species, and commences when competent larvae contact and attach
to a substrate by secreted byssal threads (Bourne et al. 1989. Be-
nigner & Le Pennec 1991). Metamorphosis and posriarval life
follows, when the larvae lose their velum (swimming organ) and
produce functional gills. Many species, such as Chamys asperri-
nuis. retain the ability to form byssus throughout life, while other
scallops, like Pecten imixiimis. cease to form byssus at around 1 5
mm shell height and live free of attachment on the sea bed (Brand
1991). Amusium balloti (Bemardi 1861) is one of these latter.
free-living scallops, but one that was also previously thought to
either lack the ability to produce byssal threads or to attach for any
significant time, even as spat (Rose et al. 1988, Cropp 1992.
Sumpton et al. 1990. Robin s-Troeger & Dredge 1993).

Amusium balloti. commonly known as the saucer scallop or
swimming scallop is a fast growing scallop that inhabits subtropi-
cal and tropical waters off the east and west coasts of Australia and
is an important component of the multi-species trawl fishery in
these areas. Some individuals recruit to the fishery (i.e.. reach legal
harvest size of 90-mm shell height) when 6 months old. although
most attain this size in 9 to 11 months (Gwyther et al. 1991).
Amusium balloti has been the subject of a number of studies on
larval development (Rose et al. 1988), wild collection (Sumpton et
al. 1990, Robins-Troeger & Dredge 1993) and hatchery produc-
tion (Cropp 1992), with a view to developing the species for aqua-
culture or stock enhancement. An essential component of either of
these latter activities is a reliable, relatively cheap and large source
of spat. While most successful scallop culture operation worldwide
utilize wild-sourced spat due to lower cost, hatchery production is
an option, and offers some advantages, such as genetic selection.
However, both wild and hatchery production of bivalve spat de-

*CorTesponding author. E-mail: pduncan@usc.edu.au

pend on attachment to some form of substrate for collection and
handling (Bourne et al. 1989, Wang et al. 1993).

According to Rose et al. (1988). newly settled A. balloti spat
crawl actively using their foot, but never appeared to attach per-
manently to substrata provided. Cropp (1992) determined that
newly settled spat lacked a firm and long-term byssal attachment,
but retained a strong and active foot that allowed them to crawl on
the substratum and detach and swim in the water column at will.
During hatchery production this apparent lack of permanent byssal
attachment resulted in significant numbers of cultured spat accu-
mulating on the tank floor after dropping from suspended collector
bags. If left on the tank floor, associated fecal matter and algal
detritus would kill the young spat (Cropp 1992). The conclusions
drawn from these hatchery observations were further supported by
data from spat collectors in the field. Attempts to obtain spat by
deploying collectors during the spawning and settlement season of
A. balloti resulted in very low numbers of captured spat (Sumpton
et al. 1990. Robins-Troeger & Dredge 1993). The most likely
explanation given for this lack of success was that A. balloti either
had a brief or non-existent byssal attachment stage.

Here we present data demonstrating that A. balloti does pro-
duce byssal threads, but unusually for a scallop, does not do so
until the dissoconch shell is produced, well after metamorphosis
and postlarval stages. We also document, for the first time, the
processes of byssal attachment and provide data on detachment
and reattachment methods, and movement of A. balloti spat.

MATERIALS AND METHODS

General Specifications

Hatchery conditioned broodstock were induced to spawn by
air-drying and heat shock using standard methods (Bourne et al.
1989). After fertilization the embryos were kept at 20°C in a 500-L
tank until hatching. Larvae were reared at 20 ± 1°C until ready to
metamorphose (i.e.. competent). Competent larvae were allowed
to settle in screen chambers, which were constructed by gluing
nylon mesh ( 1 60 jxm aperture) on to PVC pipe (250 mm diameter),

.'563

564

Wang et al.

and housed in a hatchery downweller system operated at 21 ± T'C,
pH 8.1-8.2 and 33-35%f salinity. The rearing water was changed
three times each week, followed by feeding with equal amounts of
Tahitian hochrysi.s aff. i^alhana. Pavlova lutheri and Chaeloceros
caUitniiis based on their dry weight at a density of appro.\iniately
15.000 cells mP'.

Qualitative Observations of Byssal AUachmeiU and MinemenI

Three times per week postlarvae (i.e., after imlialion of meta-
morphosis, but before evidence of a dissoconch shell) and spat
(evident dissoconch shell) were placed into petri dishes and their
movement was observed microscopically. After being left for ap-
proximately 30 min the attachment status of the immobile post-
larvae and spat was determined by subjecting individuals to gentle
water current produced by squeezing water from a Pasteur pipette.
Postlarvae and spat that were moved by the gentle water current
were considered to be unattached spat. Those that did not were
considered to be byssally attached and were photodocumented
(Sony, Mavica).

Spat attachment and movement was also qualitatively assessed
by hanging small pieces of nylon mesh (tied to 2 x 3 cm PCV
plates for support and weight) in three different ways in each of 3
replicate screen chambers (.see General Specifications), each hold-
ing 1,000 to 2,000 spat. Specifically, mesh was: (I) suspended
vertically 1cm from bottom of the screen chamber: (2) placed
vertically in contact with bottom of the screen chamber: (3) placed
in a Petri dish (5 cm in diameter with 1 cm side), that was sus-
pended 1 cm from the bottom of screen chamber. In all cases, mesh
or Petri dish was at least 1cm distant from the walls of the screen
chambers. After 24 h mesh/PVC plates/petri dishes were inspected
for settled spat and observed using a stereomicroscope. This ex-
periment was performed twice when spat had a shell height be-
tween I and 2 mm.

Spat DelachmenI

Detachment of byssally-attached spat was investigated using
two different treatments, high salinity water and a water jet. The
high-salinity treatment consisted of exposing attached spat in a
screen chamber to 409ff seawater. After 5 min. detached animals
were removed by very gentle rinsing, and counted. This procedure
was repeated after 15, 30, 60, 90, and 1 20 min. This procedure was
replicated using 3 screen chambers at the same time. The salinity
was raised by adding rock salt (Olsson's) and measured using
Horiba Water Checker (Model U-10, HORIBA Ltd., Japan). A
control group using 33%c salinity was also assessed using the same
procedure. Spat detachment using a water jet was assessed using a
small aquarium pump at a rate of 2,500 L/h (nozzle size: 0.8 cm).
The jet was directed onto spat attached to the screen chambers
suspended in water at 339f( salinity. Detached spat were rinsed
from the chamber and counted.

For all treatments, spat were hatchery reared at a water tem-
perature of 21"C. Prior to the start of the experiment, any loose
spat were gently rinsed from the screen chambers, leaving only
attached spat at approximately 750-2,000 per chamber, or 1.6—4.4
spat/cnr. Spat shell height was 1.2 mm (±0.05, SE). Following
experimental procedures, 10 detached spat from each treatment
were subsequently held in 400 ml-plastic dishes in normal seawater
(339f r) to detennine treatment effects on survival, which was assessed
by inspection under a stereomicroscope 24 h after detachment.

Spat Reatlaclimiiil

After being detached by water jet as described above, 10 spat
were held in plastic dishes holding approximately 400-ml seawater
at 21°C, 339?f, and pH 8.2. The size range of the spat was 1-1.5
mm. Spat were left to reattach for 5 min. after introduction to the
dish and attachment status was determined by subjecting spat to a
gentle water current from a Pasteur pipette. Scallops moved by the
water current, were considered unattached. Reattachment was as-
sessed after 5. 15, 30, 60, 120, 240, 360, and 720 min. using this
procedure. Three replicate samples were used. Survival was also
determined during this trial by microscopic inspection, immedi-
ately following reattachment assessment.

Quantitative Measurement of Spat Movement

Individual spat (shell height 2 mm ± 0.06, SE) were held in
plastic dishes (32 in total) containing 400 ml seawater at 23°C,
34%r and pH 8.2. Spat were left for 1 h (i.e., sufficient time to
ensure reattachment as indicated by earlier results), and then the
position of each spat was marked with permanent ink on the un-
derside of the dish. Spat position was marked again every 24 h for
seven days and the straight-line distance between the most recent
and previous position recorded. At the same time the attachment
status was checked as before and survival status of unattached spat
was assessed by microscopic inspection. Water was exchanged
daily to enhance water quality, and to minimize the effects of water
exchange on spat attachment and movement, approximately 1 ,000
ml seawater was allowed to flow through each dish in one hour.
Equivalent proportions of Tahitian Isochrysis aff. galbana, Pav-
lova lutheri and Chaeloceros calcitrans. based on dry weight at a
final concentration of 15,000 cells mP'. was added to the
flowthrough seawater to provide feed.

Statistical Analysis

GenStat (Payne et al. 2000) was used for all analyses, and
differences between treatment means were evaluated for signifi-
cance using least-significant difference testing (at a significance
level of 0.05). The water jet detached 100% of the spat in the
screen chamber within 2 min. Hence, this treatment had zero varia-
tion and was not included in the statistical analysis of detachment,
and only control (normal seawater 339^^) and hypersaline (4(Kf)
treatments were statistically compared. As the numbers of the
attached spat varied among the individual screen chambers, the
results of various detachment treatments were compared on the
basis of percentage of total number of spat detached (i.e., at 120
min). Percentage detachment values were normalized by the an-
gular transformation (arcsine of the square root of the proportions).
The transformed data were analyzed using one-way ANOVA, with
3 replicate screen chambers per treatment. In addition to analysis
of Ihe total number of detached spat at 120 minutes, percentages of
detachment in each cumulative interval were also subject to re-
peated measures ANOVA. For reattachment of young spat, the
number of reattached animals was converted to the percentage and
normalized by the angular transformation in each treatment, then
also analyzed by one-way ANOVA. For movement of young spat,
the distances of movement were analyzed by using two-way
ANOVA, with the two treatments being "days" and "'spat". The
proportions of attached (vs. moved) animals were analyzed by
using generalized linear regression analysis (McCullagh & Nelder
1989), using the binomial distribution and logit link, with the same
treatments beinu fitted.

Byssal Attachment in Amusium balloti

565

RESULTS

Qualitative Obsenations of Byssal Attachment and Mnremeut

Newly settled postlarvae crawled actively using their foot.
Non-active or immobile postlarvae were not observed to attach,
and were easily moved by a gentle water current. Spat initially
attached to the substratum with a fine and transparent byssus that
was difficult to detect using a stereomicroscope. although its pres-
ence could be inferred by resistance to the water current. Upon
reaching 1 to 2 mm. the byssus became thicker and could be
observed microscopically (Fig. I).

Shortly after byssal detachment by water jet. spat typically
retracted into their shell for se\eral seconds to ininutes. Subse-
quently a spat would extend its foot from the byssal notch, using
it to explore and crawl or attach to the substratum. Spat typically
explored and crawled for several minutes before stopping, then
continuing to explore the surrounding area with the foot. The
comet of the foot then pressed against a substrate, and seconds
later the foot was retracted into the shell leaving a byssal filament
that fixed the scallop to the substratum. Atmisium balloti spat
usually secreted two or three byssal threads. If disturbed, the spat
would extend its foot again to explore the surroundmg area and
secrete another byssus. or release existing threads, and would
crawl away using its foot and shell adduction. At about 4-5 mm,
spat started to swim and ceased byssal attachment.

Inspection of PVC plates, nylon mesh and petri dishes indicated
that 3 to 10 spat (out of 1,000 to 2,000) attached to the PVC plates
or nylon mesh in contact with the bottom screen. No spat were
found on PVC plates or nylon mesh that were off the bottom
screen and no spat were caught in any of the petri dishes.

Spat Detachment

Based on the total number of spat detached after 120 min,
one-way ANOVA indicated that hypersaline seawater (40%f) in-
duced a significantly greater proportion (98'7f ) of detachment com-
pared with washing in normal seawater Oy/ic) (75**) (P < 0.01 ).
The water jet (normal seawater) detached 100% of the spat in 1 or
2 min. Analysis of time intervals within the 120 min period (Fig.
2) indicated there was an interaction between time and treatments
(P < 0.01 ), which occun-ed in .^0 and 6(1 min intervals. Using
salinity treatments, most detachment occurred during the first 60
min (P < 0.01 ). No mortality was observed within 24 h of detach-
ment using any treatment.

Spat Reattachment

The rate of spat reattachment over time was significantly dif-
ferent (One-way ANOVA, P < 0.01 ; Fig. 3). Reattachment in-
creased linearly, reaching a plateau of around 90% at 60 min, and
remained stable up to 720 min. No mortality was recorded follow-
ing this experiment.

Qualitative Measurement of Spat Movement

The average daily distance moved by spat over seven days was
17 mm. and tended to be greater in mid trial (P < 0.01. Fig. 4).
However, there were large differences in distance moved between
spat {P < 0.01). Figure 5 shows the average daily movement of
individual spat over seven days with corresponding standard de-
viations. The longest mean daily distance travelled was 38 mm and
the shortest inean distance was 1 mm. All animals moved at least
once in seven days. Generalized linear regression analysis showed
that for total numbers of attached animal and moved animals there
was no statistical difference between different days (P > 0.2).
However, individual spat showed differences in the proportion of
times they were observed to be attached or to have moved over the
seven days (P < 0.05).

DISCUSSION

Our investigation demonstrated that A. balloti consistently se-
creted a transient byssus, contrary to, and clarifying previous re-
ports (Rose et al. 1988, Sumpton et al. 1990, Cropp 1992, Robins-
Troeger & Dredge 1993). However, unlike most scallops (Sastry
1965. Bourne et al. 1989, Benigner & Le Pennec 1991 ). there is no
evidence of the byssal attachment in A. balloti before and during
early metamorphosis. The first appearance of byssal threads occurs
after the spat begins to synthesize the dissoconch shell. Spat of
shell height less than I mm do attach to the substratum, although
it is difficult to detect byssus even under microscopic inspection
because it is very thin and transparent (future electron microscopy
may resolve this issue). The thickness and visibility of threads
increases as the animal grows.

We do. however, find similarities between A. balloti and the
reported behavior of other pectinids during byssal secretion (Be-
nigner & Le Pennec 1991), specifically the crawling, and explo-
ration phases prior to secretion, and the process of secretion itself.

Amusium balloti spat of 2-3 mm shell height secreted only 2 or
3 transient byssal filaments under our experimental conditions.
Compared with other scallops A. balloti appears to have a much
reduced bvssus attachment. Gruffydd (1978) estimated the number

"^

Figure 1. The foot, cornel, and byssus of Amusium balloti spat (shell height 2 mml (Al Foot extended. (Bl Foot retracted, b, byssal: f. fool; c.
cornet. Scale bar = 1(1 (ini.

566

Wang et al.

c
<u

E

o
a

I Control (33%o)
IHyper,saline(40%c)

15

3(J

60

90

120

'luTiL' (mui)

Figure 2. Sequential proportional detachment (mean and standard deviation of Amusiiini halliili spat (shell height 1.2 (±0.05 SEl mml from
settlement screens after exposure to normal iii'ic) and hypersaline (40"(i) seawater at cumulative time intervals of 5, 15, 30, 60, 90, and 120 min.

of byssal filaments secreted by Clilamys iskmdka to be 10-30.
while Placopecten magellanicits juveniles secrete 3-7 filaments
(Caddy 1972). Amusiwn balloti spat lose byssus when they reach
a shell height of 4-5 mm. which is smaller than for most other
scallops. For example, Pecten inaximits ceases byssal attachment
at 15 mm, Argopecten gibbus at 6-10 mm. (Brand 1991) and
Argopecten irradians at 20-25 mm (Pohle et al. 1991). Other
scallops such as Chamys asperrimus (Brand 1991) and Chlanns
farreri (Wang et al. 1993) retain the ability to form byssus
throughout life.

In culture, 1-2 mm A. balloti spat can crawl from the bottom to
substrata (mesh and PVC plates) that are in contact with the bot-
tom, although this appears to be uncommon. With our experiment
design we found no evidence that spat can swim and attach to
substrata raised above the bottom. However our experiment design

did not categorically exclude the possibilities, either, that at this
age spat were able to swim, but not able to attach to screen off
bottom, or. that spat could swim but were not able to swim verti-
cally more than 2 cm from the bottom. Future video surveillance
techniques may overcome some of these technical limitations. Ob-
servations indicated that spat begin to swim at 4-5 mm, apparently
losing the ability to synthesize new byssal filaments at the same
time. With continued growth, spat demonstrate the active swim-
ming noted in adults. According to Joll (1989). A. balloti is the
fastest and longest distance swimmer amongst the scallops.

Results also indicated that most spat moved every day. al-
though continuous movement patterns were not recorded. Since it
is likely that spat movement is not in a single direction and perhaps
more than once a day, our estimates of daily migration are likely
to be underestimates of the actual distances moved. The change in

^

OS

100
80
60 -
40
20 ^
0

15

be

bed

30

60 120

Tiine (niin)

240

360

bed

720

Figure 3. Reattachment rate (percentage with standard deviation) nX Xmusium balloti spat (shell height 1.5 (±0.115 .SE) mm) Over 720 min
following water jet detachment. Values sharing same superscript letter do not differ significantly (P > 0.05, ANOVA)

Byssal Attachment in Amusium balloti

567

Time (days)

•distaDce (mean) ram

■survival rate (%)

A percentage of moved animals )( percentage of attachment

Figure 4. Amusium balloti spat (shell height 2 (±0.06 SE) mnil. mean movement distances with standard deviation, daily proportions of moved
and attached animals, and daily survival rates over seven days. Values sharing same superscript letter do not differ significantly (P > 11.05,
ANOVA)

attachment rates over time for A. balloti young spat are similar to
those reported tor Clilatnys opercularirs (L) (Paul 1980) and Plu-
copecten magellanicus (Caddy 1972). The tinie required for most
A. balloti spat to reattach is also similar to that reported for these
species of scallop, with approximately 80% to 90% reattached
after 30 and 50 min respectively (Caddy 1972, Paul 1980).

Scallops appear to be susceptible to the effects of siltation,
particularly in relation to gills (Naidu & Scalpen 1976. Tettelbach
et al. 1988). It has been suggested that byssal attachment to el-
evated substrates, by both juvenile and adult scallops, may help
avoid burial by fine-grained sediments in areas of high near-
bottom turbidity, as well as a means of avoiding predators (Bricelj
& Shumway 1991, Pohle et al. 1991). Amusium balloti lives on
medium to coarse sandy-mud seabed (Cropp 1994) where there is

very fine sediment, but also no vertical substrate for juvenile at-
tachment. Consequently A. balloti spat probably settle directly on
the bottom with byssal attachment to coarse sand grains. As small
spat, only a few filaments are required for them to attach onto the
substrate and avoid being moved by currents, or perhaps concen-
trated and buried in areas of fine silt. The small number of fila-
ments, in addition to minimizing the energy requirements of byssal
production, would also enable more rapid detachment if the need
arose, perhaps in response to predators. However, with the growth
of young spat, sand grains are no longer effective anchors, even if
the spat could secrete more and stronger byssal threads. Interest-
ingly, after 4-5 mm, A. balloti spat rapidly develop a strong swim-
ming capability, which is also likely to be an effective means of
avoiding siltation and predation.

60

50

40

^ 30

Q 20

10

0

T

J

-i- ^

-|

-r

J

T

J

-r

T-

T < . 1 1 1 1 1 L J lJ

Mil

Hill

ill

^uDanmiTinuiui

16

21

26

31

Individual spat

Figure 5. Mean daily movement of individual Amusium balloti spat (;i = 32), (shell height 2 (±0.06 SE) mm), over 7 days, mean distance (mm)
with standard deviation. Data arranged In ascending order.

568

Wang et al.

Methods for artificial and controllable detachment of spat have
important practical applications in hatchery production. Growing
spat need to be frequently graded and transferred onto screens of
successively larger surface area and mesh aperture sizes to reduce
screen fouling and to offset increasing biomass (Heasman et al.
1994). In Pecten fuimitus. mechanical methods, such as seawater
jet and scrapers, can cause injury and subsequent high mortality,
particularly in spat less than 5 mm shell height (Heasman et al.
1994) due to their more robust attachment. However, the delicate
byssus oi A. balloti is readily broken with a gentle stream of water
does not cause mortality to the spat. A hypersaline (45%() bath was
found to be the most effective and safe means for inducing spat
detachment in both P. fumatus (>95%) (Heasman et al. 1994) and
Pinctada maxima, (>90%) (Taylor et al. 1997). This method was
also effective for A. halloti with 40%o salinity inducing 98% de-
tachment within 2 h. compared with 15'^/i in normal seawater (and
after gentle rinsing).

A prerequisite for any aquaculture operation is the reliable,
plentiful, and inexpensive supply of seed (juveniles). In most bi-
valve culture operations throughout the world, .seed is obtained
from the wild, although hatchery-produced spat is an alternative
(Bourne 2()()()). Both approaches have been used successfully to
produce scallop spat and in all documented cases, procurement of
scallop relies on the attachment of spat via byssal threads to a solid
substratum (Bourne et al. 1989. Ito 1991, Wang et al. 1993, Neima
1997, Bourne 20(J0). The results of this study demonstrate that A.
halloti secrete byssus. consisting of a few fragile filaments. In
addition, although spat are usually attached to a substratum by the

byssus, they do move frequently. These characteristics explain
why wild collection is unsuccessful, with water movement even-
tually removing most spat from collector bags, and supports the
conclusion of Robins-Troeger and Dredge (1993), that fishery
enhancement (and aquaculture) will be reliant on hatchery-
reared spat.

The results of our present study also suggest that to culture A.
Inilliili spat in the hatchery, we cannot use standard methodologies
reliant on a robust byssal connection. However, this problem can
be overcome by using screen chamber settlement systems. Down-
weller and upweller screen chamber settlement systems have been
used successfully to rear many species of bivalve, such as oyster,
clam, (Utting & Spencer 1991 ) and scallop (Heasman et al. 1998).
Our work provides the first foundations for the development of a
more reliable and commercial-scale hatchery production method
for A. halloti. which has not previously been demonstrated. The
potential for subsequent aquaculture or stock enhancement through
large-scale hatchery production of spat may then be realized.

ACKNOWLEDGMENTS

The authors thank Ms Jan Rose for supply of microalgae. Mr.
Mike Dredge and various fishermen for the supply of broodstock
and to Mr. David Mayer for advice on statistical analysis. We also
thank staff at the Bribie Island Aquaculture Research Centre and
Dr. David Hewitt for their generous help in setting up the scallop
hatchery. The authors thank Dr. Michael Heasman. Dr. Wayne
O'Connor, Dr. Zhaoping Wang and an anonymous referee for
helpful comments and advice.

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S. E. Shumway. editor. Scallop: Biology. Ecology and Aquaculture.
Developments in Aquaculture and Fisheries Science. Amsterdam:
El.sevier Science Publishing, pp. 133-223.

Bourne, N., Hodgson. C. A. & J. N. C. Whyte. 1989. A manual for scallop
culture in British Columhia. Can. Tech. Rep. Fish. Aqual. Sci.. no.
1694. 230 pp.

Bourne. N. F. 2000. The potential lor scallop culture - the next millenium.
Acjiiaciilliire huernational 8:1 13-122.

Brand. A. R. 1991. Scallop Ecology: Distributions and Bchuxiour. In: S. E.
Shumway, editor. Scallop: Biology, Ecology and Aquaculture, Devel-
opments in Aquaculture and Fisheries Science. Amsterdam: Elsevier
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Bricelj. V. M. & S. E. Shumway. 1991. Physiology: Energy acquisition and
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Caddy, J. 1972. Progressive loss of hyssus attachment with size in the sea
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9:179-190.

Cropp, D. A. 1992. Aquaculture of the saucer scallop .Aiiiii.siiim halloti.
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Cropp. D. A. 1994. Hatchery production of western Australian scallops.
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Gruffydd. L. D. 1978. The byssus and byssus glands in ChUimys isUmdica
and other scallops (Lamellibranchia). Zool. Sci: 7:277-285.

Gwyther, D., D. A. Cropp. L. M. Joll & M. C. L. Dredge. 1991. Fisheries
and Aquaculture: Australia. In: S. E. Shumway. editor. Scallop: Biol-
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eries Science. Amsterdam: Elsevier Science Publishing, pp. 835-851.

LITERATURE CITED

Heasman. M. P.. W. A. O'Connor & A. W. Frazer. 1994. Detachment of
commercial scallop Pecten fiiituitus. spat from settlement substrates.
Aqiuutiltiire 123:40l-+()7.

Heasman. M. P., O'Connor, W. A.. O'Connor, S. J. & S. Walker. 1998.
Enhancement and farming of scallops in NSW using hatchery produced
seedstock. Final Report to FRDC. NSW Fisheries. CronuUa. NSW.
Australia. 146 pp.

Ito. H. 1991. Fisheries and Aquaculture: Japan. In: S. E. Shumway. editor.
Scallop: Biology. Ecology and Aquaculture. Developments in Aqua-
culture and Fisheries Science. Amsterdam: Elsevier Science Publish-
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Joll. L. M. 1989. Swimming behaviour of the saucer scallop .Amii.siKiii
halloti (Mollusca: Pectinidae). Mar. Biol. 102:299-305.

McCuUagh. P. & J. A. Nelder. 1989. Generalized Linear Models. 2nd ed.
London: Chapman and Hall. 551 pp.

Naidu. K. S. & R. Scalpen. 1976. Settlement and survival of giant scallop.
Placopecten magellanicus. larvae on enclosed polyethylene film col-
lectors. In: T V. R. Pillay & W. A. Dill, editors. Adances in aquacul-
ture. Famam. UK: Fishing News Books Ltd. pp. 379-381.

Neima. P. G. 1997. Report on commercial scallop hatchery design. Can.
Tech. Rep. Fish. Aquat. Sci./rapp. Tech. Can. Sci. Halieut. Aquat. 62

PP

Paul. J. D. 1980. Upper temperature tolerance and the effects of tempera-
ture on byssus attachment in queen scallop. Chlainys operciilaris (L). J.
Exp. Mar. Biol. Ecol 46:41-50.

Payne. R. W.. D. B. Baird. A. R. Gilmour, S. A. Harding. P. W. Lane, D.
A. Murray, D. M. Soutar. R. Thompson. A. D. Todd. G. Tunnicliffe
Wilson. R. Webster & S. J. Welham. 2000. GenStat Release 4.2 Ref-
erence Manual. Oxford: VSN International. 782 pp.

Pohic, D. G.. V. M. Bricelj & Z. Garcia-Esquivel. 1991. The eelgrass
canopy: An above-bottom refuge from henthic predators for juvenile
bay scallops Argopeclen irradians. Mar. Ecol. Prog. Ser. 74:47-59.

Byssal Attachment in Amusium balloti

569

Robins-Troeger, J. B. & M. C. L. Dredge. 1993. Seasonal and depth
characteristics of scallop spatfall in an Australian subtropical einhay-
ment. J. Shellfish Res. 12:285-290.

Rose, R. A., G. R. Campbell & S. G. Sanders. 1988. Larval development
of the saucer scallop Amusium halloti (Bernardi) iMollusca: Pec-
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Sastry, A. N. 1965. The development and external morphology of pelagic
larvae and post-larval stages of the bay scallop. Argopecleii irradians
concentricus Say, reared in the laboratory. Bull. Mar. Sci. 15:417^35.

Sumpton, W. D.. I. W. Brown & M. C. L. Dredge. 1990. Settlement of

bivalve spat on artificial collectors in a subtropical embayment in
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Taylor, J. J., R. A. Rose & P. C. Southgate. 1997. Inducing detachment of
silver-lip pearl oyster [Pinciada maxima. Jameson) spat from collec-
tors. Aquaculiure 159:1 1-17.

Utting. S. D. & B. E. Spencer. 1991. The hatchery culture of bivalve
mollusc larvae and juveniles. Laboratory leaflet. Directorate of Fish-
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Wang. R., Z. Wang & J. Zhang. 1993. Marine Mollusc Culture. Qingdao,
China: Ocean University of Qingdao Publisher. 397 pp.

Joiinwl ofSlu'llfish Research. Vol. 21. No. 2, 571-576, 2U02.

INTRASPECIFIC VARIATION IN THE REPRODUCTIVE CYCLE OF THE TEHUELCHE
SCALLOP AEQUIPECTEN TEHUELCHUS (PELECYPODA, PECTINIDAE), IN SAN MATIAS

GULF, PATAGONIA, ARGENTINA

MATTE NARVARTE* AND MARINA KROECK

Institute) de Biologia Marina y Pesqiiera "Almirante Storni, '
Negro. Argentina

CC 104. (8520) San Antonio Oeste. Rio

ABSTRACT The reproductive cycle of the tehuelche scallop. Aaiiiiivclen leluiclclui.s. from San Matias Gulf was studied for a two
year period. Scallops were collected at monthly intervals from August 1997 to July 1999. Muscular and gonosomatic indexes were
estimated and histologic sections of the gonad were prepared and used to determine gametogenic stages and to estimate the mean oocyte
diameter. Gonosomatic index (GSI) data revealed a bimodal pattern, but the spring-summer spawn was larger and more synchronized
than the autumn spawn. GSI and histologic observations had similar patterns. Massive spawning began in December. Gametogenesis
lasted from June through late winter (September). The tehuelche scallop is a partial spawner. The spawning schedule showed
between-year tlucluations. starting at any time during September through October, depending on the year. Partial spawnings and
gonadal recovery alternated along^the summer, until March. Spawning was followed by the reab.sorption of unspawned gametes. A
resting stage in the reproductive cycle occurred from late summer (March) to mid-autumn (May). In I of the two years studied, an early
sexuatmatwation matched low temperatures. This suggests that in San Matias Gulf the reproductive cycles are not clearly synchronized
with seasonal cycles of superficial seawater temperature and that other factors, presumably the amount of food supply, may be
responsible for an anticipated gonadal maturation.

KEY WORDS: reproductive cycle, intraspecific variation. Aequipecten tehuelchus. san Matias Gulf

INTRODUCTION

The tehuelche scallop Aequipecten lehuelcluis (D'Orb., 1846)
is one of the most impoitant commercially fished bivalves in east-
ern Patagonia (San Matias and San Jose gulfs). Landings have
fluctuated from 4.700 to 100 tons during the last 30 years. Fishery
measures include minitnum commercial size, rotation of fishing
areas, and selection of catch on board (Orensanz et al. 1991 ). The
fishery operates during winter months to obtain maximum yield
and to prevent capture during reproductive periods.

The tehuelche scallop is hermaphroditic (Christiansen &
Olivier 1971). As in other pectinid species, the male gonad is
white-yellow and the female gonad is orange ("corar'). Gamete
development is easily viewed within the acini, and light varies with
the gametogenic stage (Barber & Blake 1991). Christiansen et al.
(1974) delineated the reproductive cycle of a San Mati'as Gulf
population based on irregular samplings in the region. Fecundity
ranges from 2 to 17 million eggs for scallop sizes of 35-90 mm
(Orensanz et al. 1991). The reproductive cycle of the populations
from San Jose Gulf (43°S) was studied by Lasta and Calvo ( 1978).
Partial spawnings begin in late spring and pre- and post-spawning
stages coexist as far as the end of the summer. Despite the high
value of this fishery in San Matias Gulf (41°S). the reproductive
cycle was not studied in detail until this work.

There is extensive literature on the gametogenic cycle and the
timing of spawning in many bivalve species (Giesse & Pierce
1974, Sastry 1979. Newell et al. 1982. Barber & Blake 1991). For
several pectinid species in the northern hemisphere, spawning oc-
curs at higher temperatures and later in the year in southern popu-
lations (Barber & Blake, 1991) and is often more synchronized at
higher latitudes (Bricelj et al. 1987).

The objective of this study is to describe the reproductive cycle
of the tehuelche scallop of San Matias Gulf and to compare the
reproductive patterns between years for the same population.

MATERIALS AND METHODS

Specimens of tehuelche scallop were collected from the north-
ern bank of the San Matias Gulf (Orengo, 40°32'S; 64"32'W) (Fig.
1), at a depth of 18-24 m, between August 1997 and July 1999.
Monthly samples of 20 specimens, with a mean shell height of
67.05 mm (SD: 10.1), were used to determine the gonosomatic
index (GSI) and muscular index (MI) before fixation. Scallops
were dissected; the shells removed; and the total weight (TW). soft
parts wet weight (SPW), muscular weight, and gonad wet weight
(GW) were recorded. The GSI was calculated using the equation
(Giesse & Pierce 1974, Jaramillo et al. 1993):

GSI =

GW X 100
SPW

Mean GSI was estimated for each monthly sample. To analyze
multiyear trends in reproduction, data collected in 1997 and 1999
were coupled to a GSI data set already available for the Orengo
area from 1989 and 1991, and both data sets were related to sea
water temperature. Minimal average values following high average

Gonosomatic index (GSI)

40

32

24

16

8

♦Corresponding author. E-mail: maitenarvarte@canaldig.com.ar

Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun
1997 1998 1999

Figure I. Temporal changes in the GSI iif tehuelche scallop deployed
in Playa Orengo, San Matias Gulf, from August 1997 to July 1999.
Data points are mean values ± standard deviations; n = 20 scallops per
sampling date.

571

572

Narvarte and Krofxk

values were considered as indicators of spawning (Jaraniillo et al.
1993. Avendano & Le Pennec 1997).

The mean muscular index (MI = wt. of adductor inuscle/TW
X 100) was estimated monlliK in a similar \\a\ than GSI and was
compared with GSI data.

The gonads from those 20 specimens were then processed for
histologic analysis. The whole gonad was fi,\ed in Davidson's
solution, stored in cthanol i71)'f). and then dehydrated using a
series of increasing ethanol solutions (Howard & Smith 1983).
Then, the samples were embedded in paraffin and sections 5- to
7-|jLm thick were cut and stained with Harris hematoxylin and
eosin.

Gonad tissue was quantitatively examined using the schemes of
Lasta and Calvo (1978). Dihacco et al. (1995). and Pazos et al.
(1996) to assess developmental stages. The percentage of each
gonadal stage was analyzed. The definition of each stage (early-
mid mature, ripe, partial spawn, spawning and recovery, and spent)
is provided in Table 1 . Photomicrographs were taken using a stan-
dard microscope at magnifications of 100 and 400x. Oocytes were
measured and analyzed according to Dihacco et al. ( 1995). Mean
oocyte diameter was estimated by measuring the diameter of 250
oocytes of each individual. This process was repeated for five
individuals from the total monthly sample. Only those oocytes
with clear nucleolus were considered for the analysis.

The GSI and adductor MI indexes were compared by means of
a simple ANOVA using data that had been subjected to an arcsine
transformation [x'(p)l (Sokal & Rohlf 1979). The maximum values
of the oocyte diameter were compared between years using a
simple ANOVA,

RESULTS
Gonosomatic Indices

During this two year study, major peaks in GSI were observed
in January and December 1998. Peaks were followed by decreases
in GSI. representative of spawning events (Fig. 1 ). Maximum GSI
was significantly different (F, 3y = 88.88, P < 0.001) between
these two reproductive periods. Average values of GSI ranged
between 27,58 (SD: 9,93) and 4.83 (SD: 0.85) from January to July
1998, and from 15,30 (SD: 5,17) to 4,01 (SD: 1,11) from Decem-
ber 1998 to April 1999,

GSI increased between July and October, indicative of early
stages of gametogenesis, GSI then reached maximal values from
November to January and subsequently dropped over a short pe-
riod, which indicated a massive spawn. Spawning continued to
April, when the last spawning events were detected and after
which the GSI was annually the lowest (May through June), A
resting period of 1-2 mo was observed after the autumn spawn.

The magnitude of spring and autumn spawning periods, esti-
mated by observ ing the extent of drops and peaks of the GSI.
varied interannually. particularly for the spring spawn.

Histologic Analyses

Sperm and oocytes were observed together in the gonoduct
(Fig, 2a), The male portion spawns o\er a longer period than the
female gonad (Fig, 2b) and. consequently, the female gonad de-
velopment is what determines the reproductive viability. For this
reason gonadal cycle was described only for the female portion of
the gonad.

Oocyte Characteristics

Absence of vitello was typical of the deselopmcnt oocytes, as
well as a defined nuclei (Fin, 2c), Mature oocytes had a diameter

TABLE \.

Main histological characteristics of gonadal maturity stages of the

tehuelche scallop, (ionadal stages Here determined h\ histological

analjsis and scored using a modification of Lasta and Cal>o (lyVX).

Stages

Description

Early maturation Follicles are well-defined. The lumen may occupy up
to 50% of the follicle, The intertollicular space is
highly visible,
Male: few layers of cells (germ cells and

spermatogonia) along the follicle wall. Female:
presence of germ cells, oogonia. and primary
oocytes with diameters varing between 10 and 25
|xm.

Mid-maturation The lollicles occupy up to 75"(: of the gonad.

Follicular lumen is much reduced. Interfollicular
space is still present but limited.
Male: follicles are full of spermatocytes I and II and
spermatids. Female: stalked oocytes I. 25 to 40
(jLm, protrude into the follicular lumen but still
attached to the wall (peduncled oocytes).

Ripe The follicles are completely full of ripe gametes, and

there is no interfollicular space.
Mate: spermatozoa occupy almost 100'7r of the
follicles. They are in order and oriented with the
flagella toward the centre of the follicle, Fumatc:
follicles are full of free oocytes up to 50 |xm
diameter. They have polygonal shapes, being
pressed against each other.

Spawn and Ripe gametes are being released through the

recovery gonoducts. Presence of a second series of germ

cells attached to the follicular wall.
Male: presence of spermatocytes I and II, and

spermatozoa are disordered in the follicular lumen.
Female: presence of oocyte I attached to the wall
and polygonal oocytes free and disordered in the
follicular lumen.

Partial spawn Follicles remain lull of mature gametes, but some of

them look lax because some gametes were
released. Some phagocytes are present in the
interfollicle space.
Male: spermatozoa are disordered and separated in
the follicular lumen. Female: remained oocytes
still have polygonal shapes and they are free or
attached at one side of the follicle.

Spent Most follicles are empty. Interfolliclar spaces become

very noticeable. Phagocytic cells are very
abundant. Residual gametes may or not be present,
but w ith no sians of cametosienesis.

around 50-60 |jim, with a high quantity of vitello around a con-
spicuous nuclei (Fig, 2d and 2e), Reabsorbed oocytes (both mature
and non-mature) remained attached to the follicle wall or free
inside it. They were easily distinguished by the loss of the round-
polycdric shape, typical of mature oocytes, and by the diffuse
contour of the cell membrane (Fig. 2f). The central portion of the
gonad, or the transitional zone between female and male portions,
had a high frequency of atresic oocytes inainly in the ripe stage
during August-October 1497 (Fig, 2h),

Gonadal Phases

Gonadal phases, considered as the percentage of individuals of
each gonadal stage over the monthly sample, differed between
1997 and 1998 and 1998 and 1999 (Table 1, Fi;;, 3),

Reproductive Cycle Variation of the Tehuelche Scallop

573

4^

i> ■=

>

^r.^- \

.1*^^'

^1

:ijM±

. .♦: M- ^■

«"

■^^<^:. :

■ i ■>

V .

:. J

-. ^<e

r

A

J .9

"; f

Figure 2. Photomicrosraphs of gonads of Aeqtiipecten lehueUliiis al dilfirent stages of a seasonal reproductive cycle, a. Sperm and oocytes in
the same oviduct: b, partial male spawning: c-e, maturation: f, partial spawn; g, spent; h, atresic oocytes.

In 1997. S19c of the female gonads were "ripe"" in August.
Ripeness continued into the spring months, involving 90-100'7f of
the total sample. The remaining indi\iduals were spawning and
recovering. A high percentage of individuals with atresic oocytes
(80% in August-October) were considered ripe. A massive spawn-
ing (all individuals) occurred between December 1997 and January
1998. with individuals in partial spawning and recovery during this
month. Partial spawns were observed in more than 90% of the
population in February through March 1998 and most of the in-
dividuals were spent in May through July (Fig. 3).

In August 1998. the prevalent stage was mid maturation (90%).
Proportions of total maturation during spring months (September

through December 1998) were lower in magnitude than in the
previous season, ranging from 55-85% of the sample. All indi-
viduals were in recovery in December 1998 and January 1999.
indicating a spawning event had occurred. The same stage was
observed in 70% of the individuals in February. Spent stages ap-
peared gradually, increasing between March and June 1999
(Fig. 3).

Oocyte Diameter

Mean oocyte diameter reached its ma.ximum value in Decem-
ber of both 1997 and 1998. Significant differences in the oocyte

574

Narvarte and Krofxk

Aug-97

Aug-98

oL

n wn§

n

Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun

1997

1998

1999

5 Early maturation
B Early spawn

□ Mid-maturation DRipe

■ Spawn & recovering D Spent

Figure 3. Diagram of the seasonal percentage distributions of the dif-
ferent gonadal phases in the lehuelche scallop gonads (a, male portion;
b, female portion), during August 1997 to July 1999.

diameter were found between years (F, ,, = 10.63, P < 0.05) (Fig.
4). Ma.xinuim mean value in December 1997 was 41.98 (jim (SD:
12.89), whereas a value of .36.04 ixm (SD: 16.33) was found in
December 1998.

Frequency distributions of oocyte diameters are presented in
Figure 5. Mature oocytes were representatively observed in August
1997. Proportion of mature oocytes decreased in October and in-
creased in November. The highest fraction of mature oocytes was
observed in December of both years.

Mature oocytes were almost totally absent in January 1998 and
the gonad was full of immature oocytes, indicating an intense
proliferation. Diameter of these oocytes increased in February and
they disappeared from the gonad in February and March. Two
modes ( 10 and 35 |j.m) were present in March 1998.

30

O 15

Aug

Figure 4
tehuekhus
July 1999.

To T .J. "

o T "

" T \ T T " T

1^ i\[y^ 11 ill

Oct Dec
1997

Feb Apr

Jun Aug
1998

Oct Dec Feb

Apr Jun
1999

Mean oocyte diameter of tehuelche scallops, Aiquipecten
, in Playa Orengo, San Matias (iulf during .August 1997-
Uatapoints arc mean values ± SD (;i = 5 per sampling date).

U i 50

Jan-98

0 10 20 30 40 50 60 70
Oocyte diameter (^im)

i 50

Nov-98

oL

Jan-99

0 10 20 30 40 50 60 70
Oocyte diameter (^im)

Figure 5. Frequency distribution of oocyte diameters of .4. tehuekhus
during (he period from 1997 to 1999 (columns: mean values: bars: SD;
II = 5 individuals per .sampling date.

Prevalent stages found in August and September 1998 matched
the initial cell series (early maturation). The co-e.xistence of two
generations of cells from September 1998 to March 1999 was
characteristic of partial spawnings with recovery. A great fraction
of mature oocytes disappeared from December 1998 to January
1999. In January and February, intense oocyte proliferation was
observed. Numbers of mature oocytes increased again in March
1999 and decreased abruptlv in April, indicating a fall spawning.

Iiiler-Vear Varialioiis iif (iSI and lis Relation With Water Temperature

Water temperature records of the 1 989 and 1 99 1 period showed
slight variations between years. Differences between 1997 and

Reproductive Cycle Variation of the Tehuelche Scallop

575

1998, however, were greater (Fig. 6a). Records of winter 1997-
summer 1998 varied less (A = 1°C) than records of 1998 and 1999.
The highest monthly increases (AT > 1.3°C) were recorded from
September to December for the period 1997-1998 and from Sep-
tember to January in 1998-1999.

Figure 6b represents the variations of GSI during 1989-1991
and 1997-1999 seasons. Inter-year variations were similar for the
first period. Maximum mean values oscillated between 16.15 (SD:
4.28) and 17.56 (SD: 6.02). During the 1997 and 1999 periods,
differences between years were greater, ranging from 27.58 (SD:
9.93) to 15.30 (SD: 5.17).

Muscular Energy Resen'es

Ml reached its ma.\inium mean value during winter-spring
months, before the start of the reproductive season of 1997 to 1998
and 1998 to 1999 (Fig. 7). No differences in the MI were found
between 1997 and 1998, for August, October, and November (P >
0.05). MI increased between January and July before gonadal de-
velopment (Fig. 7).

DISCUSSION

The GSI of A. relnielchiis from the San Mati'as Gulf, studied
over 2 consecutive years, indicated that this species undergoes its
maximum reproductive activity during the spring-summer
months. A small spawning occurred in autumn. Even when this
pattern was repeated during the study period, seasonality and du-
ration of spawnings were not identical between successive years.
In both the 1997 to 1998 and 1998 to 1999 seasons, a massive
spawning occurred between December and January. A second,
lower-magnitude spawning occurred in March through April 1999.
Even though a fall spawning was not supported by GSI in 1998.

Water temperature (°C)

a)

Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun

Gonosomatic index

b)

30

24

_ 18
(0

a 12

6

A

Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun

Figure 6. Inter-year variations of (a) the seawater temperature IT, C)
and (b) GSI during two study periods, 1989 to 1991 and 1997 to 1999.

»**v^ ^^^

0

Jun-97

Sep-97 Ene-98 Abr-98 Jul-98 Nov-98 F6b-99 May-99 Ago-99

-GSI

-Ml

Figure 7. Variations of MI and GSI indexes from August 1997 to June
1999.

evidence of spawning was cleariy observed in the oocyte fre-
quency distribution and mean oocyte sizes.

Temporal patterns of the reproductive condition of the tehu-
elche scallop in this study revealed some other differences between
histological analyses and GSI monitoring. The main spawning in
1997 to 1998 season was detected from January to February by
GSI and from December to January by histologic studies. In ad-
dition, the microscopy study on the female gonad of A. telmelchus
revealed the coexistence of different components of the oocyte
series in those successive years. Therefore, the tehuelche scallop
has asynchronous maturation and continuous spawnings within the
same reproductive season.

The existence of an eariy mature stage in August was con-
firmed by the oocyte diameter frequency distribution. Neverthe-
less, the great percentage of atresic oocytes in August-October
suggests that spawning did not occur. Greater than 50% atresia has
also been observed in other pectinids (Lubet et al. 1987a, Lubet et
al. 1987b, Avendatio & Le Pennec 1997, Roman et al., unpub-
lished data). Dibacco et al. (1995) suggested that during unfavor-
able environmental conditions, bivalves may have a mechanism to
reabsorb and use the high energy content of mature oocytes. As in
other pectinids (Motavkine & Varaksine 1983. Beninger 1987,
Dorange & Le Pennec 1989. Avendatio & Le Pennec 1997). the
highest levels of atresia in A. telniekluis were observed just before
spawning. These authors have suggested that atresia should be
related with an energy deficiency, caused by low food availability.
This could have occurred during the winter months in 1997 in San
Matias Gulf, when lower water temperature was recorded.

Histologic observations of the female gonad during the fall
spawning of both reproductive seasons showed a high relative
proportion of oocytes in eariy stages, whereas larger oocytes were
observed undergoing lysis. Metabolites produced by lysis of ma-
ture oocytes by phagocytic cells, present in large quantities at the
end of the reproductive period, would implicate an important en-
ergy contribution for the recovery after the reproductive season.
This phenomenon was previously observed for a variety of pec-
tmids (Dibacco et al. 1995, Avendaiio & Le Pennec 1997). Ac-
cordingly, we could infer that the second spawning of each season
was smaller than the first, and constituted a minor contribution to
that year class.

The utilization of the adductor muscle energy reserves for re-
production was cited for other pectinids (Barber & Blake 1991).
Variations of MI observed along two annual cycles in this study
showed a similar pattern, in which the energy stored in the adduc-
tor muscle is then diverted to the reproductive system, to satisfy

576

Narvarte and Kroeck

energetic demands for the synthesis of gonad components (Gould
et al. 1988). When the MI was compared between years, no clear
variations were observed between 1997-1998 and 1998-1999.
Therefore, energy demands from the muscle seemed to be similar.
However, the extraordinary GSI values in 1997-1998 suggest a
higher energy availability for the gonad development. Because
muscular reserves are limited by muscular degradation, other ex-
ogenous mechanisms could help to sustain the energy budget for
this development. A higher food availability during the spring
1997 was probably responsible for this extraordinary gonadal de-
velopment. Thompson and MacDonald (1991) have indicated that
temperate species such as Pkicopecteii iinini'lliiniciis are exposed
to phytoplanktonic "blooms" in early spring and therefore have a
short period of energy reserve accumulation. This early investment
for reproduction would depend of the environmental conditions
following the bloom (Dibacco et al. 1995).

de Vido de Mattio 1 1984) and Orensanz ( 1986) indicated that
energy for gonadal development for A. teliiielcliii.\ in the San Jose

Gulf seems to come more from food than from the energy stored
in the muscle. In the San Mati'as Gulf, a decrease of MI before
spawning indicates that this population uses both energy sources,
food and muscle. As was observed in other pectinids (Langton et
al. 1987, Barber & Blake 1991, Dibacco et al. 1995. Rheault &
Rice 1996. Avendano & Le Pennec 1997), exogenous factors, such
as temperature and food availability, may produce great variations
in the gonadal development and the spawning time of A. lehiiel-
chiis.

AKNOWLEDGMENTS

This work was supported by funds from the Europe Union-
Argentina Agreement ( 1996-1999). The authors thank the person-
nel of the IBMP"Alte. Storni" for their cooperation and assistance.
We specially thank Sandro Acosta. Tony Brochado, and Elda
Cerecera for assisting with field and laboratory activities, and Mar-
cela Pascual, Jose M. Orensanz, and an anonymous reviewer for
their constructive criticism of the manuscript.

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genesis in two populations of the Chilean molluscan bivalve, Ar-
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Barber, B.J. & N.J. Blake. 1991. Reproductive physiology. In; S. E.
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Bricelj, V. M., J. Epp & R. E. Malouf. 1987. Intraspecific variation in
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Christiansen, H. E. & S. R. Olivier. 1971. Sobre el hermafroditismo de
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Christiansen H. E., M. E. Cabrera & S. R. Brodsky. 1974. Ecologia de las
poblaciones de vieiras {Clilaniy.s tehuelcha d'Orb. 1846) en el Golfo
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reproductive. Ins. Biol. Mar. Mar del Plata. Contr. 225:17.

De Vido de Mattio, N. 1984. Variacion estacional de la composicion bio-
quimica de la vieira Chlainys tehuelcha (d"Orbigny) en el Golfo San
Jose. Centra Nac. Patagonico. Contr. 92:22.

Dibacco, C, G. Robert & J. Grant. 1995. Reproductive cycle of the sea
scallop. PUuopeclen magellaniciis (Gmelin. 1791). on northeastern
Georges Bank. J. Shellfish Res. 14:59-69.

Dorange. G. & M. Le Pennec. 1989. Ultrasctructural study of oogenesis
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Giesse. A. D. & J. S. Pierce. 1974. Pelecypoda (excluding Ostreidae). In:
Reproduction of marine invertebrates. New York: Academic Press, pp.
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Gould. E.. D. Rusanowsky & D. Luedke. 1988. Note on muscle glycogen
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Howard, D. W. & C. S. Smith. 1983. Histological Techniques for Marine
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Jaramillo, R.. J. Winter. J. Valencia & A. Rivera. 1993. Gametogenic cycle
of the Chiloe scallop (Chlamys amaiidi). J. Shellfish Res. 12:59-64.

Langton, R. W., W. E. Robinson & D. Schick. 1987. Fecundity and re-

productive effort of sea scallops Placopecten magelUmicus from the

Gulf of Maine. Mar. Ecol. Prog. Ser 37:19-25.
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reproduction de la coquille Saint Jacques {Pecten maxiinus L. ). Oceanis

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Jacques (Pecten maximus L.). Haliotis 16:17.3-180.
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nerveux et regulation de la reproduction chez les mollusques bivalves.

Paris: Editions IFREMER. 208 pp.
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In: S. Shumway, editor. Scallops: Biology, ecology and aquaculture.

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maximus. in suspended culture in Ria de Arousa (Galicia. NW Spain).

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Shellfish Res 15:271-283.
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S. Pierce, editors. Reproduction of marine invertebrates. New York:

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Journal of Shellfish Reseciirh. Vol. 21. No. 2. 577-584, 2UU2.

REPRODUCTIVE AND RESERVE STORAGE CYCLES IN AEQUIPECTEN OPERCULARIS

(L., 1758) IN GALICIA, NW SPAIN

G. ROMAN,'* M. J. CAMPOS,' J. CANO,' C. P. ACOSTA," PILAR IGLESIAS,' AND
OSCAR GARCIA'

'inslituto Espanol de Oceunografi'a. Mmisterio de Cienciu y Tecnologiu. Ceiiiro Ocecmogrdfico de A
Contna. P.O.Box 130. 15080 A Coniiia. Spain: -CMA Coron. P.O.Box 208, 36600 Vilagarci'a de
Aroiixa. Pontevedni. Spcdn: ^Departameuto de Bioqiiimica y Biologi'a Molecular. Facultad dc Fannacia.
Universidad de Santiago de Compostela. Campus Sur. A Contna. Spain

ABSTRACT A study was conducted on the reproductive and reserve storage cycles in Aequipecten opercularis (L.. 1758) from deep
waters (50-60 m) in the Ri'a de Arousa (Galicia, NW Spain). The reproductive cycle was studied by visual inspection of the gonad,
the use of the gonad condition index, the gonad dry weight of a standard queen scallop 50-mni height, the average oocyte diameter
and the percentage of the gonad occupied by oocytes. The same results were obtained with each of these methods. Following a restmg
period in autumn, which varied in length depending on the year, there was a period of recovery in winter, followed by a spawning
period lasting until the end of summer. There were several partial spawnings, followed by recovery periods until the gonad was totally
spent. Accumulated reserves in the adductor mu.scle (glycogen and proteins) and digestive gland (lipids) were used up during
gametogenesis. The weight and condition indices of both organs and their reserve substrate contents showed parallel changes
throughout the two years of the study.

KEY WORDS: Aequipecten openidari.s. Ria de Arousa, breeding season, reproduction, energy reserves

INTRODUCTION

Aequipecten operciiluris (L., H.'SS) is distributed along the
North Atlantic coast, ranging between WH and 70°N, and also
extends to the Mediterranean and the Adriatic Sea (Brand 1991).
There is one commercial fishery of this species in Galicia, which
in the 1960s caught up to 700 tonnes year"', although nowadays
the catch rarely exceeds 100 tonnes year"'.

Despite the commercial interest in A. opercidaris in Galicia,
investigation of the reproductive biology of natural populations in
this region has not been performed. Only the reserve storage cycle
and breeding season have been previously been described, by Ro-
man et al. (1996), for cultured queen scallop in the Ri'a de Arosa.

Mason (1983) and Barber and Blake (1991) reviewed the pe-
riods of gametogenesis in queen scallop populations in the North
Atlantic. However, the information provided is scant and the re-
productive behavior of geographically separated populations of the
same species may vary (MacDonald & Thompson 1988, Bricelj et
al. 1987, Devauchelle & Mingant 1991).

As gametogenesis, the storage and use of energy reserves in
scallops is also cyclical and underlies the |-eproductive cycle. The
importance of stored reserves in the reproductive cycle of pectinids
in temperate waters is well documented (see Barber & Blake 1991
for a review). In general, protein and/or glycogen are .stored in the
adductor muscle and lipids in the digestive gland. Barber and
Blake (1981) reported that in Argopecten irradiuns. the relative
importance of different parts of the body as energy storage sites is
highly adaptive, and results from genetic divergence or non genetic
adaptation to different environments within the geographical range
of the species.

This study is part of an investigation of the feasibility of cul-
tivating A.opercularis in Galicia from spat obtained from natural
environments using collectors. Studies of the reproductive behav-
ior of this species are required to optimize spat collection for
commercial purposes. The main objectives of this study are to
describe gametogenesis, the breeding season, seasonal changes in

weight of reserve storage organs and their biochemical composi-
tion in relation to the reproductive cycle, and the influence of
environmental factors on gametogenesis and spawning.

MATERIALS AND METHODS

Sliidv Area

*Corresponding author. E-mail: guillermo.roman@co.ieo.es

The largest population of queen scallops in the Ri'a de Arosa are
found in "The Channel" (Fig. I ), at a depth of between 50 and 60
m. The study was therefore centered on this area, although dis-
perse, smaller banks are found in shallower waters.

Environment

From January 1996 until December 1997 weekly recording was
made of temperature, salinity and chlorophyll a at a depth of 45 m
using a CTD. From March to September 1996 fortnightly deter-
minations were made of the paniculate organic matter, by filtering
a sample of 2 L of seawater, collected from a depth of 30 ni, using
pre weighed Whatman GFC/C filters (Whatman International Ltd.,
Maidstone. England). After drying at 60°C for 24 h. the filters
were weighed then reweighed after combustion at 450°C for 12 h.
The organic matter content was calculated as the difference be-
tween the two weights.

Animals

Adult specimens of A. opercidaris were collected between
January 26,1996 and November 18, 1997. Captures were made at
fortnightly intervals in "The Channel"" from a commercial fishing
boat, by trawling. The animals measured between 35 and 65 mm
in height, which corresponds to age year classes I and 2. The
gonads of 10 scallops were placed in Bouin"s fixative immediately
following capture. The fixative was removed by frequent washing
with 70* ethanol saturated with lithium carbonate and then stored
in 70% ethanol until being processed. Classic histologic techniques
were used; gonad was embedded in paraffin and serial sections of
ovary (5 (xm) stained using modified Wheatley"s stain. Histologic
study was only made of ovary tissue, using stereological methods
(Paulet & Boucher 1991, Pazos et al. 1996) with a Visilog 3.1.1
image analyzer. The average diameter of the oocytes (Dm) and the

577

578

Roman et al.

43'«)'

MbftG/vRCIA

43 "JS'

43°3D'

I SAVORA

43*25'

1D°5'

9° 9°55' 9°50'

Figure 1. The Channel (shadowed area) in the Ria de Arosa.

9P46'

percentage volume occupied by oocytes (OVF = % of ovary
occupied by non atretic oocytes) were calculated.

Another 30 animals were transported to the laboratory in a
coolbox and then maintained under running water for 18 h to allow
sand to be expelled. The specimens were then opened and the soft
tissue separated into 4 components: gonad, adductor muscle, di-
gestive gland, and remaining tissues. After draining for 10 min on
filter paper, the wet weight (WW) was detennined; an aliquot of
each organ was used to determine the dry weight (DW lOO'C. 24
h). We recorded the appearance of the gonad, noting if transparent

and colorless or if there were two clearly differentiated areas of
color (white or cream in the testicle, orange or red in the ovary).
The height of the shell was measured to the nearest 1 mm. using
calipers. The shells were dried (lOO'C. 24 h) and weighed. Con-
dition indices were calculated for gonad (Gci). digestive gland
(DGci). muscle (Mci) and remaining tissues (RTci) using the fol-
lowing equation:

CI organ =
1985).

100 X DWorsan/DWshell (Lucas & Beninger,

Reproduction of Queen Scallop

579

Regression equations were used to describe the relationships be-
tween shell height and the dry weight of each component, and the
weight of each component for a standard animal having a shell
height of 50 mm was calculated according to Taylor and Venn
(1979).

Aliquots of muscle, digestive gland and gonad were lyophilized
before biochemical analyses were carried out (glycogen and pro-
tein content of muscle and total lipid content of digestive gland and
gonad) following previously described techniques for lipids and
glycogen (Roman et al. 1996). Protein contents were determined
from the N content, measured using an elemental analyzer Carlo
Erba 1 108. The amount of N was multiplied by 6.25 to estimate the
amount of proteins (Ansell 1974. Beninger 1982). Grams of lipids
in gonads and digestive gland and glycogen and protein in muscle
were estimated by multiplying the percentage of each reserve sub-
strate by the previously calculated standard dry weight of each
component.

Statistical Methods

One-way ANOVA was used to test for significant differences
among the average values of the condition indices. Dm and OVF.
The normality of the distribution of the variables was tested using
the Kolmogorov-Smirnof test and the homogeneity of the vari-
ables using Bartlett's test. Where necessary, arcsine transforma-
tions (.v' = arcsin^i.v) were carried out. Correlation coefficients
were calculated for the relationships among gonad indices. Dm and
OVF, and among the condition indices of the reserve storage or-
gans, the standard dry weight and biochemical composition.

RESULTS

Environment

The temperature varied little throughout 1996, ranging between
12.7°C and 14.4°C. In 1997 very high temperatures of 17 to I8"C
were recorded in May and June. High values of 16.5 to 17°C were
also recorded from the end of October until the middle of Novem-
ber. The chlorophyll a content was low in both years, ranging
between 0.6 and 1.3 |Jig L"'; the lowest values were registered
between October 1996 and May 1997 and did not rise above 0.8
jjLg chlorophyll a L~'. Particulate organic matter ranged between
0.3 and 2.7 mg L"'. Salinity remained practically constant at 35.7-
35.8%c throughout most of the sampling period, with slight de-
creases occurring in winter to below 35.59{(. The minimum value.
34.2%t, was recorded in November 1997 (Fig. 2).

Gonad

During winter and spring of 1996 the gonads were well differ-
entiated, with testicle and ovary clearly visible in all specimens.
From July 23, 1996 onwards there was an increasing percentage
(reaching a maximum value of 90% on September 10) of queen
scallops with spent gonads, which were tlat. colorless and watery,
histologic analysis revealed empty follicles in the undeveloped
gonads. In these queen scallops that were in a period of sexual
repose, the average diameter of the oocytes and the percentage
volume occupied by oocytes was considered to be zero (Fig. 3).

Following the period with the maximum percentage of sexually
reposing animals, there was a period of gonad recovery, which
involved only part of the population, peaking (at 60%) on October
26, then a new minimum (30%) was observed on November 19.
From December 1 996 onwards there was a new period of gonad

D) 1.2

CD

1,0

r

0,8

Q-
O

O

0.6

O

04

E

O

Q.

,#•" .s^-- ,^^'~ ,.^^ ,.^^

Figure 2. Environmental conditions in The Channel.

^K-^

recovery, which this time involved the whole population, reaching
full development on January 28, 1997. Following a decrease ob-
served on February 12, which affected 40% of the population, all
animals had developed gonads. This situation persisted until the
end of summer, then from August 12 until October 28, 1997 all of
the population was in sexual repose and displayed spent gonads. In
1997, gonad recovery took place between October 28 and Novem-
ber 19.

The value of the gonad condition index (Gci) ranged between
0.1 and 2.0 (Fig. 3). Maximum values corresponded to the period
between March 13 and 26 1996 followed by a significantly lower
value on April 10. There were then a series of recoveries followed
by decreases to significantly lower values on June 4. July 2. and
August 20; minimum values were recorded coinciding with gonads
being totally spent. There was a new peak (significantly higher) on
October 20, the gonads then became totally spent before undergo-
ing recovery from November 19 onwards. The peak recorded on
January 28, 1997 was followed by a significant decrease on Feb-
ruary 12 and then again, as in 1996 there were a series of recov-
eries and decreases, with significant decreases observed on March
25, May 17, July 1, and August 12, which coincided with the

580

Roman et al.

■o

(0

c
o

O)

c

'l

nj

n
w

o
o

X

0)
■D

C

c
o

c
o
o

■D
TO

c
o

O)

E

E
Q

Figure 3. From top to bottom, a) percentage of scallops bearing gonad,
bl gonad condition index, c) oocyte mean diameter and percentage
volume of the ovary occupied by oocytes, and d) gonad dry weight of
a standard 50 mm height scallop and lipid content of the gonad.

gonads being totally spent; recovery did not take place until No-
vember 19. The decreases were assumed to be associated with
spawning.

Changes in the average diameter of the oocytes and of the
percentage volume of the ovary occupied by oocytes (OVF) were
similar (Fig. 3). Maximum values occurred in winter (February to
March 1996) then a sharp decrease was observed on April 10.
1996. The values had significantly increased by April 24 and there
was then a gradual decrease until reaching minimum values be-
tween September 10, and October 8. 1996, with two recoveries

observed on June 19 and August 6. There were significant in-
creases in both Dm and OVF between December 16 and January
14, coinciding with gonad recovery. In 1997 there were 4 peaks
observed on January 28, March 10. June 3. and July 29 followed
by decreases, which only on the latter date was not followed by
gonad recovery.

The changes in all of the parameters studied followed the same
pattern and each parameter was significantly correlated with the
others: Gci vs. Dm (/■„ = 0.588. P < 0.0001 ); OVF vs. Dm (/; =
0.772. P < 0.0001). and Gci vs. OVF {i\ = 0.716. P < 0.0(J01).

The gonad dry weight of a standard queen scallop ranged be-
tween 0.006 and 0.204 g. The seasonal variations are shown in
Figure 3. Although the mean percentage value of the total weight
was low {9.09c) the range of values varied greatly, between 0.7%
and 23.0% of the dry weight of the animal. There was a large
variation in weight, which followed the pattern of variation of Gci.

Biochemical Compositian of the Gonad

The percentage values of lipid contents are shov\n in Table 1.
The total lipid content of the gonad of a standard queen scallop
varied in a similar way to the Gci and to the standard DW. with
maximum and minimum values coinciding with the maximum and
minimum values of the condition index and the standard dry
weight (Fig. 3).

Somatic Tissues

Seasonal changes in the standard dry weight of the muscle and
the digestive gland, as well as in their respective condition indices
and in their reserve substrate contents (lipids in digestive gland,
glycogen and proteins in adductor muscle) are shown in Figure 4.
Somatic growth began in early summer and was demonstrated by
increases in dry weight, condition index and specific reserve sub-
strate content, which reached maximum values between autumn
and winter. There was then a decrease in the values of all of these
parameters.

Adductor muscle accounted for most of the w eight of the queen
scallop, on average 54.5%; the mean dry weight of the muscle
varied between 35.6% and 68.1%. representing the greatest varia-
tion in the weight of the animal. The dry weight of the muscle of
a standard animal varied between 0.273 and 0.993 g. (i.e.. the
mean dry weight varied by up to 3 times). The condition index of
the adductor muscle (Mci) varied in a similar way. ranging be-
tween 3.3 and 8.5.

Digestive gland accounted for a small proportion of the total
weight of the animal (mean. 10.3%. range 6.9%- 14.2% ). Although
the weight varied more than that of the muscle, ranging between
0.042 and 0.198 g, (i.e., varying by almost 5 times) the condition
index (DGci) ranged between 0.5 and 1.7 (i.e.. it varied by more
than three times).

The dry weight of the remaining tissues accounted for. on
average, 26.0% of the weight of the animal, ranging between
17.3% and 39. 1 % of the somatic components of the standard queen
scallop. The dry weight of the remaining tissues of the standard
specimen ranged between 0.144 and 0.310 g (i.e.. it varied by
approximately two times).

The percentage amounts of glycogen and proteins in the mus-
cle and of lipids in the digestive gland in the gonad are shown in
Table 1.

In January 1996. high values were recorded of standard dry
weight of muscle. Mci. percentage, glycogen, and total protein and

Reproduction of Queen Scallop

581

TABLE 1.
Seasonal changes in the composition of A. opercularis.

Adductor Muscle

Digestive Gland

Gonad

Date

% Lipids

% Lipids

% Glycogen

% Protein

Jan 25

57,52

28.07

14.74

71.68

Feb 28

42,70

25.27

7.28

80.19

Mar 13

32,04

25.88

5.19

80.22

Mar 26

34.96

26.63

5.32

77.34

Apl 10

27.40

27,04

4.27

7 1 .95

Apr 24

28,72

26,89

4,41

77.61

May 07

22.08

24,20

3,34

80.17

May 23

35.00

24,67

3,47

72.19

Jun05

33.75

23,30

4,49

69.38

Jun 19

44,26

27.89

4,39

76.00

Jul 02

42,77

29.14

6,11

72.5 1

Jul 23

50,55

29.41

6,84

70,64

Aug 06

54,86

25.87

7,85

69,77

Aug 20

61,02

27,05

9,21

73.42

Sep 10

48,04

29,57

16,68

67.23

Oct 08

54,81

26,71

18,02

64.44

Oct 26

49,50

22,64

18,92

69.33

Nov 19

44,34

18.86

14,03

72..30

Dec 10

41,61

21.80

12,56

70.01

Jun 14

47,20

20.61

13,03

74.83

Feb 12

62,45

23,54

8,55

74.89

Feb 25

41,8!

22.12

5,63

75.66

Mar 10

34,63

17.32

8,20

74.52

Mar 25

28,66

18.21

6,25

73.07

Abr22

25,76

18,54

2,65

77,87

May 06

31,80

22,03

6,61

77.02

May 17

25.38

23,73

2,93

76.26

Jun 03

36,65

23,73

2,76

74.26

Jun 17

39,01

18,95

5,76

78.27

Jul 01

39,31

22,31

6,36

73,45

Jul 15

44,40

22,11

8,44

—

Jul 29

49,11

23,70

8,48

—

Aug 12

52,66

23,87

11.98

—

Aug 26

57,81

30,00

13.91

72.10

Sep 09

59,36

25,67

14.74

71.58

Sep 29

57,64

23.31

14,79

72.76

Oct 28

60,75

21,28

18,68

66.25

Nov 18

62,99

19,91

14.90

68.80

glycogen content; these then decreased rapidly and minimum val-
ues were registered at the beginning of spring. During summer and
autumn there was rapid somatic growth, with maximum weight
values in November. Values of all parameters then began to de-
crease and the cycle was repeated, with minimum values occurring
at the beginning of spring 1997, before beginning to increase once
again. The percentage amount of proteins in the muscle showed a
more irregular pattern of variation, with frequent oscillations be-
tween 707f and 80% between March and September. Following a
minimum of 65% in November the values began to increase show-
ing a similar pattern to that of the previous year.

The changes in standard dry weight of the digestive gland,
DGci, percentage lipids, and total lipid content showed parallel
variation, with the pattern being very similar to that observed for
the studied muscle parameters. The greatest differences were that
in both years growth of the digestive gland began later than that of
the adductor muscle and also that in May of both years there was
a sharp increase in growth, only found in the muscle in 1997.

There was no clear pattern of variation for the remaining tis-
sues, or any correlation with the other somatic components.

There were highly significant positive correlations among con-
dition index, standard dry weight and lipid content of the digestive
gland and among condition index, standard dry weight and glyco-
gen and protein contents of the muscle (simple regression. P <
0.01). Likewise, there were statistically significant correlations
between the muscle and digestive gland condition indices (P <
0.01 ). between the "standard"" dry weight of the muscle and of the
digestive gland (P < 0.01) and between the lipid content of the
digestive gland and glycogen and protein contents of the muscle iP
< 0.01). Only the percentage amount of proteins in muscle corre-
lated negatively with the other parameters.

Spearman's correlation analysis did not reveal any significant
correlation between environmental variables and the parameters
used to study reproduction and the cycle of reserve storage.

DISCUSSION

In this study we describe the spawning cycle and the cycles of
storage and use of reserves in a population of A.opeixidahs from
relatively deep waters (50-60 m) in the Ria de Arousa.

1/05/96 1/09/96 1/01/97 1/05/97 1/09/97 1/01/98

- Digestive gland lipids (g)
Muscle glycogen (g)

- Muscle protein (g)

0,6
■ 05

0,2 S

..v<*^

.^^

Figure 4. From top to bottom, a) muscle and digestive gland conditi(m
indices, b) dr> weight of the somatic components of a standard scallop
50 mm height, and cl biochemical composition of the specific reserve
substrates of the organs of energy storage.

582

Roman et al.

Spawning Cycle

The spawning cycle of A. opercidaris was sludied by visual
observation (presence or absence of developed gonad) as well as
by quantitative (Gci) and qualitative methods (Dm and OVF) (Bar-
ber & Blake 1991).

Despite the simplicity of the first method, its use provided a
general idea of when spawning took place — from winter until the
middle of summer — there being a gradual decrease in the number
of specimens with gonad as the summer progressed, culminating in
the gonads being totally spent. There then followed a period of
gonad recovery in autumn. Taylor and Venn ( 1979) also found the
maximum number of spent scallops in summer in the Clyde Sea
Area, although both the time of appearance of animals without
gonad and the recovery period were earlier than found in the
present study. It is possible that the rough estimate provided by this
method would only be applicable in species or populations with
very marked seasonal variations and in which gonads become
totally spent.

The consistency of the results obtained b) the different meth-
ods of establishing the different processes involved in the repro-
ductive cycle (Gci, OVF, Dm) may be because Gci decreases due
to release of mature, larger oocytes. As a result, there is an obvious
decrease in the diameter of the remaining oocytes and of the per-
centage space occupied by them. Furthermore, as seen in the
graphs outlining the changes in Dm and OVF (Fig. 2), increases in
Gci coincided with cytoplasmic growth and vitellogenesis, (there-
fore larger oocyte diameter and greater percentage space occu-
pied).

Spawning was indicated by the decrease in the Gci, the de-
crease in the mean diameter and the percentage space occupied as
well as by the increase in the percentage number of spent gonads
and the decrease in "standard" dry weight and lipid content.

Optimization of spat collection in natural environments de-
pends on knowledge of the reproductive cycle, the changes in
which must be monitored over time. The consistency of results
independent of the method used allows monitoring of the repro-
ductive cycle of this species without the need for use of sophisti-
cated, expensive techniques by, simply recording the changes in
Gci.

Following spawning, we recorded a period of gonad recovery,
characterized by undifferentiated gonads, without gametes, as pre-
viously described by Allarakh ( 1979), who observed a very short
recovery period of less than a month. In this study, this period
lasted for I month in 1996 (September 10 to October S) and for
almost 2 months in 1997 (September 9 to October 28). In 1996 the
period of sexual repose was shorter because there was partial
maturation in autumn (from October 8), which was not observed in
1997. In both years gonad recovery, which involved the whole
population, started at the end of autumn or beginning of winter and
was rapid, as the first spawning look place in January; there were
then a series of successive periods of maturation and spawning that
lasted until the middle of summer. As the summer progressed, the
number of individuals participating in reproduction gradually de-
creased (1996), or all suddenly displayed spent gonads (1997).

We conclude that the reproductive strategy of this species con-
sists of a very protracted spawning period with frequent partial
release of mature oocytes, followed by recovery due to the rapid
maturation of oocytes present in the follicles, by vitellogenesis.
These ovocytes are in turn spawned while a new cohort of oocytes

matures. The successive recovery and spawning periods involve
gradually less oocytes, until the gonads are totally spent; the final
spawning is therefore not the most important. The whole popula-
tion participates in reproduction during the period of greatest ga-
metic activity, from February to July, but between August and
January, only part of the population is involved.

Descriptions of the reproductive cycle of A. opercularis in
northern areas, revised by Mason (1983) and Barber and Blake
( 1991 ), are mainly based on visual observation of gonads, although
Taylor and Venn ( 1979) used the dry weight of gonad to describe
the spawning cycle in Clyde Sea area; only Allarakh (1979) used
histologic methods to study the reproductive cycle in scallops from
Rade de Brest. In general these studies describe partial spawning
taking place at different times, and the main spawning in autumn.
Allarakh ( 1979) for example, described three natural spawnings in
Rade de Brest, the first two in January to February and June to July
and the third in September, ending with gonads being totally spent.
Soemodihardjo ( 1974) and Paul ( 1978) reported various spawning
periods in the Isle of Man. two partial spawnings in winter (Janu-
ary to February) and spring or the beginning of summer (May to
July) and the main spawning in autumn (August to October).
Amirthalingam (1928) observed spawning between January and
July in Plymouth but did not record any spawning activity in
autumn.

Our results are generally consistent with those of the authors
cited, although the more frequent sampling and the inclusion of
histologic analyses allowed confirmation that partial spawning
takes place more often than previously described in other popula-
tions and that the final spawning that resulted in the gonads being
totally spent, is not the most important, but merely the last before
gonad recovery. In summary, partial spawning forms part of the
sexual strategy consisting of staggered maturation of 4 to 3 cohorts
of oocytes that are shed when mature, throughout a long breeding
season that may span from January to July.

The generally small number of oocytes obtained by artificial
stimulation of these animals is possibly due to this behavior (Le
Pennec. 1982).

Conversion of the measurements obtained to those of a stan-
dard 50-mm animal allowed us to compare our results with those
of Taylor and Venn (1979). In one of the two years of their study,
these authors recorded a lower standard dry weight of gonad than
in this study (range between 0.087-0.105 g), whereas in the other
year the values were similar (0.170-0.198 g). In this study, the
maximum values ranged between 0.168-0.204 g. This may be
partly due to the lower frequency of sampling by Taylor and Venn
(1979), who may have recorded dry weight values as they were
rising or falling but not while at their maximum values, as we have
observed considerable increases and decreases in weight within 15
days. A similar explanation may be given for the greater number
of spawnings that were observed.

The lipid contents of scallops from the Ria de Arousa (maxi-
mum levels of 0.043 and 0.045 g in 1996 and 1997. respectively)
were considerably higher than in those from the Clyde Sea Area
(0.()LM).026 g).

Spawning took place at approximately the same times in both
years, despite the large differences in tentperature observed. Tem-
perature therefore probably does not affect spawning, as previ-
ously thought by other authors; Broom (1976) investigated the
possible relationship between spawning and changes in tempera-
ture, but with inconclusive results.

Reproduction of Queen Scallop

583

Cycles of Storage and Use of Reserves

By autumn somatic growth was completed, the muscle and
digestive gland had reached their maximum weights and had maxi-
mum levels of reserves, the breeding season was over and gonads
were spent. A new period of gonad growth began in November and
from this time onwards in both years, the accumulated reserves
began to diminish.

This decrease was associated with gametogenesis and the main
spawning that affected the whole population during most of the
breeding season. The final, partial, spawning that affects only part
of the population, is apparently carried out using external energy
enough to maintain gonad growth, without having to use the re-
serve energy stored in organs. In both years accumulation of re-
serves began (at the end of spring for muscle and at the beginning
of July for digestive gland) even though spawning was still taking
place.

The variation in the weights and indices of digestive gland and
muscle indicate their status as reserve energy storage organs; the
variation in the lipid content of the digestive gland and of glycogen
and protein in the muscle indicate that these are the substrates used
as energy reserves.

Although the percentage amount of protein in the muscle al-
ways remained high (6-1— 80'7f ), a large amount of protein was used
(ranging between 0.189 and 0.657 g). The glycogen con-
tent varied greatly (3-18%), but the total amount used was
relatively small (ranging between 0.010 and 0.185 g). As a con-
sequence of fluctuations occurring during the cycle of storage
and use of reserves, the weight of the muscle varied between
0.273 and 0.617 g and between 0.301 and 0.993 g in the first
and second years, respectively. Observations were consistent
with those of Taylor and Venn (1979) who describe a marked
seasonal variation in the weight of the adductor muscle, with
minimum values at the end of winter, gradual increases during

spring and summer and maximum values in September to October,
the weight sometimes doubling between March and October. Ac-
cording to these authors, and in agreement with our results, the
seasonal variation in weight is due to changes in protein and car-
bohydrate contents.

Taylor and Venn ( 1979) attributed the variations in lipids of the
remaining tissues to the digestive gland, although they did not
carry out specific analyses of this organ. Observed in the Ri'a de
Arousa were large variations in the lipid content of the digestive
gland (minimum values of 0.012 and 0.016 g and maximum values
of 0.133 and 0.198 g in 1996 and 1997. respectively), thereby
confirming their speculations.

In contrast with the findings of Barber and Blake ( 1981 ). who
observed that Argopecten irradians concentricus uses dif-
ferent substrates (glycogen, protein or lipids) of different origin
(muscle or digestive gland) depending on the phase of gameto-
genesis, A. opercularis apparently uses all of its reserves si-
multaneously (muscle glycogen and proteins, as well as diges-
tive gland lipids). However, these authors indicate that which
are the important substrates, where they are stored and how
their use is related to reproduction varies among species and
among different populations of the same species. It should
also be taken into account that A. irradians concentricus is se-
melparous. and has a straightforward reproductive cycle, with
one spawning after which the animal dies, whereas A. opercularis
is iteroparous, and spawns several times in one year for several
years.

ACKNOWLEDGMENTS

This study was financed by CYTMAR, project MAR95-1893-
CO3-01. The authors thank Carmen Presas, Carmen Vazquez, and
Eamon Loewe and the crew of the Chipre II. Jose Luis and Manuel
Charlin.

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tiation and on the reproductive cycle of ChUimvi opercularis L. (Mol-
lusque lamellibranche). These 3" cycle, Caen. 148 pp.

Ansell, A. D. 1974. Seasonal changes in biochemical composition of llie
bivalve Chlamys seplemradiata from the Clyde Sea Area. Mar. Biol.
26:85-99.

Amirthalingam. C. 1928. On lunur periodicity in reproduction of Peaen
opercularis near Plymouth m 1927 to 1928. / Mar. Biol. Ass. U.K.
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Barber. B. J. & N. J. Blake. 1981. Energy storage and ulilizalion in relation
to gametogenesis in Argopecien irradians concentricus (Say). / E.vp.
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Barber. B. J. & N. J. Blake. 1991. ReproducUve physiology. In: S. E.
Shumway, editor. Scallops: Biology, Ecology and Aquaculture. Am-
sterdam: Elsevier, pp. 377-428.

Beninger. P. G. 1982. Comparative biochemical study of two populations
of the clams Ruditapes decussatus (Linne) and Ruditapes phiiippi-
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Brand. A. R. 1991. Scallop Ecology: Distributions and Behaviour. In: S. E.
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Bricelj. M. v., J. Epp & R. Malouf. 1987. Comparative physiology of

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(Lamarck): mortality, growth and oxygen consumption. J. Exp. Mar.

Biol. Ecol. 112:73-91.
Broom, M.J. 1976. Synopsis of biological data on scallops Chlamys

(Aequipecten) opercularis (Linnaeus), Argopecten irradians (Lamarck),

Argopecten gibbus (Linnaeus). FAO Fish. Synops., No 1 14. 44 pp.
Devauchelle, N. & C. Mingant. 1991. Review of reproductive physiology

of scallop Pecten maximus. applicable to intensive aquaculture. Aquat.

Living Resour. 4:41-51.
Le Pennec. M. 1982. L'elevage experimental de Chlamys opercularis (L.)

(Bivalvia. Pectinidae). Vie Marine 4:29-36.
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1 87-200.
MacDonald, B. A. & R. J. Thompson. 1988. Intraspecific variation in

growth and reproduction in latitudinally differenciated populations of

the giant scallop Placopecten magellanicus (Gmelin). Biol. Bull. 175:

361-371.
Mason. J. 1983. Scallop and Queen Fisheries in the Bntish Isles. Fishing

News Books Ltd. Surrey: Farnham. 1 14 pp.
Paul. J.D. 1978. The biology of the queen scallop. Chlamys opercularis

(L.), in relation to its prospective cultivation. PhD thesis. University of

Liverpool. 127 pp.

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Paulet. Y.-M. & J. Boucher. 1991. Is reproduction mainly regulated by
temperature or photoperiod in Pecten maximiis'.' Inv. Reprinl. Dev.
19:61-70.

Pazos, A. J.. G. Roman. C. P. Acosta, M. Abad & J. L. Sanchez. 1996.
Stereological studies on the gametogenic cycle of the scallop Pecten
maximus in suspended culture in Ria de Arousa (Galicia, NW Spain).
Aqiuuiitliire 142:119-135.

Roman. G., M. J. Campos & C. P. Acosta. 1996. Relationships among

environment, spawning and settlement of Queen scallop in the Ri'a de
Arosa (Galicia. NW Spain). Aquacul. Inl. 4:225-236.

Soeinodihardjo, S. 1974. Aspects of the biology of Chlamys opercularis
(L.) (Bivalvia) with comparative notes of four allied species. PhD
thesis, University of Liverpool. 1 10 pp.

Taylor. A. C. & T. J. Venn. 1979. Seasonal variation in weight and bio-
chemical composition of the tissues of the queen scallop. Chlamys
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Jcnimal of Shellfish Resvanh. Vol. 21. No. 2. 585-590. 2002.

KARYOTYPE ANALYSIS AND CHROMOSOME BANDING OF THE CHILEAN-PERUVIAN
SCALLOP ARGOPECTEN PURPURATUS (LAMARCK. 1819)

GONZALO GAJARDO.* MARGARITA PARRAGUEZ, AND NELSON COLIHUEQUE

Laboratory of Genetics & Acjuacultiire. UniversiclacI tie Los Lagos. P. O. Box 933. Osorno. Chile

ABSTRACT The Chilean-Peruvian scallop. Argopecten purpitrahis. is the southernmost representative of the genus and has become
an aquaculture commodity in Chile as a result of 20 y of culture practice promotion after natural beds were almost exhausted by
overexploitation. Chromosome preparations of A. purpuratus D-larvae from three samples, two from commercial hatcheries, and one
from the few remaining wild populations were subjected to karyotype analysis by the usi al measurements of chromosome pairs,
Ag-NOR staining, as well as by the Hoechst 3325S/Actinomycin D counterstain and restriction endonuclease banding techniques. The
species has a modal diploid number of 2/) = 32, and the karyotype consists of 1 1 pairs of telocentric and t~ive pairs of subtelocentric
chromosomes. NF = 32. The banding techniques, attempted for the first time in the species, served as a good complement for
identifying a number of homologous chromosomes. The NOR was located terminally on the short arm and in the pericentromeric region
in three chromosome pairs. The Hoechst 33258/Actinomycin D counterstain revealed tiny fluorescent heterochromatic areas in the
centromeric region of chromosome pairs 1, 2, 4, 5. 6. 7, 8. 9. 11, 15, and 16, whereas a quenching zone was observed in chromosome
pair 6. Endonuclease restriction banding revealed pericentromeric and telomeric bands in one chromosome pair {Alu\) and interstitial
bands in other two chromosome pairs (WacIII). The karyotype and banding pattern provided in this work are likely to help an industry
seeking for improved stocks. Such information will he also valuable for further investigating both intra- and interspecific genomic
relationships in the Chilean Pectinids. where the number of species karyologically screened is still very limited.

KEY WORDS: Arf>opecleiu karyotype. Chilean scallop, genetics, banding

INTRODUCTION

Classic techniques tor chromosome analysis generally have
permitted accurate assessment of chromosome numbers and mor-
phology in a wide variety of aquatic invertebrate species (Thiriot-
Quievreux 1994). Yet the number of species investigated from the
cytogenetic point of view is rather scarce, i.e.. no more than 200 of
approximately 15.000 species in the Bivalvia class (Gonzalez-
Ti/()n et al. 2000). Recent molecular and banding techniques have
contributed to a better identification of whole or chromosome
parts, and hence to a more detailed characterization of the genome
organization in different organisms. Analysis of the distribution
and composition of heterochromatin and identification of the
nucleolar organizer regions iNORs) are among these advances
(Insua et al. 1998; Gonzalez-Tizon et al. 2000). In commercially
exploited species, these techniques have enabled the obtaining of
better karyotypes, although not fully standardized, allowing more
reliable intra- or interspecific comparisons of genetic resources
either for basic studies (evolutionary inferences) or for practical
purposes (taxonomy, chromosomal manipulations).

The Chilean-Peruvian scallop, Argopecten purpuratus. is a
functional hermaphrodite inhabiting sedimentary substrates in
sheltered bays along the Pacific Ocean from Paracas Bay. Peru
(13° South) to the Gulf of Arauco. Chile (37' South). The species
used to be a commercially important member of the subtidal
macro-invertebrate fauna along the northern part of the Chilean
coast until overfishing almost exhausted natural beds. Since 1982,
A. purpuratus has been farmed successfully and currently repre-
sents an important aquaculture product (Gajardo & Nunez 1992).
Because of its economic importance, the species has been trans-
planted to southern Chile, where it does not occur naturally. Von
Brand et al. ( 1990) ascribed it to the genus Argopecten instead of
Clilaiins as previously described based on the fact that both dip-
loid number (2n = 32) and chromosome types (4 m+ 14 m-Sm-t-
6St-i- 8t) were shared with other Pectinids. particularly Argopecten

irradians (Wada 1978). Instead, most Clilamys species studied so
far exhibit a diploid number of 2/; = 38 and few bi-armed chro-
mosomes (Komaru and Wada 1985).

A. purpuratus is replaced by other Chlamys species in the
southern Chilean coast, such as Chlamys patagonica. Chlamys
anumdi. and Chlamys vitrea. As part of a broader effort aimed at
the genetic characterization of Argopecten purpuratus. this study
seeks to provide a more reliable karyotype description and chro-
mosome pairing based on the chromosome index and banding
analysis of this important aquaculture commodity. The information
piovided is likely to help in future cytogenetic studies of intra- and
interspecific karyotype comparisons of Chilean scallops.

MATERIAL AND METHODS

Chromosome Preparations

Argopecten purpuratus D-larvae were obtained by artificial
hatching of ripe animals from three different sources. Two samples
were from commercial hatcheries. Cultivos Marines Internaciona-
les S.A (Caldera). northern Chile (24 South), and Germont S.A in
southern Chile (Calbuco; 51" South), whereas the Quintay sample
corresponds to animals collected in Laguna Verde (33° South).
This latter location is one of the few natural beds remaining in the
Central coast of Chile, close to Valparaiso.

TABLE 1.

Diploid chromosome number determination in three populations of
.4. purpuratus.

No. of
Cells

Chromosome Number

Locality Observed 25 26 27 28 29 3(1 31 32

Modal
2n

*Corresponding author. E-mail: ggajardo@ulagos.cl

Caldera

48

0 ]

1 3

4

3

7

5

23

32

Calbuco

49

1

1

5

8

11

23

32

Quintay

28

1

1

4

4

5

12

32

585

586

Gajarado et al.

TABLE 2.

Chromosome measurements in three samples of A. purpuratus. mean relative length, 9S'^e confidence interval (X ± ci). and standard
deviation ISD) of the short arm (SA) and long arm (I. A I. Chron\os(mie type according to the centromeric index iC'll.

Chromosome
No. of Pair

SA

LA

Length ± SD

(fim) T>

Locality

X ± ci (SD)

X ± ci (SD)

CI ± SD

pe

Caldera (« = 9)

1

1.64 ±04 7 (0.23)

7.75 ±0.41 (0.56

I7.47± 1.93

4.28 ±0.81 s

2

1.24 + 0.10(0.13)-'

6.37 ± 0.36 (0.48

16.26 ± 1.54

3.57 ± 0.83 s

3

1.15±0.08 (0.11)

5.60 + 0.28 (0.37

17.02 ± 1.37

3.10 ±0.63 s

4

1.10 ±0.08 (0.1 If

5.08 ±0.39 (0.52

17.87+1.74

2.88 ± 0.53 s

5

1.30 + 0.19(0.26)

4.66 ± 0.49 (0.65)

21.78 ±3.20

2.78 ±0.44 s

6

0.61 ±0.28 (0.37f

8.42 ± 0.30 (0.41

6.61 ±3.93

4.14 ±0.83 t

7

0.63 ± 0.20 (0.27)'-

7.24 ± 0.48 (0.65

8.02 ± 3.50

3.65 ±0.81 t

8

0.55 ±0.1 8 (0.24)"

5.70 ±0.35 (0.48

8.86 ±3.91

2.97 ±0.78 t

9

0.52 ± 0.22 (0.30)"

5.14 ±0.23 (0.31

9.11 ±5.20

2.64 ± 0.56 t

10

0,48 + 0.21 (0.28)"

4.71 +0.37 (0.50

9.45 ±5.44

2.40 ± 0.46 t

11

0.03 ±0.06 (0.08)

5.89 + 0.32 (0.43

0.45 ±1.34

2.80 ±0.76 I

12

0.05 + 0.10(0.14)

5.40 ±0.38 (0.51

0.87 ±2.61

2.57 ± 0.70 I

13

0.04 ±0.08 (0.11)

4.98 ±0.19 (0.26

0.69 ± 2.06

2.33 + 0.53 t

14

0.00 ±0.00 (0.00)

4.81 ±0.23(0.31

0.00 ± 0.00

2.26 ±0.57 t

15

0.00 ± 0.00 (0.00)

4.55 ± 0.29 (0.40

0.00 ± 0.00

2.15 + 0.57 t

16

0.04 + 0.10(0.13)

4.33 ±0.29 (0.39

1.02 ±3.07

2.07 ± 0.55 t

Calbuco {II = 10)

1

1.47 ±0.28 (0.39)

8.24 ±0.74 (1.05

15.08 ±3.11

5.01 ±1.84 s

2

1.28 + 0.27 (0.38)°

6.57 ±0.64 (0.91

16.23 ±4.09

4.02 ±1.. ^6 s

3

1.26 ±0.24 (0.34)

5.73 + 0.39 (0.56

17.96 + 4.37

3.64 ±1.36 s

4

1.13 ±0.22 (0.32)='

5. 18 ±0.29 (0.42

17.89 ±4.88

3. 30 ±1.23 s

5

1.00 + 0.31 (0.44)

4.75±0.83 (1.17

17.10±7.42

2.98 ±1.06 s

6

0.12 ±0.15 (0.22)"

8.07 + 0.68(0.97

1.46 + 2.67

4.34 ±1.85 t

7

0.10 ±0.15 (0.22)"

6.98 ± 0.62 (0.88

1.47 ±3.14

3.74 ±1.62 t

8

0.10 ±0.15 (0.21)"

6.30 ±0.51 (0.72

"■" 1.64 ±3.50

3.40 ±1.48 t

9

0.06 ±0.1 4 (0.1 9)"

6.07 ± 0.46 (0.65

" 1.08 + 3.41

3.27 ±1.41 t

10

0.06 ±0.1 2 (0.17)"

5.80 ± 0.30 (0.43

" 1.05 ±2.88

3.15+1.37 t

11

0.07 ±0.13 (0.19)

5.40 ±0..30 (0.43

1.33 ±3.71

2.92 ± 1 .23 t

i:

0.01 ±0.02 (0.03)

5.31 +0.24(0.34

0.19 ±0.59

2.86 ±1.25 t

13

0.00 ± 0.00 (0.00)

5.07 ±0.26 (0.37

0.00 ± 0.00

2,71 ±1,17 t

14

0.00 + 0.00 (0.00)

4.85 ± 0.26 (0.37

0.00 ± 0.00

2.58 ± 1 .07 t

15

0.00 ± 0.00 (0.00)

4.68 + 0.27(0.38

0.00 ± 0.00

2.50 ±1.06 t

16

0.01 ±0.02(0.03)

4.32 ± 0.24 (0.34

0.19 ±0.60

2.31 ±0.98 t

Quiiilay (/i = 5)

1

1.23+ 1.13 (1.13)

7.06 ±1.59 (1.60

14.69 ±13.99

1.99 ±0.42 s

t

2

1.81 ±0.20 (0.20)"

6.30 ±0.61 (0.61

22.47 ± 3.22

1.94 ±0.16 s

t

3

1.46 ±0.24 (0.24)

5.53 ±0,35 (0.35

20.84 ±2.91

1.68 + 0.15 s

t

4

1.61 +0.18 (0.18)"

4.85 ± 0.63 (0.64

25.08 ± 3.97

1 .55 ± 0. 1 2 s

t

5

1.28 ±0.28 (0.28)

4.66 ±0.72 (0.73

21.63 ±4.45

1.42 + 0.14 s

t

6

0.00 + 0.00(0.00)"

7.78 ± 1.09(1.09

0.00 ± 0.00

1.87 ±0.31 t

7

0.00 ± 0.00 (0.00)"

6.90 ±0.23 (0.23

0.00 ± 0.00

1.66±0.16 t

8

0.00 + 0.00 (0.00)"

6.57 ±0.10 (0.10

" 0.00 + 0.00

1.58 ±0.12 t

9

0.00 ± 0.00 (0.00)"

6.19±0.18(0.18

" 0.00 ± 0.00

1.49±0.13 t

10

0.00 ± 0.00 (0.00)"

5.95 ±0.18 (0.18

" 0.00 + 0.00

1.43 ±0.14 t

11

0.00 + 0.00 (0.00)

5.71 ±0.11 (0.11

0.00 ± 0.00

1.37 ±0.10 t

12

0.07 ±0.16 (0.16)

5.51 ±0.22 (0.22

1.17 + 2.61

1 .34 ± 0. 1 1 I

13

0.18 ±0.40 (0.40)

5.30 ±0.22 (0.23

2.82 ± 6.30

1.31 ±0.15 t

14

0.00 ± 0.00 (0.00)

5.18 ±0.22 (0.22

0.00 ± 0.00

1.24 ±0.09 t

15

0.00 + 0.00 (0.00)

4.69 ±0.1 5 (0.15

0.(K) ± 0.00

1.13±0.10 1

16

0.00 ± 0.00 (0.00)

4.16±0..34(0.35

0.00 ± 0.00

1 .00 + 0. 1 2 t

Values with a different letter are significantly different (Tukey, P < 0.05).

The splash method was developed in our laboratory for D-
larvae (24— 1-8 h after hatch) according to the following protocol:
initial colchicine (0.05%) treatment of a pool of D-larvae from
each locality in sea water for 2 h. then mechanical disruption of
cells, including centrifugation at 1000 rpm/10 min, hypotonic
treatment in distilled/ seawater (1:1), washing to eliminate hypo-
tonic solution (centrifuge at 800 rpm/10 min). fixation in metha-

nol-acetic acid (3:1 ) for 20 min. and washing (centrifuging twice
at 800 rpm/ 10 min). The pellet was re-suspended in 0.5 mL of
cold fixative methanol/acetic acid (3:1) and a drop splashed onto
clean slides and stained in 4% Giemsa. For diploid number deter-
minations, between 28 and 49 of the best metaphase plates (with-
out overlapping) were studied. These were then photographed on
a Nikon Labophot microscope (with epitluorescence ) for chromo-

Karyotype and Banding Pattern of A.

PURPURATUS

587

St

2n =
NF =

32
32

^'^

1

/A

3

6

7

8

1-1

ie

13

Afi

■♦..\

/.'I O' ^*

some counting. In all populations, between 5 and 10 suitable cells
were used for the estimation of the centromeric index (CI) follow-
ing the protocol of Levan et al. (1964). Chromosome measure-
ments were obtained and mean and standard deviation for CI were
calculated for each chromosome. Total chromosome relative
length (expressed as percentage) and the relative length of short
(SA) and long arms (LA) over the total length of haploid comple-
ment were also obtained. Chromosome differences within and be-
tween karyotypes were evaluated from karyo-ideograms elabo-
rated on SA and LA relative lengths (Spotomo 1985). Confidence
intervals were established for SA and LA for each chromosome
pair to assess the significance between comparisons. For chromo-
some arm number (NF) determinations, subtelocentric and acro-
centric chromosomes were considered uni-armed according to
Matthey ( 1945). This karyotype descriptor (NF). which only con-
siders the main arm of a chromosome, is quite reliable as very
often the size of the short arm of a subtelocentric chromosome is
variable due to technical artefacts during spread preparations.

Figure \. Karytitypv »( Argopeiteii piirpiiratiis. 2ii
represents 10 microns.

: 32; NF = 32. Bar Banding Methods

Howell and Black's (1980) silver-NOR staining method was
used to identify NOR-bearing chromosomes. The counterstain-

o
-I

Figure 2. Karyo-idiogram for three populations of A. purpuratus. Diagonal lines separate chromosome morphologies in metacentrics (m),
submetacentrics (sm), subtelocentrics (st), and telocentrics (t). Dotted lines show those chromosome pairs (identified by numbers) of the studied
population that are easily distinguished from the rest. FZach point in the karyo-idiogram represents a mean value (in percent! of the relative
length of the short and the long arm of 16 homologous pairs. Bars represent 95 '7f confidence intervals of the mean for short and long arms.

588

Gajarado et al.

enhanced chromosome banding technique (Hoechst 33258/
Actinomycin D; Schweizer 1981 ) was used to identify heterochro-
matic regions. Metaphase plates were photographed using a Nikon
epifluorescence microscope (filter block UV-2) with a Kodak T-
Max 100 ASA film.

For restriction endonuclease (RE) banding, RE enzymes sus-
pended in the appropriate buffer were added to one drop of an
air-dried cell suspension and covered with a coverslip. Enzyme
concentration and digestion time depended on each endonuclease
type, as follows: for EcoRl. 0.5 U/[jlL for 5 h: for B((//)HI. 0.5
U/jiL for 8 h; for Akil. 0.3 U/fJiL for 7 h: and for «(«'III. 1 .7 U/|jiL
for 5 h. Slides were incubated in a moisture chamber al 37°C,
washed in distilled water, and stained in 4% Giemsa solution for
10 min.

RESULTS

The modal diploid number of 32 was established for all three A.
purpiimtiis populations screened (Table 1 ). The CI revealed that
all karyotypes had similar chromosomal constitutions (5 pairs of
subtelocentrics and 1 I pairs of telocentrics (Table 2 and Fig. 1 ).
The karyo- ideogram showed that various chromosome pairs could
be easily identified because of size differences (pairs 1 to 7).
Interpopulational chromosome differences were evident, particu-
larly for the short arm (ANOVA. P < 0.05) for chromosome pairs
2, 4, 6. 7, 8, 9, and 10 (Table 2 and Fig. 2).

The Ag-NOR staining revealed three pairs of NOR-bearing
chromosomes (Fig. 3) with NOR location varying, i.e.. it was
pericentric in the long arm of one pair and telomeric in the short
arm of two other pairs (see Fig. 3). The number of NOR-bearing
chromosomes was in agreement with the number of nucleoli ob-
served in the interphase nucleus (3 to 6. Fig. 3b).

The Hoechst/Actinomicin D counterstain (Fig. 4) revealed a
tiny positive fluorescent heterochromatic block in the centromeric

Ag-NOR

Metapl

Metap2

Metap4

r/ldtapS

dmjk. IVIetapl

&

— » •
•

»

Poirl PolrJ PoW

^A V.-O ".'1

•- l^ \-

l- y 1-

U r ((f

If <- a

area of chromosome pairs 1,2, 4 to 9, II, 15, and 16 (Fig. 4). This
stain produced a quenching zone in the middle of the long arm of
pair 6. which corresponded to an A-T low-content chromatin se-
quence, according to the pair specificity of the Hoechst 33258.
Figure 5 shows the pattern after restriction endonuclease digestion.
Only two REs, Alid and HcieUl. produced a banding pattern in
such a way that pericentric and telomeric bands were evident in
one chromosome pair (A/id) and interstitial bands in two pairs

DISCUSSION

This work confirms the chromosome number of 2/; = 32 re-
ported for A. purpwatus (Von Brand et al. 1990; Canello et al.
1992: Alvarez-Sarret & Lozada 1992: Winkler et al. 1993). How-
ever, the chromosome formulae obtained (5 st -I- 1 1 t chromosome
pairs) differs from that reported by Von Brand et al. ( 1990), who
indicated the presence of 2 m pairs, 7 m-sm pairs, 3 st pairs, and
4 t pairs in the karyotype. Such a difference is likely to be pro-
duced by the fact that in the latter study the karyotype was tenta-
tively arranged using the size criteria without the chromosome
measurements required to determine the centromeric position. Our
results consistently show, in all three samples that were analyzed,
a chromosome complement of sub-telocentric chromosome types
(see Fig. 2). A. piirpiiratus. along with other related species, such
as A. irradians inadians (Wada 1978) and C. nohilis (Komaru and
Wada 1985), are among the few Pectinids sharing a diploid num-
ber of 32. Instead, the majority of species studied (i.e., genus
Clilamys. Pecten. Patiuopeclen. and Placopeclen) exhibit In = 38
(Thiriot-Quievreux 1994: Insua et al. 1998), with Aequipecten
opcirularis [2ii = 26) being the exception (Beaumont & Gruffydd
1974).

Molluscs and crustaceans are among the most primitive groups
considering those karyologically compared by Thiriot-Quievreux
( 1994). Nevertheless, a typical diploid number does not emerge in
most groups, Ostreidae being a special case in which a diploid
number of 2;; = 20 is common and very likely the ancestral one.
Some marine species frequently exhibit greater numbers of chro-
mosome than those of freshwater species, although in some cases,
for example, within the bivalvia, both an increase and decrease are
evident. The diploid number in most animal species ranges widely,
between 12 and 40 (White 1978). Whereas evolution to higher
chromosome numbers in certain aquatic organisms is often paral-
leled by an increase of subtelocentric and telocentric chromo-
somes, probably by fission of chromosomes (see review by

- -1 $§ «* ^^

^ 2 3 4 i

*^*t^ ^f) |< h0

t« n u

Figure .^. .Xg-NOR staining, (a) Metaphase plate showing six NOR-
bearing chromosomes. Below. NOR-bearing chromosomes obtained
from different metaphase plates, (b) Resting nuclei showing 3 to 5
nucleolus.

Figure 4. Hoescht .V'258/ Actinomycin I) counterstain. Intense lluo-
rescent bands are indicated by small arrows. Large arrows depict a
less lluorescent area in chromosome pair 6.

Karyotype and Banding Pattern of A. purpuratus

589

b
EcoRI

?

.v^r

Metap.2

Metap.5

■i

?>^

Metap.2

Metap.3

Metap4

ll

8-

Metap.7
MetafxS
Melapd —

Figure 4. RE digestion: lal BaiiiHl (no digestion): (bl EcoRl (no di-
gestion); (c) Aliil (+ digestion), photo depicts a pericentronieric band
in one pair of subtelocentric chromosome (arrows): (d) HaelU (+ di-
gestion) depicts an interstitial band in two chromosomes. Below,
banded chromosomes obtained from difTerenI metaphase plates.

Thiriot-Quievreux. 1994: 379). Such phenomenon is hkely to be
related with speciuli/ution or adaptation to a certain type of marine
environment. As mentioned previously, the diploid number in
Pectinids varies between 26 and 38; hence, the chromosome
formulae observed for A. purpuratus could be considered a de-
rivative state within the group. Although it is debatable whether
such a trend could yet be inferred in a group with few species
exhibiting standardized, or comparable, karyotypes, it is worth
mentioning that A. purpuratus is one of the southernmost Ar-
gopecten representatives. Indeed, this group originated in the tropi-
cal/subtropical region of the Caribbean and Atlantic (Von Brand et
al. 1990). A. purpuratus along with A. opercularis (Beaumont &
Gruffydd 1974) are among the few Pectinids of 14 species karyo-
typed so far with 2/i = 32 and 2n = 26. respectively (see Insua
et al. 1998).

Our results (2n and NF) and similar information from other
species within the group (reviewed by Mendez et al. 2000). sug-
gest that NF (Matthey 1945) is a valuable de.scriptor for species
comparisons and to evaluating factors affecting chromosome
shape variations such as chromosome rearrangements (White
1978). Because of technical procedures, the size of the short arm
can vary widely and this can be deduced froir) the deviation ob-
served between the average values of the centromeric index (CI ) in
Table 2 as well as from the presence or absence of short arms in
the telocentric chromosomes.

The combination of classic and banding techniques used in this
work allowed a better description of the karyotype and a more
reliable pairing of certain homologous chromosomes. Reproduc-
ible banding patterns are still uncommon in aquatic invertebrates;
hence, chromosome markers described in this work, for the first
time in A. purpuratus, are considered valuable for an improved and
reliable characterization of the species karyotype and its variations.
Three NOR-bearing chromosome pairs were preliminary identified
in the karyotype with the NOR regions, varying its location in the
chromosomes (Fig. 3). Further analysis of these chromosome re-
gions (using chromomycin A3 and a rDNA probe) will be required
to clarify a probable polymorphism for both number, position, or
NOR size in A. purpuratus. The three NOR-bearing chromosome
pairs observed in this work are within the range reported for Pte-
riomorphia subclass (1—4 pairs; Martinez-Lage et al. 1997; Insua et
al. 2000). The Ag-NOR number reported for A. purpuratus is
higher in comparison with A. opercularis. which shows only one
pair (Insua et al. 1998).

The combined use of fluorescent dyes and RE digestion al-
lowed the detection of different kinds of chromatin in A. purpu-
ratus. suggesting specific DNA composition of certain chromo-
some sectors. The Hoechst 32258/Actinomycin D counterstain
highlights heterochromatic areas in humans (Schweizer 1981) and
lower vertebrates (Colihueque et al. 2001 ). Although we observed
highly fluorescent areas in the A. purpuratus karyotype, these were
restricted to the cent)omeric regions of few chromosomes, indi-
cating that heterochromatin is not common in this species (see
Fig. 4). However, the differential response to digestion with re-
striction enzymes is also evidence of chromatin differences in A.
purpuratus chromosomes. For example, the positive detection (no
bands), at least preliminarily, of sites for Alu I (AGACT) and
HaeWl (GCiCC; see Fig. 5) indicates that these target sequences
should be highly repeated for the digestion to be evident (Sanchez
et al. 1991). It should be stressed, however, that heterochromatic
areas are few and mainly restricted to the centromeric areas as
revealed by the fluorescent banding. Digestion with RE of fixed
metaphase plates is not so common in invertebrates, with the
exception of Drosopliila (Mezzanotte 1986). Therefore, the results
of this work confirm the finding of Martinez-Lage et al. (1994),
who indicated that the digestion of bivalve chromatin produces
specific chromosome bands (Fig. 5). However, the pattern of di-
gestion is simple and restricted to few chromosomes similar to
what is observed in fishes (Sanchez et al. 1991) and mammals
(Burkholder 1989).

The fanning of the Chilean scallop was greatly promoted by the
fact that natural beds were almost depleted. Nowadays, a bloom-
ing, fast-growing industry is still far from realizing its potential
because of the limited, or unreliable, production of quality seeds
(Gajardo & Niiinez 1992). This is in part related with proper brood-
stock management. By providing a better karyological description
(quantitative measurements and banding pattern of chromosomes),
this work offers a reliable and comparable way to evaluate the
chromosome formulae of A. purpuratus and hence, a proper way
for comparing probable differences among the few wild popula-
tions remaining, or hatchery stocks, if any can be identified yet.
This is expected to assist to the management, improvement and
conservation of this economically valuable resource. At the same
time, it will help in the understanding of the processes affecting
chromosome evolution in this species as well as in the bivalvia
class.

590

Gajarado et al.

ACKNOWLEDGMENTS

Support from the European Union, INCO-DC project (contract
no. IC18-CT97-0188) is greatly appreciated. We are indebted to

Dr. Josefina Mendez, University Da Coruiia, Spain, for sugges-
tions and valuable literature. Two anonymous reviewers made con-
structive comments to improve the original manuscript.

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somic study in Chilean scaWop Argopecteii purpuranis (Lamarck 1819)
(Bivalvia, Pectinidae). Invest. Pesqueras 37:119-126.

Beaumont. A. R. & L. L. L. Gruffydd. 1974. Studies on the chromosomes
of the scallop Peclen maximii.s (L.l and related species. J. Mar. Biol.
A.«. U. K. 54:713-718.

Burkholder. G. D. 1989. Morphological and biochemical effects of endo-
nucleases on isolated mammalian chromosomes in vitro. Cliromosontii
97:347-355.

Canello. F., L. Paredes & J. Toro. 1992. Induccion a la triploidfa en el
oslion del norte Argopecten purpuratus. por medio de shock termico de
calor. Invest. Pesqueras. 37:5-1 1.

Colihueque. N., P. Iturra. F. Estay & N. Diaz. 2001. Diploid chromosome
number variations and sex chromosome polymorphism in five cultured
strains of rainbow trout i^Oncorhynchus inykiss). Aqiiaciiltiire 198:63-
77.

Gajardo, G. & J. Nufiez. 1992. Seed production, the critical factor for
mollusc farming in Chile. World Aquaculture 24:72-77.

Gonzalez-Tizon, A. M.. A. Marti'nez-Lage. I. Rego. J. Ausio & J. Mendez.
20fJ0. DNA content, karyotypes, and chromosomal location of 18S-
5.8S-28S ribosomal loci in some species of bivalve molluscs from the
Pacific Canadian coast. Genome 43:1065-1072.

Howell, W. M. & D. A. Black. 1980. Controlled silver staining of nucleo-
lus organizer regions with a protective colloidal developer: a I step
method. E.xperientia 36:1014-1015.

Insua. A.. M. J. Lopez-Pifion & J. Mendez. 1998. Characterization of
Aeqttipeclen opercularis (Bivalvia: Pectinidae) chromosomes by dif-
ferent staining techniques and fluorescent in situ hybridization. Genes
Genet. Sy.st. 73:193-200.

Insua, A., R. Freire & J. Rios. 2000. Localizacion cromosiimica y patrones
de variacidn del DNA ribosomico en moluscos bivalvos. In: J. Mendez,
editor. Los moluscos bivalvos; aspectos citogeneticos, moleculares y
aplicados. Spain: Universidade Da Corufia, pp. 77-99.

Komaru, A. & K. T. Wada. 1985. Karyotypes of four species in the Pec-
tinidae (Bivalvia: Pteriomorphia). Venus 44:249-259.

Levan. A. K., K. Fredga & A. A. Sandberg. 1964. Nomenclature for
centromeric position on chromosomes. Hereditas 52:201-220.

Martinez-Lage. A.. A. Gonzalez-Tizon & J. Mendez. 1994. Characteriza-
tion of different chromatin types in Mytilus galloprovincialis L. after
C-banding. tTuorochromc and restriction endonuclease treatment. He-
redity 72:242-249.

Marti'nez-Lage. A.. A. Gonzalez-Tizon, J. Ausio & J. Mendez. 1997.
Karyotype and Ag-NORs of the mussels Mytilus californiunus and M.
trossiilus from the Pacific Canadian coast. Aquaculture 153:239-249.

Matthey. R. 1945. L' evolution de la forniule chromosomiale chez les
vertebres. Experientia l:5(.)-56.

Mendez. J.. A. Insua & J. Lopez. 2000. Caracterizacion citogenetica en
moluscos bivalvos. In: J. Mendez. editor. Los muluscos bivalvos: as-
pectos citogeneticos, moleculares y aplicados. Spain: Universidade Da
Coruiia, pp. 15—48.

Mezzanotte, R. 1986. The selective digestion of polytene and mitotic en-
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Sanchez, L.. P. Marti'nez. C. Baeza & A. Vinas. 1991. Chromosomal het-
erochromatin differentiation in Salino iriittci with restriction en/ymes.
Heredity 6&.24\-249.

Schweizer. D. 1981. Counlerstain-enhanced chromosome banding. Hum.
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Spotorno. A. E. 1985. Conceptos y metodos en cariologia descripliva y
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Thiriot-Quievreux. C. 1994. Advances in cytogenetics of aquatic organ-
isms. In: A. R. Beaumont, editor. Genetics and evolution of aquatic
organisms. London: Chapman & Hall, pp. 369-388.

Von Brand. E.. G. Bellolio & K. Lohrmann. 1990. Chromosome number of
the Chilean scallop Argopecten puipuratns. Tohoku J. Agr. Res. 40:
91-95.

Wada. K. 1978. Chromosome karyotypes of three bivalves: the oysters,
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gopecten irradians irradians. Biol. Bull. 155:235-245.

Winkler. F., B. Ladron de Guevara, B. Estevez & L. Jollan. 1993. Induc-
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455 pp.

Jounuil ot Shellfish Research. Vol. 21. No. 2. 591-5y6. 2002.

MOLECULAR CLONING AND CHARACTERIZATION OF A FRUCTOSE- 1,6-BIPHOSPH ATE
ALDOLASE cDNA FROM THE DEEP-SEA SCALLOP PLACOPECTEN MAGELLANICUS

MOHSIN U. PATWARY,'* AKELIA WAUCHOPE,' TIMOTHY W. SHORT,^ AND
EDWARD J. CATAPANE'

^Departmeui of Biology Medgar Evers College of the City University of New York 1 150 Carroll Street
Brookhn. New York 11225: "Queens College and the Graduate Center of the City University of New
York. 65-30 Kissena Boulevard. Flushing. New York 1 1367

ABSTRACT The deep-sea scallop Phicopeclen magellanicus is an important member of commercial fisheries along the coast of
Northeastern United States and Atlantic Canada. A cDNA encoding a glycolytic pathway enzyme fructose- 1 ,6-biphosphate aldolase
was isolated from a sea scallop adductor muscle-specific cDNA library and sequenced from both directions. The full-length cDNA is
a 1627 base-pair (bp) long sequence that has a 62 bp 5' untranslated region, a 1092 bp open-reading frame, and a 47.^ bp 3' untranslated
region including a 24 bp polyA tail. The open-reading frame encodes a .^9.,^ kDa protein with 36.3 amino acids. The protein has 183
nonpolar, 94 polar uncharged, and 86 polar-charged amino acids. Several amino acids show bias for codons with G/C at their third
position. The cDNA has 28 unique restriction sites, including common restriction enzymes such as Sod, B<;mHl, 7<i</I, and B.v/EII. The
aldolase is a highly conserved protein with 66% sequence identity with that of Schistosoma mansoni, 65% with that of Drosophita
melaiwgaster. 62% with that of Homo sapiens, and 57% with that of Oryza sativa. Southern blot analysis indicates that aldolase in sea
scallops belongs to a family with 4 to 10 putative genes. Northern blot analysis shows that this gene is expressed only in adductor
muscles. Hybridization of an aldolase cDNA probe to genomic DNA from several individuals revealed restriction fragment length
polymorphisms at several loci, indicating potential use of this cDNA as a marker in genetic studies of sea scallops.

KEY WORDS: aldolase. cDNA, sea scallops, restriction fragment length polymorphisms, codon bias

INTRODUCTION

Fructose- 1 ,6-biphosphate (FBP) aldolase, technically known as
D-glyceraldehyde-3-phosphate lyase (EC 4.1,2.131, is a long-lived,
ubiquitous, glycolytic pathway enzyme. In glucose metabolism, it
catalyzes cleavage of FBP into two 3-carbon molecules, glyceral-
dehyde 3-phosphate and dihydroxyacetone phosphate (DHAP),
and in gluconeogenesis, it catalyzes the reaction in reverse order.
This enzyme is also involved in gluconeogenesis and in the Calvin
Cycle in plants. In the Calvin Cycle of higher plants, aldolases
were reported to be bifunctional (Flechner et al. 1999) because
they catalyze both the reversible condensation of DHAP to glyc-
eraldehyde 3-phosphate and DHAP and erythrose-4-phosphate to
sedoheptulose-1.7-biphosphate. Cytosolic aldolase in strawberry
was found to be related to fruit ripening (Schwab et al. 2001 ).

The aldolases are distinguished as two classes based on their
two distinct mechanisms of catalyzing glycolytic reactions, re-
quirement of divalent cations in catalysis, subunit structures. pH
optima, and substrate affinity (Rutter 1964). The two classes of
aldolases share very little in their sequence homology (Alefounder
et al. 1989. Plaumann et al. 1997) and are considered to be inde-
pendent evolutionary lineages (Flechner et al. 1999). The class I
enzymes are tetramers of 40 kDa identical subunits. each with an
active site (Lys 229) located in the core of the enzyme (Lai et al.
1974. Samson et al. 1997). These enzymes have the same molecu-
lar weight and subunit structure and catalyze the overall reaction
through Schiff base formation with ketoses of sugar substrates and
can be inhibited by borohydride reagents (Sawyer et al. 1988).
Although they are prevalent in animals and higher plants, they also
occur in green algae and a few prokaryotes grown under au-
totrophic conditions (Sauve & Sygusch 2001), The class II en-
zymes are usually dimers with certain exceptions where they are
tetramers, and are found in bacteria, yeast as well as other fungi

'Corresponding author. E-mail: mohsin@mec.ciiny.edu

and some green algae grown under heterotrophic conditions (Rut-
ter 1964, Plaumann et al. 1997). These aldolases are EDTA .sen-
sitive, stabilize the enol intermediate of the reaction by using a
divalent cation, usually Zn"* or Fe"*, and their activities are en-
hanced by monovalent cations.

In vertebrates, there are three classes of class I enzymes: aldo-
lase A (isolated from muscle), aldolase B (isolated from liver), and
aldolase C (isolated from brain). This work deals with the class I A
group. The classes have immunologic differences, different kinet-
ics, different chromosomal locations, and different gene se-
quences. Some class I enzymes are expressed only in specific
tissues. For example, aldolase A is present in muscle and red blood
cells; aldolase B in the liver, kidneys, and small intestine; and
aldolase C in neuronal tissues (Penhoet et al. 1966, Penhoet et al.
1969. Tolan et al, 1987). These enzymes were reported to be three
distinct proteins, yet are structurally closely related. Their func-
tions are different, but homologous. In invertebrates, so far no such
tissue specificity of an aldolase gene has been reported.

The sea scallops are an important fishery in the Northeastern
coastal regions of the US and in Atlantic Canada (Black et al,
1993). This species has been the subject of several genetic studies
in recent years (Volckaert & Zouros 1989, Patwary et al. 1994a.
Patwary et al. 1994b. Patwary et al. 1996. Patwary et al. 1999,
Pogson 1994, Pogson & Zouros 1994, Gjetvaj et al. 1997). As a
part of our continuing effort to characterize cDNAs in this species,
we report here a first bivalve full-length FBP aldolase cDNA and
show that it is potentially useful as a probe in genetic studies of sea
scallop and other related species. The cDNA information would
also contribute to enhancing the understanding of structure-
function relationships of aldolase genes in bivalves.

MATERIALS AND METHODS

Sea scallops were obtained from commercial beds in Nova
Scotia and from the Marine Biology Supply Center. Woods Hole.
MA. The methods of DNA extraction and cDNA library construc-
tion are as described by Patwary et al. (1996),

591

592

Patwary et al

Isolatiim of FBP Aldolase cDNA Clones

As described eariier (Patwary et al. 1999), a small portion of
adductor muscle-specit'ic Uni-ZAP XR cDNA library was plated
and 130 plaques were randomly cored and stored at 4"C in SM
buffer. Bluescript clones containing cDNA inserts were rescued
according to the supplier's protocol (Stratagene. La Jolla, CA).
inserts were partially sequenced from the 5' end. and one of these
clones was identified as a truncated aldolase cDNA. An additional
150 plaques were randomly cored from a set of new plates and
stored in SM buffer. The cDNA inserts from each of these recom-
binant Uni-ZAP XR lambda clones were separately amplified (Pat-
wary et al. 1996) by polymerase chain reaction (PCR) using SK
and KS primers (Stratagene), the PCR products were fractionated
in agarose gels, and the they were transferred onto positively
charged nylon membranes using standard protocols (Sambrook et
al. 1989). The blots were hybridi/ed with aldolase cDNA labeled
with "P-dCTP as previously described (Patwary et al. 1999). ex-
posed to X-ray films, and two positive clones were identified.
These two cDNA clones. PmC 82 and PmC 83. were rescued into
pBluescript from their recombinant uni-ZAP XR lambda \ectors
and sequenced completely from both directions. The 5' truncated
cDNA sequence end is obtained using a 5 'RACE system version
2.0 (Invitrogen Corporation, Chicago, IL).

Preparation of RNA and Northern Blotting

Total RNA for northern blotting was extracted separately from
several tissues pooled from at least six animals of both sex. The
collected tissues were immediately fixed in RNAIu/cr^'^' (Ambion
Inc. Austin. TX) and stored at -20''C. Each pooled sample was
ground in a separate baked mortar using a baked pestle. RNAs
were extracted according to the protocol that came with Ambion's
Totally"" RNA Kit. After quantification. RNA samples were
precipitated by adding salt and 95% ethyl alcohol and stored at
-180°C. The quality of RNA samples was checked in a formal-
dehyde gel containing ethidium bromide at the time of Northern
analysis. Northern blotting, preparation of '"^P probe, prehybrid-
ization, and hybridization were performed as described in Patwary
et al. (1999).

Preparation of Prohe for Restriction Fragment Length Polymorphism
(RFLP) Detection

To detect polymorphisms among sea scallops, the entire coding
region of FBP aldolase cDNA was used as a probe. The cDNA was
labeled with alkali-labile Dig- 1 1-dUTP by PCR. The 25 [x. PCR
reaction was prepared on ice. The final concentration of PCR
components were as follows: Taq polymerase buffer Ix; MgCU
1.5 mM; dATP, dCTP, dGTP 100 |jlM each; dTTP 84 |jlM; dig-
dUTP 14 fjiM; nested primers Hanking coding regions 20 ng each;
plasmids with cDNA insert 1 ng; and Tuij DNA polymera.se lU. A
Gene-Amp 9600 thermocycler (Perkin Elmer, Norwalk, CT) was
used to perform PCR reactions. The thermocycler was pro-
grammed for 31 cycles. Cycle I was at 94°C for 4 min, followed
by 29 cycles each of 94°C for 30 .sec, 55°C for 30 sec, and 72°C
for 2 min and the final cycle of 94°C for 30 sec. 55°C for 30 sec,
and 72°C for 5 min, A small portion of the labeled PCR and
unlabeled PCR (control) products were subjected to an agarose gel
electrophoresis and the slower mobility of labeled PCR products in
comparison to unlabeled control confirmed successful labeling.
The probe was cleaned using nick columns (Pharmacia Biotech,
Upsala, Sweden) and quantified by comparing hybridization with
known dig-labeled DNA on a dot blot.

Preparation of Genomic Blots

Ten micrograms of each DNA sample was digested to comple-
tion with 40 units of appropriate restriction enzymes at 37°C.
Spermidine to a final concentration of 5 mM was included in each
reaction to improve digestion. A small quantity of each sample was
fractionated in a mini agarose gel to verify the extent of digestion.
The digested DNA samples were fractionated in 0.8% regular
molecular biology grade agarose gels in TAE (0.04 M Tris-acetate,
0.0001 M EDTA) buffer together with dig-labeled molecular
weight marker III (Boehringer Mannheim). The digested DNA
samples were vacuum transferred to positively charged nylon
membranes. DNA blots were rinsed in TAE buffer, air dried, and
baked at 80°C for two hours before hybridization with the probe.

The blots were prehybridized in an oven at 40°C for three to
four hours in hybridization buffer containing deionized formamide
(50%), sodium citrate (SSC) and sodium chloride (5x each) N-
lauroylsarcosine (0.1%), SDS (0.02%), blocking reagent (2%), and
denatured yeast RNA (100 |xg/mL). Hybridization with the probe
was performed for 18 to 24 h in fresh buffer containing 10 ng/niL
denatured probe. Blots were washed twice in 2\ SSC, 0.1% SDS
for 10 min, once in 0.5x SSC, 0.1% SDS for 15 min, and once or
twice in 0.2x SSC, 0.1%. SDS for 20 min. Detection of labeling
was done according to the Boehringer Mannheim protocol.

RESULTS AND DISCUSSION

As a part of our ongoing effort to identify cDNAs that have
potential as probes in genetic studies of sea scallops, over one
hundred randomly selected phagemid clones with cDNA inserts
were partially sequenced from the 5' end. Through an amino acid
sequence homology search (BLAST P) one of these clones was
identified as the cDNA encoding FBP-aldolase. This cDNA se-
quence was highly truncated at the 5' end and it was used as a
probe to screen additional Uni-ZAP XR clones from the adductor
muscle-specific cDNA library. Two clones designated PmC 82 and
PmC 83 were isolated through this screening process. PmC 82 was
determined to have the entire open leading frame (ORF). Clone
PmC 83, although it is bigger than the first clone, is still shorter by
several bases in the 5' coding region. The clone PiriC 82 is 1546
bp long excluding the polyA tail. It has only five base pairs in its
5' untranslated region, a 1092 bp ORF and a 449 bp 3' untranslated
region. Figure 1 shows a full length cDNA that includes additional
57 bp at 5' end obtained by using the RACE system. The ORF
encodes 363 amino acid residues with a predicted molecular mass
of 39, 343 Da. These amino acid residues are 183 nonpolar. 94
polar uncharged, and 86 polar charged. There are 45 alanine and 3
tryptophan residues in the sequence, and these two are the most
heavily represented and least represented amino acids, respectively
(Table 1).

The G/C content for the coding region of the cDNA is 57.7%,
and the distribution of these two nucleotides in the gene at first,
second, and third positions are 81%, 43%, and 71.4%. respec-
tively. A similar distribution was also observed in the aldolase
gene of Thennus aqualiciis (Sauve & Sygusch 2001). The high
G/C content at the third position of the codon tend to be higher
than the overall G/C content in exons of many genes of other
organisms including humans. There is a considerable bias in
codon-usage for a number of amino acids in FBP aldolase gene.
This bias is particularly prominent in the usage of codons for
leucine, isoleucine, valine, glutamine. asparagine, lysine, and as-
partic acid (Table 1 ). For example. 25 of the 31 leucine residues
arc encoded by two codons (CUC, CUG), although there are four

FBP Aldolase cDNA in Sea Scallops 593

1 cgtgtaggcagtccttccccagaagcatccagacgcatttgtgatccagcgaaagaaccaag ATG CCA ACC TTT 74
1 M P T F 4

75 CCA CAG TAG CTG AGT CCA GAG AAG GAG GAG GAG CTC CGA AAC ATT GCC AAC GCC ATA GTA 13 4
5PQYLSPEKEEELRNIANAIV24

13 5 GCT CCT GGC AAG GGG ATC CTA GCC GCT GAC GAG TCA ACA GGT TCA GTT GGG AAG AGG TTC 194
21APGKGILAADESTGSVGKRF44

195 GCC CCC ATC AAA GTA GAG AAC ACG GAG GAG AAT CGT CGC CGG TAG AGA GAA CTG CTG TTC 254
31APIKVENTEENRRRYRELLF64

255 ACC ACC GAC AAC GCT ATC TCC GAA AAC ATC AGC GGC GTC ATC ATG TTC CAC GAA ACG TTT 314
TTDNAISENISGVIMFHETF84

315 TAG CAG AAG ACG GCC GAC GGC GTG CCC TTC ACG AAG GTT CTC CAG GAC AAG AAC ATC ATT 3 74
YQKTADGVPFTKVLQDKNI I 104

3 75 CCA GGC ATC AAG GTG GAC AAG GGT GTC GTA CCA CTG ATG GGC ACG GAC AAC GAA TGT ACC 4 34

PGI KVDKGVVPLMGTDNECT 124

435 ACA CAG GGT CTG GAT GGC CTG AGT GAG AGG TGT GCC CAG TAG AAG AAA GAT GGC GCC CAG 4 94
TQGLDGLSERCAQYKKDGAQ 144

4 95 TTT GCC AAA TGG AGG TGC GTG CTA AAG ATC CAG AAG GAG ACG CCG TCC TAC CAG GCC ATG 554

FAKWRCVLKIQKETPSYQAM164

555 CTG GAG AAC GCT AAC GTC CTC GCC CGC TAC GCC AGT ATC TGT CAA CAG AAT GGC CTG GTG 614
LENANVLARYASICQQNGLV 184

615 CCC ATC GTC GAG CCA GAG GTC CTC CCC GAC GGT GAA CAT GAT CTG GAC ACT GCA GAA AAA 674
PIVEPEVLPDGEHDLDTAEK 204

675 GCT ACA GAA CAG GTG CTT GCG TTT ACC TAC AAG GCT TTG GCC GAC CAC CAC GTG TTC CTA 734
ATEQVLAFTYKALADHHVFL 224

73 5 GAA GGG ACC CTC CTC AAA CCC AAC ATG ATC ACG GCC GGT ATG TCG TGT AGT AAG AGG GGC 7 94
EGTLLKPNMITAGMSCSKRG 244

795 ACG CCT GCC GAG AAT GCC CGC GCG ACA GTG CTC TGC CTC AGT AGG ACC GTC CCT CCC GCA 854
TPAENARATVLCLSRTVPPA 264

855 GTC GCC GGT GTG ACG TTC CTG TCC GGT GGT CAG TCA GAG GAA GAC GCG TCC ATC AAC CTC 914
VAGVTFLSGGQSEEDASINL 284

915 AAC GCC ATC AAC ACA GAC TCC GGC CGC AAA CCC TGG CCG CTG ACC TTC TCC TTC GGC CGA 974
NAINTDSGRKPWPLTFSFGR 304

97 5 GCG CTC CAG GCC AGC GTA CTC AAA ATT TGG CAG GGC AAG GAC GAA AAT GTG GCC GCA GCA 1034
ALQASVLKIWQGKDENVAAA 324

10 3 5 CAA AAG CAG CTC ACC ATG AGG GCA AAG GCC AAC GGT TTG GCA GCG CTG GGC AAA TAC CAG 10 94
QKQLTMRAKANGLAALGKYQ 344

1095 GGA GAC GCG GCG AGT GCG GCT GCA GCG GAT TCT CTG TTT GTG GCG CAG CAC GCC TAC TAG 1154
GDAASAAAADSLFVAQHAY* 363

1155 acaccagccacgcttacctccccttatctacaccatagtgtaaccctatactgaacaatgtcagagacaaattttcaca 123 3

1234 cgtttatattttaacgaacaatatcgttgttagatgcattttgatgcggtgtatgttgatgaagagattaagtaataat 1312

1313 gcatgtttttacattcggggtaacctacaatgtgatggatattaaaacatatttttagatatttttttctaggtagaaa 13 91

13 92 tctcagctctaggcataatatgtttataatgttaatgtgtgaatttgtcacctactgttttgttgacgtcagcattttg 14 7 0

14 71 tttactgtcatttatttgctttaatatcagaacaggttatgcctgtgaagaattaaataatgaatctgctataatttct 154 9
1550 gtgatccctcaattgattatactttgtcagtattaaaaaccattatatgcatgtaaaaaaaaaaaaaaaaaaaaaaaa 1627

Figure I. Nucleotide sequence of the sea scallnp lruct(>se-1.6-hiplios|)liate aldolase cDNA and the deduced amino acid seifiience of the enzyme.
The nucleotide residues are numbered from the 5' end. The amino acid residues are numbered from llrst in-frame methionine (M). The potential
polyadenylation signal is bolded. Underlines indicate primers for amplification of 3'LITR. The star indicates the stop codon. Note that the 57 bp
obtained by RACE system is added to the 5' end of the clone PniC 82.

594

Patwary et al

TABLE 1.
Codon usage in fructose-l,6-biphosphate aldolase gene.

uuu

phe

F

5

UCU

ser

S

1

UALI

tyr

\

—

UGU

cys

C

4

uuc

phe

F

K

UCC

ser

s

6

UAC

tyr

Y

9

UGC

cys

c

2

UUA

leu

L

—

UCA

ser

s

3

UAA

OCH

Z

—

UGA

OPA

z

—

UUG

leu

L

T

UCG

ser

s

1

UAG

AMB

Z

1

UGG

trp

w

3

cuu

leu

L

1

ecu

pro

p

3

CAU

his

H

1

CGU

arg

R

1

cue

leu

L

12

ccc

pro

p

7

CAC

his

H

4

CGC

arg

R

4

CUA

leu

L

3

CCA

pro

p

6

CAA

gin

Q

1

CGA

arg

R

T

cue

leu

L

13

CCG

pro

p

->

CAG

gin

Q

16

CGG

arg

R

1

AUU

ile

I

3

ACU

Ihr

T

1

AAU

asn

N

4

AGU

ser

S

6

AUC

ile

I

13

ACC

Ihr

T

9

AAC

asn

N

14

AGC

ser

S

2

AUA

ile

I

1

ACA

Ihr

T

5

AAA

lys

K

S

AGA

arg

R

1

AUG

met

M

7

ACG

Ihr

T

9

AAG

lys

K

16

AGO

arg

R

6

GUU

val

V

~>

GCU

ala

A

7

GAU

asp

D

4

GGT

gly

G

9

GUC

val

V

7

GCC

ala

A

21

GAC

asp

D

13

GGC

gly

G

13

GUA

val

V

4

GCA

ala

A

7

GAA

glu

E

10

GGA

gly

G

1

GUG

val

V

10

GCG

ala

A

10

GAG

glu

E

13

GGG

gly

G

3

In each block, the first, second, third, and fourth columns are codons. three-letter abbreviations for amino acids, one-letter abbreviations for amino acids,
and number of amino acid residues, respectively.

other codons for this amino acid. Likewise, one (CAG) of the two
codons for glutamine code for 16 of the 1 8 residues of this amino
acid. This conspicuous nonrandom usage of synonymous codons
in the FBP aldolase gene in sea scallops is consistent with the bias
found in highly expressed genes of several other organisms (Shaip
& Li 1986, Sharp et al. 1988). This strong codon-usage bias may
be the result of selection for translation efficiency and accuracy in
highly expressed genes like FBP aldolase.

The number of FBP-aldolase amino acid residues in different
organisms varies from 358 in Oriza sativa to 366 in Caenorhab-
ditis etegans. However, a wide range of organisms such as Droso-
phila. rat. and Xenopits have 363 residues as in sea scallops. Based
on the high amino acid sequence homology, we conclude that this
is a subunit of class I aldolase. Because of its essential role in
glucose metabolism, the primary structure of this enzyme has re-
mained highly conserved across kingdoms. The rate of evolution
of aldola.se was estimated to be about Wc amino acid residue
changes every 100 million years ( Sawyer et al. 1988). The muscle-
specific aldolase evolved at an even slower rate, with only about
2% amino acid residue changes per 100 x 10'" years (Freemont et
al. 1988). The BlastP search (Altschul et al. 1997) results show that
the sea scallop aldolase is approximately 669^ identical with that of
Schistosoma mansoni, 65% with that of Drosophita melaiwgaster,
64% Sahno salar, 63% with Galhis galliis and Xenopits leavis,
62%' with human muscle aldolase A. 62% with caenoihahdilis
elegans, 5T7c with Oiyza saliva, and 55% Plasmodium falciparum.

The approximate number of genes encoding FBP-aldolase in
sea scallop was established by Southern blot analysis. Hybridiza-
tion of cDNA coding region probe with a blot containing DNA
samples frotn a single sea scallop but digested with several restric-
tion en/.ymes produced signals of varying intensity in all lanes
(Fig. 2). From the number of signals we estimate that sea scallops
have a family of 4 to 10 FBP-aldolase genes. Aldolase constitutes
a medium-sized gene family in comparison to the estimated family
size of 12-15 for the actin gene (Patwary et al. 1996) and 1 to 3 for
the tropomyosin gene (Patwary et al. 19991 in sea scallops. The
organization of FBP aldolase as a small multigene family was also
observed in Euglena (Plaumann et al. 1997).

kb

21.2-

5.2-

3.5-

2.0

Figure 2. Detection of fruclose-l,6-biphosphte aldolase gene in the sea
scallops. In this Southern hyhridi/.alion. digoxigenin-labcled aldolase
coding region probe was hybridized to genomic DNA from a single
animal. Kach lane has U\ pg of DNA digested with /uiiRI (lane a),
/?<7)RV (lane b). Himl\\\ (lane ci. Sal\ (lane d). and \ha (lane el. M is
digoxigenin-labeled DNA molecular weight marker (Boehringer
Mannheim I.

FBP Aldolase cDNA in Sea Scallops

595

kb Mabcdefg

4.40- -

2.37- -
1.35- - **"

kb Mabcdefghijklmno
21.2-1

Figure 3. Northern blot analysis. A 3' untranslated cDNA region was
labeled with "P and hybridized to blots each lane with 12 fig of pooled
total RNA obtained from adductor muscles (lanes a and b), gonads
(lane cl, hearts (lane d), livers (lane e), mantles (lane fl, and gills (lane
g). M is RNA molecular marker lane.

Figure 3 shows that the size of sea scallop FBP aldolase mes-
senger RNAs in the Northern blot corresponds closely to the length
of the sea scallop FBP aldolase cDNA (Fig. 1 ). This result suggests
that the clone PmC-82 represents a FBP aldolase cDNA with a
very small truncation at its 5' untranslated region. We have used
the 3' non-translated region of cDNA as a probe in Northern
hybridization to determine the tissue specificity of aldolase expres-
sion, and obtained signals in the adductor muscle lanes only (Fig.
3). In absence of any indication of RNA degradation in the gel or
in the blot, we conclude that the aldolase gene in question is
expressed tissue-specifically in the adductor muscle of sea scallop.
This finding suggests that, like vertebrates (Penhoet et al. 1966.
Penhoet et al. 19691, sea scallops may have different class I aldo-
lase genes expressed in different tissues. The extent of structural
differentiation of these genes and the pattern of their distribution
among different sea scallops tissues remain to be studied.

We examined the utility of FBP-aldolase cDNA as a probe to
reveal polymorphisms. The probe revealed RFLPs in three loci in
an /AftT-digested blot (Fig. 4) and in two loci in a Drffl-digested
blot (data not shown), Pogson and Zouros (1994) and Pogson
(1994) obtained three types of RFLPs in sea scallops when they
used unidentified sea scallop cDNAs as probes. These were re-
striction site polymorphisms, polymorphisms caused by variable
numbers of tandem repeats (VNTR) and complex fingerprinting
patterns (Pogson 1994). In this study the FBP aldolase cDNA
probe revealed restriction site polymorphisms at the approximately
15 kb and 3 kb loci and a VNTR type polymorphism at the 5 kb
locus in the Accl digested genomic blot (Fig. 4). Each of these loci
has one or two bands, suggesting that the genomic DNA was

^ ••» ««

Figure 4. RFLPs in the region surrounding the aldolase genes in sea
scallops. Each lane has 10 Mg of genomic DNA from a different animal
obtained from live different sea scallop beds (a-c from Yarmouth, d-f
from Sable Island, g-i from Georges Bank, J-l from Brow ns Bank, and
m-o from Newfoundland) digested with restriction enzyme .icc\ and
hybridized with the complete aldolase cDNA coding region. M is
digoxigenin-labeled DNA molecular weight marker (Boehringer
Mannheim).

completely digested and that the gene lacks restriction sites within
it for the enzyme concerned. This finding of polymorphisms at
multiple loci supports the presence of multiple copies of the aldo-
lase gene probably distantly placed in the genome. Some of these
genes possibly carry one or more variable sized introns and or
VNTRs within their introns or in their fianking regions, producing
multiple alleles at a particular locus. These results demonstrate that
FBP cDNA is a useful addition to our collection of cDNAs that can
be used as a probe to reveal RFLP markers for varieties of genetic
studies in deep-.sea scallops and possibly in other related bivalves.
The isolation of the first bivalve aldolase cDNA may also assist in
understanding the mechanism of aldolase function in this unique
group of organisms.

ACKNOWLEDGMENTS

M. U. P. received support for the construction of the cDNA
library from the Department of Fisheries and Oceans. Canada
through a contract to the NRC Institute for Marine Biosciences,
Halifax, Canada. The authors thank Dr. Ellen Kenchington, De-
partment of Fisheries and Oceans, Bedford Institute of Oceanog-
raphy, Dartmouth, Canada for initiating this contract with NRC-
IMB. We acknowledge the support received from American So-
ciety for Cell Biology through a Visiting Professorship awarded to
M. U. P. and from the Office of Academic Affairs, and Health
Science Research Assistance Center, School of Science, Health
and Technology, Medgar Evers College of the City University of
New York.

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phila melanogaster. and Homo sapiens: A review of the considerable
within species diversity. Nucleic Acids Res. 16:8207-821 1.

Tolan. D. R.. J. Niclas, B. D. Bruce & R. V. Lebo. 1987. Evolutionary
implications of the human aldolase-A. -B. -C. and -pseudogene chro-
mosome locations. Am. J. Hum. Genet. 41:907-924.

Volckaert, F. & E. Zouros. 1989. Allozyme and physiological variation in
the scallop Placopecten magellanicus and the general model for the
effects of heterozygosity on fitness in marine mollusks. Mar. Biol.
103:51-61.

Journal of Shellfish Research. Vol. 21. No. 2, 597-603. 2002.

COMPARATIVE ANALYSIS OF OOCYTE TYPE FREQUENCIES IN DIPLOID AND TRIPLOID
CATARINA SCALLOP {ARGOPECTEN VENTRICOSUS) AS INDICATORS OF

MEIOTIC FAILURE

ROSALIO MALDONADO-AMPARO AND ANA M. IBARRA*

Centra de Investigaciones Biologicas del Noroeste. S.C. Aqiiaciilninil Genetics Labonitoiy. A. P. 128,
La Pa: B.C.S. 23000. Mexico

ABSTHACT Differences between diploid and triploid female mollusks in their gametogenic process had previously centered on
comparing the general gametogenic cycle, not the oocyte types in each ploidy class, which by their presence or absence might be used
as indicators of completion of specific meiotic events. In this study we compared oocyte type frequencies at three different stages of
the gametogenic cycle of tnploid and diploid catarina scallops. Regardless of age of sampling (81 d, 1 18 d, 205 d of grow out), the
most abundant oocyte type in diploids was always the vitellogenic. and in triploids the previtellogenic. The frequency of occurrence
of vitellogenic oocytes in tnploids was only 4.7-19'7f of that in diploids, and that for postvitellogenic oocytes was only from 0-8%
of that in diploids. This indicates that the oocyte stage in triploids at w hich gametogenesis was largely arrested was the previtellogenic
oocyte, at meiosis I. The principal cause for the meiotic arrest might be associated with problems during zygotene at the time of
synapsis between the three homologous chromosomes in triploids as previously suggested by other authors.

KEY WORDS: triploid, oocyte-frequency, scallop, Argopecten rentricosiis

INTRODUCTION

Previous research comparing diploid and triploid female mol-
lusk gonad development have centered on comparisons of the ga-
metogenesis cycle as described for diploids. Those studies have led
to the general conclusion that triploid gametogenesis is retarded
when compared with diploids (Allen & Downing 1986, Allen &
Downing 1990. Allen 1987, Komani & Wada 1989. Co.x et al.
1996). Whereas some studies have based the comparative analysis
of diploid and triploid gametogenic cycle in the qualitative occur-
rence of some particular type of oocyte at an age (Cox et al. 1996),
a different approach to study the abnormal gonad development in
triploid females is to estimate oocyte type frequencies in each
ploidy class at different stages of the gametogenic cycle as it has
been done for some tlsh species (Carrasco et al. 1998, Felip et al.
2001 ). The most common method of comparing diploids and trip-
loids (by gametogenesis cycle) results in that it is not clear if the
delayed gametogenesis in triploids is caused by a lower develop-
mental rate of most oogenic stages, or by an inhibitory effect
resulting in delay or an arrest of a particular stage so further
development is impaired, or by both. What is clear is that a final
consequence of the triploid condition at the end of the gametogenic
cycle is the occurrence of partial or total sterility observed for most
moUusk species, measured as the qualitative observation of low
numbers of either spermatozoa or mature oocytes (Komaru &
Wada 1989, Allen & Downing 1990, Guo & Allen 1994a. Guo &
Allen 1994b, Cox et al. 1996, Eversole et al. 1996).

As with other mollusk species, in the functional hermaphrodite
catarina icsWo^. Argopecten ventricosus. gametogenesis of triploid
scallops is apparently delayed when compared with diploids, re-
sulting in partial sterility of triploids. The partial sterility results in
fecundity being largely reduced in triploid females, with the male
gonad generally not developing further than the spermatocyte
stage, and becoming replaced by female germinal cells later in the
life cycle of tripkiids (Ruiz-Verdugo et al. 2000, Ruiz-Verdugo et
al. 2001, Maldonado-Amparo & Ibarra 2002). However, as is the
case with most other mollusk species, an oocyte stage at which

*Corresponding author: aibarra<s cibnor.mx

gametogenesis is arrested has not been clearly identified. The ob-
jective of this study is to compare oocyte type frequencies between
diploid and triploid catarina scallops at three different ages to
define if such a stage exists,

MATERIALS AND METHODS

Definition of Oocyte Types and Their Size

Because oocyte types in catarina scallop have not been previ-
ously described, we first defined the oocyte stages based on those
described by Dorange and LePennec ( 1989) and Saout (2000) for
another scallop, Pectcn maximiis. The oocyte stages described by
those authors included oogonia, previtellogenic oocyte, vitello-
genic oocyte, and postvitellogenic oocyte. An additional oocyte
stage was described for catarina scallop, mature oocytes, following
the description from Longo (1983). The definition of meiotic
stages occurring in previtellogenic oocytes of catarina scallop were
based on those described for Pecten maximus by Saout (2000).

To obtain high definition photomicrographs of each oocyte
stage, samples of diploid and triploid catarina scallop gonads were
taken from five scallops of each ploidy group at 7 months old.
These scallops were produced following the same methodology as
Ruiz-Verdugo et al. (2000). inhibiting formation of the second
polar body with cytochalasin-B (0.5 mg L"'). and grown under the
same conditions. All gonad samples were fixed similarly as for
electronic microscopy studies (Komaru et al. 1994) to be able to
obtain semi-thin (2 |jim) sections, with the following modifica-
tions. The gonads were fixed in 29c gluteraldehyde in phosphate
buffer (0.2 M) with an adjusted pH to 7.4 for 24 h. After this time,
all samples were washed twice, each during 30 min, in a washing
solution (9 g NaCI, 0.14 g KCI, 0.12 g CaCK, 0.2 g NaHCO,, 2 g
glucose, in 1000 ml distilled water). The samples were then pro-
gressively dehydrated passing them sequentially from 30% alcohol
to absolute alcohol. The rest of the histologic process was the same
as with H&E. Photomicrographs taken with an Olympus BX-4I
microscope with an integrated camera (CoolSNAP-Pro Color)
were digitalized and measurements of oocyte types diameters ot
each ploidy class were taken with the image analysis program
SigmaScan Pro5. obtaining the area by digitalizing contours and

,597

598

Maldonado-Amparo and Ibarra

estimating diameters. A total of 30 cells of each type were mea-
sured per ploidy.

Quantification of Oocyte Types

After defining the type of oocytes in catarina scallop, we pro-
ceeded to obtain oocyte types frequencies. The diploid and triploid
catarina scallops used in the evaluation of numbers of oocyte types
in this study were those used by Ruiz-Verdugo et al. (2000) for the
study of gametogenesis between triploids and diploids, hi that

study oocyte types present in each ploidy class at different ages
were not comparatively quantified. Those triploids were produced
by inhibition of polar body 2 with 0.5-mg cytochalasin-B L"', and
the scallops were reared for one year at Bahi'a Magdalena, B.C.S.
Mexico.

Gonad samples from three ages were evaluated for both ploidy
groups. Samples were taken at 81 d of grow out when vitellogen-
esis was beginning in diploids, at 1 18 d as an intermediate game-
togenesis stage in which advanced vitellogenesis was evidenced by

Figure 1. Diploid oocMus in catarina scallop. Light niicidsinpv ixlOO). (A) Oogonia (Og): (B) Previtellogenic oocytes in z\golcne-pachytene (Prev
zy-pa); (C) Previtellogenic oocyte in diplotene (Prev di): (O) \ itellogenic oocyte; (El Postvitellogenic oocyte; (Fl Mature oocyte. AC = auxiliary
cells; Ch = chromosomes in metaphase: CT = connective tissue; Cy = cytoplasm; Nc = nucleolus: Nu = nucleus; VE = vitelline envelope.

Oocyte Type Frequencies in Diploid and Triploid Catarina Scallop

599

TABLE 1.

Mean cytoplasm diameters (minimum — maximum) of each oocyte

type ill = 20) for each group, diploid (2N) and triploid (3N), in

catarina scallop (Argopecteii veiitricosiis ).

Cytoplasm Diameter, (im (range)

Oocyte Types

Diploid

Triploid

Oogonia
Previtellogenic
Vitellogenic
Postvitellogenic

4.57 (3.98-5.12) a

6.95 (5.75-8.34) a

37.34 (11.99^9.31) a

43.84 (35.25-50.54) a

4.85 (4.08-5.46) b

7.81 (7.24-8.76) b

32.77 (10.29-55.54) a

50.35 (38.77-58.93) b

Different letters between columns indicate significant differences between
ploidy classes.

the presence of large numbers of vitellogenic and postvitellogenic
oocytes, and at 205 d of grow out when mature oocyte stages
occurred in diploids and spawning had taken place in some scal-
lops. For the quantification of each oocyte type the same digitali-
zation process that was used for definition of oocyte types was
followed. For each ploidy class 9 to 10 gonads (organisms) were
randomly sampled, and for each gonad all oocyte types in 20
randomly selected acini were counted.

Statistical Analyses

Differences in size of oocyte types between ploidy groups were
analyzed with a single factor ( ploidy ) analysis of variance for each
oocyte type. Means were compared with a Duncan test. Differ-
ences in numbers of oocyte types between ploidy groups were
analyzed with a multivariate mixed ANOVA model, in which
scallops were a random effect, and age and ploidy were fixed
effects. For the only fixed interaction (ploidy by age), post hoc
comparisons were done between treatment means using a Tukey
test (Sokal & Rohlf 1981). Significance for all analyzes was es-
tablished at P < 0.05.

RESULTS

Oocyte Types in Catarina Scallop

The oocyte types occurring in diploid catarina scallop are de-
picted in Figure 1, and their mean sizes in Table 1. Those stages
included oogonia (diameter 4.57 jxm) attached to the acinus wall,
previtellogenic oocytes with an average diameter of 6.95 |xm in
which the prophase I stages of zygotene-pachytene and diplotene

could be distinguished (based on Saout 2000). The first association
of oocytes to auxiliary cells characterized vitellogenic oocytes,
with an average diameter of 37.34 ixm, but ranging from 1 1 .99 |j.m
to 49.31 |jim. Postvitellogenic oocytes, in which a clearly defined
vitelline envelope was present, had an average diameter of 43.84
(j.m. Few mature oocytes, those in which the gemiinal vesicle was
broken, were observed in these scallops, and therefoie not mea-
sured.

In triploid catarina scallop (Fig. 2, Table 1), oogonia also at-
tached to the acinus wall were significantly larger (4.85 |ji.ni) than
those in diploids. Previtellogenic oocytes (diameter 7.81 jjim) were
also larger and differed from diploids in that they were the most
abundant oocyte type, and in that they were surrounded by empty
spaces, probably a consequence of cell lyses. Vitellogenic oocytes
diameter (32.77 |j,m) in triploids was not significantly diffeient
from diploids, although in triploids most of the few observed vi-
tellogenic oocytes were still pedunculated. Postvitellogenic oo-
cytes (diameter 50.35 |jim) were larger than in diploids and free in
the lumen of the acinus. No mature oocytes were observed in
triploid scallops.

Analyses of Numbers of Oocyte Types

Both, ploidy class and age were significant effects (P < 0.0001 )
in the analysis for frequencies of all oocyte types. There was a
significant interaction between ploidy class and age, and also the
triple (random) interaction effect was significant. At 81 d and 1 18
d of grow out, diploids had significantly less oogonia and previ-
tellogenic oocytes than triploids. and more vitellogenic, postvitel-
logenic, and mature oocytes than triploids (Table 2). At 205 d
diploid and triploids practically showed no oogonia, but diploids
still had significantly less previtellogenic oocytes and more vitel-
logenic, post-vitellogenic, and mature oocytes than triploids. In
triploids evaluated at this late age the most common oocyte type
was still the previtellogenic oocyte.

Beside the differences within age for numbers of oocyte types
between ploidy groups, and the difference in the most common
oocyte type between diploid and triploid scallops at any age (Fig.
3), diploids had an increasing number of vitellogenic oocytes and
a decreasing number of previtellogenic oocytes from 81 d (15.26
and 6.89, respectively) to 118 d (29.47 and 0.7, respectively).
Triploid scallops had an increasing number of previtellogenic oo-
cytes and a decreasing number of vitellogenic oocytes during the
same period (27.82 and 2.19 at 81 d, and 85.53 and 1.4 at 1 18 d).
There was a decrease in numbers of vitellogenic oocytes found in
diploids from 1 1 8 d (29.47) to 205 d (24.64), which was paralleled

TABLE 2.

Mean numbers of oocyte types at each age and for each ploidy group, diploid (2N) and triploid (3N) of catarina scallop.

Argopecten venlricosus.

Oocyte Types

Oogonia

Previtellogenic

Vitellogenic

Postvitellogenic

Age

Ploidy

Mature

81 d

2N

1.43 b

6.89 b

15.26 c

1.07 b

0.09 a

3N

6.62 b

27.82 c

2.16a

0.00 a

0.00 a

118d

2N

0.00 a

0.66 a

29.47 e

6.62 cd

0.65 b

3N

0.35 a

85.53 e

1.40 a

0.03 a

0.00 a

205 d

2N

0.03 a

0.01 a

24.64 d

5.76 c

2.74 c

3N

0.00 a

77.06 d

4.76 b

0.47 ab

0.00 a

Different letters within columns (oocyte types) indicate significant differences between ploidy groups and ages.

600

Maldonado-Amparo and Ibarra

Figure 2. Triploid oocytes in catarina scallop, li^lil iiiki .iscopy. (A» Oogoniii !()«). and large numbers of previtellogenic oocytes in zygotene-
pachytene (Prev zy-paMx40). (Bl Closer view of Prev zy-pa (xlOO). (C) Isolated vitellogenic oocyte (Vit) 1x40). (D) Isolated postvitellogenic oocyte
(Postv) (x40). LC = Cell lysis.

by an increase in mature oocytes (0.63 at 1 18 d, and 2.74 at 205 d).
Among iriploids there was a decrease in number of previtellogenic
oocytes tYom 118 d (83.33) to 203 d (77.01 ). which paralleled an
increase in numbers of vitellogenic oocytes (1.40 at 118 d. and
4.76 at 203 d).

The significance of the triple random interaction (ploidy, age,
and organism) was caused by diffei'ences among diploid and ti'ip-
loid individuals in the number of the different types of oocytes
each showed at the diffei-ent ages evaluated, with the largest dif-
ferences occurring a]iio)ig triploid individuals for the number of
pi'evitellogenic oocytes present per individual, followed by the
number of oogonia and vitellogenic oocytes. In diploids, variation
between individuals was seen especially for numbers of vitello-
genic oocytes, but the variatioti was not as marked as that seen for
previtellogenic oocytes atnong triploids (Fig. 4),

DISCUSSION

We have dononstrated in this study that in catarina scallop the
number of late developmental oocyte stages (vitellogenic, postvi-
tellogenic, and mature) is largely reduced in triploids when com-
pared with those developing in diploids, and that the number of
early oocyte stages in triploids, especially the previtellogenic oo-
cyte, is in a large frequency from the beginning to the end of the
reproductive cycle of diploids. A large reduction in fecundity,
measured as number of mature or spawned eggs, as a consequence

of triploidy has been observed for oysters and clams (Guo & Allen
iyy4c. Utting et al. 1996), as well as for the catarina scallop
(Rui/-Verdugo et al. 2001). The sporadic occurrence of vitello-
genic oocytes in triploid scallops appears to be a random event, as
significant variation between individuals was seen. The low num-
ber of vitellogenic oocytes translates into a reduction in fecundity
of 86* at 81 d, 93%. at 118 d, a)id 81% at 203 d of growth.
However, when post-vitellogenic oocytes ai'e considered, the re-
duction in fecundity was larger, 100% at 81 d, 99% at 1 18 d, and
92%^ at 205 d.

Because the first sampling analyzed already contained diploid
scallops with advanced stages of development, not just the first
oocyte stages, we were unable to determine whether there was an
actual delay in triploid scallops in the initial occuirence of the first
oocyte stages (oogonia a)id previtellogenic oocytes) when com-
paied with diploids by oocyte frequency counts. In this study the
scallops shell height at the earliest sampling (8 Id) was close to 3
cm (Rui/,-Verdugo et al. 2000), and it is known that the first age of
sexual jiiaturation for catarina scallop when grown at the same
environment is a shell height of 2 cm (Cru/, et al. 2000). Further
.studies at ages or sizes smaller than the ones sampled in this study
are necessary to clarify whether there is an actual delay in early
oogenesis of triploid scallops when compared with diploids.

In fish, studies co)nparing numbers of oocyte types between
diploids and triploids (Carrasco et al. 1998, Felip et al. 2001 ), have

Oocyte Type Frequencies in Diploid and Triploid Catarina Scallop

601

Figure 3. Diploid lliltl and Iripioid iriniill laliii ina scallop Itinak- uiinad structure at 81 d (A & B), 1 18 d (C & D), and 2((5 d (E & F) of f>row
out. Light microscopy (x20). In diploids at 81 d lAl (here were ahundant vitelloRenic oocytes (Vit), and few previtelloyenic (Prevl; by 118 d (C)
postvitellogenic oocytes (Postv) were abundant; by 2(15 d (K) some mature oocytes (Mat) were also present. In triploids prcvitellogenic oocytes
were the most common type at all ages although a few vitellogenic (IS) were already present at 81 d, increasing in numbers by 118 d (0): some
postvitellogenic oocytes were evident by 205 d (F).

also indicated that there is a difference between ploidy groups in
the type of oocytes most frequently occurring, with the most com-
mon one in triploids being the prcvitellogenic oocyte. This points
toward the first oocyte stage at which the triploid condition results
in an inhibitory effect of further development being the previlel-
logenic oocyte, such that more advanced oocyte stages occur at
much lower frequencies. In scallops it is known that only those
previtellogenic oocytes that complete the pachytene .stage and en-

ter the diplotene stage of meiosis I will begin the process of vi-
tellogenesis and become mature (Beninger & Le Pennec 1991,
Dorange & Le Pennec 1989). Our I'esults clearly point to the fact
that oogenesis in triploid catarina scallop [Araopcctcn Yenliicosiis)
was arrested at the previtellogenic oocyte, and the reduced num-
bers of ooycte stages other than the previtellogenic type in triploids
point to the halt of oocyte development at prophase of meiosis I,
before the diplotene stage. In the male gonad part of this hermaph-

602

Maldonado-Amparo and Ibarra

81 days

o Oogonia

♦ Previtellogentc

■ Vilellogenic

o o o o o o o o o

^ o o o

-, , 1 T T I f * T ¥

123456789 123466789

Scallop No

118 days

Oogonia

Prevltellogenic

Vitellogenic

' ■ - ■
9 f » 9 — >>*>?»

-^ — $ — 4> — $ — ^ — ^ — * — * — * — 9 —
123466789 10 123466789 10

Scallop No

Oogonia

Previlellogenic

Vitellogenic

205 days

t f

2 3

-^ — tf — .ji — <r-

9 10 . ,
Scallop No.

23466789 10

Figure 4. Frequencies of oocyte types (oogonia. previteilogenic, vitello-
genic) per scallop in both, diploids and triploids, at each of the evaluated
ages.

rodite species it is known that the hiilt in spermatogenesis occurs
also at prophase I (Maldonado-Anipairo & Iban'a 2002).

Until now. the cause of the sterility in triploid mollusks had
been mostly ascribed to the inability of chromosomes to synapse,
align, and segregate. However, at least for one species, the Pacific
oyster, it has been demonstrated that synapses and segregation of
chromosomes can occur in triploid oocytes even if in a lower
number of oocytes than in diploids and in an abnormal fashion

(Guo & Allen 1994c). In other mollusk species, including the
catarina scallop, female triploids are known to be able to produce
mature oocytes, even if in largely reduced numbers when com-
pared with diploids (Guo & Allen 1994a. Utting et al. 1996. Ruiz-
Verdugo et al. 2001 ). Among some species of male triploid mol-
lusks. meiosis I and II are able to proceed through sperm formation
although in reduced numbers when compared with diploids (Ko-
maru & Wada 1990. Guo & Allen 1994a. Cox et al. 1996, Mal-
donado-Amparo & Ibarra 2002). and the produced sperm is gen-
erally aneuploid.

Because mollusks do not have to go through the completion of
meiosis I to form mature oocytes. Allen ( 1987) proposed that the
fact that oogenesis is arrested in triploid mollusks must be a con-
sequence of meiotic difficulties arising at pre-synaptic or synaptic
stages, after chromosome replication, rather than difficulties in
pairing of homologous for segregation in metaphase I of meiosis.
Our results support this same conclusion. The finding that few
oocytes proceeded to the vitellogenic oocyte in triploids points
toward the halt of meiosis being just before the diplotene stage at
prophase I. The regulatory mechanisms to explain that arrest of
meiosis remain to be investigated. One possible regulatory mecha-
nism might be associated with the known "meiotic checkpoints"
described for a number of organisms (Murakami & Nurse 1999.
Murakami & Nurse 2000. Roeder & Bailis 2000. Tarsounas &
Moens 2001). with the "recombination or pachytene checkpoint"
being one of the most interesting ones to explain the arrest of
meiosis in oocytes of triploid organisms, as it provides with a
genetic, rather than just a mechanical mechanism for the observed
arrest of further development. Meiotic checkpoints, also called
"housekeeping mechanisms", are known to act by means of protein
complexes signaling abnonnal chromosome behavior, with effec-
tor proteins acting to delay or arrest the meiotic process (Roeder &
Bailis 2000).

In conclusion, partial sterility in the female gonad of the cata-
rina scallop is related to a halt in previteilogenic oocytes. As it is
known that only those previteilogenic oocytes that complete the
diplotene stage of meiosis 1 become vitellogenic. we conclude that
the sterility is caused by problems during chromosome synapsis
and recombination, that is. during the zygotene and pachytene
stages of meiosis 1. Why some oocytes are able to complete the
vitellogenic process in spite of the demonstrated general arrest at
previteilogenic oocyte is not known, but recent studies in triploids
of other species point toward the existence of a correction mecha-
nism of trivalent and tetravalent formation when homologous pair
for recombination, which appears to be sex specific (Gui et al.
1991. Gui et al. 1992. Gui et al. 1993; Oliveira et al. 1993. Zickler
& Kleckner 1999). and explains triploids differences between
sexes in sterility.

ACKNOWLEDGMENTS

This research was supported by CONACYT grant no. 28236B
to A. M. Ibarra. The authors thank Dr. Thomas Famula. biometri-
cian from UC Davis for advice on the statistical analyses. We also
thank Carmen Rodriguez for histology support. The senior author
is a CONACYT and SEP (DECYTM) Ph.D. fellow, and the results
presented here are part of his thesis.

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Journal ofShellthh Research. Vol. 21. No. 2, 605-618. 2002.

LOW TEMPERATURE, BUT NOT AIR EXPOSURE SLOWS THE RECUPERATION OF
JUVENILE SCALLOPS, PLACOPECTEN MAGELLANICUS, FROM EXHAUSTING

ESCAPE RESPONSES

MARTIN LAFRANCE,' HELGA GUDERLEY,'* AND GEORGES CLICHE"

^ Depurtemeut de Biologie. Unhershe Laval, Quebec. P.Q.. Canada GIK 7P4: 'Ministere de

r Agriculture, des Pickeries et de rAlimentatiou. Direction de la recherche scientifique el technique.

P.O. Box 65S. Cap-cni.\-Meules. P.Q. Canada GOB IBO

ABSTRACT Marked changes in temperature a,s well as periods of air exposure are common during scallop seeding operations. We
examined whether the escape responses of cultured juvenile sea scallops Placopecten magellaiticiis (35—45 mm shell height) of the size
used for seeding were hindered by such stresses and how the performance of stressed scallops changed within the week following
thermal change. Tagged cultured scallops were either transferred to 8°C or maintained at IS^C; escape responses from starfish were
measured 8 times during the following 156 h. The second study combined the stress of air exposure (4 h) with that of transfer to colder
temperatures. Transfer of scallops from 18 to 8°C significantly reduced the clapping rates and maximum number of claps in a series
at every sampling time (12 to 156 h) following thermal transfer. After 15 min of recuperation from exhaustive escapes, the percent
initial claps was lower than that of control scallops (-40-50% vs. 60-70% ). The time spent closed after exhaustive exercise was similar
between the cold-stressed and control scallops, except at the first sampling time (6.5 and 2.7 min. respectively). E.scape performance
was not modified by 4 h air exposure. While decreased performance under cold stress could be ascribed to direct, kinetic effects of
temperature, certain aspects of the swimming response improved during the 156 h at 8"C. However, the scallops did not completely
acclimate their clapping rate to the thermal change within 6 d. suggesting that if a cold stress accompanied seeding, cultured scallops
may remain vulnerable to starfish predation during a prolonged period. Seeding operations should therefore try to minimize thermal
shock.

KEY WORDS: Placopecten iiuii;ellaiiicu.'^. escape responses, aquaculture. temperature, emersion, acclimation

INTRODUCTION

The sea scallop, PUicupeclen mugellaniciix. is a cold-water spe-
cies that does well at temperatures ranging from 5°C-15°C, with
optimum growth occurring at about 10°C (Naidu et al. 1989).
Depending on previous thermal acclimation history, temperatures
above 21-23.5°C can cause scallop mortality (Dickie 1958). Spe-
cific thermal regimes are known to influence growth, to provide
stimuli for synchronous spawning (Bonardelll et al. 1996) and to
condition predator-prey Interactions (Hatcher et al. 1996). Low
temperatures can delay larval growth and Increase larval mortality
as a consequence of longer exposure to predators (Dickie 1955,
Young-Lai & Aiken 1986). High temperatures have frequently
been held responsible for mass mortalities of scallops since the
1920s in the southwestern Gulf of St. Lawrence (Dickie & Medcof
1963). Since most of these mortalities occurred In the summer at
depths of less than 12-20 m, oscillations In the thermocllne were
likely to have led to exposure of scallops to lethally high tempera-
tures. Moreover, warm water debilitates sea scallops and Increases
their susceptibility to predation (Dickie & Medcof 1 963 ). Thus, the
sea scallop, which performs best at 10°C ± 5°C, can be exposed to
thermal fluctuations that modify Its performance In Its natural
habitat and during aquaculture operations.

Aerial exposure and handling prior to .seeding may weaken
scallops, rendering them more vulnerable to predation. In summer
and autumn, scallops can take more than 3 days to recover from
such stress (Fleury et al. 1996). While some bivalves are well
adapted for air exposure, scallops such as Pecten ma.ximus are
unable to withstand a progressive respiratory acidosis, and some
may die after approximately 72 h of emersion (Duncan et al.

♦Corresponding author. Email: Helga.Guderley@bio.ulaval.ca; Tel.: +1-
418-656-3184: Fax: -f 1-4 18-656-2043.

1994). Further, adult saucer scallops Amusium japonicum halloti
suffer appreciable mortality when exposed to air more than 2 h
(Dredge 1997), while mortality of juvenile P. maximus increased
significantly after 15 h of dry transport (Maguire et al. 1999a).
Desiccation stress and air exposure reduce both the righting and
recessing activities of juvenile scallops (Maguire et al. 1999b,
MInchIn et al. 2000). During air exposure of scallops, the adenyl-
ate energy charge (AEC) in the striated muscle decreases mark-
edly, but recovers after a few hours in aerated seawater (Maguire
et al. 1999a, Maguire et al. 1999b). When scallops. Chlamys oper-
cularis (4—5 cm in diameter), were exhausted and then allowed to
recover in the air, the adductor muscle AEC of the scallop In-
creased to 92% of its original value within 30 min. However, final
recovery of muscle energetic status only occurred In aerated sea-
water (Grieshaber 1978).

Since metabolic pathways and the contractile machinery are
temperature-sensitive (Olson & Marsh 1993), escape responses
may decrease during short-term exposure to cold. Subsequent ac-
climation to such cold temperatures Is likely to compensate for this
reduction In performance. For example, scallops living at greater
depths compensate for the prevailing lower temperatures through
an increase of membrane fluidity by adjustments of membrane
fatty acid composition (Napolitano et al. 1992). Thus the escape
responses of scallops may be temporarily reduced by changes In
temperature.

During bottom seeding operations, juvenile scallops can be
exposed to marked changes in temperature as well as periods of air
exposure. For example, the scallops may be transferred from a
warmer rearing temperature (i.e., during suspension In pearl nets)
to a cooler temperature on the bottom. Both air exposure and
thermal change during seeding operations may hinder the devel-
opment of escape responses. In this study, we examined whether
such stresses contribute to the loss of juvenile scallops after seed-

605

606

LaFrance et al.

ing. by reducing their escape response capacity or their recupera-
tion from exhausting escape responses.

MATERIALS AND METHODS

Scallop Collection, Storage, and Tagging

Spat were collected east of the Iles-de-la-Madeleine in Septem-
ber 1997 (Fig. 1). Thirteen months later, juveniles (17-23 mm
shell height) were transferred for intermediate culture in pearl nets
(35-cm square base, with a 6 mm mesh netting) at a density of
100 • net^'. From June to September 1999. the scallops were
maintained at a density of 20 individuals per pearl net (35-cm
square base, with a mesh size of 9 mm). For our experiments, we
selected individuals between 35-45 mm of shell height (maximum
dorsal-ventral distance). Hallprint™ labels (4x8 mm) were glued
on the upper valve using cyanoacrylate adhesive to identify the
scallops during sequential tests.

Cold Shock Experiment

On September 19. 1999. 64 scallops were brought in an icebox
from their suspended culture site in the Havre-aux-Maisons lagoon
to the laboratory (emersion time <10 min; see Fig. 1 ). Temperature
in the lagoon was measured hourly to the nearest 0.1 °C by a
Sealog-T V 1 .04 thermograph during this period. Water tempera-
ture in the lagoon was approximately I8°C the week before le-

trieving experimental scallops (Fig. 2A). Hence, we used 18°C as
our control treatment and 8°C to simulate the cold stress experi-
enced during transfer from suspension culture in the lagoon to the
seeding ground (Fig. 1). since autumn seeding may expose scal-
lops to such bottom temperatures. The scallops were equally di-
vided between 2 well-aerated 200-L tanks, one at 18°C (control)
and the other at 8''C (thermal stress). The photoperiod was main-
tained at natural day lengths (12 h light and 12 h dark). Salinity
ranged from 29.0-30.5%f. No food was supplied and seawater was
filtered (1 p-m) and UV-sterilized.

Cold Shock with Air Exposure Experiment

A second group of scallops was brought to the laboratory on
September 26th to compare the impact of air exposure coupled
with cold stress (thermal stress -i- air (TSA) with that of air expo-
sure alone [control -f air (CA)]. The transfer from warm ( 15.5^C)
(see Fig. 2) to low (8°C) temperature was preceded by a 4 h
emersion (18°C) during which the scallops were sprayed with
seawater every 30 min to keep them damp. As in the former
experiment. 32 scallops were placed at 8°C and 32 others were
kept at 18'C. Scallops were maintained in the same conditions as
indicated earlier.

Evaluation of Escape Responses

Scallops were put in 33 x 28 x 12 cm-basins and tested sepa-
ralelv after a minimum of 2 min without disturbance. An escape

Suspended
culture site

Seeding ground-

47.40'

47.30'

47.20'

- 47.10'

62.10' 61.50' 61.30'

Figure 1. Location of sites for sea scallop suspension culture in pearl nets (Havre-aux-Maisons lagoon l and seeding on the sea bottom (Chaine-
de la-Passe fishing ground) in the Iles-de-la-Madeleine. Inset shows location of the study site in the (iulf of St. Lawrence, eastern Canada.

Cold Stress Slows Escape Responses of Juvenile Scallops

607

E

September

Figure 2. Temperature measured in the Havre-aux-Maisons lagoon (A) between early June and early October and (B) for the week (September
19-26) that scallops of the Cold Shock with Air Exposure Experiment spent in the lagoon, while scallops of the Cold Shock Experiment were
being tested. Data were collected hourly with a precision of 0.1 C using a Sealog-T thermograph.

reaction consisted of a jumping or a swimming response following
contact with the arm of a starfish. Time, number of valve claps
(adductions) and the maximum number of claps in a series were
counted until repeated stimulation did not elicit a clap within 1 min
of the previous clap (exhaustion). Once the scallop was exhausted.
it was left in its aerated basin for 15 min. Then the escape response
was quantified a second time. Each escape parameter evaluated
during this second test was called "response after 15 min of recu-
peration". Response time, defined as the time from initial contact
of a starfish with the scallop mantle to first valve adduction, was
measured in seconds. At the end of the escape response, the scallop
was classified either as a "swimmer" or a "jumper". A "swimmer"
performed several series of claps (3 claps or more) in response to
predator stimulation, whereas a "jumper" did not clap more than
twice in a row. The escape responses of twenty-four scallops from
each treatment were measured 12, 24, 36, 48, 60, 84. 120. and 156
h after transfer. The remaining eight scallops from each treatment
were left undisturbed in their respective tanks all week. These

reference scallops (from the Cold Shock with Air Exposure Ex-
periment) allowed us to examine the effect of repeated swimming
behavior on the levels of macromolecular reserves in the muscle.
The experiments on the impact of thermal transfer were carried out
a week before those examining the combined impact of air expo-
sure and thermal change. Whenever a percentage of recuperation is
given for an escape parameter, it represents the ratio of the per-
formance during the second stimulation (after 15 min of recupera-
tion) relative to the initial response. These values are useful in that
they show the extent to which a particular response returns to
initial values after a short recuperation.

The Asterias vulgaris (radius of 5.5-7.5 ± 0.25 cm) used to
provoke the escape responses were collected in the lagoon Le
Bassin (southern end of the Jles-de-la-Madeleine) and kept in tanks
containing filtered, continuously aerated seawater either at 1 8°C or
8°C (holding capacity of -80 and 180 L, respectively) 30 h before
the first stimulation. The starfish used in a particular escape re-
sponse test were haphazardly chosen among 12 individuals at the

608

LaFrance et al.

experiiiiental temperauire. The same starfish was used for the two
tests carried out on a given scallop at a given sampling time.

Biometric Measurements and Macromolecnlar Reserves in
Adductor Muscle

Dissections of the scallops used for the Cold Shock with Air
Exposure Experiment were caiTicd out the day after the last escape
response tests. The adductor muscles were immediately frozen on
dry ice and maintained on dry ice for approximately I mo before
transfer to a -80T freezer at Universite Laval. Other soft tissues
were dried to constant mass at 60°C to determine their water
content. We calculated a '"muscle index" (muscle mass/mass of
total soft tissues minus muscle mass) to examine the relative con-
tribution of the adductor muscle. Muscle protein concentrations
were measured using the hicinchoninic acid method with bovine
serum albumin (BSA) as a standard (Smith et al. 1983). Muscle
carbohydrate levels were determined using the phenol-sulfuric-
acid method of Dubois et al. (1956). as modified by Martinez
(1991).

Statistical Analyses

Clapping behaviors were compared using repeated measures
ANCOVAs following the MIXED procedure (SAS 1999). The two
main factors. Treatment (control vs. stress) and Time (12. 24.

36 136 h after start of experiment) were considered as fixed

effects. Individuals (n = 24) nested within Treatment were con-
sidered as a random effect. Time was better defined with a spatial
power covariance structure (SP(POW) command) since it followed
an exponential distribution. The hiteraction term. Treatment x
Time, was always included in models. Comparisons were only
made between 2 treatments at a given sampling tiine using the
difference of least squares means (LSMEANS TIME x TREAT-
MENT / DIFF command). P-values associated with comparisons
between treatment means at a given time came from these least
squares means contrasts. The stability of escape performance over
time for specific treatments (ex: Time x Treatment CA) was as-
sessed by tests of effect slices (see Table 2). To meet requirements
of normality and homoscedasticity of residuals, parameters may
have been transformed using logarithms, square root or inversion
although untransformed values are shown in the figures. If a be-
havioral response after 15 min recuperation exceeded 200% of the
initial response, it was reinoved from the analysis (see notes in
Tables 1. 2. and 3).

Multiple pairwise comparisons (Scheffe) were u.sed to test for
specific differences when ANOVAs showed significant effects
(Table 4). Normality was assessed using the Shapiro-Wilk's test
and homogeneity of variances by a Brown-Forsythe's test (SAS
1999). A probability level (a) of 0.03 was used.

RESULTS

Our first experiment compared the escape behaviors of scallops
transferred from warm ( 18X) to colder water (8' C) with tho.se of
scallops maintained in 18°C. The second examined the impact of
such a thermal transfer when it followed 4 h of air exposure. Most
of the escape response parameters (except clapping time and time
spent closed) for control (control and control -I- air) scallops
showed higher values than for cold stressed (cold and cold -i- air)

scallops (Figs. 3. 4. 3. 6). The difference between control (control
+ air) and cold (cold -i- air) treatments was also apparent when
considering the scallops" capacity for recuperation 15 min after the
mitial response (Figs. 48. 4C. 4D; 6B. 6C. 60: Tables 1. 2. 3).

Cold Shock Experiment iCnnlrol r\. Cold-Stressed)

Scallops responded to the first contact with the starfish by
clapping their valves for a mean of 1.6 and 3.3 min before ex-
haustion (no clap within 1 min of the previous clap), during which
an average of 5 1 and 45 claps was observed for control and cold-
stressed scallops, respectively (Fig. 3A. 3C). Although the number
of claps only ditTered between treatments at a few sampling times,
control scallops had a significantly higher clapping rate and a
greater number of maximum claps in a series at every test (Fig. 3E.
30; Table 1 ). Control scallops kept a relatively constant clapping
time throughi)ut their successive encounters with starfish, whereas
cold-stressed scallops showed a gradual reduction of their initial
clapping time (Fig. 3C; Table 2). Initial clapping time differed
between the two treatments throughout the 168 h. Clapping time
after 1 5 min of recuperation did not vary throughout time for either
group (Table 2). Cold-stressed scallops only showed longer clap-
ping times than control scallops at 12. 60. and 156 h (Fig. 3D). In
contrast, the gap between control and cold-stressed scallops for
total number of claps, clapping rate and maximum number of claps
in a series was similar or increased after 15 min recuperation (Fig.
3B. 3D. 3H).

The changes in escape behaviors during the experimental pe-
riod provided indications of thermal acclimation (cold-stressed)
and habituation (control and cold-stressed). Cold-stressed scallops
improved some aspects of their responses while control scallops
remained constant or worsened. Control scallops decreased the
total number of claps and maximum claps in a series during suc-
cessive sampling events while cold-stressed scallops maintained
their values (Fig. 3B. 30; Table 2). Control scallops showed steady
clapping time and rate and time spent closed while cold-stressed
scallops shortened their clapping time, increased their clapping
rate and decreased the time closed (Fig. 3C. 3E; 4A; Table 2). The
other parameters shared downward (Fig. 3A) or steady trends (Fig.
3D. 3F. 3H) in both groups (Table 2).

The time during which scallops kept their valves closed after
exhaustion was similar in the treatments {P > 0.058; Fig. 4A;
Table 1 ). except at the first observation period when cold-stressed
scallops stayed shut more than twice as long as the control scallops
[P < 0.0001 ). At virtually all observation periods, control scallops
recuperated more of their initial response (i.e.. in tenn of percent-
age) than cold-stressed scallops (Fig. 4B. 4C. 4D). This was par-
ticularly clear for the total number of claps (Fig. 4B).

Cold Shock with Air Exposure Experiment (Control + Air vs.
Cold-Stressed + Air)

In this experiment, all scallops were initially exposed to air
(18°C) for 4 h to simulate conditions during transfer from culti-
vation sites to seeding grounds. Control scallops were then re-
turned to warm water ( 18°C) whereas cold-stressed scallops were
transferred to 8°C. Scallops from these treatments made an average
of 50 claps in the first escape test (Fig. 5A). At this time, the 1.5
min clapping time of control scallops was significantly shorter
than the 2.5 min time for cold-stressed scallops (P = 0.0002) (Fig.

Cold Stress Slows Escape Responses of Juvenile Scallops

609

TABLE 1.

Statistical analysis of the effects of treatment and time on escape performance of juvenile sea scallops from Cold Shock Experiment. The
scallops were transferred from pearl nets in the lagoon (18"C) to seawater at either 18 C (control! or 8 C (cold-stressed) in the laboratory to
simulate the cold stress accompanying seeding operations. p:ach scallop (;i = 24 per treatment) was measured during each sampling time (12.

24, 36, 48. 6(1, 84, 120 and 156 h).

Initial Response

df

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

1

7
7

1

7
7

I

7
7

1

7
7

1

7
7

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

2.63

4.73
1 .08

33.67
7.33
5.78

23.25
2.44
L71

48.79
3.26
0.67

3.68
4.39
3.09

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

0.11

<0.0001

0.37

<().()0()1
<0.0(M1
<0.0001

<0.()()(11
0.019
0.11

<0.0001
0.0023
0.70

0.061

<0.()0(11

0.0036

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

n.a.
n.a.
n.a.

Response After 15 min of Recuperation

df

Number of claps

Clapping time

Clapping rate

Ma.Kimum number of claps in a .series

Time spent closed

% initial number of clapst

initial clapping ratet

% initial maximum number of claps in a seriesS

1

45.44

<0.0001

7

2.31

0.026

7

1.22

0.29

1

13.33

0.0007

7

1.54

0.15

7

1.83

0.081

1

63.12

<0.0001

7

0.80

0.59

7

1.30

0.25

1

104.38

<0.0001

7

0.44

0.88

7

1.16

0.32

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

1

49.86

<0.0001

7

2.47

0.018

7

0.49

0.84

1

15.54

0.0003

7

1.11

0.35

7

0.12

0.99

1

26.39

<0.0001

7

1.33

0.24

7

1.53

0.16

t 1. t5. or §7 observations were removed from the analysis as recuperation exceeded 200%.
n.a. = not applicable.

?C). Thus, initial clapping rate was higher tor control scallops
(38.7 vs. 23.6 claps • min"'). Control and cold-stressed scallops
had. respectively, average maxima of 10 and 7 claps in a row (Fig.
5E. 5G).

One day (24 h) after re-immersion, the escape performance of
cold-stressed scallops resembled that of control scallops. The ini-
tial number of claps of cold-stressed scallops remained constant
throughout the experimental period while it gradually decreased
for control scallops (Fig. 5A; Table 2).). The initial clapping time
was only greater for cold-stressed scallops at the H4th (P = 0.032)
and the 120th h (P = 0.0002) (Fig. 5C). Initial clapping rales
remained similar between 24 and 156 h (Fig. 5E; Table 3). In spite
of the slightly greater mean values for control scallops, the 2
treatments did not differ in the initial maximum number of claps in
a series, except at the 4Sth h (P = 0.038) (Fig. 5G: Table 3).

After the 15 min recuperation, control and cold-stressed scal-
lops differed in terms of total number of claps for the first six
sampling periods (P < 0.046) (Fig. 5B). This difference was al-

leviated at 120 and 156 h after transfer, when both groups made
22-24 claps. After recuperation, the clapping time of the two
groups never differed (Fig. 5D; Table 3), even though the clapping
time of cold-stressed scallops remained steady, whereas that of
control scallops decreased slightly (Table 2). After recuperation,
clapping rate and maximum claps remained stable for control and
cold-stressed scallops (Table 2). Clapping rate tended to be higher
for control scallops (Fig. 5F). Control scallops consistently made a
maximum of 7-8 claps in a series in contrast to 4-5 claps for
cold-stressed scallops (Fig. 5H; Table 3). Overall, cold-stressed
scallops improved their performance relative to that of control
scallops with time after transfer, either sustaining their perfor-
mance as control scallops decreased (Fig. 5 A, 5B; Table 2) or
improving their performance while control scallops remained
stable (Fig. 5E. 5G; Table 2).

During the first test 12 h after re-immersion, the time spent
closed after exhaustion differed between control and cold-stressed
scallops (3.8 vs. 6.7 min. respectively P = 0.0019). Subsequently.

610

LaFrance et al.

TABLE 2.

Temporal stability of escape performance by juvenile sea scallops either maintained at 18°C (control: C), transferred from 18°C to 8°C

(thermally-stressed: TS), air-exposed for 4 h (control + air: C'A) or air-exposed for 4 h during a transfer from 15.5 C to 8°C

(thermally-stressed + air: TSA). Tests of effect slices analogous to the ANCOVAs shown in Tables I and 3. Kach scallop (h = 24 per

treatment! was measured at each sampling time (12, 24. 36, 48, 60, 84, 12(1, and 156 h).

Initial Response

df

Time X Treadiienl C 7 3.30 ().()(i:i

Time X Treatment TS 7 2.51 Odlh

Time x Treatment CA 7 3.03 ().()()42

Time X Treatment TSA 7 1.10 0..^6

Time X Treatment C 7 1.73 0.10

Time X Treatment TS 7 11.38 <().0001

Time X Treatment CA 7 4.24 0.0002

Time x Treatment TSA 7 6.50 <0.0001

Time X Treatment C 7 l.(.)4 (1.41

Time X Treatment TS 7 3.12 0.00,34

Time x Treatment CA 7 1 .93 0.065

Time X Treatment TSA 7 6.72 <0.(X)01

Time X Treatment C 7 3.13 0.0033

Time x Treatment TS 7 0.80 0.59

Time x Treatment CA 7 1 .04 0.40

Time X Treatment TSA 7 3.06 0.0040

Time x Treatment C 7 1.32 0.24

Time X Treatment TS 7 6.16 <0.()001

Time X Treatment CA 7 1.72 (1.1(1

Time x Treatment TSA 7 4.54 <0.0(1(11

Time X Treatment C n,a. n.a. n.a.

Time x Treatinent TS n.a. n.a. n.a.

Time x Treatment CA n.a. n.a. n.a.

Time x Treatment TSA n.a. n.a. n.a.

Time x Treatment C n.a. n.a. n.a.

Time x Treatment TS n.a, n.a. n.a.

Time x Treatment CA n.a. n.a. n.a.

Time x Treatment TSA n.a. n.a. n.a.

Time x Treatment C n.a. n.a. n.a.

Time x Treatment TS n.a. n.a. n.a.

Time x Treatment CA n.a. n.a. n.a.

Time x Treatment TSA n.a. n.a. n.a.

n.a. = not applicable.

Response .After 15 min
of Recuperation

df

Number of claps

Clapping time

Clapping rale

Maximum number of claps in a series

Time spent closed

% initial number of claps

% initial clapping rate

% initial maximum number of claps in a series

7

2.94

0.0053

7

0.59

0.77

7

2.93

0.0055

7

1.04

0.40

7

1.92

0.065

7

1.44

0.19

7

2.28

0.028

7

0.45

0.87

7

0.62

0.74

7

1.48

0.17

7

0.84

0.56

7

0.97

0.45

7

0.54

0.81

7

1.06

0.39

7

0.74

0.64

7

0.76

0.62

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

7

1.63

0.13

7

1.33

0.24

7

0.84

0.56

7

0.79

0.60

7

0.77

0.61

7

0.46

0.86

7

1.80

0.087

7

2.41

0.021

7

1.65

0.12

7

1.19

0.31

7

0.63

0.73

7

1.22

0.29

both control and cold-stressed scallops mainlained their valves
closed 4-7 min (Fig. 6A; Table .3). Towards the end of the obser-
vations, control scallops stayed closed longer than cold-stressed
scallops with this trend becoming significant at 156 h (f = 0.044).
As noted during the first experiment, control scallops recovered
more of their initial responses than cold-stressed scallops (Fig. 6B.
6C. 6D; Table 3). This difference was again particularly marked
for the percent initial number of claps (Fig. 6B). The overall re-
covery of control scallops was between 65-75'7r compared with
42-52% for cold-stressed scallops.

Evaluation of the Impact of Air Exposure

Statistical comparison of the escape performance (number of
claps, clapping time, clapping rate, etc.) of the control groups from
the two experiments did not reveal any differences. As the values
for each escape parameter were similar, with P-values ranging
from 0.12 to 0.94, 4 h of air exposure did not seem to affect the
escape response capacity of the scallops.

Sixty to ninety percent of the air exposed scallops responded to
the starfish within 5 sec of the initial contact whether they were

Cold Stress Slows Escape Responses of Juvenile Scallops

611

TABLE 3.

Statistical analysis of the effects of treatment and time on escape performance of juvenile sea scallops from Cold Shock with Air Exposure

Experiment. A 4 h air exposure during the transfer of scallops from pear! nets in the lagoon (15.5°C) to seawater at either 18°C (control) or

8°C (cold-stressed) in the laboratory was used to simulate the cold stress with air exposure accompanying seeding operations. Each scallop

(II = 24 per treatment) was measured during each sampling time (12, 24, 36, 48, 60, 84, 120, and 156 h).

Initial Response

df

Response After 15 min
of Recuperation

df

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

Treatment

Time

Treatment x Time

1
7

7

1

7

7

1

7

7

1

7
7

1

7
7

n.a.
n.a.

n.a,

n.a.
n.a.
n.a.

n.a.

n.a.
n.a.

2.85
2.93
1.20

7.78
6.60

4.13

1.89
3.86
4.80

3.20

2.03
2.06

0.11

2.98
3.28

n.a.
n.a.

n.a.

n.a.
n.a.

n.a.

n.a.

n.a.
n.a

0.010

0.0055

0.30

0.0077

<0.0001

0.0002

0.18
0.0005
<0.0001

0.080
0.051
0.047

0.74

0.0049

0.0022

n.a.
n.a.
n.a.

n.a.
n.a.

n.a.

n.a.

n.a.
n.a.

Numtier of claps

Clapping time

Clapping rate

Maximum number of claps in a series

Time spent closed

initial number of claps

% initial clapping ratef

initial maximum number of claps in a seriest

1

20.40

<0.0001

7

1.93

0.064

7

2.04

0.050

1

1.94

0.17

7

1.96

0.060

7

0.77

0.61

1

7.35

0.0094

7

1.03

0.41

7

0.78

0.61

1

15.94

0.0002

7

0.76

0.62

7

0.73

0.64

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

1

57.24

<0.0001

7

0.77

0.61

7

0.85

0.54

1

9.32

0.0038

7

3.24

0.0025

7

0.98

0.45

1

15.65

0.0003

7

0.75

0.63

7

1.10

0.36

n.a. = not applicable.

t observations were removed from the analysis as recuperation exceeded 200%.

cold-Stressed or not (Fig. 7C, 7D). Interestingly, scallops from the
Cold Shock Experiment (without air exposure) took more time
(5-15 sec) than those given 4 h air exposure before initiating an
escape response (Fig. 7A, 7B). Very few individuals (1 to 3 out of

24) required more than 15 sec following the initial contact with the
starfish.

In both experiments, control scallops swam in virtually all tests
(Fig. 8), while a greater proportion of cold-stressed scallops re-

TABLE 4.

Carbohydrate and protein contents in the adductor muscle, and muscle index of juvenile sea scallops that were used in repeated escape tests

or that remained inactive during the Cold Shock with Air Exposure Experiment. Mean (SE. ii). In a given line, values for treatments that

do not share the same letter differed (P < 0.05) according to Scheffe's multiple comparisons.

Control (I8°C) + Air

Thermally-stressed (8°C) + Air

Variable

Repeated Escapes Rest

Repeated Escapes Rest

Muscle indext

Carbohydrate concentration (mg • g"' wet tissue)

Protem concentration (mg • g"' wet tissue)

65.7(1.2.24)' 68.7(2.5,8)-'

10.6(0.8.24)-' 17.5(1.9,8)''

145.4 (2.0, 24)" 138.7 (3.8, 8)-'

66.4(1.6,24)-' 59.7(2.9,8)-'

11.1(1.1.24)-' 17.5(1.6,8)"

138.9(2.5.24)-' 139.6(5.3,8)°

t (M„„,„^(M,

digestive gland

■M,

oiher soft lissues

)) X 100.

612

LaFrance et al.

Initial response

Response after 15 min recuperation

0

Clapping time (min)
D
4

fi

I I I

4

-I- ^

50 E
40 -1 I
30
20
lo-

ll II

Clapping rate (no. of claps ■ min' )
40 F

tjH t }

30

20

10

0

{ I

16 iG

Maximum no. of claps in a series
12 nH

0

12 24 36 48 60 84 120 156

12 24 36 48 60 84

120 156

Time (h) after transfer

Figure 3. Effect of a transfer from 18°C to 8"C (thermal stress) vs. maintenance at 18 C (control) in seawater on the escape behaviors of juvenile
sea scallops, I'lacopecten magellaniciis. measured before and after 15 min of recuperation from exhaustive exercise. Means are shown for all
parameters. Vertical bars indicate 95% confidence limits and ;i = 24 for each treatment.

sponded with jumps. In the experiment with air exposure, jumps
were again more frequent for cold-stressed individuals, particu-
larly within the first 60 h.

Biometrics and Muscle Energetics

At the end of the Cold Shock with Air Exposure Experiment,
we compared shell characteristics and soft tissues of cold-stressed
and control scallops. Furthermore, we compared resting scallops
with those that had taken part in escape tests to assess the impact
of the repeated escape measurements on tissue masses and muscle

reserve levels. Shell mass was marginally smaller in resting scal-
lops (P = 0.040). However, we found no differences between the
adductor muscle mass {P = 0.67), digestive gland (P = 0.13) and
the mass of other soft tissues (wet and dry; P > 0.26) of scallops
that were induced to swim and those left undisturbed. Nonetheless,
the muscle index of resting scallops exposed to air prior to the cold
stress was slightly lower than that of control scallops {P = 0.05 1 )
(Table 4).

Muscle carbohydrates were decreased in scallops used for es-
cape tests (Table 4). The phasic muscle of resting scallops con-
tained about 18 mg of carbohydrates ■ g"' wet tissue in comparison

Cold Stress Slows Escape Responses of Juvenile Scallops

613

10
8
6

4 ]

2

0

Time spent closed (min)

Thermally-Stressed (8°C)

I

Control (18°C)

-1 1 r

% initial number of claps

% initial clapping rate

00 ^

C

.

90 -
80 -

70 -
60 "

1

1

1

<

1

1

. I
t

I !

! '

50 -

■'

'- .

1-

■

-

'-

n 1 \ 1 1 ] 1 1 1 1 1 r

12 24 36 48 60 84 120 156

Time (h) after transfer

Figure 4. Effect of a transfer from 18 C to 8 C (thermal stress) vs.
maintenance at 18 C (control) in seawater on (A) the time spent closed
after initial exhaustive stimulation and (B. C, D) performance after 15
min of recuperation relative to initial response for the main escape
behaviors of juvenile sea scallops. Placopecten magellanicus. Means
are shown for all parameters. Vertical bars indicate 95% confidence
limits and n = 24 for each treatment.

to 11 mg ■ g"' wet tissue for exercise(J scallops. Muscle carbohy-
drate concentrations did not vary with cold stress. The mean
muscle protein content was about 140 mg ■ g~' wet tissue (Table
4); it did not differ between control and cold-stressed scallops or
between the scallops that were forced to swim and those left un-
disturbed.

DISCUSSION

During seeding operations, juvenile scallops are often subjected
to a cold stress. Although sea scallops can live at low temperatures,
a rapid decrease in temperature will reduce the capacity to escape
predators or colonize suitable habitats. Our results indicate that
scallops cannot acclimate their clapping rate to such a cold stress
within 6 days and thus may he more vulnerable to starfish pieda-
tion. Indeed, the transfer of juvenile scallops from I8"C to 8°C
markedly slowed their rate of clapping and the maximum number
of claps in a series, but did not significantly alter the total number
of claps carried out during an escape response. Nonetheless, after
6 days at 8°C, scallops reduced the time spent closed after ex-
hausting escape responses and became similar to control scallops.
Over the 6 days of study, cold-stressed scallops reduced their
clapping time during an initial stiirulation and increased their ini-
tial clapping rate whereas control scallops remained quite constant.
Nevertheless, as the cold-stressed scallops did not attain the rates
typical of control scallops, only partial thermal compensation oc-
curred. Effectively, rate processes usually take considerable time
to acclimate when an organism is moved into a colder environment
(Bennett 1990). Dickie (1958) stated that the loss of the acclima-
tion to warm temperatures may require up to 3 mo in nature and
that sea scallops are extremely slow to recover their normal be-
havior when exposed to a sudden decrease in temperature.

A marked impact of cold stress was apparent in the recupera-
tion from escape responses. Cold-stressed scallops were less able
to recover their total claps and clapping rate than control scallops.
Fifteen minutes after exhaustion, scallops transferred to cold water
only recuperated 40-50% of their total claps relative to 60-70%
for the control scallops. In the Iles-de-la-Madeleine. predators ag-
gregate within three days after seeding of juvenile scallops (Cliche
et al. 1994). Since seed scallops are unlikely to have recuperated
from cold stress in such a short time, this may place them at a
disadvantage when faced with predators that elicit active escape
responses.

Temperature is likely to be a major determinant of predation
rate since it involves many behaviors (location, attack, capture, and
ingestion of prey) that are sensitive to temperature. Up to a certain
point, predator activity is known to increase with temperature. For
example, predation by starfish on 5-9 mm juvenile scallops shows
a (2,0 of 6.9 (between 4-15 °C) (Barbeau & Scheibling 1994). On
the other hand, scallop escape reactions also benefit from higher
temperatures (Barbeau & Scheibling 1994). Valve contraction rate
increases with temperature (Dadswell & Weihs 1990, Manuel &
Dadswell 1991, Olson & Marsh 1993). Sea scallops (5-35 mm in
shell height) increase their clapping rate between 9 and 14.5°C
(2,0 of 1.9) (Manuel & Dadswell 1991). The clapping rates of our
35^5 mm sea scallops gave Q,„ values of 2.4 (8-1 8°C) and 1.6
(8-15. 5°C) in our first and second experiments, respectively.

The thermal sensitivity of clapping rate may reside in any of its
components: latency period, time to peak force and time to relax-
ation (Olson & Marsh 1993). Thus, a greater clapping rate may
come from more rapid adductions, more rapid opening of the

614

LaFrance et al.

Initial response Response after 15 min recuperation

Number of claps before exhaustion
45 iB

40
35
30
25

'M\\\

Control(18X) + Air

Thermally - Stressed(8°C) + Air

15 ^

Clapping time (min)
3 D

2 ^

0

0

55 ^E

45

35 '

25 -
15 4

13

11

9

7
5

Clapping rate (no. of claps ■ min' )
50 F

G

T

r .

1 0 H p-

1 K5 H

1— 1-0 — m 1

n 1 r

:: t •

-<

1 ,

Maximum no. of claps in a series
H

9

T ■ ■

1 |.f-^r:;

1 1,

^^^

^:

' {{ill

3 4—. : : — r-

I

<i

(1

— 1 — 1 — 1

12 24 36 48 60 84

120

156

12 24 36 48 60 84

120

156

Time (h) after transfer

Figure 5. Escape response behaviors of juvenile sea scallops. Placopeclen magellaniciis. after 4 h air exposure and transfer to seav^ ater at 8°C
(thermal stress + air) or at 18 C (control + air) measured before and after 15 min of recuperation from exhaustive stimulation. Means are shown
for all parameters. Vertical bars indicate 95% confidence limits and n = 24 for each treatment.

valves (due to an increase of the hinge elasticity or to greater
power in the adduction), or a combination of these factors (re-
viewed by Manuel & Dadswell 1993). In some interactions be-
tween scallops and gastropod or starfish predators, the scallop's
response time was greater at a low temperature (Ordzie & Garofalo
1980). Since the liberation of active substances (e.g., saponins
(Mackie et al. 1968)) by predators and chemical sensitivity of the
scallop's tentacles may decrease with temperature, delayed preda-
tor detection may account for this longer latency period. Although
the sudden cooling reduced the scallops' escape responses, it did
not completely inhibit them. Hence, we did not observe the debil-

ity described by Dickie and Medcof (1963). In Dickie's work
(1958) with larger Placopecten magellanicus (80-100 mm), a drop
of 4°C to 7°C was enough to cause virtual immobility for pro-
longed periods, with scallops keeping their valves almost closed
with the tentacles only slightly extended for periods as long as 40
d. The smaller sea scallops we worked with were probably more
active. Effectively, swimming is more frequent among younger
and medium-sized scallops and rarely seen in >70 mm scallops
(Dadswell & Weihs 1990). Further research should assess the re-
sistance of different size classes to tluctuating temperatures
(Dickie & Medcof 1963).

Cold Stress Slows Escape Responses of Juvenile Scallops

615

Time spent closed (min)

10
8

6 ^
4

2 -\
0

Control(18°C) + Air

Thermally-Stressed(8°C) + Air

~i 1 1 1 I I I i r

% initial number of claps

% initial clapping rate

% initial maximum number of claps in a series

110
100

90

80

70 -

60 -

50

40

30

— 1 — I — I — I — I — I — I — I — I — I — I — I — I I
12 24 36 48 60 84 120 156

Time (h) after transfer

Figure 6. Escape response behaviors of juvenile sea scallops, Pla-
copecten magellanicus, after 4 h air exposure and transfer to seawater
at 8 C (thermal stress + air) or 18°C (control + airl. (A) Time spent
closed after initial exhaustive stimulation and (B, C, D) performance
after 15 min of recuperation relative to initial response for the main
escape behaviors. Means are shown for all parameters. Vertical bars
indicate 95% confidence limits and » = 24 for each treatment.

Starfish from two thennal regimes were used to provoke escape
responses. They were put in tanks at 8°C or 18°C the day before
the first escape test. Although we manipulated the predator arms to
stimulate the scallops, the amount of chemical cues emitted by
cold-stressed and control starfish may have modified escape re-
sponses. However, no differences were detected in the response
times of scallops at 8°C and 18"C.

In the Cold Shock with Air Exposure Experiment, the differ-
ences between control and cold-stressed scallops were smaller than
those in the Cold Shock Experiment. The drop of water tempera-
ture in the lagoon from 1 8°C to 15.5°C (September 23-26; see Fig.
2) may have initiated acclimatization to cold water in the scallops
remaining in pearl nets. Thus, a transfer to 8°C would have been
less of a stress during the second than the first experiment. This
suggests that seeding juvenile scallops later in fall when thermal
shock is reduced could reduce losses from predation.

The 4 h air exposure seemingly had no effects on the escape
parameters of the two groups of control scallops. This is consistent
with previous studies of Pecten niaximus showing no significant
differences between 0 and 3^ h of air emersion on various as-
sessments of scallop's quality (Maguire et al. 1999b. Christo-
phersen 2000, Minchin et al. 2000). Also, our preliminary inves-
tigations showed that the righting time of juvenile sea scallops was
not significantly prolonged by 4 h emersion (unpublished data).
The 4 h emersion period of juvenile scallops during their transfer
from the lagoon to open sea seems not to be an issue in terms of
vulnerability to predation.

Air exposure for 4 h led the scallops to react more quickly to
contact with the starfish. The occurrence of a stress such as air
exposure, even if it may not be injurious, may increase the general
excitability (Wilkens 1981). Scallops held in laboratory conditions
react more rapidly to any perturbation during the first days of their
captivity (unpublished data). As contact of a starfish near the re-
gion of the dorsal ears often triggers a swimming response (Wilk-
ens 1981), we consistently stimulated in this region, making it
unlikely that, the greater occurrence of jumps in cold-stressed
scallops was due to differences in our method of stimulation.

Total tissue mass of the control scallops was similar to that of
cold-stressed scallops in the air exposure experiment. This variable
was not affected by thermal regimen when juvenile sea scallops
were subjected to constant ( 10 'C) or Ouctuating temperatures (6-
I5°C. 8 d temperature cycle) during 48 d (Pilditch & Grant 1999).
The muscle carbohydrate content of resting scallops from the Cold
Shock with Air Exposure Experiment did not differ from that
obtained for cultured scallops of the same size, same origin and
under similar grow-out conditions (Lafrance et al., submitted).
Thus, a 4-h air exposure did not influence muscle carbohydrate
levels. The muscle of scallops that performed escape responses
contained carbohydrate levels 40% lower than that of resting scal-
lops. Thus, repeated exhausting escapes markedly reduce the scal-
lop's energy reserves in an environment with no food. Extrapolat-
ing results from laboratory to field is risky since laboratory con-
ditions can lead to abnormally high levels of stress (Pilditch &
Grant 1999). Nonetheless, repeated attacks by predators on the sea
bottom, even if not successful, could make scallops more vulner-
able to other stresses (sustained food shortage, diseases, etc.).
Hence, predator elimination (Ventilla 1982) on seeding grounds
should be considered to increase their chance of survival.

The metabolism of Placopecten mageUaiiicits is tightly coupled
to thermal fluctuations, as shown by Pilditch and Grant (1999) who
observed that metabolic rates did not acclimate to thermal oscil-

616

o

A Control ( I ,S°C)

LaFrance et al.

B Thennally-Stressed(8°C)

1(1(1 -

12 24 36 4ti 60 84 120 156

12 24 36 48 60 S4 120 156

D + 30 sec

15-30. sec

C Control(18°C) + Air DThcrmaliy-Stressed(8°C) + Mr Q 5-15. sec

100

0-5 sec

36 4S 60 84 120 156

12 24 36 48 60 84 120 156

Time (h) after transfer

Figure 7. Effect of a thermal stress (transfer from -18 C to 8 C") and a 4 h air exposure on ttie delay before initiation of escape reaction of
juvenile sea scallops stimulated by contact with a starfish. One-hundred percent of scallops corresponds to ;; = 24 for each treatment from (A,
B) Cold Shock Experiment and (C'.Dl Cold Shock with Air Exposure Experiment.

latioiis between 6'C and 13 C during 6 x 8 d cycles. On the other
hand in our study, temperature only decreased at the start of our
observations. During long term exposure to low temperatures,
scallops are likely to acclimate and at least partially compensate
for the decrease in temperature, as shown by the changes in mem-
brane fluidity during thermal acclimation of P. iiiagellcmiciis (Na-
politano et al. 1992). In a study of the time course of changes in
membrane fluidity during transfer of f. mas^eUaniciis from 13°C to
5°C. compensation of membrane fluidity through increases in the
proportion of polyunsaturated fatty acids required 1.3 lo 21 days
(Hall 1999).

This study provides information that should help planning of a
crucial step in bottom culture of sea scallops. A 4-h air exposure

does not enhance the impact of cold shock on escape responses of
Pkicopeclen inagellanicus. While it has been argued thai seeding
of juvenile scallops should be performed at cold temperatures to
decrease predation (Barbeau & Scheibling 1994), cold tempera-
tures also reduce the scallop's viability in terms of escape behav-
ior. As the scallop's escape capacities require more than 6 days
before complete themial acclimation, seeded juveniles may benefit
from operations minimizing thermal shock or from the use of an
acclimation period with no predators at intermediate seawater tem-
peratures prior to seeding operations. This need for an acclimation
period agrees with the recommendation of Barbeau et al. 1 1994).
However, since crab attacks overwhelm the anti-predator defenses
of iuvenile scallops at virtualh' all temperatures (Barbeau &

Cold Stress Slows Escape Responses of Juvenile Scallops

617

ex

a

u

u

Oh

c
o

o

Q.
O

■ Control (18°C)

n Thermally-Stressed (8°C)
D Control ( 1 8°C) + Air

■ Thermally-Stressed (8°C) + Air

12

24

36

48

60

84

120

156

Time (h) after transfer

Figure 8. Influence of a thermal stress (transfer from -18°C to 8"C) and a 4 h air exposure on the proportion ( % ) of juvenile sea scallops using
swimming behavior as a means of escape from a starfish. A "swimmer" was defined as a scallop that made at least 3 claps during most series
of claps during the stimulation (evaluated on the response after 15 niin recuperation), n = 24 for each treatment from Cold Shock Experiment
and Cold Shock with Air Exposure Experiment.

Scheihling 1944. Barbeau et al. 19%). such considerations would
not be relevant in an environment dominated by crabs.

ACKNOWLEDGMENTS

This study was supported by funds from MAPAQ (Ministere de
TAcriculture des Pecheries et de TAlimentation du Quebec) to M.

Lafrance and H. Guderley and by an operating grant from the
NSERC to H. Guderley. The authors thank the staff of the Station
Technologique Maricole des iles-de-la-Madeleine of MAPAQ for
assistance. Special thanks are extended to C. Cyr for technical
help, to H. Paradis and G. Daigle for their statistical support, and
to J. H. Himmelman and B. Myrand for their insightful comments
on the manuscript.

LITERATURE CITED

Barbeuu. M. A.. B. G. Hatcher. R. E. Scheihling. A. W. Hennigar. L. H.
Taylor & A. C. Risk. 1996. Dynamics of juvenile sea scallop (P/k-
copecten magelUmiciis) and their predators in bottom seeding trials in
Lunenburg Bay, Nova Scotia. Can. J. Fish. Aquat. Sci. 53:2494-2.'il2.

Barbeau. M. A. & R. E. Scheihling. 1994. Temperature effects on predation
of juvenile sea scallops (Placopecten magellunicus (Gmelin)) by sea
stars (Asterias vulgari.s Verrill) and crabs {Ciiini'r inciniliis Say). ./.
E.xp. Mar. Biol. Ecol. 182:27-17.

Barbeau. M. A.. R. E. Scheihling & B. G. Hatcher. 1994. Effect of prey
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Joiirmil of Shellfish Research. Vol. 21, No. 2. 619-625. 2002.

SELECTIVE INGESTION OF PELAGIC VERSUS BENTHIC ALGAE BY THE COCKLE

CERASTODERMA EDULE (LINNE, 1758)

JOSE L. RUEDA'* and AAD C. SMAAL'

' Marine and Estuaiine Ecology Unit. Department of Zoology and Entomology. The Univenfity of
Queensland. Brisbane Qld 4072, Australia: ^Netherlands Institute for Fisheries Research (RIVO), P. O.
Box 77. 4400 AB Yerseke. The Netherlands

ABSTRACT The pre-ingestive selection of microphytobenthic algae by the cockle Cerastoderma ediile was studied in comparison
with diets containing the pelagic diatom Phaeodacryliim tricornutum. Treatments with the different diets covered a range of seston
concentrations and organic content similar to field conditions. Rejection rates of C. edule exposed to the different treatments were
significantly correlated with the concentration of total particulate matter. No significant differences in total rejection rates were found
between pelagic and benthic diets. Organic rejection rate was significantly correlated with particulate organic matter of the treatments
and no significant differences were found between both diets. Selection efficiency was significantly correlated with particulate organic
matter concentration In both diets and no significant differences were found between the diets. Analysis of the pseudofeces composition
by fiow cytometry from cockles exposed to a mixed diet of microphytobenthic algae and P. iriconuilum. showed a preferential
ingestion of the pelagic diatom. Benthic species, such as small pennates and Navwula sp., were preferentially Ingested In comparison
to larger microphytobenthic species. The largest microphytobenthic species, Cylindrotlxeca sp., was significantly rejected. In general,
C. edule Is an opportunistic filter feeder that takes advantage of both pelagic and benthic algal cells.

KEY WORDS: Benthic diatoms, cockles, fiow cytometry, rejection, selection efficiency, Cerastoderma edule

INTRODUCTION

Filter feeding bivalves are able to sort particles with their gills
and labial palps (Kiorboe & M0hlenberg 1981, Newell & Jordan
1983, Newell et al. 1989, Ward et al. 1993) resulting in preferential
ingestion of organic material and rejection of inorganic particles in
pseudofeces. This preingestive selection has also been observed
within phytoplankton with preferential ingestion of some groups of
algal cells, both benthic and pelagic (Shumway et al. 1983, Bou-
grier et al. 1997, Baker et al. 1998). Preingestive selection of
particles may be influenced by Iheir chemical composition or by
their size and shape.

Some studies about preingestive selection in bivalves have
been carried out by using microscopic techniques to compare wa-
ter and biodeposits or stomach contents in different species of
deposit or filter feeder bivalves (Hummel 1985, Kamermans 1994,
Shumway et al. 1987). Flow cytometry offers opportunities for the
analysis of algal cells in experiments on the feeding behavior of
bivalves (Shumway et al. 1983, Bougrier et al. 1997. Baker et al.
1998). Differential selection of various algal species has been dem-
onstrated, using flow cytometric techniques by Peirson ( 1983) in
the scallop Argopecten irradians (Lamarck 1819) and by Shum-
way et al. (1985) in Ensis direetus (Conrad 1843). Ostrea edulis
Linne, 1758, Plaeopecten magellanicus (Gmelin, 1791) and Arc-
tica islandica (Linne, 1767). Shumway et al. (1985) showed that
the flat oyster Ostrea edulis fed with a mixture of three algae of
similar size (Phaeodacryliim tricornutum. Prorocentrum minimum.
and Chroomonas salina) preferentially ingested the dinotlagellate
Prorocentrum minimum. In contrast, Cucci et al. (1985) have
shown that individuals of MytUus edulis Linne, 1738 fed with a
mixed diet of the diatom Phaeodactylum triconuitum. the di-
noflagellate Prorocentrum, and the cryptomonad flagellate
Chroomonas .udina. showed no differential ingestion of cells from
suspension. Bougrier et al. (1997) found differences in the pre-
ingestive selection of different algal cells (mainly pelagic) by

*Correspondlng author. E-mail; J.rueda@zen.uq.edu.au

Mytilus edulis and Crassostrea virginica (Gmelin, 1791) and re-
lated to differences in the shape of the algal cells. Recently Loret
et al. (2000) have shown preferential ingestion of nanoflagellates
and cryptophytes by the bivalve Pinctada margaritifeni (Linne,
1758), by applying in situ HPLC techniques.

There is some controversy about the feeding behavior of bi-
valves, especially C. edule, exposed to benthic algae compared
with pelagic algae, or the importance of microphytobenthos as a
food source. Kamermans ( 1994) found microphytobenthic algae in
the stomachs of the filter feeding bivalves Mya arenaria (Linne,
1738), C. edule and M. edulis, although the percentage of their
abundance compared with pelagic algae, was higher in the deposit
feeders Scrobicularia plana (Da Costa, 1778) and Macoma hal-
thica (Linne. 1738). Recently two studies have been done measur-
ing ratios of C and N isotopes in different primary producers and
benthic invertebrates (Kang et al. 1999, Riera et al, 1999). These
studies concluded that C. edide may prefer to ingest microphyto-
benthos and phytoplankton compared with detritus from fragments
of benthic macro-algae and seagrasses or detritus from other ori-
gins. However, Herman et al. (2000), using also stable isotopes
ratios, suggested that suspension feeders, such as C. edule and M.
arenaria, depend almost exclusively on pelagic rather than micro-
phytobenthic algae.

The aim of this study is to quantify the pre-ingestive selection
of microphytobenthic algae in comparison with a pelagic species
by the cockle Cerastoderma edule. A mixture of benthic and pe-
lagic micro-algae occurs in the tidal flat environment of this filter
feeder bivalve. The response of cockles exposed to the different
diets was studied separately and also when both diets were mixed
in the water column. In the latter case analysis were done by flow
cytometry to analyze differential algal cell acceptance or rejection.

MATERIALS AND METHODS

Animals

In total 187 cockles (Cerastoderma edule) were used in the
different experiments. Cockles had a shell length of 29,78 mm ±

619

620

RUEDA AND SMAAL

1.27 (mean ± standard ernir) in experiments with microphyto-
benthos and 24.75 mm ± 0.46 in e.\periments with Phaeodactylwn
trkdnuttiimy. ash free dry weight was 255.75 mg ± 30.15 in ex-
periments with microphytobenthos and 259.87 mg ± 20.55 in ex-
periments with P. irkonwtum. No significant differences were
found in shell length or ash free dry weight of the animals used in
both experimental treatments. All individuals were collected from
an intertidal mudtlat situated in the Oosterschelde estuary (SW
Netherlands). Experiments were performed in spring 1997 and
summer 1999, in a field laboratory at Jacobahaven (Oosterschelde
estuary — SW Netherlands).

Shell length, dry tissue weight (DW) and ash-free dry tissue
weight for each animal were determined. After measuring the shell
length, animals were immersed in boiling water until the shell was
opened. Soft tissues were excised, dried at 70°C for minimum 48
h in a drying oven and weighed (= dry weight). Ash-free dry
weight is determined as the weight loss after ignition in a furnace
at 520"C for 3 h.

Diets and Treatments

Two different diets were used in the different experiments:
microphytobenthic algae (Benthic) and cells of Phaeodacr^htin
triconuiliiiii (Pelagic). Microphytobenthic algae were collected in
the Oosterschelde estuary. The top layer of sediment (2-3 mm)
was sampled and after sampling, coarse sediment and microphy-
tobenthos were separated by mixing them in filtered seawater.
After sedimentation of the large particles the water containing
mainly benthic algae (based on observations with microscopy) was
used for the experimental treatment. The microphytobenthic spe-
cies were benthic diatoms, including species from the genus Nitzs-
chia (length 61 ± 1 1 jxm; width 7 ± 1 ixm), Naviciila (length 46 ±
1 1 p.m: width 22 ± 3 \xm). Cytiiutiothecu (length 81 ± 5 (xm: width
23 ± 2 |xm), and small pennate cells (length 30 + 2 jjim; width 6
± 1 fj.m). The pelagic diatom P. iricunuitiiiii was cultured outdoors
in 100-1 reservoir tanks.

Pelagic and Benthic algae were added separately to filtered
seawater in different concentrations (treatments) for both diets
(Table 1 l. Each treatment was completed by adding silt particles
collected in the same location as cockles and microphytobenthos.
This silt was dried and burned at 520"C during 4 h for removing
the organic content. A coulter counter was used to determine par-
ticle concentration, which was monitored during the experiment
for differences of the particle concentration from the experimental
treatments. Total particulate matter in the different experiments
ranged from 40.2 mg.r' to 161.2 mg.l"' and the organic content
ranged from 9.08^-58.91% (see Table 1).

Analysis of Particulate Matter

Water samples of the different treatments were collected from
the control chamber (without animal) at the start of the experiment
and before collection of the biodeposits. Total particulate matter
(TPM; mg.r') was determined by filtering one liter sample onto
pre-weighed and pre-ashed Whatman GF/C filters, rinsing with
ammonium formate and drying at 70°C (minimum 24 h). Particu-
late inorganic matter (PIM: mg.l"') was estimated from the same
filters as the weight after ignition during 3 h at 450'C. Particulate
organic matter (POM) resulted from the difference of TPM and
PIM. In all weight measurements an electronic balance was used
which determined the nearest 0.01 mg. Dietary composition was

TABLE 1.

Characteristics of the different experiments f(ir each diet and

treatment. Concentrations of total particulate matter (TPM:

my • I 'l. particulate ornanit matter (POM: mg • I"'), particulate

Inorganic matter (PIM: mg • !') and percentage of organic matter

from the seston ('> OMi.

Treatment

TPM

POM

PIM

OM

code

(mg-!"')

(mg-l"')

(mgl"')

(%)

Diet with Ph,

[U'iitliutyhtni inc

>niuUini

IP

4S.y(l

4.44

44.46

9.08

^P

55.32

29.56

25.76

53.43

3p

58.82

8.18

50.64

13.91

4p

83.64

24.88

58.76

29.75

5P

92.40

17.00

75.40

18.40

6p

95.28

32.32

62.96

33.92

7p

125.84

15.04

110.80

11.95

Xp

\5234

17.66

134.68

11.59

Diet with Microphytobenthos

Im

48.68

28.68

20.00

58.92

:m

53.92

10.10

43.82

18.73

3m

72.52

13.76

58.76

18.97

4m

76.44

30.72

45.72

40,14

.sm

80.96

28.04

52.92

MM

dm

87.48

34.24

53.24

,^9.14

7ni

98.40

20.98

77.42

21.32

Hm

161.20

37.92

123.28

23.52

Mixed diet

Imx

5 1 .56

20.80

30.76

40.34

2mx

59.52

17.42

42.10

29.27

.Imx

28.05

5.87

22.18

20.92

characterized in terms of organic content by weight (9cOM =
POM X 100/TPM). POM and TPM.

Experiments with Separate Diets

A flow-through system was used in all experiments. The water
containing the different treatments was pumped to the experimen-
tal chambers from a diet-reservoir (300 L) in which a mixer and an
air pump promoted the resuspension of particles. Peristaltic pumps
(Watson Marlow) were used for pumping the water to the indi-
vidual experimental chambers. The animals were placed individu-
ally in chambers of 300-ml volume and one chamber was used as
a control for sedimentation of particles. The flow in all chambers
during the experiments was between 3 and 4 1/h. and the concen-
tration of particles in the outflow of the chambers containing ani-
mals was always more than 70% compared w ith the control cham-
ber (without animals).

Collection of pseudofeces started after an acclimation period of
3 h to each treatment. Pseudofeces rejected during 3-h periods
were collected 2 to 3 times separately for each individual. Pseu-
dofeces samples were filtered on preweighed Whatman GF/C fil-
ters and measured according to the same procedure as described
for samples of the diets. Rejection rates (mg.h"') of total (RRl,
oi-ganic (ORR) and inorganic (IRR) particulate matter were calcu-
lated. Rates were standardized to an equivalent 500 mg ash free
dry tissue cockle by calculating the expression Ys = Ye (0.5AVe)''.
where Ys: rate of standard-sized cockle: Ye: uncortected physi-
ologic rate; We: measured ash free dry weight of experimental
animal: b: allometric coefficient for clearance rate of cockles (b =
0.57; Umjtia 1997, Smaal et al. 1997).

Selective Feeding in Cerastoderma edule

621

Selection efficiency (SE) was calculated using the values for
organic fraction of seston (f = POM/TPM) and pseudofeces (p =
ORR/RR) from the different experiments. Values were calculated
as: SE = (l-(p/f)) x 100.

Experiments with a Mixed Diet

In these series of experiments, cockles were exposed to a mixed
diet and the same flow through system described in the previous
paragraph was used. The diet contained a mixture of microphyto-
benthic algae and the diatom P. triconuttum (si/e 17 ± 2 ixm). The
different size of this pelagic species, and its fluorescence charac-
teristics make it clearly distinguishable from the microphyto-
benthic species used (sizes between 30 ± 2 p.m and 81 ± 1 1 iJim).
Individuals were acclimated 3 h to the mixed diet and pseudofeces
produced by each animal were collected during periods of 1 h.
Sample analysis by flow cytometry was done directly after the
experiment. Another series of samples of pseudofeces was col-
lected in the experiment for selection efflciency measurements.

Algal composition of diet and pseudofeces was analyzed using
a flow cytometer (EurOPA: European Optical Plankton Analyser).
Standard beads (1.07 |jim, Duke Scientific, USA) were used for
calibration and optical adjustments of the EurOPA instrument. A
529 nm and 633 nm laser were used for excitation. Laser light is
scattered when a particle traverses the laser beam and is measured
in forward and perpendicular (PLS) direction. Laser light irradiat-
ing autotrophic phytoplankton is partly emitted as fluorescence.
Fluorescence emission excited by the green laser is measured in
the red (FOR) and orange (FGO) bandwidth. The number of par-
ticles processed in the flow cytometric analysis was 5.000 and
20,000. Only data derived by FGR-triggering (i.e.. fluorescent
particles) were used to distinguish between groups of particles
with different optical characteristics. The grouping or clustering of
data was calculated by using the software program Matlab ver-
sion 1.0.

The algal species studied are easy to distinguish in the scatter
plots of the graph representing FOR and PLS data. The star-shaped
form of Phaeodactytwn tricomutum occupied a large area with
values of PLS. between 1.500 and 2.200. and values of fluores-
cence FOR in general below 2.200 and above 1.900. Microphyto-
benthos were composed with mainly four subgroups (related to the
most abundant species). The benthic diatom species with lower
FOR and lower PLS was Navicula sp.. together with a group of
small pennate cells, which could not be identified with values of
PLS (1.800-2.300) and FGR (1.900-2.200). Nilzdmi species had
a higher PLS value (2,400-2,500) and higher FGR (2,500-2.700).
Cylindrotheca species displayed the highest PLS (almost 2.500)
and the highest FGR (almost 3.000).

To examine the degree of acceptance or rejection of particle
types (pelagic or benthic algae), we calculated an electivity index
(EI) (Jacobs et al. 1974). modified by Baker et al. (1998), as
follows:

EI = -[(P - S) / ((P -H S) - (2 * P * S))]

where P is the particle ratio in the pseudofeces and S is the particle
ratio in the suspension. Electivity index can range from -1.0 to 1.0.
A positive El for a given particle type indicates preferential inges-
tion (depletion of the particle type in the pseudofeces compared
with the suspension), and a negative EI indicates rejection (enrich-
ment of a particle type in the pseudofeces compared with the
suspension).

Subsaniples of the diet and pseudofeces were also fixed in
LugoPs and the algal composition was determined using micro-
scopic techniques for determination in addition to the flow cytom-
eter results.

Statistics

All statistical tests were performed using the program SYSTAT
for PC version 9.0. Multiple stepwise regression analyses were
used to test for significant relationships between physiologic rates
and parameters of the treatments. Analysis of covariance was used
for comparing the rejection rates and selecdon efficiencies of
cockles with the different diets. Electivity indices were compared
with zero using a one-sample, two-tailed, nonparametric Wilcoxon
signed-rank test. These analyses test the null hypothesis that se-
lectivity of a particular type is equal to zero (no sorting). A t-
student test was used to compare concentrations of the different
algal species in samples of the diet and pseudofeces.

RESULTS

Response to Diets

Data about quality and quantity of the experimental treatments
are shown in Table I . All treatments were above pseudofeces
threshold and the obtained physiologic values with the different
diets and treatments have been listed in Table 2. Multiple stepwise
regression analyses indicated a significant positive relationship
between rejection rate (RR) and total particulate matter (TPM)
from the treatments of both diets (Fig. 1; Table 3). The model
rejected parameters POM and %0M. Analysis of covariance in-

TABLE 2.

Physiological response of cockles with the different diets and

treatments: Selection efficiency (SE: %), rejection rate of

pseudofeces (RR: mg • h"'), organic rejection rate (ORR: nig • h ')

and inorganic rejection rate (IRR: nig • h"'). Mean value ± standard

error. Number of measurements (n).

Treatment

SE

RR

ORR

IRR

code

(%)

(mg- h~')

(mg-h')

(mg- h"')

n

Diet with Phueodactyhim

tricornumin

IP

4.9 ± 6..S

29.3 ± 1.7

2.6 ± 0.3

26.8 ±1.4

6

2p

46.1 ±4.3

38.3 ± 7.2

10.8 ±2.0

27.5 ±5.5

10

3p

13.9 ±9.4

22.3 ± 6.2

2.4 + 0.7

19.9 ±5.5

6

4p

36.8 ± 9.3

24.3 ±5.0

5.9+ 1.9

I8.4±3.1

12

^P

9.4 ±2.5

54.3 ± 5.5

8.9 ±0.7

45.3 ± 4.9

16

6p

4I.9±2.2

45.8 ± 2.7

9.1 ±0.6

36.7 ± 2.3

12

7p

26.1 ±3.4

96.4 ±8.7

8.4 ± 0.6

88.0 ±8.1

6

8p

14.5 + 2.6

80.3 + 5.5

8.0 ±0.7

72.3 ±4.9

10

Diet with Mi

icrophytobenthos

Im

31.9 + 4.1

34.7 + 2.9

13.8 ± 1.1

20.9 ± 2.2

12

:ni

18.7 ±y.4

17.2 ±3.1

2.6 ± 0.3

14.6 ±2.9

6

3ni

13.1 ±9.6

27.4 ±4.9

4.3 ± 0.6

23.2 ±4.4

6

4m

34.9 ± 2.4

42.7 ± 1.6

11.1 ±0.4

31.6± 1.5

15

."^m

34.3 ± 2.5

70.9 ± 4.5

16.2 + I.I

54.7 ± 3.7

12

6m

43.1 +3.5

47.9 + 6.4

10.1 ±0.9

37.7 ± 5.7

10

7 111

22.5+ 1.9

50.8 ± 6.8

8.2 ± 1.0

42.7 + 5.8

16

8ni

37.5 ± 7,7

41.0 ± 13.2

1 3.6 ±0.2

77.4 ± 13.0

6

Mixed diet

Imx

25.1 ±4.4

-

-;

-

10

2mx

15.6 ±3.5

-

-

-

6

3mx

6.6 ± 1.6

-

-

-

8

622

RUEDA AND SmAAL

100

o 40
20

50 100 150

Total particulate matter (mg. 1'^)

Figure 1. Rejection rate (mg ■ h ') of pseudofeces as a function of tfie
seston concentration in Ijoth diets. Pelagic: Results for treatments with
Phaeodaciyliiiii IrkornMiim (line). Bentliie: Results for treatments with
microphytohenthos (dotted line). Vertical bars indicate standard er-
ror. Sec Table 2 for number of measurements.

obtained with the benthic diet, analysis of covariance indicated that
there were no significant differences between diets (/t/ = 0,791; P
>0.05).

Response to a Mixed Diet

Selection efficiency values as a function of POM concentration
(see Fig. 3) were generally lower for the mixed diet of pelagic and
benthic cells. Higher values of SE were registered at higher values
of particulate organic matter.

Flow cytometer results showed significantly lower cell concen-
trations (expressed as '/r-age) in pseudofeces in comparison to the
diet composition (Fig. 4). and therefore significant positive dec-
livity indices (EI) (acceptance). For both diets, differences of algal
concentration in pseudofeces and diet were significant using a
t-student test (Pelagic: DF = 12, t = 14.09, P < 0.001; Benthic:
DF = 12. t = 4.86. P < 0.01 ). However, the EI of cockles was
significantly higher for pelagic (EIp^.,^,g,^. = 0.31 ± 0.01, mean ±
standard error) than for benthic cells (ElB^,„,h,c = 0.20 ± 0.03,
mean ± standard error), indicating a preferential acceptance of the
Pelagic rather than the Benthic component of the mixed diet, A
lower EI for the benthic species was due to differences in the
acceptance or rejection of the different benthic species within the
Benthic group (Fig, 5). The smallest benthic species, such as Na-
viciila sp. and the group of small pennates, were significantly
ingested, displaying higher EI values than larger species such as
Nilzchia sp. However, Cylindrotheca sp.. the largest species, was
significantly rejected, resulting in a negative EI value.

dicaled that there were no significant differences between the diets
(N = 0.2.';6; P>0.05).

Organic rejection rates (ORR) were only significantly corre-
lated to the particulate organic matter (POM) (Fig. 2, see Table 3),
with TPM and %OM as nonsignificant parameters related to ORR.
According to the regressions obtained in each case no significant
differences were found between both diets (/t/ = 1 . 1 36; P > 0.05 ).

Multiple stepwise regression analysis indicated a significant
positive relationship between selection efficiency (SE) and par-
ticulate organic matter (POM) (after log transformation) in both
diets (Fig. 3 see Table 3). Although lower values of SE were

TABLE 3.

Stepwise multiple regression analyses of physiological parameters of Cerastoderma cdiile with total particulate matter (TPM: mg • T ).
particulate organic matter (POM: mg • I'), particulate inorganic matter (PIM: mg ■ r') and percentage of organic matter from the different

diets (% OM). se: standard error.

DISCUSSION

Production of pseudofeces of cockles was significantly related
to the seston concentration in both diets. This relationship between

Pelagic diet

Benthic diet

Coefricient

Coefficient

Rejection rate (RR)
Terms retained

Constant 7,97

TPM 0.64

Terms rejected

POM

% OM

TPM X POM
Organic rejection rate (ORR)
Terms retained

Constant 2.45

POM 0.24

Terms rejected

TPM

% OM

TPM X POM
Selection efficiency (SE)
Terms retained

Constant -28.85

Log POM 19,92

Terms rejected

Log TPM

Log <7r OM

0.73

0.16

<0,01

<0.01

-1.67

<0.0I

0..S8

0.72

0.14

<0.01
<0.01

<0.0]

0.63

0.(J8

<().().5

<0.01

-0.27

<0.05

0.41

0.68

0. 1 1

<0.()l
<().01
<0.01

0.67

5.46

<0.0I

<0.01

-33.49

<0.01

19.94

0.82

3.85

<().()0.S
<0,()(1.S
<0.005

Selective Feeding in Cerastoderma edule

623

20 T

U Pelagic

I

115

£
n

c
5 10

O Benthic

o

"S.J^.

r

o

S 5

^^^^"'"'^ 1 1

o

- i°

0 10 20 30

40

Particulate organic matter (mg. 1'^)

Figure 2. Organic rejection rate of pscudofeces (nig • h ') as a function
of the organic seston concentration in botii diets. Pelagic: Results for
treatments with Pliaeodactyltiin tricornutum (line). Benthic: Results for
treatments with microphytobenthos (dotted line). Vertical bars indi-
cate standard error. See Table 2 for number of measurements.

60 .

QDiet

40

D fteudofaeces

Percentage

O

T

1

T

i

Pelagic

Algal cell

Benthic

Figure 4. Percentages of the different algal cells, from the total num-
ber of particles, both in the diet and the pseudofeces. Pelagic: Pliaeo-
dactylum trkontiitum. Benthic: Microphytobenthic algae Vertical bars
indicate standard error. In all measurements n = 14.

RR and the seston concentration has been observed in several
studies and rates were comparable with our outcomes (Iglesias et
al. 1992. Navarro & Widdows 1997, Urrutia 1997). Similarly, the
rejection rate of organic matter increased with organic content of
seston. At a constant clearance rate, filtration rate increases at
increasing seston concentrations and the digestive system reaches
maximum capacity, hence pscudofeces production increases.

Together with the production of pseudofeces, selection of or-
ganic material occurs prior to ingestion. Selection efficiency (SE)
values found in our experiments were very similar to values docu-
mented for C. edule under comparable conditions of food quality
(Iglesias et al. 1992, Urrutia 1997). The response by the cockle to
the pelagic diet did not differ significantly from the benthic diet.
The SE maximizes in C. edule as a response of an increment in the
organic content in the diet (Iglesias et al. 1992. Urrutia 1997)
reaching a maximum value in our study of 409}-. This maximum
value of gross SE (measurements based on the organic content of
pseudofeces) for the cockle is similar to those obtained in previous

60

■ Pelagic

r

efficiency (%)

O Benthic

[

o

T

.^

Selection

1

'

1 10 20

30

40

-10 J

Particulate organic matter (mg.

■')

Figure 3. Mean values of selection efficiency ( % ) as a function of the
organic seston concentration in each dietary condition. Pelagic: Re-
sults for treatments with Phaeodaclyliim tricormilum (line). Benthic:
Results for treatments with microphytobenthos (dotted line). Mix: Re-
sults for treatments with a mixed diet. Vertical bars indicate standard
error. See Table 2 for number of measurements.

studies using pelagic algae as a food source (Iglesias et al. 1992.
UiTutia 1997). The percentage of organic matter in the different
treatments covered a range between W7c-6Q%. Several authors
(Iglesias et al. 1992, Urrutia 1997) have described a decrease of the
SE in C. edule exposed to treatments above 50% organic content.
The decrease of SE values obtained in treatments containing
Benthic algae could be related to a lower acceptance or the pref-
erential rejection of some benthic species as it has been found with
Nitzchia sp. and Cylindrotheca sp.

In this study, we have documented the selection and acceptance
of microphytobenthos as a benthic food source by the cockle.
However, pelagic algae were accepted preferentially when mixed
with microphytobenthic species. Moreover, only some microphy-
tobenthic species were preferentially accepted by C. edule. Micro-

X

CD
■D

_C

>^

-^

o

LU

O '

a
ro

Q.

o
o

<

c
o

o

0)

Figure 5. Electivity indices (El) for the different microphytobenthic
species, as separate species or as a group, relative to the total concen-
tration of particles. The studied species are small pennate cells (SmP),
Navicula sp. (Nav), Nitzchia sp. (Nit), Cylindrotheca sp. (Cyl) and the
benthic group (All), which includes all microphytobenthic species.
Vertical bars indicate standard error. * Indices significantly different
than zero (f < 0.05). In all measurements n = 14.

fi24

RUEDA AND SMAAL

phytobenthos seems Ui represent a food source in several species
of bivalves and its importance for filter feeding bivalves has been
addressed previously. Kamemians ( 1994) found relutii>ns between
algal composition of the water column (30% were resuspended
benthic diatoms) and the stomach contents of the bivalves C. ed-
ulf, Mya arenaria and Mytihts edulis in the western Wadden Sea.
She concluded that selection of algae by the bivalves was absent
based on comparison of water and stomach samples. Recently two
studies measured ratios of C and N isotopes in different primary
producers and benthic invertebrates (Kang et al. 1999. Riera et al.
1999). These studies concluded that C. edute preferentially in-
gested microphytobenthos and phytoplankton compared with de-
tritus from benthic macro-algae or sea grasses fragments. How-
ever. Herman et al. (2000) indicated that C. edule depends basi-
cally on pelagic algae as a food source and benthic algae hardly
contribute to the metabolism. Our experiment showed a differen-
tial pre-ingestive selection by C edule of the different benthic
species. This may explain the actual controversy about microphy-
tobenthos as a food source for suspension feeders. More detailed
information about acceptance and/or rejection of single microphy-
tobenthic algal species is needed to clarify the actual controversy.

Results obtained in this study, using flow cytometry tech-
niques, showed a preferential ingestion of Pelagic over Benthic
species and a differential selection of the benthic species. Within
the Benthic group, some species such as Cylindrolheca sp. was
rejected in significantly higher concentrations than small pennates
or Navicida sp. The mi.xed composition of the benthic diet is a
feature that resembles the natural conditions where C. edule lives
and a preferential selection of some species may occur also in the
field. Studies on sorting of food in bivalves using flow cytometry
to determine the preferences within mixed samples are scarce
(Cucci et al. 1985. Shumway et al. I9«3. Chretiennot-Dinet et al.
1991. Bougrieret al. 1997. Baker et al. 1998). Cucci et al. (1985)
did not observe preingestive selection in mussels fed with a diet of
a diatom, a dinoflagellate and a cryptomonad. However. Bougrier
et al. ( 1997) observed in the oyster Crassostrea gigas (Thunberg,
1793) that a preferential ingestion of flagellates species occuned
compared with diatom species. Moreover, Baker et al. ( 1998) ex-
amined the ability of zebra mussels Dreissena polyiiiorphci (Pallas.
1771 ) to preferentially ingest or reject various phytoplankton spe-
cies in the Hudson River (New York). In their study, zebra mussels
selected more efficiently small algal cells, such as cyanobacteria.
explaining the changes in the Hudson River phytoplankton com-
munity in the last decade.

Accepted benthic species by C. edule had smaller and similar
sizes (Navicula sp.: measured size 46 ± 1 1 iJim; small pennates:
measured size 30 ± 2 p.m) than the significantly rejected species
such as Cylindnnheca sp. (measured size 81 ± 5 p,ni). Therefore.
a possible relation may be found with a preferential ingestion or
rejection of those algae and their size. Nevertheless, preingestive
selection of algae in bivalves could also be related to chemical
characteristics (e.g.. taste of the particles) or to morphologic char-
acteristics (e.g., shape of the particles). In this context. Shumway
et al. (1990) discussed that not only size is an important factor in
the preingestive selection of particles but also mucoid trapping and
chemoreceptors. Chretiennot-Dinet et al. (1991 ) reported that the
relative ingestion or rejection was not dependent on the size of the
algae in C. gigas and M. edulis. Bougrier et al. (1997) reported
similar results on those bivalves and no relation was found be-
tween preingestive selections of algal species with their size. Mac-

Donald and Ward ( 1994) showed that rejection is not dependent on
particle size in the scallop Placopecleii inagellauicus.

The cockle. C. edule. preferentially accepted small benthic dia-
toms such as small pennates and Navicula sp.. both also with an
elliptical smooth shape, and rejected others such as Cylindrniheca
sp.. which has a larger size and large lateral spicules. The actual
mechanisms whereby particles are selected on the gills and the
labial palps have been studied using endoscopy (Ward et al. 1991.
Ward et al. 1993) but not much information is known about their
relation with morphologic characteristics of the particles. Bougrier
et al. (1997) indicated that a differential rejection or ingestion of
diatom and flagellate species might depend on their shape and
flexibility, with a preferential rejection of species with inflexible
spicules or the presence of a non-flexible membrane (i.e., silicate
frustule). which may hamper the pass through the gill filter. Some
authors (Shumway et al. 1985. Bougrier et al. 1997) have also
detected a preferential ingestion of flagellates in comparison to
diatoms and have speculated that the presence of the silicate frus-
tules from the diatoms may appear as mineral particles to sensory
receptors of the labial palps. According to this hypothesis the
preferential selection of the pelagic diatom P. thcornutum could be
influenced because benthic species are. generally, attached to silt
particles and it may be possible that some species will have more
chance to be rejected as inorganic particles by C. edule.

In this study, similar values of selection efficiency were regis-
tered when C. edule was exposed to Pelagic and Benthic treat-
ments, and lower values obtained in mixed treatments. Riera and
Richard (1995) have reported in C. gigas a preferential ingestion of
benthic diatoms and detritus compared with phytoplankton in shal-
low estuarine waters. But they also reported that C. gigas had a
preferential ingestion of phytoplankton. when compared with
benthic diatoms and detritus, in deeper waters from the same bay.
Shumway et al. (1987) found a preferential ingestion of benthic
algae by P. magellanicus collected from deep-water populations,
however individuals from shallow water populatiiins seemed to
ingest more pelagic than benthic algae. Gut contents of P. magel-
lanicus generally reflected available organisms in the immediate
habitat. The preingestive selection of algae in some filter feeding
bi\ alves. such as C. edule. could be adapted to the composition and
the percentages of the different species available in the water col-
umn. The preferential ingestion of benthic species in comparison
with pelagic species has been observed in the mussel M. edulis in
conditions with low concentrations of pelagic algae in the water
column (Prou et al. 1994). In our experiments, microphytobenthos
is selected at the same efficiency as P. tricornutum in nonmixed
treatments. In a mixture of pelagic and benthic species, the rejec-
tion of microphytobenthos is higher and this could be related to the
dominance of the pelagic algae in the diet. Preingestive selection
of benthic compared with pelagic species has been studied previ-
ously in the oyster C. gigas by using the flow cytometry technique
(Bougrier et al. 1997). In those experiments oysters preferentially
ingested pelagic species than benthic species, in a dominant pe-
lagic species treatment. Baker et al. (1998) observed that the se-
lectivity of different phytoplankton species by the zebra mussel
Dreissena polymorplia differed depending on the complexity of
the offered suspension.

Summarizing, the pre-ingestive selection of benthic algae by C.
edule is similar and nonsignificantly lower than the pre-ingestive
selection of pelagic species, when benthic and pelagic species are
not mixed. This situation of maximum presence of benthic diatoms
in the seston could be similar to environmental characteristics of

Selective Feeding in Cerastoderma edule

625

tidal flat areas with high resuspension rates of benthic diatoms, or
seasons with low pelagic productivity (e.g.. autumn and winter).
However, acceptance of benthic algae is lower, compared with
pelagic, and not equal for the different components of the benthic
group when mixed in a pelagic dominant treatment. These condi-
tions may be present during warm periods of the year (e.g., spring
and summer) when phytoplankton blooms occur in their natural
environment. C. edule may be considered as an opportunistic filter
feeder that may take advantage of certain algal species, both pe-
lagic and benthic, in relation to their availability in the field.

ACKNOWLEDGMENTS

This work is a contribution to the ECOFLAT (Eco-Metabolism
of a Tidal Flat) project carried out under contract ENV4-ct96-

0216, jointly sponsored by the ENVIRONMENT and MAST pro-
grams of the EU. The first author has also been supported by a
Marie-Curie Training Research Grant from the European Commis-
sion within the SIMCERE project (Fair GT 97-4325). The Na-
tional Institute for Coastal and Marine Management (RIKZ). Mid-
delburg (The Netherlands) offered us the possibility to use part of
their equipment and technology. Many thanks go to researchers
from RIKZ such as Thomas Rutten and Ben Sandee for their help
in the analysis of the samples with the flow cytometer. Louis
Peperzak for his help in the identification of the algal species and
Fred Twisk and Richard Eertman for considerable support at dif-
ferent stages of this research. We also thank Dr. Pauline Kamer-
mans and Dr. Sandra E. Shumway for their comments in earlier
versions of the manuscript.

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Joiinial iif Shellfish Research. Vol. 21. No. 2. 627-634. 20U2.

REPRODUCTIVE CYCLE OF THE BIVALVE CLAMS SEMELE SOLIDA (GRAY. 1828)
(SEMELIDAE) AND GARI SOLIDA (GRAY, 1828) (PSAMMOBIIDAE) FROM CHILE

DONALD BROWN,'* BERNARDITA CAMPOS," AND H.-JORG URBAN'

^ Departiuneiito de Biologi'a. Iiistlluto de Ciencias Bioldgicas y Qiiimicas, Faciiltad de Ciencias,
Universidad de Valparaiso, casilla 5030, Valparaiso, Chile; 'Facultad de Ciencias del Mar.
Universidad de Valparaiso, casilla 13-D. Vina del Mar, Chile: ^Alfred-Wegener-Instititte for Polar
and Marine Research, Section of Comparative Ecosystem Research, Columbusstr. 27568 Bremerhaven,
Germany

ABSTRACT Coinmercial clam landings reached an average of almost 91.000 tons annually in Chile over the last decade. In spite of
the high value of this resource, few efforts have been made to understand the basic biology of the exploited species, data that might
in the future be needed to aid in their protection or even artificial culture. This study is a contribution to the knowledge of the
reproductive cycles of two valuable species. Semele solida (Gray ) and Gari xolida (Gray). Representative samples of these species were
collected at two widely separated localities in Chile and examined histologically to determine their seasonal reproductive cycles. It was
found that the species were of separate sexes, and had annual gonadal cycles. In S. solida from northern Chile, the reproductive period
extended from June 1991 to February 1992. In C. solida from southern-central Chile, the reproductive period was relatively short, from
October 1991 to February 1992. In both species, most specimens have empty gonads by March. The data obtained are useful in
developing management plans related to their reproductive periods. Relevant to culture strategies. S. solida has the comparative
advantage of a lengthy reproductive period, wherein mature individuals may be frequently encountered in nature for spawning
inductions. G. solida. with its shorter annual reproductive cycle may have the advantage of being induced to mature in artificial
conditioning systems over relatively short periods of time.

KEY WORDS: clam reproduction, reproductive cycle, bivalves. Semele solida, Gari solida. Chile

INTRODUCTION

Chilean coastal waters host very productive and diverse clam
fisheries due to the rich coastal upwelling and fasorable water
temperatures. The largest clam populations occur in the protected
bays and fjords of southern Chile, Over a number of years official
fisheries records in Chile (SERNAPESCA 1990-1999) considered
all clam species as one generic group ("clams") among which were
included the venerids Protoihaca tluica (Molina). Venus antiqita
(King and Broderip). Eiirhomalea exalbida (Chemnitz), E. lenticu-
laris (Sowerby). E. rufa (Lamarck), and the mactrid Midinia edit-
lis. Semele solida (Gray) and Gari solida (Gray) belonging to the
Semehdae and the Psammobiidae respectively, and objects of this
study are also included in this group. They are primarily exploited
in artisanal fisheries, and commercialized mostly in canned form.
G. solida is highly valued from the culinary standpoint. In 1994.
the first year of its listing as an individual species. 461.^ tons of S.
solida were harvested, declining to 2071 tons by 1999; G. solida.
recorded separately beginning in 1990, was registered at 31.37.^
tons, which declined to 9931 tons by 1999 (SERNAPESCA 1990-
1999). The only regulation for the fishery of these clams is a
minimum size limit of 53 mm for 5. solida and 60 mm for C.
solida (Subsecretari'a de Pesca 1996).

Despite their great economic value, not much research has been
done on reproduction in Chilean clams, particularly in S. solida
and G. solida. One recent report (Jerez et al. 1999) suggested that
G. .wlida in southern Chile had a continuous reproductive cycle
throughout the year, a pattern apparently common among the heav-
ily commercialized clams such as V. antiqua (Lozada & Bustos
1984) and P. thaca (Henri'quez et al. 1981 ). This also was true for
E. lenticidaris (Campos & Brown 1997. Campos et al. 1999) and
M. edulis (Jaramillo et al. 1998).

* Corresponding author. E-mail: donald.brownCgiuv.cl

Semele .solida (Fig. lA). locally termed "tumbao", occurs par-
tially buried in sand and gravel bottoms froin the intertidal (Osorio
et al. 1979) to the subtidal zone (Urban 1994). Its geographic
distribution ranges from 12°S to 47°S. (Viviani 1979). Gari solida
(Fig. IB), locally termed "culengue", occurs completely buried in
bottom sands and gravels, usually at greater depths than S. solida
from the intertidal to 15-m depth (Urban 1994). Its range of dis-
tribution along the Pacific Coast, as given by Viviani (1979) and
later by Guzman et al. (1998). was between 12°S and 47°S.

Biologic data for species of economic importance is fundamen-
tal for proposing regulatory recommendations for sustainable har-
vest of these resources over time. The obvious declines in harvest
over the years enhances the need for more information on the
reproduction and survival of these species to support efforts di-
rected towards their artificial culture, repopulation. or management
as a renewable resource in over-exploited beds.

In this study, we describe the reproductive cycles of G. solida
and S. solida by means of histologic observations of gametogen-
esis during different seasons of the year. Patterns in reproductive
cycles, including gametogenesis and resting gonadal periods were
studied in a population of S. solida from northern Chile and in a G.
solida population from central-southern Chile, representing the
first study of this nature for these two clam species in their re-
spective regions.

MATERIALS AND METHODS

Adults of each species were obtained by diving at monthly
intervals from June 1991 to July 1992. S. solida was collected in
La Herradura Bay (29°58'S) and G. solida from Coliumo Bay
(36 '32'S) (Fig. 2). Maximum anterior-posterior length of the shell
was measured on each specimen, to the nearest 0.1 mm. Matura-
tional status of the gonad was determined monthly on around 30
animals of each species. Tissue samples 5 mm in thick-

627

628

Brown et al.

FiKurc 1. Species under study. A: Semele sulida. 78 mm total length
(maximum anterior-posterior shell dimension); B: Gari solida, 89 mm
total length.

ness were obtained and fixed 24 h in Bouin's fluid and prepared by
routine histologic procedures as follows: dehydration with graded
series of ethanol. clearing in xylol and embedding in Paraplast.
Five micrometers histologic sections obtained from three levels of
each gonad separated 300 p.m, were stained with hematoxylin and
yellowish eosin, dehydrated in graded series of ethanol. cleared in
xylol and permanently mounted with Canadian balsam (Gabe
19681.

The gametogenic cycles of the two clams were followed by
describing the histologic appearance of the gonadal sections and
classifying them into different stages of maturity using a modifi-
cation of the scale proposed by Lucas (1965). Each individual was
assigned to one of the following stages based on the degree of
morphologic development of its germ cells: (dl) = initial devel-
opment or maturation; (d2) = advanced development or matura-
tion; (d.^) = complete development or maturation; (rl) = initial
regression or evacuation; and (r2) = total regression or evacua-
tion. The results were expressed as percentage frequency histo-
grams of: (1) males in each gonadal stage; (2) females in each
gonadal stage; and (3) males plus females in each stage, separately
for S. solida and G. solida during the sampling period from June
1991 through July 1992.

Figure 2. Location of collection sites for A: S. solida and B: G. Solida
on the Chilean coast.

RESULTS

Semele solida specimens measured from 38.9 to 86.0 mm and
Gari solida from 41 .4 to 88.0 mm. The two clam species were of
separate sexes, with no hermaphroditism and no sexual dimor-
phism evident. Histologic analysis of the gonad in both species
showed a multilobulate organization of the acini connected to
evacuation tubes covered by simple ciliated epithelium similar to
that observed in other bivalves (Sastry 1979). The acini consisted
of a basal lamina of variable thickness depending on the stage of
gonadal maturity. Its relative thickness was greatest in specimens
initiating gametogenesis. and in those that had spawned. In these
specimens an intra-acinar reticulum consisting of vesicular so-
matic cells and an intra-acinar space containing groups of ame-
bocytes may be found (Figs. 3A and 4F).

The cells of the male germinal line that characterize spermato-
genesis may be restricted to two zones of the gonadal acinus; ( I )
a basal region representing the early germinal line that includes
spermatogonia and spermatocytes that form a band of circular
voluminous nuclei, and recently formed round spermatids that also
form a band of small circular nuclei (Figs. 3A, 3B and 4A. 48).
that is evident in G. solida: and (2) a lumen region, representing an
advanced germinal line with spermatids undergoing cytodifferen-
tialion with heavy stained elongated nuclei, gathered by their heads
in double columns, giving a ■■feathered" appearance (Figs. 38. 3C
and48. 4C).

Reproductive Cycle of Chilean Clams

629

Fiyurt 3. A-K: Linlil plKPlomiiroyraphs ol diltiTi'iil histiilii}iic:il slanis ol male and K-,J: li'inali' ;i<inadal acini of S. saliilu Ldilciletl in La
Htrradura Bay from June I'Wl to July IW2. A-K bar = 10(( jim; F-J bar = 200 fini: A and V = initial development of maturation (dl): B and
(; = advanced development of maturation (d2); C and H = total development or maturation (d3l: I) and I = initial regression or evacuation (rl);
E and J = total regression or evacuation (r2).

630

Brown et al.

Figure 4. A-E: Lisht photomkrosniphs of different liist S'^al stases of male and F-,|: female gonadal acini of G. snlida collected in C oliumo

Bay from June 1991 to Julv 1992. A-K bar = KM) urn: K-J har = 2(10 Mm: A and F = initial development or maturati<(n (dl I; B and (; = advanced
development or maturation (d2l: f and H = total development or maturation (d3); D and I = initial regression or evacuation (rl): K and J =
total regression or evacuation lr2l.

Reproductive Cycle of Chilean Clams

631

The cells of the female germinal line in the basal region are
represented by oogonia. previtellogenic and adhered vitellogenic
oocytes. In the lumen region they are represented by pedunculate
vitellogenic oocytes as well as free oocytes (Figs. 3G, 3H. and 4G.
4H). The histologic stages of the gonads of 5. solitia and G. solidci
females are shown in Figures 3F-3J and 4F-4J. respectively.

The three sampled areas of the gonad from both species all
showed the same degree of gametogenic activity or development
of the germinal line, indicating synchronic maturation throughout
the gonad.

Semele sulida

The distribution of percentage frequencies of the different his-
tologic stages in male gonads, female gonads, and in the popula-
tion as a whole for 5. solida are given in Figures 5A. 5B and 5C,
respectively. This species presented a seasonal pattern of gonadal
development in both sexes. Males and females with developed or
mature gonads (d3 stage) as well as in initial regression (rl) (Figs.
3D and 31) were predominant from June 1991 to February 1992. In
contrast, from March to June 1992, there was a greater frequency
of individuals in total regression (r2) (Figs. 3E and 3J). Although
in both sexes the frequency of samples with gonads in initial stages
of development (dl) (Figs. 3A and 3F) was observed between
April and July 1992, the number of females in this stage was
greater, and predominated over those in total regression (Fig. 5A
vs. Figure 5B). However, during the first period, there was a small
percentage of males in total regression (r2) and in initial develop-
ment (dl) (Fig. 5A), which was a condition more apparent in
females (r2-dl; Figure 5B). Some observations not included in the
figures suggested that individuals technically considered to be in
regression could show a new wave of initial maturation beginning
at the germinal line in the gonadal acini.

The second period was characterized by the total regression
stage (r2), where all individuals had gonads with depleted acini in
March 1992 (Figs. 5A, 5B). The percentage frequency distribution
of the different gonadal stages for the population sample (Fig. 5C)
shows this tendency in both males and females.

It is of interest to point out the difference in gonadal conditions
between specimens sampled in June to July 1991 compared with
those from the same period in 1992. In 1991 a high frequency of
both sexes contained elevated numbers of specimens with gonads
in advanced and total development (d2-d3), whereas in 1992 this
condition was different, with specimens containing gonadal acini
in total regression (r2) or without advanced germinal line (Fig. 5).

Gari solida

In this species the distribution of percentage frequencies of the
different histologic stages in males, females, and the entire popu-
lation are given in Figures 6A, 6B, and 6C, respectively. There is
a periodicity in both sexes with the same general tendency.

There is a well-marked period in which advanced and totally
developed gonads (d2-d3) are observed, as well as those in initial
regression (rl) (Figs, 4D and 41) from October 1991 to February
1992. In females this period is much shorter (November 1991 to
January 1992). This condition persisted in some males until April
1992. Some observations not included in the figures showed indi-
viduals in regression during this period, which had a new wave of
initial maturation beginning at the germinal line, as observed in 5.
solida (see earlier). In a second, more extensive period, the gonads
were characterized by the occurrence of total regression and initial

development (r2-dl) (Figs. 4E and 4A, respectively) in males
from June to September 1991, and from March to July 1992 (Fig.
6A). In females the r2-dl period (Figs. 4J and 4F, respectively)
extended notably until October 1991. and from February to March
1992 (Fig. 6B).

Figure 6C shows the general frequency of the gonadal stages
for the general population, with a similar pattern to that presented
separately for males and females. From June to August 1991,
February 1991. and April to July 1992 the number of specimens in
each population sample exceeded the males and females together
because included were specimens whose total regression stage was
such that there were no cells on the germinal line differentiated
enough to pennit sex determination. In comparing the gonadal
stages of specimens obtained in June to July 1991 with those of the
same period in 1992, stages were observed that were near total
regression and initial development showing an inverse fluctuation
where regression predominated in 1991 and initial development in
1992.

Finally, it was apparent in both species that the stages of ad-
vanced or total maturity were observed in water having higher
relative temperature, whereas initial stages of development were
related to water of relatively low temperature, although our tem-
perature measurements were not extensive (Figs. 5C and 6C).

DISCUSSION

The reproductive cycle is characterized by a series of events
that in annual species comprises a reproductive period involving
the gametogenic and spawning phases and a resting period in
which there is not gametogenic activity.

Present results have shown similar values between the annual
reproductive cycles of S. solida and C. solida. where both showed
seasonal gametogenesis and spawning, followed by a resting pe-
riod without production of gametes.

The reproductive period of S. solida. from June 1991 to Feb-
ruary 1992 was longer than that of G. solida. Most of the speci-
mens showed gametogenic activity and signs of having spawned.
The majority of spawning occurred in February, and in March all
specimens had empty gonads. This point marked the initiation of
the resting period, indicated by a completely regressive condition
in the gonad, which was more marked in G. solida than in S.
solida.

We are cautious to consider the possibility of a second spawn-
ing phase during the reproductive period of 5. solida because of the
low number of animals (4) sampled and examined in October,
notwithstanding that all of them were in advanced development
(D2).

In G. solida the reproductive period was relatively short, from
October 1991 to February 1992; the spawning phase mostly occurs
in February. A majority of the specimens had empty gonads in
March 1992; then the spawning phase mostly occurs in February.
The presence of specimens with gonads in the initial state ot de-
velopment in this period may indicate possible activity in gonial
multiplication and generation of cytes without gametogenic activ-
ity that leads to massive production of differentiated gametes. The
results showed that low temperatures favored the proliferative ac-
tivity of the early germinal line, while high temperatures aided
cytodifferentiation of the advanced germinal line. This condition
was most notable in the reproductive cycle of G. solida from
Coliumo Bay. a more southeriy location. These events occurred

632

Brown et al.

a-

B

o

c

0)

3

a-
a

Semele solida. Male cycle

100%'
80%'
60%
40%
20%
0%

13 15 13 16 1

N
15 15 14 15 15 14 18 15

n

i

n

i

I
i

I

■

I

I

1

i

S

I

J JASONDJ FMAMJ J

1991 1992

Months

Semele solida. Female cycle

Months

Semele solida. Reproductive cycle

□ MR2

OMRI

□ MD3

SMD2

HMD1

□ FR2

niFR1

nFD3

SFD2

0FD1

100%

29 29 28 31

nR2

aRi

nD3

HD2

0D1

Months

Fisurc 5. nistribution of different gonadal stages In A. males: B. females; and C. males + females of S. so//V/a colleced in I.a Herradura Bay from
June 1991 to July 1992. Nvllh sea surface temperature added. The length of each area represents the percentage frequency of specimens m each
histologic stage of the gonadal acini. N = number of specimens examined.

simultaneously in both males und females showmg (expected) syn- ln>m the majority ol the specimens, a phenomenon more pro-

chrony of the reproductive cycles. nounced in S. solida. There was a predominance ot advanced

Every monthly sampling during the reproductive period stages of gonadal maturity, and also those with complete regres-

showed a few individuals having gonadal conditions differing

as an evidence that spawning had occurred. These observa-

Reproductive Cycle of Chilean Clams

633

100%-i
80%

a 60%'

40%'

20%-

0%'

B

Gari solida. Male cycle

H
5 11 11 11 14 14 13 14 14 12 7 2 7 12

I

I

■

J

mm

m

I

J JASONDJ FMAMJ J
1991 1992

Months

100%-|
80%
60%
40%
20%4

Gari solida. Female cycle

N
23 15 15 18 14 15 16 11 16 18 12 10 19 13

0%

11

iii

i

i

pn

I

Lmi

il

I

JJASONDJFMAMJJ

1991 1992

Months

Gari solida. Reproductive cycle

□ MR2

niMRI

nMD3

BMD2

0MD1

□ FR2

QFRI

DFDS

SFD2

HFDI

DR2
DRI
aD3
SD2
BD1

Months

Figure 6. Distribution of different gonadal stages in A. males; B. Females and C. males + females of G. solida collected in Coliumo Bay from
June 1991 to July 1992, with sea surface temperature added. The length of each area represents the percentage frequency of specimens in each
histological stage of the gonadal acini. N = number of specimens examined.

tions suggest intrapopulation asynchrony of gametogenic activity. firmed this asynchrony and strengthened the hypothesis of con-

with partial evacuations of gametes over a longer period. The fact tinuous gametogenesis with various cycles of gametogenic activity

that the specimens showing signs of having spawned showed a and spawning by each individual during the reproductive period,

new wave of maturation in the germinal layer of their acini, con- A difference was observed in the degree of maturity of the

634

Brown et al.

piipulation samples of 5. solida between June and July 1991 where
advanced and complete maturity were well represented; in the
same period of 1992, on the contrary, maturity was just beginning.
This difference may be explained by normal adaptation to envi-
ronmental conditions such as temperature and fcH)d availability,
which may vary within a limited range from year to year.

Although Urban and Campos (1994) suggested that the repro-
ductive cycles of S. solUla and C. soluhi were influenced by tem-
perature, Jerez et al. (1999) working on a G. solida population
from the south of Chile found the annual reproductive cycle to be
continuous without a marked resting period. Further studies are
required to evaluate seasonal variations in gonadal cycles of these
species with latitude, as they are distributed over a broad latitudi-
nal range from Callao, Peru ( 12°S) to Chile's Chonos Archipelago
(44°S). The hypothesis here is that the reproductive cycles of these
clams become shorter in populations the farther south they occur
on their distributional gradient. Some data available on other clam
species with extensive distributions support this hypothesis. Popu-
lations of Protothaca thaca (Henn'quez et al. 1981), Tagelus
dombeii ( Acufia et al. 1994) and Eiirhomalea lenlicularis (Campos
& Brown 1997) from central and north-central Chile exhibit con-
tinuous gonadal activity with various important spawning peaks
throughout the year. Nevertheless, T. dombeii from south-central
Chile showed a period of gonadal regression in the fall (Jaramillo
et al. 1998). This phenomenon is not clear across other clam spe-
cies inhabiting the south-central zone of Chile, such as Venus
antiqiia, Tawera gayi. Midinia ediilis and £/;.v/,s macha that show
continuous reproductive cycles without resting periods (Lozada &
Bustos 1984, Jerez et al. 1999).

From the practical point of view, regulation of harvesting these
clams should be based on considerations of their reproduc-
tive cycles by limiting their harvest during the major spawning
season. Consideration of the reproductive cycles is also important
in obtaining broodstock for aquaculture. Experimental studies
should prove this a feasible alternative for production or protect-
ion of the resource. In studying resource management of these
clams, S. solida shows a comparative advantage in having a
more extensive reproductive period, as mature individuals may be
encountered over an extended period. This implies that mature
broodstock would be available in nature for artificially induced
spawning (e.g.. in aquaculture experimentation) over compara-
tively lung periods. Although G. solida. in contrast, has a more
restricted reproductive period, it may be a species amenable to
artificial conditioning in aquaculture systems, given that its gonads
almost always contain high numbers of immature gametogenic
cells.

ACKNOWLEDGMENTS

The authors thank Ms. T. Jeri for sampling G. solida at
Coliumo Bay. and Ms. G. Bellolio (U. Catolica del Norte at the
time) for providing S. solida from Herradura Bay. We also thank
Dr. R. Guerra for her supervision of the histologic processing at
the U. de Valparaiso, and Mr. C. Olivares for assistance in histo-
logic analyses. Corrections and comments by anonymous review-
ers helped to improve the manuscript. The work was financed by
FONDECYT Grant 91-.S02 to B. Campos.

Acufia, E., Ch. Guisado & M. Bem'os. 1994. Cicio reproductive de Tagelus
(/o/Hfcfii (Bivalvia: Helerodonta: Solecunidae). provenienles de la bahi'a
La Herradura de Guayacan. IV Region. XIV Jornadas de Ciencias del
Mar. Chile. 131 pp.

Campos, B. & D. Brown. 1997. Aspectos reproductivos de la almeja Eii-
rhomalea lenlicularis (Sowerby) proveniente de la rada El Algarrobo
(V Region). Informe Final Proyecto DIPUV 20-95. Universidad de
Valparaiso, Chile.

Campos, B., D. Brown, L. Duran. C. Melo & J. Urban. 1999. Estudio de
edad y reproduccion del recurso almeja en la IV y V Regiones. Informe
Final Proyecto FIP 97-32, Subsecretaria de Pesca, Chile.

Gabe, M. 1968. Techniques hi.stologiques. Paris: Masson et Cie. 1113 pp.

Guzman, N., S. Saa & L. Ortlieh. 1998. Catalogo descriptivo de los mo-
luscos litorales (Gastropoda y Pelecypoda) de la /,ona de Antofagasta.
23 S (Chile). Esnid. Oceanol 1 7; 1 7-86.

Henriquez. R.. P. Barboza, R. Ramos. E. Tapia & C. Toro. 1981. Variacion
anual de la gonada de la almeja Protothaca thaca (Molina 1782):
analisis histologico. I Jornadas de Ciencias del Mar. Chile. Resiimen.
34 pp.

Jaramillo, E., E. Clasing, M. Avellanal, P. Quijon, H. Contreras. P. Rubilar
& G. Jerez. 1998. Estudio biologico pesquero de los recursos almeja.
navajuela y huepo en la VIII y X Regiones. Informe Final Proyecto FIP
96-46, Subsecretaria de Pesca. Chile.

Jerez, G.. N. Barahona. H. Miranda. V. Ojeda, D. Brown. C. Osorio, A.
Olguin & J. Orensanz. 1999. Estudio biologico pesquero de los recur-

LITERATURE CITED

SOS tawera [Tawera gayi) y culengue (Gari solida) en la X Region.
Infomie Final Proyecto FIP 97-29, Subsecretaria de Pesca, Chile.

Lozada. E. & E. Bustos. 1984. Madurez sexual y fecundidad de Venus
antiqua antiqua King & Broderip. 1835 en la bahi'a de Ancud (Mol-
lusca: Bivalvia: Veneridae). Rew Biol. Mar. Valparaiso 20:91-112.

Lucas, A. 1965. Recherche sur le sexualite des mollusques bivalves. The-
ses Doclorat. Fac. Science. Universite de Rennes. France.

Osorio, C. J. Atria & S. Mann. 1979. Moluscos marinos de importancia
economica en Chile. Biol. Pesq. Chile 1 1:3— 47.

Saslry, A. 1979. Pelecypoda (excluding Ostreidae). In: A. Giese & J.
Pearse, editors. Reproduction of Marine Invertebrates. Vol. V. New
York: Academic Press, pp. 113-292.

SERNAPESCA. 1990-1999. Anuario estadistico de pesca 1990 a 1999.
Servicio Nacional de Pesca. Chile.

Subsecretaria de Pesca. 1996. Medidas de Administracion Pesquera. Min-
isterio de Economi'a, Desarrollo y Reconstrucci6n (Chile). 32 pp.

Urban, H.-J. 1994. Adaptations of six infaunal bivalve species of Chile:
coexistence resuhing from differences in morphology, burrowing depth
and substrate preference. Arch. Fish. Mar. Res. 42:183-193.

Urban. H.-J. & B. Campos. 1994. Population dynamics of the bivalves
Gari solida. Seinele solida and Protothaca thaca from a small bay in
Chile at 36 °S. Mar. Ecol. Prog. Sen 1 15:93-102.

Viviani. C. 1979. Ecogeografi'a del literal chileno. Stud. Neotrop. Fauna &
Environ. 14:6.5-123.

Joiinuil of Shellfish Research. Vol. 21, No. 2, 635-642. 2002.

GROWTH OF NORTHERN [MERCENARIA MERCENARIA (L.)] AND SOUTHERN [M.
CAMPECHIENSIS (GMELIN)] QUAHOGS: INFLUENCE OF SEAGRASSES AND LATITUDE

K. L. HECK, JR.' - * L. D. COEN/ ' AND D. M. WILSON' ^

^Dauphin Island Sea Lab. 101 Bienville Boiilevanl. Dauphin IslanJ. Alabama 36528; 'Department of
Marine Science, University of South Alabama. Mobile Alabama 36688: ^Marine Resources Research
Institute. SCDNR. P. O. Box 12559. Charleston. South Carolina 29422: and ^Marine Science Program.
Department of Biological Sciences. University of Alabama. Dauphin Island, Alabcuna 36528

ABSTRACT To better understand how seagrasses influence the growth of northern and southern quahogs (Mercenaria mercenaria
and Mercenaria campechiensis). we collected and compared the growth rates of clams from seagrasses and adjacent unvegetated
locations along the Atlantic (Massachusetts and New Jersey) and Gulf of Mexico coasts (Alabama and Florida) using identical methods.
In particular, we sought to test hypotheses that clam growth is inlluenced not only by the presence or absence of seagrass {Tlialassia
testudinum. Halochile nrighlii. and Zosrera marina), but also by location within seagrass beds. Walford plots constructed using annual
shell-growth band analyses showed that Mercenaria spp. growth was significantly affected by the presence of seagrass habitat, although
the magnitude of this effect varied with clam age (size) and latitude. Specifically, first-year growth was significantly greater in
unvegetated than in adjacent vegetated sites, whereas a measure of lifelong growth (Ford's growth coefficient) was not significantly
different between adjacent vegetated and unvegetated sites. We hypothesize that these conflicting results may be due, in part, to
differing patterns of energy resource allocation in Mercenaria at various life cycle stages. During the first 1.5 y or so, a clam's energy
resources are allocated primarily to somatic growth, whereas in subsequent years energy is also allocated to reproduction. Therefore,
factors affecting Mercenaria growth, such as the presence or absence of submerged vegetation, should be most easily detectable in
analyses examining the first year's growth. Even though our data showed greater first-year growth at lower latitudes (Gulf of Mexico
sites versus New Jersey and Massachusetts), overall lifelong growth rates were greatest at the Massachusetts sites. We attribute this
pattern to the fact that higher first-year growth rates quickly become asymptotic in the warm waters of the Gulf of Mexico, whereas
slower but more constant growth rates are typical of clams from colder water sites. We found little evidence for the effects of location
(as measured as distance from bed edge) on growth of individuals collected from grassbeds, except where patterns of water movement
showed consistent directionality (e.g.. Gulf of Mexico sites). Given the multitude of variables that can interact to influence clam growth
rates and the unresolved variability in results from single-site studies performed in different locations, some complex, carefully
controlled experiments are still needed to elucidate the dominant factors that regulate both short and long-term clam growth.

KEY WORDS: hardclams, Mercenaria. seagrass, latitude, growth, quahog

INTRODUCTION

Growth and survival' of suspension-feeding animals may be
strongly influenced by the presence of submerged aquatic vegeta-
tion (SAV), presumably because macrophytes reduce flow and
enhance the deposition of suspended materials (e.g.. Scoffin 1970,
Orth 1977, Tegner & Dayton 1981, Fonseca et al. 1982, Harlin et
al. 1982, Eckman 1983. Peterson, et al. 1984, Dayton 1985. Wild-
ish & Kristmanson 1997), thereby altering available food supply
(Peterson & Beal 1989, Iriandi & Peterson 1991, Irlandi 1996, but
see Judge et al. 1992, Nakaoka 20(J0). However, studies of the
suspension-feeding bivalve Mercenaria mercenaria (The Noilhem
Quahog), have continued to produce conflicting results (e.g.. field-
ing 1912, Kerswill 1941, 1949. Haskin 1952; Pratt & Campbell
1956; Ansell 1968, Greene 1979, Hadley & Manzi 1984, Peterson
et al. 1984, Arnold et al. 1991, Coen & Heck 1991, Irlandi &
Peterson 1991, Judge et al. 1992, Coen et al. in prep.) with regard
to the effect of seagrasses. For example, early studies by Kerswill
(1941, 1949) found decreasing M. mercenaria growth rates as
eelgrass (Zostera marina) density increased, whereas a later study
by Peterson et al. (1984) found just the opposite.

More recent studies (Peterson & Beal 1989; Coen & Heck
1991; Slattery et al. 1991, Nakaoka 2000, Coen et al. in prep.)
found that M. mercenaria in seagrass beds sometimes grew faster,
slower, or at the same rate as M. mercenaria in unvegetated habi-
tats. Peterson and Beal ( 1989) cited two possible explanations for

"Corresponding author.

the variability in their results. First, as water velocities decrease,
the flux of suspended food particles passing by the clam's siphons
will decrease. Alternatively, reductions in velocity could enhance
the settlement of suspended materials and increase food concen-
trations near the bottom. The net effect of decreasing suspended
horizontal food flux while increasing the number of food particles
settling from the water column will determine whether the impact
of vegetation on Mercenaria growth is positive or negative (Peter-
son & Beal 1989). Subsequently, Irlandi and Peterson ( 1991 ) have
argued that measured variations in food concentration between
vegetated and nearby unvegetated areas are insufficient to explain
the observed between-habitat differences in clam growth.

There must also be an upper limit to current velocity beyond
which bottom instability inhibits feeding, either directly by smoth-
ering or indirectly by requiring the clam to expend energy rebur-
rowing to maintain a desired burial depth (Myers 1977, Orth 1977,
Turner & Miller 1991, Iriandi &Mehlich 1996, Iriandi 1996) or by
decreasing assimilation efficiency (Bricelj & Malouf 1984).
Greater bottom stability in vegetated habitats produced by the
baftling effect of seagrasses could also lead to greater growth of
Mercenaria located within seagrass beds (cf. Peterson et al. 1984;
Irlandi & Peterson 1991: Iriandi 1996, Coen, unpublished. 2000).

Location within a landscape of habitats and the associated sys-
tem states (i.e., food, flow, competitors, predators, see Micheli
1996) can also significantly affect bivalve populations. This can
result from local food depletion from upstream-feeding individuals
(e.g., Okamura 1986. Peterson & Black 1987, Frechette et al.
1989), within or among habitat conditions (e.g., Iriandi & Peterson

6,35

636

Heck et al.

1991. Kamermans 1993) or the physical activity of neighboring
animals interfering with feeding or space acquisition (Vimstein
1977, Peterson 1979, Okamura 1986. Nakaoka 2000). These can
manifest themselves under certain situations as density-dependent
effects (Grizzle & Morin 1989, Grizzle & Lutz 1989. Grizzle et al.

1992, Kamermans et al. 1992).

The growth of suspension-feeding bivalves may also be influ-
enced by lethal or nonlethal (siphon nipping, reduced foraging)
predation by numerous species, such as fishes, gastropods, and
crabs (e.g., Coen & Heck 1991, Irlandi & Peterson 1991. Ka-
mermans & Huitema 1994, Peterson & Skilleter 1994, Skilleter
& Peterson 1994. Bonsdorff et al. 1995. Nakaoka 2000). Although
the siphon tip is easily regenerated, this requires an expenditure ot
energy (see Peterson and Fegley 1986) that could otherwise be
used for shell and soft-tissue growth/repair and reproduction (e.g.,
Trevallion et al. 1970. Hodgson 1982, Peterson & Quammen 1982.
Festa 1975. De Vlas 1985, Zwarts 1986, Coen & Heck 1991.
Skilleter & Peterson 1994. but see Irlandi 1994. 1996. and Irlandi
& Mehlich 1996 for counter examples). In addition, the mere
presence of potential disturbers or predators in the vicinity of
Mercenaria may cause individuals to cease feeding and thereby
lead to reduced growth rates where predators are abundant (e.g..
Blundon & Kennedy 1982, Irlandi & Peterson 1991, Nakaoka
2000).

This study was designed to investigate how the presence of
vegetation and location within a bed can influence the growth ot
selected Atlantic and Gulf of Mexico (U.S.) populations of the
northern and southern quahogs, M. mercenaria and M.
campechiensis. We were especially interested in determining
whether 1 ) generali/'ations could be made that would apply to
populations of both the northern and southern quahog species re-
garding the effect of seagrasses on growth: and 2) whether the
distance of individual clams from the bed's edge could be an
indirect measure of habitat quality as a result of potential differ-
ences in food supply, and therefore help explain some of the con-
flicting evidence regarding the effects of seagrasses on quahog
growth rates.

MATERIALS AND METHODS

Sd/rfv Locations

M. mercenaria and M. campecliiensis populations were
sampled at six different locations in four states (Massachusetts.
New Jersey, Florida, Alabama) along a latitudinal gradient from
about 42° to 30°N. The Massachusetts sampling area was located
on Cape Cod in the Nauset Marsh system (Nauset Harbor and Salt
Pond Bay; 42°N, 70°W), whereas M. mercenaria were collected
from both vegetated (/; = 51 clams) and unvegetated (sand) sub-
strates (/I = 52 clams) in May 1987. Vegetation consisted of pure
stands of Z. marina, and water depth at low tide was less than 1.0 m.

The New Jersey sampling site was in southern Bamegat Bay,
near Marshelder Island (39°N, 72°W), and M. mercenaria were
also collected from both vegetated in = 51 clams) and adjacent
unvegetated (sand) substrates (/; = 84 clams) in May 1987. Veg-
etation was dominated by eelgrass (Z. marina), with lesser
amounts of widgeon grass {Ruppia maririma). Water depth (at low
tide) was less than 1.0 m.

Collections were also made in the Perdido Bay system (30°N.
87°W), with one site at the western end (see Fig. 1 in Coen & Heck
1991; Perdido Pass. Alabama), and one site at the eastern end of
the Bay (in the Gulf Islands National Seashore, Big Lagoon,

Florida, see Fig. 2 in Spitzer et al. 2000). Native M. campechiensis
were collected at the Perdido Pass site in April. May, and June
1987 ()i = 32 clams) and at the Gulf Islands National Seashore site
(30 N, 87 W). in June 1987 (« = 21 clams). All clams collected
from these two sites were taken from vegetated habitats because
there were no hard clams found in the unvegetated sand adjacent
to the seagrass beds. Submerged vegetation at the two sites con-
sisted of shoal grass. Halodule wriglitii (Alabama) and mixed
stands of H. wrif^lilii and turtle grass. Tlialassia testttdinum
(Florida). Water depth (at low tide) at both sites was less than 1.5 m.

Additional details on vegetation composition and physio-
chemical conditions at these study sites can be found in Heck et al.
( 1989. Massachusetts); Wilson etal. (1990. New Jersey); and Coen
and Heck (1991 ), Judge et al. ( 1992. 1993). and Wilson (1991 ) for
the Gulf of Mexico sites.

Clams were collected by hand after being located by treading at
each sampling location. Clam location within grassbeds relative to
the edge of the bed was estimated as the minimum distance from
collection point to the nearest grass bed edge along one of the four
compass directions. All Mercenaria were frozen after collection
and later were thawed to allow removal of the soft tissue without
damaging the shell. Shells were then washed and allowed to air
dry. Valves of each clam were separated, with one valve used for
age and growth estimates and the other archived (see also Wilson
1991). We assumed that all specimens collected from Atlantic
locations are Mercenaria mercenaria (L.). and all individuals col-
lected from Gulf sites are Mercenaria campecliiensis (Gmelin).

Analysis of Growlli Incremenls

For age and growth determinations, one valve from each clam
was embedded in epoxy resin before sectioning with a Buehler
Isomet"^" low-speed saw. Sections were made through the umbo to
the ventral margin, along the axis of maximum growth (Kennish et
al. 1980). A second cut was made approximately 10 tj.ni from the
first to produce a thin section. To facilitate examination of annual
growth increments, each thin section was ground and polished on
a Buehler Ecomet \\\^'^ grinder/polisher with appropriate polish-
ing compounds. During the polishing procedure, visual inspection
of the thin section under a microscope determined when the thin
section was readable (see Wilson 1991 for more detail).

When thin sections were viewed under the microscope, growth
increments appeared as alternating translucent narrow dark and
wide white (opaque) regions corresponding to periods of slow
(narrow microgrowth increments) and rapid (wide microgrowth
increments) growth, respectively (Barker 1964, Rhoads & Pan-
nella 1970, Kennish 1980. Fritz & Haven 1983, Peterson et al.
1983. 1985. Grizzle & Lutz 1988. Arnold et al. 1991. 1996. Bert
& Arnold 1995). One annual growth cycle, therefore, consists of
two opaque regions (spring and fall) and two translucent regions
(summer and winter). In older Mercenaria. there is often only one
opaque region, with the fall band being faint or absent. Annual
growth was marked at the end of each successive winter band for
Atlantic sites (Pannella ct MacClintock 1968. Rhoads & Pannella
1970, Kennish & Olsson 1975, Fritz & Haven 1983), and at the
end of each successive summer band for the Gulf of Mexico sites
(Clark 1979, Fritz & Haven 1983). Thus, one year's growth was
measured as the linear distance between consecutively marked
annual growth bands.

To obtain a quantitative measurement of annual growth, the
distance between consecutive bands was traced on paper at 60x

Influence of Seagrasses on Growth of Hardclams

637

with a camera lucida. Tracing followed the boundary between the
outer, prismatic and middle, homogeneous shell layers with the
end points located at the point where each successive annual band
crossed this boundary (rather than the point where each successive
annual band reached the outer edge of the shell), serving to delin-
eate each year's growth. Distances were measured with a cali-
brated ocular micrometer.

Visual examination of the thin sections indicated that annual
growth increments corresponding to ages >12 y could not be re-
solved with certainty (Wilson 1991, Lowell Fritz, Rutgers Univer-
sity, personal communication). In older Menenaria. annual
growth increments become narrower and more difficult to distin-
guish owing to decreased lateral shell growth (Hopkins 1941, Fritz
& Haven 1983). This slowing of lateral growth may be accompa-
nied by an increase in shell thickness, especially along the shell's
margin. This pattern of shell growth was evident in most of the
larger (i.e., older) Menenaria .specimens in this study. Visual ex-
amination of the thin sections identified clear annual growth in-
crements corresponding to ages <1 1 y. Therefore, only the first 10
annual growth increments from each individual specimen were
used for growth rate determinations.

Walford plots (see Ricker 1975) of yearly clam growth in
length were constructed for individual M. mercenaria and M.
campechiensis (see above assumption) for each site by plotting
total length in a given year (designated G,,^, ) versus total length in
the previous year (designated G,). Total length was calculated
directly from the summation of annual measured growth incre-
ments measured from thin sections. In all cases, the first data point
represents length at year two versus length at year one. "Popula-
tion" Walford plots for each site, constructed by plotting 0,^.,
versus G, values across all ages and clams from a given site,
contained a minimum of 63 and a maximum of 756 data points
(Wilson 1991).

A linear regression of G,.^, versus G, yielded two values of
interest: the slope of the regression line and its y-intercept. The
y-intercept is the growth for the first year (McCuaig & Green
1983, hereafter referred to as the first year growth). The slope (also
called Ford's growth coefficient or simply growth coefficient) is
the fraction of total growth still to be attained after the first year
(Walford 1946, McCuaig & Green 1983). The slope represents the
decrease in growth increments over age classes (Ricker 1975) and
has been used to characterize clam growth rates after the first year.
The point at which the regression line for the Walford plot inter-
sects the line of slope = I, indicates the point at which growth
stops and maximum size is reached.

Walford plots were constructed for each site to determine the
site-specific population growth coefficient and first year growth.
SAS (Version 5) regression procedure (Proc Reg) and its "Test"
option (Freund & Littell 1986) were used then to simultaneously
test for differences in growth coefficient (slope), first year growth
(intercept), and coincidence of the regression lines among pairs of
sites. Pairwise comparisons of M. inerceiuiria between vegetated
and unvegetated habitats were only analyzed for sites in Massa-
chusetts and New Jersey. Between-habitat (within site) compari-
sons were not made at the two Gulf sites because no M.
campechiensis were found in any of the suiTounding unvegetated
habitats in either Alabama or Florida.

Walford plots were also constructed for individual M. merce-
naria and M. campechiensis to determine growth coefficients and
first year growth for each clam. Ford's growth coefficient and first
year growth for each clam were then tested for the significance ot

correlations between these parameters and the linear distance to
the grassbed edge (see above). Because proper construction of the
Walford plots requires a minimum of three sets of data points (i.e.,
three G,^,, G, pairs), only those individuals having four or more
annual growth bands were used to calculate the growth coefficient
and first year growth for individual Mercenaria and sites (see
Wilson 1991 for more details).

RESULTS

Year I and Overall Growth

First year growth, calculated for each site (Fig. 1: Table 1)
ranged from a high of 2.11 cm for the Gulf Islands National
Seashore vegetated site in Florida (mixed H. wriglnii and T.
restiidinum) to a low of 1.10 cm for the vegetated site in Massa-
chusetts (Z. marina). First-year growth was significantly greater in
sand ( = unvegetated) than in seagrass at both the Massachusetts
and New Jersey sites (see Table 2).

There was also significantly greater first year growth at the
vegetated sites in New Jersey, Alabama, and Florida than at the

» -

A

4 -
2 -

-•- Slope = 1

-V- NH
-•- NHU
-*- FL
-1- HI
-»- NS

-e- Hiu

A

0
8 ■
6 -

4

n

^

^

f

Length (t)

Figure L Walford plots for all six sites constructed by plotting length
at year t,., versus length al year t for all ages of all clams combined.
Only the regression lines fitted to each site sampled and a line of slope
= 1 (solid circle) are displayed. The six lines displayed correspond to
the following sites: (1) unshaded diamond line = vegetated Gulf
Islands National Seashore, Florida site (mixed Thalassia tesludinum
and some Halodule wrightii). (2) open circle line ' = unvegetated New
.Jersey site (sand), (3) solid diamond line ♦ = vegetated Perdido Pass,
Alabama site {Halodule). (4) large + line = vegetated New Jersey site
{Zoslera marina and some Ruppia marilima), (5( large shaded triangle
line A = unvegetated Massachusetts site (mud/sand), and (6) large
unshaded triangle line 7 = vegetated Massachusetts site (Z. marina).

638

Heck et al.

TABLE 1.
Growth parameters for Walford plots calculated from clams collected at each study site.

Study Sites

Intercept
(First-Year Growth I cm

Slope
(Ford's Growth Coefficient)

Massachusetts, vegetated (Zoslera)

Massachusetts, unvegetated

New Jersey, vegetated iZostera)

New Jersey, unvegetated

Gulf Islands National Seashore, vegetated iTInilassUi)

Perdido Pass, vegetated (Halodule)

51

1.10

0.91

0.96

84

1.28

0.88

0.96

51

1.64

0.82

0.95

52

1.88

0.80

0.94

21

2.11

0.82

0.94

32

1.73

0.81

0.87

Intercept represents the first year's growth and the slope represents Ford's growth coefficient.

Massachusetts site (Table 2). and first-year growth was signifi-
cantly greater in Florida [Thalassia/Hadodule) than New Jersey
(Zostera) grassbeds. However, no significant difference in growth
was observed between clams from New Jersey and Alabama (Per-
dido Pass, Halodule) grassbeds.

Growth rates of the 291 clams measured, while not presented
individually here, varied over about an order of magnitude. For
example, first-year growth ranged from a low of 0.67 cm for a
clam at the vegetated site in Massachusetts (in Z marina) to a high
of 6.31 cm for an individual at the Perdido Pass vegetated site
(taken from H. wrightii).

Ford's growth coefficients for individual clams ranged from a
low of 0.14 for a clam at the vegetated site in Perdido Pass (W.
wrightii) to a high of 0.97 for an individual at the vegetated site in
Massachusetts (Z marina). Overall, site-specific Ford's growth
coefficients ranged from a high of 0.91 for the vegetated site
Massachusetts (Z marina) to a low of 0.80 for the unvegetated site
in New Jersey (Table 1; Fig. 1). The only significant differences in
the growth coefficients were between both the Massachusetts sites
and all other sites in New Jersey. Alabama, and Florida. These
significantly higher growth coefficients at the Massachusetts sites
were unexpected because first-year growth was lowest at these
sites.

Growth and Location in the Grassbed

There were two significant positive relationships between first-
year growth and distance to the edge of the grassbed, which were

found at the vegetated sites in Massachusetts (Z marina) and
Perdido Pass, Alabama (W. wrightii: r = 0.315 and 0.432; P < 0.05
and 0.01. respectively), indicating greater growth in year I with
increasing distance from the edge of the bed at these two sites
(Table 3). The only significant correlation between Ford's growth
coefficient for individual clams and distance from the grassbed
edge was found at the vegetated site at Perdido Pass (//. wrightii:
Table 3, /■ = -0.435; P < 0.05). a result that indicates greater
lifelong growth near the edge of the bed.

DISCUSSION

The effects of habitat (seagrass versus unvegetated substrate)
on clam growth did not vary consistently when examined by either
clam age or geographical location. With respect to age, we found
significantly greater growth during year one in clams collected
from the unvegetated habitats at both Massachusetts and New-
Jersey (Table 2). We attribute this to the fact that M. merceiwria
growth is greatest during the first 1.5 y of life, with decreasing
growth thereafter as sexual maturity is reached (Haskin 1952,
Gustafson 1955, Walker 1985. Eversole et al. 1986). This suggests
to us that habitat effects would be most evident in analyses of the
rapid growth attained in the first year. However, this finding con-
flicts with that of Iriandi and Peterson (1991), who found faster
growth of juvenile clams (mean size of 2.6-2.87 cm in length)
planted for up to 6 months in seagrass (vs. sand), and who con-
cluded that "'. . . results should dispel any remaining doubts about

TABLE 2.
Pairwise comparisons of first-year growth and Ford's growth coefficient among study locations.

First-Year Growth

Ford's Growth Coefficient

Pair-Wise Comparisons

N
Site 1/Site 2

F Value

Significance Level

{P <)

F Value

Significance Level

(/»<)

Massachusetts, vegetated vs. unvegetated 5I/S4

New Jersey, vegetated vs. unvegetated 5l/.'i2

Massachusetts, unvegetated vs. New Jersey, unvegetated 84/52

Massachusetts, vegetated vs. New Jersey, vegetated 51/51

Gulf Islands National Seashore vs. Perdido Pass 21/32

Massachusetts, vegetated vs. Gulf Islands National Seashore 51/21

Massachusetts, vegetated vs. Perdido Pass 51/32

New Jersey, vegetated vs. Gulf Islands National Seashore 51/21

New Jersey, vegetated vs. Perdido Pass 51/32
All four vegetated subsites (GINS. MA. NJ. and PP) 21/51/51/32

7.90

9.61
78.80
55.47

5.92
89.21
38.03
19.98

0.83
35.38

001
0.01
0.01
0.01
0.05
0.01
0.01
0.01
Not significant
0.01

3.10

3.72

50.71

37.28

0.29

22.66

21.10

0.00

0.62

13.00

Not significant
Not significant

0.01

0.01
Not significant

0.01

0.01
Not significant
Not significant

0.01

Comparisons were performed on the regression equations calculated for the Walford plots at each location using the test option in the regression procedure
of SAS version 5.

Influence of Seagrasses on Growth of Hardclams

639

TABLE 3.

Pearson product moment correlations between first-year growth and Ford's growth coefficient and proximity of clams to the edge of the

grassbeds at each study site.

First-Year Growth

Ford's Growth Coefficient

Correlation

Significance

Level

Correlation

Significance Level

Study Sites

n

Coefficient

(^O

Coefficient

(P <l

Massachusetts {Zostera)

51

0.32

0.02

-0.14

0.34

New Jersey (Zjisiera)

51

-0.05

0.72

0.01

0.95

Gulf Islands National Seashore

(Thatassia)

21

-0.16

0.50

0.15

0.53

Perdido Pass (Haloduk)

32

0.4.3

0.01

-0.43

0.01

the potential for seagrass in relatively high-energy environments to
promote faster growth of suspension feeder living buried in the
sediments." Perhaps the key to resolving the differences in our
results and those of Irlandi and Peterson (1991) is differences in
flow regimes among sites examined. These differences could also
reflect some inherent latitudinal differences between conditions in
North Carolina and those conditions further north (see results of
Ansell !968. or life-long growth reported by Slattery et al. 1991 ).
For example, at our tw o more northern sites, the winter dieback of
seagrasses may impact clam growth seasonally to a greater extent.
than that which more typically occurs in lower latitudes.

Pair-wise comparisons of first year growth between Massachu-
setts, New Jersey, and Gulf of Mexico (Florida and Alabama) sites
revealed significant differences in all but the \egetated New Jersey
versus Perdido Pass comparison. The observed pattern of generally
increasing first-year growth with decreasing latitude was expected,
and is consistent with patterns reported more than 30 years ago by
Ansell (1968).

Significantly higher values of Ford's growth coefficient were
found for all comparisons of the vegetated site in Massachusetts
with the other vegetated sites (Table 2) and in the comparison of
the Massachusetts versus the unvegetated New Jersey sites. We
attribute these differences to slower, but steady lifelong growth in
Massachusetts, with clams eventually attaining a larger final size,
in contrast to the more rapid year 1 growth that quickly becomes
asymptotic in New Jersey, Alabama, and Florida. This is consistent
with a previous study of both Mercenaria species (Jones et al.
1990), which found rapid, but variable growth in Gulf of Mexico
M. campechiensis populations that was often, but not always,
greater than rates measured for Atlantic coast populations of M.
mercenaria. Jones et al. (1990) also found life spans to be typically
shorter in Florida than elsewhere. Based on our finding of rapid
early growth, and the results of Jones et al. (1990). it appears that
Gulf of Mexico M. campechiensis typically have high but variable
growth rates and often shorter life spans than their Atlantic coast
congener.

We found no significant differences in life-long growth at ei-
ther the Massachusetts or New Jersey sites (Table 2) when com-
paring clams in seagrass with those in adjacent unvegetated areas,
although as noted above, we could only consider growth in the first
10 years of life. This result agrees with those of Slattery et al.
(1991). who found no significant differences in lifelong growth
between vegetated and unvegetated habitats at the same study sites
in Massachusetts and New Jersey. However. Slattery et al. (1991)
found that life-long clam growth in North Carolina was greater in
vegetation than adjacent sand, just as Peterson and colleagues had
previously reported (Peterson et al. 1984, Peterson & Beal 1989,

Irlandi & Peterson 1991. but see Nakaoka 2000 for short duration
results).

It is unfortunate that we could not find clams in unvegetated
areas at our Gulf of Mexico sites to better determine whether there
are generally observable latitudinal trends in the effects of vegeta-
tion on clam growth, or whether North Carolina conditions are
unique for Mercenaria (e.g.. Peterson & Beal 1989, Nakaoka
2000). Later studies at the same sites (Coen et al.. unpublished
data) also did not find clams in sand and recorded similar low
densities within grassbeds. Some studies in\olving experimental
planting of juvenile clams in the northern Gulf of Mexico, which
controlled for location in the grassbed, genetic differences, filter
feeder abundance, and sublethal predation. have reported shifts in
growth rates between vegetated and unvegetated substrates de-
pending on season and year. For example. Coen and Heck ( 1991 )
found greatest growth at the same Alabama site in sand than at the
grass edge or bed center, whereas Coen et al. (manuscript in prepa-
ration) found that growth effects varied with location within the
bed, although the relative importance of different effects some-
times varied with season at the same two Alabama and Florida
sites.

Correlations between the growth coefficient and first-year
growth and the distance of clams relative to the grassbed edge
were significant only at the Perdido Pass Thalassia-Halodule site
and for first year growth only at the Massachusetts Zosiera site
(Table 3). As Irlandi and Peterson (1991) note, until recently these
results would have been unexpected because the prevailing view
has been that bivalve growth is positively correlated with flow
rates or food flux (Belding 1912, Kerswill 1949, Haskin 1952,
Pratt & Campbell 1956, Greene 1979, Hadley & Manzi 1984,
Bricelj et al. 1984, Arnold et al. 1991, Bock & Miller 1994,
Wildish & Kristmanson 1997, but see Irlandi & Peterson 1991,
Judge et al. 1992. Lenihan et al. 1996), and slower current veloc-
ities with increasing distance into the seagrass bed should lead to
reduced growth (e.g., Fonseca et al. 1982). However, Peterson et
al. (1984). Irlandi and Peterson (1991), and Judge et al. (1993)
have all indicated that food supply may actually be greater in
vegetated areas primarily resulting from the availability of sus-
pended benthic diatoms and epiphytes. The latter work, along with
Coen and Heck (1991). and Coen et al. (manuscript in prepara-
tion), also evaluated position in the grass bed and its affect on
growth. An additional explanation for variable growth between
vegetated and adjacent unvegetated areas was suggested by Irlandi
and Peterson ( 1 99 1 ). who found differential growth on alternate
edges of seagrass patches, possibly as a consequence of variation
in sediment stability.

Because current velocity and direction were not measured, veri-

640

Heck et al.

ficution of whether distance to the grassbed edge as recorded cor-
responded to distance from the source of prevailing currents was
not possible. However, it did not appear that currents flowed in a
consistent pattern across the beds in Massachusetts, New Jersey,
and Florida but rather varied greatly in their point of origin. The
Perdido Pass site had the clearest directionality of currents and the
"leading (flood tide) edge" of the bed was easily determined
(Judge etal. 1992. 1993). perhaps helping to explain the significant
relationship found there between growth and proximity to the
grassbed edge (Table 3). In addition, any shifts in seagrass bed
boundaries over a clam"s lifetime, or over a significant portion of
the growing season because of winter dieoffs. could also affect our
one time distance estimates from the point of collection to the edge
of the bed and weaken the calculated correlations. Given the nu-
merous articles that discuss seagrass patch dynamics, this potential
problem is likely (e.g., Irlandi 1997. Robbins & Bell 2000). Our
previous work has also suggested that siphon nipping can signifi-
cantly impact clam growth (e.g.. Coen & Heck 1991. Coen et al..
manuscript in preparation)

An additional factor whose importance is difficult to assess
here or in previous studies is the impact that harvesting may have.
Both the New Jersey and Massachusetts populations are subject to
recreational harvesting, and commercial harvesting was observed
at the New Jersey site. In contrast, there is no recreational or
commercial harvesting at either of the Alabama or Florida sites,
resulting in a large part from, very low natural densities. To the
extent that harvesting is biased toward different-sized individuals
or to the extent that desirable sized clams are found and collected
in specific locations within the beds, results from harvested popu-
lations could bias our conclusions. For example, we typically ob-
served, as have others, how aggregated hard clams often are. with
similar age-classes within a high-density patch. In addition, har-
vesting changes the number of filter feeders and potential com-
petitors, which is another variable of potential significance that
could not be controlled.

CONCLUSIONS

Our results clearly showed significantly greater first-year, but
not life-long, growth in sand than in seagrass in Massachusetts and
New Jersey habitats. Therefore, whether one concludes that the
presence of seagrass significantly affects clam growth at our study

sites depended on clam age and the amount of time considered. As
reported previously by others, we found strong evidence for
greater first-year growth in M. campechiensis. but we also found
higher life-long growth rates in our most northern M. inercenaria
population, and we found little evidence that location (i.e.. distance
to nearest bed margin) within the seagrass bed was consistently
related to either short or long-term growth rates.

Although we attempted to account for one potentially con-
founding factor, location within a seagrass bed. we were unable to
control for siphon nipping, a factor that seems to produce signifi-
cant effects on clam growth only some of the time (Coen & Heck
1991. Irlandi & Peterson 1991. and Coen et al.. manuscript in
preparation vs. Kamermans & Huitema 1994. Irlandi & Meh-
lich 1996. Iriandi 1996. and Nakaoka 2000). nor could we control
for the effects of harvesting on filter feeder abundance. Given the
complexity of factors that interact to affect clam growth rates over
Meirenaria's broad geographic range, and past conflicting results,
it is clear that additional, extensive field experiments using the
same design are still needed. At present, we are still unable to
make accurate predictions about the effects of seagrasses on bi-
valve growth rates and related post-settlement survival.

ACKNOWLEDGMENTS

This work was supported in part by NSF (EPSCOR) Grant No.
Rl 1-8996152 to Kenneth L. Heck. Jr.. and fellowship support for
D. Marc Wilson was pro\ ided by the NOAA Office of Sea Grant.
Department of Commerce, under Grant No. NA85AA-D-SGOO.'i
(Project E/O-16). the Mississippi-Alabama Sea Grant Consortium,
and the University of Alabama as well as the Mobil Foundation.
Inc. and Shell Oil Company Foundation. The authors thank Drs.
W. W. Schroeder. A. C. Benke. and M. Judge for their assistance
with various drafts of this manuscript. Thanks are also extended to
David Nadeau. Naomi Mitchell, and Rebecca Rolen for assistance
in the field and in the laboratory, to Randy Schlude for her guid-
ance and help with data processing and analysis, and to Lynn
Bryant and Carolyn Wood for assistance with manuscript prepa-
ration. Special thanks are also extended to Lowell W. Fritz for his
assistance in verifying annual growth bands on thin sections. This
is Contribution #341 from the Dauphin Island Sea Lab and Con-
tribution #492 from the Marine Resources Research Institute.
SCDNR.

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Journal of Shellfish Research. Vol. 21. No. 2, 643-648. 2002.

REPRODUCTION OF CALLISTA CHIONE L., 1758 (BIVALVIA: VENERIDAE) IN THE
LITTORAL OF MALAGA (SOUTHERN SPAIN)

C. TIRADO.' C. SALAS.'* AND J. I. LOPEZ"

'Deparlaniento Bi()loi>ia Animal. Faciihiul de Ciencias. Universidad de Malaga. E-29071- Malaga.
Spain: 'Delegacion Provincial de Agricultura v Pesca. Avda, Aurora 47. E-29071- Malaga. Spain

ABSTRACT The reproduclive cycle of Callisui chione Linnaeus. 1758 was studied using histology and changes in fiesh dry weight,
in the littoral of Malaga (southern Spain), from June 1999 to May 2000. Histologic study of the gonads showed a long reproductive
period, with spawning throughout the year. Three peaks of spawning have been observed through histology as well as flesh dry weight
variation. The first one lasts from February to March and is accompanied by the highest decrease of weight; the second one is in spring,
with the highest percentage of population in spawning but with a lower decrease of weight, and the third one is in summer. The latter
peak represents a new activation of the gonads from postactive stages, without passing through a resting period. The absence of a
resting period in the reproductive cycle of the studied population could be influenced by the mild seawater temperatures and high levels
of chlorophyll a (attributable to the occurrence of upwellings) in the littoral of Malaga. The decrease of the average size in the
population of Malaga from 1981 (70 mm) to 1999-2000 (54 mm length), points out an overexploitation of this resource. The above
data, together with the removal of the close of season since 1990 in Malaga province, make it necessary to regulate a close of season
in this area. We propose a close of season from February to March, months during which there was the most intense release of gametes
in the population.

A'£)' WORDS: Callisiu chione. histology, biomass, reproductive cycle, fishery

INTRODUCTION

Callista chione Linnaeus. 1758 is an Atlantic-Mediterranean
species ranging in the Atlantic from the southwestern British Isles
to Morocco, and also in the Canary, Madeira, and Azores islands
(Tebble 1966, Poppe & Goto 1993). and in the Mediterranean. It
lives in fine and clean sand, from low tide down to ISO m (Poppe
& Goto 1993).

Most of the studies on this species regard growth (Hall et al.
1974. Cano 1981, Forster 1981. Valli et al. 1983-1984, Strada &
Zocco 1985, Valli et al, 1994). Because C. chione is a commercial
species, some studies in Mediterranean waters were conducted in
relation with the monitoring of infections by protozoans (Bravo et
al, 1990, Canestri-Trotti et al. 1998. Canestri-Trotti et al. 2000)
and heavy metals (Belmonte & Grasso 1986. Valli et al, 1994).
Others were related with the influence of the dredge design on the
size of the individuals captured and on the damage caused to the
associated macrofauna (Gaspar et al. 1999). and with the fishery of
this species in the Gulf of Trieste (Italy) (Del Piero 1994). Some
few studies have addressed physiologic aspects. Charles et al.
(1999) analyzed the selective utilization of bacteria and microalgae
by C. chione. Cano ( 1983) analyzed different indexes of condition
over a year in Malaga.

Although C chione is a target-species of local fisheries in the
whole Mediterranean area, there are few studies on reproduction.
Valli et al, (1983-1984) presented preliminary data on the repro-
duction of C. chione in the Gulf of Trieste (northern Adriatic) later
completed in Valli et al. (1994). Nicotra and Zappata (1991) ana-
lyzed the ultrastructure of the mature sperm and spermatogenesis
from an Italian population.

In southern Spain, C. chione. locally known as "concha fma,"
is a very popular shellfish with a considerable consumer market
and an average of about 599 tons/year from 1985-1996. according
to the data of regional authorities. Most of 90'7r of this amount was

*Corresponding author: Tel,: 34-952-131857; Fax: 34-952-132000;
E-mail: casanova@uma,es

obtained and consumed in the littoral of Malaga, Moreover, there
is an significant illegal market and a quantity of catches that could
be of the same order of magnitude as the official catch, but is not
taken into account by official statistics. The law in Andalucia (an
autonomous region including eight southern provinces of Spain)
rules that the fishing season for C. chione must be closed from 1
February to 30 September (order of Consejerfa de Agricultura y
Pesca, November 12, 1984), However, the provincial delegations
are authorized to change it within this interval; in the case of
Malaga, the close of season has been removed since 1990. The
latter decision has been justified by the absence of biologic studies
on the reproductive cycle of the species in the area, together with
the fact that Malaga is a tourist area, which implies greater demand
for shellfish species during almost all year, pailicularly in summer.
The absence of previous studies on the reproductive cycle of C.
chione in southern Spain together with the overexploitation of this
resource because of the absence of close of season, led the regional
authorities of fishery (Consejerfa de Agricultura y Pesca) to pro-
mote this research. This is part of a project on the reproductive
cycles of the most important commercial bivalves of Andalucia
supported by the Consejerfa de Agricultura y Pesca (Department of
Fishery) and entrusted to D,A,P, enterprise (Tirado & de la Rua
2000),

MATERIALS AND METHODS

A total of 3,882 specimens of C. chione were examined and
measured for shell length, ranging from 23-89 mm. The samples
were collected from June 1999 to May 2000, with monthly fre-
quency from October to February and with fortnightly frequency
in the other months. The specimens were captured using a dredge
with a toothed aperture, teeth length of 26 cm, and 6.7 cm of mesh,
usual among the fishermen of the area. The samples were taken in
Fuengirola (36°28'N, 4''43'W) (Fig. 1), at 20 m depth, in a sandy
bottom.

To evaluate the possible influence of environmental factors on
the cycle, the temperature of the seawater at 20 m depth was
measured. Samples of water (2 L) were taken from the bottom for

643

644

TiRADO ET AL.

36° 30'

4° 45"

Figure 1. Saniplinj; area.

determination of chlorophyll a. Pigment analyses were carried out
by filtering the water through Whatman GF/C glass filters. The
pigments of the retained cells were then extracted with acetone for
twelve hours in cool, dark conditions, following the recommenda-
tions of Lorenzen and Jeffrey (1980). Concentrations of chloro-
phyll a were calculated using the trichromatic equations of Jeffrey
and Humphrey (1973).

A total of 3.371 specimens were used for the analysis of tlesh
dry weight variation (about 200 specimens/sample). The length of
every specimen was measured, and the soft parts were then pulled
out of the shell, placed in the drying stove at 100°C for 24 h, and
weighed to the nearest milligram. Two different indexes of con-
dition were applied, the flesh dry weight/L' variation, and thai
proposed by Crosby and Gale (1990) Condition Index (CI) flesh
dry weight x 1.000/volume of the internal cavity of the shell.

The regression of flesh dry weight on the length was calculated
for each sample to estimate the variation in biomass of a standard
individual, based on the logarithmic transformation of Ricker's
function W = aL'' (Ricker 1975), where W is the weight. L is the
length, a is the ordinate at origin, and b is the slope.

The histologic study was performed on 51 1 specimens (usually
30 per sample), with shell length ranging between 23-85 mm. For
the histologic processing, specimens were anesthetized with
MgCK, fixed in 10% formaldehyde, embedded in paraffin, sec-

tioned at 10 |jLm. and stained with hematoxylin of Carazzi and
eosin, and a trichromic staining (V.O.F according to Gutierrez
1967) of hematoxylin of Carazzi. light green, orange G. and acid
fuchsine. The stages of gonad development were scored according
to the scale proposed by De Villiers (1975) for Donax serra
Roding 1798 in South Africa: cytolized, preactive, active, spawn-
ing, and postactive |the equivalent stages from Seed (1969) and
Boyden ( 1971 ) are provided in Table 1 ).

The test of Kolmogorov-Smimov and Kendal and Pearson's
rank correlations included in the program SPSS 8.0. were used to
check the distribution of the data. Cross correlation between both
contlition indexes and percentage of spawning with seawater tem-
perature and chlorophyll a levels were calculated to assess the
influence of the environmental factors on the reproductive cycle.

RESULTS

Sex Ratio

The sex of the specimens of C. cluone cannot be distinguished
macroscopically by the color of the gonads. Therefore, sex deter-
mination must be made microscopically. A total of 51 1 specimens
were microscopically examined, but it was impossible to deter-
mine the sex of some individuals in several months. These samples
were not considered for the sex ratio estimation. From the remain-

TABLE 1.

Different scales of gonad condition.

Authors

De Villiers (197.';)
Seed (1969)
Boyden (1971)

Cytolized
Resting G. (0)
Indeterminated G (I)

Scales of Gonad (G) Condition

Preactive

Developing G ( 1.2.3)
Developing G.

Active Spawning
Ripe G. (5)
Ripe G (ill)

Postactive
Spawning G. (4.3)
Spawning (IV)

Spawning G (2.1)
Resting G. (V)

gonad.

Reproduction of Calusta ch/one Linnaeus. 1758

645

ing 272 individuals. 125 (45.96%) were males and 147 (54.04 7p)
females. The sex ratio for all them can be considered as 1:1 (x~ =
0.012. P>0.95) (Fig. 2).

Sexual Cycle

Biomass Analysis

The variation of flesh dry weight/L' ratio during the annual
cycle is shown in Figure 3. The mean values of both variables.
flesh dry weight and size (L'), were considered. The standard
deviations were between 10.59^8.32%. A broad size range (Fig.
4) can be observed in the samples, which explains, in part, these
differences. Also, a broad weight range can be observed in most of
the samples (Fig. 5), which is related, in part, with the presence of
different stages of development of the gonads.

From June to October. C. chione shows two decreases of the
above ratio (Fig. 3). with a small increase in August. During the
autumn months, there is another increase, followed by a decrease
from January to March. During the spring, there is a continuous
slight increase, before the first spring decrease.

The other index (CI) shows less pronounced increases and de-
creases (Fig. 6). Two decreases can be detected, one of them from
late August to December and the other one from January to March.

To minimize the bias introduced by the somatic growth of
individuals during the cycle and by the variation in the size of the
specimens between successive samples, the variation of flesh dry
weight was estimated for a standard individual of 54-mni length,
taking into account the regression lines for every sample (Table 2).
This size is close to the mean size of the population.

Figure 7 shows a major decrease in the flesh dry weight from
January to March, although there are also drops from June to
September. It is interesting to observe the difference in flesh dry
weight between the first half of June 1999 and second half of May
2000. which could indicate that the sexual cycles are out of phase
between contiguous years. In autumn, the values remain more or
less stable. The two major increments of the flesh dry weight of the
standard individual were registered between March and April and
between the two samples of June.

Ganietogenic Cycle

Data from the histologic study are presented in Table 3. which
shows the total number of specimens analyzed and their develop-
ment stages, according to the scale of De Villiers (1975). The
studied population of C. chione shows continuous spawning
throughout the year, with values higher than 30% of the population
in 12 of the 17 samples examined. The whole population was
spawning in May, while the lowest percentage of spawning (30%
of the sample) was registered in the first half of September (Fig. 8).

If we considered the absence of any individual in cytolized or

% 100

Ag Sp Oc Dc Fb Ap My

Figure 3. Flesh dry weight (FDW)/Lenglh (I/) ratio throughout the
year of study. Bars sho» standard deviation.

postactive stage, the main period of sexual activity lasts from
February to June. In February, the presence of active individuals
points to the beginning of the active period (Table 3). There is not,
however, a true resting period because of the existence of spawn-
ing in the population at any time of the year. The regression of the
gonads begins in June and July, with the occurrence of individuals
in postactive stage, which were predominant from the second half
of September to December, together with the presence of some
individuals in cytolized stage.

The ganietogenic cycle is asynchrononic in the population,
which is evidenced by the presence of at least two developmental
stages in nearly all the samples. An asynchrony is also detected in
the individuals attributable to the coexistence of areas with differ-
ent stages in the same gonad.

Several cohorts of ovocytes can be detected throughout the
year, together with the direct step from postactive to active stage,
without a previous cytolized and preactive phases.

F^nvironmental Factors

The maximum temperature (Fig. 9) was registered in the sec-
ond half of August (22'C) and the minimum (13.8°C) in January.
The maximum of chlorophyll a (Fig. 9l levels occurs in the first
half of August 2000 and the second peak in the first half of May
1999. Between these extremes, we observed several minor peaks,
at the end of summer (first half of September), in autumn (Octo-
ber), and at the end of winter (March).

Coefficients of correlation of Pearson were estimated between
temperature and FDW/L^ and temperature and CI, because the
variables showed a normal distribution (according to the test of
Kolmogorov-Smimov). However, between percentage of popula-
tion in spawning and temperature, chlorophyll a levels, and the
different condition indexes, the coefficients of correlation of Ken-

Jn Jn Jl Fb Mr Ap Ap My My
Figure 2. Relative frequency (% ) of sexes during the year of study.

Ag Sp

Ap My

Figure 4. Monthly average length (L) throughout the year of study.
Bars show standard de\iation.

646

TiRADO ET AL.

M 7000
^ 6000

a 5000

4000

TABLE 2.
Linear regression calculated for each month.

Figure 5. Monthly average flesh dry weight (FDVV) throughout the
year of study. Bars show standard deviation.

dal were estimated, because the percentage of spawning dataset
showed a non-normal distribution.

These coefficients of correlation have been calculated simulta-
neously and with one and two months of delay. The temperature
was directly correlated, with one month of delay, with CI (r =
0.629. P < 0.05). In the same way, the temperature was also
directly correlated, with a delay of two months, with the FDW/L'
of the population (r = 0.573. P < 0.05). A significant coefficient
of Kendal's correlation has been obtained between the temperatuie
and the tlesh dry weight of the standard individual, with a month
of delay (tau = 0.394, P < 0.05). No other correlation was sig-
nificant.

DISCUSSION

Sex Rath)

Callista chione is a species that does not show sexual dimor-
phism, and the sex ratio is 1:1. Our data regarding the sex ratio are
similar to those of Valli et al. (1994) in the Gulf of Trieste, who
obtained percentages of 46.25% males and 53.759r females. Also,
we have not found any hermaphrodites in the studied population.

Reproductive Cycle

According to the histologic data. C. chione from the littoral of
Malaga had individuals in spawning stage throughout the year
(Fig. 8), while in the Gulf of Trieste (northern Adriactic) the
spawning extends from February to September (Valli et al. 1983-
1984). or from March to September (Valli et al. 1994). This dif-
ference is easily explained by the much lower winter seawater
temperatures (in the order of 6°C) registered in the Northern Adri-
atic. However, the high amount of phytoplankton and zooplankton
of the Gulf of Trieste resulted in the absence of a resting period,
because the individuals remain in a preactive stage during autumn
and winter months (Valli et al. 1994J. In Malaga, most of the

W (L = 54

Months

Lni

Regression

^ines

R-

R

n

mm)

June

62.93

Y

=

2.927 X

-1.774

0.967

0.984

128

1983.59

June

66.20

V

=

3.017 X

-1.837

0.908

0.953

94

2445.84

July

59.23

V

=

3.218 X

-2.204

0.935

0.967

197

2346.59

July

50.40

V

=

3.205 X

-2.192

0.958

0.979

200

2286.74

Aug

51.10

V

=

3.297 X

-2.371

0.969

0.985

202

2194.50

Aug

52,94

V

=

3.294 X

-2.358

0.966

0.983

200

2229.42

Sept

51.57

y

=

3.261 X

-2.363

0.958

0.979

199

1934.74

Sept

49.49

V

=

3.345 X

-2.510

0.945

0.972

201

1928.17

Oct

48.90

Y

=

3.533 X

-2.837

0.952

0.976

200

1917.97

Nov

56.56

Y

=

3.423 X

-2.645

0.974

0.987

152

1930.79

Dec

51.30

Y

=

3.524 X

-2.820

0.979

0,990

200

1928.28

Jan

62.00

V

=

3.246 X

-2.309

0.900

0.949

203

2057.40

Feb

51.75

Y

=

3.115 X

-2.150

0.998

0.999

198

1 760.88

Mar

47.30

Y

=

2.265 X

-0.762

0.511

0.715

200

1451.32

Apr

55.04

Y

=

3.449 X

-2.675

0.938

0.968

199

1997.13

Apr

53.06

Y

=

3.168 X

-2.141

0.919

0.959

200

1982.24

Mav

56.38

V

=

3.137 X

-2.124

0.972

0.986

199

2045.41

May

53.30

y

=

3.335 X

-2.451

0.972

0.986

200

2123.80

LM = average length; R- = coefficient of determination; R = coetTicient
of correlation; /I = number of observations; W (L = 54 mm) = weight of
a standard individual of 54-mm long.

individuals remained in postactive and spawning stages during the
autumn and winter months (Fig. 8). Cano ( 1983) studied different
condition index in C. chione from the littoral of Malaga, detecting
only one strong decrease of dry weight (between January and
February of 1981). Other species from southern Spain, such as D.
truncuhis. D. veniistus. and D. semislriatus, show very long
spawning periods, but in these species, there was at least one
month without spawning (Tirado & Salas 1998. Tirado & Salas
1999).

The mild seawater temperature (between 13.8°C-23°C) and the
high concentration of phytoplankton (because of the presence of
upwellings) in the littoral of Malaga (Fig. 9) would favor, prob-
ably, a long reproductive cycle and the absence of a resting period.
The temperature is the most influential factor, according to the
coefficients of correlation. It is correlated with weight increments,
a consequence of development of the gonads.

According to the hypotheses of most authors, the gamete re-
lease seems to be controlled by such physical environmental vari-
ables as changes in temperature, salinity, or photoperiod (Sastry
1979 and references therein). An alternative hypothesis is that
phytoplankton induces spawning (Ruiz et al. 1992). Starr et al.

220

200-1

180

160

140

120

100-

80-

60

40

Jn Jl Ag Sp Oc Dc Fb Ap My

Figure 6. Index of condition of Crosby & Gale (CI I: tlesh dry weight

X 1,(H)()/ volume of the internal cavity of the shell, throughout the year Figure 7. Variations in Hesh dry weight in a standard

of study.

Ap My
Callista chione

animal 54-mm long.

Reproduction of Callista chione Linnaeus. 1758

647

TABLE 3.
Developmental stages of the gonad over the year.

Months

Pr

EA

Ps

June

1

11

20

32

July 1

6

24

30

July 2

I

4

9

1

13

1

29

Aug 1

6

3

9

1

10

30

Aug 2

9

1

II

9

30

Sept 1

4

1

14

11

30

Sept 2

4

4

1")

30

Oct

3

6

21

30

Nov

7

5

18

30

Dec

I

9

20

30

Jan

1

14

15

30

Feb

4

1

19

30

Mar

2

3

4

19

2

30

Apr 1

1

29

30

Apr 2

3

27

30

May 1

30

30

May 2

1

29

30

C = eytolized; Pr = preactive; EA = early active; A = active; S =
spawning; Ps = postactive; n = number of observations.

(1990) showed that blooms of phytoplankton should be sufficient
to induce spawning in tnussels as well as in urchins. The spawning
of Crassostrea gigas in El Grove (Galicia, northwestern Spain),
with water temperature below 18°C. was correlated with phy-
toplankton bloom (Ruiz et al. 1992).

Although the chlorophyll a levels in the littoral of Malaga
during this study did not show significant correlation with any
analyzed index or variable, the coincidence of some decreases in
weight (Figs. 3. 6) with peaks of chlorophyll a (Fig. 9). seem to
indicate some influence.

The data of biomass showed three important decreases: be-
tween January-March, second half of August-September, and
June and July (Figs. 3. 7). The latter was not reflected in CI (Fig.
6). The first drop is coincident with the start of activation of the
gonad and with the increase of percentage of population in spawn-
ing (Fig. 8). At the first half of summer, there are high percentages
of individuals in postactive stage, which begin a new gonadal
activation in the second half of July. This direct step from post-
active to active stages without a previous eytolized phase, has been
found in Donax tnincidus (L. 1758) from the littoral of Malaga
(Tirado & Salas 1998) and seem to be related with the presence of
peaks of chlorophyll a in this area (Fig. 9). Ansell (1961) reports

DC BPrilEASA HS SPs

% 100

Figure 8. Relative frequency of different stages of development of the
gonads in C. chione.

Figure 9. Seawater temperatures and changes in concentration of
chlorophyll a in seawater throughout the year of study.

that in the population otChamclea striatiila from Kame Bay (Mill-
port), the ovary passes directly from the spawning condition to an
early stage of development, because of the development of young
ovocytes in the ovary before the end of spawning.

Also, according to the data for the standard individual (Fig. 7).
the main decrease of flesh dry weight was registered in winter;
whereas, those of summer seem to be less intense. Although the
percentage of the population spawning in spring was the highest of
the year (Fig. 8). the resulting decrease of biomass was less than in
winter or summer ( Fig. 3 1. This is consequence of the fact that the
emissions from winter were more intense, with implication of the
entire gonad; whereas, in spring and summer, there were only
partial spawnings. The latter, together with the coexistence of dif-
ferent stages of development in the same gonad, could indicate that
the spring/summer spawning is at least the second one for an
individual during the cycle. In the field, it is difficult to know if a
particular individual has more than two spawnings by reproductive
cycle. In the laboratory, the venerid Chamelea striatula spawns
repeatedly at intervals throughout the spawning season (Ansell
1961). In the littoral of Malaga, two spawning periods per indi-
vidual were detected in D. mmculus: whereas, in other such spe-
cies as Donax vemistus and Donax semistiiatus, only one spawn-
ing per individual and cycle was observed (Tirado & Salas 1998.
Tirado & Salas 1999).

The asynchronic gametogenic cycle in the population is re-
flected by the high standard deviations of the flesh dry weights
(Fig. 5), the existence of several cohorts of ovocytes, and the
coexistence of several stages of development in the same gonad.
The coexistence of different stages has been found in many bi-
valves from temperate areas, among them, Chamelea striaiiila
(Ansell 1961), Donax serra (De Villiers 1975), Tapes rhomboides
(Morvan & Ansell 1988), D. mmculus (Tirado & Salas 1998). D.
venustus and D. semistriatus (Tirado & Salas 1999).

Although the total of captures of C. chione in the littoral of
Andalucia is high (about an average of 599 tons/year from 1985-
1996). in the last few years (from which statistical data have been
published) this volume has decreased to values of about 188 tons
in 1995. or about 259 tons in 1996. On the other hand, the average
length of the Malaga population has decreased from about 70 mm
in 1979-1981 (Cano 1981) to 54 mm in 2000 (present study). All
these points indicate an overexploitation of this resource. More-
over, the recorded data on growth indicate a slow growth of C.
chione. Hall et al. ( 1974) registered a growth of 2 mm by year in
the Gulf of Trieste; Forster ( 1981 ) suggested that a specimen of
Plymouth reaches 9 cm in 40 y. The above data make it an urgent
necessity to provide a close of season that, according to the data of
this study, must be February-March period during which there was
the most intense release of gametes in the studied population.

648

TiRADO ET AL.

ACKNOWLEDGMENTS

The aullidis thank David Lope/, Daniel Gome/, and M"
Jose Garci'a-Patino for helping in the laboratory process. This
study is part of a project supported by the Junta de Andalucia,
(Department of Fishery). M" Dolores Atienza. general manager
of the Department of Fishery is thanked for her trust in our
work and the permission to publish. We are grateful to Manuel

Castanon (Provincial Manager of Fishery) for his encourage-
ment in this research. The project was entrusted to D.A.P.
enterprise, which is thanked for the use of facilities given for
realization of the work and publication of the results. We
are grateful to Ildefonso Marquez and Manuel Aguilar for
their management and the facilities given for the development of
this research. Eva Garcia is thanked for her help with the refer-
ences.

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INGESTION, DIGESTION, AND ASSIMILATION OF GELATIN-ACACIA MICROCAPSULES
INCORPORATING DEUTERIUM-LABELED ARACHIDONIC ACID BY LARVAE OF THE

CLAM VENERUPIS PULLASTRA

S. NOVOA,' D. MARTINEZ,' J. OJEA,' P. SOUDANT," J.-F. SAMAIN,' J. MOAL,' AND
J.-L. RODRIGUEZ^ *

^Centra de Ciiltivos Marinas de Rihcideo. CIMA. Conselleria de Pesca, Marisqueo y Acuicultiira. Miielle
de Ponilldn s/n 27700 Rihcideo, Spcuii: -(Jniversite de Bretague Occidentcde. lUEM. UMR 6539
LEMAR, Teclmopole Brest-Iroise. Place Nicolas Copernic, 29280 Plouzane. France: DRV/A.
Laboratoire LPI. IFREMER Centre de Brest. BP 70. 29280 Plouzane. France: and '^Departmento de
Bioquinucu y Biol. MoL, Facultad de Veterinaria. campus univ. s/n, 27002 Lugo, Spain

ABSTRACT Olive oil gelatin-acacia microcap.sules (GAMs) enriched with deuterium-labeled arachidonic acid (*AA) were prepared
and fed to Veiienipis pullaslra larvae. In a first experiment, larvae were either starved or ted these microcapsules (*AA-GAM) for 12
h. The *AA-GAM-fed larvae incorporated *AA and oleic acid (OA) in their neutral and polar lipids. The incorporation yield was
around 5% for *AA in neutral and polar lipids and 2% and \'7c for OA in neutral and polar lipids, respectively. In a second experiment,
larvae were fed for 7 days with a mixed algal diet supplemented with or without *AA-GAM. The microcapsules were ingested and
digested without any harmful effect on larvae. OA was incorporated preferentially in neutral lipids whereas *AA was equally
distributed in neutral and polar lipids. The incorporation yield was higher in the second experiment probably in relation to the presence
of algae. However, the incorporation rate of both tracer fatty acids decreased with time in both lipid classes. *AA dropped form 16-2%
in both neutral and polar lipids and OA decreased more rapidly in polar (16-3%) than in neutral lipids (33-15%). These changes in
the incorporation yield could correspond to the growth slowing down at the approach of the metamorphosis or to a saturation in the
levels of fatty acids because the percentage of natural arachidonic acid stayed stable in neutral lipids or tended to decrease in polar
lipids. The easy fabrication and use of GAM associated to the direct measurement of a deuterated fatty acid by gas chromatography
is a promising tool for studying lipid metabolism in mollusks.

KEY WORDS: clam, Venerupis piilliisiru, nutrition, microcapsules, larva, fatty acids

INTRODUCTION

The production of living microalgae (LMA) as food for the
larvae and spat of bivalves in commercial hatcheries accounts for
approximately 30% of operating costs (Coutteau & Sorgeloos
1992). Because of the high cost and unpredictability of the algal
culture, the development of artificial diets for bivalve mollusks
was attempted by several investigators (Jones et al, 1974). Micro-
capsules of the cross-linked protein-walled type were used to ex-
amine aspects of protein (Langdon & Siegfried 1984) and carbo-
hydrate (Kreeger et al, 1996) metabolism of bivalves and gelatin-
acacia microcapsules were investigated in lipid nutrition (Langdon
& Waldock 1981, Chu et al. 1987, Numaguchi & Nell 1991,
Knauer & Southgate 1997a, 1997b), Some potential alternatives,
such as dried microalgae, microalgal pastes, lipid emulsions, and
microcapsulated or yeast-based artificial diets (for revision, see
Coutteau & Sorgeloos 1993; Robert & Trintignac 1997), have
shown promising results. The total or partial substitution of mi-
croalgae by artificial particles has been proposed, where, ideally,
nutritional requirements of bivalves should be satisfied using diets
whose composition could be precisely controlled. The gelatin-
acacia microcapsules (GAMs) are not a suitable vehicle to deliver
complete artificial diets to the larvae of bivalves because only
water-insoluble nutrients can be encapsulated using this method.
However, they may be a useful tool in studies addressing aspects
of the lipid nutrition in addition to being a suppleinent for mixed
microalgal diets poor in some essential fatty acids, GAM could be
a useful supplement if sufficient quantities of LMA were unavail-

*Corresponding author. E-mail address: jrodritsiugo. usees

able to feed spat (Numaguchi & Nell 1991), GAMs are simple to
produce, relatively easy to use. and can be prepared rapidly on
demand. However, the potential of GAM as a substitute for LMA
under large-scale culture conditions must still be assessed (Knauer
& Southgate 1997a). A number of studies have been undertaken to
prove GAM ingestion, digestion, and assimilation and have shown
that GAM were readily digested by bivalves (Chu et al, 1982,
Southgate 1988) and that the lipids supplied were assimilated with
high efficiency (Knauer & Southgate 1997c). The data generated
in such studies will facilitate the further development of suitable
artificial diets for the larvae of marine bivalves. The knowledge of
the nutiitional requirements of the larvae of cultured bivalves is
necessary to improve the efficiency of algal diets used in hatch-
eries and to design convenient artificial diets. Previous studies
pointed out the energetic role of lipids during the larval develop-
ment of mollusks (Holland 1978), as well as the changes in the
composition of the polyunsaturated fatty acids (PUFAs) occurring
in neutral and polar lipids during embryogenesis. This nutritional
transition suggests that a metabolic control takes place in devel-
oping larvae (Marty et al. 1992). It is probable that this control
results from a selective incorporation of dietary fatty acids by
acyltransferases, indicating a preferential incorporation of long-
chain PUFA, especially the 22:6(n-3) in the case of the larval
development of Pecten maximus (Marty et al. 1992). This study
was proposed as an attempt to confirm the assimilation of the fatty
acids incorporated in GAM and their bioconversion in endogenous
fatty acids of neutral and polar lipids in the larvae of marine
bivalves. The data generated could indicate whether GAM could
serve as a tool in studies of lipid metabolism in marine bivalves as
well as demonstrate their use as a nutritional supplement in mixed
microalgal-microcapsulated diets.

649

650

NOVOA ET AL.

MATERIALS AND METHODS

Chemicals

Lipid Standards and Reagents

Deuterium-labeled arachidonic acid (FA-503 arachidonic acid-
5,6,8,9, 11, 12. 14. 15-ds)wa.s obtained from BIOMOL Research
Laboratory. Inc. (Plymouth Meeting. PA). Identification and quan-
tification were based on standard fatty acid methyl ester mixture
(Sigma-Aldrich. Chemical Spain). Internal standard, Tricosanoic
acid (C2.'':0). was purchased from Nu-Chek-Prep. Butylated hy-
droxytoluene (BHT) and fluorescein isothiocyanate (FITC) were
supplied by Sigma Chemical Co. (St. Louis. MO) and sodium
hydrogen bicarbonate was from Merck (Damistadt. Germany).

Solvents

Hexane. chloroform, and methanol were high-performance liq-
uid chromatography (HPLC) grade from Merck (Darmstadt. Ger-
many). Boron trifluoride (10%. w/w) in inethanol (BF,) was ob-
tained from Supelco (Bellefonte. PA).

Fabrication of G A Ms

Two different sets of GAM were fabricated following a modi-
fied method described by Rodriguez et al. (1992).

HTC-GAM

GAMs were prepared with 500 (jlL of cod liver oil and by later
addition of FITC. Briefly. 500 (xL of cod liver oil was homog-
enized with antioxidant BHT (5%. w/v). Then. 500 mL of cod liver
oil plus antioxidant was emulsified with a 1:1 mixture of a 2%
(w/v) solution of gelatin and a 2% (w/v) solution of acacia, which
had been made up separately in distilled water. The emulsion was
stirred in a Virtis Tempest IQ" blade homogenator programmed at
.30.000 rpm for 2 min The pH of the mixture was reduced until the
coacervation pH (4.3). by the drop-by-drop addition of dilute HCl.
The mixture was stirred for 40 min and pH was then raised to 9.3
by the addition of dilute NaOH. The resulting GAM suspension

Figure L Larva of V. piillastra observed under fluorescence micros-
copy. Accumulation of yellow lluorescence indicates ingestion of mi-
crocapsules. (Author: .I.-L. Rodriguez)

Figure 2. Digestion of microcapsules throughout a period of 20 min.
Sequence A-C shows the disappearance of the yellow fluorescence
inside a larva of V. piillaslra. (.Author: J.-L. Rodriguez)

was poured into 500 mL of cold distilled water and kept in a
refrigerator for 2 h. Previously a .solution of FITC 0.25% (w/v) in
100 mM sodium hydrogen bicarbonate (NaHCO,) was prepared.
Two milliliters of stock microcapsules were incubated at 4"C for
12 h with 0.5 mL of FITC solution diluted with 2.5 inL of 100 mM
NaHCO, solution. The mean diameter of FITC-GAM was 4.0 ±
1 .0 (Jim (n = 100). Two milliliters of FITC-GAM was used to feed
a batch of 23-day-old Venenipis piillastra larvae and subsequently
were observed under fluorescent microscopy.

Ingestion, Digestion, and Assimilation of GAM

651

340

320

|300

=5 260
240 -
220
200 J

100

^ 60

I

d 20 -

□ AB2 Feeding algal laivae
-■- AB2 Feeding 'AA -GAM + algal larvae

Q AB2 Feeding algal laivae

■ AB2 Feeding "AA -GAM + algal larvae.

9$°„ 96°;

16

20

22

23

25

Figure i. Shell length (fim) of the AB2 batch, fed with algal (open squares) and *AA-GAM + algal (solid squares). Survival rate (%) of the same
batch fed with algal (open bars) and with *AA-GAM -h algal (solid bars). Results are expressed in (im (%); Mean ±. SD, n = 100.

Deuterated Arachidonic Acid (*AA)-GAM

GAMs were prepared with olive oil supplemented with deute-
rium-labeled arachidonic acid. Briefly. 30 (xL of olive oil was
homogenized with 5 mg of deuterium-labeled arachidonic acid and
antioxidant BHT (5%. w/v). Deuterated *AA-GAM was obtained
following the method previously described. The resulting GAM
suspen.sion was poured into 250 mL of cold distilled water and
held in a refrigerator for 2 h. The mean diameter of *AA-GAM
was 3.5 + 0.9 ixm (;? = 100) and the stock suspension of GAM
was kept at 4°C and shaken daily.

Feeding Experiments

A population of V. piillastra D-larvae termed AB2 was selected
by its good growth and survival rates and distributed in 500-L
tanks at a density of 5 larvae-mL"'. Larvae were fed daily with a
microalgal mixture of Pavlova lutheri (Droop). Isochnsis aff. gal-
bana Green (clone T-iso; Tahiti Isochiysis). Skeletoneina coslaliim
(Greville), and Chaetoceros calcitrans (Tanako) at a density of
15/15/15/15 cells-M-L-'.

Experiment 1

Larvae 14 days of age from the AB2 culture were collected and
transferred at the same density into two 6-L flasks for a 24-h
experiment. One batch was starved whereas the other was fed with
60 |xcap-|a.r' *AA-GAM. *AA-GAM-fed larvae had seawater
renewed 12 h after feeding, and no food was supplied then. Twelve
hours later, both fed and starved larvae were collected on a 45-(xni
mesh.

Experiment 2

Larvae 17 days of age from the same AB2 culture were dis-
tributed in two 150-L tanks at a density of 4 larvaemL"' and fed

daily with two different diets for 7 days, either a microalgal diet of
P. lutheri. I. aff. galhana. S. costatum, C. calcitrans. and Tetra-
selmis suecica (Butcher) at a density of 20/20/15/15/10 cellspiL"'
or a diet consisting of the same microalgal mixture supplemented
with 5 |jLcap-|jiL"' *AA-GAM.

Samples for Fatty Acid Analyses

Larvae from the 24-h experience were collected as indicated
above. Larvae from the 7-day experience that were 18. 19. 21. and
24 days of age were collected 4 h after feeding on a 45-(j.m mesh
('( = 1).

All larval samples were crushed and stored in a CHCl,-MeOH
mixture (2:1. v/v) under nitrogen at -30°C until fatty acid analysis
could be pert'ormed. Samples of microalgal mixture (n = 3) and
microcapsules {ii = 3) were filtered on GF/F (Whatman) glass
fiber filters and stored similar to larvae before analysis.

Fatty Acid Analysis

The separation of the polar and neutral lipids was performed by
micro-column liquid chromatography as described by Marty et al.
( 1992). Total lipids were evaporated to dryness and dissolved three
times using 500 |xL of chloroform/Tnethanol (98:2). Neutral and
polar lipids were separated on a silica gel 6% (w/w) hydrated
microcolumn (30 x 5 mm) using chloroform/methanol (98:2) and
methanol successively as eluting solvents. The fractions were col-
lected under nitrogen in screw-capped flasks containing a known
amount of 23:0 as internal standard for quantitative determina-
tions. Fatty acid composition and quantification of polar and neu-
tral lipids were determined using gas chromatography (CG). after
purification of fatty acid methyl esters (FAMEs) by HPLC.
FAMES from the neutral and polar lipid fractions were transes-

652

NOVOA ET AL.

terit'ied with \0% (w/w) BF, in melliunol (Metcalfe and Scliniit/
iy61 ) for 10 niin at lOO'C. After cooling. FAMEs were extracted
with hexane. The organic phase was evaporated under nitrogen and
dissolved in chlorofomi/methanol (98:2) for purification by HPLC
(Hennion et al. 1983. modified). FAMEs present in each lipid
fraction were injected in a CG System HP6890 series equipped
with a split/splitless injector, a tlame ionization detector, and a
DBWAX capillary column (30 m x 0.25 mm ID; 0.2-|xm film
thickness). The carrier gas was H^, at an initial pressure of 80 kPa.
The oven was programmed to stay at the initial temperature of
60°C for 2 min. rise from 60 to 160X at a rate of 5()°C/min. stay
for 2 min and from 160 to 170°C at 1.5°C/min. then 170-185°C at
2°C/min. next 185-240^C at 3°C/min. and tmally remain at 240'=C
for 10 min. Injector and detector temperatures were 230°C and
250°C. respectively. The flow rates of compressed air. hydrogen
and make up gas (nitrogen) were 300. 30. and 20 niLiiiin"'. re-
spectively. Fatty acids were identified by comparison of their re-
tention times with those of standards. The C:X (n-Y) notation was
adopted, where C was the number of carbons. X the number of
double bonds, and n-Y the position of the first double bond from
the terminal methyl group.

TABI.K 1.

Fatty acid composition of the deuterium-labeled
microcapsules (*AA-GAM).

Incoi-poration yield was calculated using the following equa-

(AA-GAM„-algae„)ng FA x lar\'ae"

*AA-GAM ( % )

Fatty Acid

Mean

SD

Mean

14:U

0.061

().()03

0.028

16:0

10.830

0.066

4.920

18:0

2.606

0.075

1.182

20:0

0.390

0024

0.176

22:0

0. 1 3 1

().()t)7

0.059

24:0

0.073

0.027

0.032

16:1 (n-9)

0.171

0.018

0.077

16:1 (n-7)

0.872

0.036

0.395

18:1 (n-9)

65.691

0.310

29.863

18:1 (n-7l

2.481

0.493

1.145

20:1 (n-9)

0.326

0.019

0.147

18:2 (n-6)

10.056

0.333

4.564

18:2 (n-4)

().()()()

0.000

0.000

18:3 (n-6)

0.000

0.000

0.000

18:3 (n-3)

0.749

0.029

0.339

18:4 (n-3)

0.000

0.000

0.000

20:2 (n-6)

0.000

0.000

0.000

20:.^ (n-6)

(),()()()

0.000

0.000

20:4 (n-6)-d«

5.363

0.238

2.428

20:4 (n-6)

0.000

0.000

0.000

20:4 (n-3)

0.000

0.000

0.000

20:5 (n-3)

0.000

0.000

0.000

22:4 (n-6)

0.000

0.000

0.000

22:5 (n-6)

0.000

0.000

0.000

22:5 (n-3)

0.000

0.000

0.000

22:6 (n-3)

0.076

0,012

0.034

TO.SAT.

14.215

0.208

6.453

TO.MONO

69.541

0.728

31.627

TO.POLY

16.244

0.520

7.366

(n-3)/(n-6)

0.053

0.001

0.002

ng/larvae

45.446

AA-GAM (ng/larvae)
SD

0.002
0.583
0.118
0.011
0.004
0.009
0.002
0.033
3.804
0.37 1
0.010
0.497
0.000
0.000
0.028
0.000
0.000
0.000
0.192
0.000
0.000
0.000
0.000
0.000
0.000
0.003
0.711
4.210
0.711
0.000
5.613

X 100

incorporation %:

(AA-GAM-algae) ng dietary FA x larvae

where the numerator is equal to fatty acid difference between
larvae fed the microcapsules + algae and larvae starved or fed the
algae and the denominator is the fatty acid supplied by the micro-
capsules.

RESULTS

Ingestion and Digestion of Microcapsules

After feeding on FITC-GAM for 4 h. larvae were observed
under fluorescent tDicroscopy. The ingestion of FITC-GAM was

TABLE 2.

Fatty acid composition of the diets supplied in experiment 2: algal

diet (mixted niieroalgae only) and *A.4-GAM + algal diet (mixted

microalgae supplemented with microcapsules).

,\lgal Diet (ng/larvae)

*AA-GAM + Algal Diet
(ng/larvae)

Fatty Acids

Mean

SD

Mean

SD

Values in hold are. respectively, the major fiitty
(n-9)] and the deuterium-labeled ;inichid(inic ac
Results are expressed in percentages (%) and
(ng/larva) in experiment 1. Values are means

icid pre.sent in olive oil |18:1
id added |20:4 (n-6)-d8|.
in ng supplied to each larva
+ SD (« = 3).

14:0
16:0
18:0
20:0
22:0
24:0

16:1 (n-9)
16:1 (n-7)
18:1 (n-9)
18:1 (n-7)
20:1 (n-9)

16:2 (n-7)
16:2 (n-4)
16:3 (n-6)
16:3 (n-4)
18:2 (n-6)
18:2 (n-4)
18:3 (n-6)
18:3 (n-3)
18:4 (n-3)
18:5 (n-3)
20:2 (n-6)
20:3 (n-6)
20:4 (n-6)-d«
20:4 (n-6)
20:4 (n-3)
20:5 (n-3)
22:4 (n-6)
22:5 (n-6)
22:5 (n-3)
22:6 (n-3)
TO.SAT.
TO.MONO
TO.POLY
ne. Diet/larvae

15.220
14.1(.)5
0.694
0.189
0.109
0.122

0. 1 8 1
19.401
7.766
1.289
0.083

2.356
0.692
0.000
0.964
4.876
0.216
0.623
5.152
6.767
1.663
0.048
0.074
0.000
1.407
0.052
4.015
0.043
0.964
0.105
6.047
30.975
29.582
37.027
98.304

1 .520
1.670
0.290
0.245
0.014
0.046

0.029
1 .695
0.648
0.142
0.012

0.236
0.045
0.000
0.137
0.308
0.029
0.040
0.354
0.575
0.146
0.027
0.007

o.ooo

0.095
0.007
0.155
0.008
0.007
0.032
0.145
3.403
2.470
1.979
8.358

15.223
14.590
0.811
0.207
0.115
0. 1 25
0.000
0.188
19.440
10.710
1.401
0.097
0.000
2.356
0.692
0.000
0.964
5.326
0.216
0.623
5.185
6.767
1.663
0.048
0.074
0.239
1.407
0.052
4.015
0.043
0.964
0.105
6.050
31.611
32.701
37.753
102.785

1 .520
1.721
0.300
0.244
0.014
0.047

0.030
1.698
0.997
0.175
0.012

0.236
0.045
0.000
0.137
0.345
0.029
0.040
0.357
0.575
0.146
0.027
0.007
0.019
0.095
0.007
0.155
0.008
0.007
0.032
0.145
3.461
2.846
2.034
8.845

Values in bold are, respectively, the major fatty acid present in olive oil [18: 1
{n-9)) and the deutenumdabeled arachidonic acid added [20:4 (n-6)-d8].
Data are means ± SD (« = 3).

Ingestion. Digestion, and Assimilation of GAM

653

V ' p/vl Liaxvae starved LP

(b)

p*.

Lam

U) *AJ

V-GAM I

^P

18-

10-

14-

12-

10-

*o

8-

1

so

0-
<*-

'

c

^

'-I

\
4

f

\

R

i

, Js .

i.

i-V,

2-

2B

(c)

pA~| Larvae starved LN

(d)

pAl Larvae *AA-GAM LN

JU

^

s

o

^
^

O OS

JUiiM

ae

Figure 4. Partial chromatograms of FAMEs prepared from larvae of V. puUastra corresponding to retention time between 26 and 28 min. Figure
shows the incorporation of 20:4(n-6)d8 in polar (b) and neutral (d) Upids in larvae fed with microcapsules with respect to starved larvae (a) and (c).

proved by an accumulation of yellow fluorescence (the dye color)
inside the larva (Fig. 1 ). A sequence of photographs taken through-
out a period of 20 min showed the decrease of yellow fluorescence
in the digestive gland of a larva fed with microcapsules, due to
digestion (Fig. 2).

Growth and Survival

Throughout the 7-day experiment, larvae were sampled with
the daily water renewal to determine growth (anterior-posterior
shell length) and survival. Growth and survival were similar for the

654

NOVOA ET AL.

batches fed either microalgae supplemented with microcapsules or
the microalgal diet (Fig. 3).

Fatty Acid Composition of*AA-GAM

The mean fatty acid compositions of *AA-GAM are listed in
Table 1 . The fatty acid profile of microcapsules fabricated with 50
fj,L of olive oil and 5 mg of deuterated arachidonic acid 20:4
(n-6)-dis showed a predominance of 18:l{n-9) (65.7 ± 0.31%), 16:0
(10.8 ±0.07%). 18:2(n-6)(10.1 ± 0.33%). and 20:4{n-6)-dH (5.4 ±
0.24%). A single microcapsule contained 3.8 pg total fatty acids.

Dietary Fatty Acid Supply

The diet consisting of *AA-GAM exclusively was used in the
24-h experiment and supplied 60 GAMjjiL"', which corresponded
to 29.86 nglarva"' for the 18:l(n-9) and 2.43 nglarva"' for 20:
4(n-6)-ds (Table 1).

Table 2 shows the fatty acid composition of the two different
diets (algal diet and *AA-GAM-supplemented diet) used in the
7-day experiment. The first supplied 7.77 nglarva^' of 18:l(n-9)
and the second supplied 10.71 ng-larva"' of 18:l(n-9) and 0.24
nglarva"' of 20:4(n-6)-ds.

Assimilation of Microcapsules in the 24-h Experiment

The FID-CG analysis allowed the separation and the quantifi-
cation of deuterated *AA in polar and neutral lipis of larvae (Fig.
4). Larvae fed *AA-GAM exclusively showed incorporation of
20:4(n-6)-ds in polar and neutral lipid fractions (0.10 and 0.12
ng-larva"'. respectively). The amount of the major fatty acid in
olive oil, 18:l(n-9), also increased in neutral and polar lipids when
compared with that of starved larvae: 1.08 ng-larva"' versus 0.33
in neutral lipids and 0.66 ng-larva"' versus 0.28 in polar lipids
(Fig. 5a). Incorporation yield percentages of 20:4(n-6)-dg were

nearly equal in both lipid fractions (4.30% for polar lipids and
4.80% for neutral lipids), whereas for 18:l(n-9) they were 2.53%
and 1.31% for neutral and polar lipids, respectively (Fig. 5b).

Fatty Acid Composition of iMrvae Fed with Different Diets

Larvae fed with -^AA-GAM-supplemented diet showed an in-
crease in the incorporation of the two monitored fatty acids [20:
4(n-6)ds and 18:l(n-9)| in both lipid fractions when compared
with those fed the microalgal diet (Fig. 6).

With regard to 20:4(n-6)-ds. incorporation profiles were similar
in both lipid fractions throughout the 7-day experiment (16.3% for
neutral lipids and 16.8% for polar lipids between days of culture 18
and 19). Assimilation percentages were higher during the early
days of the culture and decreased in the late days from 16.3 to
5.6% in neutral lipids and from 16.7 to 6.1% in polar lipids
(Fig. 7).

Incorporation of 18:l(n-9) was better in neutral than in polar
lipids. Throughout the experience, the incorporation yield de-
creased in both fractions, although the phenomenon was more
pronounced in polar lipids (Fig. 7).

The Hpid content, the percentage of neutral and polar fractions
and the fatty acid composition of larvae were similar in both 7-day
experiments (Table 3). Supplementation with microcapsules did
not affect the incorporation of the essential PUFA provided by
microalgae. However, larvae fed microcapsules appeared to have a
higher FAME content if compared with larvae fed microalgae
only.

DISCUSSION

Numerous artificial particles have been tested to supplement or
partially replace the living algal diet for mollusks. These included
yeast (Epifanio 1979). fiour (Albentosa et al. 1999). dried algae

(a)

18:l(i>-9)

20:4<ih«)d8

□Starved LN
■*AAGAM Det LN
n Starved LP
■ •AA-GAM Det LP

1.2
10
0.8
06

f 04
02

□Starved LN
■*AAGAM Det LN
D Starved LP
■*AAGAM Det LP

(b)

18:l(iv-9)

20;4(ik6) d g

DLN

dln

Figure 5. Bar diagrams showing the incorporation of both traced fatty acids in experiment 1. In (a), comparison of their contents as neutral and
polar lipids in starved larvae and those fed *AA-GAM, expressed in ngflarva. In (b), incorporation rates as neutral and polar lipids in larvae
fed *AA-GAM, expressed in percentages. In all cases, n = 1.

t

Ingestion, Digestion, and Assimilation of GAM

18:l(n-9) neutral lipids 20:4(n-6)d8 neutral lipids

655

19 21 23

Days of larval development

25

0.09
0.08-

— _— algae
-•-•AA-GAM

+ algae

0.07

0.06

0.0Si

/

0.04-

y

s

003

0.02-

0.01-

1

0-

J

17

19

21

23

25

Days of larval development

2.5

1.5

18:l(n-9) polar lipids

-algae

- •.^A-GAM + algae

'^ '

1

0.5
0

17 19 21 23

Days of larval development

20:4(n-6)ds polar lipids

0.1

— ~— algae

0.09 - — ■— *AA-GAM + algae

0.08-

0.07 ,

^0.06 X

^0.05 /

^0.04-
c0.03i

/

0.02 H

/

0.01

/

17

19

21

23

25

Days of larval development

18:l(n-9) total lipids

20:4(n-^d8total lipids

£;
1)

10
9
8

7
6
5
4
3
2
1
0

17 19 21 23

Days of larval development

25

0.18
0.16-
0.14-
0.12 1
g 0.1
I 0.08
"|>0.06
0.04
0.02
0

-r— algae

-m— 'AA-GAM -f algae

17 19 21 23

Days of larval development

25

Figure 6. Dynamics of 18:l(n-9) and 20:4(n-6)d„ in neutral, polar, and total lipids of larvae fed microalgae and ""AA-GAM + microalgae during
experiment 2.

(Laing & Millican 1991). emulsions, and liposomes (Coutteau at
al. 1996. Caers et al. 1999. 2000, Soudant at al. 2000). Davalop-
mant of particles like gelatin-acacia spraybeads (Buchal & Lang-
don 1998) was mostly devoted to identifying essential molecules
and to understanding their metabolism. In this article, we report
data on the incorporation efficiency of the essential PUFA AA
using GAM. Because mollusks contain AA in their corporal fatty
acid, we incorporated deuteratad AA (*AA), which can be easily
discerned from the natural one by direct GC analysis, allowing a
good estimation of its assimilation. This straightforward method-
ology avoids the use of radioactivity and its additional measure-

ments. Indeed, it may be applied to other essential fatty acids.
Although deuteratad metabolites of AA were not detected in our
study, their determination is possible with this methodology.

Small-scale GAM fabrication is easy and suitable for experi-
mental studies on metabolism, in particular for lipids, which are
easily encapsulated. Olive oil was chosen as lipidic support be-
cause its composition is very simple. Oleic acid (OA) represents
659!- of the total fatty acid present in olive oil. Moreover, olive oil
lacks most very long-chain PUFA that are essential for marine
mollusks (Trider & Castell 1980, Enright et al. 1986, Soudant et al.
1996). Therefore, the fatty acids from this oil would not interfere

656

(a)

Polar Hpids

NOVOA ET AL.

(b)

Nei<rd6]ifb

liD21

18:l(n-9) 20:4(n-6)d8 18:l(n-9) 20:4(n-6)d8

Figure 7. Incorporation rates of 18:l(n-9) and 2():4(n-6)(i„ in polar and neutral lipids of larvae fed witli *AA-GAM + microalgae. Ih = I)

with larval development. The size of GAM obtained was compat-
ible with mollusks" ingestion and they did not show harmful effect
on larvae. On short-term observation (7 days), no deleterious effect
was observed on growth. Growth and larval mortality were similar
both in the presence and in the absence of GAM in our experi-
ments.

The gradual disappearance of the fluorescent labeling observed
in the digestive system of larvae (Fig. 2) was probably due to the
digestion and assimilation of microcapsules by the larvae. In pre-
vious studies, Sudan red-stained GAM faded in the stomach and
digestive diverticula of Cnissiislrea viri;iiuca larvae during the
early 24 h after feeding, and disappeared completely within 4<S h
(Chu et al. 1982). Also, Crassostrea gigas spat were demonstrated
to assimilate '''C-labeled lipids incorporated in corn oil GAM
(Knauer & Southgate 1997c).

Digestion and assimilation were eventually confirmed by trac-
ing the deuterated arachidonic acid supplied by *AA-GAM. Re-
sults showed that V. piillastra larvae were able to incorporate this
fatty acid into their endogenous lipids as both neutral and polar
fatty acids. The results obtained in both 24-h and 7-day experi-
ments established incorporation of 20:4(n-6)-dx. which could only
be detected in larvae if *AA-GAM had been assimilated. How-

ever, larvae fed with *AA-GAM also showed an increase of OA.
the other tracer fatty acid of GAM, in both neutral and polar lipids
when compared with those starved or fed algae.

Contrary to neutral lipids, which may correspond to transient
storage of ingested food, polar lipids are mostly located in mem-
branes. Thus, incorporation in polar lipids better indicates true
assimilation in both experiments. These results confirmed inges-
tion, digestion and assimilation of the fatty acids furnished by
GAM.

With regard to the second experiment, it is noteworthy that
essential PUFAs supplied by algae were found to be accumulated
at the same level in neutral and polar lipids of larvae regardless of
the diet. This shows that the incorporation of PUF.A pro\ ided b\
the algae was not modified by the microcapsules, probably mean-
ing that the amount of algae filtered by the larvae did not decrease
by the presence of GAM. During the time course of this experi-
ment, the polar lipid content increased continuously, which re-
sulted in a regular growth. The neutral lipid content showed a clear
drop between day 18 and 19 and then tended to reach a maximum
at day 21. This may suggest the existence of a high energetic
demand al this point of the larval development.

Fatty acid incorporation rates were observed to vary depending

TABLE 3.
Dynamics of the essential PUFAs and the total FAMEs in polar and neutral lipids during experiment 2.

18 Day"
Algae

19 Day

21 Day

24 Day

Algae

Algae

+ *AA-GAM

Algae

Algae

-f *AA-GAM

.Algae

Algae

■f *AA-GAM

Polar lipids

20:4 (n-6)

0.378

0.453

0.595

0.731

0.922

1.105

0.982

20:5 (n-3)

0.552

1,160

1.349

2.381

2.679

3.506

3.397

22:6 (n-3)

2.744

3.474

4.284

6.942

7..396

9.574

10.109

Total FAME (

ig/larvae)

13.939

19.378

24.951

37.501

44.109

51.. ^62

53.533

Neutral lipids

20:4 (n-6)

0.316

0.298

0.304

0.963

1.095

1.054

1.119

20:5 (n-3)

3.875

1.182

1.124

4.136

4.341

4.792

5.302

22:6 (n-3)

2.437

1.324

1.418

4.232

4.403

4.163

5.073

Total FAME (

ng/larvae)

53.935

18..';41

21.209

62.576

71.264

56.544

69.745

Total lipids

Total FAME (

ng/larvae )

67.874

37.919

46.16

100.077

115.373

107.906

123.278

Percent neutral lipids

79.463

48.896

45.947

62.528

61.768

52.401

56.575

' Data are expressed as mean for three analysis.

Ingestion. Digestion, and Assimilation of GAM

657

on the fatty acid supplied, as well as on the stage of larval devel-
opment. OA incorporation was mainly observed in neutral lipids
whereas *AA was equally distributed in neutral and polar lipids.
OA is a monounsaturated fatty acid, thought to play a mainly
energetic role in the reserve lipid fraction. The equal distribution of
*AA in polar and neutral lipids is more surprising since, like
vertebrates, some marine bivalves {Pecten maximus) have shown a
preferential incorporation of AA in polar lipids, especially in the
phosphatidyl inositol class (Soudant et al. 1998). However in V.
pidlastra. the natural AA supplied by the food was found at a
similar level in neutral and polar lipids of 24-day-old larvae. The
preferential location of arachidonic acid in polar lipids may be
species specific. Furthermore, it is known that the fatty acid com-
position of diet is largely reflected in neutral lipid composition and
to a lesser e.xtent in polar lipids of animals. This is because polar
lipids are regulated according to the needs of structural mainte-
nance of membranes. Fatty acids supplied by the food would be
first incorporated in neutral lipids and then, depending on the
needs, transferred to polar lipids. The percentage of AA in polar
lipids of mollusks fed with algae tended to decrease in the course
of the experiment, perhaps because those needs were satisfied by
the algae or, maybe, the needs of the larvae just dropped naturally
with development. This would also explain the weak incorporation
of *AA in polar lipids in the case of larvae fed with *AA-GAM.
OA and *AA incorporation in both neutral and polar lipids was
high during the early days of the experience but decreased toward
the later days. These data coincided with the fact that the amount
of neutral and polar lipids per larva leveled off beween days 2 1 and
24. corresponding to a decrease in growth rate and lower biosyn-
thesis needs. Moreover, the decrease of the natural AA percentage
in polar fatty acids and its stable percentage in neutral lipids may
explain the decrease of incorporation of tracer fatty acids from

GAM. It is during this period that larvae are ready to undergo
metamorphosis and change into spat.

Comparing the values of incorporation of both fatty acids sup-
plied by GAM between experiments, we observed that incorpora-
tion increased in the second experiment. This may be explained by
the presence of microalgae in the second experiment, which fa-
cilitated the retention of artificial GAM either by favoring the
filtration or by adhesion of GAM on algae. Similar results were
obtained by Knauer and Southgate (1997b). who reported an ef-
ficiency increase from 57-809^ when Dunuliella was added to
GAM.

An appropriate dosage of lipid components incorporated in
microcapsules could be useful as a nutritional supplement for
lipid-poor microalgal diets. The possibility of using microcapsules
containing labeled fatty acids also allows us to trace the molecules
and to study their distribution and possible metabolic conversion to
structural and reserve lipids. This would contribute to go deeply
into both energetic and structural needs for fatty acids in mollus-
kan larval development, and to design nutritionally optimal lipidic
diets to obtain spat of good quality.

ACKNOWLEDGMENTS

This work was supported by a financial aid from the Secretaria
de Estado de Universidades. Investigacion y DesarroUo. Plan Na-
cional I -I- D. and Proyect MAR96-1876. A part of this study was
supported by a grant of the Conselleri'a de Pesca, Marisqueo y
Acuicultura of the Xunta de Galicia, Spain, to S. N. The authors
thank J. F. Samain and the colleagues of IFREMER. Centre Brest,
for supplying their theoretical and practical aid. The authors would
like to thank warmly the technical staff of CIMA. Ribadeo. for
their generous work and technical assistance.

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Jniinuil of Shellfish Resecirch. Vol. 21. No. 2. 659-672, 2002.

ENHANCING HARD CLAM (MERCENARIA SPP.) POPULATION DENSITY IN THE INDIAN
RIVER LAGOON, FLORIDA: A COMPARISON OF STRATEGIES TO MAINTAIN THE

COMMERCIAL FISHERY

WILLIAM S. ARNOLD,'* DAN C. MARELLI,' MELANIE PARKER,' PHILIP HOFFMAN,'
MARC FRISCHER,- AND JOHN SCARPA^

^Florida Fish and Wildlife Coiisc nation Commission. Florida Marine Research Institute. 100 Eighth
Avenue SE. St. Petersburg, Florida. 33701-5020: 'Skidaway Institute of Oceanography. 10 Ocean
Science Circle. Savannah, Georgia. 3141 1: Harbor Branch Oceanographic Institution. 5600 U.S.
Highway 1 North. Ft. Pierce. Florida. 34946

ABSTRACT Hard clams of the genus Mercenaria support an important commercial fishery in the Indian River Lagoon on the east
central coast of Florida. The fishery is relatively young but has proven to be quite sporadic, with two periods of e.xceptional landings
(mid-1980s and mid-1990s) bounded by periods of almost complete fishery collapse. In response to a request from commercial fishery
participants, three strategies for enhancing the abundance of harvestable hard clams in the lagoon were compared. The first strategy
involved harvesting adult clams from a low-density population and transplanting them at high density in a concentrated area, in an
effort to increase fertilization efficiency and thereby maximize reproductive success. That approach does not appear to be viable in the
Indian River Lagoon because mortality of transplanted clams approached 100% and because Indian River hard clams display a
remarkably high incidence (>80%l of gonadal neoplasia. Neoplasia reduces the life span of Indian River hard clams relative to their
northern congeners and probably reduces fecundity of those clams that do survive. The second strategy involved seeding juvenile clams
at very high densities (843-7165 m~' depending upon seed size), again with the intent of maximizing fertilization efficiency but with
the additional goal of maximizing residual reproductive value. Various planting treatments were tested in an effort to minimize
monality of seed clams, but losses were still high (generally >70%1, and the yield did not appear to justify the cost. The final strategy
involved spawning large numbers of hard clams in the laboratory, fertilizing the resultant eggs, and immediately releasing the larvae
at a predetermined location in the lagoon. Large numbers of larvae did appear to survive the 8-day lar\'al life span, but it remains to
be seen whether those larvae will translate into harvestable hard clams. In any event, enhancement of hard clam populations appears
to be feasible only when the density of naturally occurring clams is so low that reproducfive viability is compromised. Otherwise,
natural reproductive potential will swamp any artificial efforts at population enhancement.

KEY WORDS: Mercenaria. Indian River Lagoon, population enhancement, spawner transplant, seeding, larval release

INTRODUCTION

Hard clams of the genus Mercenaria support an economically
and culturally important fishery in the Indian River Lagoon on the
east central coast of Florida. This is a relatively new fishery that
developed in the early 1980s (Fig. 1) and was originally centered
near the town of Grant in southern Brevard County (Fig. 2). The
clam population that supported the original fishery collapsed in the
late 1980s, probably in response to decreased salinity caused by
the release of St. Johns River floodwaters into the Indian River
basin (Barile and Rathjen 1986). Another substantial set of hard
clams was detected in the northern Indian River Lagoon, between
Cocoa and Titusville. in the early 1990s. That population sup-
ported a profitable and productive tlshery throughout the mid-
1990s (Fig. 1), but the population again collapsed and again the
proximate cause appeared to be decreased salinity resulting from
above-average rainfall in the watershed. As of the year 2001, the
fishery for naturally occurring hard clams in the Indian River
Lagoon has remained depressed, although a small number of fish-
ermen continue to pursue the few clams that are available.

The Indian River is a shallow, narrow, bar-built lagoonal sys-
tem that stretches from Oak Hill to Stuart along the central Atlantic

Present Address of Dan C. Marelli; Florida State University. 036 Mont-
gomery. Tallahassee. Florida. 32306-2310.

Present Address of Philip Hoffman: Pinellas County Department of Envi-
ronmental Management. Environmental Resources Management Division.
300 South Garden Avenue. Clearwater. Florida. 33756.
*Corresponding Author: E-mail bill.amoldta'fwc.state.fl.us

coast of Florida. For our study, we considered only that area of the
Indian River Lagoon (IRLl system that is located north of Sebas-
tian Inlet (Fig. 2) and that is composed of the Indian River (IR). the
Banana River (BR), and Newfound Harbor. Prehistorically. the
IRL was a single continuous basin, and the IR and BR were con-
nected to the south at Dragon Point and to the north through
Banana Creek. In recent years, development and bridge construc-
tion have resulted in the subdivision of the IRL into a series of
basins that are defined by the causeways that span the lagoons.
Water exchange between basins is restricted to the areas under the
bridges that connect the causeway dikes (Evink & Morgan 1982).
Additionally, the pattern of water exchange between the IR and BR
has been altered in the last 40 years. Development of the Kennedy
Space Center essentially severed the Banana Creek connection
between the two lagoons (McCall et al. 1970). and construction of
the Canaveral Barge Canal in the eariy 1960s (Yusof 1987) created
a new connection south of Banana Creek.

Reported clam landings from the Indian River Lagoon for the
past 20 years suggest that at least under certain conditions, the
capacity for production of hard clams in the lagoon is substantial.
However, no single basin of the lagoon appears to be consistently
productive. Instead, an individual basin may support a dense clam
population for several years, after which the population collapses
and remains depressed until another major set of Juvenile clams
occurs either in that basin or in another area of the lagoon. Envi-
ronmental conditions can vary substantially among basins, creating
a potential mismatch between successful spawning events (Hes-
selman et al. 1989) and the environmental conditions necessary to
support the survival and development of that spawn.

659

660

Arnold et al.

2000

0'°" ^0!°' 0'° o'o s<>!°" ^<i^ o^' ^9)^ s<?>^ v<i^ ^'5.'* o'*' o* ^<4■<' s".* ^o^

^qf O-^- sof'- T?-

Year

Figure 1. ConinuTcial hard clam {Merceiwria spp.) landings from Brevard Cmmly, Florida. Data from \')M) through 19S5 were provided by the
National Marine Fisheries Service. Data from 1986 onward were provided hj the Florida Marine Research Institute's Fisheries Dependent
Monitoring Program.

Salinity is one of the key environmental variables affecting the
success of hard clam populations. Adult clams are not well adapted
to salinities below 20 practical salinity units (p.s.u.), and embry-
onic and juvenile clams tend to be even more sensitive (Castagna
& Chanley 197.^). In the Indian River Lagoon, salinity may range
from less than 13 p.s.u. (Barile & Rathjen 1986) to more than 40
p.s.u. (Young & Young 1977) with extreme conditions causing
even those limits to be exceeded. Furthermore, the salinity condi-
tions in one basin of the lagoon may be ideal for clams, whereas
those in an adjacent basin may be inimical to clam survival (e.g.,
McCall et al. 1970, Banle & Rathjen 1986). Thus, a suitable match
between environment and biology may be very localized, which is
why a commercially successful hard clam spawn is a rare event in
the Indian River Lagoon.

The diversity of water quality conditions in the lagoon may
create difficulties for the natural clam populations occupying the
IRL, but it also may create an opportunity for enhancement of
those populations. Although it is difficult to predict when condi-
tions will be suitable for reproduction, recruitment, growth, and
survival of hard clams, gross conditions (e.g., salinity, dissolved
oxygen, turbidity; Arnold et al. 2000) can be evaluated from the
results of frequent water-quality monitoring activities. Such moni-
toring programs are ongoing in the lagoon, under the auspices of
the St. Johns River Water Management District and the Florida
Department of Agriculture and Consumer Services. These pro-
grams make it possible to identify water-quality conditions suit-
able for clams, although it is not possible to ensure that tho.se
conditions will occur at a time and place coincident with a spawn-
ing event.

A variety of options are available to increase clam abundance

in the lagoon. Here, we compare three approaches that might be
effectively applied in the IRL. The spawner transplant approach
involves harvesting mature animals and subsequently replanting
them in an area that is either more suitable for survival and repro-
duction or that will allow larvae to disperse to such areas (e.g..
Carter et al. 1984). Generally, animals are harvested troin a rela-
tively large area (square kilometers) and replanted into a much
smaller area (square meters), thus concentrating potential spawners
in an effort to increase reproductive success. This strategy has been
used in efforts to increase the population abundance of a variety of
organisms, including hard clams (e.g.. Carter et al. 1984). bay
scallops (Peterson et al. 1996). and abalone (Tegner 1992). The
rationale for the seeding approach is similar to that for the spawner
transplant approach: the animals are concentrated in a small area in
the hope of increasing reproductive success. Seeding differs from
spawner transplants in that young, generally prereproductive ani-
mals are planted (e.g., Marelli & Arnold 1996). Thus, the residual
reproductive value (Ricklefs 1979) of the planted animals should
be ma\imi/ed relative to a spawner transplant operation that may
include a variety of age classes of the target organism. Seeding as
an enhancement strategy also has a rich history in the population-
enhancement literature, including numerous efforts involving hard
clams (e.g.. Castagna & Kraeuter 1977. Walker 198.3, Peterson
et al. 1995, Marelli & Arnold 1996). The third approach dis-
cussed herein involves the release of recently fertilized eggs
directly into the lagoon, thereby circumventing the spawning pro-
cess entirely. This .strategy has been tested with abalone (Preece
et al. 1997. Shepherd et al. 2000). but to our knowledge had not
been tested with marine bivalves such as the hard clam prior to our
study.

Clam Population Enhancement in Florida

661

Oak Hilt

NASARFtr-

Max Brewer Mem Cswy/
Tltusville '

Atlantic
Ocean

^, Cape
' Canaveral

Pineda Cswy<- -^ _, .

Dragon Point ♦^•rgau Gallie Cswy.

-Or V*
^, \\

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Melbourne : . ^

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Sebastian Inlet

Figure 2. Indian River Lagoon, Florida, showing locations of spawncr transplant and seeding studies in the Indian River and Banana River
lagoons. Star indicates site where adult clams were collected for the spavvner transplant study. Closed circles indicale sites of the spawner
transplant and seeding studies in the Indian River lagoon and of the spawner transplant study in the Banana River lagoon.

MATERIALS AND METHODS

Spawner Transplants

In the spawner transplant eoiiipoiient oi this study, we har-
vested adult clams of a variety of sizes, marked the shells with

spray paint to allow for later identification, and then replanted the
clams into an area closed to shellfish harvest. The objective of this
project was to concentrate reproductively mature clam.s to maxi-
mize the number of successfully fertilized eggs (Levitan 1995).
During October 1998 (fall relay), we used professional clam har-
vesters to gather 5,000 clams from an area of the Indian River

662

Arnold et al.

Lagoon north of Titusville (Fig. 2). The harvested clams were
returned to shore, where the shells were allowed to dry for several
hours and then labeled with yellow spray paint for later identifi-
cation. The labeled clams were split into two groups of 2.500; the
following day. the clams in one group were planted at a site in the
Indian River and clams in the other group at a site in the Banana
River. To transplant the clams, we poured them over the side of
our research vessel as we traversed the extent of a 100-m" study
plot demarcated at each corner by a crab trap float. Similar trans-
plants were conducted during January (winter relay; blue paint I.
April (spring relay; green paint), and August 1999 (summer relay;
red paint) to assess the best season for conducting transplant op-
erations.

Sampling of the adult relay plots was conducted 2 wk after each
relay event and again 3. 6. 9, and 12 mo after each relay event
(Tables I and 2). thus allowing us to assess reproductive status and
mortality during each season of the year for each transplant date.
To sample, we thoroughly hand-raked the contents of 20 randomly
selected 0.25-m" quadrants within each plot, returned all recovered
hard clams to the research vessel for identification and counting.
and subsampled 15 randomly selected clams from each plot (2-wk
samples excepted) for later reproductive analysis. The location of
each sample quadrant was marked with a PVC stake to ensure that
plots were not resampled. Note that the Indian River spawner
transplant plots could not be effectively sampled after the passage
of Hurricane Irene in October 1999 because the sample plots were
destroyed by that storm. The Banana River spawner transplant
plots appeared to be unaffected.

Clam samples for reproductive analysis were processed accord-
ing to the following procedures. Live animals were returned to the
laboratory, where the gonad was excised from each animal and
stored for 24 hours in a solution of 5*^ fomialdehyde in seawater.
Large gonads were removed from the fixative solution after about
four hours, lacerated to ensure penetration of the fixative, and
returned to the fixative for the remaining 20 hours. Afterwards, the
gonads were thoroughly rinsed in tap water to remove fixative,
dehydrated through a series of alcohol concentrations, and infil-
trated with JB-4 mounting plastic. Two 3.5-|j.m sections, separated
from each other by at least 50 p,m, were then cut from each
embedded gonad using a diamond blade microtome and the sec-
tions mounted on labeled glass slides. Mounted sections were
stained with hematoxylin and eosin. covered, and stored for later
microscopic analysis.

Gonad sections were examined under a binocular light micro-
scope and each sample assigned a qualitative ranking of gonad
developmental stage as described by Arnold et al. (1997) and
summarized in Table 3. This ranking scheme is a composite of
gonad development ranking schemes previously used by
Loosanoff (1937), Jaramillo et al. (1993). and Walker and Hef-

fernan ( 1994). with the addition of categories for early spawning
and for unreadable samples.

Seeding

On October 13. 1998. we planted three size-classes of seed
clams under four protective conditions at our study site in the
Indian River lagoon. Seed clams were obtained from the Division
of Aquaculture. Harbor Branch Oceanographic Institution. Ft.
Pierce. Florida. Seed size-classes were 2 mm mean shell height
(SH = maximum distance from umbo to ventral margin). 8 mm
mean SH. and 16 mm mean SH. Protective conditions included no
cover, oyster shell cover, plastic mesh netting cover, and a com-
bination of oyster shell and plastic mesh netting cover. Oyster shell
was purchased from a commercial aggregate company and aver-
aged approximately 5 cm in maximum shell diameter. Fifteen-
millimeter mesh plastic netting (Vexar) was purchased from a
commercial aquaculture supply company. Two replicates of each
size-class by protective-cover combination were deployed, for a
total of 24 treatments, each of which was assigned to an individual
l-m" plot. We planted approximately 7.165 2 mm SH clams (27 g
wet weight). 2.340 8 mm SH clams (259 g wet weight), or 843 16
mm SH clams ( 1 19 g wet weight) in each plot, which equated to
approximately $30 of clam seed per plot. Before planting, all
clams in the 2 and 8 mm SH size-classes were marked with tet-
racycline (Marelli & Arnold 1996). and the valves of all clams in
the 16 mm SH size-class were painted so that we could identify
them later.

On October 27 and 28, 1998, we used a hydraulic suction
dredge (e.g.. Peterson et al. 1983) to sample five 0.0278-m" cores
from each replicate plot to determine initial planting mortality. On
November 1 I and 12. 1999. we again collected five 0.0278-m"
suction dredge samples from each replicate plot to estimate mor-
tality and shell growth after one year. The location of each repli-
cate was determined by using a string grid to ensure that the
replicates within each plot did not overlap.

luinal Release

The larval release study was designed to determine the feasi-
bility of directly introducing fertilized clam eggs into the lagoon
and allowing them to grow and disperse as a natural population.
This approach to clam population enhancement allows us to cir-
cumvent the expensive and labor-intensive process of growing
clams in the laboratory, while still ensuring that large numbers of
fertilized eggs will be available in the natural environment. Lar\ al
release also allows us to target areas of the lagoon that are suitable
for the growth and survival of clams and to rapidly respond to
changing conditions. However, to be able to determine the success

TABLE 1.
Planting and sampling dates for the hard clam (Merceiiaria spp.l spawner transplant stud> in the Indian River lagoon.

Season

Plant Date

2-wk Sample

3-mo Sample

6-nio Sample

9-mo Sample

12-mo Sample

Fall

10/28/98(100)

11/9/98(127)

2/3/99(107)

4/28/99(114)

8/10/99(57)

10/26/99(17)

Winter

1/27/99(100)

2/3/99 (.'59)

4/28/99(238)

8/10/99(53)

10/26/99(18)

N/A (N/A)

Spring

4/20/99(100)

4/28/99 080)

8/10/99(131)

10/26/99(38)

N/A(N/A)

N/A(N/A)

Summer

8/3/99(121)

8/10/99(171)

10/26/99(6)

N/A(N/A)

N/A(N/A)

N/A (N/A)

Numbers in parentheses indicate sample si/e of hard clams collected on that date. Note that on each initial seasonal sampling date, clams were randomly
sampled from all of those harvested, whereas on later dates sample size reflects the number of clams actually collected from each study plot.

Clam Population Enhancement in Florida

663

TABLE 2.
Planting and sampling dates for the liard clam (Mercenaria spp.) spawner transplant study in the Banana River lagoon.

Season

Plant Date

2-wk Sample

3-mo Sample

6-mo Sample

9-mo Sample

12-mo Sample

Fall

10/28/98(100)

11/9/98(73)

2/3/99 (73)

4/28/99 (96)

8/11/99(97)

10/25/99(3)

Winter

1/27/99 ( 100)

2/3/99(200)

4/28/99(144)

8/11/99(91)

10/25/99(16)

1/25/00 (2)

Spring

4/20/99(100)

4/28/99(161)

8/11/99(87)

10/25/99(31)

1/25/00(10)

4/25/00 ( 1 )

Summer

8/3/99(121)

8/11/99(229)

10/25/99(20)

1/25/00(7)

4/25/00(9)

8/16/00(2)

Numbers in parentheses indicate sample size of hard clams collected on that date. Note that on each initial seasonal samphng date, clams were randomly
sampled from all of those harvested, whereas on later dates sample size reflects the number of clams actually collected from each study plot.

of this enhancement strategy, it is necessary to be able to track and
sample the animals during the planktonic phase of their life.

Spawning

The larval release strategy requires the production of large
numbers of viable hard clam embryos that can be successfully
transported to and released at the site targeted for enhancement.
Adult hard clams were collected from various areas of the Indian
River Lagoon on several dates during 1999. The clams were trans-
ported to a holding area at Harbor Branch Oceanographic Institu-
tion, where they were conditioned in preparation for spawning.
Conditioning consisted of holding the clams for several months in
a small lagoon on the Harbor Branch campus. On the day before
spawning, the clams were transferred from the holding lagoon to a
refrigerated storage area and held overnight. On the following day.
the clams were removed from the storage area, placed in equally
spaced rows on each of three spawning tables, and submerged in
approximately 10-15 cm of 28 p.s.u. seawater. During the next six
hours, the clams were exposed to cycles of cool and warm water
and induced to spawn. As each individual clam initiated spawning,
the sex of the animal was identified and the clam was removed
from the table and isolated in containers with other clams in small
groups of males or females. The clams continued to spawn within
the containers, and the resultant eggs were pooled and concen-
trated on a 35-jjim-mesh sieve and exposed to a sperm concentra-
tion adequate to ensure fertilization of all eggs. Total egg produc-
tion and fertilization success were determined microscopically,
after which the developing embryos were transferred to 20 L

TABLE 3.

Gonad staging scheme for female hard clams {Mercenaria spp.)

collected from the Indian River Lagoon, including female clams

transplanted to study sites in the Indian River and Banana River

lagoons and sampled during various times of the year, and from

their undisturbed conspecifics.

Reproductive

Numerical

Status

Description of Gonadal Tissue

Stage

No Data

Tissue unreadable

0

Inactive

Gonad tissue undifferentiated

1

Developing

Tissue differentiated, eggs present

2

Ripe

Tissue full of eggs

3

Early spawning

Eggs being shed, but follicles still
full in appearance

3.5

Spawning

Many eggs shed, follicles appear
partially empty

4

Spent

Follicles nearly empty

5

aquarium bags and transported to the study site for immediate
release.

Larval Tracking

Hard clam embryos were transported from the spawning facili-
ties at Harbor Branch to our study site in the Banana River lagoon
and released at 20.30 EDT on May 16. 2001. at a site approxi-
mately 1 .75 m deep. Water temperature at the site was 28°C and
salinity was 22 p.s.u.. whereas the temperature of the water in
which the larvae were transported was 25.8"C and the salinity was
27.7 p.s.u. Before larval release, five subsurface drifters (Davis
1985. Hitchcock & Arnold, unpublished data) were deployed in a
box-and-one pattern (one drifter at each comer of a 10 m x 10 m
box. with a single drifter in the center of the box), and their initial
positions were recorded using a differential Global Positioning
System. Then, at a depth of approximately 0.5 m. the clam larvae
were gently poured from the bags into the center of the drifter
array. Gradual mixing between the transport water and the la-
goonal water was allowed in an attempt to minimize osmotic
shock.

During daylight hours on May 16, we collected thirteen 200 L
water samples from the targeted release area to determine the
prerelease concentration of hard clam larvae in the study basin. On
May 17 and 23. 2000. post-release water samples were collected to
determine the distribution and density of the larval mass. For the
May 17 sampling, when the larval mass was predicted to be rela-
tively concentrated, the subsurface drifters were visually located
and the position of each drifter recorded. Sample collection loca-
tions for hard clam larvae were then selected based upon the lo-
cation and distribution of the subsurface drifters. On May 23. after
diffusive processes were anticipated to have spread the larvae
throughout the study basin, samples were collected at each of 23
grid nodes equally distributed throughout the basin.

On each sampling date, samples of hard clam larvae were ob-
tained by using a Jabsco Model 34600-0000 diaphragm pump to
collect volumes of water that ranged in size from 100-400 L,
depending upon the projected density of larvae. Water was
pumped through a 150-jim-mesh sieve to remove large objects,
and then captured in a 63-|xm-mesh plankton net. Each resultant
sample was removed from the cod end of the plankton net and
carefully distilled to a volume of approximately 30 mL, then trans-
ferred to a 50-mL screw-cap centrifuge tube. labeled, and placed
on ice until arrival at the laboratory, where it was frozen at ap-
proximately -5°C. Within one month of the completion of the
study, all of the frozen water samples were sent to the Skidaway
Institute of Oceanography for determination of the presence and
abundance of clam larvae. Samples were analyzed for the presence
of hard clam larvae using a previously developed genetic probe

664

Arnold et al.

that is both quantitative and Menenaria-speafic (e.g.. Frisctier et
al. 20001.

RESULTS
Spawner Transplants

At botli tlie Indian River and Banana River study sites, the
mortahty of relayed hard clams was severe, particularly during the
summer and early fall of 1999. Also at both sites, considerable loss
of clams from the plots was associated with the initial transplant.
At each site, on all dates, we transplanted an average of 25 clams
m"-. and within 2 wk the densities for all plantings had decreased
by more than 50% (Fig. 3). After the initial transplant event and
the loss of clams associated with that event, clam densities stabi-
lized throughout the winter, spring, and early summer at both sites
(Fig. 3). During late summer and early fall of 1999, the clams
experienced substantial mortality, possibly as a result of decreased
salinity associated with Hurricane Irene (Fig. 4). As noted previ-
ously, that storm destroyed our study plots in the Indian River. It
also appears to have had a severe detrimental effect on the clams
planted in the Banana River, as clam density decreased substan-

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Fidurt 3. Mean density of hard clams (Mercenaria spp.) on various
sampling dates after transplantation during (A) fall IW8; (B) winter
IVW; IC) spring 1999; and (I)) summer 1999 al the Indian River
lagoon (filled bars) and Banana River lagoon (open barsi study sites.
Sec Figure 2 for the location of each study site. Note that at the Indian
River study site, all four study plots were destroyed l)y Hurricane
Irene during October of 1999 and no further sampling was conducted
after that date. F>ror l>ars represent one standard deviation. On the
November 9, 1998, sampling date, samples were pooled and no stan-
dard deviation was calculated.

lially in all Banana River plots between the August I 1 and October
25, 1999. sample dates (Fig. 3). Hurricane Irene swept through our
study area on October 16. 1999, and salinity near our Banana River
study site decreased to a study-period minimum of less than 15
p.s.u. at the end of October 1999 (Fig. 4|.

The high levels of mortality that we obsei-ved in our transplant
plots may have been inlluenced by the inability of clams (espe-
cially large clams) to reburrow following initial harvest. The In-
dian River study site was characterized by a soft sand/mud sub-
strate that appeared to provide little resistance to burrowing clams.
Upon re-sampling that site two weeks after the fall transplant. 26%
of all clams collected remained on the surface, and three months
after the fall transplant 20% of all clams collected still remained on
the surface. In contrast, the Banana River study site was charac-
terized by a hard sand bottom that appeared to provide consider-
able resistance to clams attempting to burrow. Two weeks after the
Banana River fall transplant. 47% of all clams collected remamed
on the surface, and three months after the fall transplant 34%^ of all
clams collected remained on the surface. At both sites, failure to
huiTow was related to clam size. A comparison of the mean SH ot
buried versus unburied clams at each site two weeks after trans-
plant during both fall and winter, indicated that the clams that
failed to burrow were significantly larger than those that success-
fully reburrowed (/-test, see Fig. 5A. B. E. and F for respective P
values). We detected no significant difference in SH between bur-
ied and unburied clams at either site three months after transplant
(Fig. 5C and D). although only clams transplanted during the fall
were compared. During the spring and subsequent sampling epi-
sodes, we discovered few clams at either site that were both alive
and unburied.

After the initial episode of transplant mortality, overall mortal-
ity of relayed clams did not appear to be size-related. At the Indian
River study site, the size distribution of hard clams did not differ
significantly among sampling dates (Simultaneous Test Procedure;
Sokal & Rohlf 1995) except during the summer transplant study
(Fig. 6). During the final sampling episode (October 26. 1999) of
the summer transplant study, the size distribution of planted clams
differed significantly from the size distribution recorded during the
previous two sampling dates and appears to have shifted towards
a preponderance of small clams (Fig. 6). For all four of the trans-
plant episodes at the Banana River study site, a significant shift in
clam size distribution was detected for the October 25. 1999.
sample date, and for all sample dates subsequent to October 25.
relative to all sample dates preceding October 25 (Fig. 7). The only
exception to this pattern was from the spring transplant study, for
which the size shift was not detected until the January 25 sampling
episode (Fig. 7).

During each seasonal harvesting event, a subsample of 15
clams from each plot was returned to the laboratory for analysis of
reproductive condition and for a comparison with control samples
collected from the natural population on the same date. However,
the results from only the fall planting date at both study sites are
included in the present analysis because that is the only planting
date for which adequate sample numbers were available for all
sample dates from both sites. The female clams in the control
samples had a pattern of reproductive development typical of In-
dian River hard clams (Hesselman et al. 1989). During fall and
winter, when Hesselman et al. (1989) reported that spawning oc-
curred in Indian River hard clams, most clams that we sampled
(control and transplant) were either spawning or were spent (Fig.
8). During spring, the season of peak spawning in Indian River

Clam Population Enhancement in Florida

665

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Sampling Date

Figure 4. Salinity recorded at the Indian River lai>uun (closed triangles) and Banana River lagoon (closed circles) study sites during September
1998 through October 2(((MI. The dotted line provides a 20 p.s.u. reference. Data courtesy of the St. Johns River Water Management District
Surface Water Quality Monitoring Program.

hard clams (Hesselman et al. 1989). most of the clams from the
control sample were ripe, and lesser proportions were either
spawning or spent. In contrast, most animals collected from the
Indian River and Banana River transplant plots during spring were
in some stage of spawning. Finally, during summer the vast ma-
jority of control and transplant clams that we sampled were in the
spent condition, which agrees well with the observation of Hes-
selman et al. (1989) that most Indian River hard clams are repro-
ductively spent during summer.

Gonadal neoplasia is extensive in Indian River hard clams
(Hesselman et al. 1988, Bert et al. 1993), and this condition ap-
pears to be related to hybridization between the two species of
Mercenaria (M. mercenaria and M. cainpechiensis) that occupy
the lagoon (Bert et al. 1993). We recorded neoplasia in 85% of the
male clams and almost 92% of the female clams that we collected
from the natural clam population of the lagoon. Male clams suf-
fered 93% and 86% neoplasia when harvested after transplant to
the Indian River and Banana River, respectively. Female clams
suffered 96% and 93% neoplasia when harvested after transplant
to the Indian River and Banana River, respectively.

When all samples of clams collected for reproductive analysis
during the course of the spawner transplant study were pooled,
there were significantly more female than male clams (0.53 males:
1.00 females, x" = 42.13, df= 1, P< 0.0001 ). However, , sex ratio
was dependent upon clam size-class. For all clams that were < 60
mm SH, we detected no significant difference in sex ratio (0.75
males: 1.00 females, x" = 1-43, df = \.P = 0.232). In contrast,
for clams > 60 mm SH, the sex ratio was significantly skewed

towards female clams (0.50 males:
= I, P< 0.0001).

1.00 females, x" = 42.89, df

Seeding

Mortality of 2 mm hard clams was substantial within 2 wk of
planting under all treatment conditions (Table 4). Only under mesh
protection did the 2 mm size-class suffer less than 90% mortality,
but even with mesh protection the small clains experienced a mean
mortality of 85.6%. Survival of clams in the 8 mm size-class was
not much better: those clams also experienced >90% mortality in
the open plots and at least 50% mortality within 2 wk after being
planted in the remaining plots. In contrast, clams in the 16 mm
size-class experienced <10% mean mortality in the mesh plots and
a mean mortality of 1 1.3% in the open plots. However, those clams
suffered 30.4% mean mortality under the combined protective
cover and >70% mean mortality in the shell plots.

Hurricane Irene also severely impacted our seed clam plots.
Nevertheless, on November 1 1, 1999, we attempted to reconstruct
the experimental plots and we did conduct suction dredge sam-
pling of the reconstructed plots. We found no live clams in the seed
plot I y after planting. However, we cannot determine whether that
lack of clams was due to the effects of Hurricane Irene or due to
factors independent of the hurricane.

iMrval Release

Spawning

On May 16. 2000. approximately 550 million hard clam eggs
were spawned and collected. The eggs were then exposed to an

666

Arnold et al.

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Size-Class (mm)

Figure 5. Percent frequency of occurrence of hard clams {Mercenaria
spp.) at each of the Indian River lagoon and Banana River lagoon
study sites during fall and winter sampling events. Filled bars repre-
sent the percentage of clams that were recovered on the surface of the
plots, and open bars represent the percentage of clams that were bur-
ied upon recovery. (A) Indian River fall planting, 2-wk sampling: (Bl
Banana River fall planting, 2-wk sampling: (C'l Indian River fall plant-
ing. 3-mo sampling; (D) Banana River fall planting, 3-mo sampling:
(E) Indian River winter planting, 2-wk sampling; and (F) Banana
River winter planting, 2-wk sampling. The /' values included in each
plot represent the probability (Mestl that the mean size of clams re-
covered on the surface was not significantly different than the mean
size of clams that were buried upon recovery. \ P < 0.05 indicates a
statistically significant difference in the mean size of surficial vs. bur-
ied clams.

amount of sperm sufficient to ensure fertilization of all eggs as
determined by microscopic examination. Spawning occurred in
three "batches" beginning at 1200 EDT and ending at 1600 EDT.
Transport from Harbor Branch (1800 EDT departure) to the Ba-
nana River release site (2030 EDT release) required approximately
2.5 h, so the clams ranged in age from 4.5 to 8.5 h at the time of
release.

Larval Release

Larvae were released at a site in (he Banana River lagoon
between SR 520 and SR 528 (28°23.32()'N latitude, 80°37.95rw
longitude) at 2030 EST on May 16, 20(.)0 (Fig. 2). During the next

24 h. the drifters that tracked the water mass within which the
larvae were released were transported towards the west until they
approached the western shore of the lagoon (Fig. 9). As the drifters
approached the shoreline, they gradually swung around to the
north, but four of the five drifters contacted the bottom, hung up,
and were retrieved. The fit'th drifter passed through a small bridge
at the western end of the SR 528 causeway and was retrieved to
prevent its loss.

Analysis of water samples collected on May 16, before the
release of cultured larvae, detected no evidence of naturally oc-
curring hard clam larvae in the study basin. On May 17. after our
larval release during the evening of the 16th, we detected larvae at
only one sampling station (Fig. 9), probably because the larval
mass remained tightly constrained in the area around that station.
By May 23, the larval mass had spread throughout the basin (with
the exception of the southeast comer), although several peaks of
abundance were detected. Two of these peaks were in the north-
west comer of the basin, where larval density exceeded 7 veligers
L^' (Fig. 10).

DISCUSSION

We tested three strategies for enhancing the abundance of har-
vestable hard clams in the Indian River Lagoon. Florida. The first
strategy involved harvesting adult clams from low-density popu-
lations and concentrating them in high-density patches in an effort
to increa.se fertilization success and the production of viable larvae.
This strategy does not appear to be cost-effective because most of
the clams that we transplanted did not survive the 1 2-mo moni-
toring period. Furthermore, the vast majority of the clams that we
transplanted were infected with gonadal neoplasia, a disease that
progressively reduces fecundity and probably proceeds to a fatal
outcome (Yevich & Barry 1969, Hesselman el al. 1988, Eversole
& Heffernan 1995). The second strategy involved planting small
seed clams at relatively high densities, again with the intention of
creating concentrated patches of reproductively active clams. This
approach has one anticipated advantage (a longer life span for the
spawners) and one unanticipated advantage (avoidance of gonadal
neoplasia) when compared with the spawner transplant strategy.
However, the first advantage may be offset by the high rates of
mortality experienced by most size-classes of the seed clams, and
the second advantage may be temporary because the seed clams
become increasingly susceptible to gonadal neoplasia, as they
grow older (Bert et al. 1993). Although survival rates were rela-
li\ely high for the largest size-class of clams (16 mm mean SH),
the considerable cost of those clams ($0,036 each) reduces the
cost-effectiveness of this strategy. The third strategy involved cir-
cumventing the entire process of natural fertilization by releasing
already fertilized eggs directly into the lagoon. Our results suggest
that this strategy may be effective, but more information is needed.
It appears that large numbers of clam larvae survived to an age at
which settlement can be reasonably expected (8 days to set in
culture conditions during May using Indian River water; B. Leem-
ing, personal communication). However, the ultimate fate of those
larvae has not been determined, and successful settlement needs to
be demonstrated in vivo for this approach to have any validity.

The common currency with which to gauge the success of each
of these strategies will be an increase in the abundance of hard
clams available for harvest by the fishermen. Hard clams in the
Indian River Lagoon require approximately 18 months to achieve
the legal harvest size of 2.54 cm in shell width (Arnold et al. 1996).

Clam Population Enhancement in Florida

667

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, -H-

--J|-

J ^

,„ II 1

1

II III. .1. ,

40 -

30 -

C)

1,

20 -

„ 1, 1 .

10 -
0

, J . M

, ll,

ll

1 1

— Jl — -1 ,

40 -

30 ■

D)

1 1

10
0 •

L

i. J J. 1.

h.,

-b^ (f> if> «f> «p fcO 6") -VO ^b 4« 4% ,jO 7

Hard Clam Size-Class (mm)

Figure 6. Percentage of live hard clams {Mercenaria spp.) recorded
within various size-classes during each seasonal sampling event fol-
lowing the (A) fall: (B) winter; (Cl spring: and iDl summer transplants
in the Indian River lagoon. For each size-class category within each
transplant season, sampling dates are plotted from left to right. Thus,
the leftmost bar within each size-class represents the sample collected
on the original date of harvest, and the rightmost bar represents the
sample collected on the final sampling date. See Table 1 for sampling
dates and sample size for each date. Note that samples were collected
on only five dates for the winter transplant, four dates for the spring
transplant, and three dates for the summer transplant because of the
impacts of Hurricane Irene, which destroyed the sample plots on Oc-
tober 16, 1999. Otherwise, missing bars indicate that no clams were
collected from that size-class on that date.

For the adult transplants, it would have been possible to detect
adult offspring as early as spring 2000, assuming that clams trans-
planted in fall 1998 spawned very soon after transplant. That is a
reasonable assumption considering that a fall spawn has been de-
scribed for Indian River hard clams {Hesselman et al. 1989) and
was similarly indicated by our fall 1999 reproductive data. For the
seeding study initiated in fall 1 998, we would have expected that
maternally derived offspring would be available for harvest no
earlier than fall 2000. Hard clams as small as 27 mm SH are
reproductively active in the Indian River Lagoon (Hesselman et al.
1989), but clams are generally male during the first year of life
(Loosanoff 1937). Thus, we would not have expected egg produc-
tion from the 16 mm size-class of seed clams for at least 6 months
post-planting (i.e., spring 1999). For the larval release study con-

OT

E
_2

o

>

o

0)

c

0)

o

i.
V

ifs' u<>= <s'-^ ^~ fcO" fcV .vV ^y
Hard Clam Size-Class (mm)

Figure 7. Percentage of live hard clams {Mercenaria spp.l recorded
within various size-classes during each seasonal sampling event fol-
lowing the (A) fall; (B) winter: (O spring: and (I)) summer transplants
in the Banana River lagoon. For each size-cla.ss category within each
transplant season, sampling dates are plotted from left to right. Thus,
the leftmost bar within each size-class represents the sample collected
on the original date of harvest, and the rightmost bar represents the
sample collected on the final sampling date. See Table 2 for sampling
dates and sample size for each date. Missing bars indicate that no
clams were collected from that size-class on that date.

ducted in May 2000. surviving animals would have been expected
to achieve harvest size during fall 2001. Information from the
fishery and preliminary results from our own sampling efforts
provide no evidence of a substantial yield of harvestable hard
clams, as might be expected from the above timetables. Of course,
our study was conducted on a much smaller scale than would be
necessary to realize a significant contribution to future year-classes
(McHugh 1981). Our primary objective was to experimentally
compare three possible approaches to hard clam population en-
hancement in the lagoon. From those results, we hoped to be able
to choose a single approach that has the greatest likelihood of
success and then to apply that approach on a .scale appropriate for
success.

For the Indian River hard clam fishery, success is a quantifiable
parameter. The primary fishing grounds fall within the boundaries
of Brevard County, and commercial clam harvest in that county is
strictly regulated. Each clammer must be licensed, and only 500
licenses are allocated for the fishery. The goal of our enhancement
work is to provide the clam harvesters with a resource base that

668

Arnold et al.

Undifferentiated VP77^ Developing i^^^^ Ripe ^^ Early Spawning I 1 Spawning ILLUiU Spent

8

8 7 6

9 11 13 13 8

10 2

100

O

c
cr

(U

c

0
O

0)

Q.

80 -

60

40 -

20 -

Control IR BR Control IR BR Control IR BR Control IR BR Control IR BR
Fall 1998 Winter 1999 Spring 1999 Summer 1999 Fall 1999

Season

Figure 8. Reproductive stages of female hard clams (Mercenaria spp.l colltclid on various seasonal sampling dales after the fall transplant.
Stages are as described in Table 3. Numbers across the lop of the plot are sample sizes. Control, clams that were harvested from the natural
population during each season: BR, clams thai «ere transplanted to the Banana River lagoon study site during fall and sampled during each
season; IR, clams that were transplanted to the Indian River lagoon study site during fall and sampled during each season.

would allow them to survive a nadir in the abundance of naturally
occun-ing clams. If it is assumed that $20,000 is a minimum ac-
ceptable annual income for each clam fisherman and that the sale
price for each clam is $0.20, then it can be estimated that 50
million harvestable clams must be produced each year to satisfy
that harvest goal. Using the larval release strategy as an example.
it is apparent that 50 million larvae must be released and must all
survive to harvest size to meet that goal. Similarly, if only K/f ot

TABLE 4.

Percent mortality of hard clam iiUenenaria spp.l seed 2 wk after

being planted under various protective conditions at the Indian

River study site.

Clam

Open

Mesh

Shell

Mesh/Shell

Size-Class

Plots

Plots

Plots

Plots

2 mm (rep 1 )

46.6

86.9

91.0

92.5

2 mm (rep 2)

MS.d

84.2

92.5

98.3

2 mm (mean)

97.3

85.6

91.8

95.4

8 mm (rep 1 )

91.7

53.8

71.7

84.9

S mm Irep 2l

')7..S

88.9

62.5

90.2

8 mm (mean)

94.6

71.4

67.1

87.6

16 mm (rep 1 )

2().y

5.2

65.2

48.7

16 mm (rep 2)

1.7

7.6

80.9

12.2

16 mm (mean)

11,3

6.5

73.0

30.4

Protective treatments are listed across the top of the table and the results
from replicate treatments (and the mean of the paired replicates) are pre-
sented within the body of the table.
The boldface rows represent the mean of the two observations.

the larvae survive to produce clams of legal harvest size, then 5
billion larvae must be released. We have been able to consistently
produce approximately 50 million eggs per spawning table per day
during subsequent tests of the larval release strategy, and we have
used four spawning tables per day during tho.se trials. At that rate,
it would require 25 spawning days to produce 5 billion larvae.
Carriker (1961) estimated the mean survival rate of hard clam
larvae to be 2.6% when rates of flushing were low. but the mini-
mum survival rates that he reported were 0.1% or less. At those
minimum larval survival rates it would be necessary to increase
daily larval production by a factor of five and to increase the time
span of larval release to approximately 50 days to achieve our
stated goal. That may be possible, but it would require a broad-
scale effort that includes participation by the clam fishermen. Ad-
ditionally, our best-case larval production estimates require the
provision of 800 broodstock per day, because we place approxi-
mately 200 clams on each spawning table (equivalent to about 1.35
female clams with a 2: 1 ratio of females to males). Considering the
present status of the hard clam fishery in the Indian River Lagoon,
the limited availability of broodstock to support egg production
may prove to be a serious problem that limits the effectiveness ot
the larval release strategy. Moreover, broodstock availability may
also be adversely affected by the high incidence of gonadal neo-
plasia in Indian River hard clams. Large clams, which are eco-
nomically less valuable than small clams and are therefore more
available for use in the spawning program, are relatively rare in
Indian River waters and produce fewer eggs than would be pre-
dicted based upon allometric considerations alone (e.g.. Peterson
1983. 1986). The clams that we have successfully used in previous
spawning efforts generally fall within the "topneck" commercial

Clam Population Enhancement in Florida

669

Banana River
Larval Distribution
May 17, 2000

^ Release Point

Drillers

• 5/16/00

» 5/17/00

» 5/18/00
Larval Concenlration

0.2 (Veligers/L)

04

06

— -- 08

.... t

1.2

Osceola
Co.

Stuc y Area

0.5 Kilometers

Merritt
Island

Figure 9. Contour plot of tht distribution of hard clam {Menenaria spp.) larvae as estimated by sampling conducted on May 17, 2000, in the
Banana River lagoon, Florida. Data are presented as number of clam larvae I^ ', and larval concentrations are depicted at the location of each
sample station. Also plotted are the locations of the subsurface drifters that were deployed during the evening of May 16, 2000.

classification (average SH = 60 mm). There is a strong market
demand for these clams, and seafood processors are reluctant to
sell them to us even at a premium price for fear of upsetting
previously established buyers. During times when native Indian
River broodstock are readily available from seafood processors
because of high levels of harvest in the natural fishery, there is
little need for an enhancement program, and the contribution that
could be realized from enhancement would he swamped by natural
production (Kassner & Malouf 1982).

Our estimate of the yield of harvestable clams from larval
release does not take into account post-settlement losses, which
typically exceed 80% and may approach 100% under some con-
ditions (Gosselin & Qian 1997). However, hard clam populations
continue to thrive in Florida and throughout the eastern seaboard
of the United States, so conditions suitable for survival must occur.
Perhaps it is more appropriate to consider our enhancement efforts
within the context of natural clam densities in the lagoon. During
the early 1980s in the area near Grant and during the mid- 199()s in
the area north of Cocoa, clams were extremely abundant and peak
densities exceeded 10 clams m~~ (Arnold, unpublished data).
However, during the time frame of the present study, clams were
practically nonexistent in the vicinity of the Banana River study

site (Arnold et al. 1997) and were similarly rare in the vicinity of
the Indian River study site. As a result, the artificial densities that
we initially established in each planting study (spawner transplants
and seeding) substantially exceeded the background density at
each study site. The 550 million eggs that we contributed during
our larval release study equate to an average contribution from the
spawn of more than 75 female "cherrystone" size hard clams
(Bricelj 1992). assuming 100% fertilization of naturally spawned
eggs. Two factors increase the value of the fertilized eggs that we
released. First, it is unlikely that 100% fertilization efficiency is
realized in the natural environment (Levitan 1995). Second, be-
cause of the prevalence of gonadal neoplasia, egg production in
Indian River hard clams may be considerably less than that pre-
sented by Bricelj (1992) for northern US waters. If we estimate
mean production to be I million eggs per female, and we estimate
a fertilization efficiency of 1% at the low clam densities («l m~")
currently found in the lagoon, then the number of larvae that we
released is equivalent to the number of larvae produced by ap-
proximately 55000 female clams or a bed of >800(X) clams as-
suming a 2:1 female:male sex ratio.

We did not anticipate the rate of loss of transplanted clams that
was actually realized during this study. Similar transplants have

670

Arnold et al.

Banana River

1 1 L..iid
-^r Release Poinl

Larval Distribution
IVlay 23, 2000

Larval Concentration
0 (Veligers/L)

2

5

^'^ 7

c^ —

") I /l 'Merritt /
i, N Island^'

i\

Oraftge v\-'i~^^

7 Cap

1 Q^

e
averal

Osceol;
Co

Brevard \
Co. V

i

Atlantic
Ocean

•

i

■ s

udy Area

\

\

0.5

0 0.5 Kilometers

W A

n-"

Merritt
Island

Fijjurt 10. Cimlour plot of the distribution of hard clam {Mercenaria spp.l larvae as estimated bv sampling conducted on Ma> 23, 2000, in the
Banana River lagoon, Florida. Data are presented as number of clam larvae 1 ', and larval concentrations are depicted at the location of each
sample station.

been conducted in other areas throughout the range of Merceiuiria.
either for eventual harvest of the relayed clams (e.g.. Rice et al.
2000) or to increase or expand larval production (e.g.. Kassner &
Malouf 1982, Ganz 1991). However, the high frequency of go-
nadal neoplasia in the clams that we collected for transplant will
mitigate against the success of this approach. Gonadal neoplasia is
common in Indian River hard clams (Hesselman et al. 1988). par-
ticularly in the northern lagoon where we collected clams for trans-
plant (Bert et al. 1993). Although gonadal neoplasia has been
reported in hard clams collected from northeastern US coastal
waters (e.g.. Barry & Yevich 1972), the frequency of occurrence
was less than 5% versus greater than 80'7r in our study. That
disease appears to substantially reduce the reproductive potential
of hard clams (Hesselman et al. 1988) and probably contributes to
the relatively short life span of hard clams in Indian River waters
(Jones et al. 1990). Considering that the reproductive potential of
Mercenaria increases with age (Bricelj & Malouf 1980. Peterson
1983. 1986), the high frequency of gonadal neoplasia in Indian
River hard clams appears to render spawner transplants an inef-
fective strategy for enhancing the abundance of harvcstable clam
populations in the lagoon.

In all cases, gonadal neoplasia was more prevalent in the trans-
planted clams than in their undisturbed conspecifics, and the dif-

ference in the frequency of neoplasia between the transplants and
their undisturbed conspecifics was greater in the Indian River than
in the Banana River. This difference was minor and may reflect
sampling bias related to differences in the frequency of neoplasia
that have been reported for various size-classes and genotypes of
hard clams (Bert et al. 1993). Nevertheless, gonadal neoplasia was
considerably more prevalent in clams collected during our study
than in clam collections reported by either Hesselman et al. ( 1988)
or Bert et al. (1993). Neoplasia is more common in hybrid hard
clams (Bert et al. 1993) and hybrid clams are more common in the
northern Indian River Lagoon where we collected our transplant
animals (Bert & Arnold 1995). Our spawner transplant study
would perhaps have been more successful if we had collected
clams from more southerly Indian River waters, but we were lim-
ited in our choice of harvest sites by the availability of clams.

Seeding as a means of increasing the abundance of hard clams
has been attempted in various areas throughout the range of Mer-
cenaria. including both coasts of Florida (Men/el & Sims 1962.
Menzel et al. 1976. Marelli & Arnold 1996). Georgia (Walker
1985). North Carolina (Peterson et al. 1995). Virginia (Castagna &
Kraeuter 1977). New York (Flagg & Malouf 1983). and Rhode
Island (Rice et al. 2000). With the exception of the work in North
Carolina (Peterson et al. 1995), these efforts have met with limited

Clam Population Enhancement in Florida

671

success due to the high rate of loss of seeded clams even when
protective measures are used. In North Carolina, relatively large
seed clams (14—22 mm shell length) were planted at relatively low
density (I m"") in shell hash habitat in late fall, resulting in 35%
survival after 14 months (Peterson et al. 1995). However, clam
density below 5 m~~ is considered to be inadequate for commercial
harvest in Florida waters (Arnold et al. 2000), and increasing the
density of planted seed clams might result in a loss of economic
viability attributed to this approach. Moreover, achieving even the
limited enhancement goals that we have set for our project (50
million harvestable clams) would require seeding almost 15.000
hectares of submerged land with almost 1.5 billion clams. Even if
the available clam hatcheries could produce that many clams, the
cost of the clams alone would be exorbitant. At least for the 500
hard clam fishermen currently licensed to work Brevard County
waters, seeding for direct harvest does not appear to be a cost-
effective means of ensuring a minimum annual income, although
that approach may be feasible for projects of a smaller scale.

We are familiar with only one application of larval release as a
means of stock enhancement. Shepherd and colleagues (Preece et
al. 1997, Shepherd et al. 2000) released various densities of aba-
lone larvae at several sites in South Australia and monitored their
survival. They found that because larval and post-larval survival
was density-dependent, the releases of relatively low densities of
larvae were more successful than were the high-density releases.
The overall conclusion of those authors (Shepherd et al. 2000) was
that larval release was not a viable strategy for abalone stock
enhancement because of the density-dependent nature of larval
mortality. Those results, and results from our own analyses of
diffusive processes acting on artificially introduced hard clam lar-
vae (Hitchcock & Arnold, unpublished data), suggest that a point
release of the larvae is not the best strategy. Instead, higher sur-
vival rates may be obtained by spreading the larvae throughout the
target basin, thereby enhancing diffusive processes that will take
place anyway (Hitchcock & Arnold, unpublished data). We will
test that approach in future experiments.

Efforts to enhance the population abundance of commercially
important marine molluscs have been ongoing for decades, and the

published record of those efforts indicates that success has been
rare if not nonexistent. Clearly, efforts to enhance even moderately
dense populations are superfluous, as the reproductive potential of
the natural population is sufficient to swamp any directed enhance-
ment efforts. Only when population density is very low, such as it
would be in an essentially collapsed population, might such efforts
yield success. At that point, we shift from an enhancement effort
to a restoration effort, and the goals of the project shift from
increasing the abundance of an ecologically functional population
to restoring reproductive viability in an ecologically dysfunctional
population (Arnold 2001 ). Nevertheless, user groups and manage-
ment agencies continue to request that population enhancement
efforts be undertaken, and we will continue our efforts to deter-
mine if or under what conditions we can meet those requests.

ACKNOWLEDGMENTS

Kate Hagner, Tracy Idocks, Micah Humphrey, and Melissa
Harrison of the Florida Marine Research Institute provided invalu-
able field and laboratory assistance. Commercial clam harvesters
Perry McMahon, Bill Leeming, Peter Barile. Doug Telgen, Mason
Bowen. Bill Bowler, John Condos, David Panizzi, Jay Anderson.
George Vincent. Allen Ellingham, Richie Luck, George Rotsch.
James Horst, Pete Roy. and Riley Bergman assisted with clam
collections for the spawner transplant study. Frederico Prahl,
Sandy Zeiner, Richard Baptiste. and David Vaughan of the Divi-
sion of Aquaculture, Harbor Branch Oceanographic Institution
were essential to successful spawning and fertilization of hard
clam eggs. Gary Hitchcock of the University of Miami assisted
with larval tracking and was instrumental in the design and de-
ployment of the shallow-water drifters. Winnie White (FMRI) pre-
pared the maps and provided GIS support. Dick Moravec (FMRI)
provided assistance with vessels and vehicles, and Gerry Bruger
(FMRI) ensured that our electronic database management was
seamless and uneventful. This study was made possible by the
generous financial support of the commercial clam harvesters of
Brevard County. Florida. This is Harbor Branch Oceanographic
Institution contribution number 1483.

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Joiinwl of Shellfish Rcxeanh. Vol. 21. No. 2. 673-676. 2002.

ABUNDANCE OF OCEAN QUAHOG, ARCTICA ISLANDICA, ASSESSED BY UNDERWATER

PHOTOGRAPHY AND A HYDRAULIC DREDGE

STEFAN AKI RAGNARSSON* AND GUDRUN G. THORARINSDOTTIR

Marine Research Institute, Skiilagata 4. P.O. Box 1390, 121 Reykjavik. Iceland

ABSTRACT Abundance of ocean quahog (Arcrica ishindica) was estimated with underwater photography and a hydraulic dredge at
15 ni depth in Onundarfjordur. NW Iceland. Abundance estimates based on counts of siphons from the underwater photographs were
much higher than from analysis of the dredge catches. A total of seven taxa were identified from photographs, of which only ocean
quahogs and brittle stars were found m sufficient abundance for further analysis. The large scale spatial distribution of ocean quahogs
and brittle stars was contagious, where the data corresponded well with a negative binomial distribution but differed significantly from
a Poisson distribution. The Morisita index of dispersion was used to analyze distribution patterns within each photograph. These
analyses revealed that even on small spatial scales the distribution of ocean quahog could be contagious.

A.£}' WORDS: ocean quahog. distribution, abundance, underwater camera, hydraulic dredge. ArcUca islaiulica

INTRODUCTION

The ocean quahog, Arcticii islciiulica, is an important commer-
cial species in Icelandic (Thorarinsdottir & Einarsson 1996) and
US waters (Kennish & Lutz 1995). Stock assessments of ocean
quahog have generally been performed using hydraulic dredges.
However, accurate quantitative abundance estimates cannot be
provided if the efficiency of the dredge is not known. Besides,
efficiency may vary with environmental conditions and between
different types of hydraulic dredges (Eleftheriou & Holme 1984,
Kennish & Lutz 199.5. Anonymous 1998). Abundance of ocean
quahog has also been estimated quantitatively from grab samples
(Amtz & Weber 1970. de Wilde et al. 1986) but because of the
small surface area the grab collects, relative to the low abundance
of ocean quahogs in sediments, this laborious method requires a
very large sample size to obtain reasonable quantitative estimates.

Many studies have been performed in the last decade using
underwater photographic techniques to estimate abundance of epi-
faunal bivalves, such as scallops (Langton & Robinson 1990.
Stokesbury & Himmelman 1993. Goshima & Fujiwara 1994) and
deep burrowing crustaceans, based on counts of burrows (Chap-
man et al. 1975. Hughes & Atkinson 1997). However, to our
knowledge only one photographic study has estimated abundance
of infaunal bivalves (Wigley & Theroux 1970). Estimation of
abundance from photographs is more difficult for infaunal bivalves
than for epifaunal bivalves because identification depends solely
on siphon characteristics and the protruding parts of the shell.
However, siphon characteristics can be very distinctive and infau-
nal bivalves have been identified to species and genus level by
using these features (Siferd & Welch 1992).

The stock size of ocean quahogs off Iceland has been estimated
using a hydraulic dredge (EiriTcsson 1988, Thorarinsdottir &
Einarsson 1996). The stock size in Onundarfjordur NW Iceland
was assessed by this method in 1994 (Thorarinsdottir & Einarsson
1996). Assessments conducted with dredges provide only infor-
mation about the larger scale (>10 m") distribution patterns of
ocean quahogs. However, variation in distribution patterns may
occur on much smaller spatial scales (O.I-IO m"^). At these scales,
distribution of ocean quahogs could be influenced by small scale
variation in sediment grain size, bottom topography, and the pres-
ence of other infaunal bioturbators.

*Corresponding author. E-mail: steara@hafro.is

Assessments of ocean quahog stocks using underwater photog-
raphy has the clear advantage that quantitative information on
abundance of ocean quahogs can be assessed more rapidly and
cheaply compared with surveys conducted with hydraulic dredges.
The aim of this study was to assess whether underwater photog-
raphy can be used to estimate the abundance of ocean quahogs and
secondly to assess how reliable these estimates are by comparing
them with estimates obtained with hydraulic dredge within the
same area. Furthermore, large and small scale distribution patterns
of ocean quahogs and brittle stars were investigated.

MATERIALS AND METHODS

The study was performed in July 1999 in Onundarfjordur NW
Iceland (66°02'N-23°34'W) at a depth of 15 m. A 35 mm verti-
cally oriented underwater camera (Photosea lOOOA) loaded with a
roll of 10 m (250 exposures) of 200 ASA filtn (KODAK) and an
obliquely oriented 150 watt strobe (Photosea I500S) were placed
on a steel frame. A videocamera directly connected to a monitor
was also attached to the frame. While the vessel was drifting, the
frame was repeatedly (at 3()-sec intervals) lowered until the trigger
weight touched the bottom, activating the camera and the flash to
take a photograph (at a speed of l/lOO sec). Because the flash from
the strobe could be clearly seen in the video monitor, the camera
equipment was hauled up =2 m immediately after each photograph
was taken. Photographs were taken at a constant distance (0.9 m)
from the bottom (the distance from the camera to the trigger
weight) and therefore the area of seabed covered in each photo-
graph was always the same (0.46 m"). The position of the start and
the end of each photographic transect was located by a global
positioning system.

To analyze the photographs, they were displayed using a slide
projector onto a board ( 144 cm x 100.5 cm), the size of the dis-
played image matching the size of the board. The board was di-
vided into 24 squares (i.e., sampling units), each of 0.019 m" and
all animals on the photographs were counted and identified to the
lowest taxonomic level possible. To estimate numbers of ocean
quahogs per photograph, all siphons were counted. The siphon of
the ocean quahog is very short and consists of inhalant and exhal-
ant apertures, which lie close to each other on the inner fold of the
mantle. These are the only parts of the bivalve that can be seen on
the sediment surface (Fig. 1 ).

Only two species were found in sufficient abundance for sta-
tistical analysis, ocean quahogs (Arcticci islaiulica) and brittle stars

673

674

Ragnarsson and Thorarinsdottir

Figure 1. Underwater phulugruph of the seabed showing siphons of
ocean quahogs.

(Ophiiiroidea). The large scale distributions of the ocean quahog
and brittle stars were compared witli Poisson (random) and nega-
tive binomial (contagious) distributions using a chi-square test
(Elliot 1977). To calculate the goodness-of-fit (chi-square) of the
Poisson distribution, only variance and average abundance statis-
tics are required. The negative binomial distribution is based on
two parameters, arithmetic mean and the exponent k, which is
related to the spatial distribution, and is calculated iteratively using
the maximum-likelihood equation (Elliot 1977). In the negative
binomial distribution, the variance is greater than the mean, indi-
cating that the distribution of individuals is patchy, whereas if it is
the same as the mean, the distribution is random (Poisson). The
significance of departures from random and negative binomial
distributions were tested using chi-square tests. The extent of con-
tagion in the distribution of ocean quahog was furthermore as-
sessed using the variance to mean ratio (Elliot 1977).

The abundance of ocean quahogs was in general sufficiently
high to allow analysis of small-scale distribution patterns within
each photograph using the Morisita index of dispersion (Elliot
1977). This index is ideal for this analysis because it is indepen-
dent of the mean and total number of individuals per photograph
and the number of sampling units (i.e., photographic squares) was
always the same. The index is greater than one for a contagious
distribution, less than one for a regular distribution, and equal to
one for a random distribution. Data for analysis were based on
abundance of individuals within each of the 24 squares of the
photograph. Photographs with less than 20 individuals were not
considered suitable for this analysis.

A single cage hydraulic dredge was used to make a compara-
tive investigation of the abundance of ocean quahogs in the same
area where the underwater photography took place. The dredge

had an overall dimension of 590 x 103 x 230 cm. with a 150 cm
wide cutting blade. The bar spacing in the dredge was 34 mm. To
determine ocean quahog densities, the distance covered by the
dredge was calculated. Ocean quahogs from the catch were
weighed, and the total catch weight was divided by the size of the
area covered in the tow (5 min duration) to give biomass caught in
kg m"". Individuals in a subsample of 25 kg were counted, mea-
sured and weighed. The towing speed was 2.4 nautical miles per
hour.

RESULTS

Six invertebrate taxa were identified from a total of 180 seabed
photographs. Of these only ocean quahogs [Aixlica isUimlicu) and
brittle stars (Ophiuroidea) were found in high numbers (Table 1).
The brittle stars could not be identified down to species level from
the photographs. The remaining species, Asterias rubens (starfish),
Biiccinum undatum (whelk), Cucttmaria frondosa (sea cucumber),
and Hyas araneus (spider crab) were all found with mean abun-
dance less than O.I m"". Ocean quahog was present in 97% of the
photographs (max = 148 m~"; Fig. 2a) and brittle stars in 71% of
photographs (max = 252 m~~; Fig. 2b). The large scale distribu-
tion patterns of ocean quahogs and brittle stars agreed with the
negative binomial distribution but differed significantly from the
Poisson distribution (Table I ). Furthermore, the variance-to-mean
ratio exceeded unity for both ocean quahog and brittle stars (Table
I ). The findings from both these analyses suggest that the large
scale distribution of ocean quahogs and brittle stars were patchy.

The Morisita index of dispersion ranged greatly between pho-
tographs. In 90% of photographs, the Morisita index ranged from
0.68 to 1.62 for ocean quahogs and from 0.46 to 2.58 for brittle
stars. These results indicate that dispersion patterns within photo-
graphs can be very variable i.e., in some photographs the disper-
sion patterns of ocean quahogs were highly contagious whereas in
others more regular (Fig. 3).

The estimated mean biomass of ocean quahogs in the dredge
catch was 1.6 kg m"". equivalent to 14 indiv. m"- (mean live
weight of an individual caught by the dredge was 1 18 g). Because
of the selectivity of the hydraulic dredge, 85% of the clams caught
ranged in shell length between 70 to 95 mm, the mean shell length
was 82 mm, and no clams <30 mm were observed. Ocean quahog
was the only bivalve species found in the dredge samples.

DISCUSSION

This study shows that underwater photography can be used to
estimate the abundance of infaunal bivalves, although it may be
limited to areas dominated by a single species because identifica-
tion depends solely on siphon characteristics. Bivalves of size
comparable to ocean quahog have not been found in Onundarf-

TABLE 1.
Analysis of the distribution patterns of ocean quahogs and brittle stars.

Poisson

Negative

Binomial

Mean

V/M

df (n = 1)

Chi-square

df

k

Chi-square

Ocean quahog
Briltle star

.«;2.s

43..'^

13.5
28.9

177
128

1343.64*
4682.8*

4
5

3.68
l.OS

6.74
10.37

The goodness of fit of the observed frequency (Jistrihiitioiis compared wilh the negative binomial distribution and the Poisson distribution were assessed

with the chi-square test.

* P < 0.005. mean, mean number of individuals nr'; V/M. variance to mean ratio.

Abundance of Ocean Quahog

675

Ocean quahog

CM

E

0)

E

3

0 20 40 60 80 100 120 140 160 180

Brittle star

':W^UjiyL

0 20 40 60 80 100 120 140 160 180

Photograph frame number

Figure 2. Abundance of ocean quahogs (a) and brittle stars (bl. per
each successive photographic frame.

jorSur in previous surveys (Thorarinsdottir & Einarsson 1996).
suggesting that all siphons seen on the photographs belonged to
ocean quahog. Several smaller sized species have been recorded in
Onundarfj6r9ur (Gardarsson et al. 1980) but the siphons of these
are likely to be too small to be seen on the photographs.

The abundance estimates of ocean quahog based on dredge
catch (14 m"~) were about four times lower than based on the
photographs (53 m""). The proportion of individuals (i.e.. effi-
ciency) caught by the dredge relative to the total number of indi-
viduals in sediments (based on counts from photographs! was
therefore 21%. Although this can be regarded as a crude method to
calculate efficiency, Thorarinsdottir and Ragnarsson (2001) ob-
tained a very similar estimate (19%) using the same hydraulic
dredge in a different area.

Dredge efficiency may vary with sediment type, current speed,
the ratio warp length:water depth, towing speed, hydraulic pres-
sure of the jet, vertical distribution of the clams, and the size of the
dredge. This may explain why efficiency estimates can differ so
greatly between different studies (Medcof & Caddy 1971. Anony-
mous 1998).

Ocean quahogs are known to periodically burrow deeper in
sediments until the siphons can no longer be seen on the sediment
surface (Taylor 1976). Prolonged absence of food (e.g.. during
winter) has been suggested as one of the factors thai triggers them
to bury deeper in sediments (Oeschger 1990). Ocean quahogs are
able to reduce their metabolic rate to 1% of the aerobic rate,
enabling them to remain deeply buried without feeding over long
periods of time (Theede et al. 1969, Oeschger 1990). Clearly, if a
large proportion of the ocean quahogs are deeply buried, stock
assessment with underwater photography or hydraulic dredges will
always underestimate abundance.

Recent evidence suggests that the vertical movements of ocean
quahogs within sediments are seasonal in Icelandic waters. Si-
phons were seen on the sediment surface from April to September

2000 but not between the period of November 2000 to February

2001 (Thorarinsdottir, unpublished data). These findings suggest
that ocean quahogs can burrow to greater depths during winter but
may emerge to the sediment surface and start feeding when sea-
water temperature rises and the levels of phytoplankton biomass

X

0)
■D

C

2.0

- 1.5

^ 1.0

0.5

Ocean quahogs Brittle stars

Figure 3. Small-scale distribution patterns (within each photograph)
of ocean quahogs and brittle stars assessed with the Morisita index of
dispersion. The box plot shows the range in the distribution of the
Morisita index in all photographs with more than 20 individuals. The
box and whiskers represents 50 and SO'/r of the data, respectively.
Mean (dotted line) and median (solid line) are shown as well.

increases during summer (Thordardtittir & Eydal 1996). If this
proposition remains true, this may explain why catches of ocean
quahog per hour towed tend to be much lower in winter than in
summer as the catchability of this bivalve is likely to be lower
while deeply burrowed in sediments (Thorarinsdottir unpublished
data). It is therefore likely that only a small proportion of indi-
viduals were deeply burrowed in sediments when this study was
performed.

The present study demonstrates that ocean quahog and the
brittle stars were contagiously distributed. Analysis of the distri-
bution patterns within each individual photograph revealed that
even on such small scales, the distribution of ocean quahogs and
brittle stars can be highly contagious. In some photographs this
was caused by the presence of physical features such as stones,
which limited their distribution, whereas on other photographs, the
underlying reasons for an aggregated distribution were not clear.

Patchy distribution of brittle stars is a common feature widely
reported in other studies (e.g.. Warner 1971, Piepenburg & Juterzenka
1994). Density-dependent effects (Fujita & Ohta 1990. Summers
& Nybakken 2000). predation pressure (Aronson 1989). and local
heterogeneity in environmental conditions (Summers & Nybakken
2000) have been suggested to explain contagion of brittle stars.

Stock assessments performed with underwater photographic
techniques may provide more accurate quantitative estimates of
ocean quahog abundance than using dredges. However, such in-
vestigations performed during winter months may greatly under-
estimate the abundance of ocean quahogs because a large propor-
tion of individuals may be buried deeply in sediments. Further-
more, the small surface area covered by each photograph requires
a relatively large sample size to obtain reasonable quantitative
estimates. This method is not without limitations and may only be
useful in areas where no large infaunal bivalve other than ocean
quahog occurs, as identification depends solely on siphon charac-
teristics. Future research should focus on investigating those factors
that can influence stock assessment of ocean quahog such as vertical
movement in sediments and the efficiency of hydraulic dredges.

ACKNOWLEDGMENTS

We would like to thank Karl Gunnarsson. Elena Guijarro Gar-
cia, James Begley, Loma Taylor, and Astj^or Gi'slason for provid-
ing comments that improved this manuscript and Loma Taylor for
her help with the statistics. We also want to thank Elena for her
assistance in the field and the crews of the vessels Stundvis and
Skel IS.

676

Ragnarsson and Thorarinsdottir

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Journal of Slwllfhh Research. Vol. 21, No. 2. 677-684, 2002.

TETRAPLOID INDUCTION BY MEIOSIS INHIBITION WITH CYTOCHALASIN B IN THE
DWARF SURFCLAM, MULINIA LATERALIS SAY: EFFECTS OF TEMPERATURE

STEFANO PERUZZI AND XIMING GUO*

Haskin Shellfish Research Ltiboralory. Institute of Marine and Coastal Sciences, Rutgers University,
6959 Miller Avenue. Port Norris, New Jersey 08349

ABSTRACT Tetraploidy, which is useful for the production of all-triploids. can be induced in marine hival\es by manipulating
meiosis. In this study, we tested the effects of temperature on cytochalasin B (CB)-induced meiosis inhibition in a model bivalve, the
dwarf surfclam, Mulinia lateralis Say. Newly fertilized eggs of the dwarf surfclam were treated with CB (0.75 mg/L) at proper times
to block the release of polar body I ( PB 1 ) or polar bodies I and 11 ( PB 1 &2) at 19'C, 24°C, and 28'C. Inhibition of PB I produced largely
triploid and aneuploid-tetraploid (3.7n to 3.8n) larvae, and sometimes small percentages of pentaploids. Inhibition of PB1&2 produced
primarily pentaploids and occasionally triploids and aneuploid-tetraploids. Most of the larvae with ploidy levels higher than triploid
developed as trochophores only and died a few days after fertilization. At Day 25 post-fertilization, four tetraploids (29!-) were detected
among 196 juvenile clams in one of the eight PB1&2 groups. No tetraploids were observed in the eight PBl groups produced. In
general, higher (28'C) temperatures did not improve tetraploid production measured as survival to juvenile stage. Low temperature
(19'C) made CB treatment less effective, producing all diploid juveniles by Day 17. This work indicates that tetraploidy can be
tolerated in M. lateralis and scope for further work on this species therefore exists.

KEY WORDS: tetraploidy, triploidy. cytochalasin, temperature, clam. Mulinia lateralis

INTRODUCTION

Tetraploid induction in bivalves has been widely investigated
because of its potential to provide an ideal approach to triploid
production. All-triploid Pacific oysters have been produced by
mating tetraploids and diploids (Guo et al. 1996). Triploids are of
interest in shellfish aquaculture for their superior growth and itn-
proved meat quality. Since the original work on triploidy by Stan-
ley et al. ( 1981 ) with the American oyster (Crassostrea virginica).
triploids have been produced in over 20 bivalves by manipulation
of polar body I (PBI) and polar body 2 (PB2) as reviewed by
Beaumont and Fairbrother ( 1991 ) and Guo (1999).

Viable tetraploids have been produced with variable success in
fish (reviewed by Pandian & Koteeswaran 1998). and in amphib-
ians (Humphrey & Fanhkauser 1949, Fischberg 1958. Reinschmidt
et al. 1979. Nishioka & Ueda 1983). In bivalves, tetraploid larvae
have been produced by several methods, including meiotic and
mitotic inhibition, meiotic inhibition of gynogenetically activated
eggs, and cell fusion (Beaumont & Fairbrother 1991. Guo 1991).
However, tetraploid embryos rarely survived beyond metamorpho-
sis in bivalves. In the Pacific oyster Crassostrea gigas. tetraploid
gynogenesis produced high percentages (95*^) of tetraploid em-
bryos, but no viable juveniles were later obtained (Guo et al.
1993), Similar results were obtained by inhibition of mitosis I
(Guo et al. 1994), inhibition of PBl (Stephens 1989, Guo et al.
1992a. 1992b). and blastomere fusion (Guo et al. 1994). There are
probably many other failed attempts that have not been reported.

A small number of viable tetraploids have been obtained by
manipulating meiosis in blue mussel (Scarpa et al. 1993). Manila
clam (Allen et al. 1994). and zhikong scallop (Yang et al. 2000).
These sporadic successes have been difficult to reproduce and no
breeding populations of tetraploids have been established by ma-
nipulating meiosis in eggs from diploids. A different method of
tetraploid induction was developed in the Pacific oyster in 1993,
when inhibiting PBl in eggs from triploids produced 2000 viable
tetraploids (Guo and Allen 1994). This method produced small

*Corre.sponding author. E-mail: xguo@hsrl.rutgers.edu

nuinbers of tetraploids in the pearl oyster Pinctada martensii (He
et al. 2000) and the eastern oyster (Supan et al. 2000). Large
numbers of tetraploid spat were subsequently obtained in the Pa-
cific (Eudeline et al. 2000) and eastern (Guo et al. 2002) oysters,
demonstrating the reproducibility of the Guo-Allen method. Al-
though the Guo-Allen method is reproducible, it is limited to
species in which triploids produce significant numbers of eggs.
Therefore, effective methods for direct induction of tetraploids
from diploids are needed in some species.

In the search of new candidate species for tetraploid induction
and of conditions that might favor its production, we examined the
suppression of PBl alone and both PBI&2 in the dwarf surfclam.
Mulinia lateralis, by use of cytochalasin B (CB) at different tem-
peratures. Different levels of tetraploids have been produced by
inhibiting PBl at different temperatures in the Pacific oyster
(Stephens 1989. Guo 1991 ). We expect that higher or lower treat-
ment temperatures might affect cellular properties and chromo-
some segregation patterns, possibly enhancing the effectiveness of
the chemical and finally improve survival of tetraploids.

Several characteristic of the dwarf surfclam make it suitable as
model for genetic investigations in inarine bivalves: a short gen-
eration time, a gametogenic activity that occurs over much of the
year under cultured conditions, a relatively high reproductive rate,
a sex differentiation easily discernible through the shell of sexually
ripe specimens, easy conditioning, and small space requirements
(Calabrese 1969),

MATERIALS AND METHODS

Broodstock and Gamete Collection

Adult M. lateralis were collected from wild populations of
Rhode Island, They were conditioned in recirculating seawater at
20°C (salinity 30 ppt) for 2-4 wk before use. During this period,
they were fed daily with Isochiysis fitdhana (C-ISO) at densities of
80-100.000 cells/niL. For natural spawning, ripe clams were se-
lected, air-dried for 1 h and induced to spawn in individual beakers
containing filtered (1 |j,m) and UV-sterilized seawater at 24°C.
Clams that did not respond to thermal stimulation were dissected

677

678

Peruzzi and Guo

for gamete collection. Eggs were passed through a lOO-fxm-nytex
screen to remove any tissue debris, collected, and rinsed on a
20-jjim screen. Sperm were passed through a 20-|i,m screen, and
their motility was checked under microscopy before fertilization.
The total number of eggs per female was estimated under micro-
scope by counting small aliquots, and the eggs checked for spon-
taneous development or possible contamination.

Artificial Fertilization, Chemical, and Thermal Treatments

Sperm were added to egg suspension at about 3-10 sperm/egg.
Fertilization and incubation were conducted using filtered seawa-
ter with a salinity of .^0 ppt at 19-28°C according to the experi-
mental design.

Fertilized eggs were treated with CB. dissolved in dimethyl-
sulfoxide (DMSO) at a final concentration of 0.75 mg/L. All CB
treatments started approximately at 5-7 min post-fertilization (PF).
The control group was used to gauge the timing of the treatment at
all times. For PBl inhibition, treatments lasted approximately 15-
20 inin or until about 75'7f of the untreated eggs released PBl in
the control group. For the retention of both PB1&2, CB treatments
were extended for an additional period of 15-20 min, or until the
majority of untreated eggs in the control group were ready for the
first mitosis (as indicated by the formation of the first polar lobe).
After each treatment, the eggs were separated from the chemical
by passing them onto a 20-|xm screen and gently rinsed with 0.5%
DMSO in seawater. Finally, they were suspended in fresh seawater
and left undisturbed.

Experimental Design

Experiments were performed using pooled eggs from 4-6 fe-
males and pooled sperm from three males. Soon after fertilization
the gametes were divided into three groups: in the first group,
fertilized eggs were allowed to develop as controls, and in the
second and third group the eggs were treated with CB to block PB 1
or both PB1&2.

Treatments were tested at normal (24°C). high (28-C). and low
( 19°C) temperatures. All experiments were replicated three times
except for the experiment at low temperature that was conducted
twice. Control groups were exposed to DMSO only at the same
temperature as the treated groups and for as long as the longest
treatment lasted.

Lanal Culture and Growth-Out

Larvae were reared in 20-L buckets at 22-24°C and fed daily a
diet of Isocliiysis galbana at densities of 100-120,000 cells/mL
starting at 24 h PF. Larvae were cultured at a maximum density of
20 larvae/mL and reached metamorphosis in approximately 8-10
days at a shell length of 180-250 jjim. Culture water was com-
pletely changed every 48 h, and larvae were collected and washed
gently on screens of proper size. D-stage larvae were separated
from trochophores by using a 44-jjim screen placed on top of a
25-|j.m screen. At Day 2, the percentages of development (D-
larvae and/or trochophores) were calculated from at least 100 in-
dividuals per group. Larval survival was determined for all groups
at each water change.

After metamorphosis, M. lateralis specimens were cultured in
upwellers and then in trays contained in a well-aerated recirculat-
ing seawater system at a temperature of 19-20°C. Changes of

culture water (approximately 1/5 of total volume) were performed
every other day.

Determination of Ploidy

Ploidy status of larvae (Day 1-14 PF) and ju\eniles (Day 17-
55 PF) was determined by flow cytometry with DAPI staining
according to Allen and Bushek (1992). For larvae, several hundred
were sampled after concentrating on a nytex screen and mixed
with a DAPI/DMSO stain (Guo et al. 1993). Samples were kept
frozen at -80°C and thawed to room temperature before analysis.
They were vortexed, passed three times through a 26-gauge
needle, and then filtered through a 25-|jim-mesh filter. Larvae were
analyzed in pooled samples (/; = 500-1000). whereas juveniles (/;
= 100) were analyzed individually. Juvenile samples were pre-
pared by mincing the whole body in a 1.5-mL test tube. Haploid
sperm and diploid cells from untreated clams were used as stan-
dards at all times.

Statistical Analysis

Fertilization level was determined as percentage of divided
eggs at 2— f h PF. Cumulative survival at Day 2 (D-stage), Day 7.
Day 17. and to juvenile was calculated relative to the number of
fertilized eggs. Statistical analyses were performed using SYSTAT
10 (SYSTAT Inc.). Percentage data for fertilization and survival
were arcsine-transformed and analyzed by ANOVA followed by
Tukey's HSD post-hoc comparison. All differences were accepted
as significant when P < 0.05.

RESULTS

Meiotic and Mitotic Events

At temperatures of 24°C. control eggs started to release PB 1
around 12 to 15 min PF, and PB2 at 30-35 min PF. At higher
(28°C) or lower (19°C) temperatures, timing was respectively
shortened or delayed by 10-15 min. Nevertheless timing of PBl
and PB2 release varied among replicates according to egg quality.
In CB-treated groups, no release of PBs was observed during
treatments, and meiotic events were restored 3 to 5 min after
complete removal of the chemical. Untreated eggs reached mitosis
40-50 min PF except at low temperatures ( 19°C), where eggs were
slightly retarded in development and started cleaving at 55-65 min
PF. As expected, mitotic events in CB-treated eggs, particularly in
PBI&2 groups, were delayed compared with their control.

Survival and Development

Treatment temperatures did not significantly affect fertilization
level within any of the experimental groups, and there was no
difference among treated or control groups at any given tempera-
ture. Overall, fertilization level ranged between 75% and 99% and
varied among replicates (Table 1 ).

At Day 2, no differences in survival were found between con-
trol and treated groups at 24°C or 28°C. At 19°C. the percentage
development in the PB1&2 group was 21%. which was signifi-
cantly lower than in control (53%. P = 0.009) and PBl (51%. P
= 0.01) groups. At 28°C, eggs in most of the PB1&2 groups
survived as trochophores only and did not develop any further,
unlike most of the control or PBl groups where variable propor-
tions of D-larvae (ainong all larvae) were found (Table 2). At this
temperature, survival in the PBl group was generally low (13-

Tetraploid Induction in the Dwarf Surfclam

679

TABLE 1.

Inhibition of PBl or PB1&2 in M. lateralis under different

temperatures: number of eggs used; fertilization level; percent

survival of fertilized eggs to Day 2, 7 and 17; and the number of

juvenile obtained.

TABLE 2.

Inhibition of PBl or PB1&2 in M. lateralis under different

temperatures: percentages of D-larvae and trochophores observed in

experimental groups at Day 2.

Group

D-larvae ( % )

Trochophores ( % )

Eggs Fertilization
xlOOOl {9c)

Day 2

Day 7

{%)

Day 17

(%)

Juvenile

(/I)

Group (

Low temperature (19°C)
LTl-C

100

0

Low temperature

(19X)

LT2-C

100

0

LTl-C

88

90

58

35

6

5.000

LTl-PBl

81

19

LT2-C

103

84

49

25

0.7

600

LT2-PB1

84

16

LTl-PBl

86

86

51

-)2

1.6

1,200

LT1-PB1&2

73

27

LT2-PB1

70

89

52

32

0.8

480

LT2-PB1&2

75

25

LTI-PBI&2

136

90

21

10

0.2

300

Normal temperature

I24''C)

LT2-PB1&2

83

93

21

4

0.2

L50

NTl-C

100

0

Normal temperature (24°C)

NT2-C

100

0

NTl-C

251

97

100

63

56

50.000

NT3-C

100

0

NT2-C

278

99

90

20

0

0

NT 1 -PBl

51

49

NT3-C

158

94

87

66

0.2

350

NT2-PB1

74

26

NT 1 -PBl

1,045

94

21

8

4

2,000

NT3-PB1

85

15

NT2-PB1

500

99

98

48

20

8,000

NT1-PB1&2

26

74

NT3-PB1

144

90

40

43

0.6

102

NT2-PB1&2

41

59

NT1-PB1&2

1,105

94

15

-)

0.8

800

NT3-PB1&2

71

27

NT2-PB1&2

405

99

63

24

0.1

300

High temperature (2

8°C)

NT3-PBI&2

194

89

40

5

0.3

104

HTl-C

0

100

High temperature

(28°C)

HT2-C

22

78

HTl-C

323

91

S"

0

0

0

HT3-C

92

8

HT2-C

113

84

29

0.8

0.3

218

HTl-PBl

25

75

HT3-C

265

94

69

53

4

1.000

HT2-PB1

23

77

HTl-PBl

420

89

13

0.7

0.3

200

HT3-PB1

35

65

HT2-PB1

91

84

34

1

0.1

59

HTI-PB1&2

17

83

HT3-PBI

163

91

15

3

0.3

78

HT2-PB1&2

0

100

HT1-PB1&2

280
156

90

75

IS"

0

0

0
0

0
0

HT3-PB1&2

0

100

HT2-PB1&2

HT3-PB1&2

182

93

4"

0

0

0

' Trochophores only.

34%) and extremely variable in the control {5-69'7c). In one con-
trol group (HTl-C). all the larvae developed as trochophores
only. Overall, percentages of D-larvae at 24°C were significantly
higher in control groups (100%) than in PBl (70%, P = 0.01)
and PB1&2 (46%, P = 0.002) groups, whereas no differences
were found between treated groups. At lower temperatures
(19°C), again control groups had higher percentages of D-larvae
(100%) than PBl (82%, P = 0.001) or PBI&2 (74%. P < 0.001)
groups. D-larvae percentages were not analyzed for the high-
temperature experiment where 100% trochophores were observed
in all PB1&2 groups and one control. Trochophores in the CB-
treated groups appeared to be deformed and/or swam in a cir-
cular motion.

Larvae in all groups metamorphosed between Day 7 and Day
10. At Day 7 and until Day 17. survival of larvae did not differ
among treated and control groups at any temperature and varied
among replicates (Table 1 ). Percentage survival to juvenile was
generally low within the high-temperature groups, averaging 2.2%
and 0.2% in the control and PBl groups, respectively. An unex-
pected and complete mortality was encountered after Day 7 in one
of the control groups (NT2-C).

In general, the number of surviving juvenile and juvenile clams
(Days 16-54) varied greatly among replicates but equally among
groups (Table 1 ).

Ploidy of Larvae and Juveniles

Ploidy of larvae was analyzed on Day 1 and Day 2 to detect
possible difference due to differential mortality. No differences in
ploidy composition were found between Day 1 and Day 2, and
only data from Day 2 are presented.

Ploidy of 2-day-old larvae from treated groups varied greatly
among treatments and replicates, ranging from diploidy to penta-
ploidy (Fig. lA-D). In particular. PBl inhibition produced vari-
able proportions of triploid. aneuploid-tetraploid and some penta-
ploid larvae (Fig. lA and B), whereas pentaploids were most com-
monly observed and dominant ploidy in PB1&2 groups. The
aneuploid-tetraploid peaks were between 3.6-3.8n, sometimes
overlap with 4n. One PB1&2 group had distinctive aneuploid
(3.7n) and pentaploid peaks (Fig. IC), whereas others had clear
diploid, triploid, tetraploid and pentaploid peaks (Fig. ID). After
separation of D-stage larvae and trochophores, the flow cytometric
analysis of isolated larvae, showed that all or nearly all tetraploid
and pentaploid larvae developed as trochophores only (Fig. 2B
and D), whereas virtually none or small proportions (Fig. 2C
and E) were detected among the D-stage larvae. Control groups
from treatments at 19°C and 24°C contained diploid larvae
only, unlike controls at 28"C that showed variable proportions
of diploid, triploid and pentaploid larvae in two of the three rep-
licates (HTl and HT2). indicating some influence of tempera-
ture on meiotic events. In general, there was no clear pattern that
higher or lower temperatures affected the proportions of poly-

680

Peruzzi and Guo

300 ■

count

1 ^"

2*0 ■

2n

ll

JOO ■

llll

160 ■

ll

i

too

\

i 1

[ 1

50 -

Q

■

lll>ll»<ll<lllf«l

200 ?50 3O0

DMA content

150 200 260 300 350 400

DMA content

450 500

i>mw>H<iipijil iH ii«l<i<l|lM I III I r

00 150 200 250 300 360 400 460 600

DIMA content

300 H

count

soo •

5n

4

D

«0 •

i

300

2n i

200 ■

1 ffl

100 ■

_J

M^ I.

■■'■'f"'""i"'

5

■ ""r'-v., , , , 1 1

go 100 160 20a 250 300 350 400 450 500

DMA content

Figure 1. Flow cytometry analysis of 2-day old MiiUnia larvae from inhibition of PBl (A, B) and PB1&2 (C, D) showing various ploidy levels
induced.

ploids among the treated groups, although precise characterization
of ploidy composition was difficult in this study because of
the occurrence of aneuploids and tremendous variation among
replicates.

The majority of trochophores died within the first .■? days, al-
though some survived to Day 4. Ploidy analysis of larvae was
performed several times before metamorphosis and showed virtu-
ally no detectable aneuploid. tetraploids. or pentaploids among the
surviving larvae after Day 4. Diploids remained the most frequent
ploidy in most groups. Distinctive triploid peaks were observed in
most PBl groups at 24°C and 28°C.

After metamorphosis, individual measurements of juvenile and
juvenile clams (Days 16-.'i5l showed variable percentages of dip-
loids and triploids in CB-treated groups (Table 3). Of 100 juvenile
sampled from one of the PB 1&2 groups, two were tetraploid [29c:
Table 3 and Fig. 3B). To confirm this finding, another group
{n = 96) of individuals were analyzed and resulted again in 2% of
tetraploids. This group contained juvenile with a measured shell
height of 1.5-2.5 mm (Fig. 3A).

At juvenile stages, mean percentages of triploids in PB 1 groups
did not differ significantly between 24°C and 28°C (Table 3). Eggs
treated with CB to inhibit PB1&2 produced viable triploid juvenile
in the experiment at 24 'C only. No triploids were found among
any of the surviving juvenile in the experiment at low temperature
(19°C). Three triploids were found among 100 juvenile in one of

the control groups (HT2-CI at 28°C, thus confirming the results
obtained during early rearing.

DISCUSSION

A low percentage of tetraploid clams (2%) was obtained in one
of the eight groups treated to inhibit PB1&2. Although the tetra-
ploid production was low in frequency and inconsistent among
replicates, this result provides the first evidence that tetraploid
Mtilinia can survive beyond metamoiphosis and reach juvenile
stage. This finding provides another example that a small number
of viable tetraploids can be produced by manipulating meiosis in
normal zygotes. Similarly, small numbers of tetraploids have been
obtained in the blue mussel (Scarpa et al. 19931. Manila clam
(Allen et al. 1994). and zhikong scallop (Yang et al. 2000).

The production of tetraploids from inhibition of both PBs is
unusual. Mature eggs of marine bivalves are arrested at prophase
I. Before fertilization, eggs of oysters and clams are actually tet-
raploids and. theoretically, pentaploids should be produced after
fertilization and successful inhibition of PB1&2. The production of
pentaploids by blocking both PBs has been demonstrated in the
Pacific oyster (Cooper & Guo 1989), in blue mussel (Scarpa et al.
1993), and in this study. Two possible explanations exist why
tetraploids were produced in PB1&2 groups. First, it is possible
that the CB treatment produced pentaploids. which subsequently
reverted to tetraploids through chromosome loss. There is indica-

Tetraploid Induction in the Dwarf Surfclam

681

800 -

count

A

500 -

5n

4

400 -

i

300 -

2n

1

200 -

1

ft

100 -

1

3n

2

4n

3

s

0

^44«M

^

^3

^■i^-^-^y

5

50

100

150

200 250

DNA content

300

350

400

450

500

count

soo -

zoo 250

DNA content

B

300 350 400 450

I'

E

yf^^

2C

) um

1 'ffip^WwM"^* ■

\

> II 11*11*11 i-iM^Ha

^

^Si

1

Figure 2. Flow cytometry analysis of 2-day old Mulinia larvae from I'B1&2 inhibition: A, a sample containing both trochophores and D-larvae;
B, trochophores only; C: D-stage larvae only; D: a representative abnormal trochophore; and E: a representative D-stage larva.

682

Peruzzi and Guo

tion that tetraploid oysters may revert to triploids or triploid/
tetraploid mosaics (Guo et al. 2002). However, reversion is
thouglit to be rare event in triploid and tetraploid oysters (Allen et
al. 1997). Secondly, it is possible and even likely that the CB
treatment for the inhibition of PBI&2 was not 100% effective and
that only PBl was inhibited in some of the eggs (Guo et al. 1992b,
Scarpa et al. 1993). In fact, treatments targeting specific meiotic
events are rarely 100% effective because of unsynchronized de-
velopment of zygotes, which is why triploid induction is rarely
100% effective (Lu 1986. Allen et al. 1989). Therefore, the tetra-
ploids we obtained might be from accidental inhibition of PB 1
alone. Interestingly, however, treatments specifically targeting
PBl did not produce viable tetraploids in this study.

Inhibition of PBl is known to result in a complex segregation
pattern that leads to the production of triploids, tetraploids and
variable proportions of aneuploids larvae (Guo et al. 1992a,
1992b). These findings were later confirmed in zhikong scallop by
Yang et al. (2000). Our study showed that PBl inhibition had
similar effects producing a comparable spectrum of nuclear DNA
content in 2-day-old trochophores and D-larvae from CB-treated
eggs. Triploids and aneuploids were most commonly observed
unlike tetraploids that were rarely detected by flow cytometric
analysis. The pentaploid larvae were most likely produced from
the incidental inhibition of both PBl and PB2. Although their
ploidy status was not confirmed by chromosome counting, the
putative aneuploids (approx. 3.7n) were either hypertriploid or
hypotetraploid and possibly produced through tripolar segregation
patterns under CB treatment (Guo et al. 1992b). Aneuploids were
not found among control groups. Variable proportions of aneup-
loids were also observed by tlow cytometry after PB 1 inhibition in
zhikong scallop (Yang et al. 2000) and confirmed by chromosome
counting. Similar results were obtained on other mollusks by pre-
vious workers (Guo et al. 1992a, 1992b, Scarpa et al. 1993, Allen
et al. 1994).

Our results show that the higher ploidy levels observed by flow
cytometry were mainly represented by populations of tro-
chophores, which declined rapidly over the same period. Clear-
ly, few if any tetraploid embryos survived to D-stage in most
groups. The proportion of aneuploids and pentaploids declined
abruptly during the first week, and only diploids and triploids
were detected among the surviving juvenile. A similar decrease
in the proportion of pentaploids and increase in triploids in CB
groups was observed in other studies (Scarpa et al. 1993, Guo
& Allen 1994). Generally, aneuploidy probably causes imbalance
of gene dosage and is often lethal in mollusks with survival de-
pending on specific tolerance to chromosome loss or gain
(Guo & Allen 1994, Wang et al. 1999). The failure of tetraploid
and pentaploid larvae to survive beyond early developmental
stages and metamorphosis has been explained by the unbalanced
cytoplasm/nucleus ratios or the cell-number (egg-volume) defi-
ciency hypothesis (Guo 1991, Guo & Allen 1994, Guo et al. 1994).
Guo's hypothesis states that the cleavage of eggs of a given
size with large tetraploid (or pentaploid) nuclei results in reduc-
tions in cytoplasm/nucleus ratio or cell number, both fatal for
further development in bivalves. Accordingly, high percentages
or large numbers of tetraploids (up to 100%) have been pro-
duced using larger eggs from triploids in the Pacific (Guo & Allen
1994, Eudeline et al. 2000) and eastern (Guo et al. 2002) oysters.
Supan et al. (2000) also reported the production of tetraploid
eastern oysters, but specific numbers and percentages were not
available.

Under our experimental conditions, high or low temperature
did not improve tetraploid induction as measured by viable tetra-
ploids produced. Effects on ploidy of early larvae were not clear
partly because of the tremendous variation within treatments. The
presence of large proportions of aneuploids made ploidy determi-
nation by flow cytometry difficult. We did not do chromosome
counting in this study and had no accurate estimates what chro-
mosome numbers were actually induced under different tempera-
tures. Nevertheless, ploidy of surviving juveniles indicates that
high (28°C) and normal (24°C) temperatures produced similar
percentages of triploids and that low ( 19'C) temperature made CB
treatment ineffective. Low temperatures did not produce viable
polyploids in this study. This result would suggest that a lower
temperature might influence the cellular properties and chromo-
some segregation patterns in this species. In a previous study in the
Pacific oyster, high levels of tetraploids were produced under low
temperatures (Stephens 1989). High temperatures (28 C) nega-
tively affected the development of larvae deriving from eggs
treated to block both PB1&2 and finally provoked complete inor-
tality. It is likely that this result was induced by a combination of
long chemical treatment and high temperature. Generally, control
groups exposed at the same temperature and for the same duration
did produce viable larvae. However, the same temperature was
partially effective in blocking PB release and induced 3% triploidy
in one control group. Poor egg quality might have favored these

TABLE 3.

Inhibition (if PBl or PB1&2 in M. lateralis under different

temperatures: age (in days), number, and ploidy composition of

Mulinia juveniles as determined b) flow cytometrj.

Age

Juvenile

2N

3N

4N

Group

(days)

in)

(%>

(%)

(%)

Low temperature (19"C)

LTI-C

17

100

100

0

0

LT2-C

16

100

100

0

0

LTI-PBl

17

100

100

0

0

LT2-PB1

16

100

100

0

0

LT1-PB1&2

17

100

100

0

0

LT2-PB1&2

16

100

100

0

0

Normal temperature (24°C)

NTI-C

25

100

100

0

0

NT2-C

na"

0

(1

0

0

NT3-C

27

100

100

0

0

NT 1 -PBl

25

100

84

16

0

NT2-PB1

25

100

64

36

0

NT3-PB1

27

102

100

0

0

NTI-PBI&2

25

1%

89

9

-)

NT2-PBlcS:2

25

100

83

17

0

NT3-PB1&2

27

104

99

1

0

High temperature (28°C)

HTI-C

na

0

0

0

0

HT2-C

54

100

97

3

0

HT3-C

51

100

100

0

0

HTI-PBl

55

44

31

69

0

HT2-PBI

54

59

88

12

0

HT3-PBI

51

50

98

1

0

HT1-PBI&2

na

0

0

0

0

HT2-PBI&2

na

0

0

0

0

HT3-PB1&2

na

0

0

0

0

' na, not available because of complete mortality.

Tetraploid Induction in the Dwarf Surfclam

683

300
count

250
200
150
100
50

B

-

i

^ iH#w,.

j

1 ■■.■'i. -f .Xir-i -t f

50

100

150

DNA contenl
Figure. 3. Flow cytometry analysis of 25-day-old Mulinia clam: A, representative juvenile clams analyzed; B: analysis of a diploid (1) and a
tetraploid clam (2).

conditions too. Heat shocks in the range of 32^0°C are most
commonly required in blocking the release of PBs in other mol-
lusks (Quillet & Panelay 1986, Yamamoto & Sugarawa 1988, Guo
et al. 1994).

In summary, this study indicates that temperature has little
effects on the final outcome of tetraploid induction in M. lateralis.
Temperature affects the liming of meiotic segregation and there-
fore should affect tetraploid induction frequency. We could not
delect such effects in this study probably due to random variation
in treatment and eggs quality. Considering the low induction ef-
ficiency (2%), manipulating meiosis may not be a viable approach
to tetraploid production in this species. Nevertheless, this study
provides the first evidence that tetraploids are viable in M. lateralis

and offers encouragement for further research on tetraploidy in this
species.

ACKNOWLEDGMENT

We are grateful to Dr. Huiping Yang for assistance with clam
culture and sampling and Dr. Standish K. Allen Jr. for constructive
comments. The authors thank Dr. Timothy Scott for providing
clam broodstock. This work is supported by a grant from the New
Jersey Sea Grant Consortium (R/BT-20()1) and by a grant from
New Jersey Commission on Science and Technology's R&D Ex-
cellence Program (No. 00-2042-007-20). This is IMCS/NJAES
Publication No. 2002-18 and NJSGC No. 02-495.

684

Peruzzi and Guo

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Joiirniil of Shellfish Research. Vol. 21. No. 2. 685-690. 2002.

CYTOGENETIC STUDY OF OSTREA CONCHAPHILA (MOLLUSCA: BIVALVIA) AND
COMPARATIVE KARYOLOGICAL ANALYSIS WITHIN OSTREINAE

ALEXANDRA LEITAO,' - RAQUEL CHAVES," SARA SANTOS," PIERRE BOUDRY,'
HENRIQUE GUEDES-PINTO," AND CATHERINE THIRIOT-QUIEVREUX'*

^ Laboratoire de Genetiqiie et Pathologie. Station de I'lnstitut Frangais pour la Recherche et
I' Exploitation de la Mer (IFREMER). B.P. 133. 17390 La Tremhlade. France: ~Departamento de
Genetica e Biotecnologia. ICETA-UTAD. Vila Real. Portugal: ' Oh.se rvatoi re Oceanologique, Universite
Pierre et Marie Curie, Centre National de la Recherche Scientifique. B.P. 28. 06230
Vdlefranche-sur-Mer. France

ABSTRACT Chromosome preparations of the Olympia oyster Ostrea conchaphila Carpenter were studied using conventional
Giemsa. silver staining, and C-banding techniques. The karyotype consists of six metacentric (1. 2. 4, 6. S. and 10) and four
submetacentric (3. 5, 7, 9) chromosome pairs. The silver-stained nucleolus organizer regions (Ag-NORs) were terminally located on
the short arms of the submetacentric pair 5 (569f of cases) and on the long arms of submetacentric pair 7 (6% of cases). Constitutive
heterochromatin was observed as telomeric C-bands on the short arm of the NOR-bearing chromosome pair 5 and as centromeric blocks
of several chromosome pairs. Comparative analysis of patterns of karyotype, Ag-NORs, and C-bands of this species and of five other
flat oysters, Ostrea angasi. O. cliilensis. O. denselamellosa. O. ediiiis. and O. piielchaiui. for which data have been previously
published, were performed, allowing the inference of cytota.\onomic relationships within Oslreinae.

KEY WORDS: Oslrea conchaphila. cytogenetics. cytota,\onomy. Ostreinae

INTRODUCTION

Studies on oyster cytogenetics have been performed so far on
26 species of Ostreacea (see Nakaniura 1985, leyama 1990,
Thiriot-Quievreux 2002). The first data only concerned chromo-
some number and gross morphology (Ahmed & Sparks 1967,
Menzel 1968). Later, morphometric measurements of chromo-
somes enabled the comparison among karyotypes at the interspe-
cific and intraspecific level (e.g., Ladron de Guevara et al. 1996. Li
& Havenhand 1997). During the last decade, the development of
banding techniques has allowed the fine characterization of indi-
vidual chromosomes (e.g.. Leitao et al. 1999a).

According to the morphologically based classification of Harry
(1985), which is currently used, the family Ostreidae includes
three subfamilies, that is, Lophinae, Ostreinae, and Crassostreinae.
These oysters are sequential hermaphrodites and contain both
broadcast spawners (Crassostreinae) and brooders (Lophinae and
Ostreinae). Recent techniques such as inolecular phylogenetic
analysis provided novel insights into oyster evolution and system-
atics (Littlewood 1994, Jozefowicz & 6 Foighil 1998, 6 Foighil &
Taylor 2000). Karyological analysis among cupped oysters, the
Crassostreinae (Leitao et al. 1999b). has proven complementary to
these approaches and has provided additional evolutionary infer-
ences.

Among the flat brooding oyster species, the Ostreinae, five
species have been previously karyologically investigated: Ostrea
edulis (Linne) (Thiriot-Quievreux 1984). O. denselamellosa (Lis-
chke) (Insua & Thiriot-Quievreux 1991), O. puekluma (Orbigny)
(Insua & Thiriot-Quievreux 1993), O. chilensis (Philippi) (Ladron
de Guevara et al. 1994), and O. angasi (Sowerby) (Li & Haven-
hand 1997).

The Olympia oyster, O. cimchaphila (Carpenter 1857). previ-
ously known as O. hirida (Carpenter 1864). has been studied by
Ahmed and Sparks (1967) and Ahmed (1973) using squash tech-
niques and tentative grouping of chromosomes. Ostrea con-

*Conesponding author. E-mail; thinotteobs-vlfr.fr

chaplula. native to the western United States and Canada, ranges
from the southeast Alaska to Baja California (in tidal channels,
estuaries, bays, and sounds). Commercially important in the late
19th century, this species was cultured in the state of Washington
until near-collapse of the industry in the 1950s (Baker 1995).

In the present work, the karyotype, nucleolus organizer regions
(NORs), and constitutive heterochromatin distribution were stud-
ied in Ostrea conchaphila (Carpenter 1857) and a comparison with
previously published karyological data on the five other flat oyster
species mentioned above was performed to analyze cytotaxonomi-
cal relationships within Ostreinae.

MATERIALS AND METHODS

Specimens of the Califomian Olympia oyster Ostrea con-
chaphila (GO) were imported from the Pacific Institute (Olympia,
WA). Oysters were strictly confined to the quarantine facilities of
the IFREMER hatchery of La Tremhlade, Charente-Maritime,
France, according to international recommendations. After repro-
duction, the progeny (Gl ) used in this experiment was reared in the
same quarantine facilities for at least 5 mo before sampling.

Whole juvenile animals (ca. 2.5 cm length) were incubated for
7-9 h in a 0.005% solution of colchicine in seawater. The gills
were then removed by dissection and treated for 30 min in 0.9%
sodium citrate in distilled water. The material was fixed in a
freshly prepared mixture of absolute alcohol and acetic acid (3:1 )
with three changes of 20 min each. Fixed pieces of gill from each
individual were dissociated in 509J: acetic acid with distilled water
solution. The suspension was dropped onto heated slides at 44'C
and air-dried (Thiriot-Quievreux & Ayraud 1982).

For conventional karyotypes, gill preparations were stained
with Giemsa (4%, pH 6.8) for 10 min. The silver-staining method
for NORs was performed on unstained slide preparations accord-
ing to the procedure of Howell and Black (1980). This method
only detects those NORs that were active at the precedent inter-
phase (Miller et al. 1976). Chromosomal Ag-NORs can serve as
characters for inferring phylogenetic relationship (e.g., Amemiya

685

686

Leitao et al.

& Gold. 1990). Constitutive heterochromatin regions (C-bands)
were revealed using the method of Sumner ( 1972) with the coun-
terstain propidium iodide. The evolutionary significance of the
heterochromatin has previously been discussed in vertebrates (e.g..
Hsu & Arrighi 1971. Saffery et al. 1999. Chaves et al. 2000).

Images of Giemsa-stained metaphases and C-banding were ac-
quired with a CCD camera (Axioplan, ZEISS) coupled to a ZEISS
Axioplan microscope. Digital images were processed using Adobe
Photoshop 5.0 (Windows) using functions affecting the whole of
the image only. Microphotographs of Giemsa stained metaphases
and C-banding were taken with a ZEISS Axioplan microscope.
Digital images were processed using Adobe photoshop 5.0 (Win-
dows). Microphotographs of suitable NOR-stained metaphases
were taken with a ZEISS III photomicroscope.

After karyotyping, chromosome measurements of 10 suitable
metaphases were made with a digitizer table (Summa Sketch II)
interfaced with a Macintosh. Data analysis was performed with an
Excel macro-program. Relative length was expressed as 100 times
the absolute chromosome length (in (xm) divided by the total
length of the haploid complement. Centromeric index was calcu-
lated by dividing 100 times the length of the short arm by the total
chromosome length. The arm ratio was determined (length of short
arm divided by length of long arm). Both centromeric index and
arm ratio are given because each expresses centromere position
and allows comparison with other karyological studies. Terminol-
ogy relating to centromere position (m: metacentric, sm: submeta-
centric) follows that of Levan et al. (1964).

To elucidate similarities between Ostreinae species, a hierar-
chical agglomerative flexible clustering program was used (Lance
& Williams 1966). Both NOR and centromeric index information
of O. conchaphila and five previously studied Ostreinae species
were used to cluster species. The Manhattan metric was used to
discriminate and then to associate individual species. Manhattan
distance appears appropriate to this kind of combination of quan-
titative (centromeric index values) and qualitative (NORs posi-
tions) data and to measure an association between individual ob-
jects (species) (Legendre & Legendre 1998).

RESULTS

Analysis of 60 mitotic metaphase spreads from 15 individuals
of O. ctmchaphila confirmed the diploid chromosome number of
In = 20. scored by Ahmed and Sparks (1967). For karyotyping.

the chromosomes of 21 well-spread metaphases were paired on the
basis of chromosome size and centromere position. From these, the
10 best spreads were used for chromosome measurements and
classification (Table 1). The karyotype (Fig. I A) consists of ten
chromosome pairs. Pairs I, 2, 4, 6. 8. and 10 were metacentric.
Pairs 3. 5. 7. and 9 were submetacentric.

The Ag-NORs were examined in another 122 metaphases from
10 animals. A variable number of one to three Ag-NOR chromo-
somes were identified (Fig. IB). The NOR site was located ter-
minally on the short arms of the submetacentric pair 5 and on the
long arms of the submetacentric chromosome pair 7. The most
frequent case (56% of observed silver-stained metaphases) was
one active silver-.stained NOR chromosome in pair 5. The Ag-
NORs located on pair 7 occurred in few cases (6%).

Constitutive heterochromatin was observed in 31 karyotypes
made from well-spread C-banded metaphases from 13 animals.
Telomeric C-bands were always observed on the short arm of the
NOR-bearing chromosome pair 5. In addition, centromeric blocks
were also found in chromosome pair 2 in 84% of observed
metaphases. pairs 1, 4. and 5 in 68%. pairs 6 and 8 in 58% of the
C-banded karyotypes and in fewer cases in pairs 3. 7. and 9 (35%),
and in pair 10 (26%) (Fig. IC).

To compare the karyological data from O. concluiphlki and
from the other five flat oyster species previously studied, ideo-
grams (Fig. 2) were constructed from relative length and centro-
meric index values of O. conchaphila (see Table 1 ). O. ediilis
(after Leitao 2000. French population of La Tremblade hatchery.
Charentes Maritimes. France), O. aiigusi (after Li & Havenhand
1997). O. chilensis (after Ladron de Guevara et al. 1994), O.
clenselamcllosa (after Insua & Thiriot-Quievreux 1991), and O.
pnelchana (after Insua & Thiriot-Quievreux 1993). The location of
Ag-NORs was also included because chromosomal NOR have
been used as characters for inferrmg hypothesis of cytotaxonomic
relationships (e.g.. Amemiya & Gold 1990, Leitao et al, 1999 b).

The comparison of the relative length and centromeric index of
the 10 chromosomes pairs of the studied species showed that pair
1 was similar among all species, pair 2 was also similar except for
O. puelchuna, pair 3 and 4 were similar except for O. conchaphila.
but taking into account the close relative length and the standard
deviation of pair 3 and 4 of O. conchaphila. they may be inverted.
Pair 5 was variable among species, pairs 6 and 7 were identical
except for O. denselamello.sa. but in this case, the pairs 6 and 7
cannot be inverted because of their different relative length and the

TABLE 1.
Chromosome measurements and classification in U) cells of Ostrea conchaphila.

Chromosome
Pair No.

Relative Length

Arm

Ratio

Centromeric Index

Mean

SD

Mean

SD

Mean

SD

Classification

1

12.77

0.99

2.-W

0.21

42.12

1.78

111

2

1 1 .60

0.35

2..'i4

0.18

43.87

1.89

m

3

10.64

0.57

1.26

0.20

27.76

2.86

sm

4

10.54

0.50

2.37

0.27

41.94

2.81

m

5

10.47

0.87

1.72

0.16

34,05

2.28

sm

6

9.88

0.78

2.48

0.25

42.81

2.67

m

7

9.46

0.38

1.26

0.16

27.56

2.59

sm

8

9.30

0.58

2.53

0.39

43.59

3.92

m

9

8.79

0.63

1.52

0.25

31.27

3.37

sm

10

6.56

0.65

2 4.^

0.26

42.18

2.60

ni

Cytogenetics of Ostrea conchaphila

687

St l\ M H U

1 2 3 4 5

V

Ki

10

■». j^

3

K>,

¥

■.>i».

;

aK

iX

10

y

1

1

y< t^ n

10

Figure 1. Karyotypes of Ostrea conchaphila. A, Conventional Gicmsa staining; B, silver-stained nucleolus organizer regions (Ag-NORs); C,
C-banding. Note the simultaneous presence of Ag-NOR and C-bands in a telomeric position on the short arms of pair 5 (arrows) and the
centromeric heterochrnmatic blocks on chromosome pairs 1, 2, 4, 5, and 9. Scale bar = 5 )im.

C-bandiiig of pair 6. Pair 8 was variable among species. Pair 9 was
identical except for O. aiif;asi and pair 10 was variable.

A statistical analysis based on Ci and NORs (Fig. 3) high-
lighted the clustering of O. ediiUs and O. angasi and of O. dense-
lamellosa and O. ehilensis with O. conchaphila placed near this
cluster. O. puekluma is separated from the other species by the
highest dissimilarity.

DISCUSSION

This is the first report on karyotype after chromosome mea-
surements and NORs and C-banding patterns of the Olympia oys-
ter. The diploid chromosome number In = 20 observed is char-
acteristic of the genus Ostrea and is common throughout the Os-
treacea (Nakamura 1985, Thiriot-Quievreux 2002). The karyotype.

688

Leitao et al.

Relative length

edulis

120

100

80

60

40

20

7 8 9 10

Chromosome pair

Figure 2. Ideograms of six flat oysters constructed from relative
length and cenlromeric index values. Stippled chromosome: metacen-
tric, white chromosome: suhmetacentric. striped chromosome: suhte-
locentric, hiack chromosome: telocentric. Circles indicate Ag-NORs,
dark circles the most frequent case.

O.den

O.Chi

O.con

O.pue

O.edu

O.ang

Figure 3. Hierarchical agglomerative flexible clustering of Ostrea spp.
O. den: Ostrea deiiselamellosa; O. chi: O. chileiisis; O. con: O. con-
chaphila: O. pue: O. piiekhana; O. edu: O. edulis; O. ang: O. angasi.

including six metacentric and tour sulimetacentric cliromosome
pairs, and the NOR and C-band distribution differ from the other
ostreinid species studied. The comparison of the relative length
and centromeric index of the 10 chromosome pairs of the studied
species shows that, if one postulates that shared chromosome pairs
with the same relative length and centromeric index may be con-
sidered as primitive, pairs I, 3. and 4 are primitive and pairs 5. 8.
and 1 0 the most derived. However, these chromosome homologies
should be confirmed by other banding techniques.

The comparison of karyotypes and location of Ag-NORs
among species highlighted first the chromosome similarity be-
tween the European species O. edulis and the Australian and New
Zealand species O. angasi. already pointed out by Li and Haven-
hand (19971. Their karyotypes differ slightly {5m. 5 sm in O.
edulis and 5m. .^ sm. 2 st in O. angasi). but the phenomenon of
variation in the number of submetacentric and subtelocentric chro-
mosomes have been reported in French populations (Thiriot-
Quievreux 1984). More striking is that the most frequent Ag-NOR
patterns are similar in both species.

The isolated karyotype of O. puelchana is remarkable because
of the single telocentric chromosome. The occurrence of telocen-
tric chromosomes has been only seen in one other species of Os-
trcidae. Dendrostrea folium (Lophinae) (leyama 1990).

The three other flat oysters bear high karyotype resemblance,
that is. .seven metacentric and three submetacentric pairs for O.
denselamelosa and O. chilensis and six metacentric and four sub-

Cytogenetics of Ostrea conchaphila

689

metacentric pairs for O. conchaphila. Their NOR chromosomal
location revealed that there is a higher resemblance between the
NOR patterns of O. chilensis and O. conchaphila than between
these two species and O. denselameUosa. O. chilensis and O. con-
chaphila showed terminally located NORs on the short arms of
one chromosome pair and on the long arms of another chromo-
some pair. On the contrary, in O. denselamelosa. Ag-NORs were
always terminally located on the short arms of chromosome pairs.

Data on constitutive heterochromatin distribution only con-
cerned three species, O. denselameUosa (Insua & Thiriot-
Quievreux 1991), O. angasi (Li & Havenhand 1997), and O. con-
chaphila (this study). Centromeric C-bands were observed in chro-
mosome pairs 3. 6, 8, 9, and 10 in O. angasi and in pairs 6. 8, 9,
and 1 0 in O. denselameUosa. Occasional C-bands were seen on the
centromere of pairs 4 and 7 in O. angasi and on telomeres of pairs
3, 5, 6, 8, 9, and 10 in O. denselameUosa. A substantial proportion
of the eukaryote genome consists of constitutive heterochromatin.
This genomic fraction includes, among other repetitive sequences,
satellite DNA. Sequence analysis of these repeats suggests that the
sequences are rapidly evolving, and hence they are valuable as
evolutionary markers; consequently, constitutive heterochromatin
analysis can give insights about the phylogeny relationships of
related species (Saffery et al. 1999. Chaves et al. 20001. The ob-
servation in O. denselameUosa and O. conchaphila of the simul-
taneous presence of Ag-NORs and C-bands on telomeric position
in the same chromosome pair, that is, pairs 3 and 8 in O. dense-
lameUosa and pair 5 in O. conchaphila. might corroborate the
close karyological relationship between these two species noted
above.

The cytotaxonomic relationships pointed out here are incon-
gruent with the morphologically based classification of Harry
(1985), who stated that O. chilensis and O. angasi were junior
synonymous of O. puelchana in the subgenus Eoslrea of the genus
Ostrea and that O. edulis and O. denselameUosa were included in
the subgenus Ostrea ss. The species O. liirida was considered as a
junior synonymous of O. conchaphila in the genus Ostreola. Li

and Havenhand ( 1997) have also previously disagreed with Harry
(1985), placing O. angasi as a separate species, very close to O.
edulis.

Our results show greater congruence with molecular phyloge-
netic analyses of the Ostreinae, based on partial mitochondrial 16S
rDNA (Jozefowicz & 6 Foighil 1998) and nuclear 28S rDNA (6
Foighil & Taylor 2000) datasets. This is most evident for O. edulis
and O. angasi. where a sister species relationship for these Euro-
pean and Australian flat oysters is strongly supported by both
karyological and gene tree data. The ostreinid mitochondrial gene
trees place the six karyologically-characterized flat oysters into
two clades: one containing (among other taxa) O. puelchana. O.
conchaphila. and O. denselameUosa. the other composed of O.
edulis. O. angasi. and O. chilensis. With the exception of posi-
tioning of O. chilensis. which in our study is closer to O. dense-
lameUosa, these results are in broad agreement with the topology
generated by our statistical analysis based on Ci and NORs.

All Ostreinae species are of the brooding type with an extended
planktotrophic larval development with the exception of O. chil-
ensis, which shows a greatly abbreviated pelagic phase (Walne
1963). This peculiarity is not reflected at the karyological level.
However, O. puelchana is the only brooding oyster with a distinct
dwarf male and it shows a unique phenomenon of settling the
larvae on an expansion of the anterior shell margin (Pascual et al.
1989). These unique morphologic features could be related to the
karyological isolation of O. puelchana.

ACKNOWLEDGMENT

This work was partially supported by a Ponuguese grant from
the Ministry of Science and Technology (FCTI): SFRH/BPD/
1582-2000. We are grateful to S. Lapegue and D. Cheney for
supplying live oysters. The authors thank S. Sabini and S. Heu-
rtebise for excellent technical assistance, R. Ben Hamadou for
statistical analysis, V. Thiriot for collaboration in Fig. 2, P. Chang
for English editing, and D. 6 Foighil for constructive comments.

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Journal of Shellfish Raeunh. Vol. 21. No. 2, 691-695. 2002.

THE INHERENT EFFICIENCY OF OYSTER DREDGES IN SURVEY MODE

ERIC N. POWELL,'* KATHRYN A. ASHTON-ALCOX,* JOSEPH A. DOBARRO.^
MEAGAN CUMMINGS,' AND SARAH E. BANTA'

' Haskin Shellfish Research Lcihoratoiy, Rutgers University, 6959 Miller Ave.. Port Norris. New Jersey
and 'Rutgers University Marine Field Station. Great Bay Blvd.. Tuckerton. New Jersey 08087

ABSTRACT To develop a quantitative stock assessment for the New Jersey oyster (Cra.v.vo.sv/ca virginica) seed beds, oyster dredge
efficiency was measured on 10 different oyster beds in the Delaware Bay. Depending on size class and location, mean dredge efficiency
for market-size oysters varied from 7.8 to >8,'i'}j-. The sampled beds could be allocated into two groups, one characterized by low dredge
efficiency and the other characterized by high dredge elTiciency. The low-efficiency group. Group 1. had mean dredge efficiencies for
market-size oysters that ranged from 10.9 to 19.5%. The high-efficiency group. Group 2. had mean dredge efficiencies for market-size
oysters that always exceeded 45%. A strong tendency existed for market-size oysters to be captured with higher efficiency than smaller
oysters. In addition, live oysters tended to be captured with higher efficiency than boxes (articulated valves). Although a conclusion
cannot be reached unequivocally, the differential in dredge efficiency observed between Group 1 and Group 2 beds may represent the
difference between dredge efficiencies on beds routinely fished and those not routinely fished. An effect of salinity regime cannot be
excluded as a possible explanation, however. The differential in dredge efficiency between the two bed groups, about a factor of 4.5
for market-size oysters indicates that variations in bed consolidation may have a large influence on dredge efficiency and may
significantly bias estimates of abundance if not taken into account in stock assessments.

KEY WORDS: oyster, dredge efficiency, stock assessment, survey, fishery

INTRODUCTION

Dredges are frequently used survey tools. Knowing the effi-
ciency of the dredge is, therefore, paramount in a quantitative
estimate of stock abundance. Dredge efficiencies have been evalu-
ated for a number of bottom-dwelling commercial species, includ-
ing .scallops. Placopecten magellanicus and Zygochlamys patag-
onica (Giguere and Brulotte 1994, Lasta and Iribame 1997), surf
clams, Spisula solidissiina (NEFSC 2000a), ocean quahogs, Arc-
tica islandica (NEFSC 2000b), and blue crabs, Callinecies sapidiis
(Volstad et al. 2000).

The stock assessment for the New Jersey oyster seed beds uses
a standard 1.27-m oyster dredge (Fegley et al. 1994). Quantifica-
tion of this survey depends upon knowing the efficiency of the
dredge under survey conditions. Oyster dredges are not among the
most efficient of sampling gear. Estimates of dredge efficiency
range between 2 and 32% in survey mode (Chai et al. 1992). As
used by the industry in the normal routine of fishing, the dredge
efficiency consistently falls into the lower portion of this range
(Banta et al. in press).

To develop a quantitative stock assessment for the New Jersey
oyster seed beds, we performed a series of measurements of dredge
efficiency for a standard oyster dredge (Fig. I ). Because previous
estimates had varied over a wide range, we conducted these mea-
surements on a number of oyster beds covering a range of salinities
and degrees of fishery impact to evaluate whether changes in bed
environment and fishing history affect dredge efficiency.

METHODS

Field Program

Dredge efficiency measurements were conducted in summer
1999 and summer 2000 on 10 different oyster beds in the Delaware
Bay. Eight of these were in New Jersey waters and two were in
Delaware waters (Fig. 2).

Normally, three separate experiments were conducted on each
bed. Time constraints limited the number to less than three in

*Corresponding author. E-mail: eric@hsrl.rutgers.edu

several cases. Each experiment was conducted in an identical man-
ner in the following way.

The oyster boat F/V Howard W. Sockwell carried out a l-miii
dredge tow using a standard 24-tooth 1.27-m dredge (Fig. I).
Tooth length was approximately 44 mm, and the mouth opening
was 1.27 m X 31 cm. The bag consisted of 17 rows of 50.8 mm
rings. During the dredge tow, a data logger recorded DGPS posi-
tion and time at 5-s intervals. A second boat, the RA' Zephyrus,
ran immediately parallel but about 5 m off the oyster boat. A buoy
was dropped from the R/V Zephyrus at the point immediately
opposite dredge deployment and another immediately opposite
dredge retrieval as the tow progressed.

The dredge haul was brought on board and u one-bushel sample
taken for analysis. The remainder of the haul was measured volu-
metrically and discarded overboard. A full oyster dredge holds
about 12 bushels of material. Presumably, dredge efficiency de-
clines as this volume is approached. In this study, dredge volumes
exceed 8.5 bushels at only two sites, Bennies and Arnolds, where
dredge volumes routinely fell between 9 and 10 bushels. However,
efficiency estimates tor these sites did not materially diverge from
efficiency estimates for other sites, suggesting that tow distances
were short enough so that dredge capacity did not influence mea-
sured efficiency.

A buoyed 23-m transect line was dropped from the R/V
Zephyrus near the first buoy and the line payed out towards the
second buoy. Limitations in visibility prevented divers from rou-
tinely sampling within the tow path. As a consequence of the need
to maintain a consistent sampling protocol, the diver line was
positioned about 5 m from the dredge path so that all diver samples
were taken near to and in parallel with the tow path. Twelve
collection sacks were affixed at equal intervals along this line.
Divers were then deployed to sample along the transect line using
an 0.5- X 0.5-m quadrat (0.25 m'). A random 0.25-m" sample was
taken at the location where each collection sack was affixed.
Divers attempted to retrieve all of the loose bottom material, in-
cluding all live oysters and boxes (dead, articulated valves). Col-
lection was facilitated by the use of small hand-held scratch rakes.
The divers were instructed to take only the loosely consolidated

691

692

Powell et al.

DELAWARE RIVER

Figure 1. A standard 1.27-m New Jersey oyster dredge.

material on the surface that would normally be taken by the
dredge. In most cases, quantitative retrieval was simple because
the consolidated portion of the bed was near the surface. In a few
cases, unconsolidated shell extended downwards for some dis-
tance. In these cases, the diver took the upper portion of the shell
until the collection sack was filled.

Luhoratory Analyses

Each bushel sample and each diver sample was sorted into live
oysters, bo,\es, and shell and other debris and the respective vol-
umes measured. The longest dimension of each oyster and box >2()
mm was measured. Swept area was calculated for each dredge tow
from the 5-s position logs and the dredge width.

Population abundance (A') is normally related to survey abun-
dance UiJ by a catchability coefficient: A' = qii^. The conversion
requires four parameters: A. the area of the survey; il. the distance
traveled per tow; ir the cross-section sampled by the dredge; and
('. the efficiency of capture by the dredge (NEFSC unpubl.). Thus,
(/ = A/dw Me. Becau.se, in this study, the dredge boat and diver
samples were individually normalized to a 1 nr area {Aldw = 1 ).
catchability q becomes the reciprocal of efficiency e. and was
calculated as follows;

/ ,Z, number of oysters or boxes {diver sample)
number of diver samples * 0.23 m'
'' ( luimber of oysters or boxes (busheiy * # bushels
in' dredge swept area

NEW JERSEY

OVER THE BAR
LOWER MIDDLE

DELAWARE

Figure 2. Location of the 10 oyster beds where dredge efficiency mea-
surements were conducted in survey mode. Banta et al. (In press)
obtained estimates of dredge efficiency under commercial use from
New Beds.

Statistical Analysis

For statistical analyses, we used Spearman's rank correlation
and ANOVA on ranked data. When appropriate, differences within
the ANOVA were resolved with comparisons that used the least
squares means. For some statistical analyses, live oysters and
boxes were split into three size classes: juvenile (20-63.5 mm),
submarket (6-^..'>-76.2 mm), and market (>76.2 inm).

RESULTS

Mean values of q for each seed bed are provided in Table 1.
Values of q for live oysters ranged from 1.54 (an efficiency of
64.9%) to 11,27 (an efficiency of 8,9%). With one exception, all
the size classes of oysters and boxes were correlated with one
another (Table 2), The efficiency of collection of shell debris
(disarticulated shell and shell fragments), however, was much
more rarely correlated with the efficiency of collection of live
oysters or boxes and the correlation coefficients were consistently
lower. Divers had difficulty determining when to stop digging out
shell and shell fragments on some beds, and this uncertainty in
diver collection resulted in xariation in the computed catchability
for debris among diver samples.

Visual observation of Table 1 suggests that the sampled beds
can be divided into two groups, those with relatively high values
of q (low dredge efficiency) and those with relatively low values
of q (high dredge efficiency). This observation was confirmed by
ANOVA analysis comparing the efficiency of collection of mar-

Inherent Efficiency of Oyster Dredges

693

TABLE 1.

Mean values of q (the reciprocal of dredge efficiency! for eacli of the size classes of live oysters and boxes, total live oysters, total boxes,

and debris.

Live

Oysters

Boxes

Oyster Bed

Juveniles

Submarket*

.Markets

Total Live

Juveniles

Submarkets

Markets

Total Box

Debris

Group 1

Arnolds

10.30

2.22

6.46

9.26

10.05

8.81

10.04

9.74

7.47

Cohansey

12.80

7.96

7.81

11.27

17.62

50.54

1 1 .40

18.83

37.23

Over the Bar

9.63

5.88

5.14

7.61

6.81

12.58

5.58

7.33

6.93

Lower Middle

9.33

7.69

6.23

8.80

8.09

7.92

13.40

8.31

15.26

Ship John

10.16

12.06

9.15

10.40

12.79

9.91

13.17

11.88

50.12

Group 2

Shell Rock

4.16

2.84

1.99

3.70

5.37

4.55

2.44

4.71

12.45

Nantuxent Pt.

3.86

1.98

-

3.30

2.32

2.48

-

1.98

6.06

Bennies

2.57

2.31

1.17

2.32

6.70

4.01

5.48

5.58

5.20

New Beds

2.04

3.12

0,64

2.13

2.88

2.08

-

2.71

9.65

Egg Island

-

-

in

1.54

15.41

6.00

20.31

16.99

14.47

Debris includes disarticulated shells and other shell debris. Dash indicates situations where diver sampling did not provide an adequate catch of that
variable to permit an estimate of dredge efficiency.

ket-size oysters between oyster beds (Table 3). Note that, in gen-
eral, the beds Arnolds. Cohansey. Over the Bar. Lower Middle,
and Ship John, hereafter termed Group 1 , are usually significantly
different from the beds Shell Rock. Bennies. New Beds, and Egg
Island, hereafter termed Group 2, in this analysis (Table 3). Mar-
ket-size live oysters were not collected at Nantuxent Point, but
perusal of the remaining data in Table 1 suggests that, had they
been, Nantuxent Point would have fallen within the latter group of
beds.

The average values of q for Groups 1 and 2 are shov\ n in Table
4. With the exception of market-size boxes and debris, the effi-
ciency of collection of live oysters and boxes on Group 1 beds is
significantly lower (a higher q) than the efficiency of collection of
live oysters and boxes on Group 2 beds.

The efficiency of capture of market-size oysters was higher
(lower q) than for submarket-size (P = 0.04) and juvenile oysters
P = 0.003). The latter two were not significantly different. The
averages recorded in Table 4 also suggest that the efficiency of
capture of live oysters is somewhat higher than boxes. In fact,
values of q were significantly lower for all live oysters relative to
all boxes (P = 0.002), live submarket oysters relative to submar-
ket boxes (P = 0.04), and juvenile live oysters relative to juvenile
boxes (P = 0.006). Market-size live oysters and boxes did not
differ significantly, although the mean of the former falls well

below the mean of the latter, especially for Group 2 beds. Accord-
ingly, boxes were collected with a lower efficiency than live oys-
ters overall.

The two groups of beds differ in aveiage salinity. Group I beds
are upbay of Group 2 beds. However, the intensity of fishing also
follows the salinity gradient. Visual inspection of samples showed
that oysters were tnuch more clumped in samples from Group 1
beds as a consequence of the much lower frequency of dredging
that has historically occurred on these beds. Clumping and reef
consolidation might decrease dredge efficiency. If so, a correlation
might exist between the amount of dredging on the bed during the
preceding year and our measurement of dredge efficiency. We
evaluated the significance of dredging using Spearman's rank cor-
relations between the number of bushels taken per bed in 1 999 and
2000 versus the measured value of ^. The number of bushels taken
is a reasonable surrogate for the total swept area of dredging
(Banta et al., in press). All correlations were negative in accor-
dance with the hypothesis that a higher value of q (lower dredge
efficiency) should coincide with lower harvest rates. However,
only the correlation with market-size live oysters was significant.

DISCUSSION

Oyster dredge efficiency varied over a wide range among the
oyster beds sampled in the Delaware Bay. The range measured

TABLE 2.
P-values from Spearman's rank correlations between the efficiency of capture of the various groupings of live oysters, boxes, and debris.

Submarket

Market

Total

Juvenile

Submarket

Market

Total

Live Oysters

Live Oysters

Live Oysters

Boxes

Boxes

Boxes

Boxes

Debris

Juvenile live oysters

0.0013

0.0001

0.0001

0.0001

0.0001

0.0048

(.1.000 1

0.1289

Submarket live oysters

0.0022

0.0005

0.0007

o.ooos

0.0073

0.0002

0.0003

Market live oysters

0.0001

0.0001

0.0001

0,0019

0.0001

0.0167

Total live oysters

0.0001

0.0001

0.0047

0.0001

0. 1 1 28

Juvenile boxes

0.0001

0.0003

0.0001

0.0231

Submarket boxes

0.0030

0.0001

0.0136

Market boxes

0.0001

0.3216

Total boxes

0.0174

694

Powell et al.

TABLE 3.
/"-values from comparisons of least squares means for the efficiency of capture of market-size live oysters among the sampled beds.

Over

Ijower

Ship

Shell

New

Egg

Cohansey

the Bar

Middle

John

Rock

Bennies

Beds

Island

0.5529

0.5360

0.6912

0.2775

Arnolds

0.0310

0.0057

0.0207

0.1967

Cohansey

0.2597

0.3290

0.5651

0.0100

0,0019

0.0093

0.0972

Over the Bar

0.7895

0.1312

0.1478

0.0356

0.0645

0.4315

Lower Middle

0.1.590

0.0645

0.0121

0.0351

0..3017

Ship John

0.0061

0.0014

0.0057

0.0539

Shell Rock

0.3778

0.3680

0.7253

Bennies

0.7784

0.3337

New Beds

0.3111

No market-size oysters were collected at Nantuxent Point, hence this bed is not included in the table. Boxed area delineates the comparisons between
Group 1 and Group 2 beds discussed in the text.

encompasses dredge efficiencies higher than those recorded by
Chai et al. (1992) in the Chesapeake Buy. In that study, dredge
efficiencies varied from 2 to 32%. In this study, depending on size
class and location, mean dredge efficiency for market-size oysters
varied from 7.8 to >85%. Such high efficiencies are achieved when
the dredge is used in survey mode, with short one minute tows that
do not result in the complete filling of the dredge. The oyster
fishery, as it routinely fishes, rarely achieves a dredge efficiency
above 5% (Banta et al., in press) because the tows are longer and
the dredge is routinely full when retrieved.

The range of efficiencies measured is large. Extreme values,
whether high or low, probably are due to patchiness in the sampled
area. Diver samples were not taken from the dredge tow path, but
rather along a transect run parallel and close to the dredge tow
path. Nevertheless, the sampled beds could readily be allocated
into two groups, one characterized by low dredge efficiency and
the other characterized by high dredge efficiency. The low-
efficiency group, Group 1 , had mean dredge efficiencies for mar-
ket-size oysters that ranged from 10.9 to 19.5%. The high-
efficiency group had mean dredge efficiencies for market-size oys-
ters that always exceeded 45%.

A strong tendency existed for market-size oysters to be cap-
tured with higher efficiency than smaller oysters. Presumably, a
greater tendency exists for the smaller oysters to pass between the
dredge teeth or through the rings of the collection bag and, thus,
not be collected. Dredge efficiencies were particularly low for
juveniles, many of which may be attached to smaller pieces of
shell that are poorly sampled. Very likely, dredge samples rou-
tinely result in a significant bias against juveniles. In this study.

market-size oysters were captured with about twice the efficiency
of juveniles.

In addition, live oysters tended to be captured with higher
efficiency than boxes. The difference was highly significant, par-
ticularly in Group 2 beds where boxes tended to be captured with
an efficiency of about one-third the efficiency of living oysters.
Two possible reasons exist for the lower capture efficiency of
boxes: I ) collection by dredge may result in disarticulation. This
possibility is not supported by experiments designed to evaluate
this source of disarticulation, however (Powell et al. 2001); 2)
some boxes taken by divers may be deeper in the reef than the
dredge normally samples. Efficiency of collection of debris was
significantly correlated with efficiency of collection of total boxes
in accordance with this hypothesis (Table 2); however, the effi-
ciency of collection of debris was also correlated with some live
oyster variables. Thus, a conclusive explanation for the variation in
efficiency of capture between boxes and live oysters is not pro-
vided by the present analyses.

Box counts are routinely used as a method to estimate mortality
rates in shellfish populations (Merrill and Posgay 1964, Fegley et
al. 1994, Christmas et al. 1997). The differential in dredge effi-
ciency measured in this study could result in a significant bias in
the live:dead ratio and a significant underestimation of mortality
rate from box counts if diver collections are unbiased. On the other
hand, Christmas et al. (1997) observed that disarticulation often
took more than one year, whereas mortality estimates from box
counts usually assume that boxes were produced in the current
year. This bias would tend to counterweigh the potential bias im-
posed by dredge efficiency. However, Powell et al. (2001) ob-

TABLE 4.

Mean values of q (the reciprocal of dredge efficiency) for each of the size classes of live oysters and boxes, total live oysters, total boxes, and

debris for two groups of beds.

Live

Oysl

ers

Boxes

Oyster Bed

Juveniles

Subniarkets

Markets

Total Live

Juveniles

Subniarkets

Markets

Total Box

Debris

Group 1
Group 2
P value

10.46
3.33
0.0009

6.89

2.57
0.04

6.93
1.54
0.0001

9.40
2.83
0.0002

11.26
6.78
0.04

18.98
4.03
0.0008

11.00
8.85
NS

11.47
6.50
0.02

21.49
9.55

NS

Group 1 contains Arnolds. Cohansey. Over the Bar, Lower Middle, and Ship John. Group 2 contains Bennies. Shell Rock, Nantuxent Point, Egg Island,
and New Beds. Debris includes cultch and other debris. P values record the results of ANOVA analysis comparing the two groups with respect to the
variable listed as the column heading. NS. not significant (a = 0.05).

Inherent Efficiency of Oyster Dredges

695

served that disarticulation rates might be higher in the Delaware
Bay than observed in Chesapeake Bay by Christmas et al. ( 1997).
Consequently, the potentially offsetting biases in the interpretation
of box counts cannot, as yet, be fully evaluated.

Why Group 1 beds yielded such low dredge efficiencies in
comparison to Group 2 beds cannot unequivocally be identified.
Group 1 beds are all upbay of Group 2 and, thus, exist at lower
average salinities. Unfortunately, Group I beds also have been
impacted less over the long term by dredging because effort in the
Delaware Bay oyster industry also follows the salinity gradient,
with lower effort on the lowest salinity beds (HSRL 2000, Fegley
et al. 1994. Banta et al., in press). We attempted to assess the
influence of dredging using catch data for 1999 and 2000, without
much success. However, whether the catch data for the year before
sampling is the correct estimator of the effect of dredging is ques-
tionable. Visual observation, for example, shows that oyster
clumps are larger and contain more living oysters on these upbay
beds. These clumps, very likely, are more firmly attached to the
underlying bed than are the oysters on Group 2 beds. Greater bed
consolidation on the upbay beds (Group I) would reduce the ef-
fectiveness of the teeth in scraping shell material up into the
dredge and, consequently, reduce dredge efficiency.

Oyster catchers normally report that catch rates are lower on
beds that have not been fished for a time and that catch rates

improve after repeated dredging over a few days. Very likely, this
repeated dredging breaks the bottom up and results in a substantial
increase in the efficiency of capture. Although the conclusion can-
not be reached unequivocally, it seems most likely that the differ-
ential observed between Group 1 and Group 2 beds represents the
difference between dredge efficiencies on beds routinely fished
and those not routinely fished. The differential is large, about a
factor of 4.5 for market-size oysters, indicating that variations in
bed consolidation may have a large influence on dredge efficiency
and may significantly bias estimates of abundance if not taken into
account in stock assessments.

ACKNOWLEDGMENTS

Special thanks to Larry Hickman, Captain of the FA^ Howard
W. Sockwell and to Bivalve Packing for providing the F/V How-
ard W. Sockwell and logistical support for this study. Special
thanks also to Royce Reed, Captain of the RA' Zephyrus and to the
New Jersey Department of Environmental Protection for providing
vessel support for the dive team and help in sample collection. We
particularly thank the dive team. Jack Keith. Dave Andrews. Geoff
Graham, Bill Dixon, and Jessica Vanisko, who collected the 324
diver samples that constituted the quantitative collections for this
study.

LITERATURE CITED

Banta, S. E., E. N. Powell & K. A. Ashton-Alcox. in press. Evaluation of
dredging behavior by the Delaware Bay oyster fishery. North Am. J.
Fisheries Management.

Chai, A-L., M. Homer. C-F. Tsui & P. Goulletquer. 1992. Evaluation of
oyster sampling efficiency of patent tongs and an oyster dredge. North
Am. J. Fisheries Management 12:825-832.

Christmas, J. P., M. R. McGinty, D. A. Randle, G. F. Smith, & S. J. Jordan.
1997. Oyster shell disarticulation in three Chesapeake Bay tributaries.
J. Shellfish Res. 16:115-123.

Fegley, S. R., S. E. Ford, J.N. Kraeuter, & D. R. Jones. 1994. Relative
effects of harvest pressure and disease mortality on the population
dynamics of the Eastern oyster [Crassostrea virginica) in Delaware
Bay. Final Report # NA26FL0588 to the National Oceanic and Atmo-
spheric Administration, Rutgers University, Haskin Shellfish Research
Laboratory.

Giguere, M. & S. Brulotte. 1994, Comparison of sampling techniques,
video and dredge, in estimating sea scallop iPUicopecten mageltaniciis.
Gmelin) populations. / Shellfish Res. 13:25-30.

Lasta, M. L. & O. O. Iribame. 1997. Southwestern Atlantic scallop (Zy-
gochlamys patagonica) fishery: assessment of gear efficiency through
a depletion experiment. J. Shellfish Res. 16:59-62.

Merrill, A. S. & J. A. Posgay. 1964. Estimating the natural mortality rate of

the sea scallop {Placopecten magellanicus). ICNAF Res. Bull. 1:88-
106.

NEFSC. 2000a. 30"' northeast regional stock assessment workshop (30'"
SAW): Stock assessment review committee (SARC) consensus sum-
mary of assessments. Northeast Fisheries Science Center Reference
Document 00-03. 477 pp.

NEFSC. 2000b. 31"' northeast regional stock as.sessment workshop (31"'
SAW): Stock assessment review committee (SARC) consensus sum-
mary of assessments. Northeast Fisheries Science Center Reference
Document 00-15. 400 pp.

NEFSC. unpubl. 34"' northeast regional stock assessment workshop (34'''
SAW): Stock assessment review committee (SARC) consensus sum-
mary of assessments. Northeast Fisheries Science Center Reference
Document 02-06. 346 pp.

Powell, E. N., K. A. Ashton-AIcox. S. E. Banta & A, J. Bonner. 2001.
Impact of repeated dredging on a Delaware Bay oyster reef / Shellfish
Res. 20:961-975.

V0lstad, J. H., A. F. Sharov, G. Davis & B. Davis. 2000. A method for
estimating dredge catching efficiency for blue crabs, Callinectes sapi-
diis. in Chesapeake Bay. US Fish Wildlife Service Fisheries Bull. 98:
4I0-f20.

Joiinwl of Shellfish Research, Vol. 21. No. 2. 697-705, 2002.

NOVEL REPEAT ELEMENTS IN THE NUCLEAR RIBOSOMAL RNA OPERON OF THE FLAT
OYSTERS OSTREA EDULIS C. LINNAEUS, 1758 AND O. ANGASI SOWERBY, 1871

E. KENCHINGTON,'* C. J. BIRD," J. OSBORNE," AND M. REITH"

^ Department of Fisheries & Oceans. Bedford Institute of Oceanography, 1 Challenger Drive. P.O. Bo.x
1006. Dartmouth. NS. B2Y 4A2. Canada; 'Institute for Marine Biosciences. National Research Council
of Canada. Nil Oxford Street. Halifax. NS. B3H 3ZI. Canada

ABSTRACT The nucleotide sequence of the ribosomal RNA operon. from the 18S rRNA gene through ITS-1. 5.8S rRNA gene, and
ITS-2, wa.s determined for single clones of the flat oysters Ostrea edulis from Europe and O. angasi from Australia. The genie regions,
the 18S and 5.8S rDNAs, were Identical between the two species and displayed a high degree of similarity with available sequence
from Crassostrea gigas, C. nippona, Saccostrea commercialis. and 5. cucullala. In lTS-1. the Ostrea species had a characteristic repeat
region, with subrepeats of 54 base-pairs (bp) (a) flanking a 27-bp fragment {b). where h remained constant while a changed by 1 bp
through successive Iterations ia' . a"). In O. edulis. the pattern was a. b, a', b. a", whereas In O. angasi It was a. b. a". Apart from the
difference in repeats, the ITS-1 of the two Ostrea species was identical except for a single substitution and two 2-bp Insertions/
deletions. lTS-2 was Identical between the two Ostrea species except for a single substitution and a 3-bp insertion/deletion, and
contained a neariy complete repeat of a near the 5' end. These ITS sequences were markedly dissimilar to available sequence from the
other oyster species examined, and the repeat region was unique to Ostrea. with possible functionality In transcription. On the grounds
of sequence similarity. It Is suggested that O. edulis and O. angasi are conspeclfic.

KEY WORDS: rDNA sequence, ITS. Oslrea

INTRODUCTION

The taxonomy and systematics of oysters is far from being
resolved (Carriker & Gaffney 1996), and confusion over the rec-
ognition of species persists, although the recent application of
genetic techniques has been very successful in providing valuable
characters to address phylogenetic relationships of the major spe-
cies groups and families (Buroker et al. 1979a, Buroker et al.
1979b, Brock 1990, Banks et al. 1993. Anderson & Adlard 1994,
Littlewood 1994, Boudry et al. 1998, Jozefowicz & 6 Foighil
1998, 6 Foighil et al. 1999, 6 Foighil & Taylor 2000). Among the
remaining contentious issues is the question of the phylogenetic
relationship between Ostrea edulis Linne, 1 758, the European flat
oyster, and O. angasi Sowerby, 1871, the Australian flat oyster.

In the most recent comprehensive revision of the living oysters,
Harry ( 1 985 ) reduced O. angasi to a synonym of the Southern
Hemisphere species O. piielcluina D'Orbigny, 1841, placing it in
a different subgenus from O. edulis based on larval morphological
and developmental characters. However, this classification has not
been universally accepted (e.g., Jozefowicz & O Foighil 1998),
and further data have since become available to suggest a closer
relationship between O. angasi and O. edulis than had been sup-
posed. Comparison of karyotypes, nucleolus organizer regions,
and constitutive heterochromatin from chromosome preparations
of O. angasi and O. edulis shows a very high degree of similarity,
which is not seen between O. angasi and its supposed Southern
Hemisphere conspecifics O. puelchana and O. chilensis Philippi,
1845, or between O. angasi and the Indo-West Pacific O. dense-
lamellosa Lischke, 1869 (Li & Havenhand 1997). Partial mito-
chondrial 16S rRNA gene sequences position O. edulis and O.
angasi in a well-supported clade with a low degree of DNA se-
quence divergence relative to the other conspecifics (Jozefowicz &
O Foighil 1998). These results are also supported by partial 28S
rRNA gene sequences (O Foighil & Taylor 2000). Furthermore,
the concept of O, puelchana (previously thought to be native to

*Corresponding author. E-mail; Kenchlngtone@mar.dfo-mpo.gc.ca

Argentina) as a single circumglobal species in the Southern Hemi-
sphere has been convincingly challenged by partial mitochondrial
16S rRNA (Jozefowicz & 6 Foighil 1998) and 28S rRNA gene
sequences (O Foighil & Taylor 2000). These data support resur-
rection of the regional taxa in the southern hemisphere, namely O.
chilensis in Chile and New Zealand, O. angasi in Australia and O.
algoensis Sowerby. 1871 in South Africa.

To date, genetic information on the living oysters has focused
on the rRNA gene (rDNA) array. In the Bivalvia, and in many
other taxa, the rRNA genes have been useful in inferring phylo-
genetic and taxonomic relationships (e.g.. Field et al. 1988, Ghis-
elin 1988, Nakamura 1989, Rice et al. 1993, Kenchington et al.
1994. Kenchington et al. 1995, Sterner & Muller 1996. Campbell
et al. 1998, Frischer et al. 1998). These genes are co-transcribed,
producing a single transcript consisting of the 18S, 5.8S. and 28S
rRNA coding regions (or their homologues), separated by two
internal transcribed spacers (ITS-! and ITS-2) with an external
transcribed spacer (ETS) located upstream of the 18S rRNA gene
(Fig. 1). The transcribed precursor rRNAs are separated by inter-
genic spacers (IGS or NTS) consisting of transcribed and non-
transcribed spacer sequence. This rRNA cistron is tandemly re-
peated head to tail at one or more chromosomal sites termed
nucleolus organizer regions (NORs) (Hadjiolov 1985). The pri-
mary transcript is cleaved via a series of processing reactions into
the 18S. 5.8S and 28S rDNAs, which along with approximately 85
ribosomal proteins and 5S rDNA, form the 40S and 60S pre-
ribosomal subunits (Hadjiolov 1985).

Regions of the rRNA cistron are under varying degrees of
functional constraint and therefore provide a wide range of phy-
logenetic resolution. The IGS, a noncoding region, is under the
least selective constraint and is therefore expected to show the
greatest sequence divergence, rendering it useful for intraspecific
comparisons. The ITS regions are also rapidly evolving, but to a
lesser degree than the IGS. In contrast, the coding region of the
rRNA repeat unit, that is. the I8S. 5.8S. and 28S genes, is highly
conserved within and among species both in sequence and in sec-
ondary structure, with the 18S being the most conservative over a

697

698

Kenchington et al.

ITS-2

ETS 18S

5.8S

28S

ribosomal RNAs

transcribed regions; 40 ribosomal proteins

Figure 1. A schematic of the ribosomal RNA (rRNAl cistron indicat-
ing the genie regions in black and the noneoding regions in white.

broad taxonomic range (Appels & Honeycutt 1986). However,
within the IBS rRNA gene, there are both conserved and variable
regions associated with its secondary structure. Although some
regions of this gene are conserved across all eukaryotes. other
regions (loops) can be highly variable. Therefore, it has been sug-
gested that this gene is valuable for phylogenetic investigations at
a variety of taxonomic levels (Sogin et al. 1986). particularly when
the time since divergence of the taxa is not known. A similar
argument has been made for the much larger 28S rRNA gene
(Hillis & Dixon 1991).

Here, we examine the nuclear rRNA cistron for sequence dif-
ferences in the 18S. ITS-1, 5.8S. and ITS-2 regions to provide
additional support for the level of differentiation between O. an-
gasi and O. edulis. In the course of our studies, novel repeat
elements were identified in the ITS which are described and dis-
cussed in context of their possible evolution.

MATERIALS AND METHODS

Sample Location

Specimens of O. angasi were collected from Cloudy Bay La-
goon, south end of Brany Island. Tasmania. Australia on June 23.
1993 by Dr. John Thomson and shipped alive to our laboratory.
Specimens of O. edulis were obtained from Department of Fish-
eries and Oceans research stock, originally imported from Europe
via the United States to Canada during the late I97()s and used as
broodstock at a hatchery in Port Medway, Nova Scotia. Animals
were dissected and voucher specimens of the shells were archived
at the Bedford Institute of Oceanography. Dartmouth, Nova Scotia.

DNA Extraction and Purification

High-molecular-weight DNA was extracted from the adductor
muscle of a single animal of each species according to the protocol
of Rice and Bird ( 1990). Briefly, tissue was ground in liquid ni-
trogen, proteins were removed by mixing with an equal volume of
phenol:chloroform:isoamyl alcohol, followed by a final wash with
an equal volume of chloroform:isoamyl alcohol to remove the
phenol residues, and DNA was precipitated with sodium acetate in
cold ethanol. The DNA was further purifled on a cesium chloride
gradient by high-speed centrifugation.

DNA Amplification

The 18S rRNA gene, ITS-1, 5.8S rDNA. and ITS-2 regions of
the rRNA cistron were amplified as two fragments, one approxi-
mately 1825 base pairs (bp) and corresponding to the 18S rRNA
gene, and the second approxnnately 1220 bp and containing the
ITS spacers and 5.8S sequences. Synthetic oligonucleotides cor-

responding to conserved regions of the 18S rRNA gene and ap-
pended to restriction endonuclease sites were used as amplification
primers for the first fragment (Bird et al. 1992). Deoxy-uracil
monophosphate (dUMP) primers, with four trinucleotide repeat at
the 5' ends and complementary to the coding strand of the 18S
(dUMP 18SF: 5'-CALICAUCAUCAUGGTTTCCGTAGGTGAA-
CCTGC-3') and the 28S rDNAs (dUMP 28SR: 5'-CUACUACU-
ACUAATATGCTTAAGTTCAGCGGGT-3'). were used to am-
plify the second fragment. The change in protocol to the dUMP
primers was purely a matter of convenience because these primers
were available in our laboratory at the time for another project (i.e..
Patwary et al. 1998). Amplification was performed in a Perkin-
Elmer (Irvine, CA) DNA Thermal Cycler programmed for an ini-
tial cycle of 5 min. at 94°C followed by 30 cycles of 2 min at 37°C.
3 min at 72°C, and 1 min at 94'C. plus a final cycle of annealing
for 2 min at 37°C and one of extension for 5 min at 72"C. The
same amplification conditions were used for each of the primer
pairs and a negative control (no DNA template) was included in
each run.

Cloning Polymerase Chain Reaction Products

Amplified DNA containing the 18S rRNA gene was ligated
directly into the plasmid sequencing vector pCR 1000 using the
TA Cloning'"''^ kit, following manufacturer's instructions (Invitro-
gen Corp.. Carisbad, CA). Recombinant plasmids were maintained
in the OneShot'^' Escherichia coli INVaF' cells provided in the
kit. Transformants were grown on YT agar plates supplemented
with kanamycin (100 p-g mL"') and were detected by blue/white
selection in the presence of X-gal and IPTG (Gold Biotechnology,
St. Louis. MO).

Amplification products for the second fragment containing the
ITS spacers and 5. 88 gene generated with the dUMP primers were
purified using the Geneclean If" elution kit (BIO/CAN Scientific.
Mississauga. ON). The purified DNA was directionally cloned into
Epicurean competent cells (Stratagene. La Jolla. CA) using the
CLONEAMP"' System (Life Technologies, Rockville, MD) for
rapid cloning of amplification products.

The size of the inserts was screened by protoplasting (Sekar
1987), followed by digestion with Noll (New England Biosystems,
Mississauga, ON) and Kpn\ (Pharmacia, Peapack, NJ). A single
colony was inoculated into 2xYT medium, following the protocols
for a large-scale plasmid preparation (Maniatis et al. 1982).

DNA Sequencing and Alignment

Single clones of purified plasmid DNA containing the I8S
rRNA gene were subjected to dideoxy sequencing according to the
USB Sequenase version 2.0 protocol (USB Corporation, Cleve-
land, OH). Amplification primers plus the oligonucleotide primers
described in Bird et al. ( 1992) were used to sequence the complete
I8S rRNA gene in both directions. dITP was substituted for dGTP
in the sequencing protocol to resolve compressions caused by sec-
ondary structure.

Purified DNA containing the ITS spacers and 5.8S rRNA gene
was sequenced on an ABI 373 Automated DNA Sequencer, using
the PRISM '^' Dye Terminator Cycle Sequencing Kit (PE/ABI;
Applied Biosystems. Foster City. CA) according to the manufac-
turer's instructions. M13 universal forward (5'-GTAAAACGA-
CGGCCAGT-3') and reverse (5'-TTCACACAGGAAACAG-3')
primers (LISB) were used for initial sequencing. The DNA was
sequenced completely in both directions by using a set of internal

Repeat Elements in Ostrea ITS Sequence

699

18S (end)

Fl

ITSl

F2

F3

F4

5.8S

F5

F6

ITS2

F7

F8

ITS I

Rl

R2

R3

5.8S

R4

R5

1TS2

R6

R7

TABLE 1.

Forward (F) and Reverse (R) Primers used to sequence the ITS-1,
5.8S, and ITS-2 regions of the rRNA cistron.

-GGTTTCCGTAGGTG AACC-3 '
-CTCCGTCGCCTTAAATGC-3'
-GATTCCGAGCTCACGGAC-3'
-CGTTTCACGTACATCAATAGT-3'
' -TCTAAGTGGTGG ATC ACTCG-3 '
■CATTGAACATCGACATCTTG-3'
-CTCCGTCGCCTTAAATGC-3' (m repeat </)
-CAAGTACGGACGGCGACG-3'
■CGCCGTCGGGA AG ACGG-3 '
-GCTCGGAATCACCGA A AGT-3 '
-ACTATTGATGTACGTGAAACG-3'
■CGAGTGATCCACCACTTAGA-3 '
'-ACGCACCTGGCTGCGCTC-3'
-G ATTAGTGTTGTACG AGGCG-3 '
-GCCATCCGTCCGAGCAGACG-3'

primers that overlapped one another, providing at least 4 and up to
8 sequence streams from separate reactions tor most regions of the
fragment (Table 1 ). A consensus sequence from these reactions
was produced via the software program Sequencher''^^' (Gene
Codes Inc., Ann Arbor, MI).

Sequences were aligned with CLUSTALW (Thompson et al.
1994) and then checked manually. Repeat regions were identified
by visual inspection, and a BLAST search (Altschul et al. 1997)
was performed on these elements. Our data were compared with
sequences downloaded from GenBank: Ostrea ediilis 18S rDNA
(Accession No. U88709); Saccostrea conunercialis Iredale &
Roughley. 1933, ITS-1, 5.8S rDNA (L28702); Crassostrea gigas
Thunberg, 1793, ITS-1, 5.8S rDNA, ITS-2 (AF280609.
AF280610): C. nippona Seki, 1934, ITS-1, 5.8S rDNA
(AB041760); C. cuciillata G.P. Deshayes, 1836, 18S (AJ389634).

RESULTS

Ostrea edulis Versus O. angasi

Nucleotide sequences of the rRNA operon, from the 18S rDNA
through ITS-2, were similar in Ostrea edulis and O. angasi. Both

the 18S (1823 bp. not shown) and 5.8S (139 bp. Fig. 2) rRNA
genes were identical in their entirety. The ITS sequences were also
very similar except for a large (81 bp) insertion/deletion in ITS-1
(Fig. 3), so that this sequence was 546 bp in O. edulis and 465 bp
in O. angasi. Otherwise, ITS-1 differed between the species by a
single substitution and two short (2-bp) insertions/deletions, while
ITS-2 differed by only a single substitution and a 3-bp insertion/
deletion, for a length of 476 bp in O. edulis and 479 bp in O.
angasi (Fig. 4).

Further inspection of the ITS-1 sequence revealed the presence
of three distinct regions (Fig. 3): (1) a short 5' end of approxi-
mately 1 10 bp, which contained the single substitution and a 2-bp
insertion/deletion; (2) a tract of repeated elements which differed
in their number and arrangement between the species but were
otherwise nearly identical in sequence; and (3) a post-repeat frag-
ment of approximately 225 bp which had the second 2-bp inser-
tion/deletion. The repealed elements were composed of two sub-
repeats (a = 54 nucleotides and b = 21 nucleotides) that occurred
in tandem, with /; always flanked on both sides by a (Fig. 4).
Whereas the /' sequences were conserved within and between re-
peats and between species, the a sequences of O. edulis showed a
point mutation between a and a' . and a second mutation between
a' and a". In O. angasi. a' and the second b were missing, and the
single /' sequence was flanked at its 3' end by a" (Fig. 3). In O.
edulis. the repeat sequence formed the pattern a. b. a', b. a".
whereas in O. angasi it took the form of a, b, a".

A 49-nucleotide portion of a also appeared in the ITS-2 se-
quences near the 5' end (Fig. 4). This element was missing 3 bp at
the 5' terminus and 2 bp at the 3' terminus, but otherwise differed
froin (( in only 2 bp. plus a third bp in O. angasi which represented
the only point mutation in ITS-2 between the species.

Comparison with Other Oyster Species

Both the 18S and 5.8S rRNA genes displayed considerable
similarity with those of other oysters of the genera Crassostrea and
Saccostrea. Our O. edulislangasi 18S rDNA sequence differed
from Cras.wstrea gigas (GenBank AB()64942) at 43 sites over
1824 bps for 97. 6*^ similarity, and from Saccostrea cucullata
(GenBank AJ389634) by 49 sites over 1755 bp for 96.5% simi-
larity. However, another sequence of O. edulis 18S rDNA from

0. edulis /angasi CAACTCTAAGTGGTGGATCACTCGGCTCGGGGGTCGATGAAGAGCGCAGCCAGCTGCGTG 60

C. gigas CAACTCTAAGTGGTGGATCACTCGGCTCGGGGGTCGATGAAGAACGCAGCCAGCTGCGTG 60

C. nippona CAACTCTAAGTGGTGGATCACTCGGCTCGGGGGTCGATGAAGAGCGCAGCCAGCTGCGTG 60

Saccostrea CAACTCTAAGTGGTGGATCACTCGGCTCGGGGGTCGATGAAGAGCGCAGCCAGCTGCGTG 60

********************^

r**********1

r******* ****************

0. edulis/angasi AATTAATGTGAATTGCAGGACACATTGAACATCGACATCTTGAACGCACATGGCGGCCTT 120
C. gigas AATTAATGTGAATTGCAGGACACATTGAACATCGACATCTTGAACGCACATGGCGGCCT- 119
C. nippona AATTAATGTGAATTGCAGGACACATTGAACATCGACACCTT 101

Saccostrea AATTAATGTGAATTGCAGGACACATTGAA 89

************************************* *********************

0. edulis /angasi CGGGTAACTCCCGAGGCTCACGTCTGTCTGAGGGTCGGC 159

C. gigas CGGGTAACTCCCGAGGC-CACGTCTGTCTGAGGGTCGGT 157

C. nippona

Saccos t rea

***************** ********************

Figure 2. Nucleotide sequence of the 5.8S rRNA gene from the oysters Ostrea edulis, O. angasi, Crassostrea gigas (GenBanl< Accession Numbers
Af280609, AF2806I0), C. nippona (GenBanIi Ab()41760l, and Saccostrea (GenBanl< L28702). Asterisks identify points of identical sequence
among the available data. Hyphens indicate points of nucleotide deletion/insertion and incomplete sequence at the 3' end in C. nippona and
Saccostrea. Numbers refer to the number of nucleotides from the 5' end.

700 Kenchington et al.

O.edulis ATTAACAAAACAAGGAAACAACCAAGGTCGTTGAGACGTGT--CTCTCTCGTCATAGCCC 58

o'. angasi ATTAACAAAACAAGGAAACCACCAAGGTCGTTGAGACGTGTGTCTCTCTCGTCATAGCCC 60

***♦***<■*********♦* ♦*♦*♦*♦*♦*******♦***♦ *♦**♦****♦**♦**♦*

0. edulis GCGACCAGGTCGTGTGGAACGGGCGAGAGAAGGCCAATCGCCGTCCCCGCGGCGCCTTGG 118

O. angasi GCGACCAGGTCGTGTGGAACGGGCGAGAGAAGGCCAATCGCCGTCCCCGCGGCGCCTTGG 12 0

:***■* + ********************■******■***********■*"*

**********

a b

o.edulis GCCGTCGTAGCCGAACAGGCTCCGTCGCCTTAAATACAGACGA|aTGGGAAACTCGTCGCC| 17 8
O. angasi GCCGTCGTAGCCGAACAGGCTCCGTCGCCTTAAATACAGACGAATGGGAAACTCGTCGCC 180

*******■*■■*■*■*********** + ** + ***■*■****■*

r + **** + **************

a'

o.edulis |gtcttcccga|cggcgccttgggcagtcgtagccgaacaggctccgtcgccttaaatacag 23

0. angasi GTCTTCCCGA

190

b a"

o.edulis acgaIatgggaaactcgtcgccgtcttcccgaIcggcgccttgggcagtcgtagccgaacag 238

O. angasi CGGCGCCTTGGGCAGTCGTAGCCGAACAG 219

***+****+***********+i

0 edulis GCTCCGTCGCCTCAAATACAGACGAGCTCGACTTTCGGTGATTCCGAGCTCACGGACACG 3 58

0. angasi GCTCCGTCGCCTCAAATACAGACGAGCTCGACTTTCGGTGATTCCGAGCTCACGGACACG 279

0. edulis GCATTTGGTCACCGAATATGACGCCAGGCATCGGCGGTCGTGGGGCCGACGTTATAAGCA 418

0 . angasi GC ATTTGGTCACCGAATATGACGCCAGGCATCGGCGGTCGTGGGGCCGACGTTATAAGCA 3 3 9

r*** + *********-il

.J.*^***********************************!

O. edulis CCCTGGTATGAACGTTTCACGTACATCAATAGTTTTGATCTTGGCGCGGGGGGAAAAACA 478

0. angasi CCCTGGTATGAACGTTTCACGTACATCAATAGTTTTGATCTTGGCGCGGGGGGAAA--CA 397

o.edulis CTCCTCGCGGTCGCGTCCGGCGTTCTTGTTGCGCGCCGAGACCGGCCGAATTTGGCATCT 53 8

0. angasi CTCCTCGCGGTCGCGTCCGGCGTTCTTGTTGCGCGCCGAGACCGGCCGAATTTGGCATCT 457

** + ** + ******■*********

►******************♦******+*****++*****

O.edulis CTTTTTGA 546
O. angasi CTTTTTGA 465

r * * * *

Figure \ Nucleotide sequence of tiie ITS-1 region from the oysters Ostrea edulis and O. auaasi. Asterisks identif> points of identical sequence
and iiyphens indicate points of sequence deletion. Numbers refer to the number of nucelolides from the 5' end. The 54-bp repetitive element (a)
is indicated in the O. edulis sequence by shading, and nucleotide changes in its second («') and third (a"» iterations are underline: the 24-bp
subrepeat (/)) is outlined.

GenBank (U88709). differed from our O. edulislangasi sequence because of numerous insertions downstream from the repeat region

in If) sites and was identical with the other oysters al 8 of those (Fig. 5). For ITS-2. both Ostrea sequences were shorter than that

sjtes. of Crassostrea gigiis. which was 61 1 bp (Fig. 4). Overall sequence

Similarly, the 159-bp 5.8S iRNA genes of O. edulisi angasi similarity of the ITS-2 region between the Ostrea species and C.

showed a high degree of similarity with the Crassostrea gigas gigas was on the order of 82%: conserved regions at the 5' and 3'

sequence, which differed at only four positions (Fig. 2; 97.5% ends were identified. In C. gigas. the area corresponding to a

similarity). Partial sequence from C nippona and Saccostrea com- contained six differences from a apart from the absent terminal

merciatis was also nearly identical with that of O. edulislangasi. nucleotides, including only one of the two point mutations in the

By contrast, considerable variability was evident among the O. edulis sequence. A BLAST search (Altschul et al. 1997) on the

spacer sequences of the oyster genera (Figs. 4 and 5). None of the repeat elements could not identify similar sequence in other or-

other ITS-1 sequences showed repeat elements, and similarity was ganisms.
low in the region corresponding to the a and /' elements. However.

the sequences were more conservative in an area coincident with DISCUSSION
the a" repetitive element in O. edulis and O. angasi. Lacking

repeats, the sequences from Cra.^sostrea and Saccostrea were Nuclear ribosomal sequences are often assumed to be homog-

shorter (445-520 bp) than in O. edulis (546 bp). although the enized within individuals and populations ot a species through

length of the C. nippona sequence approached that of O. edulis concerted evolution processes (Hillis & Davis 1988). Homogem-

Repeat Elements in Ostrea ITS Sequence

701

O.edulis GAAACTATCCATCGGACA CCTGGGTTTGTCCGCCTTGGGCCGTCGTAGCCGAAA 54

O.angasi GAAACTATCCATCGGACA CCTGGGTTTGTCCGCCTTGGGGCGTCGTAGCCGAAA 54

C.gigas GAAACTATCAATCGACTAAAATTTCATTATTCTAGTCGCCTTGGGCCGTCGAAGCCTCTC 6 0

*********

r * *

r****** ***** ****

0 . eduli s A-GGCTCCGTCGCCTTAAATGCAGACCGACGCCC GCTCGA 93

O . angasi A-GGCTCCGTCGCCTTAAATGCAGACCGACGCCC GCTCGA 93

C. gigas ACGGCTCCGTCGCCTTAAATGCAGACCGATGACCAAAGAAACGGCTCTATTGGTGACTCG 120

* *■■*■■*■■*■* + *■*■****■*■ + *■* + ***■*■** + * + + * **

t * + *

0. edulis GAGCGTCGCCGG-TCTCACCGCCTATGGGTTTCC CCCACGGTAGAAGGCTT--CTCG 147

O.angasi GAGCGTCGCCGG-TCTCACCGCCTATGGGTTTCC CCCACGGTAGAAGGCTT--CTCG 147

C.gigas GTGCCTCGGCGGGACGAAACACGTCTCGACTTCCACTCTTTTCGCTAAACTCTTATCCCG 180

0. edulis AACGTCTGCTCGGACGGATGGCGA- -GAGGAT-CGGGG AGGGCGTTGA CG 194

O . angasi AACGTCTGCTCGGACGGATGGCGA- -GAGGAT-CGGGG AGGGCGTTGA CG 194

C. gigas AACGGCTGCTCGGTCGGACGGCGAAGGAGGACGCGGGGTGCGAGGGCGCAGAAAAAGACG 240

+ **■*■ ******** **** ***** ***** ***** ****** ** **

0 . edulis GCAACGCGGTCTGGTCT TGACGACCGA ACCGA 22 6

O. angasi GCAACGCGGTCTGGTCT TGACGACCGA ACCGA 22 6

C. gigas GCAACGCGGTCTGGCCACATACGCGAAGCTCCGGCTATAGCGGCGGGTCTCGACCATCAA 3 00

************** * * ** * * * * *

0. edulis GAGCGTGCCCGAAGCCC CTTACACCAAACT CGAACCTCGCCATTCGCTTT 276

O.angasi GAGCGTGCCCGAAGCCC CTTACACCAAACT CGAACCTCGCCATTCGCTTT 27 6

C. gigas AAGCGTGCCCGAAAGTGATGCAAACTCGCAACAAACTTCCCCGGACTCTCCAT-CGCTTT 3 59

************ ** ** ****** * *** **** ******

O . eduli s GTCCTTGC - -GGTGCAAGTACGGACGGCGACG TACCGAGG GGGGAGCCGGGG 32 6

O. angasi GTCCTTGC --GGTGCAAGTACGGACGGCGACG TACCGAGGAGGGGGGAGCCGGGG 329

C.gigas GGCCTTGCCTGGCCTTAGTACGGATAACAAAGGCGGTTAAAGAAGGAGATCGTTTCGCTG 419

* ****** ** ******** * * * ** ** *

* + *

O . edul i s CGGA TCGAGAACGGC GCAAGTTGGAGC AGGTGTCGCGAG 3 65

0. angasi CGGA TCGAGAACGGC GCAAGTTGGAGC AGGTGTCGCGAG 3 68

C.gigas CAGAGTCCTGCCATTCAAAAAGGGGCTGAAGAGTCAACAGGGGCTCAAGGGCATCGCGAG 47 9

*** ******* * * **** *+ *******

0 . eduli s GAGGGCTCGCCG GCCGCCTCGTACAACACTAATCATGC CTCA 4 07

O.angasi GAGGGCTCGCCG GCCGCCTCGTACAACACTAATCATGC CTCA 410

C.gigas GGATCCGCGTTTTAAGAAAAAAAGCGCGGACCTCGATAAAACCAAAAACGGGACAACCAA 53 9

****** **** ********* **

0. edulis TGATTCCTCAA CTCCGACCTCAGATCAGGCGAGACTACCCGCTGAACTTAAGCATAT 4 64

O.angasi TGATTCCTCAA CTCCGACCTCAGATCAGGCGAGACTACCCGCTGAACTTAAGCATAT 467

C. gigas TTTTTTTTCAATCACTCCGACCTCAGATCAGGCGAGACTACGCCCTGAACTTAAGCATAT 599

* ** **** *************************** * ****************

O. edulis
O.angasi
C . gigas

TAGTAGTAGTAG
TAGTAGTAGTAG
CACTAAGGGCAG

476
479
611

Figure 4. Nucleotide sequence of the ITS-2 region from tlie oysters Ostrea edulis, O. angasi. and Crassostrea gigas (GenBank Accession Number
F28((610). Asterisks identify points of identical sequence and hyphens indicate points of sequence deletion/insertion. Numbers are the number
of nucleotides from the 5' end. The segment that corresponds to the repetitive element (a) from the ITS-1 region of Ostrea is indicated by shading
of the O. edulis sequence, and the two substitutions within it are underline, as is a third substitution of O. angasi.

zation is most likely to occur if the rRNA genes occur in a single
tandem array on one chromosome. In both O. cdiiUs and O. angasi.
two NORs are located terminally on chromosome pairs 9 and 10
(Thiriot-Quievreux & Insua 1992, Li & Havenhand 1997). Addi-
tionally, intraspecific variation in the number of NORs per cell and
the size (copy number) has also been identified in O. edulis

(Thiriot-Quievreux & Insua 1992). From this, one would predict
that sequence variation would exceed homogenization. However,
sequence divergence in the rRNA genes is very low between O.
angasi and O. edulis.

The sequences for the coding regions of the I8S and 5.8S
rDNAs were identical between the Ostrea species. This similarity

702

Kenchington et al.

O. edulis
O. angasi
C.gigas
C.nippona
Saccostrea

ATTAACAAAACAAGGAAAC - - AACCAAGGTCGTTGAGACGTGT- -CTCTCTCGTCATAGC 5 6

ATTAACAAAACAAGGAAAC- -CACCAAGGTCGTTGAGACGTGTGTCTCTCTCGTCATAGC 58

ATTAACAAAACAAAATCGG-GGCTCGTTGGCAAGAGCGACCGAGAGGCTAACGCAA--AA 57

ATTAACAAAAAAAGC ATCG -GGCTCGTCGGC A - GAGC ACCGAGGACGGC AACGAAACGAA 5 8

ATTAAC AAAC AAAATGGGGCAAACCATTGGC AAACCATCTCATGGTCG AGATGTAACGGT 6 0

O . edu lis
O. angasi
C. gigas
C.nippona
Saccostrea

CCGCGACC AGGTCGTGTGGAACGGGCGAGAGAAGGCC AATCGCCGTCCCCGCGGCGCCTT 116

CCGCGACCAGGTCGTGTGGAACGGGCGAGAGAAGGCCAATCGCCGTCCCCGCGGCGCCTT 118

ACACAGCCGAGG-G-TTGTTGTT AAAGTCG- -GATCGACCTCG-AACGCCCA 104

ACACAGCCGAGG-GGTTGCTGCTGTTTGGCAAGTGTCG--TGTCGACCTCT-AACGCCGA 114

CCAAACCCCGGA-GCCCGTGACC AGGTCG- -CGCGGA- -TGG-GGTGCGGA 105

O. edulis
0. angasi
C. gigas
C. nippona
Saccostrea

gggccgtcgtagccgaacaggctccgtcgccttaaatacagacg»1atgggaaactcgtcg| 1 7 6

gggccgtcgtagccg aac aggctccgtcgccttaaatac ag acgaatggg aaactcgtcg 1 7 8

GCG ACC AGGTCGCTAGGCCGAGGG - ATCC AGGCGAACTCGCTGGGGGGGTTTA 156

GCGACCAGGTCGCAAGGCCGAGGG-ACAC-GGCGA-CTCGCTTGGGGGGGTCGGTTCAAA 171

GAGAAAACGCTATCTCTTTCGGTC - GTAC CTCGTTGGCAGAGGCG 149

o. edulis |ccgtcttcccga|cggcgccttgggcagtcgtagccgaacaggctccgtcgccttaaatac

0. angasi CCGTCTTCCCGA

C.gigas TTTCCTCT

C.nippona GCCTTTCCCCCC

Saccostrea CAACCGA

236
190
164
183
156

O. edulis
0 . angasi
C. gigas
C. nippona
Saccostrea

agacgaIatgggaaactcgtcgccgtcttcccgaIcggcgccttgggcagtcgtagccgaac

cggcgccttgggc agtcgtagccgaac

cagcgccttgggccgtcgaagccttcc

cagcgccttgggccgtcgaagcctttt

gagagccttgggcagtcgcacccgaac

296
217
191
210
183

O. edulis
O. angasi
C. gigas
C.nippona
Saccostrea

j^ GGCTCCGTCGCCTCAAATACAGACGAGCTCGACTTTCGGTGATT 341

ft GGCTCCGTCGCCTCAAATAC AGACGAGCTCGACTTTCGGTGATT 262

T GCTCCGTCOCCTTAAGTACAG ACGAGCTCGACC GCGACC 231

TTTCTTCGGAACAAAGGGCTCCGTCGCCTTAAGTACAGACGAGCTCGACC GCGACC 266

- GGCTCCGTCGCCTTAAATTC AG ACGAGCTCGACC -

--GCGACC 22 4

O. edulis
0. angasi
C. gigas
C.nippona
Saccostrea

CCGAGCTCACGGACACGGCATTTGGTCACCGAATAT GACGCCAGGCATCGGCGG- - 3 9 5

CCGAGCTCACGGACACGGCATTTGGTCACCGAATAT GACGCCAGGCATCGGCGG - - 316

CCGGGCTTCCGGAACGGTGGGTCGCTAAAAC AAAAC AAAAAGC ACTCGGC ATCGTCGGGG 291

CCGGGCTTCCGGAACGGTGGGTCGC- -AAACAAAACTCATAGCACTCGGCATCGTCGGGA 324

C-GGGCTTT-GACGGGGAAGGTCGC AAAAC ACCGCTCGGCATCGTCGG- - 270

O. edulis TCGTGGGGCCGACGTTATAAGCACCCTGGTATGAACGTT-TCACGTACATCAATAGTTTT 454

O. angasi TCGTGGGGCCGACGTTATAAGCACCCTGGTATGAACGTT-TCACGTACATCAATAGTTTT 375

C. gigas TGGAAACCTCGACGTTATAAGCACCCGAGTATGTACTTTCTCACGTATACCAACAGATTT 3 51

C. nippona GGTTGGCCTCGACGTTATAAGCACCCGAGTATGTACTTTCTCACGTATACCGATAGCTTT 384

Saccostrea TC ATTGTTCCGATGTTGTAA - CGCCCGAGTATGTAC ATTCTCACGTATACGAAATCCTTA 329

O. edulis
O . angasi
C. gigas
C .nippona
Saccostrea

GATCTTGGCGCGGGG GGAAAAA C 477

GATCTTGGCGCGGGG GGAAA C 396

CT-TACACAAAACCCGGGAAGGCGGAGTCGGCAAG GCTCTTG 3 92

TTAC ACACAAN AACCCGGGGGGCGGAGTCGGGAGGCAGGCAATTAAGTCCTC AACTCTGC 444

TTTTA - ACGAGGA - - AGGGGTCGGGAGCAGGCAAAG TCTCTCGGC 3 71

0. edulis
0. angasi
C. gigas
C.nippona
Saccostrea

ACTCCTCGC GGTCG-CGTCCGGCGT-TCTTGTTGCGCGCCGAGACCGGCCGAATTT- 531

ACTCCTCGC GGTCG-CGTCCGGCGT-TCTTGTTGCGCGCCGAGACCGGCCGAATTT- 4 50

CCTTTTTTC GGGGGTCGGCCGGCGTACCTCGTTGCGCGCCGCGACCG ACCGAATT- - 447

TTTTTTTTCTCGGGGGGTCGGCCGGCGTACCTCGTTGCGCGCCGAGACCGACCGAATT-- 502

TTTTCTTTCCTCGGTCG-CGTCAGGCGTACTTCGTTGCGCGCCTCGACCGGCCGAATCAC 4 30

O. edulis
0. angasi
C . gigas
C .nippona

GGCATC-T-CT-TTTTGA 546

GGCATC-T-CT-TTTTGA 465

GGCATCATACTATTTTGA 465

GGCATCTTATTTTTTTGA 520

Saccostrea TTTGGCATC-

-TAT--TGA 445

Figure 5. Nucleotide sequence of the ITS-2 region from the oysters Ostrea edulis. O. angasi. and Crassoslrea gigas (GenBank Accession Number
F28n60<)l. Asterislis identify points of identical sequence and hyphens indicate points of sequence deletion/insertion. Numbers refer to the number
of nucleotides from the 5' end. The 54-bp nucleotide repetitive (a) elements in (>. edulis are indicated by shading of the O. edulis sequence. The
27-bp nucleotide repeat (ft) is outlined on the same sequence.

Repeat Elements in Ostrea ITS Sequence

703

O.angasi CTTTCGCTCTCGTCCTTGCGTGCACGTCTCGACCTGTGGAGACTGCTCTCTGGGCAGTGG 720

O. edulis CTTTCGCTCTCGTCCTTGCGTGCACGTCTCGACCTGTGGATACTGCTCTCTGGGCAGTGG 720

O.edulis (Littlewood 1994) 6

O.angasi CCGCTAACTGCGAGTGCGCACGGGGTGGGAGGCGTGGCGGTGCCCGATGGTCAGTGGCGA 7 80

0. edulis CCGCTAACTGCGAGTGCGCACGGGGTGGGATGCGTGGCGGTGCCCGATGGTCAGTGGCGA 780

O.edulis (Littlewood 1994) 6

ATCGGTCGGGCGTCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGTGCG
ATCGGTCGGGCGTCCACCCGACCCTTCTTGAAACACGGACCAAGGAGTCTAACATGTGCG
(Littlewood 1994) G

840
840

O. angasi

O. edulis

0. edulis

Figure 6. Partial nutk'otide sequence of the 28S rRNA gene showing variable positioning between the oysters Oslrea angasi (GenBank Accession
Number AF137()46) and O. edulis (GenBank AF137047) as used by 6Foighil and Taylor (2(l()(ll and as determined by Littlewood (1994).

prompted a re-examination of the partial 28S rRNA gene se-
quences produced for O. edulis by Littlewood (1994) and those
cited by 6Foighil and Taylor (2000) for O. edulis and O. angasi
(deposited in GenBank). The sequences by 6 Foighil and Taylor
(2000) show three variable positions between the taxa over 911
nucleotides. However, the Littlewood ( 1994) sequence for O. edu-
lis does not differ from O. angasi at those positions (Fig. 6).
Therefore, it would appear that O. angasi and O. edulis may have
identical nucleotide sequences for the coding regions of the rRNA
cistron given that the partial data from the 28S rRNA gene were
taken from the variable domains (Dl. D2, D3). The 10-bp differ-
ence between the O. edulis 1 8S rDNA sequence of this study and
U88709 from GenBank is not considered further as the latter is not
published and the methodology used to infer it is not known. The
I8S rRNA gene was expected to be the most conserved of the
coding regions and therefore is unlikely to differ so greatly within
a species.

Variation was delected in the non-coding internal spacer re-
gions. The ITS-1 and ITS-2 both show sequence divergence be-
tween O. edulis and O. angasi irrespective of the repeat elements
in ITS- 1. However, the differences between O. edulis and O. an-
gasi in ITS-1 are small compared with those observed between
congenerics Crassostrea gigas and C. nippona (Fig. 5). The major
difference between the Oslrea species is in the repeat unit of the
ITS-1.

Repeat elements in the ITS-1 spacer have been identified pre-
viously (Long & Dawid 1980), including those with the same
subrepeat pattern Ui. h. a), (van Herwerden et al. 1999). However,
it is not known if the repeating elements in these oysters have a
function. Those found in the IGS region of both Drosophila niela-
nogaster (Kohorn & Rae 1982) and Xenopus laevis (Moss 1983)
have an influence on the level of transcription (Busby & Reeder
1983. Grimaldi & Di Nocera 1988). The ITS-1 region has an
ability to form ribonucleoprotein complexes with cellular proteins
and this domain is critical to the efficient maturation of the pre-
cursor RNA (Lalev et al. 2000). It is likely that the repeat element
in Oslrea will have some effect on transcription. The secondary
structure formed in O. edulis is more thermally stable than that of
O. angasi. Further, the conserved nature of the base repeat and the
repetition of the a element in the ITS-2 strengthens the case for
functionality of these eleiuents.

In other parts of the genome, repeat regions commonly arise
through a preferential gain of a few repeat units at one end of the
tandem repeat array, generating a minisatellite sequence (Jeffreys
et al. 1994). Slip-stranded mispairing (Levinson & Gutman 1987)
is commonly proposed as a model for VNTR repeat formation,
expansion and contraction. Chance mutations are necessary for

repeat expansion and these produce a few tandem repeats that
facilitate the first strand slippage event (Messier 1996, Taylor &
Breden 2000). However, different mechanisms may be responsible
for the formation of repeats with motifs longer than 5 bp (Jeffreys
et al. 1994). Taylor and Bi-eden (2000) have described a general
model for minisatellite birth that results in the formation of a locus
with long repeats flanked by one unit of the original noncontiguous
repeat. The appearance of differently sized repeats in the ITS-1 of
O. edulis and O. angasi can be most expeditiously explained by
unequal or misaligned crossing over. Depending on the ancestral
configuration, the a' . b pair has been either lost or gained in one of
the two taxa since their geographic separation.

The implication of these data is that O. edulis and O. angasi are
conspecific as suggested by Jozefowicz and O Foighil (1998).
These findings parallel the example of Saccostrea connnenialis
and S. glomerata (Anderson & Adlard 1994), in which the taxa are
morphologically similar, yet geographically separated, with iden-
tical flanking 18S. ITS-1. and 5.8S regions. Although O. angasi
and O. edulis have identical coding regions, some variation was
detected in the ITS-1 and in the ITS-2, primarily through the
deletion of repeat elements in O. angasi. However, this level of
.spacer variability is minor compared with that observed among the
Crassostrea species.

As with the Saccostrea species studied by Anderson and Ad-
lard ( 1994), the species in this study are also geographically sepa-
rated. Ostrea angasi is found in Australia, from Western Australia
along the southern coast to Tasmania and north along the east coast
to the Clarence River in New South Wales (Thomson 1954). Os-
trea edulis is mainly European, occurring from Norway to Moroc-
co, including the Mediterranean Sea and the Black Sea (Carriker &
Gaffney 1996). As put forward by Jozefowicz and O Foighil
(1998). this incongruity can be explained by undocumented an-
thropogenic transport. Although O. angasi is widespread in Aus-
tralia, it is not common throughout its range (Thomson 1954) and
its distribution is centered around the area first colonized by Eu-
ropeans. Such a scenario would suggest that the ITS- 1 of O. anga.'ii
underwent a deletion of the a', b region in the approximately
200-300 y after the transfer of O. edulis to Australian waters.
However, as only one individual of each Ostrea species was se-
quenced, we do not know whether the number of repeats in the
ITS-1 varies intraspecifically. Further investigation into intraspe-
cific variation in the ITS-1 may identify a European locale from
which O. angasi originated and evolved. Closer examination of
late Pleistocene deposits at the Largs site in the Lower Hunter
Valley, New South Wales, which reportedly contain specimens of
O. cmgasi. may also shed some light on this question. In the in-
terim, we submit that O. edulis and O. angasi are con.specific.

704

Kenchington et al.

ACKNOWLEDGMENTS

We thank Dr. John Thomson (then of HOTAC, Hobart.
Tasmania. Atistralia) for sending specimens of O. angasi. We

also thank Dr. P. T. O'Reilly and B. Vercaemer (Bedford
Institute of Oceanography) for providing useful com-
ments during the preparation of this manuscript. This is NRCC
42365.

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Joiinwl of Shellfish Research. Vol. 21. No. 2. 707-714. 2002.

ULTRASTRUCTURAL AND HISTOCHEMICAL CRITERIA FOR DETERMINING NORMALITY
IN MATURE OOCYTES OF THE PACIFIC OYSTER CRASSOSTREA GIGAS

MARIA EUGENIA VALDEZ-RAMIREZ,'* ANNE DONVAL,' AND MARCEL LE PENNEC^

^Cenlro de Investigaciones Biologicas del Noroeste, S. C. (CIBNOR) - Programa de Acuicidtiini
Marina. P. O. Box 128. La Paz. B. C. S.. 23000. Me.xico: -Institiit Universitaire Eiiropeen de la Mer
ilUEM) - UMR CNRS 6539 Technopole Brest-Iwise. 29280. Plouzaue. France

ABSTRACT In hatcheries of bivalve molluscs, female gametes are assessed according to the moiphologic stages of oocytic devel-
opment. Three stages of oocytes immature, mature, and overripe were studied. We identitled each category, based on ultrastructural
features of the vitelline coat, cytoplasmic membrane, and organelles. Vitelline mclusions in the ooplasm were identified by ultra-
structural and histochemical methods. Cytologic alterations involved in the typical degenerative process of mollusc oocytes were
observed, including initial damage in the cytoplasmic membrane, rupture and degeneration of the vitelline coat, and damage in ooplasm
and vitelline globules. All of these characteristics seem to be associated with the natural process of oocytic degeneration, but they can
also be the consequence of handling during reproduction by anitlcial means.

KEY WORDS: oocyte, ultrastructure, histochemistry. Cnissoslrea gigas, reproduction

INTRODUCTION

Poor quality of gametes is believed to be one of the main causes
of irregularities of embryonic and larval development in bivalve
mollusc hatcheries (Raven 1966, Galtsoff 1964, Dohmen 1983,
Wilson et al. 1996, Gerard et al, 1997, Le Pennec et al. 1998). In
aquaculture, the quality of the gametes is defined by its capacity to
be feitilized and consequent development of a viable larva (Kjors-
vik et al. 1990). Morphologic criteria are commonly used for iden-
tification of female gametes of good quality. Three types of oo-
cytes can be identified at spawning: mature oocytes with a round
form and healthy appearance, immature oocytes with a pear shape
and heterogeneous appearance, and overripe or atretic oocytes
(Dorange & Le Pennec 1989. Paulet et al. 1992), This is a simple,
quick, and inexpensive method. Nevertheless, these criteria are not
sufficient to assess the viability of spawnings of Crassostrea gigas
(Valdez-Ramirez et al. 1999). Results obtained from hatchery pro-
duction reveal great variations that do not correlate with the
amount of mature or normal oocytes. Germ cells maturation can be
disturbed by multiple endogenous and exogenous factors in bi-
valve molluscs (Pipe 1987, Dorange & Le Pennec 1989, Paulet et
al. 1992). A visual inspection does not confirm that mature oocytes
will develop after fertilization.

Daniels et al. (1973) described the normal and abnormal char-
acteristics of female gametes of Crassostrea virginica in the cy-
toplasm components, in particular, yolk granule distribution. This
study suggests that knowledge of the gamete cytology is signifi-
cant to evaluate the reproductive capability of the species. For
Pecten maximus, Dorange and Le Pennec (1989) described, by
means of transmission electron microscopy (TEM), the ultrastruc-
tural features in the stages of oocyte development and the natural
process of oocytic degeneration. These authors showed that, at the
ultrastructural level, cytologic alteration could be observed in the
plasmic membrane, nuclear envelope, and mitochondria that were
not detectable by gross morphologic observations.

The main purpose of this study is to address some questions
related to the quality of the oocytes of C. gigas. Specifically, we
tried to identify and describe the cytologic and cytochemical

*Corresponding author. E-mail: gvaldez(8'cibnor.mx

anoinalies that most commonly affect the viability and quality of
female gametes.

MATERIALS AND METHODS

Samples of Crassostrea gigas (Thunberg 1793) were obtained
from the Bay of Brest, Brittany, France during its natural spawning
period. June and July. Oocytes were obtained by draining of go-
nads. The oocytes were kept in seawater for one hour to recover
their shape, as they are compressed in the gonads. Some gametes
were fertilized to obtain embryos and larvae. Fragments of gonads
of different specimens v\'ere also used in this study.

Sample Preparation for Transmission Electron Microscopy

Samples (oocytes, embryos or fragments of gonads) were fixed
for one hour in 2.59^ glutaraldehyde buffered with 0.2 M sodium
cacodylate adjusted to 1 100 mOsm and pH 7.2 (Cross & Mercer
1993). Postfixation was completed in 1% OsOj using the same
buffer for 60 min at 4°C. The samples were rinsed, followed by
progressive dehydration in ethanol baths (70%, 95%, and 100%).
They were embedded in resin (SpuiT 1969). Resin polymerization
was completed at 60°C for 48 h.

Semi-thin (1 |Jim) and ultrathin (60-70 nni) sections were cut
with glass and diamond ultramicrotome knives (Ultracut-S Leica)
according to the method of Cross and Mercer (1993). The semi-
thin sections for study under light microscopy were stained with
0.5% toluidine blue. The ultrathin sections were contrasted with
uranyl acetate and lead citrate stains (Reynolds 1963). Observa-
tions of ultrathin sections were made with a Transmission Electron
Microscopy (TEM) (JEOL 100 Cx). In addition, a few semi-thin
sections were also treated using techniques for the identification of
vitelline globules.

Detection of Lipids

Before postfixation, some samples of oocytes were treated in a
solution of methanol-chloroform (1:1 ) at 60X for 12 h to extract
the lipids (E samples). They were postfixed using the same pro-
cedure described above. The identification of the lipid globules
was made by comparing sections from the same batch of oocytes
from which lipids were not extracted (NE samples).

707

708

Valdez-Ramirez et al.

Detection of Proteins

The study of basic proteins was made on semi-thin sections
treated with the 1.5% periodic acid at 40°C for one hour. They
were stained by Ponceau 2r at 40°C for 3 h (Gori 1977). The
protein globules revealed by staining were compared with the in-
clusions observed in ultrathin sections of samples from the same
batch, by size, form, and their location in the cytoplasm of the
oocyte.

Detection of Carbohydrates

The characterization of carbohydrate reserves in the oocytes
was obtained by a PAS reaction (Periodic acid-Schiff) (Gabe
1968). Negative "glycogen" controls were prepared with amylase
at 37°C for 3-6 h. After several rinses, these sections were treated
at the same time as the positive glycogen controls.

RESULTS

Mature Oocyte

Mature or normal oocytes, measuring from 60 to 65 p.m. con-
tain a bulky nucleus (Fig. 1: 1). The germinal vesicle, in the
prophase of first meiotic division, shows regular contours, ap-
proximately 25 |j,m in diameter. The nucleolus, when visible, is
generally eccentrically located. Nuclear pores are sometimes ob-
served. The cytoplasmic membrane bear numerous regular mi-
crovilli embedded in a vitelline coat (oolemma) (Fig. 1:2). The
fibrillar structure of the vitelline coat appeared highly electron-
dense at the periphery (Fig. 1: 2). This was observed in all the
samples of unfertilized (Fig. I: 2) oocytes and in those still in place
in the gonad (Fig. 1: 1). Observations made of the vitelline coat
ultrastructure in oocytes before fertilization (Fig. 1: 2), at the mo-
ment of fertilization (Fig. 1: 3). and 3 h after fertilization (Fig. 1:
4) showed no real differences in electron-density. The perivitelline
space is sometimes visible between the vitelline coat and the plas-
mic membrane (Fig. 1: 5).

In the cytoplasm, mitochondria, endoplasmic reticulum, and
many vitelline inclusions are visible (Fig. 1:5). Mitochondria are
very common and are distributed homogeneously. Their size is
variable, the longest measuring approximately 0.5 |a.m. The lamel-
lae of the endoplasmic reticulum are not abundant in the cyto-
plasm. Cistemae of endoplasmic reticulum are sometimes visible
in the vicinity of the mitochondria. Some dictyosomes are also
present.

Cytoplasmic inclusions accumulated during vitellogenesis are
numerous and are varied in types. They occupy the larger part of
the cytoplasm, differing in size. form, and density. By coupling
ultrastructure study with cytochemical observations, the chemical
nature of the inclusion was identified. Three types of inclusions
were detected (Fig. 2: 1 to 8) and are described in the following
three sections.

Type /, Lipid Inclusions

On semi-thin sections of the NE samples, the lipid inclusions
(stained with Sudan black) occupy the larger part of the cytoplasm,
and their distribution is homogeneous (Fig. 2: 1 ). At the ultrastruc-
ture level, comparison of the samples with or without lipid extrac-
tion (Fig. 2; 2 and 2: 3). shows that these compounds correspond
to the largest inclusions, reaching a diameter of approximately I
(j,m. Their contour is regular and. in most cases, they are enclosed
by the granular endoplasmic reticulum. A higher electron density

in the center than at the periphery characterizes these inclusions
(Fig. 2: 2).

In several NE samples, the lipid globules were partially or
completely absent (Fig. 2: 4 and 5).

Type II, Glycoprotein and Carbohydrate Inclusions

Round organelles, from 0.2 to 1 \^.m in diameter, are sur-
rounded by a membrane. The homogeneous contents are visible in
ultrathin sections. They may be dispersed in the cytoplasm, but
they are mainly present at the periphery (Fig. 2: 5). The distribu-
tion and location of these reserves in ultrathin sections correspond
to positive PAS reaction (Fig. 2: 6) and to the globules stained pink
by Ponceau 2r (Fig. 2; 8) on the semi-thin sections. This suggests
that type II inclusions are glycoproteinaceus in nature.

Glycogen detection by comparison between PAS staining in
semi-thin sections (Fig. 2: 6) and the negative control (Fig. 2: 7)
does not allow clear observation of this compound in the mature
oocyte. However, we note a slightly more intense staining on
oocytes untreated with amylase, which suggests the presence of
glycogen.

Type III, Lysosome Inclusions

Distribution and occurrence of the lysosome inclusions de-
tected by Ponceau 2R (Fig. 2: 8). are comparable way as type II
inclusions. Certain differences were observed in MET. Type III
inclusions are an irregular structure, and their location is unusual.
They are provided with a single external membrane. They are
heterogeneous in size. form, and electron density (Fig. 1 : 2 and I:
5) and always abundant, with a maximum size comparable to the
lipid inclusions. Their heterogeneity contrasts with other inclu-
sions of relatively homogeneous forms.

Ultrastructure Anomalies of the Oocytic Degeneration

Different degenerative alterations are observed (Fig. 3: I to 5).
Degeneration in the female gamete can present a large increase of
the perivitelline space (Fig. 3: I to 3) and a dilatation of the base
of the microvilli. In some cases, the vitelline coat is detached and
no microvilli are seen at the periphery (Fig. 3: 4). In the cytoplasm,
mitochondria seem to be the first oocytic organelles involved in
the degenerative processes (Fig. 3: 3). Other vitelline inclusions
can also deteriorate, but lysosome bodies stay intact (Fig. 3: 4 and
5). The most severe damage observed in the cytoplasm include
large vacuoles enclosing the vitelline bodies and cytoplasmic com-
ponents, which are degraded to a lesser or greater level and form-
ing dense globular masses. At this stage, intact lysosomal bodies
are common (Fig. 3: 5).

Other Cytologic Alterations

In some cases, mainly in free oocytes, the vitelline coat can be
partially or completely detached from the plasmic membrane (Fig.
4: 1 and 2). In addition, the rupture of the plasmic membrane is
sometimes seen (Fig. 4: 3), but the contents of the mature oocyte
are not degraded.

DISCUSSION

Mature oocytes of C. gigas are comparable to those in other
bivalve mollusc species, such as Spisula solidissima (Longo &
Anderson 1970a. Longo & Anderson 1970b). C. virginica (Daniels
et al. 1973). and Pecten maximus (Dorange & Le Pennec 1989.
Devauchelle et al. 1997), In oocytes of C. gigas, the vitelline coat

Criteria for Determining Normality in Mature Oocytes of the Pacific Oyster

709

Figure. 1. Ultrastructural characteristics of the normal oocytes (1) Mature Crassoslreu nigus oocyte prior to breal<down of nuclear envelope
(prophase I stage) in the gonad. Scale: 5 nm. (2) Mature unfertilized C. gigas oocyte. Fibrillary structure of the vitelline coat: dense zone on the
surface. Mitochondria and vitelline inclusions: lipids (type II, glycoproteinaceus (type II), and lysosome (type III). Scale: 5 Mm. (3) Vitelline coat
of the oocyte at the moment of fertilization. Cytoplasmic and vitelline coat density (^). Scale: 1 fim. (4) Vitelline coat density (^) in fertilized
oocyte (3-h-old embryo). Scale: 5 nm. (5) Organelles and vitelline inclusions in ooplasm: lipids (type I), glycoproteinaceus (type II), and inclusions
of the lysosomal type (type III). Scale: I urn. Legend: Vc, Vitelline coat; Pm, Plasmic membrane; Vi, Vitelhne inclusions; M, Mitochondria; N,
Nucleus; Ps, Perivitelline space; Mv, Microvilli; Sp, Spermatozoa; L, Lipid inclusions; Ly. Lysosome type inclusions; Gl, Glycoprotein inclusions.

structure seems different because of the tightness of the fibrillar
net at the periphery. In this study, we did not detect modifications
of the vitelline coat after fertilization, as observed in other bivalve
molluscs; Pecten maximus (Dorange et al. 1989. Casse, 1995).
Mytilus edulis (Humphreys 1962), other invertebrates, (Dohmen

1983. Epel et al. 1984. Pashchenko & Drozdov 1998). or in fish
(Yemeryanova 1980. Kjorsvik et al. 1990). In these species, cor-
tical granules take part in the formation of the fertilization mem-
brane. It is recognized that this membrane is a barrier that prevents
polyspermy, but most molluscs apparently do not form a fertiliza-

710

Valdez-Ramirez et al.

©

*-v

(»'

fi*

La

i

®

La

1ft »Ly 5
^i • ••

S Gi n. .

i^^^jjf I

® .

(D \

■if

^^:?*'

*«*

I

Figure 2. Tht vitellint inclusion^, identification and line ^lrul■ture (1) Lipid inclusions stained In Soudan black in semi-thin section. Scale: 10
Mm. (2) Lipid inclusions in ultralhin section are enclosed In the granular endoplasmic reticulum. They present a higher electron density in the
center than at the periphery. Scale: 1 fim. (3) LUtrathin section after extraction of the lipids. Scale: 5 fim. (4) I'ltrathin section showing lipid
Inclusions partially or completely absent in NE preparations. Scale: 5 pm. (5 1 Distribution of the different types of vitelline inclusions in ultrathin
sections. Scale: 5 fim. (6) Carbohydrate granules (type III in semi-thin sections (>-) after staining with the PAS. Scale: Id fim. (7| Negative control
in glycogen detection by reaction with amylase for 3-5 h al 37 C. The less dense bottom suggests the presence of glycogen. Scale: III fim. (8|
Glycoprotein inclusions, type II, (>■) and probably type III (^) on semi-thin sections stained by Ponceau 2r for 3 h at 41) C. Scale: 10 (jm. Legend:
L, Lipid inclusions; GI, Glycoprotein inclusions; Ger, Granular endoplasmic reticulum; Lge, Lipid inclusions after extraction; La, Lipid absents.

Criteria for Determining Normality in Mature Oocytes of the Pacific Oyster

711

w*

'mm

<

, ^

" #

1

Ps

Vc

h-

/

* "'' . w

Figure 3. Ultrastructural anomalies of the oocyte; oocytic degeneration (1) Vitelline coat deterioration (^), normal vitelline coat in another
oocyte (^1. Scale: 1 (im. (2) Vitelline coat, advanced stage of degeneration (^). Perivitelline space increases. Ooplasm and organelles may be
modified at the periphery of the oocyte. Normal structure of vitelline coat (-*). Scale: 1 pm. (3) Degeneration in the ooplasm. Only mitochondria
are damaged (^1: their shape is modified, the cristae disappear, and their content becomes clearer. Scale: 1 ym\. (4) Advanced degeneration in
the ooplasm. The altered organelles are more abundant in the cytoplasm. Deteriorated vitelline coat is observed separated at some places from
the plasmic membrane (-»). Scale: 1 pm. (5) Advanced oocytic degeneration. Plasmic membrane is not visible. Vitelline coat surrounds the
contents of the oocyte ( -> ). In the cytoplasm, organelles are often grouped in vacuoles ( >• ). Degeneration of the organelles is important ( ^ 1. Intact
type III inclusions are abundant. Scale: I pm. Legend: Vc, Vitelline coat: Pm. Plasmic membrane; Vi, Vitelline inclusions; M, Mitochondria; Ps,
Perivitelline space; Mv, Microvilli; L, Lipid inclusions; Ly, Lysosome type inclusions.

tion membrane. The precise mechanism for preventing polyspermy
is not clearly understood (Dohmen 19831. The formation of this
membrane in oocytes of C. gigas could be explained as a specific
reaction (Thierry & Rambourg 1974).

Three types of vitelline inclusions were identified in the mature
oocytes of C. gigas. Lipid inclusions, type I are abundant and
easily locatable globules in the cytoplasm because of their homo-
geneous form. We observed a partial or total vacuolization of the

contents of these globules in some oocytes. Vacuolization is not
the result of extraction from an extended period of dehydration in
elhanol because, in the same batch of oocytes, one finds normal
oocytes. If a technical problem is excluded, the assumption is that
this is a variable state in the maturity of oocytes. Steele (1998)
shows comparable inodifications of these globules that coincide
with other deteriorations in the cytoplasm. Dorange (1989) shows
that the density of lipid inclusions can be correlated to the stage of

712

Valdez-Ramirez et al.

©.

Pm

Vc

7

Figure 4. Other ultrastructural anomalies of tlie oocytes ( I ) Rupture of vitelline coat in mature oocytes. Plasmic membrane is not brol<en.
Cytoplasm and vitelline inclusions are not modified its normal aspect. Scale: I pm. (2) Another example of the rupture of vitelline coat in mature
C. gigas oocytes. Scale: 1 pm. (3) Rupture of the vitelline coat and plasmic membrane in mature C. gigas oocytes can take place at the gonad.
In the cytoplasm, the organelles are intact. Cellular remains are observed between the oocytes. Scale: 5 pm. Legend: Vc, Vitelline coat; Pm,
Plasmic membrane.

oocytic maturity in P. iiui.xiiinis. This also seems to be the case for
C. gigas.

Lipid inclusions with a granular contour have been described in
several species of bivalves such as C. virgiiilcu (Daniels et al.
1973), C. gigas (Steele 1998), and gastropods such as, Ilyanassa
spp. (Gerin 1976) and Acmaea spp. (Kessel 1982). Daniels et al.
(1973) and Steele ( 1998) suggest that it could be lipoprotein gran-
ules associated with the endoplasmic reticulum.

Glycoprotein was detected in type 11 inclusions that were
stained by the protein and carbohydrate specific reaction in semi-
thin sections. This type of inclusion has been noted in C. virginica
(Daniels et al. 1973) and in the mussel Mytihts ediilis (Albertini
1985). Positive staining by PAS in semi-thin sections seems to
confirm the presence of glycogen. However, this must be checked
at the ultrastructural level (reaction. Thierry & Rambourg 1974.
for example). According to Tazawa et al. (1985) and Tazawa et al.
(1986), these reserves represent the first source of energy during
the first modifications in embryonic development of C. gigas.

Type III vitelline inclusions suggest lysosomal bodies. The
presence of hydrolases (lysosomal enzymes) has been demon-
strated in cytoplasmic globules oocytes of bivalves; P. maximus
(Lubet et al. 1987, Dorange & Le Pennec 1989, Casse 19951.
Rangia cuneata (Marsh et al. 1981), Mytiliis cdiilis (Pipe & Moore
1985). and the gastropod Litlurina littovca (Moore et al. 1982).
Based on multiple observations, lysosomal bodies that are present
during the degenerative stage of P. maximus oocytes were clearly
identified by Lubet et al. (1987). Since its enzymatic activity is

demonstrated, its presence in the oocytes of C. gigas must reveal
different functions because it is always identifiable in the mature
and degenerative .stages.

The main causes of the poor viability of the gametes detected
In the present study are related to the oocytic degenerative process.
This is a phenomenon usually described in studies of the repro-
duction of fish, invertebrates in general, and the bivalve molluscs
in particular (Lubet et al. 1987, Dorange et al. 1989. Paulet 1990,
Nliba et al. 1992. Sarojini et al. 1994, Widowati 1994. Steele
1998). Some serious alterations can be identified by direct exami-
nation of the samples using optical microscopy, but initial damage
cannot be detected by means of morphologic observation. Ultra-
structural techniques allow observation of different stages of de-
generation. The first modifications involving the vitelline coat and
plasmic membrane do not modify the form of the oocyte, which
suggest that these gametes are not excluded during a morphologic
examination. When degradation reaches the cytoplasm, oocyte
shape is not modified in all cases. Therefore, damage may not be
delected by simple microscopic observation.

It is important to mention that, according to our observations,
this last stage does not seem to correspond to the same process of
degeneration as the preceding one, but this cannot be confirmed
now. First, damages may indiscriminately affect the periphery ot
gametes. Second, damage orients itself only to the content of the
organelles, not to the periphery. These vacuolization already have
been described in the typical oocytic degeneration process for
bivalve molluscs P. maximus (Lubet et al. 1987, Dorange & Le

Criteria for Determining Normality in Mature Oocytes of the Pacific Oyster

713

Pennec 1989). Mytiliis ediilis (Pipe 1987). and Pinna nobilis (De
Guulejac et al. 1995). In these species, modification coincide with
degeneration of the vitelline membrane. This is not the general
case observed. A study following oogenesis and oocytic lysis by
ultrastructure techniques is required to explain this difference. The
only morphologically identifiable degenerative stage corresponds
to a severe degree of damage. In this stage, vitelline inclusions and
organelles are grouped without order and they are degraded, form-
ing masses of different density in cytoplasm. This causes important
modification of the mass, density, and gamete form.

Another cytologic damage that obstructs larval development
has been demonstrated in this study. It is the breaking of the
vitelline membranes, without apparent alterations in cytoplasm.
These damages are probably caused during the collection and han-
dling of the gametes, but it is not possible to be certain that these
damages are revealed in the oocyte morphology.

In conclusion, this study demonstrates that oocytes with normal
external appearance can, in fact, display biochemical deficiencies
or in-eversible and lethal cytologic damages. These anomalies can
explain the poor development of embryos and larvae. Although
observation of these defects is difficult, its detection and quanti-
fication in light microscopy is possible by means of the Trypan
blue exclusion test, as studied by Valdez-Ramirez et al. ( 1999) for
oocytes of this species.

ACKNOWLEDGMENTS

The authors thank lUEM and IFREMER/Brest (France).
CONACYT (Mexico) and CIBNOR (La Paz. Mexico) for support
and facilities for this work. Ira Fogel at CIBNOR edited the En-
glish text and M. A. Le Mercier Gerardo Hernandez edited the
illustrations.

LITERATURE CITED

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Thesis. University of Caen, France.

Casse, N. 1995. Some aspects of embryology oi Peclen imiximus L. (mul-
lusca. bivalvia). PhD. Thesis. University of Bretagne Occidentale.
France.

Cross, P. C. & K. L. Mercer. 1993. Cell and tissue ultrastructure: a fimc-
tional perspective (Ultrastructure cellulaire et ti.ssulaire. approche lonc-
tionnelle). De Boeck Universite, Bruxelles, Belgium.

Daniels. E. W.. A. C. Lonwell, J. M. McNiff & R. W. Wolfgang. 1973.
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JuKmal of Shellfish Research. Vol. 21. No. 2, 715-718. 2UU2.

AN INTRINSIC MEMBRANE PROTEIN IN OYSTER SPERM STIMULATES SPAWNING
BEHAVIORS IN CRASSOSTREA VIRGINICA: IMPLICATIONS FOR AQUACULTURE

PATRICK RICE,' SAMMY M. RAY,' SHERRY D. PAINTER," AND GREGG T. NAGLE"*

^Department cf Marine Biology. Te.xas A&M University at Galveston. Galveston. Texas 77553; 'Marine
Biomedical Institute and Department of Anatomy & Neiirosciences. University of Texas Medical Branch.
Medical Research Biiildhig. Galveston. Texas 77555

ABSTRACT Pheromones are thoughl to play a critical role in triggering spawning in oysters, but none have been identified to date.
Male and female oysters are stimulated to spawn by oyster sperm. Male oysters are also stimulated to spawn by oyster eggs, but females
are not. Earlier studies suggest that the spawning activity associated with sperm may be membrane-bound. As a first step toward
isolating and characterizing a spawning pheromone in sperm of the oyster Crassostrea virginica, we used: (1) an extraction and
purification procedure for preparing oyster sperm membranes; (2) a method to selectively remove extrinsic sperm membrane-associated
proteins; and (3) abioassay to monitor the robust, repetitive adductor muscle contractions that occur concurrently with oyster spawning.
This report presents evidence that the candidate oyster sperm pheromone is a heat- and trypsin-sensitive intrinsic membrane protein.
A synthetic or recombinant fragment of the pheromone could be used in the aquaculture industry to induce spawning in oysters.

KEY WORDS: bivalve. Crussostrea virv^iiiica, mollusk, oyster, pheromone, sperm

INTRODUCTION

Spawning in the oyster. Crassostrea virginica Gmelin. has
been known to be a group reaction for more than a century and
involves the relea.se of sperm and eggs by a number of oysters.
Although spawning in C. virginica can be influenced by a seasonal
rise in water temperature, male and female oysters are more readily
stimulated to spawn by oyster sperm; male oysters are also rapidly
stimulated to spawn by oyster eggs (Galtsoff 1938b). Sperm can
induce spawning in oysters that do not respond to increased tem-
perature stimulation. For example, approximately 55% of oysters
that failed to react to increased temperature spawned immediately
upon addition of spenii (Galtsoff 19.38b). A minimum concentra-
tion of -100-150 sperm per milliliter of seawater will induce an
all-or-none spawning reaction that cannot be inhibited or stopped
once it has begun. In physiologically ripe females, the presence of
oyster sperm normally initiates ovulation and rhythmic contrac-
tions of the adductor muscle, and the latent period to spawning is
relatively independent of the concentration of sperm (Galtsoff
1930, Galtsoff 1938b). In males, the latent period to spawning is
usually shorter compared with females (Galtsoff 1940).

The active factor(s) that stimulates spawning in C. virginica is
associated with the sperm and is not a soluble factor released with
sperm (Galtsoff 1938b). As a first step toward characterizing the
active factor in oyster sperm responsible for stimulating male and
female oysters to spawn, we employed a simple extraction and
purification procedure for preparing oyster sperm membranes, a
method to selectively remove extrinsic sperm membrane-as-
sociated proteins, and a bioassay to monitor adductor niu.scle con-
tractions that normally occur concurrently with oyster .spawning.

A major obstacle to purifying and characterizing the factor(s)
responsible for spawning in C. virginica is that spawning is re-
stricted to a relatively brief reproductive season, and the year-
round bioassay of sperm membrane fractions has not been pos-
sible. Therefore, a bioassay was used based on the observation that
oysters initiate a series of strong, rhythmic contractions of the
adductor muscle in response to sperm entering the mantle cavity
via the incurrent siphon (Galtsoff 1938a, Galtsoff 1938b). In ripe

females, this vigorous and repetitive clamping of the shells nor-
mally results in the ejection of a cloud of eggs to a distance of 30
cm or more. This activity is thought to result in a relatively wide
and uniform distribution of eggs, improving their chances of fer-
tilization by sperm. In this report, we present evidence that the
oyster sperm factor(s) that stimulates the vigorous and repetitive
adductor muscle contractions observed concurrently with spawn-
ing is a heat- and trypsin-sensitive intrinsic membrane protein.

MATERIALS AND METHODS

Animals

*Corresponding author. E-mail: gtnagle@utmb.edu

For tissue extractions, oysters (Crassostrea virginica) were col-
lected from April to June 2000 and 2001 from Galveston Bay,
Texas.

For bioassays, animals were collected from Galveston Bay
year-round and temporarily kept on ice; the shell height ranged
from approximately 38 to 52 mm. To avoid rapid increases in
temperature that could induce spawning, oysters were first accli-
mated to room temperature (20-22''C) out of water and cleaned to
remove external biolouling. Approximately 60 oysters were sub-
sequently transferred to individually numbered clear glass, screw
top beakers with lids (Qorpak; Fisher Scientific) containing 500
niL of artificial seawater (ASW; Instant Ocean"". Aquarium Sys-
tems. Mentor, OH) at 25 ppt (20-22X), and acclimated for at least
12 h prior to bioassays. Containers were continuously aerated by
an in-house air system using a pipette projecting through a hole in
the beaker lid. Oysters that were used in bioassays in late summer
and early fall were potentially ripe but may have spawned out,
whereas those collected at later times were probably completely
spawned out. The majority of bioassay animals used in these stud-
ies were presumed to be males since the largest oysters, which are
typically females, do not fit into the beakers.

Tissue Extraction and furifuation of Oyster Gonad Membranes

The entire visceral mass was removed from sexually mature C.
virginica. and most of the associated mantle, gill, and palp tissue
were excised; the sex was determined by microscopic observation
of gametes, and male gonads were stored at -70"C until they were
used. A flow diagram showing the extraction and purification

715

716

Rice et al

scheme is shown in Figure 1 . The amount of visceral mass tissue
used (range: 3.0-9.5 g) was sufficient to: (1) purify an excess
amount of sperm membranes: and (2) obtain maximal responses in
the bioassay. Visceral mass tissue was homogenized in 100 mL of
ice-cold Buffer A (20 mM Tris-HCl. 2 niM ethylenediaminetet-
raacetic acid |EDTA]. 1 mM phenylmethylsulfonyl fluoride. pH
7.4) using a Polylron homogeni/.cr (Brinkmann), and ccnlrifuged
for 10 mm at .S.OOO x t; (4"C). The resulting low-speed PI pellets
were resuspended hy brief homogeni/ation in 7.5 mL of Buffer A
and centrifuged for 10 min at 5.000 x g (4°C). The combined
low-speed SI supematants were subsequently centrifuged for 60
min at 100.000 x i; (4''C) to generate high-speed P2 pellets (mem-
brane fraction) and S2 supernatants.

Exiraclioii of Extrinsic Membrane Proteins Using Sodium Carltonate

The standard procedure used to selectively remove extrinsic
proteins from membranes using sodium carbonate (pH I 1.5) was
essentially that described by Fujiki et al. (1982). P2 pellets con-
taining male gonad membranes were resuspended by brief homog-
enization in 30 mL of ice-cold 0.1 M sodium carbonate (pH 11. 5)
and incubated for 30 min on ice. Each sample was subsequently
layered on a 3 niL cushion of 0.3 M sucrose. 10 mM Tris-HCl. pH
7.4 in an ultracentrifuge tube and the membranes were pelleted by
centrifugation for 60 min at 1 50,000 x t; to generate high-speed P3
pellets (containing intrinsic membrane proteins) and S3 superna-
tants (containing extrinsic membrane proteins) (Fig. 1 ). These
were stored at 4"C. P3 pellets were resuspended by brief homog-
enization in ASW (25 ppt) immediately prior to bioassay.

In .some experiments. P3 pellets were heated at either 6S"C (30
min) or al lOO'C ( 10 min) prior to bioassay.

Keiluelion. Alkylation. and Trypsin Digestion

To break the disulfide bonds in sperm intrinsic membrane pro-
teins present in the P3 pellets, a procedure was used for denatur-
ation, reduction and alkylation of proteins with 4-vinylpyridine
(4-VP) that was essentially that described by Coligan et al. (1997).

TVlale gonad homogenates

I 5,000s (lOmin)

I ' 1

Fl pellet SI supernatant

5,000 g (lOmin)

PI pellet

SI supernatant (pooled)

100,000 S (60 min)

P2 peUet

S2 supernatant

I

P3 pellet

0.1 M sodium carbonate, pH 11.5 (30 min)
150,000 g (60 min)

S3 supernatant

Bioassay

1

Heat treatment

I

Bioassay

1

Reduction/AII^Iation/Trypsin

I

Bioassay

Kijjurc 1. FloH diagram of the extraction and differential centrifuga-
tion procedures used to purify membrane fractions from male Cras-
sostrea virginica (jonads. The P3 pellets thai Here enriched in sperm
membranes were either bioassayed directly or treated further and
then Itioassayed.

P3 pellets containing male gonad membranes were gently homog-
enized in 10 niL of ice-cold denaturing buffer (6 M guanidine-HCl.
0.5 M Tris-HCl, 2 mM EDTA, pH S.2) to obtain a well-dispersed
suspension, and the solution was bubbled with N^ gas at room
temperature (20-22°C) for 15 min. Following addition of 2-mer-
caploclhaiiol and incubation for an additional 30 min. 4-VP was
added to the solution and incubated in the dark for 60 min. The
reduced and alkylated samples were then dialyzed (Slide-A-Lyzer
Dialysis Cassette: 10 kD MW cutoff: Pierce) against 0.5 M NaCl
overnight al 4°C. The dialysate was subsequently digested with
trypsin (Type III: Sigma; 37°C for 24 h) by established methods
(Coligan et al. 1997) and bioassayed.

Bioassays

Due to the brevity of the reproductive season, most shell clo-
sure assays were performed when oysters were either partially or
completely spawned out. Shell closure assays were performed on
oysters whose valves were open and mantles were extruded, sug-
gesting that filtration was occuning. Beaker lids were carefully
removed to permit sample addition, and aeration was maintained
throughout the assays. P3 pellets were resuspended by brief ho-
mogenization in 10 mL ASW (25 ppt). and 1.0-mL aliquols (i.e.,
one-tenth of one resuspended P3 pellet) were injected proximal to
the incurrcnt siphon of oysters, and each vigorous closure of oyster
valves was visually monitored during the subsequent assay period.

Additional experiments performed outside of the reproductive
season specifically examined whether P3 pellets also stimulated at
least minimal spawning in addition to shell closure. Oysters were
initially observed for 5 min prior to injection of samples and the
number of shell closures was recorded: oysters that exhibited large
numbers of spontaneous shell closures prior to bioassays were not
used. Following addition of sample (resuspended P3 pellet frac-
tions), the number of shell closures was recorded, and material that
was ejected during the 20-min period was examined microscopi-
cally for the presence of eggs or sperm. Since sperm are released
through the excurrent siphon whereas eggs are released through
the incurrent siphon, the direction of gamete release was noted.
When spawning was detected visually and microscopically, oys-
ters were subsequently sacrificed and the sex was confirmed by
examining gonad smears for the presence of sperm or eggs.

Statistics

For each bioassay animal, the number of shell closures was
determined, and the mean (± S-) for all replicates was calculated.
The null hypothesis of equal means (H|| = ji,, = p., = ...p-^jwas
tested using an F statistic generated during an analysis of variance
( ANOVA ) to determine whether a comparison of treatment sample
means was valid. If the null hypothesis was rejected, then a mul-
tiple comparison of the treatment sample means was accomplished
using a Fisher's protected least-square-difference (Fisher's PLSD)
post-hoc analysis.

RESULTS

To confirm and extend previous observations by Galtsoff
( 1938b). freshly isolated sperm were diluted in ASW and found to
stimulate a series of shell closures (shell clapping events; >4 in 5
min) in all cases (h = 3/3), as expected. Furthermore, when
freshly isolated sperm were snap-frozen on dry ice. thawed, and
assayed, the thawed sperm were still capable of stimulating a series
of shell closures (range: 2-4 in 5 min) in all cases (/; = 4/4).

Oyster Spawning Pheromone

717

Importantly, these observations suggest that the factor(s) thai
stimulate shell closure activity during spawning are resistant to at
least one cycle of freezing and thawing. This suggests that male
gonads could be dissected and stored at -7()°C during the repro-
ductive season and thawed for experiments at a later date. After
selective removal of extrinsic membrane proteins from freshly
isolated sperm, aliquots of resuspended high-speed P3 pellets con-
taining intrinsic membrane proteins were assayed. The P3 material
(1 niL = 10<7r of total sample) stimulated shell closure activity
(range: 3^ in 5 min) in 7 of 9 oysters (78%). whereas aliquots of
high-speed S3 supernatants (10% of total volume) containing ex-
trinsic membrane proteins stimulated shell closures in only 2 of 1 1
assays (18%). When male gonad tissue that had been frozen at
-70°C for more than six months was subjected to the same puri-
fication procedure, that is. extraction and selective removal of
extrinsic membrane proteins followed by differential centrifuga-
tion, similar results were obtained: resuspended P3 pellets ( 1 mL
= 10% of total) stimulated vigorous, repetitive shell closures
(range: 3-10 in 3 inin) in 8 of 9 assays (89%). The data indicated
that the activity was due to an intrinsic membrane factor(s) and not
to extrinsic membrane proteins. In control experiments, the solu-
tion that was used to strip extrinsic proteins off of membranes (0. 1
M sodium carbonate, pH 11.5) did not stimulate shell closure
responses in any oysters (;i = 21).

A 20-minute bioassay period was used in all subsequent shell
closure bioassays. The number of shell closures in control assays
using ASW was 1 .2 ± 0.35 (mean ± S-: ii = 32 oysters tested; Fig.
2A). After selective removal of extrinsic membrane proteins from
male gonad membranes, 1-mL aliquots of resuspended P3 pellets
(12 mg protein/mL: 10% of total sample) were found to stimulate
an average of 10.8 ± 3.8 (mean ± S^: n = 60 oysters tested) shell
closures within 20 min (Fig. 2B). After resuspended P3 pellets ( 12
mg protein/mL) were heated at 68°C for 30 min, the number of
shell closures was not significantly different (1 1.4 ± 3.4: mean ±
S-; P = 0.82; n = 5 oy.sters tested; Fig. 2B). However, when
resuspended P3 pellets (12 mg protein/mL) were heated at 100°C
for 10 min, the number of shell closures decreased by 41% (6.4 ±
1.7; mean ±S-:P< 0.05; /; = 10 oysters tested: Fig. 2B). To test
whether the active factor(s) was a protein. P3 pellets were resus-
pended in denaturation buffer, reduced and alkylated, dialyzed.
and digested with trypsin; in this case, the number of shell closures
decreased by 68% (3.5 ± 0.8; mean ± S-: P < 0.0001; n = 58
oysters tested) (Fig. 2B).

Additional experiments that were performed outside of the re-
productive season, when the majority of animals had presumably
already released most or all of their spawn, examined whether P3
pellets also stimulated detectable spawning in addition to shell
closure. After selective removal of extrinsic membrane proteins
from male gonad membranes, aliquots of resuspended P3 pellets
were observed to stimulate spawning in three males (;; = 3 of 80
experiments). In each case, sperm were released through the ex-
current siphon and were visualized microscopically. These results
suggested that an intrinsic sperm membrane protein(s) stimulated
spawning as well as shell closure activity.

DISCUSSION

Mass spawning of oysters in an aquaculture setting requires at
least one male in the tank to stimulate other male and female
oysters to spawn. In some instances, the spawning operation is
delayed for several hours or is not possible at all due to lack of a

o
o

A

B

No Membranes

Membranes

14 -

12 ■

10 .

8

T

6 -

4 -

T

2 -

n

1 " 1

ASW Control Untreated Heat (68 C) Heal(100 C) Red/Alk/Tr

Treatment

Figure 2. An intrinsic factor in sperm menihrane stimulates spawnin;;
in Crassostrea \irf;iiiica: spawning is accompanied by a series of shell
closures (shell clapping) that have been used to assess the presence of
the activity. The graph shows the average number of closures (± .S-) in
animals during a 2(l-min period following exposure to either: artificial
seawater (ASW) alone: or ASW to which treated or untreated mem-
brane fractions (resuspended P3 pellet) were added. (A) The average
number of shell closures is low when oysters are incubated in ASW
alone. (B) The average number was increased when untreated sperm
membranes were added. Heating the membrane fraction before it was
bioassayed reduced the a\erage number. The effect was statistically
significant at UMl t (P < (UI5), but not at 68 f iP = 0.82). Membranes
were also reduced and alkylated to break disulfide bonds, dialyzed,
and then digested with trypsin. The reduction in the mean number of
closures is significant (/' < (l.tKMII), supporting the suggestion that the
factor is an intrinsic protein in the sperm membrane.

single spawning male to trigger general spawning of a tank of
brood oysters. In the British Isles, there is a need for a synthetic or
recombinant pheromone for routine 48-h exotoxicity tests that are
in widespread use there. Presently, commercial hatcheries in the
British Isles purify fresh sperm from C. iiifiii.y Thunberg to stimu-
late spawning for exotoxicity tests, but would prefer to avoid hav-
ing sperm from another oyster in the water for the assays (S.
Steele, Royal Holloway Univ. of London, pers. comni.). The avail-
ability of a synthetic or recombinant oyster spawning pheromone
would: (1) avoid introducing sperm into the water during exotox-
icity tests: (2) allow for a pathogen-free method for inducing
spawning in oysters; (3) allow males and females to be induced to
spawn separately, providing pure gametes for use in genetic ma-
nipulation for selective breeding of oysteis with disease resistance,
fast growth, etc.: and (4) simplify and standai'di/e oyster hatchery
operations.

As a first step toward characterizing the sperm-associated
pheromone responsible for stimulating spawning in oysters, we
used a method for selectively stripping extrinsic proteins off mem-
branes that does not affect the disposition of integral components
such as transmembrane and lipid-anchored proteins (Fujiki et al.
1982). It is the procedure most widely used to extract extrinsic (or
adsorbed) proteins from organelle membranes, and has enabled
investigators working on integral membrane proteins to efficiently
remove soluble contaminating proteins, and to distinguish periph-
eral membrane proteins (those desorbed in the presence of sodium

718

Rice et al

curhon;ite) from integral menibrune proteins (those that remain
membrane-associated following treatment) (Coligan et al. 1997).

Following sodium carbonate extraction (Fujiki et al. 1982) of
freshly isolated oyster sperm membranes, the purified membranes
(P3 pellet) enriched with intrinsic membrane proteins caused vig-
orous, repetitive shell closures in 78% of oysters tested; in con-
trast, the S3 supernatant, which was enriched with extrinsic mem-
brane proteins, stimulated shell closures in only 18% of oy.sters
tested. Similar results were obtained using male gonad tissue
stored at -70"C for more than 6 mo. These initial data suggested
that the sperm-associated pheromone was retained in sperm mem-
branes, and that sodium carbonate treatment did not remove the
shell closure-stimulating activity from sperm membranes.

More extensive experiments were conducted to test the heat
and trypsin sensitivity of the candidate oyster sperm pheromone
(Fig. 2). After removing extrinsic proteins from sperm membranes,
vigorous and repetitive shell closure activity was still present in
purified sperm membranes (P3 pellet fractions). Incubation of
these P3 membranes at lOO^C resulted in a significant reduction in
this activity. Likewise, breaking disulfide bonds in intrinsic sperm
membrane proteins followed by trypsin digestion resulted in a
significant reduction of this activity, suggesting that the active
factor(s) is a transmembrane or lipid-anchored membrane protein
in the sperm membrane.

Little is known about the identity of any invertebrate or verte-
brate water-borne peptide/protein pheromones or their receptors.
Painter and colleagues (1998, 1999, 2000) were the first to deter-
mine the structure and biologic activity of a water-borne peptide
pheromone in invertebrates; the 58-residue attractant ("attractin")
in Aptysia has been cloned (Fan et al. 1997) and its 3-D structure
predicted (Schein et al. 2001). Water-borne peptide pheromones
(e.g., E/--1, Er-2) and their receptors have been best characterized
in the protozoan Eiiplotes raikovi; the Er receptors, which prob-
ably arise by alternative splicing from the same gene for the Er
pheromones, each contain a copy of a pheromone sequence se-
creted by the cell (reviewed in Luporini et al. 1996).

In contrast with soluble peptide/protein pheromones. oyster

spawning presumably involves the direct binding of an intrinsic
sperm membrane-associated protein(s) with membrane-associated
receptors in neighboring oysters. This is based on the observation
that oysters initiate a series of strong, rhythmic contractions of the
adductor muscle in response to sperm entering the mantle cavity
via the incurrent siphon (Galtsoff 1938a, Galtsoff 1938b). Detailed
information regarding the nature of the sperm membrane protein(s)
and its receptor are lacking, however. Therefore, a complete un-
derstanding of the molecular mechanisms underlying spawning
activation will not be achieved until the sperm-associated mem-
brane protein(s) and its receptor are structurally characterized and
cloned.

A molecular mechanism for spawning has been proposed for
other mollusks. It has been demonstrated in abalone that hydrogen
peroxide causes gravid male and female abalones to spawn and
that this effect may result from a direct activation of the enzyme-
catalyzed synthesis of prostaglandin endoperoxide. The latter is a
direct precursor of prostaglandins and thromboxanes: all three may
be involved in regulating spawning in abalone (Morse et al. 1977).
Hydrogen peroxide also induces synchronous spawning in male
and female mussels {Mytihis) and in other molluscan species
(Morse et al. 1977).

Lastly, regardless of the molecular mechanisms underlying
sperm-induced spawning in oysters, the response of female oysters
to sperm is not species-specific; sperm from C. virginica. C. gigtis.
or C. angulata Lamarck have been shown to stimulate spawning in
females of all three species (Nelson 1931, Galtsoff 1932, Galtsoff
& Smith 1932). This suggests that a synthetic or recombinant C
virginica spawning pheromone could be used in the aquaculture
industry to induce spawning in several conmiercially important
species,

ACKNOWLEDGMENTS

The authors thank B. Clough. S. Black, and C. Binz for tech-
nical assistance and an anonymous reviewer for helpful comments.
Supported by Texas Advanced Technology Program grant
()04952-(M02-1999 to G.T.N. , S.D.P., and S.M.R.

LITERATLl

Coligan. J. E.. B. M. Dunn. H. L. Ploegh, D. W. Speicher & P. T. Wing-
field. 1997. Current Protocols in Protein Science. New 'I'ork. NY: John
Wiley & Sons Inc.

Fan, X., B. Wu, G. T. Nagle & S. D. Painter. 1997. Molecular cloning of
a cDNA encoding a potential water-bome pheronional attractant re-
leased during Aplysia egg laying. Mol. Brain Res. 48:167-170.

Fujiki, Y., A. L. Hubbard, S. Fowler & P. B. Lazarow. 1982. Isolation of
intracellular membranes by means of sodium carbonate treatment: Ap-
plication to endoplasmic reticulum. J. Cell Biol. 93:97-102.

Galtsoff P. S. 1930. The role of chemical stimulation in the spawning
reactions of Ostrea virginica and Ostrea gigas. Proc. Ncitl. Acad. Sci.
16:555-559.

Galtsoff P. S. 1932. Spawning reactions of three species of oysters. J.
Wa.'ih. Acad. Sci. 22:65.

Galtsoff, P. S. 1938a. Physiology of reproduction of Ostrea virginica. I.
Spawning reactions of the female and male. Biol. Ball. 74:461—486.

Galtsoff, P. S. 1938b. Physiology of reproduction of Ostrea virginica. II.
Stimulation of spawning in the female oyster. Binl. Bull. 75:286-307.

Galt.soff P. S. 1940. Physiology of reproduction of Ostrea virginica. III.
Stimulation of spawning in the male oyster. Biol. Bull. 78:117-135.

Galtsoff, P. S. & R. O. Smith. 1932. Stimulation of spawning and cross-
fertilization between American and Japanese oysters. Science 76:371.

RE CITED

Luporini. P.. C. Miceli. C. Ortenzi & A. Vallesi. 1996. Ciliate pheromones.
Prog. Mol. Subccll. Biol. 17:80-104.

Morse. D. E.. H. Duncan, N. Hooker & A. Morse. 1977. Hydrogen per-
oxide induces spawning in mollusks, with activation of prostaglandin
endoperoxide synthetase. Science 196:298-300.

Nelson. T. 1 93 1 . Stimulation of spawning in the American oyster by sperm
of the Portuguese oyster. Anal. Rec. 51:48.

Painter. S. D., B. Clough. R. Garden, J. V. Sweedler & G. T. Nagle. 1998.
Characterization of Ap/y.s/a attractin, the first invertebrate peptide
pheromonal attractant. Biol. Bull. 194:120-131.

Painter, S. D., B. Clough. D.-B. G. Akalal & G. T. Nagle. 1999. Charac-
terization of attractin. a pheromonal attractant in .Xplvsin. Invert. Re-
prod. Devel. 36:191-194.

Painter. S. D.. D.-B. G. Akalal. B. Clough. A. J. Susswein. M. Levy & G.
T. Nagle. 2000. Characterization of four new members of the attractin
family of peptide pheromones in .Aplysia. Soc. Neurosci. Ahstr. 26:
1166.

Schein. C. G. T. Nagle. J. Page. J. V. Sweedler, Y. Xu. S. D. Painter & W.
Braun. 2001. Aplysia attractin: Biophysical characterization and mod-
eling of a water-bome pheromone. Biophxsical J. 8 1 :463-472.

JoiinuU of Shellfish Research. Vol. 21, No. 2. 719-723, 2002.

FACTORS AFFECTING SPERM MOTILITY OF TETRAPLOID PACIFIC OYSTERS

QIAOXIANG DONG,' BENOIT EUDELINE," STANDISH K. ALLEN, JR.,' AND
TERRENCE R. TIERSCH'*

^Aqiiacuhure Research Station. Louisiana Agricultural Experiment Station. Louisiana State University
Agricultural Center, Baton Rouge. Louisiana 7OH03: 'Whiskey Creek Shellfish Hatcheiy. Tillamook.
Oregon 97141: ^Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine
Science, Gloucester Point, Virginia 23062

ABSTRACT Factors such as osmotic pressure, extender solution, addition of caffeine, and pH have been shown to affect sperm
motility in aquatic species. We evaluated the effects of 18 osmotic pressures, two extender solutions, seven caffeine concentrations,
and a pH range of 3 to 14 on motility of sperm from tetraploid Pacific oysters, Crassostrea gigas. Motility was highest at 1000
mOsmol/kg (mean ± SD: 83 ± 14%). Calcium-free Hanks" balanced salt solution yielded significantly higher sperm motility than did
artificial seawater. Sperm motility increased with caffeine concentrations to 20 niM (81 ± 12%) and decreased when concentrations
were higher than 50 mM (55 ± 20%). Highest motility was obtained at a pH range of from 4 to 12; values outside this range yielded
no motility. Addition of 10 mM caffeine to the different pH treatments also enhanced motility significantly. Overall, calcium-free
Hanks' balanced salt solution at 1.000 mOsmol/kg. the addition of 10 mM caffeine, and a pH of around 10 could he used to enhance
speiTn motility of tetraploid Pacific oysters. Our findings would assist the use of motility assays to evaluate the effectiveness of various
refrigeration or cryopreservation procedures, especially outside of the peak spawning season, when sperm motility can be low and
variable.

KEY WORDS: Crassostrea gigas. sperm motility. pH. caffeine, osmotic pressure, tetraploid. cryopreservation

INTRODUCTION

MATERIALS AND METHODS

Factors such as osmotic pressure (Bates et al. 1996), extender
composition, pH, temperature (Lahnsteiner et al. 1997, Sunitha
and Jayaprakas 1997), and additives such as caffeine (Scheerer and
Thorgaard 1989, Tiersch et al. 1998) have been shown to affect
sperm motility in aquatic species. Most studies have been con-
ducted on teleosts (e.g.. Moiisawa et al. 1983a. Morisawa 198.^b).
and studies on invertebrates mainly focus on sea urchin (Morisawa
et al. 1990), polychaetes (Pacey et al. 1994), and ascidians
(Yoshida et al. 1992, Yoshida et al. 1994). Little is known about
the effects of these factors on oyster sperm, especially on sperm
from tetraploid oysters, which possess four sets of chromosomes
instead of the noimal diploid two sets.

Tetraploid oysters create opportunities for genetic improve-
ment, including direct production of triplnid (sterile) seedstocks by
crossing with normal diploids. Refrigerated and frozen storage of
tetraploid oyster sperm will be a critical tool for commercial-scale
application of tetraploid stocks and for developing tetraploid
breeding programs. Although subjective, motility estimation is the
technique used most cotnmonly to assess sperm quality of fish and
shellfish (Piironen 1993, Tiersch et al. 1994, Koupal et al. 1995).
Motility has been used to assess the sperm quality of oysters (Pa-
niagua-Chavez et al. 1998), but its application with tetraploid oys-
ter sperm is unexplored. The objective of this study was to develop
procedures for evaluation of sperm quality to assist the overall goal
of sperm storage for tetraploid Pacific oysters, Crassostrea gigas.
Specifically, we evaluated the effects on sperm motility of:
(1) osmotic pressure; (2) extender solution; (3) caffeine, and (4)
pH. Our findings indicate that these factors can alter the motility of
tetraploid oyster sperm collected late in the spawning season. To
our knowledge, this is the first study to systematically characterize
sperm motility of tetraploids of an aquatic species.

♦Corresponding author. E-mail: ttiersch@agctr.lsu.edu

Tetraploid oysters were obtained in September and October
2001 from Whiskey Creek Shellfish Hatchery (WCSH) (Tilla-
mook, Oregon) and were shipped chilled by overnight delivery to
the Louisiana State University Agricultural Center, Aquacullure
Research Station (ARS). Water samples from WCSH had an os-
molality of 873 mOsmol/kg as measured by vapor pressure o.s-
mometry (model 5500, Wescor Inc.. Logan, UT) at the ARS.
Sperm were collected by dry stripping of the gonad (Allen &
Bushek 1992). Undiluted nonmotile sperm were equilibrated in 30
|j.L of test solutions at 23'^C for 2 min before assessment of mo-
tility. Sperm motility was estimated at 200x magnification using
darkfteld microscopy (Optiphot 2, Nikon Inc.. Garden City, NY)
and was expressed as the percentage of cells actively moving in a
forward direction.

Throughout the experiments, two extender solutions were used:
artificial sea water (ASW) (Fritz Super Salt, Fritz Industries, Inc.
Dallas. TX) and calcium-free Hanks" balanced salt solution (C-F
HBSS) (Paniagua-Chavez et al. 1998). All chemicals (except
ASW) were of reagent grade (Sigma Chemical Corporation., St.
Louis, MO). Osmolality was measured with a vapor pressure os-
mometer.

In our first study, the effect on sperm motility of ASW of 18
different osmolalities ranging from 30 to 1400 mOsmol/kg was
evaluated with a total of 20 oysters in four trials, for which oysters
were received on August 24, August 30, September 19, and Sep-
tember 26. The second study compared ASW and C-F HBSS at 13
different osmolalities with five oysters, which were received on
October 16. In the third study, the effect of caffeine was evaluated
at seven concentrations (2 to 100 mM) with eight oysters from two
shipments received on October 10 and October 16. In the fourth
study, a pH range of from 3 to 14, with and without caffeine, was
evaluated with three oysters that were received on October 10.
Sperm from individual oysters was used for all studies (samples
were not pooled). Within this manuscript, extender solutions at

719

720

Dong et al.

specific osmolalities such as ASW at 1 .000 iiiOsriiol/kg are abbre-
viated as ASW 1 ()()().

Data were analyzed using one-way or two-way analysis ot
variance. Tukey's honestly significant difference procedure was
used to test for differences (a = 0.05) among results for osmo-
lalities, caffeine concentrations and pH levels (.SA.S Institute
1991).

RESULTS

Among the osmolalities tested, sperm Irom tetraploid Pacific
oysters remained immotile when diluted with A.SW below 51)0
mOsmol/kg (Fig. I). Motility increased from 12 ± 12% (mean ±
SD) in ASW500 to 50 ±11% in ASW670. There was no signifi-
cant difference among sperm motilities activated with ASW at
670, 700. 750, and 800 mOsmol/kg {P > 0.05). Although sperm
motility was not significantly different among ASW at 900, 950,
1,000. and 1.100 mOsmol/kg. ASWIOOO elicited the highest mo-
tility 83 ± 14% (Fig. 1). Motility decreased significantly when
osmolality was greater than 1,100 mOsmol/kg.

Motility in C-F HBSS was significantly higher than that in
ASW (P < 0.0001) (Fig. 2). Each extender showed the highest
motility at 1,000 mOsmol/kg, which agreed with the results of the
previous experiment. But motility in C-F HBSS 1000 (81 ±9%)
was double that in ASWIOOO (40 ± 22%). Other osmolalities of
C-F HBSS also enhanced spemi motility. For example, motility in
C-F HBSS670 (50 ± 10% I was six times greater than that in
ASW670 (8 ± 13%) (Fig. 2). Because the oysters used in this
experiment were received later in the spawning season than those
used for the first study, lower motilities overall were observed for
ASW.

Based on the results of the second experiment. C-F HBSS at
1 ,000 mOsmol/kg was used for caffeine assessments. Motility in-
creased with caffeine concentrations of from 2 mM (60 ± 13%) to
20 niM (81 ± 12%) and decreased when concentrations were
higher than 50 niM (55 ± 20%) (Fig. 3). The lowest motility (49 ±
10% I was in the control treatment: C-F HBSS 1000 without the
addition of caffeine, but additions of 2, 4. and 6 niM caffeine were

100

100

o

80

60

40

20

ghi

■-■^■1

gh

ooooooooooor^oooooo
cooooooor^oiflor^omoooo

Osmolality (mOsmol/kg)

Figure 1. Percent motility (mean ± SI)) of tetraploid Pacific oyster
sperm activated in artificial seawaler at 18 different osmotic pressures.
Bars sharing a letter were not significantly dilierent (/' > 0.(15).

O

Osmolality (mOsmol/kg)

Figure 2. Percent motility (mean ± SD) of tetraploid Pacific oyster
sperm activated in .\S\\ (filled bars) and C-F HBSS (open bars). Bars
with an asterisk indicate a significant difference {P > (1.05) between
ASW and C-F HBSS.

not significantly different from the control (P > 0.05). Motilities in
10 and 20 niM caffeine were significantly higher than in other
concentrations (P < 0.05), but they were not different from each
other iP > 0.05) (Fig. 3).

Based on these lesults, C-F HBSS 1000 with and without 10
mM caffeine were used for pH assessment. Motility was highest at
a pH lange of from 4 to 12: values outside this range yielded no
motility (Fig. 4). The highest motility was in pH 10.5 (67 ± 6%),
followed by pH 10 (63 ± 6%). The addition of 10 mM caffeine to
these pH treatments enhanced motility significantly (P < 0.001)

100
80

g 60

I"

'^
O
S 40

20

a 3

H H I I

4 6 10 20

Caffeine (mM)

50

100

Figure 3. Percent motilit> (mean ± SD) of tetraploid Pacific oyster
sperm activated with the additicm of caffeine into C-F HBSS at 1.0(10
m()smol/kg. Bars sharing a letter were not significantly different iP >
().()5).

Motility of Tetraploid Oyster Sperm

721

o

00
80

* *
II T

.

ih\]

60

1 Jll

f-

* *

40

•

20
n

T

CO oi^o^T^^*^^

pH

Figure 4. Percent motility (mean ± SDl of tetraploid Pacific oyster
sperm in C-F HBSS at 1,(100 mOsmol/kg with different pH treatments.
Open bars, C-F HBSS without addition of caffeine; filled bars, C-F
HBSS with lU mM caffeine. Bars with an asterisk indicate a significant
difference (P > 0.05) between presence and absence of caffeine.

(Fig. 4). Motility was 90 ± 0% in pH II. 87 ± 6% in pH 10.5. and
88 ± 3% in pH 10. However, there was no significant difference in
sperm motility across the pH range of from 7 to 12 (P > 0.05).

DISCUSSION

Sperm activation is a complex process in which roles are
played by many factors, including osmotic pressure, extender com-
position, membrane structural changes, and extracellular and in-
tracellular pH. Previous studies have suggested that different spe-
cies have different sperm activation mechanisms. Basically, in fish
there seem to be two major factors influencing sperm activation:
changes of osmotic pressure and changes of ionic concentration.
For instance, studies on muskellunge Esox inasqiiinongy (Lin &
Dabrwoski 1996), channel catfish ktalunis piinclatiis (Bates et al.
1996), and Asian catfish Clarias macrocephalus (Tan-Fermin et
al. 1999) suggested that sperm motility was initiated by a reduction
of osmotic pressure. However, activation of sperm from salmonids
and cyprinids was caused by changes in concentrations of ions
such as potassium and calcium (Morisawa et al. 1983a, Billard &
Cosson 1992). The mechanism of initiation of sperm motility has
been most studied in salmonids although much remains unknown,
especially at the intracellular level.

Motility studies in other taxa such as invertebrates are limited
and the associated mechanisms remain largely unknown. There are
less than 40 references addressing oyster sperm motility in any
way since 1970. Most use motility as a criterion to evaluate fer-
tilization or in toxicological assays to evaluate waste eftluents or
heavy metals. Factor that affect sperm motility such as osmolality,
extender composition. pH, and temperature are briefly mentioned
in these studies and others on sperm cryopreservation, and there-
fore the information is fragmented and dispersed. The present
study was designed to address factors affecting the sperm motility
of tetraploid Pacific oysters and provided a more detailed and
systematic approach.

Osmolality, as mentioned above, plays an important role in the

activation of fish sperm. Osmolalities of -1000 mOsmol/kg (the
full-strength salinity of sea water. 32 ppt) and 670 mOsmol/kg
were previously used for sperm activation and extender solutions
for C. gigas (Yankson & Moyse 1991. Kurokura et al. 1990).
Other species were studied with sea water of ambient environmen-
tal salinity. The first investigation of sperm motility across a wide
range of osmolalities was with the diploid eastern oyster. C. vir-
ginica (Paniagua-Chavez et al. 1998). High sperm motility (90%)
was observed across a range of 600 to 1,500 mOsmol/kg (Pa-
niagua-Chavez & Tiersch 2001). In the present study, sperm mo-
tility of tetraploid Pacific oysters was also observed across a wide
range of osmotic pressures (500-1400 mOsmol/kg). but the high-
est motility was limited to 1.000 mOsmol/kg. although the oysters
were conditioned in seawater at 873 mOsmol/kg. Whether osmotic
pressure plays the major role in oyster sperm activation remains
unknown: however, motility was suppressed in tetraploid sperm of
C. gigas at osmolalities of lower than 500 mOsmol/kg. Suppres-
sion was reported to occur at less than 22 mOsmol/kg in C. vir-
ginica (Paniagua-Chavez et al. 1998). This might correspond to the
habitat of these species in natural environments. Crassostrea gigas
prefers higher and more stable salinities (15 to 33 ppt) (Kusuki
199! ) than does C. rirginica. which normally occurs from 5 to 40
ppt (Galtsoff 1964, Wallace 1966).

Natural seawater and ASW are most commonly used as ex-
tender solutions for spenn of marine organisms including estuarine
organisms like oysters. Other extenders such as DCSB4 (Bougrier
& Rabenomanana 1986), HBSSI990 (Zell et al. 1979), and C-F
HBSS640 (Paniagua-Chavez et al. 1998) with specific ionic com-
positions have also been successfully used for oyster sperm. The
removal of calcium from HBSS was found to enhance motility in
sperm of diploid eastern oysters (Paniagua-Chavez et al. 1998) and
tetraploid Pacific oysters (this study) and was also superior to
ASW. Contrary to the situation in salmonids. in which sperm
motility is initiated by a decrease in potassium concentration upon
release into fresh water (Morisawa et al. 1983a). an increase of
potassium concentration (200 mM) was found to have an activat-
ing effect on diploid Pacific oyster sperm (Faure et al. 1995). The
same study showed no effect of the increase of potassium on sperm
of the king scallop Pecten nui.xinuis. but an increase in motility was
observed with media lacking sodium. These results may indicate a
species-specific response for ionic effects on bivalve sperm mo-
tility. Also, changes in ion concentration, rather than absolute con-
centration, may act as the trigger of initiation of sperm motility.

Caffeine has been used as motility stimulant to optimize the
recovery and quality of thawed spermatozoa in mammalian species
(Correa & Zavos 1996. Park & Sirard 1996). Few studies have
addressed use of this chemical in sperm of aquatic species and
there are no reports for oysters. The present experiment showed a
significant increase in sperm motility with the addition of 10 mM
caffeine, while concentrations above 50 niM reduced motility, A
previous study in the razorback sucker Xyraiichen texamts showed
increased motility in refrigerated sperm after the addition of 5 mM
caffeine, but not in thawed sperm (Tiersch et al. 1998). We did not
evaluate the addition of caffeine with thawed sperm in this study,
but fertilizing capacity of cryopreserved semen of rainbow trout
Oncorhynchus mykiss was improved when eggs were fertilized in
a buffered saline solution containing 5 mM theophylline, a chemi-
cal relative to caffeine used to prolong and intensify sperm motility
(Scheerer & Thorgaard 1989).

Alkaline pH has been found to be conducive to sperm activa-
tion in aquatic species (Thorogood & Blackshaw 1992. Sunitha &

722

Dong et al.

Jayaprakas 1997. Ciereszko et al. 2001 ). and pH value.s between 7
and 9 have been used in most studies for oyster sperm (e.g.. Zell
et al. 1979, Paniagua-Chavez et al. 1998). Study of the king scallop
(Faure 1996) found that gonadal pH was more acidic than seawater
pH and suggested that the acidity of the genital tract maintained
the spermatozoa in a quiescent state. A reduction of sperm motility
was reported in P. maximus and C. gigas upon decrease of pH in
seawater (Faure 1996). In the present study, pH values below 7
induced a significant reduction in spenn motility although there
was variation among individual oysters. The highest sperm motil-
ity was observed at a pH of 10, which agrees with findings for the
Japanese pearl oyster Pinctada fucata (Yu et al. 1998). although
that study found an interaction between salinity and pH. In that
study, sperm motility could not be activated by seawater at a
salinity of 30 ppt and a pH of 8.0, but motility was greater than
80% when pH ranged from 9.0 to 11.5. Sperm from tetraploid
Pacific oysters showed motility over a wider pH range in the
present study. It appears that relative influence of most factors
affecting sperm motility of bivalves are species-specific.

Finally, the main practical interest in tetraploidy is for the
production of triploids by mating with diploids. The induction ot

tetraploidy in oysters was first reported in 1994 (Guo and Allen.
1994). Studies of the factors affecting sperm motility of tetraploid
oysters have only now begun as reported here. Our experiments
indicate that use of C-F HBSS at 1,000 mOsmol/kg as an extender,
the addition of 10 mM caffeine, and a pH of around 10 can be used
to enhance sperm motility of tetraploid Pacific oysters. This would
assist the use of motility assays to evaluate the effectiveness of
various refrigeration or cryopreservation procedures, especially
outside of the peak spawning season when sperm motility can be
low and variable such as in this study. The effectiveness of these
conditions in improving fertilization rates requires further study.
Future research is required to evaluate differences between sperm
of diploid and tetraploids within and among aquatic species.

ACKNOWLEDGMENTS

The authors thank Y. Li for laboratory assistance and J. Dock-
stader for help with data analysis. This study was supported by
funding from the USDA-SBIR program and the Louisiana Sea
Grant College Program. This work was approved for publication
by the Director of the Louisiana Agricultural Experiment Station
as number 02-66-04.37.

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.fnurnal of Shcllfi'ih Rfsi-arch. Vol. 21. No. 2. 725-732. 2002.

AN IMPROVED PROCEDURE TO COUNT PERKINSUS MARINUS IN EASTERN

OYSTER HEMOLYMPH

AMY D. NICKENS,' - JEROME F. LA FEYRE,'* ERIC S. WAGNER,'
TERRENCE R. TIERSCH'

^ Cooperative Aquatic Animal Health Research Proi^ram. Department of Veterinary Science. Louisiana
State University- Agricultural Center. Ill Dalrymple Building. Baton Rouge. Louisiana 70803:
'Acjuaculture Research Station. Louisiana Agricultural. Experimental Statiim. Louisiana State University
Agricultural Center. 2410 Ben Hiir Road. Baton Rouge. Louisiana 70820

ABSTRACT Perkitisiis marimis infeL'tion intensity in Crassostrea virginica can be quantified without killing of oysters by deter-
mining parasite density in hemolyniph samples incubated in fluid thioglycollate medium (FTM). The goal of this study was to improve
existing protocols for counting of P. inarimis in oyster hemolymph. Specifically, the objectives were to examine the effects on parasite
number and diameter of: 1) adding supplements to FTM such as lipid and oyster extract; 2) incubating with various FTM preparations
with and without agar or beef extract; 3) incubating with various hemocyte densities (lO*^. 10'', and 10' hemocytes/mL of FTM) in a
constant FTM volume; 4) incubating with different volumes of FTM (0.2 mL, 1 .0 mL, 5.0 mL. and 25.0 mL); and 5) sodium hydroxide
digestion of cellular debris. From these results, an improved hemolymph protocol was developed. The diameters and numbers of
enlarged parasites or hypnospores in hemolymph of 20 oysters measured by the improved protocol and the standard FTM hemolymph
assay of Gauthier and Fisher were compared. Finally, the standard and improved protocols were compared with the FTM body burden
assay. The diameter of hypnospores from samples processed with the improved protocol (26 ± 13 |a.m) was significantly greater than
the diameters from samples processed with the standard protocol ( 10 ± 4 (jim). The number of hypnospores in samples processed with
the improved protocol (8.6 x 10' + 3.3 x 10') was significantly greater than the numbers in samples processed with the standard
protocol (1.9 X 10' ± 3.4 x 10'). Results of the body burden assay were significantly correlated with results of the standard hemolymph
assay and with results of the improved hemolymph assay. The coefficient of determination (r = 0.7602) and slope (O.yi 189) of the
regression of the FTM body burden assay against the improved FTM hemolymph assay was improved from the coefficient of
determination (0.5543) and slope (0.61257) of the regression of the FTM body burden assay against of the standard FTM hemolymph
assay.

KEY WORDS: dermo. Perkinsus iiuiriim.s. FTM hemolymph diagnostic assay. Crassosuea virginica

INTRODUCTION

Use of a protociil Id quantify the presence of the pathogenic
protozoan Perkinsus marums by determining the number of en-
larged parasites, or hypnospores. in hemolymph samples of eastern
oysters. Crassostrea virginica. allows estimation of infection in-
tensity without killing oysters (Gauthier & Fisher 1990). There-
fore, infection intensity in the same animal can be monitored over
time. In the existing protocol, hemolymph from the adductor
muscle sinus is removed, centrifuged. and the cell pellets contain-
ing oyster hemocytes and parasites are incubated in fluid thiogly-
collate medium (FTM) for 1 week. After incubation, cellular de-
bris is digested with sodium hydroxide (NaOH). The hypnospores
are stained with LugoFs solution and their numbers per mL de-
termined. This FTM hemolymph assay has been useful to follow
the progression of the disease in individual oysters under different
environmental conditicnis (Fisher et al. 1992. Ragone Calvo &
Burreson 1994).

Gauthier and Fisher ( 1990) proposed additional advantages of
the FTM hemolymph assay, including the detection of early in-
fections and the measurement of systemic infections rather than
localized infections. In their initial study, the FTM hemolymph
assay detected many infections misdiagnosed as negative by Ray's
FTM tissue assay. In Ray's FTM tissue assay, a piece of oyster
tissue (e.g.. mantle tissue in Gauthier and Fisher's 1990 study) is
incubated in FTM for about a week, the tissue is then smeared on
a slide, and the parasites stained with Lugol's solution and the
intensity of infection are estimated using a semi-quantitative scale

♦Corresponding author. E-mail; jlapeyre(a>agctr.lsu.edu

(Ray et al. 1953, Ray 1954a, Ray 1954b). Bushek et al. (1994),
however, found no evidence that the FTM hemolymph assay was
more sensitive than the FTM tissue assay in detecting low P.
inarimis infections when both mantle and rectal tissues were used
in the tissue assay. Moreover, correlations between FTM tissue
and fTM body burden assays were always higher than correlations
between FTM hemolymph and FTM body burden assays. The
body burden assay measures the number of parasites in the whole
oyster and is considered the most sensitive and accurate diagnostic
assay for P. marinus (Bushek et al. 1994, Fisher & Oliver 1996,
Oliver et al. 1998).

A quantitative competitive polymerase chain reaction
(QCPCR) assay for P. marinus was recently shown to be more
sensitive than the FTM hemolymph assay, detecting infections in
24 oysters compared with 22 infections with the FTM hemolymph
assay (Yamall et al. 2000). Diagnosis of P. marinus in oyster
hemolymph by QCPCR detected as many infections as with the
FTM body burden assay, suggesting that hemolymph is suitable
for determining P. marinus infection in oysters. Whereas QCPCR
is an effective assay and specific for P. marinus. it is also expen-
sive and requires technical expertise and equipment in molecular
biology to perform as indicated by Yarnall et al. (2000). The ITM
hemolymph assay in contrast is easy and inexpensive to perform.
There is thus a need to improve the sensitivity and accuracy of the
FTM hemolymph assay.

Potential problems with FTM assays have been previously
noted and include insufficient parasite enlargement, parasite
clumping, and parasite adherence to the walls of centrifuge tubes,
each of which can lower parasite counts (Bushek et al. 1994,
Fisher & Oliver 1996). Poor parasite enlargement may be caused

725

726

NiCKENS ET AL.

by exhaustion of necessary nutrients in FTM or tissue (Ray 1954a,
Bushek et al. 1994, Fisher & Oliver 1996). In addition, the pro-
tocols used in FTM hemolymph assay varied greatly among re-
searchers in sample preparation (e.g., centrifugation speed),
sample processing, and in parasite counting protocols (Gauthier &
Fisher 1990, Ragone Calvo & Burreson 1994, Bushek et al. 1994.
Oliver et al. 1998). It is likely that these recognized problems and
differences in protocols account for the poor performance (i.e.,
sensitivity and accuracy) of the FTM hemolymph assay.

The goal of this study was to improve the protiicol for counting
of P. marinus in oyster hemolymph through systematic examina-
tion of components of the standard protocol described by Gauthier
and Fisher (1990). The objectives were to examine the effects of:

( 1 ) adding supplements to FTM, including lipid and oyster extract;

(2) adding various FTM preparations (with and without agar or
beef extract); (3) incubating with various hemocyte densities (10\
10", and 10^ hemocytes/mL of FTM) in a constant FTM volume;
(4) incubation of hemocytes in different volumes of FTM (0.2 mL,
1.0 niL, 5.0 mL, and 25.0 mL); and (5) NaOH digestion of cellular
debris on hypnospore diameter and number.

MATERIALS AND METHODS

Oysters

Eastern oysters were collected from Hackberry Bay |29°23'54"N,
90°28"W) in the spring of 1999, placed in 16 mm mesh shellfish
cages (Aquatic Eco-Systems, Inc., Apopka, FL). and hung in the
water from docks at the Louisiana Sea Grant Oyster Hatchery
(29°12'30"N, 90°02'30"W) in Grand Isle, Louisiana, an area en-
zootic for P. marinus. In the summer and fall of 1999, oysters were
transported to the Department of Veterinary .Science at Louisiana
Slate University, Baton Rouge, and were placed in an indoor re-
circulating system equipped with 1 p.m and 10 jxm cotton filters in
polypropylene filter cartridges and an ultraviolet light. Water was
maintained at 15 ppt with hw-Marinemix Professional sea salts
(Hawaiian Marine Imports Inc., Houston. TX) and 25"C.

General Procedures

All chemicals were from Sigma Chemical Co. (St. Louis, MO)
unless otherwise indicated. A notch was ground into the edge ot
oyster shells near the adductor muscle and 3 mL of hemolymph
were withdrawn from the adductor muscle sinus. Hemolymph
samples were transferred to 3 mL snap-cap tubes and immediately
placed on ice to prevent hemocyte clumping. The number ot
hemocytes per mL of hemolymph was determined using a Neu-
bauer Bright-Line hemocytometer (Reichert, Buffalo, NY).
Hemolymph samples from each oyster, containing 10" hemocytes,
were added to 1.5 mL microcentrifuge tubes, centrifuged at 800 x
g for 10 min, and the supernatant was discarded. All samples were
prepared in triplicate. Pellets were resuspended in 1 mL of alter-
native fluid thioglycoUate medium (AFTM) (Sigma number A
0465) prepared according to the manufacturer's instructions and
supplemented with 16 g/L of hw-Marine Professional sea salts and
50 jjLg/mL chloramphenicol. This solution, referred to as AFTM
throughout this report, did not contain agar, unlike FTM (Sigma
number T 9032), which contains 0.75 g/L of agar. Samples were
layered with 10 ixL of nystatm (5,000 Units/niL) to prevent fungal
growth. Test tubes were stored in the dark at room temperature for

seven days, allowing parasites within hemocytes to enlarge to
hypnospores. After incubation, samples were centrifuged at 1500 x
g for ten min and the AFTM supernatant was discarded. Pellets
were resuspended in 1 mL of 2 N NaOH and incubated in a 60°C
water bath for 1 to 2 h to digest hemocyte debris. Samples were
centrifuged to remove NaOH and hypnospores were rinsed three
times with 0.1 M phosphate-buffered saline (PBS) containing 0.5
mg/niL bovine serum albumin (BSA). In a previous study, it was
found that BSA decreased parasite clumping and improved para-
site recovery by reducing the number of parasite that adhered to
the test tube walls (Coates et al. 1999). Samples were stored at 4''C
in 1 mL of PBS supplemented with BSA and 2 mg/mL of sodium
azide. At the time of counting, samples were centrifuged, and 900
p,L of supernatant were removed. Each 100-p,L sample was trans-
ferred to a separate well of a 96-well tissue culture plate. Fifty
microliters of each sample was transferred to wells containing 50
|xL of PBS with BSA to form a 1 ; I dilution. Samples were serially
diluted in this manner until a 1;64 dilution was reached. Lugol's
solution (50 |jiL; 0.012 g/mL of potassium iodide and 0.008 g/mL
of iodine in water) was added to each well to stain the samples.
Tissue culture plates containing samples were centrifuged at 200 x
g for 5 min to form a monolayer of hypnospores on the plate
bottoms to facilitate counting. Numbers of hypnospores were re-
corded at 200-X magnification using an inverted microscope (Cari
Zeiss, Inc., Thornwood. NY) from wells containing 100 to 400
hypnospores. Cells were counted at 400x when they were too
small to identify at 200x. The diameter (jji.m) of 50 hypnospores
from each sample was measured with an ocular micrometer, and
the number of hypnospores per 10" hemocytes was calculated.

Experiments

Effects of Supplemented AFTM on Hypnospore Diameter
and Number

Hemolymph was collected from five oysters as described
above. AFTM, supplemented with 5% lipid concentrate (Gibco,
Gaithersburg, MD), 5% oyster extract, or 5% oyster saline, was
added to 10" hemocytes from each oyster. Oyster extract was
prepared by homogenizing whole oyster tissue in oyster saline at a
concentration of 0.2 g wet tissue per mL, centrifuging at 10,000 x
g for 1 5 min, removing the oyster extract (supernatant), and storing
h at -20°C. Oyster saline (0.95 g/L CaCl,-2H,0, 1.46 g/L MgSO^,
2.18 g/L MgCU-6H,0, 0.67 g/L KCl, 11.61 g/L NaCl, and 0.35
g/L NaHCO,) was included as a control. Samples were incubated,
processed, counted, and measured as de.scribed above. Lipid was
found to increase hypnospore diameter and was added to FTM in
all further experiments.

Effect of FTM Types on Hypnospore Diameter and Nunilier

Hemolymph was collected from 15 oysters as described above
and five formulations of FTM, supplemented with 16 g/L of hw-
Marine Professional sea salts, 50 |j.g/mL chloramphenicol, and 5%
lipid concentrate, were added to 10" hemocytes samples. The for-
mulations compared were Bacto tluid thioglycoUate medium (Bee-
ton Dickinson and Company, Franklin Lakes, NJ. Difco number
0256154), Bacto thioglycoUate medium with K agar (Difco num-
ber 0607 178), Bacto fluid thioglycoUate medium with beef extract
(Difco number 0697179), alternative fluid thioglycoUate medium
(Sisimu number A 0465), and thioglycoUate medium (Sigma num-

Improved Hemolymph Procedure to Count P. marinus

727

ber T 9032) (Table 1). Samples were incubated, processed, mea-
sured, and counted as described above.

Effect of Heniocyte Density on Hypnospore Diameter and Number

Hemolymph was collected from 15 oysters as described above
and 10^, 10'\ and 10^ hemocytes from each oyster were incubated
in I mL of AFTM supplemented with 5*^^ lipid concentrate. For
this experiment, the number of hypnospores per hemocyte was
calculated, and hypnospore diameter was measured as described
above.

Effect of AFTM Volume on Hypnospore Diameter and Number

Hemolymph was collected from 15 oysters as described above.
Hemocytes (10") were incubated in 0.2. 1.0, 5.0, or 25.0 mL of
AFTM supplemented with 5% lipid concentrate. Samples were
processed, measured, and counted as described above.

Effect of NaOH Digestion of Samples on Hypnospore Diameter
and Number

Hemolymph samples from 15 oysters were processed with and
without NaOH digestion after incubation in AFTM supplemented
with 5% lipid concentrate. Samples processed without NaOH di-
gestion were centrifuged at 1500 x g for 10 min to remove the
AFTM supernatant. Cell pellets were rinsed three times with sterile
artificial seawater (hw-Marinemix Professional) at 15 ppl and con-
taining 0.5 mg/niL BSA. Samples processed with NaOH digestion
were treated as described in the General Procedures section.

Effect of Protocol on Hypnospore Diameter and Number

From the results of the above experiments, an improved pro-
tocol for counting of P. marinus in oyster hemolymph was devel-
oped and compared with the "standard" protocol of Gauthier and
Fisher (1990). Major differences between the standard and im-
proved protocols are summarized in Table 2. Briefly, hemolymph
samples were collected from 20 oysters infected with P. marinus.
Hemolymph samples were divided into two equal aliquots, one
aliquot to be processed with the standard protocol and the other to

TABLE 2.

Differences between the standard protocol and the improved
protocol to count Perkinsus marinus in oyster hemolymph.

Component

Standard

Improved

Type of FTM
FTM Supplement
Rinsina solution

FTM (with agar)
None
Distilled water

Alternative FTM (without agar)
5% lipid concentrate
Phosphate-buffered saline
(0.1 M) with 0.5 mg/mL
bovine serum albumin

be processed with the improved protocol. Hemocytes (10") from
samples processed with the standard protocol were incubated for
seven days in I mL of FTM (Sigma number T 9032) supplemented
with 16 g/L of hw-Marinemix Professional sea salts, 50 p.g/mL of
chloramphenicol, and layered with 10 p.L of nystatin (5000 Units/
ml). Hemocyte debris was digested with 1 mL of 2 N NaOH, and
hypnospores were rinsed three times with 1 mL of distilled water.
Samples were centrifuged, 900 p,L of supernatant were removed,
and each 100-p.L sample was transfened to a separate well of a
96-well plate, where it was serially diluted in distilled water and
stained with LugoPs solution. Standard protocol centrifugation
speeds were increased to the centrifugation speeds of the improved
protocol to exclude their effects on parasite recovery. Hemocytes
(10") from samples processed with the improved protocol were
incubated for seven days in 1 ml of AFTM (Sigma number A
0465) supplemented with 5% lipid, 16 g/L of hw-Marinemix Pro-
fessional sea salts, 50 p.g/mL of chloramphenicol, and layered with
10 p-L of nystatin (5000 Units/mL). Hemocyte debris was digested
with 1 niL of 2 N NaOH and hypnospores were rinsed once with
1 mL of distilled water supplemented with 0.5 mg/mL BSA and
twice with I mL PBS supplemented with 0.5 mg/mL BSA.
Samples were centrifuged, 900 p,L of supernatant were removed,
and each 100-p,L sample was transferred to a separate well of a
96-well plate, where it was serially diluted in PBS supplemented
with BSA and stained with Lugol's solution. After processing
samples with both protocols, the number of hypnospores was
counted 200x magnification in wells containing 100 to 400 hyp-

TABI.E 1.
Components of different brands of fluid thioglycollale media.

Difco"

Sigma"

Bacto FTM With
Potassium Agar

Bacto FTM With
Beef Extract

Component

Bacto FTM

Alternative FTM

FTM

Beef extract

5g

—

—

Yeast extract

5g

5g

5g

5.00 g

5.0 g

Casein digest

15 g

15g

15g

15.00g

15.0 g

Dextrose

5.5 g

5g

5.500 g

5.50 g

5.5 g

Sodium chloride

2.5 g

—

2.5 g

2.50 g

2.5 g

Potassium chloride

—

2.5 g

—

—

—

L-Cystine

0.5 g

0.5 g

0.5 g

0.50 g

0.5 g

Sodium thioglycollate

0.5 g

0.3 mL"

0.5 g

0.50 g

0.50 g

Agar

0.75 g

0.45 g

0.75 g

—

0.75 g

Resazurin

0.001 g

0.00! g

0.001 g

—

0.001 g

' Decimal places reported as suggested by manufacturer.
' Thioglycollic acid.

728

NiCKENS ET AL.

nospores after dilutions. The diameter of 30 hypnospores was mea-
sured from each hemocyte sample.

Comparison of Standard and Improved FTM Himolymph Assay
Against FTM Body Burden Assay

The standard and improved FTM protocols for counting P.
marinus in oyster hemolymph (as described above) were compared
with the FTM body burden assay for determining the number of
parasites in whole oyster and a regression line was calculated for
each comparison. The regression lines were compared with deter-
mine if infection intensities obtained from the improved
hemolymph protocol were more closely correlated with infection
intensities obtained from the body burden assay than those ob-
tained from the standard hemolymph protocol.

The 20 oysters from which hemolymph was removed for com-
parison of the standard and impriived hemolymph protocols, were
processed using modification of the body burden assay of Fisher
and Oliver (1996) (Coates et al. 1999). Brietly. each oyster was
removed from the shell, blotted dry. and homogenized with a
hand-held Biohomogenizer, a stainless-steel rotor/stator emulsify-
ing instrument. {Biospec Products, Inc.. Batlesville. OK. catalog
number 1 28 1 ) in 20 niL of sterile artificial seawater prepared at 15
ppl with hw-Marine Professional sea salts. One milliliter of ho-
mogenate was transferred to 9 mL of AFTM (Sigma number A
0465) supplemented with 16 g/L of hw-Marinemix Professional
sea salts, 50 jjig/mL of chloramphenicol 5% lipid, and layered with
100 jiL of nystatin (5000 Units/mL), and incubated for seven days.
Samples were centrifuged. the supernatant removed, and 10 mL of
2 N NaOH added for 4 to 5 h at 60°C to digest the oyster tissue.
Hypnospores were rinsed once with 10 mL of distilled water
supplemented with 0.5 mg/mL BSA and twice with 10 mL PBS
supplemented with 0.5 mg/mL BSA. Samples were centrifuged, 5
niL of supernatant were removed, 50 (jiL of each sample were
transferred to a separate well of a 96-well plate, where it was
serially diluted in PBS supplemented with 0.5 mg/mL BSA and
stained with Lugol's solution. The number of hypnospores was
counted (200x magnification) in wells containing 100 to 400 hyp-
nospores after dilutions. The number of hypnospores per gram of
oyster tissue was calculated for each oyster and compared with the
number of hypnospores per 10** hemocytes determined by the stan-
dard and improved hemolymph protocols using linear regression
analysis.

Slalislical Analysis

Statistical analysis was pertoniied using SAS Version 8.0 soft-
ware (SAS Institute, Inc. Gary, NC). Only hemolymph samples
containing at least 50 hypnospores (per triplicate) were used data
analysis. Data were log transformed and analyzed with a random-
ized block design (blocked by oyster). Tukey's post-ANOVA test
was used to examine differences among treatments. Differences
were considered significant at P < 0.05. All data were reported as
mean ± standard deviation. To compare the standard and improved
FTM hemolymph assays against the FTM body burden assay, two
linear regressions were calculated using log-transformed data.

RESULTS

Effects of Supplemented AFTM on Hypnospore Diameter and Sumher

The diameter of hypnospores from samples incubated in AFTM
supplemented with lipid concentrate (27.3 ± 11.5 (Jtm) was sig-

nificantly greater (P < 0.0001 ) than the diameter of hypnospores
from samples incubated in AFTM supplemented with oyster ex-
tract (18.8 ±9.4 (Jim) or with saline (16.5 ± 10.2 jim) (Fig. I). No
significant difference in hypnospore diameter was found between
samples incubated in AFTM supplemented with oyster extract or
with saline. No significant difference was found in the hypnospore
numbers between samples incubated in AFTM supplemented with
lipid concentrate (1.9 x 10^ ± 7.1 x lO"*), oyster extract (1.8 x 10^
± 4.7 X 10^) or the saline control (2.2 x 10'^ ± 6.6 x 10"*).

Effeet of FTM Types on Hypnospore Diameter and Sumher

No significant differences were found for h\ pnospore diameter
or hypnospore number among samples incubated in different for-
mulations of FTM (Table 3). The absence of agar in the Sigma
alternative fluid thioglycollate medium facilitated sample process-
ing because the viscous layer that generally collected over hypno-
spore pellets after centrifugation when samples were incubated in
FTM with agar was eliminated.

Effect of Hemocyte Density on Hypnospore Diameter and Sumter

The diameter of hypnospores from samples that received 10'
hemocytes per ml of AFTM (19.9 ± 8.8 p.m) was significantly
greater (P < 0.001 ) than the diameter of hypnospores from samples
that received 10" hemocytes per ml (15.3 ± 8.7 p.m) and 10^
hemocytes per ml (15.2 ± 12.1 p-m) (Fig. 2). No significant dif
ference was found in hypnospore diameters between samples that
received lO*" hemocytes per ml and lO' hemocytes per ml. No
significant difference was found in the number of hypnospores per
hemocyte among samples that received 10'' hemocytes per ml
(0.06 ± 0.15 hypnospores/hemocyte), 10'" hemocytes per ml (0.67
± 2.26 hypnospores/hemocyte) and 10^ hemocytes per ml (0.03 ±
0.07 hypnospores/hemocyte).

Effect of AFTM Volume on Hypnospore Diameter and Number

The diameter of hypnospores from samples that were incubated
in 5.0 mL (24 ± 12 (jim) and 25.0 mL (28 ± 13 fj.m) of AFTM was
significantly greater (P < 0.0001 ) than the diameter of hypnospores
from samples incubated in (1.2 mL ( 13 ± 6 p.m) and 1.0 mL ( 16 ±
8 |jim) of AFTM (Fig. 3A). However, the number of hypnospores
in samples incubated in 25.0 mL of AFTM (4.5 x K)-* ± 6.9 x lO^*)
was significantly lower iP < 0.0002) than the number of hypno-
spores in samples incubated in 0.2 mL (9,3 x lO"* ± 1.1 x 10"), 1.0

Saline Lipid Oyster Extract

Figure 1. Hypnospore diameter (mean ± SD. »! = 5) after incubation in
AFTM supplemented with lipid concentrate, oyster extract, and saline
(control). Hemocytes (10") were incubated for 7 days in 1 niL of
supplemented AFTM. Treatments sharing a letter were not signifi-
cantl> different.

Improved Hemolymph Procedure to Count P. marinus

729

TABLE 3.

Average values (mean + SD, n = 9| of hypnospore diameter and

number of hypnospores per 10" hemocytes after incubation in

different types of FTM.

Type of FTM

Hypnospore
Diameter (^ml

Number of
Hypnospores

Difco Bacto FTM

Difco Bacto FTM with K agar

Difco Bacto FTM with beef

extract
Sigma alternative FTM
Sigma FTM

21 + 11

S.3 X lo-* ± 1.1 X nr

■o

20± 12

1.8 X 10' ± 3.3 X 10'

in

19+11
18± 11
22 ± 13

1.2 X 10' ± 1.7 X 10'
2.0 X 10' ± 4.0 X 10'
1.8 X 10' ±3.1 X 10'

a
ai
O

c
a
>.

niLd.Sx 10'±2.7x 10'). atid 5.0 niL ( 1.2 x lO' ± 1.5 x 10') of
AFTM (Fig. 3B). No signitlcant difference was found in the num-
ber of hypnospores among samples incubated in 0.2. 1.0, and 5.0
niL of AFTM.

Effect of NaOH Digestion of Samples on Hypnospore Diameter
and Number

No significant difference was found in hypnospore diameter
between samples processed with NaOH digestion (24 ± 17 (xm) or
without it ( 19 ± 9 ixm). No significant difference was found in the
number of hypnospores between samples processed with NaOH
digestion (1.8 x 10"* ± 2.6 x lO"*) or without it (2.6 x 10"" ± 3.8 x
10''). Sodium hydro.xide facihtated counting by reducing cellular
debris.

Effect of Protocol on Hypnospore Diameter and Number

Hypnospore diameter from samples processed with the im-
proved protocol (26 ± 13 (j.m) was significantly greater (P <
0.0001: ;; = 20) than the diameter of hypnospores from samples
processed with the standard protocol (10 ± 4 (j.m). Hypnospore
numbers in samples processed with the improved protocol (8.6 x
10' ± 3.3 X 10') were significantly greater (P < 0.0001) than the

3: 30

£
o

E
5 20

o
a

o 10

c

Q.

T ^

b

1x10* 1x10*

Hemocytes/ml

1 xlO'

Figure 2. Effect of hemocyte density ( KF, 10'', or lO' hemocytes per ml
of AFTM) on hypnospore diameter (mean ± SD, n = 13). Hemocytes
were incubated in 1 mL of AFTM supplemented with 5% lipid for
seven days. Means sharing a letter were not significantly different.

50

— 40

E
E.

I 30
« 20

10

400

w
S
o

a

(A

O

c

>; 200 H

|_g_y

_L

0.2

25

1 5

FTM Volume (ml)

Figure 3. Hypnospore diameter (.A) and number (B) (mean ± SD. n =
10) after incubation in 0.2. 1.0. 5.0. and 25.0 niL of AFTM. Hemocytes
(10'') were incubated for seven days in 1 niF of AFTM supplemented
with 5% lipid. Means sharing a letter in each panel were not signifi-
cantly different.

number of hypnospores in samples processed with the standard
protocol (1.9 x 10' ± 3.4 x 10'). Hypnospores from hemocytes
processed with the improved protocol readily settled to the plate
bottom and could be easily counted. Hypnospores processed with
the standard protocol were difficult to count because they re-
mained suspended in the wells and the media column had to be
scanned vertically as a consequence (Fig. 4).

Comparison of Standard and Improved FTM Hemolymph Assays with
FTM Body Burden Assay

Using linear regression, comparison of the infection intensity
determined by the body burden assay (log of hypnospores per
gram of oyster tissue) and the infection intensity determined by the
standard hemolymph assay (log of hypnospores per 10''
hemocytes) showed that the correlation was highly significant [P <
0.0001) with a coefficient of determination (r) of 0.5543 and a
slope for the regression line of 0.6126 (Fig. 5A). Using linear
regression, comparison of the infection intensity determined by the
body burden assay and the infection intensity determined by the
improved hemolymph assay showed a highly significant correla-
tion (P< 0.0001 ) with an improved coefficient of determination {r
= 0.7602) and slope (0.91 19) (Fig. 5B). This increased coefficient
of determination indicated a reduction in variance with the im-
proved FTM hemolymph assay.

730

NiCKENS ET AL.

•

*

•

•

• •

•

•

•

»

•

•

•.

'

*• ^ ,'

Jk

•

•

##

•

•#

# ♦

•

•

« #

•

«

•

• ^ t

•

• •

♦

Figure 4. Light micrographs of Perkiitsiis marinus hypnospores after
incubation and processing with the standard and improved protocols
at 200x magnification. Bar represents 30 pm. A, Hypnospores were
small (7-13 pm) and not clearly visible after incubation and processing
with the standard protocol. B, Hypnospores were larger (15—44 pni)
and more visible after incubation and processing with the improved
protocol.

DISCUSSION

Use of the improveiJ protocol to count P. marinus in
heniolymph resulted in a 167% increase in hypnospore diameter
and a 358% increase in hypnospore number over the standard
protocol. The improved protocol also facilitated sample processing
and counting. These improvements were caused by modifications
of the standard hemolymph protocol, such as the addition of lipid
concentrate to AFTM. the use of PTM without agar, and the ad-
dition of BSA to rinsing solutions. These modifications resulted in
larger hypnospores, decreased sample viscosity, increased hypno-
spore recovery, and reduced cellular debris. Because processing.
identification, and counting of hypnospores were facilitated, the
improved protocol was simpler and more accurate than the stan-
dard protocol for counting of hypnospores in hemolymph. As a
result, the coefficient of determination {r) of the linear regression
between P. marinus log,,, numbers in oyster body and P. marinus
login numbers in hemolymph increased from 0.554 to 0.760 when
the improved hemolymph protocol instead of the standard
hemolymph protocol was used to count P. marinus in hemolymph.

Results from previous studies have suggested that nutrients
from FTM and oyster tissues are used by enlarging parasites and
that the ratio of FTM to oyster tissue may be critical for optimal
hypnospore enlargement (Ray 1954a. Bushek et al. 1994. Fisher &
Oliver 1996). The failure of parasites in highly infected oyster
tissue to fully enlarge in FTM was proposed to be a result of
exhaustion of necessary substances in medium or tissue (Ray

iy54a). Hypnospore enlargement was also reported to be greatest
in the FTM tissue assay, intermediate in the FTM hemolymph
assa\ and least in the FTM body burden assays and therefore
related to the volume of oysters sampled per ml of FTM (Bushek
et al. 1994). Moreover, parasite enlargement was inversely related
to infection intensity. Using P. marinus cultured in vitro, parasite
enlargement in FTM can be significantly increased by addition of
various nutrients including lipids (Wagner et al. 2001). Despite
these observations, the effects of supplementing FTM with nutri-
ents such as lipid or oyster extract and the effects of the ratio of
FTM to oyster nutrients (e.g.. from hemocytes) on parasite en-
largement and numbers had not been studied and therefore needed
to be investigated to improve the FTM hemolymph assay.

Addition of lipids to AFTM significantly increased hypnospore
diameter by 66% over the saline control, and facilitated sample
processing and counting because larger hypnospores were more
easil\ pelleted during centrifugation. Lipids were added because
the accumulation of numerous lipid droplets in hypnospores is
characteristic of this life stage and may be critical to enlargement
(Perkins & Menzel 1966. Perkins 1996). It was recently confirmed
that lipids are taken up and stored in hypnospores (Soudant et al.
2000). This study found that fluorescent lipid analogs were pri-
marily stored in cytoplasmic lipid droplet after 24 h of incubation
but that after 24 h. fluorescence appeared in the membrane and
cytosol of hypnospores.

The mean diameter of hypnospores in AFTM supplemented
with 5% oyster extract was only 14% greater than the diameter of
hypnospores in AFTM supplemented with oyster saline. It was
surprising that the addition of oyster extract did not induce sig-
nificant cell enlargement over saline as oyster extract would be
expected to contain high concentrations of nutrients. Although the
components of the oyster extract were not examined, it is likely
that the water-soluble extract contained only small amounts of
oyster lipids, which may account for the minimal enlargement of
hypnospores.

Determining the effects of the ratio of infected hemocyte num-
ber to AFTM volume on hypnospore formation helped formulate
the optimal combination of nutrients, from oyster and AFTM, to
promote maximal parasite enlargement. In this study the number of
infected hemocytes incubated in a con.stant volume of AFTM was
adjusted, as was the volume of AFTM that had a constant number
of infected hemocytes. Hypnospores from the lowest hemocyte
density ( 10"' cells per ml of AFTM) had the greatest enlargement
indicating hypnospores in this treatment had more nutrients avail-
able from AFTM. Unfortunately, this low density may not provide
an adequate sample size for determining parasite infection inten-
sity. Using a higher number of hemocytes while maintaining
hemocyte density (e.g.. 10* per 10 mL) requires a larger volume of
FTM. which in turn may affect the assay. To address this potential
effect of volume on the assay. 10'' hemocytes were incubated in
various volumes of AFTM. Hypnospores from 10'' hemocytes in-
cubated in the largest AFTM volumes (5 and 25 mL) had the
greatest enlargement, but there was also a significant decrease in
the number of hypnospores from samples incubated in 25 mL of
AFTM. indicating that smaller cells may be lost during processing
of these volumes. Moreover, large volumes of AFTM requires the
purchase of larger amounts of media and antibiotics, which in-
creases cost. The most practical and optimal ratio was set at 10''
hemocytes to 1 mL of AFTM. At this ratio of infected hemocytes
to AFTM volume, parasites had adequate nutrients available for

Improved Hemolymph Procedure to Count P. marinus

731

a>

3
M
(0
*^

E
re

«

I.

o

a
«
o

c
a

>.

X

o
o

y = 0.61 26x + 4.4344

♦ ♦

|6

LOG Hypnospores/10 Hemocytes

y = 0.9119x + 2.8232
R^ = 0.7602

3
5 0

B

LOG Hypnospores/10 Hemocytes

Figure 5. Linear regression lines, formulas, and coefficient of determinations for the comparison of the log of the numher of hypnospores per
10'' hemocytes determined hy the standard FTM hemolymph protocol and the log of the numher of hypnospores per gram of oyster tissue
determined by the body burden assay (A), and for the comparison of the log of the number of hypnospores per 10'' hemocytes determined by
the improved FTM hemolymph protocol and the log of the number of hypnospores per gram of oyster tissue determined by the body burden
assay (B).

enlargement in a small AFTM volume while minimizing protocol
cost and hypnospore loss.

Perkinsus marinus in hemolymph has exclusively been re-
ported in numbers of parasite per ml of hemolymph (Gauthier &
Fisher 1990. Bushek et al. 1994. Oliver et al. 1998. Yamall et al.
2000). The volume of hemolymph collected per oyster for use in
the assay in these past studies varied by as much as a factor of 1 0.
affecting the assay performance. Most parasites, however, are
found within the phagosomes of hemocytes (Ray 1954a, Perkins
1996) and the density of hemocytes in hemolymph of bivalves can
vary considerably with factors such as temperature, disease and
feeding (Feng et al. 1977, Ford & Tripp 1996). Perkinsus may.
therefore, be better expressed in number of parasites per number of
hemocytes (e.g.. per 10'' hemocytes).

The coefficient of determination (?~) of the linear regression of
P. marinus log,,, numbers in oyster body on P. marinus logm
numbers expressed per 10^ hemocytes was 0.760 (n = 20). When
the numbers of P. marinus for the same hemolymph samples were
expressed per ml of hemolymph instead of IC hemocytes. the
coefficient of determination was 0.776 (n = 20) and higher than
all. except for one previously reported r. An r of 0.53 (n = 12)
for Texas oysters sacrificed 24 h after collection and an r of 0.89
(/? = 12) for animals held for ten days at high temperature and
salinity before sampling were reported by Gauthier and Fisher
(1990). An r of 0.675 (n = 25) was reported by Yamall et al.
(2000). An r of 0.241 (n = 100) for oysters from Apalachicola
Bay, Florida and an /' of 0.771 in = 100) and 0.738 (/i = 100)
for oysters from Virginia and New York were reported by Oliver
et al. (1998).

The use of FTM hemolymph assay is not recommended for
predicting the number of parasites in whole oyster with light P.
marinus infections (<l.000 parasite/g tissue) (Bushek et al. 1994,
Oliver et al. 1998). In our study, the intersects (i.e., y when x =
0) of the linear regression for the number of parasites in whole
oyster was 665 (log,o 665 = 2.823) or 1834 (log,o 1834 =
3.2635) depending on whether hemolymph parasite number was
expressed per hemocyte or per ml. Hence, parasite numbers per g

tissue in whole oyster would need to be greater than these numbers
for the hemolymph FTM assay to be able to detect any parasite.
This is in agreement with a Bushek et al. (1994) study in which a
high percentage of oysters with parasite numbers below 1.000 P.
marinus per g (wet) tissue were diagnosed as negative with the
FTM hemolymph assay. The limitation in sensitivity of the FTM
hemolymph assay is less of a concern for Gulf coast oysters than
for oysters from the Northeast and central Atlantic coast because
P. marinus prevalence in Gulf oysters in most sites is close to
100% as determined by the FTM tissue assay. This assay typically
detects infection intensities when they are greater than 1 ,000 para-
site/g wet tissue (Bushek et al. 1994. Soniat 1996, Fisher et al.
1996).

No statistical differences in hypnospore diameters or numbers
were found among the FTM formulations tested, indicating that
none of the FTM formulations provided more nutrients for parasite
uptake than others. After incubation of parasites in media com-
posed of individual PTM components. Ray (1954a) found that
yeast extract combined with dextrose or casitone were the nutrients
responsible for parasite enlargement. All FTM formulations tested
in the present study contained approximately equal concentrations
of yeast extract (5.0 g/L). dextrose (5.5 g/L), and casitone (15.0
g/L). which explains why no increase in hypnospore diameter or
number was found among the FTM formulations tested. Incubation
of hemocytes in FTM without agar, or AFTM, did simplify the
processing and counting of samples. A viscous layer often forms
over the cell pellets during rinsing steps before and after NaOH
digestion when FTM with agar is used to enlarge P. marinus cells
(La Peyre. personal observation, Oliver et al. 1998). This layer is
made of indigestible agar because the use of AFTM eliminated this
layer, thereby simplifying sample processing and reducing the risk
of losing hypnospores in the discarded layer. Sample counting was
also greatly simplified because I ) debris including indigestible
agar that can interfere with hypnospore counting was eliminated,
and 2 ) hypnospores readily settled on the bottom of wells of tissue
culture plates forming monolayers and could be easily counted. In
contrast to earlier studies, the need to scan for parasites vertically

732

NiCKENS ET AL.

through the column was eliminated (Choi et al.l989, Gauthier &
Fisher 1990. Bushek et al. 1994). Moreover, parasite suspensions
could be serially diluted in 96-well plates and counted at the ap-
propriate dilution faster than they could be prepared at the appro-
priate dilution and counted on filter paper by the technique t)f
Oliver and Fisher { 1996) or counted with hemacytometers as used
by Choi et al. (1989). Although agar is traditionally used in the
microbiological medium FTM to preserve anaerobic conditions
and keep bacteria suspended throughout the media for maximizing
the use of nutrients (Hitchens 1921). it was clear from our study
that the conditions produced by this viscosity are not necessary to
enlarge P. maiimis parasites. AFTM was therefore selected for
parasite enlargement in the improved FTM hemolymph protocol.
In an evaluation of methods to diagnose P. marinus. NaOH
digestion was used to process hemolymph and oyster tissue. It was
found that NaOH digestion facilitated the counting of hypnospores
from oyster tissues and from hemolymph. but it was noted that
NaOH may not be needed for the hemolymph technique because it
adds time and labor to the assay (Bushek et al. 1994, Fisher &
Oliver 1996). In our study, NaOH digestion of hemolymph
samples simplified and reduced the time spent counting. By elimi-
nating debris, cells formed a monolayer on cell culture plate bot-
toms eliminating the need to scan vertically for hypnospores
caught in debris. In past studies, it was noted that hypnospores
became sticky forming clumps and adhering to the sides of test
tubes after NaOH digestion and upon rinsing (Choi et al. 1989.
Bushek et al. 1994, Fisher & Oliver 1996). In our study, the loss
of hypnospores as a result of clumping and adherence to the side

of test tubes was alleviated by adding BSA to rinsing solutions as
recommended by Coates et al. (1999).

In conclusion, simple modifications to the standard protocol for
counting of P. iiuirinus in oyster hemolymph improved the tech-
nique. The addition of lipid to AFTM. the use of FTM without
agar, and the use of BSA in rinsing solution facilitated sample
processing and counting. The availability of an improved protocol
to count P. marinus in hemolymph will be useful in monitoring the
dynamic change of parasites in hemolymph in infected individual
oysters under various conditions. The role of heinocytes in the
infection process or in the elimination of the parasites (e.g.,
hemocyte killing assay) can also be investigated more accurately
with this assay.

ACKNOWLEDGMENTS

The authors thank the faculty and staff of the Louisiana State
University Department of Veterinary Science. The authors thank
Rachel Gross-Delbos, Dr. J. Geaghan, and Rujun Teng of the
Louisiana State University Experimental Statistics Department for
statistical advice. The authors thank Yanii Li, Siow-Hui Hau,
Chiew-Hong Lee. and Chwan-Hong Foo for technical assistance.
The authors thank Dr. John Supan for providing oysters used in
this study. This work was funded by the Louisiana Sea Grant
College Program and National Sea Grant Oyster Disease Research
Program. The authors thank Ron Becker for his support. Approved
by the Director of the Louisiana Agricultural Experiment Station
as manuscript number 02-64-0333.

LITERATURE CITED

Bushek. D.. S. E. Ford & S. K. Allen Jr. 1994. Evaluation of methods using
Ray's fluid ihioglycollate medium for diagnosis of Perkiiisiis mariniis
infection in the eastern oyster, Crassoslrea virginica. Ann. Rev. Fish
Dis. 4:201-217.

Choi, K.-S.. E. A. Wilson. D. H. Lewis. E. N. Powell & S. M. Ray. 1989.
The energetic cost of Perkinsns marinus parasitism in oysters: quanti-
fication of the thioglycoUate method. J. Shellfisli Res. 8:125-131.

Coates. G. M.. R. K. Cooper & J. F. La Peyre. 1999. Improvement of the
whole-oyster procedure for enumerating Perkinsns niarinns in oyster
tissues. J. Sliellftsh Res. 18:328.

Feng, S. Y.. J. S. Feng & T. Yamasu. 1977. Roles of Mylilus enruscns and
Crassostrea gigas blood cells in defense and nutrition. In: L. Bulla &
T. C. Cheng, editors. Comparative pathobiology. vol. 3. New York:
Plenum Press, pp. 31-67.

Fisher, W. S., J. D. Gauthier & J. T. Wmstead. 1992. Infection intensity of
Perkinsns disease in Crassostrea virginica (Gmelin. 1791) from the
Gulf of Mexico maintained under different laboratory conditions. J.
S/k'///;.s/! Res. 11:363-369.

Fisher, W. S. & L. M. Oliver. 1996. A whole-oyster procedure for diag-
nosis of Perkinsns marinus disease using Ray"s fluid thioglycollale
culture medium. / Shellfish Res. 15:109-117.

Fisher. W. S.. J. T. Win.stead. L. M. Oliver. H. L. Edmiston & G. O. Bailey.
1996. Physiologic variability of eastern oysters from Apalachicola Bay.
Florida. J. Shellfish Res. 15:543-553.

Ford. S. & M. R. Tripp. 1996. Disea.ses and defense mechanisms. In: V. S.
Kennedy and R. I. E. Newell, editors. Eastern oysters. College Park.
MD: Maryland Sea Grant College. University of Maryland, pp. 581-
660.

Gauthier, J. D. & W. S. Fisher. 1990. Hemolymph assay for diagnosis of
Perkinsns marinus in oysters Crassostrea virginica (Gmelin. 1741 I. ,/.
Shellfish Re.t. 9:367-371.

Hitchens. A. P. 1921. Advantages of culture media containing small per-
centages of agar. J. Infectious Dis. 29:390-407.

McLaughlin. S. M. & M. Faisal. 1999. A comparison of diagnostic assays

for detection of Perkiiisus spp. in softshell clam Mya nrenaria. Aqua-
culture 172:197-204.

Oliver. L. M.. W. S. Fisher. S. E. Ford. L. M. Ragone Calvo. E. M.
Burreson. E. B. Sutton & J. Gandy. 1998. Perkinsns marinus tissue
distribution and seasonal variation in oysters Crassostrea virginica
from Florida. Virginia and New York. Dis. Aquat. Org. 34:51-61.

Perkins. F. O. & R. W. Men/el. 1966. Morphological and cultural studies
of a motile stage in the life cycle of Dermocystidinm murmuni. Proc.
Natl. Shellfish As.soc. 56:23-30.

Perkins. F. O. 1996. The structure of Perkinsns marinus (Mackin, Owen
and Collier. 1950) Levine. 1978 with comments on taxonomy and
phytogeny of Perkinsns spp. / Shellfish Res. 15:67-87.

Ragone Calvo, L. & E. M. Burreson. 1994. Characterization of overwin-
tering infections of Perkinus marinus (Apicomplexa) in Chesapeake
Bay oysters. J. Shellfish Res. 13:123-130.

Ray. S. M.. J. G. Mackin & J. L. Boswell. 1953. Quantitative measurement
of effect of disease caused by Dernu>cystidium marnnnn. Biol. Hull.
Mar. Sci. Gulf Carihh. 3:6-33.

Ray. S. M. 1954a. Biological studies of Derntocysticlium mariiium. a fun-
gus parasite of oysters. Rice Institute Pamphlet. Special Issue. Houston:
The Rice Institute.

Ray. S. M. 1954b. Studies on the occurrence of Dermocystidinm m young
oysters. Natl. Shellfisheries Assoc. Conv. Add 1953:80-88.

Soniat. T. M. 1996. Epizootiology of Perkinsns marinus disease of eastern
oysters in the Gulf of Mexico. / Shellfish Res. 15:35-43.

Soudant. P., F.-L. E. Chu & Y. Marty. 2000. Lipid class composition of the
protozoan Perkinsns marinus. an oyster parasite, and its metabolism of
a fluorescent phosphatidylcholine analog. Lipids 35:1387-1395.

Yarnall. H. A.. K. S. Reece. N. A. Stokes cS: E. M. Burreson. 2000. Quan-
titative competitive polymerase chain reaction assay for the oyster
pathogen Perkinsns marinus, J. Parasitol. 86:827-837.

Wagner. E.. S. Casas & J. F. La Peyre. 2001. Induction of hypnospore
formation and /oosporulation of Perkinsns marinus cells from long
term cultures. Ac/uaculture. 2001. Book of Abstracts, page 670.

Journal of Shellfi.'.li Research. Vol. 21. No. 2, 733-741. 2002.

OYSTER BIOMASS, ABUNDANCE, AND HARVEST IN NORTHERN CHESAPEAKE BAY:

TRENDS AND FORECASTS

STEPHEN J. JORDAN,'* KELLY N. GREENHAWK,' CAROL B. MCCOLLOUGH,'
JESSICA VANISKO,' - AND MARK L. HOMER'

^Maiylond Department of Natural Resources, Paul S. Sarbanes Cooperative Oxford Lxiboratory. 904
South Morris Street. Oxford, Maryland 21654; 'University of Maryland. Chesapeake Biologic
Laboratory. P.O. Box 38ni Solomons, Maryland 20688

ABSTRACT We applied time series of fishery-dependent and fishery-independent data to develop indices of relative biomass and
estimates of absolute abundance and biomass for the Maryland oyster population. The principal objectives for this work were to specify
a baseline and determine trends relative to the Chesapeake Bay Program's goal to achieve a 10-fold increase in the standing stock of
oysters in the Bay from a 1994 baseline. Population biomass varied by 3-fold from 1986-2001. with most of the variation caused by
changes in the stock of market-sized oysters. There were also important spatial differences in population structure and trends over a
gradient of salinity. Lagged correlations between small oyster biomass and either niarket-si,^ed oyster biomass or landings were not
statistically significant but suggested that most of the market-si/ed oysters and those harvested were four to five years old. The index
of relative biomass for market-sized oysters proved to be a useful predictor of annual harvests in Maryland. We used this relationship
to estimate absolute abundance and biomass of oysters for the time series. The latter estimates may be biased negatively with respect
to true abundance and biomass because of biases in both the fishery and the fishery-independent monitoring program. The index of
relative biomass will be a suitable measure of population status relative to the oyster restoration goal only so long as the population
remains at relatively low levels. Because the index has a theoretical maximum of about 75'7r of the 10-fold goal, a quantitative estimator
will be required for higher population levels.

KEY WORDS: oysters, Crassostrea virf^inica. stock assessment, biomass. Chesapeake Bay

INTRODUCTION

MATERIALS AND METHODS

Oysters once supplied the most valuable fishery in Chesapeake
Bay (Christmas & Jordan 1991), but after the mid-1980s, a de-
clining oyster stock could no longer suppoil historical levels of
harvest. The principal reasons for reduced harvests from 1987 to
2001 were (1) high mortality rates of oysters caused by the para-
sitic diseases MSX [Haplospuridiitm nel.soni) and Dermo (Per-
kinsus marinus) and (2) low rates of spat settlement throughout
most of this period (Krantz undated. Maryland DNR 2001). In
addition to economic losses to the fishery, the depletion of oyster
standing stocks has been thought to ( 1 ) reduce the quantity and
quality of shell-bar habitats for oysters and associated epifauna
(MacKenzie 1996) and (2) impair water quality by loss of the
oyster population's capacity to filter particulate matter (Jordan
1987, Newell 1988. MacKenzie 1996).

In June 2000. the interjurisdictional Chesapeake Bay Program
established a goal to increase oyster populations 10-fold by 2010,
from a 1994 baseline. Our objectives for the work presented here
were to (I) quantify the 1994 baseline, (2) compare the current
population with recent and historical populations, (3) develop
methods to measure consistently the status of populations with
respect to the goal, and (4) examine relationships between long-
term fishery-independent surveys and annual harvests. In addition
to meeting these objectives, we have developed methods for esti-
mating absolute abundance, biomass and fishing mortality from
these data. Only data from the Maryland portion of Chesapeake
Bay were used in our analysis. We are working with scientists in
Virginia on a combined, bay-wide assessment of the oyster popu-
lation.

*Corresponding author. E-mail: sjordan@dnr.state.mo

Oyster Surveys

Size frequency, disease, mortality, and spat density data were
collected from oyster dredge samples at 43 sites (Fig. 1 ) in October
to November from 1990 to 2000 (in a few cases, 42 or 44 sites
were sarnpled). The standard dredge has an opening 80 cm wide
and a capacity of approximately 2.5 bushels (the volume of a
Maryland oyster bushel is =46 L, or about 475 oysters at the
minimum market size of 76 mm). Dredge tows were taken from
areas of the oyster bars with relatively high concentrations of shell,
as detennined by the sampling crew with a sounding pole. Our
observations indicated that tow times and boat speed (hence the
area swept) tended to be consistent, but these variables were not
formally controlled. From 1990 to 1996, five replicate dredge tows
were made at each site; data were collected from a 0.2 bushel
subsample of all material in each dredge sample. From 1997 to
2000, two tows were made at each site, with a 0.5 bushel sub-
sample taken from each tow. All live oysters in the subsamples
were counted and measured, with measuretiients recorded in 5 mm
size classes. Boxes (articulated oyster shells without tissue remain-
ing) also were counted and measured. All spat were counted in
each sample. Sample data were standardized to a fixed volume
(one bushel, as defined above) of all material retained by the
dredge. A complete description of this survey, known as the Modi-
fied Fall Survey (MFS) can be found in Smith and Jordan ( 1993).

A more extensive survey of 300 to 400 sites has been con-
ducted in the fall of each year since the 1970s. In this survey,
typically only one dredge tow is tnade; all live oysters, spat, and
boxes from 0.5 bushel of material are counted. The oysters are not
measured individually but are classified as markets (>76 mm),
smalls (age l-l- and older oysters <76 mm), or spat. Means and
ranges of shell heights for each class are estimated visually by an
experienced technician and recorded (Homer et al. 1996).

733

734

Jordan et al.

Low

■-^i^

+ Medium ^%^i^ ,>

High /5^' J

^.

Figure 1. Maryland MFS sites in Chesapeake Bay and tributaries.
Symbols identify classification of sites by long-term mean salinity: Low
<I2 ppt. Medium 12 to 14 ppt. High >14 ppt.

Length-Weight Analysis

A relationship between dry tissue weight and shell height was
developed to convert size-frequency data to biomass. During the
1999 fall survey. 10 oysters were collected from each of 42 sites
(one of the 43 survey sites did not have sufficient live oysters) and
returned to the laboratory for processing. The oysters were selected
to be representative of the size range encountered at each site. In
the laboratory, the oysters were measured (bill to umbo curved
shell height in mm), then shucked carefully, retaining all meat and
shell liquor. Each oyster was put into a tared aluminum weighing
boat, weighed, dried to constant weight at 85"C. and then re-
weighed. Wet and dry tissue weights were recorded in g. to the
nearest mg.

The relationship between dry tissue weight and shell height was
quantified by linear regression of the log|(,-transfornied variables.
Indices of relative biomass were calculated by applying the length-
weight regression equation to each 5 mm size class of live oysters
(excluding spat), then multiplying the number of oysters in that
size class by the predicted weight. The midpoint in mm of the size
class was used as the nominal shell height for the estimated weight.

The weights were summed over all size classes within each rep-
licate subsample. resulting in 1629 individual observations of the
index over 43 sites and ten years. Indices of relative abundance
were calculated similarly using total counts of oysters (excluding
spat) within each subsample. Indices of biomass and abundance
were calculated separately for small (<72 mm) and market (>72
mm) oysters, and for all oysters combined. The legal minimum
size for harvested oysters in Maryland is 3 inches (76 mm), but the
nearest size class in which survey data are recorded is 72 to 77
nmi. We examined the underlying variation in the indices by two-
way analysis of variance, accounting for the fixed effects of site,
year, and site-by-year interaction.

Relationships Between Fishery-Dependent and
Fishery-Independent Data

We u.sed linear regression to determine whether the fishery-
independent data could predict commercial harvests. Harvest data
were obtained from the Maryland Department of Natural Re-
sources. Because the oyster season is open from October to March.
landings are reported for the year the season closes, whereas the
MFS data applicable to the annual harvest are collected in October
through November of the previous year. For this reason, the analy-
ses reported here are indexed to the harvest year, that is. the 198,'i
MFS corresponds to the 1986 harvest, and the biomass index for
1986 is derived from data collected in the fall of 1985. However,
when referring to the fishery-independent data per se. we use the
year in which the data were collected. Annual harvest totals, re-
corded in bushels, were regressed against the annual mean biomass
index for market oysters and the annual abundance index for mar-
ket oysters. Nine years of data, from harvest years 1991-1999,
made up the initial calibration data set. We then used dredge
survey data from 1985-1989 for the bars sampled by the MFS to
calculate the biomass indices and perform a hindcast test of the
harvest prediction model. The 1985-1986 harvest was 1.56 million
bushels, nearly 20 times greater than the 1993-1994 harvest. Also,
the index previous to 1990 was based on mean sizes for market
oysters rather than individual measurements. Prediction of the
1985-1986 and subsequent harvests, therefore, would provide a
stringent test of the robustness of this simple model. Once the
model was calibrated and validated by hindcasting, we recalibrated
over the entire time series, and forecast landings for the 1999-2000
and 2000-2001 harvest seasons.

Quantitative Estimation of Standing Stocks

The success of the market oyster biomass index in predicting
annual landings suggested that we could use this relationship to
quantify the oyster population. We made the key assumption that
differences between predicted and reported landings were entirely
caused by differences in rates of fishing mortality. Smith and
Jordan (1993) estimated a mean exploitation rate of 0.53 (53% of
the market stock harvested) for Maryland oysters for the 1990 to
1991 season. They projected size-frequency data from the 1990
MFS to 1991, assuming a mean growth rate of 20 mm per year and
subtracting 1991 observed natural mortality, estimated from box
counts as [boxes/(boxes -i- live oysters)], from each size class. The
exploitation rate then was estimated by difference between the
projected relative abundance (without exploitation) and that ob-
served from the 1991 survey. Because the ratio of reported to

Oyster Biomass in the Chesapeake Bay

735

predicted harvest for that year was approximately unity, we com-
puted the instantaneous rate of fishing mortahty (F) for the time
series as

F = -logf

■ 0.53

where H = reported harvest and H = predicted harvest. Estimates
of harvestable stock size (S) for each year were computed as S =
H ■ e^ (in units of bushels). Estimates of total population abun-
dance (M excluding spat) were computed as

N, = N,„ + N,„[ —

where the subscripts i. in. and .v indicate total, market, and small
oysters, respectively. R refers to the relative abundance estimates
from the fishery-independent survey, and

N..,

a,S

where a, = lo^-"^"'"" '^"-'\ /?_ js the relative abundance of market
oysters in each size class, R, is the total relative abundance of
market oysters, and L, is the nominal length for each size class of
market oysters. The last equation estimates the number of oysters
of a given size per bushel, derived by linear regression from tabu-
lated counts (unpublished data. Maryland Department of Natural
Resources). Absolute population biomass was estimated by mul-
tiplying the proportion of oysters in each size class from fishery-
independent size frequency distributions by total absolute abun-
dance iN^ calculated as above) and the nominal weight for each
size class:

«, = 2

R,

N,W,\ -

where B, is total population biomass. W, is the nominal weight in
g for each 5 mm size class. /?, is the relative abundance in each size
class, and R, is the total relative abundance over all size classes.

Recruitment

Spat (young of the year oysters) are counted in each sample
taken during fall oyster surveys. Although annual mean spat counts
have been used for many years as an indicator of year class
strength (Meritt 1977, Homer et al. 1996, Krantz undated), they are
not always reliable indicators of subsequent recruitment to the
population because of high, variable rates of early juvenile mor-
tality (Newell et al. 2000) and the impacts of diseases on submar-
ket oysters. Counts of small oysters (>1 y old and <76 mm in shell
height) may be better indicators of recruitment to adult and ex-
ploitable stocks than spat counts. Therefore, we examined the po-
tential of small oyster counts and biomass to predict landings, as
well as biomass of market oysters in subsequent years, by regress-
ing landings and market oyster biomass against 1- to 5-y lags of
the small oyster indices.

InPuence of Salinity

Recruitment of oysters to the population and disease-related
mortality are positively correlated with salinity in northern Chesa-

peake Bay (Jordan 1993. Calvo et al. 1996). To examine the effects
of .salinity on the biomass index, we assigned each of the MPS sites
to one of three salinity zones based on 1 990 to 2000 mean salinity
measured during the fall at the time oyster samples were taken
(Fig. 1). These zones were defined as low (mean salinity <I2 ppt),
medium (12-14 ppt). and high (>14 ppt). At salinity <12 ppt. H.
nelsoiii infections occur rarely, if ever; Perkinsus marinus infec-
tions, although chronic in this zone, are associated with low to
moderate mortality rates: and recruitment rates of both small and
market oysters are typically very low, except in areas where natu-
ral recruitment has been supplemented by transplanted seed oys-
ters. In the medium salinity zone. H. nelsoni epizootics are spo-
radic, occurring only in drought years, mortality associated with P.
marinus is moderate to high, and recruitment is variable. In the
high-salinity zone. H. nelsoni infections tend to be enzootic, mor-
tality rates associated with P. marinus infections are consistently
high, and recruitment, although variable, tends to be higher than in
the lower salinity zones.

RESULTS

Length-Weight Relationship

The linear relationship between the log of dry tissue weight and
the log of shell height was quantified as log,o(dry tissue weight) =
2.06[log|„(shell height)] - 3.76. with tissue weight in g and shell
height in mm; /; = 410. r" = 0.50, P < 0.0001 (Fig. 2). Removal
of a few outlying observations or weighting the regression for
uneven distribution of the dependent variable on the independent
variable made only trivial differences in the parameter estimates
and the coefficient of determination. Therefore, population-scale
estimates of biomass should be accurate, even though the relation-
ship does not have satisfactory precision for estimates at smaller
scales (individuals or small samples).

Variability of Biomass Indices

In the two-way analysis of variance models, sites, years, and
site-by-year interactions explained 85 to 919^ of the variation in
index values for small oysters, market oysters, and the total popu-
lation; all effects were significant (Table I). The residual 9 to 15%
of overall variation was caused by differences between replicate
samples.

T \ 1 r

40 50 60 80 100

150

SHELL HEIGHT (mm)

Figure 2. Relationship of oyster dry tissue weight to shell height.

736

Jordan kt al.

TABI.E 1.

Analysis of variance results for examining sources of variability in
bioniass indices of total, market, and small oysters, 1990 to 1999.

hindcast

Source of Variation

Degrees of Freedom

P Value

Total Biomass

(Mean = 1 14.4 g dry weight per bushel, r
Site 43

Year 9

Site X year 378

Total 1628

Market Oyster Biomass

(Mean = 63.7 g dry weight per bushel, )' =
Site 43

Year 9

Site X year 378

Total 1630

Small Oyster Bioniass

(Mean = 44.8 g dry weight per bushel. /•" =
Site 43

Year 9

Site X year 378

Total 1630

= 0.91. II = 1629)

S.'i.96 <0.(10()1

89.10 <0.0001

18.73 <0.0001
28.10 <0.0001

0.85. II = 1631

46.54 <0.0001

94.46 <0.0001

8.88 <0.0001

16.24 <0.0001

0.89, 11 = 1631)

64.60 <0.0001

23.31 <0.0001

15.81 <0.0001

22.74 <0.0001

Relationships Between Fishery-Dependent and
Fishery-Independent Indicators

There was a significant linear relationship between the mean
relative biomass of market oysters sampled in October through
November of each year and the quantity of oysters harvested dur-
ing the commercial season (generally October I through March 3 1 .
with minor variations). The resulting regression equation was
log,o(y) = 1.64(log,oX) + 2.39. where y = predicted annual har-
vest in bushels and x = mean relative biomass of market oysters
((' = 0.89; Fig. 3). This regression was developed from 10 y
(harvest years 1991-2000) for which we had replicate samples and
size frequency data. A hindcast of this model for 1986 to 1990
landings, using only counts and mean sizes of market oysters,
yielded reasonable estimates. Over the 15 harvest years from 1986
to 2000. the mean absolute difference between predicted and re-
ported landings was IS.S^'r (minimum 3.7%. maximum 57.6%.
standard deviation 17%). The poorest prediction was for 1987, a
period of sharply declining relative biomass associated with an
epizootic of MSX disease. An earlier validation of the model used

O Si

< o
_l o

500
400

300^
200

100

calibration

• ■■ predicted
— reported

fore-
cast

35 40 50 60 70 80 90

MEAN MARKET OYSTER BIOMASS
gdw/bushel

Figure 3. Relationship between annual reported oyster landings and
annual mean market oyster biomass index (g dry weight of oyster
tissue per bushel of dredged material). Both axes are logarithmic, r- =
0.89.

1990 1992 1994 1996
HARVEST YEAR
Figure 4. Predicted and reported oyster landings, 1986 to 2001.

a regression of nine years of data along with biomass from the
1990 Fall .Survey to predict 1999 to 2000 oyster landings at
370,000 bushels; reported landings were less than 1% gi-eater
(380.(J00 bushels). The prediction for 2000 to 2001 landings was
331.000 bushels, about 5% less than reported (348,000 bushels).
With the model validated by hindcasts and forecasts, it was ap-
propriate to use the entire time series to estimate model parameters
(Fig. 4).

Biomass as an Indicator of Population Size

The biomass index for the total population showed more than
3-fold variation over a 15-y period (Fig. 5). Most of this variation
was caused by changes in the market oyster index, which varied by
more than 6-fold. Small oyster biomass was more stable, varying
by a factor of 2.4. The 1 994 baseline index for the total population

small oysters
I market oysters

300

n 200

?

O)

CO
(O

< 100

o

CD

86 88 90 92 94 96 98 00

YEAR

Figure 5. Biomass indices (g dry weight of oyster tissue per bushel of
dredged material) for small, market, and total oysters, 1986 to 2001.

Oyster Biomass in the Chesapeake Bay

737

— abundance
-•- biomass

[260

220
200
180
160
140
120
100
80

a. CD
^ >

1985 1987 1989 1991 1993 1995 1997 1999 2001

SURVEY YEAR

Figure 6. Annual means of relative biomass and relative abundance,
1985 to 2001.

(84.4 g dry weight of oyster tissue per bushel of dredged material)
was the lowest in the time series, largely because of a very low
index of market oysters (34.8), yet small oyster biomass in 1994
(49.6) was close to the 13-y mean (51.4).

Although there were similarities between trends in relative bio-
mass and relative abundance from 1986 to 2001, there were im-
portant divergences (Fig. 6). The sharp rise in abundance from
1 997 to 1 998, for example, reflects the large spat set observed in
1997, followed by a sharp increase in the abundance of small
oysters in 1998. The biomass also increased from 1997 to 1999.
but more slowly. Sharp declines in biomass from 1985 to 1989 and
from 1990 to 1992 were associated closely with disease epizootics.

Trends in biomass varied considerably over three salinity zones
(Fig. 7). In the low-salinity zone, population biomass was moder-
ate and relatively stable, whereas in the midsalinity zone, biomass
declined almost steadily for nine years, followed by partial recov-
ery from 1995 to 1999. In the high-salinity zone, biomass de-
creased sharply from 1986 to 1989 and did not show sustained
recovery. Differences between the zones were even more evident
when expressed as mean biomass per individual over the full time
series (Fig. 8).

Estimates of Fishing Mortality, Total Mortality, Absolute Abundance,
and Absolute Biomass

Estimated instantaneous annual rates of fishing mortality varied
from 0.23 in harvest year 1997 to 1.29 in 1989. Instantaneous
annual total mortality for the market oyster stock ranged from 0.57
in 1997 to 1.67 in 1989 (Table 2, Fig. 9). These ranges corre-

400

(0

<

o

CQ

z
<

UJ

S

^ 300

in

E 200

■o

100:

4

1 .

1
.ow i Medium High

s12 1

12-14 >14

I 1

L "

1

1^

,:■.■ r

i

i .

;l

1 ■ '

m

86 00 86 00 86 00

HARVEST YEARS 1986-2000
Figure 7. Mean relative biomass by .salinity zone, 1986 to 2001.

SALINITY
ZONE

Figure 8. Mean relative biomass per individual oyster by salinity zone.
Error bars are standard errors.

sponded to annual exploitation rates of 21 to 72'7r and annual total
mortality rates of 43 to 92% for the market stock.

Our estimates of total oyster abundance from harvest years
1991 to 2001. for which size-frequency data were available,
ranged from 2.66 to 6.29 x 10** individuals, with a mean of 4.78 x
10**. Total population biomass over the same time period ranged
from 2.41 to 8.64 x 10" g dry tissue weight, with a mean of 5.74
X lO'^ g (Fig. 10). Mean biomass per individual oyster (biomass
divided by abundance) was 1.18 g. ranging from 0.82 to 1.42 g
over the time series (Fig. 11). For reference, the predicted dry
weight of a minimum-sized market oyster (76.2 mm) is 1.31 g.

Recruitment

Lags of small oyster biomass (0-5 y) were not significantly
correlated (Spearman /■, P > 0.05) with market oyster biomass or
annual oyster harvests from 1986 to 2001. The strongest positive
correlation (r = 0.58, P = 0.06) was between harvest and a 4-y
lag of small oyster biomass (Fig. 12).

DISCUSSION

We estimated total oyster population abundance and biomass
for Maryland's portion of Chesapeake Bay from 1986 to 2001.
Although these estimates appear to represent population trends
accurately, there are several sources of uncertainty and potential
bias, which are discussed below. Until these uncertainties can be
reduced by further assessment and analysis, we recommend use of
the indices of relative biomass reported here as more reliable mea-

TABLE 2.

Summary statistics for instantaneous rates of Tishing mortality (F),

natural mortality (M), and total mortality (Z) for harvest years 1986

to 2001 (H = 16).

Variable Minimum Maximum Mean Standard Deviation

F
M
Z

0.23
0.11

O.-'i?

1.29
0.91
1.67

0.71
0.39
I. II

0.2.^
0.22
0.^2

738

Jordan et al.

86 88 90 92 94 96 98 00
HARVEST YEAR

Figure 9. Estimated instantaneous annual mortality rates, 1986 to
2001; F = Fisliing mortality, M = natural mortality.

sures of changes in the oyster population than our estimates of
absolute abundance and biomass.

Indices of relative biomass and abundance rely on the principal
assumption that the fishery-independent monitoring program ac-
curately represents trends in the oyster population as a whole. The
MFS. the source of data for index calibration, was designed spe-
cifically for consistent assessment of trends in oyster relative abun-
dance, population structure, mortality and disease status (Smith &
Jordan 1993). For these purposes, fixed sites, sampled consistently

Figure
oyster.

1993 1995 1997 1999

HARVEST YEAR

II. Estimated annual means of absolute biomass per individual
1981 to 2002.

3

^

9UU,UUU :

;

700,000

500,000

<

O

QQ

300,000 ':
100,000

over time, are appropriate. The inonitoring sites are widely dis-
tributed throughout the oyster-producing areas of Maryland, and
evidence presented here suggests they provide data representative
of real trends in the population.

Unbiased, quantitative estimates of population size generally
require spatially randomized sampling, a condition not satisfied by
the data used in our assessment. Our estimates of absolute biomass
and abundance also depend on the untested assumption that dif-
ferences between predicted and reported oyster landings are
caused solely by variations in fishing mortality. Both of these
considerations introduce bia.ses and inaccuracies of unknown mag-
nitude to the quantitative assessment. Preliminary estimates of
population abundance we derived entirely from fishery-inde-
pendent data sources resulted in higher estimates of population
abundance than those derived from fishery-dependent estimates.
The fishery-independent estimates ranged from 1.1 to 1.7 x 10"^
oysters in 2000 to 2001 . compared with 0.6 x \(f oysters estimated
for the same period by the methods reported here. We conjecture
that neither the fixed-station monitoring program nor the fishery
landings data adequately represent potentially large numbers of
oysters scattered at low densities in marginal habitat areas. Areas
with low densities of oysters would not be targeted by the fishery
because the catch per unit of effort would be very low. Similarly,
the fixed monitoring sites were selected to represent typically pro-
ductive areas of high quality habitat. We plan to collect quantita-
tive data from a variety of habitat types during 2002 to get better

1991 1993 1995 1997 1999 2001

HARVEST YEAR

<0 0.31

cx:

LL 0.2-

o'

-0.1^

12 3 4 5

12 3 4 5

SMALL OYSTER BIOMASS
LAG (YEARS)

Figure 10. Estimates of absolute biomass and abundance, 1991 to Figure 12. Correlations between lags of small oyster biomass and (A)
2002. reported landings and tB) market oyster biomass.

Oyster Biomass in the Chesapeake Bay

739

estimates of oyster densities, especially in areas of marginal hiabi-
tat. Until further refinements can be made to the quantitative as-
sessment, we view it as yielding minimal estimates of oyster abun-
dance and biomass.

The equation relating shell height to dry tissue weight used to
compute the biomass index explained 50% of the variation be-
tween individual oysters. Morphology, growth, and physiologic
conditions can vary greatly in C. virginica, depending on specific
habitat conditions, health, and other factors (Carriker 1996, Ford &
Tripp 1996). The condition of each oyster weighed and measured
for this study was assessed both by visual inspection (scored 1-8.
"watery" to "fat," according to standard procedures used in our
laboratory) and by calculating the percentage of water in the tis-
sues from the difference between wet tissue weight and dry tissue
weight. Inclusion of either of these condition indicators as a co-
variate in the length-weight regression improved the total /•" to >
0.70. Because oyster condition is not measured routinely in popu-
lation surveys, we could not account for this source of variation in
biomass indices. We conclude that the length-weight equation has
low precision for predicting the biomass of individuals or small
groups of oysters, but has sufficient accuracy for estimating bio-
mass at population or sub-population scales.

Harvest Predictions, Fishing Mortality, and Recruitment

The close relationship between reported annual landings and
the biomass index for market oysters has provided a method for
predicting total annual harvest early (mid-November) during each
oyster season (October-March). This capability is helpful to man-
agers of the oyster fishery, who are often asked by the press and
other interested parties to make these predictions. This relationship
also has provided a quantitative link between relative and absolute
abundance and biomass of oysters, and a way to estimate fishing
mortality rates. Because natural mortality (the ratio boxesdive oys-
ters plus boxes) is observed directly during the annual monitoring
program, we now have estimates of total mortality for market-
sized oysters. These relationships are being used, along with other
parameters, to calibrate a population dynamics model for Mary-
land oysters.

Ulanowicz et al. ( 1980) developed a multiple regression model
to predict Maryland oyster harvests four years into the future from
data on spat settlement and management effort (transplantation of
seed oysters) in previous years. The time series of data used in
their model was from the 1960s and 1970s, a period when parasitic
diseases were not major influences on oyster population dynamics.
During the period of the present study, variations in annual land-
ings have depended strongly on oyster mortality rates, which in
turn have depended primarily on variations in disease intensity
(Gieseker 2001).

Instantaneous annual rates of fishing mortality (F) varied from
0.23 to 1.29 from 1986 to 2001, corresponding to exploitation rates
of 2 1 to 72% of the market oyster stock harvested each year. The
mean exploitation rate of market-sized oysters over 16 y was 51%-
(F = 0.71). This value of F contrasts with that estimated by
Rothschild et. al. (1994) at 1.3 (73% exploitation rate) for 1990;
our estimate of F for that year was 0.76 (53%). Their estimate of
F apparently included some natural mortality (M) because their
estimated value of M was 0.15, compared with our measured value
oi M = 0.22 for 1990. The resulting total mortality of the market
stock for 1990 was 0.94 (61%) in this study, compared with 1.45
(77%) reported by Rothschild et al. (1994). Besides underestimat-

ing natural mortality. Rothschild et al. (1994) estimated mean age
at first capture at 2.6 y (legal ininimum length 76 mm), and the
mean age of the harvest (85 mm mean length) at 3.0 y. Growth
curves we derived from size-frequency data for thousands of
Maryland oysters collected during surveys from 1990 to 2000
predicted that the average 76 mm oyster was 4.0 y old, and the
average 85 mm oyster was 4.5 y old. This disparity in age-at-
length determinations could explain the higher rates of total mor-
tality and fishing mortality estimated by Rothschild et al. (1994)
using a modified Beverton-Holt formulation (Ehfhardt & Ault
1992), because overestimation of the growth coefficient would
yield a higher rate of total mortality. Our recruitment analysis
suggested that oysters are most likely to be harvested at ages of
four to five years in Maryland, consistent with our growth-based
predictions of age at first capture and mean age of capture, and
with findings by Ulanowicz et al. (1980).

Our estimates of total mortality assume that instantaneous rates
of fishing mortality and natural mortality are additive, that is, these
sources of mortality do not occur simultaneously within a harvest
year. This assumption cannot be strictly true, but mortalities asso-
ciated with P. imiriiins and H. nelsoni dominate the natural mor-
tality term and generally occur from May to October (Ford &
Tripp 1996). Because the fishery operates from October to March,
interaction between the two mortality terms should not be a major
source of error, but probably is a source of positive bias in the total
mortality estimates. We plan to test this assumption more rigor-
ously in our continuing stock assessments. Among the uncertain-
ties in estimating mortality, we note the questionable accuracy of
determining annual rates of natural mortality from box counts. For
example, experimental data presented by Christmas et al. (1997)
indicated that roughly 50% of boxes remained intact for more than
two years in northern Chesapeake Bay, suggesting that our surveys
overestimated annual mortality. Counteracting biases might in-
volve (1) mechanical disarticulation of boxes by the dredge used
for sampling, or (2) pre-mortem deterioration of the hinge ligament
in diseased and dying oysters, which could hasten disarticulation
compared with the chemically-killed oysters used in the Christmas
et al. (1997) study.

Effects of Salinity on Population Structure and Biomass

The sharp differences between trends in oyster biomass in
high-, medium-, and low-salinity zones partially reflect different
rates of recruitment and natural mortality. In high salinity areas,
infections with H. nelsoni and P. nuiriniis are enzootic, with
chronically high rates of mortality. Relatively few oysters survive
to market size in this zone, but higher rates of spat settlement and
recruitment to the sub-market stock tend to maintain populations
of small oysters. Populations in the medium salinity zone undergo
epizootics of H. nelsoni and attendant mortality episodes during
extended dry periods, when salinity rises to within the tolerance
range for the parasite (13-15 ppt; Paraso et al. 1999). Infections
with P. marinus are enzootic in this zone except during very wet
periods; mortality can be high even in the absence of H. nelsoni.
but oysters generally survive to larger sizes than in the high-
salinity zone. Spat settlement is moderate and episodic in these
mid-salinity areas. In the low-salinity zone, H. nelsoni is absent or
very rare, and although P. marinus can reach high prevalence in
these populations, oyster mortality rates are lower than in high-
and mid-salinity zones. Spat settlement and natural recruitment are
sporadic and must be supplemented by transplants from other re-

740

Jordan et al.

gions of the Bay or from hatcheries to sustain these harvested
subpopulations. Occasionally, freshets cause significant oyster
mortality in the areas of lowest salinity. The net long-term results
of these dynamics are as follows: (1) relatively stable subpopula-
tions of larger oysters in the low-salinity zone; (2) subpopulations
that tluctuate in size and abundance in the mid-salinity zone; and
(3) subpopulations in the high-salinity zone that are typically com-
prised mostly of submarket oysters, which can reach high abun-
dance in some areas. Jordan (1995) used multivariate analysis to
classify Maryland oyster bars into six groups based on several
population attributes; salinity was the most important independent
variable associated with population structure in that analysis.

Oyster Biomass and Abundance Relative to the Chesapeake Bay
Restoration Goal

In terms of the relative biomass index, the 1994 baseline Mary-
land oyster population (from which a 10- fold increase is to be
gained by 2010) was 84.4 g dry tissue weight per bushel of
dredged bottom material. An index of 844, therefore, would indi-
cate achievement of the goal. In 2001. the index was 1 17. about 1.4
times the baseline, or 14% of the goal. By combining the length-
weight equation and the equation for estimating the number of
oysters per bushel, we estimate that a Maryland bushel cannot
contain more than about 640 g dry weight of oyster tissue. For this
reason, the index of relative biomass cannot realize the desired
value of 844 with current sampling methods. Although the biomass
index is a satisfactory indicator of trends as long as the oyster
population remains at low levels, quantitative assessment will be
essential for determining attainment of the goal. Working with
Virginia scientists, we expect to establish a bay-wide baseline and
annual assessments of absolute abundance and biomass by the end
of 2002.

These analyses would not have been possible without relatively
long time series of consistent monitoring data. Earlier publications
based on MFS data (Smith & Jordan 1993, Jordan 1995) charac-
terized the first 2-4 y of the data set ( 1990 — 1993), but relation-
ships such as those depicted in Figures 3, 4 and 12, along with our
estimates of absolute oyster abundance and biomass, would be
difficult to elicit without data spanning many years. Significant
temporal trends in ecosystems as large and variable as Chesapeake
Bay can be difficult to detect without decades of consistent data
(Vaas & Jordan 1991, Jordan & Vaas 2000), an important consid-
eration in the design and maintenance of large-scale monitoring
programs. In turn, such monitoring programs, including the re-
sources to manage, analyze and interpret the data, are essential
wherever society has established specific goals for rehabilitation of
natural resources and ecosystems.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the NOAA
Chesapeake Bay Stock Assessment Committee (NA07FU0539),
and by the Maryland Department of Natural Resources (DNR).
Many people from DNR's Shellfish Division, especially Roy
Scott, John Hess, Mickey Astarb, Mitchell Tamowski, and Cap-
tains John Collier and Lee Daniels contributed to field data col-
lection. Connie Lewis of DNR provided oyster landings data. Jim
Uphoff s thoughtful review of a draft of this article was most
helpful. The authors thank our colleagues from Virginia, Roger
Mann and Jim Wesson, for their enthusiastic collaboration in our
stock assessment efforts, and Kennedy Paynter from the University
of Maryland for his support and encouragement. Copies of the
following literature are available from the first author of this ar-
ticle: Christmas and Jordan ( 1991 ), Gieseker (2001 ), Homer et al.
( 1496). Krantz (undated), Maryland DNR (2001 ), and Smith and
Jordan (1993).

LITERATURE CITED

Calvo, G. W., R. J. Fagan, K. N. Greenhawk, G. F. Smith & S. J. Jordan.

1996. Spatial distribution and intensity of Perkinsus marinits infections
in oyster recovery areas in Maryland. J. Shellfish Res. 15:381-389.

Carriker. M. L. 1996. The shell and ligament. In: V. S. Kennedy, R. I. E.

Newell & A. F. Eble, editors. The Eastern oyster Crassosrrea virginica.

College Park. MD: Maryland Sea Grant College Publ. UM-SG-TS-96-

01. pp. 7.5-168.
Christmas, J. F. Jr. & S. J. Jordan. 1991. Choptank River Oyster Mortality

Study. Annapolis, MD: Maryland Dept. of Natural Resources CBRM-

HI-91-1.
Christmas. J. F.. M. R. McGinty, D. A. Randle. G, F. Smith & S. J. Jordan.

1997. Oyster shell disarticulation in three Chesapeake Bay tributaries.
J. Shellfish Res. 16:115-123.

Ehrhardt, N. M. & J. S. Ault. 1992. Analysis of two length-hased mortality
models applied to bounded catch length frequencies. Trans. Am. Fish-
eries Soc. 121:115-122.

Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms. In:
V. S. Kennedy, R. I. E. Newell & A. F. Eble, editors. The eastern oyster
Crassoslrea virginica. College Park. MD: Maryland Sea Grant College
Publ. UM-SG-TS-96-01, pp. 581-A60.

Gieseker, C. 2t)OI. Year 2()00 Maryland Oyster Disease Status Report.
Oxford, MD: Maryland Department of Natural Resources Technical
Report FS-SCOL-01-1.

Homer, M. L., R. Scott & G. E. Krantz. 1996. Maryland Oyster Population
Status Report: 1995 Fall Survey. Oxford, MD: Maryland Department
of Natural Resources Report MDDNRSPl-96.

Jordan. S. J. 1987. Sedimentation and remineralization associated with

biodeposition by the American oyster Crassoslrea virginica (Gmelin).
Ph.D. Dissertation. College Park, MD: University of Maryland, 2(X) pp.

Jordan, S. J. 1995. Population and disease dynamics of Maryland oyster
bars: a multivariate classification analysis. / Shellfish Res. 14:459-
468,

Jordan, S. J. & P. A. Vaas. 20()0. An inde.\ of ecosystem integrity for
Northern Chesapeake Bay. Environ. Sci. Policy 3:S59-S88.

Krantz. G. E. undated. Oyster recruitment in the Chesapeake Bay 1939 to
1993. Oxford, MD: Maryland Department of Natural Resources, 15 pp.

MacKenzie. C. J. Jr. 1996. Management of Natural Populations. In: V. S.
Kennedy. R. I. E. Newell & A. F. Eble. editors. The eastern oyster
Cras.wsirea virginica. College Park. MD: Maryland Sea Grant, pp.
707-721.

Maryland DNR. 2001. Maryland Oyster Population Status Report, 1996-
2000 Fall Surveys. Annapolis, MD: Maryland Dept. of Natural Re-
sources, Annapolis.

Meritt, D. W. 1977. Oyster spat settlement on natural cultch in the Mary-
land portion of the Chesapeake Bay (1939-1975). UMCEES Special
Report No. 7, Cambridge. MD: University of Maryland, Horn Point
Laboratory.

Newell. R. I. E. 1988. Ecological changes in Chesapeake Bay: are they the
result of over-harvesting the American oyster. Crassoslrea virginica!
In: M. Lynch & E. C. Krome. editors. Understanding the estuary:
advances in Chesapeake Bay research. Solomons. MD: Chesapeake
Research Consortium, pp. 536-546.

Newell, R. I. E., G. S. AKspach Jr., V. S. Kennedy & D. Jacobs. 2000.

Oyster Biomass in the Chesapeake Bay

741

Mortality of newly metamorphosed eastern oysters {Crassosm-a vir-
ginica) in mesohaline Chesapeake Bay. Marine Biol. 136:665-676.

Paraso. M. C. S. E. Ford. E. N. Powell. E, E. Hofmann & J. M. Klinck.
1999. Modeling the MSX parasite in Easter oyster iCrassostrea vir-
ginica) populations. 11. Salinity effects. / Shellfish Res. 18:501-516.

Rothschild. B. J.. J. S. Ault. P. Goulletquer & H. Heral. 1994. Decline of
the Chesapeake Bay oyster population: a century of habitat destruction
and overfishing. Marine Ecol. Prog. Series 1 1 1:29-39.

Smith. G. F. & S. J. Jordan. 1993. Monitoring Maryland's Oysters: a
Comprehensive Analysis of Modified Fall Survey Data 1990-1991.

Oxford. MD: Maryland Department of Natural Resources Report
CBRM-OX-93-03.

Ulanowicz. R. E.. W. C. Caplins & E. A. Dunnington. 1980. The forecast-
ing of oyster harvest in central Chesapeake Bay. Esniarine. Coastal
Shelf Sci. 11:101-106.

Vaas. P. A. & S. J. Jordan. 1991. Long term trends in abundance indices
for 19 species of Chesapeake Bay fishes: reflection of trends in the Bay
ecosystem. In: New perspectives in the Chesapeake System: a research
and management partnership. Proceedings of a Conference. Chesa-
peake Research Consortium Publ. 137.

Journal of Shellfish Research. Vol. 21, No. 2. 743-747, 2002.

EXPERIMENTAL EFFECTS OF WATER TEMPERATURE ON THE GAMETOGENIC
DEVELOPMENT OF BROODSTOCK IN THE OYSTER, OSTREA CHILENSIS

ANDREW G. JEFFS,' ' B. J. DUNPHY." AND R. M. G. WELLS"

^ National Insliliile of Water and Atmospheric Research. P.O. Bo.x 109-695. Auckland. New Zealand:
'School of Biological Sciences. University of Auckland, Private Bag 92019 Auckland, New Zealand

ABSTRACT A protocol for reliably conditioning broodstock of the Chilean oyster, Osirea chilensis. is required because the
commercial aquaculture of this species has been constrained by the low and variable number of naturally spawning adults. Therefore,
the gametogenic response of hatchery conditioned O. chilensis broodstock under differing temperature regimes ( 10°C, 15°C, and 20°C)
was investigated, and compared with changes in a wild population over the same time. The overall loss of gametes, particularly ova.
through spawning and phagocytosis was significantly higher in oysters held at 20°C and these oysters tended to increase early
oogenesis and decrease early spermatogenesis. There was also greater variability in the abundance of reproductive products, particu-
larly female products, among oysters at the higher experimental temperature. At IO°C and 15°C. oysters had similar levels of
gametogenic products to wild oysters. Over the experimental period when natural water temperatures remained low the wild oysters
also increased early spermatogenesis and oogenesis, but there was little loss of gametes or phagocytosis. The results indicate that
warmer water temperatures may be involved in synchronizing and initiating oogenesis, and could be a useful tool for resetting the
gametogenic cycle in broodstock. Unlike many other species of cultured oysters, it appears that conditioning broodstock of O. chilensis
may best be undertaken at low water temperatures.

KEY WORDS: Chilean oyster, Ostrea chilensis. conditioning, gametogenesis, reproductive cycle. New Zealand

INTRODUCTION

The lack of a large and reliable supply of larvae is a major
impediment to commercial aquaculture of tfie Chilean oyster. Os-
irea chilensis (Philippi 1845), in a number of countries including
South America and New Zealand (Lepez 1983. Utting 1987, Va-
lencia Camp 1990, Hickman 1992. Jeffs 1995). The development
of hatchery techniques for artificially conditioning and inducing
larval production in broodstock at any time of the year would
overcome this obstacle. However, the factors influencing the re-
productive cycle of the Chilean oyster are poorly understood and
consequently the development of effective hatchery techniques
remains in their infancy (Jeffs & Creese 1996). There are only
three published reports of preliminary attempts in Chile to condi-
tion broodstock of this oyster, all of which have produced mixed
results (Ramorino 1970, Chaparro 1990, Wilson et al. 1996). The
first unsuccessful attempts to condition and spawn Chilean oysters
in a laboratory were made with minimal control over water tem-
perature and feeding (Ramorino 1970). A preliminary study by
DiSalvo et al. ( 198.3a), DiSalvo et al. (1983b) induced some oys-
ters to spawn earlier than in the wild by using artificially raised
temperatures. Similar results were obtained with control over both
water temperatures and feeding by Chaparro (1990). He found that
some broodstock held at 17°C and 20"C matured and spawned
spermatozoa and eggs, however, oysters held at 14°C matured and
spawned spermatozoa, but did not spawn eggs. Broodstock held at
I4°C and given a higher feeding allocation was the only experi-
mental treatment in which oysters maintained their reproductive
condition.

Field research in New Zealand has suggested that low winter
water temperatures (<I2°C) play an important role in increasing
adult gonad condition and subsequent larval production (Wester-
skov 1980. Jeffs et al. 1996. Jeffs & Hickman 2000). The aim of
this research is to experimentally assess the role of water tempera-
ture on the gametogenic cycle of the Chilean oyster while attempt-
ing to control other variables, such as food availability, that may
also affect reproductive activity. The broodstock for the research
came from a wild population of oysters in the Manukau Harbour.

in northwestern New Zealand that has previously been identified
as having high natural fertility (Jeffs et al. 1996).

MATERIAL AND METHODS

Experimenlal Setup

On June 20, 1993. 216 oysters of a size known to be capable of
brooding larvae (£50 mm shell height; Jeffs et al. 1996. Jeffs et al.
1997a) were randomly collected from a natural bed of oysters in
the Manukau Harbour (see Fig. 1 ) and transported to the labora-
tory. The oysters were scrubbed to remove debris and then ran-
domly assigned to six 300-1 rectangular plastic conditioning tanks.
Oysters were suspended in the tanks in plastic trays and each tank
was filled with 5 p.m filtered seawater at the ambient temperature
of the Manukau Harbour that was I3,rc. Over the next 48 h the
water temperature of three sets of two tanks was adjusted slowly to
each of the three experimental temperatures of 10°C, 15°C. and
20°C. Every 24 h the seawater in the tanks was changed with 5 |jim
filtered seawater al the experimental temperature. The water tem-
peratures in the tanks were monitored using maximum to mini-
mum mercury thermometers that were reset at each water change.
Tanks were aerated continuously via an airstone. With each water
change, the tanks of oysters were all fed an equal quantity of mixed
monoalgal cultures of Chaetoceros gracilis. Isochiysis galbana.
Pavlova httberi. and Thalassiosira pseudonana that were in excess
of daily requirements. This was judged by the presence of remain-
ing food in the water at the end of the 24-h period. If the water was
cleared prior to this time additional food was added to all of the
tanks. On five occasions when live algal cultures were insufficient
Celsys^'^' Algal 161 microfeed consisting of dried Tetraselmis
suecica resuspended in seawater was also fed. All of these mi-
croalgal species are known to be nutritious to Ostrea chilensis or
bivalves generally (Walne 1970, Brown et al. 1989, Laing 1991).
After 50 days the broodstock from each tank were harvested and
processed for histology.

Wild Population Samples

Oysters were collected from the population in the Manukau
Harbour to provide a comparison with the results for the experi-

743

744

Jeffs et al.

Figure 1. Map showing location of Chilean oyster population used for
this study.

mental broodstock. At the start (day 1). mid-point (day 28) and
conclusion (day 50) of the experiment about 70 oysters (>50 mm
shell height) were collected randomly from the Manukau Harbour
and processed for histology. Ambient water temperature in the
harbor was recorded at each sampling time.

Histologic Processing

Each oyster was carefully opened and a note made of the pres-
ence of larvae in the brood chamber. A 5-mm thick section of
tissue was excised from each oyster parallel to the anterior-
posterior axis between the labial palps and the posterior adductor
muscle and then fixed in Bouin's solution. Tissue sections taken in
this manner for this species are known to contain gametes repre-
sentative of the whole gonad (Jeffs 1998). Permanent microscope
slide mounts were made of the gonad material once it had been
stained with haemotoxylin and eosin to differentiate the reproduc-
tive tissues.

Histologic Analysis

Each slide was examined under a compound microscope at up
to xlOO magnification and the presence of different types of re-
productive products were analyzed by a semi-quantitative method
developed and verified specifically for this oyster (Jeffs 1998. Jeffs
1999). The method relies on assigning a score of 0-3 (0 = absent.
3 = abundant) to the abundance of reproductive features found
that are associated with different aspects of the gametogenic cycle.
Five stages of spermatogenesis (spermatogonia, primary and sec-
ondary spermatocytes, spermatids, and spermatozoa) and three
stages of oogenesis (oogonia, oocytes, and ova) were scored. Ad-
ditionally, the release of ova and spermatozoa from the lumen of
the follicles ( = gamete loss or spawning) and the abundance ot
reabsorption of unspawned ova and spermatozoa ( = phagocytosis
or reabsorption) were scored in the same manner that has previ-

ously been verified (Jeffs 1998. Jeffs 1999). The amount of con-
nective tissue surrounding the follicles was also scored, providing
an inverse measure of the overall quantity of reproductive material
present in the follicles (Jeffs 1998. Jeffs 1999). Finally, a visual
estimate was made of the percentage of male reproductive prod-
ucts over the entire gonad section.

Statistical Analyses

Statistical comparisons were made for the scores of the game-
togenic attnbutes of oysters among the three wild sampling events,
the three temperature treatments and between the replicate pairs of
treatment tanks using Kruskal-Wallis tests (Sokal & Rohlf 1995).
The 1 2 response variables consisted of those reproductive attrib-
utes measured under the microscope. Bonferroni corrected signifi-
cance levels were used for the tests to control for inflated type 1
errors caused by multiple significance testing.

RESULTS

Water Temperatures

Recorded water temperatures in Manukau Harbour were
13.1"C at the initial collection of broodstock. 1 1.6°C at the day 28
of the experiment, and 1 1 .9 'C at the completion of the experiment.
After the initial water temperature change over 48 h the water
temperatures in the experimental tanks did not fluctuate more than
± 1 -C from the treatment temperatures for the remaining duration
of the experiment.

Brooding

At the completion of the experiment five oysters (6.9'7c) were
brooding larvae in the 10=C treatment (3 in one tank and 2 in the
other). Three oysters (4.2%) were brooding in the I5°C treatment
(3 in one tank and none in the other). No oysters were brooding in
the 20-C treatment.

Samples of oysters taken from the wild population found 5 out
of 70 oysters (7.1%) were brooding larvae at the outset of the
experiment, 5 out of 73 oysters (6.8%) were brooding at day 28
and 6 out of 73 oysters (8.2%) were brooding at the end of the
experiment.

Statistical Analyses

Analyses showed that there was no difference in the reprtxiuc-
tive attributes of oysters held in the two tanks used in each ex-
perimental treatment. Therefore, the results for the two tanks were
pooled for each experimental treatment. Statistical comparisons
were made of the scores for the reproductive attributes of oysters
among the three experimental temperature treatments, and among
those sampled from the wild over the duration of the experiment
(Fig. 2 and Fig. 3). For the experiment, significant differences
were found among the scores for connective tissue (P < 0.000001 )
and phagocytosis iP < 0.000005). For oysters sampled from the
wild, significant differences were found among the scores for per-
centage'^male (P < 0.001). spermatogonia iP < 0.0005). primary
spermatocytes (P < 0.0001 ) and oocytes (P < 0.0001 ). The overall
trends in the data were explored by plotting mean values for the
variables with their standard errors (Figs. 2 and 3).

Spermatogenesis

Over all of the oysters examined the male reproductive prod-
ucts were generally more abundant than female reproducti\e prod-

Effect of Temperature on Chilean Oyster Gametogenesis

745

100-
80-

S? 40-

20-

3-

I 2-

CO

E
I 1-

■5.

S. 1-

peO.001

p<0 0005

p<0 0001

Wild

Experimental

0 28 50

Day of Experiment

10 15 20

Water temperature ( °C )

Figure 2. Mean scores (± standard error) of male gametogenic char-
acteristics and percent male of Chilean oysters plotted against either
time of sampling (wild population), or experimental water tempera-
ture (experimental population). Signincant ditTerences between scores
as identified by Kruskal-Wallis tests are indicated (ns = not signifi-
cant).

ucts. In the wild population of Chilean oysters there was a signifi-
cant increase in the proportion of male reproductive material ac-
cumulating in the gonad {P < 0.001). This was associated with a
trend for male reproductive attributes, especially for the early sper-
matogenesis stages (spermatogonia (P < 0.0005), primary sperma-
tocytes ^P < 0.0001 ) and secondary spemiatocytes) to increase in
abundance over the 50-day experimental period as the natural wa-
ter temperatures decreased. At the end of the experimental period
oysters in the three temperature treatments contained a similar
abundance of male reproductive products as oysters in the wild
population. However, there was a general trend for the abundance
of male reproductive products, especially the early spermatogen-
esis stages, to decrease slightly and for the variability in the abun-
dance of reproductive products to increase with increasing experi-
mental temperature.

Oogenesis

On average ova were more abundant among all of the experi-
mental and wild oysters than oogonia or oocytes. These earlier
stages of oogenesis tended to increase in the wild population of
Chilean oysters over the study period. For example there was a
significant increase in oocytes among wild oysters over the period
(P < 0.0001). At the end of the experiment the oysters in three
temperature treatments contained a similar abundance of female
reproductive products as oysters in the wild population. The abun-
dance of the early stages of oogenesis (oogonia and oocytes) in
oysters tended to increase with higher experimental water tem-
perature. The abundance of ova in the 20^C treatment, however,
tended to be lower than for the other temperatures, and for the wild
population. The variability in abundance of female reproductive
products was also greatest in the 20°C treatment.

8

p<0.0001

p<0.000001

Wild

p<0,C00005

Experimental

28

Day of expenment

15

Temperature ( C.)

20

Figure 3. Mean scores (± standard error) of female gametogenic of
Chilean oysters plotted against either time of sampling (wild popula-
tion), or experimental water temperature (experimental population).
Significant differences between scores as identified by Kruskal-Wallis
tests are indicated (ns = not significant).

746

Jeffs et al.

Gamete Loss and Phagocytosis

The recent loss of gametes, the amount of connective tissue and
phagocytosis of reproductive products was at similar lovv* levels
among the three wild samples of oysters suggesting that gamete
materials were accumulating in the gonads over this period. How-
ever, for the experimental oysters the recent loss of gametes, and
especially the amount of connective tissue and the amount of
phagocytosis of reproductive products all tended to increase with
increasing temperature and especially at 20°C.

DISCUSSION

Low water temperatures have been widely implicated in con-
trolling the production of female gametes in Ostica chilensis
(Westerskov 1980. Winter et al. 1984. Jeffs et al. 1997a. Jeffs et
al. 1997c. Jeffs 1999). Field studies within New Zealand surmised
that water temperatures of < 1 2''C acted to increase the gonad con-
dition and the development of ova in local Chilean oyster popu-
lations (Westerskov 1980. Jeffs et al. 1997b. Jeffs 1999, Jeffs &
Hickman 2000). The results of our laboratory study revealed that
the effect of temperature on gonad development was consistent
with the patterns observed in wild populations of this oyster (Jeffs
& Cree.se 1996, Jeffs et al. 1997a). However, a considerable
amount of variability remained in the gametogenic response of
individual oysters to experimental temperatures, a pattern that ap-
pears to be a feature of this species (Jeffs et al. 1997b).

In this study male and female gametes were accumulated in the
gonads of wild oysters experiencing cold winter water tempera-
tures and there was little overall loss or phagocytosis of gametes.
The accumulation of reproductive products in winter/spring pre-
ceding a minor spring/summer peak in spawning has been ob-
served in other wild populations of this oyster (Jeffs et al. 1996.
Jeffs 1999. Jeffs & Hickman 2000). The experimental oysters ex-
hibited a similar response to lower water temperatures, with oys-
ters held at 10°C and 15°C also accumulating similar amounts of
developing reproductive material to wild oysters. In both the wild
and experimental oysters at 10°C and 15°C early spermatogenesis
had increased. In wild oysters there were signs that early oogenesis
also increased. A similar pattern has also been observed in the
Olympia oyster. Ostrea lurida. where experimental broodstock
held at I2"C increased their gonad condition, while those at I8°C
and 2r"C lost condition through the gonad cycle being rapidly
advanced (Santos et al. 1993). Similar results have also been found
for Chilean populations of O. cliileiisis maintained in hatcheries
over a range of temperatures (Chaparro 1990, Toro & Morande
1998). Such a response is common to many other species of bi-
valves held at elevated temperatures (Utting & Spencer 1991, Ut-
ting & Millican 1997). This rapid advance of the gonad cycle
would also explain the absence of brooding oysters among the
broodstock oysters held at 20°C in our study because these oysters
would have already spawned or reabsorbed their gametes, whilst

those at lower temperature were still developing. This was evi-
denced by the increases in the loss of gametes, connective tissue
and phagocytosis observed amongst our experimental oysters at
20°C. and previously identified among.st oysters taken from wild
populations during periods of similar warm water conditions (Jeffs
et al. 1996. Jeffs et al. 1997a. Jeffs et al. 1997c. Jeffs et al. Jeffs
1998. Jeffs et al. 1999).

The results for these two Ostrea species are in contrast to the
widely cultivated Pacific oyster. Crassostrea gigas. the European
oyster. O. edidis, and the American oy.ster. C. virginica. which are
known to undergo gonad development at warmer water tempera-
tures, (e.g.. 18"C to 24°C) and commercial broodstock condition-
ing relies on using these warmer temperatures (Aboul-Ela 1960,
Quayle 1969. Mann 1979. Muranaka & Lannan 1984. Dinamani
1991. Santos et al. 199.3, Castagna et al. 1996). Within this tem-
perature range the experimental O. chilensis in this study lost
condition through increased gamete loss and phagocytosis and
decreased early spermatogenesis. There was also a trend for early
oogenesis to increase, but this has previously been associated with
rapid initiation of early gametogenesis after the extensive loss of
gametes and subsequent rapid phagocytosis of remaining gamete
material at warm water temperatures (Jeffs 1998, Jeffs 1999, Jeffs
& Hickman 2000). All of these patterns of gonad change observed
in our study are consistent with those previously observed in popu-
lations of wild oysters in warm water temperatures during sum-
mer-autumn (Jeffs 1998, Jeffs & Hickman 2000).

The results of this study suggest that manipulating water tem-
peratures has the potential to be an effective method of controlling
broodstock development in O. chileiisis. Unlike other species of
commercial oysters, cold-water temperatures appear to be impor-
tant in stimulating early spermatogenesis and oogenesis and the
accumulation of developing gametes much needed for effective
conditioning of broodstock. Warm water temperatures appear to
rapidly advance the development and loss of developing gametes
(through spawning and phagocytosis) already held in the gonads,
and this is followed by the rapid increase in oogenesis. Therefore,
warm water temperatures have the potential to be used for advanc-
ing the development of gametes in broodstock conditioned at cold
temperatures toward spawning and for restarting the gametogenic
cycle from early gametogenesis in broodstock in attempt to create
greater synchrony among individual broodstock.

ACKNOWLEDGMENTS

The authors thank Geoff McAlpine for sagely oyster advice. Jo
Evans and Simon Hooker for helping to establish the algal cul-
tures, and Beryl Davy for the histologic preparations. Logistic
support from Bob Creese and the Leigh Manne Laboratory made
this research possible. Martin von Randow undertook the statistical
analyses. This work was supported by the New Zealand Founda-
tion for Science. Research and Technology.

Aboul-Ela. I. A. 1960. Conditioning Ostrea ediilis from the Limfjord for

reproduction out of season. Mcddelelser Fret Danmarks Fiskeri - Of;

Huvuuders0gelser 2: 1-15.
Brown. M. R.. S. W. Jeffrey & C. D. Garland. 1989. Nulrilional aspects of

microalgae used in maricultuic; a literature review. CSIRO Mar. Udi.

Rept. Ser. 205:1-44.
Castagna, M., M. C. Gibbons & K. Kurkowski. 1996. Culture: application..

In: V. S. Kennedy. R. 1. E. Newell & A. F. Able, editors. The Eastern

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Oyster. Cnissoslreci virginicti. Maryland: Maryland Sea Grant Publi-
cations, pp. 675-690.

Chaparro. O. R. 1990. Effect of temperature and feeding on conditioning of
Ostrea chilensis Phillipi 1845 reproductors. Aqua. Fish. Manag. 21:
399^05.

Dinamani. P. 1991. The Pacific Oyster Cras.soslrea gigas (Thunberg.
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Joiinwl of Shellfish Reseurch. Vol. 21, No. 2, 749-756. 2002.

HISTORICAL CHANGES IN INTERTIDAL OYSTER {CRASSOSTREA VIRGINICA) REEFS IN A
FLORIDA LAGOON POTENTIALLY RELATED TO BOATING ACTIVITIES

RAYMOND E. GRIZZLE,'* JAMIE R. ADAMS,' AND LINDA J. WALTERS-

^Jackson Estituhne Laboratory, University of New Hampshire, Diirliain. New Hampshire 03S24;
^Department of Biology, University of Central Florida. Orlando, Florida 32816

ABSTRACT Research in the late 19y0s showed that some intertidal eastern oyster (Cra.ssosrrea viiginica. Gmelin) reefs in Mosquito
Lagoon within the Canaveral National Seashore. Florida had dead margins consisting of mounded up. disarticulated shells. It was
hypothesized that heating activities were the cause of the damage because all the reefs were adjacent to major navigation channels. To
investigate this, we characterized the history of the appearance of dead margins and other reef changes using aerial photographs taken
between 1943 and 2000. Imagery analyzed included prints (black & white, color, or color IR) from 1943, 1951, 1963, 1975. 1988, and
1995, and digital imagery from 2000 (USGS 1:12,000 digital onho-quarterquads). at scales from 1:6.000 to 1:24.000. Prints were
scanned at a resolution sufficient to yield 1-m pixels. After scanning, each set of images was georeferenced to the year 2000 imagery
using Arc View and Arclnfo GIS software. All reefs found to have dead margins based on 1995 and 2000 aerials were visited in 2001
and 2002 to confirm the presence and extent of dead areas. This provided ground-truthing for the "signature" (a highly reflective,
light-colored area adjacent to darker-colored live reef) to be used to detect the appearance of dead margins in the historical aerials. The
earliest appearance of dead margins was in the 1943 aerials on one reef adjacent to the Intracoastal Waterway (ICW). a major
navigation channel. The total number of reefs affected and areal extent of dead margins steadily increased from 1943 through 2000.
In 2fK10. 60 reefs (of a total of -400 in the Park) had dead margins, representing 9. 1'^f of the total areal coverage by oyster reefs in
the Park. Along the ICW. some reefs migrated away from the channel as much as 50 m and in 2000 consisted mainly of empty shells
mounded up a meter above the high water mark. In contrast, many reefs in areas away from navigation channels showed little change
over the 57-year period. This historical analysis provides strong (although correlative) evidence that boating activity has had dramati-
cally detrimental effects on some oyster reefs in the study area. Ongoing studies are aimed at further testing this hypothesis and
elucidating the causal mechanisms involved.

KEY WORDS: eastern oyster. Crassosirea virginica, reefs, aerial photographs, remote sensing, boating activities

INTRODUCTION

The eastern oyster, Crassosirea virginica. forms extensive
reefs subtidally and intertidally along the eastern US coast (Bahr &
Lanier 1981. Burrell 1986). Intertidal reefs make up the dominant
form from North Carolina to the northern end of the Mosquito
Lagoon in east central Florida. Most of Mosquito Lagoon in this
area is within the boundaries of Canaveral National Seashore
(CANA). the present study area. Here, the intertidal oyster reef
constitutes a major habitat type (Grizzle 1990). Within CANA.
reefs are managed for their ecological importance and are har-
vested commercially and recreationally (Walters et al. 2001).

Previous research within CANA characterized reef distribution
and abundance patterns (Grizzle 1990) and focused on manage-
ment issues (Grizzle & Castagna 1995). Based on an analysis of
1995 aerial imagery and subsequent field surveys, it was discov-
ered that numerous reefs had dead margins consisting of disarticu-
lated shells mounded up several decimeters above the adjacent
living reef (Grizzle, pers. obs.). This pattern differed from the
well-documented, long-term growth pattern of a dead middle area
surrounded by living oysters (the "senescent stage" of Bahr &
Lanier 1981) because the dead zones were along the margins of the
reefs. Also, the dead margins consisted mainly of well-packed
shells instead of a shell/sand/mud mixture as typically found in the
dead middle area of senescent reefs. Further analysis showed that
all reefs with dead margins occurred adjacent to channel areas that
were heavily used by boats, including the federally maintained
Intracoastal Waterway (ICW) that runs along the western edge of
CANA. It was hypothesized that boating activities may be respon-
sible, at least in part, tor the dead margins.

This study was initiated as part of a larger project aimed at
assessing the potential impacts of boating activities on oysters.
Emphasis in the present report is on historical changes in oyster
reefs based on aerial imagery, and the results of associated studies
will be reported in future publications. The major objectives of this
report are: (1) describe the present (2000) distribution of oyster
reefs in CANA; (2) characterize historical ( 194.3 to 2000) changes
in the CANA reefs based on aerial photographic imagery, with an
emphasis on those reefs with dead margins in 2000; and (3) relate
historical reef changes to environmental factors, with an emphasis
on boating activities.

METHODS

Study Area

*Corresponding author. E-mail: ray.grizzle@unh.edu

The Study area was in northern Mosquito Lagoon in east-
central Florida (Fig. I ) and restricted to the Canaveral National
Seashore (CANA). a National Park unit that is an example of a
relatively stable barrier beach/lagoonal ecosystein (Grizzle 1990,
Walters et al. 2001 ). Most of the Lagoon within CANA is a com-
plex system of shallow open water areas and nearly 100 mangrove
(Rhizophora mangle a.n(X Avicennia genninansydommaied islands.
Oyster reefs occur in intertidal areas, often adjacent to seagrass
(mainly Halodide wriifhtii) beds that are extensive in some areas
(Morris et al. 2000). Water depths are <1 m in most areas and the
annual salinity range is typically between 25 and 35 psu. occa-
sionally increasing to 40 psu (Grizzle 1990). Mosquito Lagoon is
the northernmost body of water in the Indian River Lagoon system
(IRL) that has been described as the richest and most diverse
estuary in the continental United States (see references in 1995
special issue of Bulletin of Marine Science. Vol. 57; also see
Walters et al. 2001). The ecological importance of this area has

749

750

Grizzle ht al.

; _ ,.,

•';

' (

> 4

5 > -

-^^

HwT

7

9 .

o
o

ELDORA

Figure 1. Northern portion of Canaveral National Seashore, showing most ol 40<l mapped o\sti-r reels (each shown as a hiack polygon
exaggerated 2x horizontally) based on year 2000 imagery. Nine study groups used in historical ( 144,^2000) assessment are indicated. Note that
"live reefs" and "dead margins" are combined in this figure.

been demonstrated by the US Environmental Protection Agency in
listing the entire IRL as an Estuary of National Significance, and
by the State of Florida in classifying it as a Florida Outstanding
Waterway and Aquatic Preserve, the highest level of State protec-
tion.

Aerial imagery of the types and sources indicated in Table I
were used in this analysis. Prints were converted to digital format
following the "soft-copy photogrammetry" methods outlined in
Finkbeiner et al. (2001). Each print was scanned at a resolution
sufficient to yield 1-m pixels. After scanning, each set of images
was referenced to the year 2000 orthorectified digital imagery

TABLE 1.
Aerial imagery used in present study.

Year

Type

.Source

Scale

1943

Black & white prints

St. Johns River Water 1
Management District

16,000

1951

Black & wliite pnnls

Volusia County 1

16.000

1963

Black & white prints

Volusia County 1

24,000

1975

Color prints

Volusia County 1

24.000

1988

Color prints

Volusia County 1

24,000

1995

Color infrared prints

University of New Hampshire 1

6,000

2000

Digital

US Geological Survey 1

24.000

using sufficient reference points to remove distortion from the
photographs. Year 2000 imagery provided enough detail so that
link points could easily be established to all historical photos. The
link files were created in ArcView by doing a side-by-side com-
parison of photos and creating points on each to reference corre-
sponding locations. This method created "from" and "to" points
that were used in Arclnfo to warp the historical photos and register
them to a UTM coordinate plane. On average, 35 to 40 points were
established for each photo pair. This allowed the polynomial order
of transformation to be varied enough to compare different levels
of warping in Arclnfo. The higher the order of the polynomial, the
more distortion can be removed. Warping results from order i to
6 were compared and it was determined that third order transfor-
mations were sufficient to match these itnages to their year 2000
counterparts. Such results were anticipated due to the low vertical
relief of the target area. Vertical relief is one of the primary sources
of distortion in aerial photography.

Live reefs initially were identified based on the following cri-
teria (i.e.. "signature"): dark margins with a lighter middle area,
round to in'egular in shape, and with a smooth te.xture (Fig. 2).
These criteria were applicable regardless of imagery type. In color
and color IR photos, the darker zone of the reef was typically olive
10 olive-gray in color. As an example of the overall image, round
reefs resembled a donut with the center being lighter in color than
the outer ring. In stark contrast to the live reef signature, dead

Historical Changes in Oyster Reefs

751

Figure 2. Examples of signatures for live reef (LR; reefs enclosed in boxes lor one year only of each pair of aerials) and dead margins (DM
indicated by arrows) from different kinds and qualities of imagery. Group I — 1995: LR signature with dark margin not evident; Group 1 — 2000
typical LR signature of dark margin surrounding lighter central region, but low quality imagery. Group 7 — 1963: 100% LR; Group 7 — 1995
LR with extensive DM (bright white) along outer edge of reef. Group 8 — 1963: three LR reefs in low quality imagery caused by poor sun angle
and surface reflectance; Group 8 — 1995: three LR reefs as in 1963, but outer reef with extensive DM in 1995.

margins appeared in all types of imagery as a high reflective,
light-colored (typically bright white) area adjacent to the darker-
colored live reef (Figs. 2 and 3).

Using these interpretation criteria, eight preliminary maps were
produced: a basemap showing all oyster reefs (minimum size -10
m") in CANA based on 2000 aerials, and one map for each of the
seven aerials from previous years showing only those reefs that
had dead margins in 2000. It should be noted that only a subset of
all reefs in CANA was mapped using the pre-2000 imagery. This
is because emphasis with respect to historical changes was on
those reefs that had dead margins in 2000. To make assessment of
historical changes inore manageable, reefs with dead margins in
2000 were arranged into nine groups based on location (Fig. 1 ). All
nine groups were quantitatively analyzed but only representative
aerial imagery is shown here. Due to variations in size of the reefs,
a constant map scale could not be used across all nine groups. Each
group boundary was positioned to emphasize changes from year to
year on an individual group basis and to show sufficient detail.
Special care was also taken to include any partial reefs so that area
calculations would remain consistent.

Field surveys were conducted in November 2001 and March
2002 to ground-truth the reef maps produced using the 2000 aeri-
als. Ground-truthing included assessment of initial reef signature
criteria and mapping accuracy. In November 2001, all reefs ini-
tially mapped with dead margins were visited to determine the
accuracy of the interpretations of live reef and dead margins.
Based on this survey, a revised set of reef maps was produced. A
second field survey was conducted in March 2002, to assess the
revised maps with an emphasis on mapping accuracy. Following

the general recommendations of Congalton and Green (1999) and
Finkbeiner et al. (2001 ), multiple points on several individual reefs
were chosen for field inspection. At each point, latitude and lon-
gitude (using a differential geographic positioning system, DGPS)
were recorded, and bottom type (dead margin, live reef, or non-
reef) identified. Each logged point was plotted on the revised maps
to determine locational and interpretational accuracy.

RESULTS AND DISCUSSION

Reef Signature and Ground-Truthing

A field survey in November 2001 indicated that the initial reef
signature criteria were sufficient for correct identification of nearly
all of the reefs based on 2000 aerials. Live reefs surrounded by
bottom features (e.g.. light-colored sand, dark mud) that contrasted
with the reef signature were accurately mapped. Interpretation of
reef boundaries was difficult only when bottom features (e.g.,
some seagrass beds, gray sands and muds) that resembled live
reefs occurred adjacent to the reef. It should also be noted that in
some areas on some aerials, reefs appeared a uniform gray, either
darker or lighter than surrounding areas (e.g., see "Group
I — 1995" and "Group 8 — 1963" reefs in Fig. 2). These departures
from the typical signature were apparently a result of the photo
being taken during low tide when the reefs were exposed. In some
cases, these aerials were also of poor quality caused by excessive
glare off the water surface and thus poor light penetration of the
water column (e.g.. "Group 8 — 1963" reefs in Fig. 2). Dead mar-
gins were dramatically visible in all but the poorest quality aerials
because of high reflectivity of the mounded up and sun-bleached

752

Grizzle et al.

i^tmti- "^DM

Figure 3. Dead margins (DM) in 20(12 showing typical relation to live reef (LR). A) Long, arching reef from (iroup 6; note presence of LR on
both sides of DM. B) .Small reef from Group 4 with DM and scattered LR behind it. Cl Large cluster of DM in Group I resulting from long-term
migration away from ICVV from 194.V-2()00. Dl DM being colonized in middle bj macrophytes; mound of dead shells extended nearly 1 m above
the high vvaler line and was adjacent to navigation channels on tv^o sides, resulting in the rounded shape. E) and F) Typical live reefs with
no DM.

shells (Fig. 2). Dead margins were only difficult to interpret when
they occurred adjacent to light-colored sand or sand/shell mixtures.
Hence, the initial reef signature criteria described above were
found to be valid.

Remote sensing of various types of coastal benthic habitats
using aerial photography is becoming an important tool for detect-
ing environmental change (Finkbeiner et al. 2001 ). Much of this
work has been on seagrasses (e.g., Ferguson et al. 1993, Morris et
al. 2000, Vimstein 20001 but research on other habitat types, in-
cluding oysters, has been published. One of the earliest published
studies mapped the reefs in this study area using aerial photo-
graphs taken in 1984 (Grizzle 1990). A similar survey was con-
ducted in Georgia in the late 1970s (Harris 1980). There are also
ongoing studies involving aerial photography of oyster reefs in
other areas of Florida and South Carolina, but we are aware of no

published literature on this work. Hence, although aerial imagery
has been used in previous oyster reef research, the published lit-
erature is meager. Finkbeiner et al. (2001) suggest a general pro-
tocol for mapping coastal habitats, but to our knowledge no pub-
lished description of an interpretive signature for intertidal oyster
reefs exists. Such a description is essential for further development
of a standard protocol for mapping oyster reefs.

It should be noted that the suggested signature for live reefs
includes potential "dead centers" (see Introduction section) as part
of the live reef. This study did not discrminiate between different
densities of live oysters. However, there were discernable differ-
ences between dead centers and areas of different densities of live
oysters, suggesting that useful information on oyster abundance
may be obtained using aerial imagery. The overall pattern of a
darker outer region surrounding a lighter-colored middle derives at

Historical Changes in Oyster Reefs

753

least in pan from the fact that higher densities of live oysters
typically occurred at the inargins of CANA reefs compared with
middle areas (Grizzle & Castagna 1995). This pattern should be
expected in general for older reefs in the "senescent stage" de-
scribed by Bahr and Lanier ( 1981 ) but it may also be the pattern
for most large reefs. Increased growth at the margins also has been
reported for reef-forming blue mussels. Mytihis ediilis (e.g.. New-
ell 1990; Svane & Ompi 1993). In the only relevant study on
oysters we are aware of. Powell et al. ( 1987) characterized small-
scale differences in distributions of oysters on reefs but did not
report an "edge effect." Further work is needed on small spatial
scale patterns detectable by aerial photographs so that remote sens-
ing techniques can be developed to monitor characteristics reflect-
ing overall reef "health" in addition to just total areal coverage of
"live" reef.

To further assess interpretational accuracy as well as locational
accuracy, 405 individual points on 56 reefs were logged and plot-
ted in March 2002; the second field visit to the study site. Overall,
this assessment again (as did the November 2001 field survey)
verified that interpretation accuracy was nearly 100%. It also
showed that location accuracy was within expected ranges. Of the
405 points visited, identified and plotted, 94% were within 5 m of
the actual DGPS-determined location (Table 2). DGPS is generally
considered to have an accuracy of about 5 m with 95% confidence.

Oyster Reef Distribution Within CANA in 2000

Approximately 400 live reefs ranging in size from 10 nr to
4180 m' were mapped using year 2000 aerials, and nearly all reefs
occurred in the northern half of the study area (Fig. 1). Although
not quantified, there was a strong decreasing trend in areal cover-
age by live oyster reefs from north to south, reflecting the amount
of tidal influence in the area (see Grizzle 1990 for further discus-
sion). The total bottom area within CANA covered by live reefs in
2000 was 12.3 ha. Live reefs showed a wide range of overall
morphologies and spatial orientations, including all three types
described by Kennedy and Sanford 1999 (based on Stenzel 1971 ):
string (perpendicular to shore), fringe (parallel to shore), and patch
(small, compact form). String reefs and fringe reefs occurred along
the edges of many of the major tidal channels. Patch reefs typically
occurred away from the channels, sometimes in groups that re-
sulted in dendritic patterns, with reefs separated by winding open
water areas only a few meters wide.

In 2000. dead margins occurred on about 60 reefs covering 1.12
ha and representing 9. 1 % of the total oyster reef areal coverage in
the Park (Fig. 1, Table 3). All reefs with dead margins occurred
adjacent to major channel areas, including narrower channels prob-
ably not affected by wind waves and broader expanses with suf-
ficient fetch for development of waves. On an individual reef

TABLE 2.

Locational accuracy data showing percent of individual plotted
points within the indicated (1 m, 2 m, etc.) accuracy level.

Reef

# points

Accuracy

level

type

plotted

0 m

1 m

2m

Sm

live

1.^7

KM)

no

116

125

dead

161

119

140

145

1.54

live -1- dead

298

219

250

261

279

Percent accuracy:

73.4

S.VM

87.6

93.6

TABLE 3.

Total coverage in hectares for dead margins and live reef in all nine

groups by year, and Park-wide in year 2000 only. See Figure 4 for

bar charts of data by group by year.

All of
Year 194.3 1951 1963 1975 1988 1995 2000 CANA

TOTAL Dead 0.02 0.10 0.26 0.29 0.55

TOTAL Live 6.55 5.28 4.71 5.73 5.39

Live + Dead 6.57 5.38 4.97 6.02 5.95

% Dead 0,3 2.0 5.5 5.1 10.3

0.85 1.12 1.12

3.60 4.06 12.28

4.46 5.18 13.39

23.7 27.6 9.1

basis, the dead margins ranged from <10% to -100% of each reefs
total areal coverage. Hence, some reefs only suffered minor losses
while others appear to have been completely killed.

Historical Changes in CANA Oyster Reefs

There was a consistent increase in areal coverage by dead mar-
gins from their first appearance on a reef near the ICW in 1943
through 2000. when about 60 reefs were affected throughout the
study area (Fig. 4. Table 3). In 1951, dead margins covered only
0.10 ha of bottom area, and all but one (Group 5) were located
along the ICW. In most other areas away from the ICW, dead
margins first appeared in 1975 or 1988 and showed a steady in-
crease in areal coverage over time. As already noted, by 2000,
dead margins occupied 1.12 ha of bottom area representing 9.1%
of the total oyster reef areal coverage in the Park.

Some reefs were remarkably dynamic over the 57-year study
period, with the most dramatic changes occurring in nearly all
reefs adjacent to the ICW (e.g.. Group 1 in Fig. 5). In these areas,
there was a general movement away from the ICW and an increase
in areal coverage by dead margins over time. For example, some
reefs in Group 1 moved a distance of approximately 50 m away
from the ICW between 1943 and 2000. In contrast, none of the
reefs in groups located away from the ICW moved by more than
a few meters over the 57-year period, including those with dead
margins. Also, the general shape, areal coverage, and location
remained relatively stable for many reefs over the period (e.g.,
most reefs in Group 5. Fig. 5).

An obvious and probably the most serious limitation on use of
historical imagery is that ground-truthing is limited. For this study,
the 2000 aerials were ground-truthed with respect to interpretation
and mapping accuracy. Information from this effort was used to
infer the level of confidence placed on historical mapping. There
was a wide range of overall quality of the historical aerials caused
by differences in sun angle, water clarity, tidal stage, and other
factors. This variability affected the mapping process but there is
no way to quantitatively assess the effect. A technique that seemed
to make the mapping process more accurate for the historical im-
agery was to analyze a single reef or group of reefs beginning with
the ground-truthed 2000 maps and proceeding backwards in time.
It was also found that for a year with poor quality imagery, ex-
amination of the previous and subsequent years made identifica-
tion of the reefs easier on that imagery. Thus, while there is no way
to rigorously assess accuracy of historical imagery there may be
ways to improve the process.

Based on 1986 imagery. Grizzle (1990) presented a distribution
map of oyster reefs in this study area. This map was revised based
on 1995 imagery by Grizzle and Castagna (1995). The maps pro-

754

Grizzle et al.

Group 1

E_ looou
S acrij

on m:

1 r

—

^

p,

■ Dead
3 Live

3H

1943 1951 1963 1976 1988 1995 2000

Group 2

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1943 1951 1963 1975 1988 1995 2000

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Figure 4. Areal coverage by live reef and dead margins from nine study groups over 57-year period, and all groups combined in last graph.

duced for both studies were very similar with respect to total rates this conclusion, and suggests that other than an increase in

number of reels (-400) and overall reef distribution pattern. How- dead margins there was probably little change in total oyster reef

ever. Grizzle and Castagna (1995) noted a wide disparity in re- coverage in the Park between 1986 and 1995.

ported total areal coverage by oyster reefs: 31.7 ha in 1986/1990.

and 14.0 ha in 1995. They further noted that the higher figure was Potential Causes of Historical Reef Changes

incorrect, and was an artifact of the low-resolution GIS program

used in 1990 that assigned all mapped reefs a minimum size of This historical assessment was designed to provide a partial test

0.05 ha. Inspection of the 198.^ aerials used in this study corrobo- of the hypothesis that boating activities have been a causal factor

Historical Changes in Oyster Reefs

755

Figure 5. Ihrec of nine reef groups sliiiHing (lilltrcnces in reef dynamics over the 57-year study period. Li\c reefs outlined with narrow black
line: dead margins (DM) Hlled in. Circles show Hrsl occurrence of I)M for that group (m43 lor tJroup 1. 1951 for Group 5. 1975 for Group 6.

in the occurrence of dead margins on some oyster reefs in the study
area. Data presented here support this hypothesis in several ways.
and yield no information for rejection of it. Moreover, the present
analysis indicates that for some reefs the existing (2000 to 2002)
dead margins represent only a vestige of the areal extent of those
reefs historically.

The major findings that implicate boating activities are associ-
ated with the fact that all of the approximately 60 reefs with dead
margins identified for the 57-year study record (1943 to 2000)
occurred immediately adjacent to major navigation channels. Data
available since 1986 show an average annual increase of about
10% in the number of boats registered in the two counties (Volusia
and Brevard) that border the Mosquito Lagoon (Hart et al. 1994).
coinciding with an average annual increase of about 9% in bottom
area covered by dead margins from 198S to 1993 (Table 2). There
were over 51.000 registered boats in Volusia and Brevard counties
in 1998, nearly twice the state average (J. Stewart, pers. com.). The
extent of dead margins increased by 16.4% from 1995 to 2000.
Such a correlation between registered boats, and thus potential
boating activity, and dead margins does not of course require a
cause-and-effect relation. However, if boating activities were in-
volved in the observed increases in dead margins, such a correla-
tion would be expected. Assuming boating activities were in-
volved, what might be the actual mechanisms causing the dieoffs
at the margins?

The importance of water movement (including tidal currents
and waves) to oyster reefs was recognized by Grave (1905), and
subsequent research has provided several hypothetical cause-and-

effect relations but \u> quantification of them (see reviews by Bahr
& Lanier 1981. and Kennedy & Sanford 1999: see additional
discussion later). Bahr and Lanier (1981) suggested that vertical
growth rate and morphologic form of intertidal reefs were strongly
affected by wave action. Waves (and currents) transport sediment
to and from the reef, and in some cases eventually result in sedi-
ment buildup and loss of oysters in the central region (see more
discussion in the Introduction section). Bahr and Lanier aLso noted
(p. 57) that "■. . . a water current or wave energy regimen above a
certain threshold level will prevent the development . . ." of a reef.
And they showed (p. 34) a photograph of "shell debris character-
istic of high energy beach shores . . .'" that resembled the mounded
up dead margins observed in the present study. Wave energies of
some magnitude are clearly capable of destroying an existing reef.
However, we are not aware of research that quantifies the rela-
tionships involved, nor the actual mechanisms involved.

Hypothesized inhibitory mechanisms for reef development in-
clude; vertical accretion of the reef surface to an elevation too high
in the intertidal caused by waves (Churchill 1920; Bahr & Lanier
1981). smothering and substrate instability caused by excessive
sediment transport (Marshall 19541. and inhibited larval settlement
caused by sediment ("grit") movement (Gunter 1979). Analysis of
historical aerials for reefs along the ICW in this study also suggest
that a reef can be physically moved by wave energies, resulting in
a range of responses from slow migration to total destruction,
much in the fashion that barrier islands migrate shoreward (termed
"'transgression") under increased wave energies (Davis 1996). As
already discussed, this study was not designed to test particular

756

Grizzle et al.

causal mechanisms for observed reef declines. They are the topic
of ongoing studies by Linda Walters and colleagues (e.g.. Wall et
al. 2002. Walters et al. 2002) and will be reported elsewhere. This

study, however, does provide strong (although correlative) evi-
dence that boating activity has had dramatically detrimental effects
on some ovster reefs in the studv area.

LITERATURE CITED

Balir, L. N. & W. P. Lanier, lys L The ecology of inlertidal oyster reefs of
the South Atlantic coast; a community profile. U.S. Fish and Wildlife
Service. Washington. DC: Office of Biological Services. FWS/OBS-
81/15.

Burrell. V. G. 1986. Species profiles: life histories and environmental
requirements of coastal fishes and invertebrates. American oyster.
Washington. DC: US Fish and Wildlite Service. Office of Biological
Services. Biol. Rep. 82:11 -,57.

Churchill, E. P., Jr. 192(1. The oyster and the oyster industry of the Atlantic
and Gulf coasts. Bureau of Fisheries Document 890. Appendi.x VIII.
Report of the US Fisheries Commission for 1919. pp. 1-.51.

Congalton. R. G. & K. Green. 1999. Assessing the accuracy of remotely
sensed data: principles and practices. Boca Raton. FL: Lewis publish-
ers. 137 pp.

Davis. R. A.. Jr. 1996. Coasts. Upper Saddle River. NJ: Prentice Hall. 274
pp.

Ferguson. R. L.. L. L. Wood & D. B. Graham. 1993. Monitoring spatial
change in seagrass habitat with aerial photography. Pboto)^. Eiiginefi:
Rem. Sens. 59:1033-1038.

Finkbeiner M.. B. Stevenson & R. Seaman. 2001. Guidance for benthic
habitat mapping; an aerial photographic approach. Charleston. SC:
NOAA Coastal Services Center. (NOOA/CSC/201 17-PUB).

Grave. C. 1905. Investigations for the promotion of the oyster industry of
North Carolina Rep. U.S. Comm. Fish. 29:249-315.

Grizzle, R. E. 1990. Distribution and abundance of Cwssoslrea virginica
(Gmelin. 1791) (eastern oyster) and Mercenaria spp. (quahogs) in a
coastal lagoon. J. Shellfish Res. 9:347-358.

Grizzle. R. E. & M. W. Castagna. 1995. Final report— Oyster reef moni-
toring program in Canaveral National Seashore. Titusville. FL; Na-
tional Park Service. Canaveral National Seashore. 14 pp.

Gunter, G. 1979. The grit principle and the morphology of oyster reefs.
Proc. Natl. Shellfish. Assoc. 69:1-5.

Harris. C. D. 1980. Survey of the intertidal and subtidal oyster resources of
the Georgia coast. Brunswick. GA; Department of Natural Resources.
Coastal Resources Division. 44 pp.

Hart. A. W.. D. L. Myers. T. M. O'Keefe & M. G. Thorn. 1994. A boater's
guide to the Indian River Lagoon. Melbourne. FL; National Estuary
Program. 85 pp.

Kennedv. V. S. & L. P. Sanford. 1999. Characteristics of relatively une.\-

ploited beds of the eastern oyster, Crassostrea virginica. and early
restoration programs. In; M. W. Luckenbach, R. Mann & J. A. Wesson,
editors. Oyster reef habitat restoration; a synopsis and synthesis of
approaches. Gloucester Point. VA: Virginia Institute of Manne Science
Press, pp. 25-46.

Marshall. N. 1954. Factors controlling the distribution of oysters in a
neutral estuary. Ecology 35:322-327.

Morris. L. J.. R. W. Vimstein, J. D. Miller & L. M. Hall. 2000. Monitoring
.seagrass changes in Indian River Lagoon, Florida using fixed transects.
In: S. A. Bortone. editor. Seagrasses; monitoring, ecology, physiology,
and management. Boca Raton. FL; CRC Press, pp. 167-176.

Newell. C. R. 1990. The effects of mussel (.Mylilus edulis. Linnaeus. 1758)
position in seeded bottom patches on growth at subtidal lease sites in
Maine. J. Shellfish Res. 9:1 13-1 18.

Powell. E. N.. M. E. White, E. A. Wilson & S. M. Ray. 1987. Small-scale
spatial distribution of oysters (Crassostrea virginica) on oyster reefs.
Bull. Mar. Sci. 41:835-855.

Stenzel. H. B. 1971. Oysters. In: R. C. Moore, editor. Treatise on inver-
tebrate paleontology. Part N (Bivalvia). Boulder. CO; Geological So-
ciety of Amenca. pp. N953-N1224.

Svane. I. & M. Ompi. 1993. Patch dynamics in beds of the blue mussel
Mvtilus edidis L.: effects of site, patch size, and position within a patch.
Ophelia 37:187-202.

Vimstein. R. W. 2000. Seagrass management in hidian River Lagoon.
Florida: dealing with issues of scale. Pacific Cons. Biol. 5:299-305.

Wall. L.. L. Walters. K. Johnson. N. Martinez & R. Grizzle. 2002. Re-
cruitment of the oyster Crassostrea virginica on intertidal reefs adja-
cent to waters with intense boating activity in the Indian River Lagoon.
Florida. National Shellfisheries Association 94th Annual Meeting.
Apnl 14-18. Mystic. CT. J. Shellfish Res. 21:415-416.

Walters. L.. A. Roman, J. Stiner & D. Weeks. 2001. Water resources
management plan, Canaveral National Seashore. Titusville. FL: Na-
tional Park Service, Canaveral National Seashore. 224 pp.

Walters, L., K, Johnson, L. M. Wall, N. Martinez & R. Grizzle. 2002. Shell
mo\ ement and juvenile survival of the oyster Crassostrea virginica on
intertidal reefs adjacent to waters with intense boating activity in the
Indian River Lagoon. Florida. National Shellfisheries .Association 94th
Annual Meeting, Apnl 14-18. Mystic. CT. J. Shellfish Res. 21:439.

JoKi-iwI of Shellfish Research. Vol. 21, No. 2. 757-762. 2002.

BIOCHEMICAL COMPOSITION OF SPONDYLUS LEUCACANTHUS BRODERIP, 1833

(BIVALVIA: SPONDYLIDAE) AND ITS RELATIONSHIP WITH THE REPRODUCTIVE CYCLE

AT ISLA DANZANTE, GULF OF CALIFORNIA, MEXICO

SONIA RODRIGUEZ-ASTUDILLO,* MARCIAL VILLALEJO-FUERTE,
FEDERICO GARCIA-DOMINGUEZ, AND RAFAEL GUERRERO-CABALLERO

Institute) Poliieciiico NacioiuiL Centra Intenlisciplinaiio de Ciencius Marinas. La Paz. Baja California
Stir. Mexico, A.P. 592 C.P. 23UUU

ABSTRACT The monthly variation of the biochemical composition was studied for one year in relation to the reproductive cycle in
a population of S/xiiulxlus leucacmuhus. a commercially important species in the Gulf of California. Carbohydrate, lipid, and protein
concentrations were determined in the adductor muscle, gonad, and digestive gland. Protein was the main constituent in the gonad,
digestive gland, and adductor muscle. Lipids were important in the digestive gland, whereas in the gonad they were moderately
represented and in the muscle they showed low and virtually invariable concentrations during the study period. Carbohydrates were
found largely in the adductor muscle and the digestive gland, whereas the gonad showed lower concentrations. S. leucacantlms has a
seasonal reproductive cycle with a reproductive inactivity period at the end of the autumn and throughout the winter. Gametogenesis
starts at the end of the winter, continuing during springtime, and ending in the summer, where 90% of organisms are ripe, coinciding
with the highest lipid concentration in the gonad. Spawning takes place in early autumn. It is concluded that this species has a
conservative reproductive strategy because it largely uses energy reserves for gamete development, in addition to energy obtained from
food, to supplement its energetic expenditure.

KEY WORDS: Spondylus leucucunthiis. reproductive cycle, biochemical composition. Gulf of California

INTRODUCTION

In the Gulf of California, Spondylus leucacanthus and other
bivalve species, including Megapitaria aurantiaca, Argopecten
ventricosus. Lxropecten nodosus. and Spondylus calcifer are of
great importance because of the high commercial value of soft
parts, adductor muscle, and shell. (Villalejo-Fuerte & Muneton-
Gomez 1995, Skoglund & Mulliner 1996. Villalejo-Fuerte &
Garcia-Dominguez 1998, Muneton-Gomez et al. 2001). The ex-
ploitation of commercially important bivalve populations in the
Gulf of California has fostered a number of studies focused on
reproductive and feeding aspects (Baqueiro et al. 1982, Villalejo-
Fuerte & Ochoa-Biiez 1993, Villalejo-Fuerte & Cebailos-Vu/quez

1996. Felix-Pico et al. 1997, Villalejo-Fuerte & Garcia Dominguez
1998. Villalejo-Fuerte et al. 2002).

In general, reproduction in marine bivalves is associated with
food availability and nutrient reserve storage in specialized organs
(Gabbott 1975, Bayne 1976. Barber & Blake 1981. Boadas et al.

1997. Claereboudt & Himmelman 1997). When organisms reach
reproductive maturity, growth slows down as a result of the re-
productive effort, and the biochemical composition may change
according to the reproductive requirements iLodeiros et al. 2001 ).
Those species where energy storage and gainete production cycles
overlap temporally are considered "opportunistic"; in contrast,
those in which both cycles are clearly separated in time because of
the use of previously stored energy reserves for gonad develop-
ment are known as "conservative species" (Bayne 1976).

Gonad development implies an intense metabolic activity, in-
cluding the storage of large amounts of lipids in the developing
eggs, either at the expense of glycogen reserves previously accu-
mulated in storing tissues, at the expense of food supply, or from
both sources (Gabbott 1975), A decrease in glycogen and protein
contents coupled with a rise in lipid content during gonad devel-
opment has been described for the pectinids Chlamys septemra-
diaki (Ansell 1974), Pecten maximus (Comely 1974), Argopecten

*Corresponding author. E-mail: srodrig@ipn.mx

irradiims (Hickey 1978. Barber & Blake 1481). Chlumys opcrcu-
laris (Taylor & Venn 1979). Plucopectcn imigellanicus (Robinson
et al, 1981 ), and Himiites giganteus (Lauren 1982). The influence
of temperature on nutrient-transfer regulation from reserve-storing
organs to the gonad has also been described for A. irradians and
Patinopecten caurinus (Sastry & Blake 1971. Barber & Blake
1981. MacDonald & Bourne 1987). The potential relationship be-
tween morphophysiologic indices and the storage and use of nu-
trient reserves during reproduction was described for wild popu-
lations of A. circularis (Villalejo-Fuerte & Ceballos-Vazquez
1996) and S. calcifer (Villalejo-Fuerte et al. 2002),

In the Gulf of California. S. leucacanthus banks have been
overexploited at depths of less than 15 m (Baqueiro et al. 1982),
but beyond 40 m there are banks that have not been affected by
commercial fisheries. Studies of this population focused on the
reproductive cycle (Villalejo-Fuerte & Garci'a-Domi'nguez, 1998)
and gut content have been conducted in relation to reproduction
and phytoplankton abundance (Muneton-Gomez et al. 2001). The
lack of infonnation on protein, lipid, and carbohydrate content in
storage organs and its potential association with the reproductive
period in this species, analyzed from a seasonal perspective, were
the reasons for the development of this research.

MATERIALS AND METHODS

An average of 36 specimens of S. leucacanthus with a range in
shell height from 40 to 94 mm (mean of 60 mm) were collected
each month, from June 1 996 to May 1 997. south of Isla Danzante
t26°55'-26°30'N and 112-1 1 1"40'W) in the Gulf of California
(Fig. 1 ). using a net at 40 m of depth.

For the biochemical analysis. 16 specimens were used each
month. In the laboratory, specimens were cleaned, eliminating
water from the pallial cavity, and were washed with distilled water.
Then, soft parts were separated from the shell and weighed, fol-
lowed by the dissection of the adductor muscle, gonad, and diges-
tive gland. Each organ was weighed separately and oven-dried at
100°C for 24 h. The dry tissue was pooled and homogenized and

757

758

RODRl'GUEZ-ASTUDILLO ET AL.

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RESULTS

Hiochemical Coiiiposilidii

.11.20 '"°'*'

Fifjurt 1. Location of the sampling area in the \icinity of Danzante
island, (iulf of California.

then was ground into a powder. Subsaniples ol this homogenate
were taken for the biochemical determination.

Protein determination was conducted using the method by
Lowry et ai. (1951). Carbohydrates were determined by Hewitfs
method (1958). Lipid extraction was performed using the method
by Folch et al. (1957) and a modification of Bligh and Dyer's
method (1959).

Energetic substrate concentration mg/g dry weight curves were
interpreted as follows: An increase was considered as indicative of
accumulation and a decline as indicative of substrate use in the
corresponding organ.

The reproductive cycle analysis included an average of 20
specimens per month. The degree of gonad development was de-
fined from histologic section (7 jxm thick) stained with the hema-
toxylin and eosin technique (Humason 1979), assigning one of the
following categories proposed by Villalejo-Fuerte and Garci'a-
Domingue/. (1998) for this species, undifferentiated, developing,
ripe, spawning, and spent. Botlom water temperature was recorded
at the time of collections.

A Spearman's rank conelaiion analysis irj was used to deter-
mine the relationship of the gametogenic cycle stages with protein,
lipid, and carbohydrate concentrations in the adductor muscle, di-
gestive gland, gonad, and water temperature. Before performing
the correlation analysis, logarithmic transformations were per-
formed for energetic substrates, and arc-sin transformation were
conducted for percentages of (he reproductive cycle stages, aimed
at obtaining data normality and homoscedasticity (Sokal & Rohll
1995). Only tho.se results where a significant correlation was found
are reported below.

The trend in the protein concentration curve is similar for the
three organs analyzed. During the summer, protein concentration
reaches high values and remains relatively unchanged. In autumn,
there is a noticeable decline, with low concentrations remaining
until the end of this season. In the winter, protein concentration in
the gonad and digestive gland shows a recovery, reaching high
values in mid-winter and decreasing by early spring. In the muscle,
proteins show a steady increase that lasts until mid-spring, reach-
ing high values also in the digestive gland and gonad, then de-
creasing at the end of the spring (Fig. 2A).

Lipid concentration in the adductor muscle did not show no-
ticeable variations during the study period. In the digestive gland,
high concentrations were found at the beginning of the summer,
which declined throughout this season and remained low, although
with some variations, during the autumn. In winter, there is a clear
recovery, reaching the peak value in mid-winter and then declin-
ing, with some fluctuations, afterwards and during the spring. In
the gonad, the highest lipid concentration was recorded during the
summer, with lower values in all other seasons (Fig. 2B).

During the summer, carbohydrate concentration drops in the
three organs analyzed (August), \Mth drops being more pro-
nounced in the muscle and gonad. In late summer and early au-
tumn, a considerable rise occurs in both gonad and muscle, re-
maining unchanged in the gland. During autumn, there is a decline
in the muscle and gonad, remaining low in early winter whereas
there is a considerable increase in the digestive gland (November);
at the end of the autumn, carbohydrates decrease, reaching low
\alues at the beginning of the winter. During the winter, carbohy-
drates in the muscle and digestive gland rise steadily, reaching
peak values during the spring. In the gonad, there is a slight in-
crease in mid-winter, decreasing afterwards until mid-spring, then
recovering at the end of this season (Fig. 2C).

Temperature

During the study period, bottom water temperature ranged from
17 to 24.5'C. the highest values recorded in July (24.5 C) and
October 1996 (24°C). The lowest temperature occurred in January
1997 ( 17'C; Fig. 2D). Spawning took place during a temperature
rise from 22.5 to 24°C. and gametogenesis occurred when tem-
perature rose from 17 to 18"C (Fig. 2D).

Reprodiicliie Cycle

The bivalve population enters a reproductive inactivity stage at
the end of the autumn (undifferentiated stage near lOO'/f in No-
vember), high values being maintained (around 70%) during the
winter. The reproductive activity (gametogenesis) starts at the end
of the winter, and in springtime 100% of the population is under-
going this stage. The first ripe individuals appear at the end of the
spring, and in the summer 90% of the population is ripe. The
population spawns in the autumn (90% in October). Gamete re-
sorption is rapid because the post-spawning stage only takes place
in early winter with low percentages (Fig. .^A-D).

DISCUSSION

111 general, proteins were the main constituent of the gonad,
digestive gland, and adductor muscle. Lipids were important in the
digestive gland, whereas in the gonad they were highest betore

Biochemical Composition of Spondylus leucacanthus

759

500

A

-m- Muscle -^ Gonad -*- D Gland

400

B

O N

-^ Gonad -^ D Gland

D

Figure 2. Variation of biochemical components in Spondylus leuca-
canthus. A. proteins: B, lipids; C carbohydrates in muscle, digestive
gland, and gonad: and D, bottom water temperature at Danzante is-
land, (iulf of California.

JJASONDJFMAM

Figure 3. Reproductive cycle stages in Spondylus leucacanthus. A, inac-
tivity; B, gametogenesis; C, maturity; D, spawning; E, post-spawning.

spawning and in the muscle they showed low and virtually un-
changed concentrations during the study period. Carbohydrates
were found chiefly in the adductor muscle and digestive gland,
whereas the gonad showed lower values. These same trends in
energetic substrate concentration were found in Pcclen ituLxiiiiKs
(Strohmeier et al. 2000). During gonad maturation of many marine
bivalves, an increase of biochemical compounds is observed in the
female gonad, followed by a drop during spawning (Ansell 1974,

760

RODRIGUEZ-ASTUDILLO ET AL.

Taylor & Venn 1979. Barber & Blake 1981. Suntlel & Vahl 1981.
Pazo.s et al. 1996).

The slight variation of proteins durini; the summer, when the
population is ripe, suggests a low demand of this energetic sub-
strate during this stage. The trend in the lipid curve for the diges-
tive gland suggests that lipids are used during the ripe stage: how-
ever the correlation obtained is low (r^ = 0.55) and nonsignificant.
Lipid transfer from the digestive gland to the ovary has been
demonstrated in Chlamy.s Iwricia (Vasallo 1973). Furthermore,
digestive gland carbohydrates show a significant correlation with
the ripe stage (i\ = 0.83, P < 0.05). indicating its use during this
stage.

It is likely that during the last gamete growth stage, food is also
used as an energy source. In this same population, ripe organisms
have been found in July, coinciding with a peak in food availabil-
ity, which may provide a portion of the nutrients required for the
gonad's final ripening (Mufteton-Gomez et al. 2001). Similar be-
havior was reported for Pkwopecten magellanUiis. which reside
southeast of Terranova (Thompson 1977). In other bivahe species
like Doiui.x villatiis and Telliua tenuis, the accumulation of reserve
glycogen seems to be related with the season when the highest
phytoplankton biomass occurs (Ansell & Trevaillion 1967. Ansell
1972). American oysters (Crassostrea virginica) held at 14 to
19°C were able to exploit a large phytoplankton bloom and syn-
thesize glycogen (Ruddy et al. 1975).

In S. leucacanthus. spawning is an event that demands a heavy
energy expenditure, requiring the use of protein and carbohy-
drate from the adductor muscle, the gland, and the gonad. How-
ever, it is in this latter organ that a significant correlation is found
(,-^ = -0.63, P < 0.05). Lipids do not appear as an important
source in this stage. Based on the analyses of morpho-physiologic
indexes, gut content, and phytoplankton abundance in the envi-
ronment, the required energy expenditure during spawning in
this species had been suggested to be partially supported by re-
serves stored in the muscle, the digestive gland, and the gonad
(Villalejo-Fuerte & Garci'a-Domi'nguez 1998, Muiieton-Gomez et
al. 2001).

When spawning ends, lipids and proteins remain low whereas
carbohydrates are stored in the digestive gland, providing the en-
ergy required for the resorption of residual gametes during the
stage known as spent that occurs at the end of the autumn. In
Crassostra virginica. the concentration of neutral lipids decreased
considerably during the spawning (Trider & Castell 1980) in C.
giga.^ was observed an increased in lipid during the gametogenesis
phase (Deslous-Paoli & Heral 1988).

When the population enters reproductive inactivity (undiffer-
entiated stage) in early winter, there is a reorganization in the
gonad (Villalejo-Fuerte & Garci'a-Dominguez 1998); during this
stage, carbohydrates from the digestive gland are used in the first
place (i\ = 0.59, P < 0.05), followed by proteins stored in the
digestive gland and the gonad.

Gametogenesis starts at the end of the winter and in early
spring. Energy for the initial gamete development is provided by
lipids from the digestive gland, an event occurring similarly in
Spondylns culcifer (Villalejo-Fuerte et al. 2002). Proteins from the
three organs analyzed are used afterwards, most probably during
the gamete final development stages. In this respect, this species
has been shown to undergo rapid gamete growth at the beginning
of gametogenesis. followed by a slowdown at the end of this stage
(Villalejo-Fuerte & Garcia-Dominguez 1998).

Temperature has been reported to influence the gametogenic

cycle through the regulation of nutrient transfer from reserve-
storing organs (Sastry & Blake 1971. Barber & Blake 1981, Mac-
Donald & Bourne 1987). Our findings suggest that nutrient trans-
fer and use takes place when temperature varies as a result of the
summer to autumn (spawning) and winter to spring (developing)
seasonal changes. This had been previously suggested for this
species by Villalejo-Fuerte and Garcia-Dominguez (1998) and for
A. circidaris (Villalejo-Fuerte & Ceballos-Vazquez 1996). The
reserve storage process takes place when the lowest temperatures
occur.

The biochemical composition of marine bivalves is affected by
exogenous factors, including food availability and temperature, as
well as by endogenous factors such as reproduction (Gabbott 1975,
Bayne 1976, Barber & Blake 1981. Boadas et al. 1997, Claere-
boudt & Himmelman 1997). "Opportunistic species" use the en-
ergy directly obtained from food for gonad development, and en-
ergetic substrate storage and gamete production cycles may over-
lap temporarily, whereas "conservative species" use previously
stored energy reserves (Bayne 1976).

Based on the findings of this study, it is confirmed that 5.
leiicacantlnis has a markedly seasonal reproductive cycle. This
species shows a reproductive inactivity period that is well delim-
ited throughout the year, characterized by the accumulation of
energy reserves in the adductor muscle, the digestive gland and the
gonad. The reproductive cycle stages that demand a higher expen-
diture of reserves are developing and spawning. According to these
features, it can be concluded that this species has a conservative
reproductive strategy, which is a characteristic of iteroparous spe-
cies that largely use energy reserves, supplemented by energy
obtained from food for energetic expenditures.

Similar strategies have been identified in other bivalves.
ScupluiiTO broughtonii behaves as a typical conservative species,
with gametogenesis taking place during the winter and spring at
the expense of reserve glycogen stored during the autuinn. and
spav\ning occurring during the summer (Park et al. 2001); by
contrast. Cmssosln-a gigcis was shown to behave as an opportu-
nistic species, given that gametogenesis starts simultaneously with
reserve accumulation and proceeds until gonadic maturity is
reached, coinciding with the highest level of biochemical compo-
nents followed by a rapid drop of these substances during spawn-
ing (Rang et al. 2000). Argopecten ventricosus preferentially uses
food available in the environment when it is abundant but makes
use of reserves in the adductor muscle when food is scarce (Luna-
Gonzalez et al. 2000). In Cerastodenna edide. gametogenesis may
occur at the expense of glycogen reserves or simultaneously with
glycogen storage, depending on the amount of available food (Na-
varro et al. 1989).

ACKNOWLEDGMENTS

The authors thank the Instituto Politecnico Nacional (National
Polytechnic Institute, IPN) for facilitating the economical re-
sources to perform this research work (Project 200229), to
IPN's Comision de Operacion y Fomento de Actividades Ac-
ademicas (Commission for the Operation and Promotion of Aca-
demic Activities) for grants given to M. Villalejo-Fuerte and F.
Garci'a-Dominguez, and to Maria Elena Sanchez Salazar for her
assistance in translating and editing the English version of this
article.

Biochemical Composition of Spondyws leucacanthus

761

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Journal of Shellfish Research. Vol. 21, No. 2, Ibi^lhl. 20U2.

SEASONAL DISTRIBUTION OF THE OYSTER OSTREA EDULIS (LINNAEUS, 1758) LARVAE IN

THE BAY OF MALI STON, ADRIATIC SEA

ANA BRATOS.' JAKSA BOLOTIN," MELITA PEHARDA,' JAKICA NJIRE"

'Colleiiiiiin Rai^iisinuiii, Cira Caricii 4. 20000 Duhnnnik, Croatia: 'Institute of Oceanofiiuphy and
Fisheries, D. Jiide 12, 20000 Dnivovnik. Croatia: ^Institute of Oceanoi^rapliy and Fislieries. S. I.
Mestrovica 63, 21 000 Split, Croatia

ABSTRACT Study of spatial and temporal distribution of European flat oyster larvae in relation to temperature and salinity has
conducted over a two-year period at four study sites in a Mali Ston Bay, largest bivalve production area in the Eastern Adriatic Sea.
In 2()()0. significant number of larvae was noted in a period from May till September, with a peak in July at all study sites. Lower
number of flat oyster larvae in 2001, than in 2000, were noted at three study sites, while larvae appeared to be similarly abundant in
both years at site Bistrina. Maximal recorded number of larvae was 5029 ind/m' at site Bistrina in August 2001. Our results show
between year and between site variations in distribution of oyster larvae.

KEY WORDS: huahe, oyster, Ostrea ediilis. larvae, Adriatic Sea

INTRODUCTION

The Bay of Mali Ston is the largest bivalve production area in
the Eastern Adriatic Sea. with a tradition of collection and aqua-
culture that e.\tends for tew centuries, and according to some au-
thors, even from the time of the Roman Empire (Basioli 1968).
Today, production is still based on small family farms. Most im-
portant species cultured in this region is European flat oyster Os-
trea ediilis (Linnaeus. 1758), and according to Benovic (1997),
about 1.2 million oysters per year were produced in the 1 980s. In
the early 1990s, due to the war situation in this region, aquaculture
production was largely neglected and it is only in last few years
that interest has been generated to improve oy.ster aquaculture in
Mali Ston Bay as a result of market demands caused by developing
tourism industry. According to Simunovic (2001), current annual
production is only 300 thousand pieces of oysters.

European flat oyster is one of the few indigenous bivalve spe-
cies in European commercial aquaculture. Since the mid-
nineteenth century, in other European countries flat oyster beds
have progressively shrunk making this species a scarce and ex-
pensive item (Cano et al. 1997). Naturally occurring flat oyster
beds are of great interest as a natural source of seed for achieving
a steady and viable culture of O. ediilis (Cano et al. 1997). This
gives Mali Ston Bay exceptional importance since it is one of the
rare places in the Mediterranean where both an adequate collection
of O. edulis spat and subsequent aquaculture are still possible.

A previous study conducted in Mali Ston Bay, described two
oyster spawning periods, one in the spring and the other in the fall
(Morovic & Simunovic 1980). Between 1985 to 2000, local farm-
ers noticed insufficient attachment of oyster spat. Change was
especially evident in relation to fall-spat settlement, which accord-
ing to farmers, did not occur in some years (Maskaric 2001, pers.
com.). Therefore, a study of spatial and temporal distribution of
oyster larvae in relation to environmental characteristics was un-
dertaken with the objective of improving collection of oyster spat
and its aquaculture in this region and to gain a better insight into
possibilities of spat collection that could potentially be used for O.
edulis aquaculture in other parts of Mediterranean.

Study Site

Mali Ston Bay is an extended and ramified bay situated be-
tween the mainland and the Peljesac peninsula (Fig. 1 ). It is rela-

tively shallow (maximal depth = 26 m) and characterized by
strong marine currents and underwater freshwater springs, which
along with precipitation, lower the salinity of the bay and bring
organic matter from the surrounding terrestrial area (Simunovic
1981). Abundant and constant sedimentation influences formation
of soft-mud sediments. Owing to the high level of fresh water
input, the concentration of nutrients is high, but there are no signs
of eutrophication (Vukadin 1981, Caric et al. 1992). Analysis of
zooplankton community has indicated that the bay is a naturally
moderate eutrophic ecosystem (Lucie & Krsinic 1998). As a con-
sequence, production in the entire bay area is high, providing a rich
diet for filter feeders (Bahun 1981). To protect this unique eco-
sy.stem and its bivalve aquaculture production. Mali Ston Bay was
declared a re.serve in the sea.

MATERIALS AND METHODS

Investigation was conducted at four sites within the Bay of
Mali Ston; Bistrina (9 m), Soca (8 m), Krstac(15 m), and Bjejevica
(14 m) (Fig. 1). in a period from May 2000 until March 2002.
Samples were collected by vertical tows of plankton net (1.5 m
long, 56 cm in diameter, and 125 pim in mesh size) for 8 to 15 m.
depending on a maximal depth of a study site. Sampling was
conducted a few times a month, depending on the number of larvae
in the water column and weather conditions, and was more inten-
sive during the summer period. Collected samples were preserved
in 4% formaldehyde solution and analyzed on a microscope in a
laboratory within 24 h after the collection. Oyster larvae were
determined according to Rees (1950) and Loosanoff et al. (1966).
and their numbers were calculated per cubic meter of seawater.
Temperature and salinity were measured with the WTW multiline
hydrographical probe at three depths on each site: surface, mid
water column and one meter above the bottom. Degree of asso-
ciation between larval number and environmental parameters was
determined with Pearson's rank correlation analysis.

RESULTS

The surface temperature ranged between 7.0°C (Soca, January
2002) and 26.9"C (Bjejevica. August 2000). The rise in surface
temperature above 2 PC occuned in late May and temperature did
not drop below 2 1 °C before mid September, in both years and at
all study sites. It is interesting to note that in 2000, surface tem-
perature was higher in December than in November. In early fall

763

764

Bratos et al.

Fi)>ure 1. Ijpcation of Mali Slon Bay and four study sites: Bistrina.
Soca, Krstac, and Bjeji'xica.

of 200 1, surface temperature decreased slowly and was very simi-
lar in September and October at all study sites, that was followed
by exceptionally low surface temperatures that dropped below
10°C in December and January.

In the late spring and early summer periods, due to stratification
of a water column, bottom temperature is a few degrees lower than
surface temperature. Toward the end of the summer, isothermia
was observed in a water column, while during the fall and winter
periods near bottom temperatures were higher than surface or mid
water column temperatures (Table 1 ).

The surface salinity values ranged between 26.6 psu (Bistrina,
May 2001) and 39.7 psu (Soca, March 2001). High variations in
surface salinity were observed at Bistrina and Soca in a period
from October till May and in Krstac and Bjejevica from October
till February (Fig. 2). Near bottom layer was characterized with
higher salinity values, than surface and mid water column layer,
throughout the year.

European flat oyster larvae were present in the Bay of Mali
Ston in varying numbers throughout the year. Maximal number of
larvae recorded in a given month, and average surface temperature
recorded in that month, are shown in Figure 3. High numbers of
Ostrea I'tlulis larvae were recorded in samples collected from May
till September, while larvae were present in some samples, but not
abundant, from November till April. Absence of larvae was pri-
marily noted in samples collected between January and March
2002 al all study sites.

In year 2000, the highest larval numbers were recorded in July
at all sites, with the highest value being recorded in the cove of
Bjejevica (4875 ind./m') in late July. Minimal, maximal and mean
values for number of O. edtili.s larvae recorded in study period are
shown in Table 2. In 2001, maximal number of larvae collected in
each month appeared to be lower than in 2000 at sites Soca,
Krstac, and Bjejevica. At site Bistrina, the highest number of oys-
ter larvae was recorded in August 2001 (5029 ind./nr') while the
number of larvae observed in other months of that year appears to
be similar to values obtained for oyster larvae in year 2000.

There was a statistically significant positive correlation (P =
0.05) between number of O. editlis larvae and surface temperature
and mid water column temperature (Table 3). Correlation between
larvae number and near bottom temperature was significant only at

TABLE 1.
Water temperature at all four study sites from May 2001 to March 2002.

Temperatu

re CO

Bistrina

Soca

Krstac

Bjejevica

Month

Sur.

Mid.

Bot.

Sur.

Mid.

Bot.

Sur.

Mid.

Bot.

Sur.

Mid.

Bot.

May

21.0

20.0

18,7

19.9

19.7

19,5

20.6

18.2

18,2

19.7

18.4

17.6

June

23.6

22.8

21.8

23.3

22.8

21,9

22.8

214

19,8

22.7

21.3

19.9

July

23.1

23,0

22.3

23.3

23.0

22.8

234

22.9

21,3

23.3

22.7

21.7

August

26.1

26,0

26.1

26,0

25.8

25.7

26.6

25.7

18.4

26.9

26.1

17.9

September

21.6

21,2

21.1

21 1

21.1

21.0

21.1

20.9

20.7

20.6

20.4

20.0

October

18.8

19.8

20.7

18,7

18.7

20.2

18.7

20.1

20.5

November

154

17.9

17,8

12-2

17.3

17.5

13.8

17.5

18.0

12.8

18.3

17.9

December

18.4

174

17.4

13.8

17.0

17.2

16.8

17.7

17.6

15.3

15.6

16.0

January

12.0

13.7

14.7

114

13.1

15.8

12.4

14,7

15.8

12.8

154

15.5

February

12.0

12,3

13.8

114

12,5

14,3

11.5

14,1

14,3

11,8

13.3

14.5

March

14.4

14.0

14,5

14.5

14,2

14,2

14,1

14,3

14,0

14,0

14,0

14.0

April

14,4

14.9

15.2

14.5

14.7

14.9

14.9

14,7

14,7

14.5

14,4

14.7

May

21.1

17.7

17.5

20.2

184

17.8

18.2

17.0

16,8

18.3

17,1

16.8

June

21.0

20.5

19.8

21.2

21.1

20.5

20.6

20.4

20,1

20,8

20.4

19.9

July

24.4

24.2

22.9

24.3

24.2

22.6

24.5

23.8

19.9

24.6

23.8

18,8

August

25.5

23.9

21.8

25.4

23.8

21.7

25.0

24.7

20.0

25.3

23.7

20.0

September

20.0

20,0

19.9

20.1

19.8

19,7

20.3

20.1

19.6

20.2

19.7

19.3

October

20.0

20.2

20.5

19.9

20.1

20,2

20.0

20,6

20.4

20.0

20.5

20.4

November

13.8

15.6

16.3

13,8

15,2

16 1

15,2

15,6

16 2

14 1

15 3

16,0

December

9.5

9.8

10,6

8.^)

9,0

10,6

1(1.0

10,7

10,6

10 1

10,1

10.3

January

9.3

10.6

11,1

'),!

9,5

11,1

9.3

10,6

11,1

i),()

10,6

11.0

February

11.5

12.6

13.1

114

11.8

12,7

11.8

12.0

12.5

11,6

11,6

12.4

March

13.5

134

13.3

13.4

13.2

13,3

12.8

13,1

13.2

13.0

13,0

134

Seasonal Distribution of Ostrea edlilis Larvae

765

2000 2001 2002

~Sur. -■— Mid. -*-Bol~|

40
^ 36

"i 30
" 28

b. Snca

'---^^^'^^^''^7^^^-^^

May
Jun
Jul
1 Aug
Sep
Oct

1

s ^ 1 1 III ^ II S 1 oM 1
2001 2002

5

]_._Sur. -.-Mid. -»-Bot, ]

d. Bjejevica

-Sur. -*-Med,

Figure 2. Time trend over a two-year study period for surface (Sur. I,
mid water column (Mid.) and near bottom (Bot.l salinity (psu) at four
study sites: (a) Bistrina, (b) Soca. (c) Krstac, and (dl Bjejevica.

Iwo shallower sites — Bistrina and Soca. Salinity values were not
correlatecJ with number of oyster larvae observed in samples, ex-
cept at site Soca for near bottom salinity and at site Krstac for mid
water column salinity.

DISCUSSION

Traditionally, the Bay of Mali Ston was well known as an area
where the European flat oyster has two spawning peaks per year.
one in the late spring (May and June) and other in the autumn
(September and October) (Morovic & Simunovic, 1980). How-
ever, during the last 15 years, change in spawning season, was
observed by local farmers. Results of this research confirm their
observations. Although larvae were present throughout the year.

2000 2001 2002

.?»* ^.^ s> ^"cf" cC" ,j'^ o* ■,* **■ ^' ?^' -S** S^ V* ■^'f" *" <f^ ^ -f <,f ^

2000 2001 2002

[^MLaTVaemax — »— TempSui]

^'^V^ -P ^'^!fl<i^^'^<:^\^<,S'-^^'-^-f S^fjV'd^^o'o'*' V^*,}*

■ Larvae max — •— TemSur

d. Bjejevica

2000 2001 2002

■ Larvae man — * — TemSui

Figure 3. Time trend over a two-year study period for number of flat
oyster larvae (ind/m') and temperature ( C) at for study sites: (a)
Bistrina, (b) Soca, (c) Krstac, and (d) Bjejevica.

the quantities suitable for commercial collection occurred several
times during the warmest season, between May to September.
Therefore, our results point out that in the suminer period of 2000
and 20(H. two previously known spawning peaks were combined
to form a single, longer spawning period. Cano et al. (1997) also
observed only one oyster spawning peak in Mar Menor (Spain)
where significant oyster larvae numbers appeared at temperature

TABLE 2.
Number of Ostrea ediilis larvae (ind./m') with respect to location.

Location

Records (n)

Maximum

Mean ± St.dev.

BistilnLi

61

5029

473 ± 805

Soca

59

2651

247 ± 459

Krstac

60

3993

352 ± 592

Bjejevica

."iS

4875

366 ±312

766

Bratos bt al.

TABLE 3.
Pearson correlation matrix for Oslrea edulis larvae and environmental parameters considered at four stud) sites in Bay of Mali Ston.

Location

Temp. S

Temp. M

Temp. B

Sal. S

Sal. M

Sal. B

Bislrina
Soca
Krstac
Bjejevica

0.495***
0.604***
0.690***
0.423**

0.501**
0.575**
0.654*<
0.369*

0.444*
0.604*
0.501*
N.S.

N.S.
N.S.
N.S.
N.S.

N.S.

N.S.

-0.451*

N.S.

N.S.

-0.484***

N.S.

N.S.

Temp. S. = surface temperature; Temp. M. = mid water column temperature; Temp. B. = temperature one meter above the bottom; Sal. S. = surface
salinity; Sal. M. = mid water column salinity; Sal. B. = salinity one meter above the bottom. Level of significance: * 0.01 < p < 0.05; ** 0.001 < p
< 0.01; *** p < 0.001; N.S. = non-significant.

from I5°C to 19°C, and maximum larvae number was noted at
temperature between 24°C and 26"C.

The rise in mean temperature above 21°C in Mali Ston bay in
early June correlates with the increased number of oyster larvae:
over 500 individuals per n\ at all sites from mid-June of 2000. The
number of larvae reached the highest level in July, when the mean
temperature of the water column was between 20' C and 24"C.
When the mean water column temperature dropped to values be-
tween 12°C and 18°C, larval presence became negligible. The new
spawning cycle and increase in larval numbers began again in
April of 2001, correlating with the temperature increase above
14°C. This high dependence of initiation and duration of gameto-
genesis in Ostrea edulis on water temperature was already noted in
many previous studies (e.g.. Hrs-Brenko 1971, Sastry 1975, Mann
1979, Wilson & Simons 1985). In the summer of 2001 larval
distribution was different than in summer of 2000. At sites Soca,
Krstac, and Bjejevica lower number of larvae was collected in
2001 than in 2000. At site BLstrina, highest number of oyster larvae
was observed in August 2001 while in other months larval distri-
bution was very similar with respect to two observation years.

According to Morovic and Simunovic ( 1980), ma.ximum num-
ber of flat oyster larvae in Mali Ston bay in 1976 occurred in the
first days of September and the minimum in August, while high
numbers were also found in June and July, pointing out between
year variation in distribution of oyster larvae.

Maximal number of larvae noted in this study is significantly
different than values noted by Morovic and Simunovic (1980).
These authors noted over 75 thousand oyster larvae in I m^' at site
Krstac in May of 1974, while maximal number of larvae noted in
this study was only 5029 ind/m\ Observed differences might be
attributed to reduced size ot the flat oyster brood stock, water
temperature, food availability, and water quality. Value obtained
by Morovic and Simunovic (1980) is also high when compared
with the maximal number of oyster larvae recorded by Hrs-Brenko
(1977) at several locations in Northern Adriatic where only up to

1000 ind/m' were recorded. Further on. Cano et al. (1997) also
recorded a significantly smaller number of flat oyster larvae (6,000
ind/m') at one location in Spain.

In relation to environmental parameters recorded m this study,
there was no apparent difference between year-temperature and
salinity in the period between May and September that could ex-
plain the difference in numbers of flat oyster larvae observed in
2000 and 2001. In both years salinity in the bay was lower in
comparison to open Adriatic waters (Vukadin 1981) and was
within optimal values for larval survival. However, since flat oys-
ters are filter feeders and phytoplankton is generally known to be
the main source of nutrition for bivalve filter feeders (Dame 1996),
possible explanation might be related to difference in distribution
of phytoplankton biomass. According to Nincevic (unpublished
data), phytoplankton biomass in Mali Ston Bay was lower in sum-
mer of 2001 than in summer of 2000. Another possible explanation
for the reduced number of larvae in 2001 might be presence of
unidentified species of ctenophore within the bay (Bolotin 2002,
unpublished data). According to Dame (1996), ctenophores and
jellyfish are the most commonly reported bivalve larval predators.
However, at this point it is not possible to explain why the number
of larvae decreased at three study sites in 2001 but did not de-
creased at site Bistrina.

To provide an explanation for observed differences in larvae
distribution, further research should be conducted at all study sites,
measure other environmental parameters, such as chlorophyl a and
nutrients, as well as identification of ctenophore species. Obser-
vation of gonad maturation and settlement success should also be
included to obtain a complete picture on reproductive effort and
settlement of European flat oyster in this unique bay and to im-
prove the aquaculture production.

ACKNOWLEDGMENT

The authors thank the Ministry of Science and Technology of
the Republic of Croatia for funding this project.

LITERATURE CITED

Bahun, S. 1981. Hydrological and geological relations in the of Bay Mali
Ston — Review. Zbornik radova Savjetovanja "Malostonski zaljev
prirodna podloga i drustveno valoriziranje". Dubrovnik. pp. 22-26.

Basioli. J. 1968. Shell-breeding on the eastern shores of the Adriatic.
Pomorski zhomik. 6:179-218.

Benovic, A. 1997. The history, present condition, and future of the mol-
luscan fisheries of Croatia. In; C. L. MacKenzie Jr.. V. G. Burrell Jr.,
A. Rosenfield & W. L. Hobart, editors. The History, present condiuon,
and future of the molluscan fisheries of North and Central America and

Europe. Volume 3. Europe NOAA Technical Report NMFS 129. US
Department of Commerce, pp. 217-226.

Cano, J., M. Jose-Rosique & J. Rocamora. 1997. Intluence of Environ-
mental parameters on Reproduction of the European Flat Oyster iOs-
ireci I'dulis L.) in a Coastal Lagoon (Mar Menor, Southeastern Spain I.
Jiiunuit of Molluscan Studies 63:187-196.

Caric, M., N. Jasprica & D. Vilicic. 1992. Nutrient and chlorophyll con-
centracions in Gruz and Mali Ston Bays (southern AdriaUc). Rapp.
Comm. Int. Mer. Mcdii. 33:367.

Seasonal Distribution of Ostrea eduus Larvae

767

Dame. R. F. 1996. Ecology of marine bivalves — an ecosystem approach.

CRC Marine Science Series. 254 pp.
Hrs-Brenko, M. 1971. Observations of occurrence of several bivalves in

the Northern Adriatic Sea. In: D. J. Crisp, (ed.). Proc. 4th European

Marine Biology Symposium, pp. 45-53.
Hrs-Brenko, M. 1977. Uzgoj lifinki i mladih skoljaka u mrijestilistima.

Ichryologia. 9:85-100.
Loosanoff. V. L., H. C. Davis & P. E. Chanley. 1966. Dimensions and shapes

of larvae of some marine bivalve molluscs. Malacologia. 4:376-383.
Lucie, D. & F. Krsinic. 1998. Annual variability of mesozooplanklon as-
semblages m Mali Ston Bay (Southern Adriatic). Peiiodiciim Biol-

ogonim. 100:43-52.
Mann, R. 1979. Some Biochemical and Physiological aspects of Growth

and gametogenesis in Crassoslrea gigas and Ostrea edulis Grown at

Sustained Elevated Temperatures. / Mar. Biol. Assoc. U.K. 59:95-1 10.
Morovic, D. & A. Simunovic. 1980. A contribution to the knowledge of the

variations in the larvae of oyster Ostrea edulis L. in the area of the Bay

of Mali Ston. Acta Adriatica 21:195-201.

Rees, C. B. 1950. The identification and classification of lamellibranch
larvae. Hull. Bull. Mar. Ecol. 5:5-104.

Sastry, A. N. 1975. Physiology and Ecology of Reproduction in Marine
Invertebrates. In: F. J. Vemberg, editor. Physiological Ecology of Es-
tuarine Organisms. Columbia, SC: University of South Carolina Press,
pp. 279-299.

Simunovic, A. 1981. Biolosko-ekoloska istrazivanja jestivih skoljkasa
Malostonskog zaljeva. Zbornik radova savjetovanja "Malostonski zal-
jev prirodna podloga i drusneno valoriziranje. " JAZU. znanstwni sav-
jet za zastitu prirode. Dubrovnik. pp. 252-267.

Simunovic, A. 2001. Stanje i problemi uzgoja kamenice i dagnje u MaL
ostonskom zaljevu. Hnatska vodoprivreda 10:31-35.

Vukadin. I. 1981. Hidrografska svojstva Malostonskog zaljeva i susjednog
mora u periodu 1980-1981. godine. Zbornik radova Savjetovanja
"Malostonski zaljev prirodna podloga i drustveno valoriziranje". Du-
brovnik. pp. 52-65.

Wilson, J. H. & J. Simons. 1985. Gametogenesis and Breeding of Ostrea
edulis on the west Coast of Ireland. Aquaculture. 46:307-321.

Journal of Shellfish Research. Vol. 21, No. 2, 769-775, 2002.

OBSERVATIONS ON THE EGG CAPSULES AND HATCHLINGS OF THE KNOBBED WHELK,
BUSYCON CARICA (GMELIN, 1791) IN COASTAL GEORGIA

ALAN J. POWER,** ELLIE COVINGTON,' TODD RECICAR,' RANDAL L. WALKER,' AND
NELSON ELLER'

^Marine Extension Senice, Shellfish Research & Aqiiacultiire Lahorataiy. University of Georgia. 20
Ocean Science Circle. Savannah. Georgia 31411; 'Marine Sciences Program, Savannah State
University, Savannah. Georgia 31404

ABSTRACT Despite the commerciiil importance and abundance of Icnobbed whelks on the east coast of the United States, very little
life history information exists for juveniles in the egg capsule and the first few critical weeks post hatching. As a result, various
intertidal sandy-mud flats around Wassaw Sound. Georgia were monitored from early April 2001 when copulating and egg-laying
whelks were first observed to early June 2001 when most egg capsules had opened and the hatchlings had begun to disperse. During
this spawning event, egg strings were collected, the length of the embryonated (mean = 339.86 mm) and unembryonated (mean =
199.67 mm) portions were measured, and the number of embryonated capsules per string (mean = 89.76) was determined. The middle
section of the embryonated portion comprised the largest capsules (mean height = 22.12 mm, mean width = 31.11 mm, mean
thickness = 6.03 mm) and had the highest number of encapsulated embryos with an average of 46 (up to 99). Egg strings were also
hatched in the laboratory with the intention of examining the effects of diet (meat, macroalgae, and microalgae) and temperature (20,
25, and 30"C) on the growth and survival rates of newly hatched juveniles. Optimum growth and survival conditions resulted in those
reared on a meat diet in aquaria maintained at 20°C.

KEY WORDS knobbed whelk. Biisycon carica. egg capsules, hatchlings

INTRODUCTION

The knobbed whelk, Busycon carica (Gmelin 1791). is a com-
mon prosobranch gastropod (Family Melongenidae) along the east
coast of the United States from Cape Cod. Massachusetts to Cape
Canaveral. Florida on sandy mud. in shallow water to 4.6 m (Reh-
der 1981). The reproductive cycle of B. carica has not been de-
termined in Georgia, however. Walker (1988) reported spawning
in the spring and fall based on the appearance of egg strings and
copulating whelks. In North Carolina (Magalhaes 1948) also found
biannual spawning between May to June and September to No-
vember. In South Carolina. B. carica was reported to reproduce in
the fall (October to November) and spring (April) (Stevens 1976).
Stevens (1976) suggested that the fall reproductive period was
more important than the spring based on a comparison of body
weights, digestive gland weights, and ovarian indices. Weinheimer
(1982) reported that B. carica from South Carolina are not re-
stricted to one or two breeding seasons, but reproduce from Sep-
tember to May. In Virginia, spawning was reported between mid
August and November (Castagna & Kraeuter 1994).

Fertilization occurs internally in Bicncon (Weinheimer 1982).
Males are equipped with a large muscular penis, and the female
oviduct is composed of a series of chambers and glands. Fertilized
eggs are surrounded by a transparent viscous mass of albumin and
are laid in protective disciform egg capsules arranged on a helicoid
string (D'Asaro 1997). Several whelk species are reported to mi-
grate to favorable egg laying sites during the breeding season
(Power & Keegan 2001). In coastal Georgia, egg strings are com-
monly encountered near the low water mark on intertidal sandy
mud flats. Ram et al. ( 1982) determined an egg deposition rate of
1.9 ± 1.5 h/capsule in the laboratory. According to Magalhaes
(1948) long strings are usually laid over several days.

Egg strings in Virginia were reported to contain between 42
and 121 capsules (Castagna & Kraeuter 1994). The average em-

*Corresponding author. E-mail: alanpowr(s>arches. uga.edu

bryos per egg capsule in Chesapeake Bay ranged from 46 to 67
(Harasewych 1982). Castagna and Kraeuter ( 1994) determined the
average number of embryos per capsule varied according to posi-
tion on the egg string, averaging 52 in the middle section and
decreasing in the beginning and terminal portions. The female
always begins by anchoring one end of the egg string deep into the
substrate by several structurally modified capsules to prevent the
string from being washed ashore (Magalhaes 1948). Ram (1977)
and Ram et al. (1982) induced spawning in the knobbed whelk by
stimulation with nervous system extractions. Spawning always be-
gan with a number of unembryonated capsules, a choice dictated
by genome. It has not been determined how the female adjusts the
number of anchoring capsules. Studies have found that the egg
capsules in the anchoring portion are typically thinner walled and
closely spaced (Harasewych 1982). The number of unembryonated
capsules in this anchoring portion has been reported to range from
8 to 22 (Castagna & Kraeuter 1994). from 13 to 17 (Ram et al.
1982). and to average 21 (D'Asaro 1997).

The young pass the veliger stage within the egg capsule and
einerge as fully developed miniature "adults." In Virginia. Casta-
gna and Kraeuter ( 1994) found that egg strings deposited in the fall
hatched from mid-March through early-May in the following year.
The average size for hatchling juveniles is -4 mm in length (Ma-
galhaes 1948, Castagna & Kraeuter 1994).

Most studies that have been conducted were on adult whelks
(Morton 1987). Very little information exists about the ecology of
post hatchling knobbed whelks. As an adult, the knobbed whelk is
a predator of bivalve species. There has been no research published
on the habitat and nutritional requirements of juveniles in the wild.
In Virginia, juvenile whelks were hatched and raised inside poly-
propylene bags in the laboratory to estimate post hatching growth
rates (Kraeuter et al. 1989). There was limited information pro-
vided on rearing conditions (i.e.. the temperature and diet). Length
measurements were taken five times during the first year and one
to three times per year for the subsequent nine years. The juveniles
were fed live clams when they reached -20 mm in length. Growth

769

770

Power et al.

rates averaged 13.2 mm/y over the ten-year period, however ju-
veniles grew from 4 to 36.5 mm (32.5 mm/y) in the first year. The
only other published growth study on juvenile knobbed whelks
was by Magalhaes (1948). She maintained juveniles in the labo-
ratory for one month only and observed one individual added 1 .5
mm in length in 22 days. This experiment attempts to determine
the optimum food source for juvenile knobbed whelks with respect
to growth and survival rates. Temperature is also considered since
warmer temperatures foster accelerated growth.

MATERIALS AND METHODS

Weekly field trips to intertidal sandy-mud fiats around Wassaw
Sound, Georgia were carried out from April 2001, when copulat-
ing and egg-laying whelks were first observed, to early June 2001
when most egg capsules had opened and the hatchlings had begun
to disperse. On each occasion, notes were taken on the behavior
(i.e., copulating, depositing eggs, and feeding). If a whelk was in
the process of laying, an estimate was made of the number of
capsules already produced, her position was taken with a hand held
global positioning system, and the location was physically marked
with a metal stake. Once the strings were completed, the number
of egg capsules per string was determined (excluding the anchor-
ing section). The condition of the egg capsules was also monitored
regularly to determine the larval developmental period in the field.
At the end of May 2001, egg strings were collected and re-
turned to the Shellfish Research & Aquaculture Laboratory on
Skidaway Island. Ten strings were selected to examine the capsule
morphometries, length, width, and thickness, as defined by
D'Asaro (1997). The length of unembryonated and embryonated
sections of each string was determined with a fish measuring
board. Each string was then divided into four portions: the unem-
bryonated anchor, and an inner, middle, and outer portion of the
embryonated section. Five capsules were randomly selected from
each section, the dimensions were measured and the capsules were
opened to count the developing embryos.

Egg strings were also maintained at the laboratory in an open
system raceway until hatching began. Hatchling juveniles were
collected, measured for length, and isolated for growth rate ex-
periments using diet and temperature variables. Three dietary treat-
ments were chosen for the growth study: macroalgae, microalgae,
and meat. Sea lettuce. Ulva sp., was harvested from Wassaw
Sound and the Skidaway River for the macroalgae series. Benthic
microalgae (species unknown) was cultured on mats in a closed
system saltwater tank using nylon mesh as a substrate. The third
diet consisted of chopped meat: clams, Menenaria mercenaria
(L.). brittle stars, Ophiophni^miis wwdemani (Lyman), blood-
worms. Glycera dibranchUita (Ehlers). and mussels, Geukensia
demissa (Dillwyn), depending on what was available. Temperature
was included in the design. At the time of hatching, water tem-
peratures averaged 25°C (taken from the nearby Skidaway River),
therefore three glass aquaria were set up in temperature controlled
rooms, at 20, 25, and 30''C. For the duration of the experiment, the
salinities were maintained at 'i5%c. Plastic jars (120.6 x 69.8 mm)
were used to contain the juveniles. To help water circulation, thirty
2-mm holes were drilled in the top and bottom of the jars and 15
on each side. There were three replicates for each combination of
diet and temperature treatment resulting in 27 containers, each
holding 50 haphazardly chosen hatchlings. Jars were labeled and
temperatures and salinities in the aquaria were tested and main-
tained regularly. The water in the aquaria was aerated, and

changed monthly. All the juveniles were provided with an abun-
dance of food, which was cleaned out and replaced approximately
every three days. Juvenile shell length (to the nearest 0.01 mm)
and body weight (to the nearest 0.(MOI g) were recorded every two
weeks using an electronic calipers and balance. The animals were
first blotted dry by placing on a paper towel. Dead juveniles were
removed and counted to determine mortality rates. The experiment
was conducted over an eight-week period.

Statistical analysis of growth and survival data was pert'ormed
using SAS (SAS Institute, Inc.. Gary, NG). The two main effects
(temperature and diet) and a nested effect (containers) on growth
(length and weight) were analyzed by performing a nested factorial
using GLM (General Linear Model) SAS. Survival data were arc-
sine transformed prior to analysis [A" = arcsine V (.v/lOO)]. Two
main effects were again analyzed (temperature and diet), with
repeated measures of both factors using a GLM (SAS). An a level
of p = 0.05 was used to determine if significant difference in
mean juvenile length, weight, and survival occuned between dit-
ferent diets and temperature treatments.

At the termination of the eight-week laboratory growth experi-
ment, we returned to the sites of egg deposition in Wassaw Sound
to search for juveniles that had presumably dispersed on hatching.
A circular area with a diameter of approximately 4 m was searched
around approximately twenty marked egg cases. The goal was to
obtain juveniles to compare the growth obtained in the laboratory
with growth in their natural habitat. Methods for searching con-
sisted of scooping up handfuls of the surface layer of sediment at
low tide and sieving on a 1.40 mm screen. Submerged sites at
lower tidal levels were searched by gently feeling the surface of
the sediment.

RESULTS

The abundance of copulating whelks peaked in eariy April
2001 and was observed with decreasing frequency throughout the
rest of the month. This period coincided with a dramatic increase
in local water temperatures (Fig. 1). Often copulation was ob-
served even while the female was in the process of depositing egg
capsules (Fig. 2). Typically the larger female lay partially buried in
the substrate and was surrounded by several smaller sized males
(average of 3, but up to 9 observed), A large variation in the rate
of egg deposition was observed: 1 to 23 capsules per day (7.69 ±
2.12. mean ± SE). This is a coarse estimate since the egg strings
were observed once per week. An approximate total area of 2.500
m- was searched for whelks and eggs. By the end of April, most
egg strings were complete and whelks were less abundant on the
sandy-mud flats. From a total of 51 complete egg strings (0.02 m"),
the number of embryonated egg capsules per egg string averaged
89.76 ± 3.61, and ranged from 40 to 157. Egg capsules began to
soften and deteriorate quickly, the escape aperture was open on all
capsules examined on June 4, 2001 (approximately six weeks
later). While all capsules were open at this time, only one third of
those examined were empty, the remainder still held the fully
developed hatchlings.

The average length of the unembryonated section tor the ten
egg strings selected for morphometric analysis was 199,67 ± 15.21
mm (ranged from 130 to 275 mm). For the embryonated section
the average length was 339.86 ±26.17 mm (ranged from 245 to
450 mm). The number of unembryonated capsules per egg string
ranged from 6 to 26 (mean = 13.67 ± 1.97). The number of
developing embryos per egg capsule ranged from 0 to 99. The

Knobbed Whelk Eggs and Hatchlings in Georgia

771

Temperature
Salinity

Mar-OO May-00

May-01

Jul-01

Sep-01

1-00 Sep-00 Nov-00 Jan-01 Mar-

Time (month)

Figure 1. The mean ni(mthl> water temperature and salinity of the Skidaway River, Georgia from January 2000 to November 2001 (vertical bars
indicate ± 1 standard error from the mean).

middle portion of the embryonated section of the egg strings had
longer, thicker, and wider capsules with a higher number of em-
bryos per egg capsule. The inner and outer portions averaged 23.41
± 2.77 embryos per capsule, with capsule dimensions of 20.20 ±
0.47 mm in length. 27.26 ± 0.69 mm in width and 5.66 ± 0.09 mm
in thickness. The middle portions had an average of 45.86 ± 3.95
embryos per capsule, with capsule dimensions of 22.12 ± 0.41 mm
in length, 31.11 ± 0.56 mm in width, and 6.03 ± 0.14 mm in
thickness.

^'igure 2. Small male knobbed whelk, Hiisytiin larica. copulating with
a larger female that is also in the process of depositing an egg string.

In the laboratory, the egg strings maintained in raceways also
began to hatch in early June 2001. On hatching the juveniles
averaged 5.60 ± 0.02 mm in shell length and 23.10 ± 0.24 mg in
weight {n = 540). These were haphazardly assigned to the three
different diet (meat, algae and diatoms) and temperature (20, 25,
and 30°C) treatments. Figure 3 and Figure 4, present the growth
rates of these juveniles for a period of eight weeks, in terms of
shell length (mm) and weight (mg), respectively. Figure 5 presents
the percentage survival rate at the termination of the experiment
for each temperature and diet treatment. Growth in terms of length
and weight was significantly affected by diet (P = 0.003 and
0.001. respectively). Duncan's Multiple Range Test revealed no
difference in the growth of whelks fed on the macroalgae and
microalgae diets (macroalgae; mean length 5.67 mm, mean weight
21.29 mg; microalgae: mean length 5.65 mm, mean weight 21.47
mg). however those fed on the meat diet were significantly larger
(mean length 6.35 mm. mean weight 27.81 mg). Survival was not
significantly affected by diet (P = 0.2084). There was a signifi-
cant (P = 0.001 ) temperature effect on survival. Duncan's Mul-
tiple Range Test revealed no difference in survival at temperatures
25 C and 30°C (15.31% and 7.73%). but a much greater survival
rate occurred at 20"C (69.50%). Percent survival rates decreased
with temperature increase for all dietary treatments (Fig. 5).

It proved very difficult to locate juvenile knobbed whelks on
the intertidal flats in August and September. The best approach
proved to be feeling the surface layer of sediment when a shallow
depth of water co\ ered the flats. Even with this approach only nine

772

Power et al.
B.

20''C

2 4 6

Time (weeks)

as^c

2 4 6

Time (weeks)

° 2 4 6 o 20 25 30

Time (weeks) Temperature ("C)

Figure 3. A-C. Ttie mean shell length (mm + SE) of knobbed whelks reared in the laboratory on macroalgae, microalgae and meat diets at
temperatures of 20, 25. and 30 C. respectively, for eight weeks post hatching. D: The overall growth rate (shell length, mm) per diet and
temperature treatment over eight weeks.

juveniles were recovered (Table 1 1, from a total area of approxi-
mately 250 m- (-0.04 m""). The wild juveniles located, averaged
a much larger size (mean shell length = 21.2 mm) than those
reared in our laboratory treatments.

DISCUSSION

Copulation occurred throughout April 2001 during which av-
erage seawater temperatures increased rapidly front a niean ot
15.3°C in March 2001 to 20.8X in April 2001. Walker (1988)
observed six pairs of copulating whelks in Wassaw Sound in
March 1980 (mean water temperature 14.3°C). We do not believe
that whelks commenced spawning earlier than April in 2001 . since
intertidal flats were thoroughly searched throughout March 2001
as part of an ongoing whelk mark and recapture study. Another
spawning event may take place in Georgia, when seawater tem-
peratures drop to a similar range in the fall. Along the eastern coast
of the United States, the reported spawning period of the knobbed
whelk is progressively later as one moves northwards: March to
April in Georgia (Walker 1988. present study) and South Carolina
(Stevens 1976). May to June in North Carolina (Magalhaes 1948).
and mid August to November in Virginia (Castagna & Kraeuter
1994).

In the natural environment, the timing of the reproductive pe-
riod is usually synchronized with conditions that are most favor-
able for maximization of juvenile survival and continuity of the
species (Sastry 1986). Gastropod egg capsules are preyed on by

fish, crustaceans, other prosobranchs. polychaete worms, and even
shorebirds (Penchaszadeh et al. 2000). Predator activity typically
increases in the warm temperatures of the early summer months.
However, these months also permit a more rapid embryonic de-
velopment of encapsulated larvae that may reduce overall losses. A
rapid developmental period of six weeks was observed in the
present study, which concurs with findings for the pear whelk in
Florida (6 wk: Kent 1983). This is however, significantly lower
than the six-month period ( mid summer to following spring) re-
ported for the same species in the cooler northern waters of Vir-
ginia (Castagna & Kraeuter 1994). Embryonic development of
encapsulated knobbed whelks has been noted to temporarily arrest
and resume after egg capsules were refrigerated for four days at
0 C (Dr. DeEtte Walker, pers. comni.. UGA Dept. of Genetics).
According to Hain and Amaud (1992) embryonic development for
related species can be up to thirty times longer in Antarctic than in
tropical waters.

Although most egg capsules in the field were open in early
June, two-thirds of all capsules examined still held hatchlings. A
gradual dispersal has also been noted for juveniles of the lightning
whelk, with many found within and between the opened capsules
(D'Asaro 1997). Juveniles may use the structure as a refugium.
Egg strings also provide a substratum for algae, diatoms and even
other invertebrates in their juvenile stages and could therefore also
serve as a food source for newly hatched whelks.

In this study the hatchlings (» = .'540) averaged 5.60 mm in

Knobbed Whelk Eggs and Hatchlings in Georgia

773

B.

20X

-Macroalgae
-Microalgae
-Meat

2 4 6

Time (weeks)

25°C

2 4 6

Time (weel<s)

C.

30°C

-Macroalgae
-Microalgae
-Meat

2 4 6

Time (weeks)

20 25 30

Temperature (°C)

Figure 4. A-C. The mean Hel weight (mg + SE) of knobbed whelks reared In the laboratory on macroalgae, microalgae and meat diets at
temperatures of 20, 25, and 30C, respectively, for eight weeks post hutching. D: The overall growth rate (wet weight, mg» per diet and
temperature treatment over eight weeks.

shell length (ranged from 3.31 to 6.96 mm). This is larger than the
4 mm previously recorded for whelks in North Carolina (Magal-
haes 1948) and Virginia (Castagna & Kraeuter 1994). Similarly to
residence time within the capsules, the si/.e at hatching may also be
influenced by environmenlal factors including water temperature.

In this study, there was a large range in the number of encap-
sulated embryos per egg capsule (ranging from 0-99). The middle
section of the einbryonated portion comprised the largest sized
capsules (mean length 22.12 mm, mean width 31.11 mm. mean
thickness 6.03 mml and contained the highest number of embryos
(mean = 46). The inner and outer portion capsules contained
about half the number of embryos (mean = 23) and were smaller
(averaging 21.45 mm and 18.96 mm in length. 29.27 mm and
2.S.26 mm in width, and .'i.60 mm and 5J\ mm in thickness,
respectively). The middle section of egg strings in Virginia was
reported to contain a slightly higher average of 52 embryos, but the
number of embryos in the outer capsules was not provided (Casta-
gna & Kraeuter 1994). In shape, egg capsules were taken to
roughly approximate a prism on an elliptic base. The following
formula was used to obtain the mean number of embryos per unit
of capsule space for the middle, inner and outer portions of egg
strings = (# embryos)(4) / (tt) (length) (width) (thickness). Em-
bryos were packed closer in the middle capsules ( 14.07 cm"') than
in either the inner (8.45 cm"') and outer (10.95 cm"') portions.

There is typically a wide variation in the number of embryos
occurring in egg capsules within the family Melongenidae, e.g..
the lightning whelk is reported to have between 25 and 200 (Perry

& Schwengel 1955), the channeled whelk between 73 and 130
(Conklin 1907). and the pear whelk between 9 and 50 (Odhner
1927, D'Asaro 1997). The number of knobbed whelk egg capsules
per egg string averaged 90 and ranged from 40 to 157. which is
similar to the range 42-121 reported from Virginia (Castagna &
Kraeuter 1994). This species appears to have more egg capsules
per egg string than the channeled whelk (up to 70: Conklin 1907),
but less than the lightning whelk (up to 175: Perry & Schwengel
1955), which could be expected in relation to size (shell length)
differences between the species (channeled: <19 cm; knobbed:
<22.9 cm; lightning: <40.6 cm, Rehder 1981). Miloslavich and
Dufresne (1994) have directly related the size (shell length) of
female Bitcciimm undatwn whelks depositing egg capsules to the
number of capsules, the size of the capsules, and to the number of
developing embryos within. According to Spight and Emlen
( 1976), the fecundity of an organism increases with an increase in
size and age.

From the growth and survival data presented for juveniles at the
laboratory, it is clear that a carnivorous diet is optimal post hatch-
ing. Cannibalism at this stage was also observed in the treatments
provided with macroalgae and microalgae diets. Morton (1987)
suggested that juveniles of Hemifusus titha might consume one
another outside of the capsules to enhance the survival of an in-
dividual in the transition time needed to discover natural prey.
Survival rates were much reduced at 25''C and 30''C indicating that
the temperature was approaching the upper physiologic tolerance.
Most tropical marine organisms cannot survive at temperatures

774

Power et al.

100

*•*

a.

>
E

3

■ Macroalgae

DMicroalgae

ZIMeat

20 25 30

Water Temperature (°C)
Figure 5. The percentage survival rate of knobbed whelks reared in the laboratory on macroalgae, microalgae and meat diets at temperatures
of 20, 25, and 30 C, respectively, for eight weeks post hatching.

higher than 35''C (Kinne 19631. Juveniles that hatched in June
2001 would have experienced temperatures up to 30°C on the
intertidal flats, but would also have had the ability to bury them-
selves in the substrate. Chaitanawisuti and Kritsanapuntu (1998,
1 997 1 found that the highest growth rates of hatchery reared ju-
venile spotted Babylon. Babyiim'm areolata (Link 1807) whelks
occurred in treatments provided with sand substrate and flow
through seawater. while the lowest occurred in those with no sand
substrate and static seawater. In our experimental treatments, both
water flow and a sand substrate were not provided and may have
led to sub-optimal growth and survival rates. The lack of substrate

TABLE 1.

Juvenile Busycoii carica whelks captured on intertidal flats

(previously marked sites of egg string deposition! al Wassaw Island

(0.04 individuals m') in August/September, 2001.

Weight (mg)

0.2

0.4

0.3

0.4

0.6

0.6

0.5

0.4

1

0.5

Length (mm)

Width (mm

17.6

7.5

21.1

9.4

Ig.g

8.3

19.7

8.8

22.7

9.7

22.5

9.7

21.8

9.8

20.2

8.8

26.3

11.5

Means:

21.2

9.3

has been shown to induce interactive energy expenditure by juve-
nile queen conchs (Siddal 1984).

Catterall and Poiner (1983) describe small juvenile colonies
(area of 2-15 m in diameter) of the gastropod Stronibiis lulntanus
with between 5 to 100 individuals per meter squared. These colo-
nies may persist for up to two years and become larger in area and
less dense as members grow and mature. This type of spatial
segregation may also occur for juvenile knobbed whelks in the
wild. It is likely that juveniles spend a large proportion of time
completely buried in the sand, particulariy when the tide retreats,
which would explain our difficulty in locating them. We only
found juveniles at the surface of the sediment after the tide had
advanced over the intertidal flats. In the laboratory the largest sized
juvenile at the termination of our rearing experiment (8 weeks
later) was approximately 12.5 mm. The average size of the juve-
niles found in the wild was much larger at 21.2 mm. These wild
juveniles may have resulted from the previous fall spawning event
and would therefore be approximately eight months old, which
resembles the annual year one growth rate of 34.5 mm reported by
Kraeuter et al. (1989) in Virginia.

ACKNOWLEDGMENTS

This work was supported by the University of Georgia Marine
Extension Service and by the National Sea Grant program with
funds provided to Savannah State University through the Minority
Serving Institute Program. The authors thank Ms. Carolyn Belcher
for performing the statistical analysis. Thanks to Mary Sweeney-
Reeves and Dodie Thompson for assisting in the field.

Knobbed Whelk Eggs and Hatchlings in Georgia

775

LITERATURE CITED

Castagna, M. & J. Kraeuter. 1994. Age, growth rate, sexual dimorphism
and fecundity of the knobbed whelk Busycon carcia (Gmelin. 1791 1 in
a western mid-Atlantic lagoon system. Vii)>inia J. Shellfish Res. 13:
581-585.

Catterall. C. & [. Poiner. 1983. Age- and se,\-dependent patterns of aggre-
gation in the tropical gastropod Sirombiis luhuwnis. Mar. Biol. 77:171-
182.

Chaitanawisuti, N. & A. Kritsanapuntu. 1997. Effects of stocking density
and substrate presence on growth and survival of juvenile spotted
Babylonia areolata Link 1807 (Neogastropoda: Buccinidae). J. Shell-
fish Res. 16:429-433.

Chaitanawisuti. N. & A. Kritsanapuntu. 1998. Growth and survival of
hatchery-reared juvenile spotted Babylonia areolata Link 1807 (Neo-
gastropoda: Buccmidae) in four culture conditions. J. Shellfish Res.
17:85-88.

Conklin. E. 1907. The embryology of Fulgur. A study of the influence of
yolk on development. Philadelphia: Proc. Acad. Nat. Sci. pp. 320-360.

D'Asaro, C. 1997. Gunnar Thorson's world-wide collection of prosobranch
egg capsules. Melongenidae Ophelia 46:83-125.

Hain, S. & P. Amaud. 1992. Notes on the reproduction of high Antarctic
mollusks from the Weddell Sea. Polar Biol. 12:303-312.

Harasewych. M. 1982. The evolution and zoogeography of the subfamily
Busyconinae (Gastropoda: Melongenidae). Ph.D. Dissertation. Univer-
sity of Delaware. 216 pp.

Kent. B. 1983. Patterns of coexistence m Busyconine whelks. J. E.xp. Mar.
Biol. Ecol. 66:257-283.

Kinne, O. 1963. The effects of temperature and salinity on marine and
brackish water animals. 1 . Temperature Oceanogr. Mar. Biol. A. Rev.
1:301-340.

Kraeuter. J.. M. Castagna & R. Bisker. 1989. Growth rate estimates for
Busycon carica (Gmelin. 1791) in Virginia. J. Shellfish Res. 8:219-
225.

Magalhaes. H. 1948. An ecological study of snails of the genus Busycon at
Beaufon. North Carolina Ecol. Monog. 18:380-407.

Miloslavich. P. & L. Dufresne. 1994. Development and effect of female
size on egg and juvenile production in the neogastropod Buccinuin
cyaneum from the Saguenay Fjord. Can. J. Fish. Aquatic Sci. 51:2866-
2872.

Morton, B. 1987. Juvenile growth of the South China Sea whelk Hemifitsus
tuba (Gmelin) (Prosobranchia: Melongenidae) and the importance of

sibling cannibalism in estimates of consumption. J. Exp. Mar. Biol.
Ecol. 109:1-14.

Odhner, N. 1927. Eikapseln von Hemifusus colosseiis Lamarck. Arch.
Molluskenkunde. Frankfurt 59:355-357.

Penchaszadeh. P.. F. Botto & O. Iribarne. 2000. Shorebird feeding on
stranded giant gastropod egg capsules of Ailelomelon brasiliana (Vo-
lutidae) in coastal Argentina. J. Shellfish Res. 19:901-904.

Perry, L. & J. Schwengel. 1955. Marine shells of the western coast of
Florida. Ithaca: Palaeontol. Res. Inst. 318 pp.

Power, A. & B. Keegan. 2001. Seasonal patterns in the reproductive ac-
tivity of the red whelk, Neplunea antiqua (Mollusca: Prosobranchia) in
the Irish Sea. J. Mar. Biol. Assoc. U.K. 81:243-250.

Ram. J. 1977. Hormonal control of reproduction in Busycon laying of egg
capsules caused by nervous system extracts. Biol. Bull. 152:221-232.

Ram. J.. M. Ram & J. Davis. 1982. Hormonal control of reproduction in
Busycon: II. Laying of egg-containing capsules caused by nervous
system extracts and further characterization of the substance causing
egg capsule laying. Biol. Bull. 162:360-370.

Rehder, H. 1981. National Audubon Society Field Guide to North Ameri-
can Seashells. New York: Alfred A. Knopf. 894 pp.

SAS Institute Inc. 1989. SAS users guide: Statistics: Version 6. Gary: SAS
Institute. Inc.

Sastry. A. 1986. Pelagic lar\'al physiology and ecology of benthic marine
invertebrates in the context of the Indian Ocean. In: M. Thompson. R.
Sarojini & R. Nagabhushanam. editors. Biology of benthic miuine
organisms: techniques and methods as applied to the Indian Ocean.
Rotterdam: A. A. Balkema. pp. 387^01.

Siddal, S. 1984. Density-dependent levels of activity of juveniles of the
queen conch Stromhus gigas Linn. J. Shellfish Res. A:fn-1A.

Spight, T. M. & J. Emlen. 1976. Clutch sizes of two marine snails with a
changing food supply. Ecology 57:1162-1 178.

Stevens. B. 1976. A study of the seasonal changes in component mdices
and lipid contents of the digestive gland, ovary, and fool of Busycon
carica. M.S. Thesis (unpublished). College of Charleston.

Walker. R. 1988. Observations on intertidal whelk {Busycon and Busycoty-
pus) populations in Wassaw Sound. Georgia J. Shellfish Res. 7:473-
478.

Weinheimer. D. 1982. Aspects of the biology of Busycon carica (Gmelin.
1791) in waters off South Carolina with emphasis on reproductive
periodicity. MS. Thesis (unpublished). College of Charleston.

Jimnuil ofSheUfish Research. Vol. 21. No. 2. 111-179. 2002.

RAPA WHELK RAPANA VENOSA (VALENCIENNES, 1846) PREDATION RATES ON HARD
CLAMS MERCENARIA MERCENARIA (LINNAEUS, 1758)

DARIO SAVINI," JULIANA M. HARDING,"* AND ROGER MANN"

' School of Ocean Sciences. Universiry of Wales-Bciniior. Menai Bruli^e. Anglesey. LL59 5EY.
Wales, United Kingdom: 'Virginia Institute of Marine Science. Department of Fisheries Science. P.O.
Box 1346, Gloucester Point. Virginia 23062: "'Universitd degli Studi di Pavia. Departimento di Genetica
e Microhiologica. Via Sunt' Epefanio. 14. 1-27100 Pavia. Italy

ABSTRACT The recent discovery of adult veined rapa whelks Rapanu venosa (Valenciennes, 1846) in the Lower Chesapeake Bay,
U.S.A. offers cause for both ecological and economic concern. Adult rapa whelks are large predatory gastropods that consume bivalves
including commercially valuable species such as hard clams. Mercenaria mercenaria (Linnaeus. 1758). Laboratory feeding experi-
ments were used to estimate daily consumption rates of two sizes of whelks feeding on two size classes of hard clams. Large rapa
whelks (shell length. SL >10l mm) are capable of consuming up to 2.7 g wet weight of clam tissue daily, equivalent to 0.8% of their
body weight. Small whelks (60-100 mm SL) ingest an average of .^.6'7f of their body weight per day.

KEY WORDS: rapa whelk. Rapaini venosu. hard clam. Mercemiria mercemirui. prcdation. Chesapeake Bay

INTRODUCTION

The veined rapa whelk, Rapana venosa. (Valenciennes 1846;
Gastropoda: Muricidae) was discovered in the Hampton Roads
region of the Chesapeake Bay, USA, in the summer of 1998 (Har-
ding & Mann 1999). The species is native to the Sea of Japan.
Yellow Sea, East China Sea and the Bohai Sea (Tsi et al. 1983) but
was introduced to the Black Sea in the 1940's (Drapkin 1953) and
has since spread to the Aegean Sea (Koutsoubas & Voultsiadou-
Koukoura 1990) and the Adriatic Sea (Ghisotti 1974). Recently a
female specimen together with egg masses was found in the Rio
del Plata, an estuary between Argentina and Uruguay in South
America (Pastorino et al. 2000).

The predatory activity of rapa whelks in the Black Sea is con-
sidered by Zolotarev (1996) to be the prime reason for the deci-
mation of native Black Sea oyster, scallop and mussel populations.
Given this history, there is both ecological and economic concern
for the future of shellfish stocks in the Hampton Roads region of
the Chesapeake Bay. Hard clam, Mercenaria mercenaria, popula-
tions are of particular concern in that the Hampton Roads region
supports a substantial local commercial hard clam fishery. Labo-
ratory feeding experiments were used to quantify daily feeding
rates for two size classes of adult rapa whelks offered hard clams.

MATERIALS AND METHODS

Twelve adult rapa whelks, collected from the lower Chesa-
peake Bay, USA, between March and May 2000, were separated
into two different size classes: small (60-100 mm shell length
(SL), the inaximum dimension from the apex of the spire to the end
of the siphonal canal) and large (101-160 mm SL). Rapa whelks
were maintained individually in 60 x 40 x 30 cm plastic net cages
submerged in a shallow flume (250 x 70 x 30 cm) with a constant
flow of unfiltered York River water as described in Savini (2001 ).
The bottom of each cage was covered with 15 cm of clean hard
sand substrate. Rapa whelks were starved for 48 hours prior to the
addition of hard clams (prey) to each enclosure. Each whelk was
given five small (50-70 mm maximum dimension, hereafter shell

*Corresponding author. E-mail: jharding@vims.edu; Tel: +1-804-684-
7302; Fax: +1-804-684-7045

height, SH) and five large (71-100 mm SH) hard clams as poten-
tial prey. Clams were arranged in the experimental cages so that
whelks initially had the same probability of encountering each size
of prey (i.e., whelks at the center of a circle with clams of alter-
nating size classes spaced evenly around the circumference).

The experimental fiume was covered with a fixed plastic net to
prevent escape of the whelks and maintained on a 14/10 h natural
light/dark schedule. Water temperature and salinity data were col-
lected daily from the flume for the 38 day duration of the experi-
ment (June 1 1 to July 18, 2000). Experimental cages were exam-
ined daily and the empty shells of all prey were removed and
measured. Clams that were consumed were replaced daily with
clams of similar dimensions thus maintaining constant prey avail-
ability.

A size range (30-100 mm SH) of fifty hard clams was selected
from the pool of potential prey items and used to create size-
weight relationships for the prey. Individual hard clams were mea-
sured (SH, mm) and weighed (g) prior to the removal of soft tissue.
Clam soft tissue was weighed (wet weight, g) to the nearest 0.1 g.

Data Analyses

Significance levels for all statistical tests were established at
P = 0.05 (( priori. Bartlett's test for homogeneity of variance and
the Ryan-Joiner test for normality were used prior to analyses.
When appropriate, Fisher's test was used for post-hoc multiple
comparisons.

Feeding Rates

The numbers of clams consumed by each size class of whelks
during the entire experimental period were compared using a one-
way ANOVA with individual whelk as a factor. The number of
clams consumed satisfied the assumptions of homogeneity of vari-
ance and normality without transformation. Daily feeding rates
were calculated for each whelk by dividing the total number of
clams consumed during the experimental period by the duration of
the experiment (38 days).

Consumption on a Weight-Weight Basis

Clam wet and dry tissue equivalents consumed by whelks were
compared using a two-way ANOVA with whelk size class and

777

778

Savini et al.

individual whelk as factors. Tissue equivalent data satisfied the
assumptions of homogeneity of variance and normality without
transformation.

RESULTS

Average water temperature during the experimental period was
26°C (±I°C). Average salinity was 21 ppt (±1 ppt). During the 38
day experiment, the six small whelks ate a total of 19 clams while
the six large whelks consumed a total of 15 clams. There was no
significant difference in the total number of clams eaten by small
and large whelks (ANOVA. F = 0.67; P > 0.05). Small rapa
whelks did not show any clear size preference when offered hard
clams as prey (Fig. I ) although small whelks consumed a total of
1 1 small clams and 8 large clams. It should be noted that 5 of the
1 1 small clams were consumed by one individual. Large rapa
whelks consumed large clams more frequently than small clams (4
small clams vs. 1 1 large clams; see Fig. I).

Clam Size-Weight Relationships

Clam tissue wet and dry weights were plotted in relation to
shell height and used to calculate shell height-wet tissue weight
relationships for hard clam prey. These relationships were used to
calculate wet tissue equivalents for each clam consumed by an
individual whelk and were described with the following equations:

Loc (CWWgt) = -3.93 + 2.77 * (Log SH); R"

0.96

where CWWgt is clam tissue wet weight (g) and SH is clam shell
height.

Rapa Whelk Size-Weight Relationships

Rapa whelk tissue wet weight was plotted in relation to shell
length and used to calculate a shell length-wet tissue weight re-

100 80 60 40 20 0
Percentage of small clams consumed

0 20 40 60 80 100
Percentage of large clams consumed

Figure 1. Percentage of small (50-70 mm SH) and large (71-100 mm
SH) hard clams eaten in each experimental cage by each rapa whelk
during the whole experimental period (June 1 1 to July IS, 2000). .\) N.
1-6 = small whelks (60-100 mm SL), B) N. 7-12 = large whelks (101-
160 mm SL).

1000

100

• Small rapa whelks (60-100 mm SL)
O Large rapa whelks ( 10 1 - 1 60 mm SL)

1 10 100

Rapa wtielk body wet weight (g)

1000

Figure 2. Daily Ingested clam wet weight (g) In relation to rapa wet
weight (gl observed in laboratory feeding experiments during .lune
and .July. 2000. The solid diagonal line represents clam consumption
equal to the body weight of the predator (whelk) or a 1:1 consumption
relationship on a prey wet weight: predator wet weight basis. Points
abo\e the line indicate prey consumption at a rate greater than one
while points below the line indicate daily consumption rates less than
the body weight of the predator.

lationship for whelk predators. This relationship is based on 150
animals (80-165 mm SL) collected from lower Chesapeake Bay.
USA between October 1999 and July 2000 (Harding and Mann,
unpublished data):

WWWgt = 6.4908 * e(0.0229 * SL).

R-

0.69

where WWWgt is whelk tissue wet weight (g) and SL is whelk
shell length (mm).

On the basis of tissue wet weight, large whelks consumed
significantly more prey flesh tissue than small whelks (ANOVA.
F = 4.45, P < 0.05). Individual small whelks ate proportionately
more hard clam tissue on a clam wet weight: whelk wet weight
basis than large whelks (Fig. 2). Maximum daily clam consump-
tion rates of 5.b7r of body wet weight were recorded for small
whelks as compared to 1 .69^ of body wet weight for large whelks.

DISCUSSION

Large rapa whelks (101-160 inm SL) are able to consume up to
2.7 grams of clam tissue (wet weight) per day or 0.8% of their
body weight per day at water temperatures of approximately
26' C. In contrast, small rapa whelks (60-100 mm SL) ingested an
average 3.6% of their body weight every day, which is more than
four times that observed for larger rapa whelks at similar water
temperatures on a weight-specific basis. Edwards and Huebner
(1977) suggest that temperature affects feeding rate in the moon
snail Piiliuices by increasing predators' metabolic rate, and thus
the reqturement for a larger amount of food. The present investi-
gation was conducted during warmer months and is probably in-
dicative of the maximum feeding activity of rapa whelks. There is

Rapa Whelk Predation on Hard Clams

779

considerable variation in reported ingestion rates for predatory
gastropods with values up to 25% of its body weight per day
reported for the moon snail, Polinices duplicatus (Thorson 1971 ).

The hard clam fishery in the lower Chesapeake Bay is already
in decline. Hard clam landings during 1999 were less than 10% of
landings during 1973 (Virginia Marine Resources Commission.
Newport News, VA). The observed decline in hard clam stocks
may be related to increased anthropogenic impacts on the Chesa-
peake Bay ecosystem in the past 20 years including overfishing,
water pollution and disease. Habitat changes are considered the
major threats to estuarine ecosystem (Smith et al. 1999). The su-
perimposition of a novel invading predator on this already stressed
population has clear ecological and economic implications.

Vimstein (1977), found that particularly in Chesapeake Bay.
densities of infaunal species are not controlled by competitive
interactions for food or space but mainly by the action of predators.
If the introduction of Rapana venosa into the lower Chesapeake
Bay results in a large scale successful invasion, rapa whelks could
have a serious negative impact on the density and distribution ot
the native hard clam population in the lower Chesapeake Bay. At
this time we do not have a good estimate of the resident population
of rapa whelks in the Chesapeake Bay but is possible to use our
data for a hypothetical calculation to estimate potential impact of
the whelk on the clam population. The rapa whelk distribution in
the Chesapeake Bay, which extends from the mouth of the Rap-

pahannock River in the North, to the Chesapeake Bay Bridge
tunnel in the southeast and to the Lafayette River in the south
(Harding & Mann 1999, Mann & Harding 2000), is within the
historic distribution of M. nierceiiaria (Roegner & Mann 1991).
The 1999 summer fishing season for hard clams in the lower
Chesapeake Bay produced a harvest of 27388 kg or approximately
3,040,000 individual clams. Based on the predation rates observed
in this study, a population of 1000 rapa whelks in the lower Bay
could reduce this yield by between 0.3 to 0.9%.

ACKNOWLEDGMENTS

This work was supported by funds troni the National Oceanic
Atmospheric Administration, National Sea Grant Aquatic Nui-
sance Species Research and Outreach Program. Virginia Sea
Grant, the Virginia Saltwater Commercial Fishing Development
Fund, and the Commonwealth of Virginia. It was completed by the
senior author during study for a M.S. degree at the University of
Wales, Bangor, UK. Financial support for the senior author's visit
to the Virginia Institute of Marine Science was provided by the
Drapers Company, London. The authors thank Melissa South-
worth, Erica Westcott, Stephanie Haywood, Rhonda Howlett,
D. Bryn Jones, and Catherine Ware for assistance in general labo-
ratory procedures during the study. This is contribution number
2487 from the Virginia Institute of Marine Science,

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Polinices duplicatus preying on Mya arenaria at Barnstable Harbor,

Massachusetts. Ecology 58:1218-1236.
Ghisotti, F. 1974. Rapana venosa (Valenciennes) nuova ospite adriatica'.'

Conchiglie Milano 10:125-126.
Harding, J. M. & R. Mann. 1999. Observations on the biology of the

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Chesapeake Bay. / Shellfish Res. 18, 9-17.
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J,„anal of Slwlltlsh Research. Vol. 21, No. 2, 781-785. 2002.

ECONOMIC ANALYSIS OF A PILOT COMMERCIAL HATCHERY-BASED OPERATION FOR
SPOTTED BABYLON, BABYLONIA AREOLATA LINK 1807, JUVENILES IN THAILAND

N. CHAITANAWISUTI,* S. KRITSANAFUNTU," AND Y. NATSUKARl'

'Aquatic Resourcfs Research Institute. Cluilaloiigkoni University. Baiii>kuk. Thailand 10330:
-Department of Bioproducts, Prince of Songkla University (Suratani campus). Suratani. H4I00:
^Department (if Fisheries. Nagasaki universit^■. I -14. Bunkyo-Machi. 852-8521 Japan

ABSTRACT An economic analysis of constructing and operating a pilot hatchery and ntirsery for the commercial production of
spotted babylon. Babylonia nreo/aw. juveniles in Thailand was presented on the basis of proven practical techniques and published data
from larval culture trials. The total initial investment requirement for the construction of a spotted babylon hatchery producing 1.2
million juveniles per year was 9.310.5 USS. Annual ownership costs were estimated to be 2.497.5 US$. with annual operating costs
of 5.31 1.8 USS. Total annual cost for the juvenile production (Hatchery) phase of spotted babylon was 7,809.3 USS, and the co.st of
producing 1.2 million juveniles in this hatchery design was estimated at 6.09 USS per 1.000 juveniles. Under the basic assumptions
in this study (juvenile production of 1.2 million/year), a selling price of 13.8 USS per 1.000 juveniles results in a positive cash flow
by year 2. Price and survival sensitivity resulted in substantial variability in net returns. This pilot hatchery operation is marginally
feasible under these conditions if costs can be lowered considerably by improving growth and survival.

KEY WORDS: Spotted babylon. Babylonia areolara. economic analysis, hatchery-based operation

INTRODUCTION

Spotted hahylon. Bahyhmla areolala (Link), are promising a
new aquacultuial marine gastiopod in Thailand. Fast growth, high
survival, and low feed conversion ratio (FCR) in grow-out culture
provide this species with the biologic production and market char-
acteristics necessary for a profitable aquaculture venture (Chai-
tanawisuti & Kritsanapuntii 1999a. Chaitanawisuti & Kritsan-
apuntu 1999b). Considerable interest has developed recently re-
garding the commercial culture of spotted babylon in Thailand
because of a growing demand and an expanding domestic market
for seafood, as well as a catastrophic decline in the natural spotted
babylon population in the Gulf of Thailand. An accurate economic
assessment of culturing spotted babylon in Thailand is required
before producers can make informed decisions regarding the po-
tential of this enterprise. A lack of economic data can be an im-
portant constraint to the successful development of spotted baby-
lon aquaculture in Thailand. A financial investment analysis de-
scribes the relationships between yield (growth and survival),
market price, fixed and variable costs, and profitability indicators.
From 1998 to 2000, Chulalongkom University conducted a col-
laborative research and development project with the National Re-
search Council of Thailand (NRCT) to develop the land-based
aquaculture system for large-scale hatchery and grow-out opera-
tions for spotted babylon in Thailand. Thereafter, the methods and
techniques are intended to transfer for the economically hatchery-
based operations in Thailand. This study was the first attempt to
present estimates of the cost of producing juvenile spotted baby-
lon, B. areolata. under a successful management scheme. The
specific objectives were performed to identify the investment re-
quirements, annual ownership and annual operating costs associ-
ated with the hatchery, and the net returns, returns on investment
and cash flow according to selected survival and prices of juvenile.

MATERIALS AND METHODS

Hatchery Operation

A pilot commercial-scale hatchery in this study is designed to
produce a total of 100,000 juveniles in 30-day production cycle for
transfer to growout phase of culture. The design and operation of

the hatcheiy was based upon the techniques of Chaitanawisuti and
Kritsanapuntu (1997). The data necessary to estimate investment
requirements for the hatchery were mainly gathered from a pilot
research. The broodstock spawned and reared the larvae up
through the post-set stage to 1.0 cm juvenile. The process involves
manipulating adult snails until natural spawning occurs. The egg
capsules are collected and placed in rearing tanks, where they
develop into veliger larvae. The set are then placed in rearing units
in which algae-enriched water is offered once daily. The algae are
cultured from unicellular algal flask cultures of preferred species
{Chiietocfros culcltrans and Tetraselmis sp.) in a controlled sys-
tem. The average monthly productions of egg capsule and veliger
larvae are approximately 8,180 and 7,1 16,600, respectively (Chai-
tanawisuti & Kritsanapuntu 1999b). The initial stocking density
rate was specified at 500 larvae L"' of water. The entire hatchery
process require 14-18 days to go from spawning to newly settled
juveniles ready for placement in the nursery system. This method
requires a substantial level of capital investment and technical
expertise to produce a consistently high quality of algae supply.
The nursery is a critical link in the spotted babylon culture process.
Placing hatchery juveniles directly into the grow-out system may
induce unacceptably high mortality levels. The nursery provides an
intermediate step that nurtures hatchery-reared Juveniles to a size
less vulnerable to the stress of grow-out operation. In addition,
growing juveniles to the size required for the grow-out stage
within the intensive hatchery environment would likely not be cost
effective. The length of time (30-43 days) required by a nursery to
produce 1.0-cm juveniles from newly settled juveniles. Based on
this pilot hatchery, an average survival of veliger larvae to 1.0 cm
juveniles is approximately LS'/r.

Financial Analysis

Financial analysis was based on investment cost, production,
and marketing data from the pilot-scale trials. The components of
the economic analysis include the following.

For the initial investment, the building used for hatchery op-
erations included a 300-nr space made of concrete floor and sheet
zinc roof with no window and wall for reducing construction cost
and entrance of natural light. The hatchery design consists of three

781

782

Chaitanawisuti et al.

TABLE 1.

Initial investment requirements for hatchery production of spotted
bab) Ion. li. areolulu. ju\eniles.

Number,

Investment,

Percent of

Item

(unit)

(US$)

Total Cost

Land

1,149.4

12.35

Buildine (300 nr)

1

3,448.3

37.04

BroodstiK-k lanks (3 x 3 x 0.7 m)

3

229.9

2.47

Lanal reanng tanks (500 Ll

30

689.7

7.40

Nursery tanks (5(H) L)

15

344.8

3.70

Alaal reanng tanks (500 L)

10

229,9

2.47

Mass Algal re;inng tanks (3 ton)

5

689.7

7.40

Aeration system

1

229.9

2.47

Water supply and drainage

1

574.7

6.17

Storage tanks (20 m')

2

459.8

4.94

Algal laboratory

1

804.6

8.65

Hatchery equipment

1

459.8

4.94

Total

9.310 5

100

rectangular broodstock rearing tanks (3.0 x 3.0 x 0.7 m canvas
tanks), 30 cylindrical larvae rearing tanks (500-L plastic tanks), 20
cylindrical (nursery tanks (500-L plastic tanks), 15 conical algae
culture tanks (500-L plastic tanks), and 4 mass algae culture tanks
(4-ton concrete tanks). A small 3.0 x 3.0 m room is also included
for algae culture and stock tnaintenance. Sand filter rated at 150 L
per minute are specified for filtering water before its initial use in
the hatchery. Two sets of 2-horsepo\ver pumps rated seawater used
are necessary to transport water through the system. Two 10-ton
canvas tanks were used as seawater stocking lanks. The two-
horsepower blower is designed to provide a high volume of dis-
solved oxygen, low pressure, and uncontaminated air necessary for
larval survival and to keep food suspended in the water column
after it has been introduced into the larval culture tank. Operating
a hatchery requires the regular monitoring of water-quality vari-
ables such as pH. salinity, water temperature, ammonia, nitrite, and
nitrate levels, and these parameters are maintained at satisfactory
levels for optitnum growth and survival. A refractometer is speci-
fied for salinity measurements and a seawater test kit is specified
for the determination of important water-quality parameters. Other
designated equipment includes a refrigerator for the storage of

TABLE 3.

Estimated annual operatin;; costs (Baht) for hatchery production of
the spoiled babylon, B. areulata, juveniles.

Item

Cost

(US$)

Percent of
Total Cost

Repairs and maintenance

Hired labors (2 full time)

Feed

Broodstock purchase

Electricity

Interest on operating cost

Total operating cost

408.0

7.68

2.758.6

51.94

724.1

13.63

413.8

7.79

827.5

15.58

179.6

3.38

5.311.6

100

supplemental feed ingredients and algae stocks. A binocular mi-
croscope IS needed for the daily inspection of the quantity of food
and growth of the larvae. PVC pipes and fittings are used to
transport seawater and air from their sources to the culture system.
Additional miscellaneous equipment also is specified for the daily
operation of the hatchery. Interest rates for capital cost is based on
2000 bank loan rate of 3.5% for business enterpnse.

Annual ownership costs mainly consisted of annual deprecia-
tion and interest on investment. These costs are fixed and incurred
in the long run regardless of whether the facilities are operated.
Annual depreciation was based on the expected useful life of each
equipment item. A zero salvage value was assumed on all items
constituting the facilities. The hatchery building, canvas tanks, and
plastic tanks were assumed to have a useful life of 10 years be-
cause of the seawater environment. The life expectancies of equip-
ment ranged from 3-5 years. Interest rates are based on 2(.)(;)0 bank
loan rates of 3.5% of the original prices for all investment items.

Annual operating costs are incurred upon actual operation of
the hatchery and include repairs and maintenance, labor, feed,
utilities, and interest on operating capital. The annual cost of re-
pairs and maintenance for the hatchery was computed as 5.0% of
purchase price. Two labor requirements were estimated based on
the particular needs for full-time operation of the hatchery. Labor
cost for each individual was calculated at a rate of 1 14.9 USS per
month, without fringe benefits. Feed cost is based upon the as-
sumption that larvae are fed microalgae for 14 days of the cycle.

TABLE 2.
Estimated depreciation, interest charges, and repairs and maintenance of hatchery production for B. areolala juveniles.

Economic

Annual

Annual Interest

Annual Repair and

Item

Cost (USS)

Life (yrl

Depreciation (USS)

Charges (USS)
40.2

Maintenance (USS)

Land

1,149.4

1

1.149.4

-

Buildings

3.448.3

10

344.8

120.7

172.4

Broodstock tanks

229.9

10

22.9

8.0

11.5

Larval-rearing tanks

689.7

10

68.9

24.1

34.5

Nursery tanks

344.8

10

34.5

11.9

17.2

Algal tanks

229.9

10

22.9

8.0

11.5

Mass algal tanks

689.7

10

68.9

24.1

34.5

Aeration system

229.9

3

76.6

26.6

11.5

Water supply and drainage

574.7

3

191.5

20.1

28.7

Storage tanks

459.8

10

2.000

16.1

22.9

Algal culture laboratory

804.6

10

80.5

28.2

40.2

Hatchery equipment

459.8

5

45.9

1.6

22.9

Total cost/year

9,310.3

2,153,1

344.4

408.0

Economic Analysis of Spotted Babyxon Hacthery

783

TABLE 4.

Estimated annual costs for hatchery production of spotted bahylon.
B. areolala. ju\eniles.

TABLE 6.

Gross return for hatchery production of spotted babylon. B.
areolata, juveniles at selected survival rates and selling prices.

Item

Cost (US$1

Percent of Total

Selling Price |IS$ per 1.000 Juveniles)

Ownership costs

Depreciation 2.153.1

Interest on investment 344.4

Total ownership cost 2,497.5

Operating costs

Repairs and maintenance 408.0

Hired labor 2,758.6

Feed 724. 1

Broodstock purchase 413.S

Electricity 827.6

Interest on operating capital 179.6

Total operating cost 5,311.8

Total annual cost 7,809.3

27.57

4.41

3 1 .98

5.23
35.33

9.28

5.29
10.59

2.30
68.02
100

Thereafter, feed is principally composed of the fresh meat of fish.
The cost of purchasing and caring for broodstock was estimated to
be 4.6 US$/kg of spotted babylon. Based on the production sce-
nario, the hatchery designed in the present study requires 40 kg of
broodstock. This estimate was based on the assumption that ap-
proximately 52,200 larvae are produced from one female brood-
stock and an average of 1.5% survival of juveniles was expected.
Electricity is used for operating the various pumps and lighting
units in the hatchery. The average charge of electricity was as-
sumed to be 68.9 US$ per month. Interest rates are based on 2000
bank loan rates of 3.5% per year for all depreciable items that
compose the hatchery.

Return Analysis

Net returns and returns on investment for hatchery production
were computed at final survivals ranging from 1.0-3.5% and the
selling price of juvenile ranging from 9.2-22.9 USS per 1.000
juveniles. Gross return was computed for each level of survival
and each selling price. Net return was calculated from the gross
return minus the total annual cost. Return to capital and manage-
ment was computed for each level of survival and each selling
price by subtracting annual operating cost from gross returns. Sub-
sequently, return on investment was estimated by dividing returns

TABLE 5.

Estimated total annual cost for production of spotted babylon, B.
areolata. juveniles at selected survival rates.

Survival
Rate ( % »

Annual Production
(.Juvenllel

Annual Costs

(USS)

Cost per 1,000
Juveniles (USS)

0.5
1.0
1.5
2.0
2.5
3.0
4.0

426.996
853.992
1.280.988
1.707.984
2,134.980
2.561,976
3,415.968

7,809.3
7,809.3
7,809.3
7.809.3
7.809.3
7.809.3
7.809.3

18.29
9.14
6.09
4.57
3.66
3.05
2.29

Survival rate is calculated from veliger larvae to juveniles of 1 .0-cm shell
length with an average monthly egg capsule and veliger production of
8.180 and 7.116,600, respectively.

i%)

9,2

13.8

16.1

18.4

22.9

0.5

3.92S.3

5.892.5

6.874.6

7.856.7

9,778.2

1.0

7.856.7

11,785.0

13.749.3

15.713.4

19,556.4

1.5

11,785.1

17,677.6

20,623.9

23,570.1

29,334.6

2.0

15,713.4

23,570.2

27,498.5

31.426.9

39,112.8

2.5

19,641.8

29,462.7

34,373.2

39,283.6

48,891.0

3.0

23,570.2

35,355.3

41,247.8

47,140.3

58,669.3

4.0

31.426.9

47,140.4

54.997. 1

62,853.8

78,225.7

Gross return was calculated for each level of survival and selling price.

to capital and management by initial capital investment (Fuller et
al. 1992). An annual production budget was developed from the
variable and fixed costs, and cash flow budgets were developed to
examine profitability in relation to the timing of expenditures and
earning. Net cash flow was determined by projecting estimated
revenues and costs over a 10-year period because an aquaculture
enterprise would be an attractive investment opportunity if it were
profitable within this period. The initial investment was charged in
the first year, and costs in subsequent year included annual vari-
able and fixed costs. (Rubino 1992, Head et al. 1996).

RESULTS

Total investment requirements for construction of the hatchery
were 9,310.5 USS (Table 1). The building was the largest cost
component (37.04%) of the hatchery. The rearing tank. land, water
supply and storage tanks, and algal culture tanks are the second
most expensive items in equipping the hatchery, representing
13.57%, 12.35%. 11.11%. and 9.87% of total investment, respec-
tively. These five components of the hatchery represent 83.94% of
total investment requirements for production of spotted babylon
juveniles. Annual ownership costs were estimated to be 2.497.5
USS with annual depreciation and interest of 2.153.1 and 344.4
USS. respectively (Table 2). The annual operating cost is estimated
to be 5,311.8 USS. Hired labor was the largest cost component
(51.94%) of the operating cost, followed by electricity, feed, and
repairs and maintenance of which comprised 15.58%, 13.63%. and
7.68%, respectively (Table 3). Total annual cost for the juvenile

TABLE 7.

Net return for hatchery production of spotted babylon, B. arenlata,
juveniles at selected survival rales and selling prices.

Selling Price (USS per 1,000 Juveniles)

(%)

9.2

13.8

16.1

18.4

22.9

0.5

-7,556.3

-19.168

-934.7

47.4

1.968.9

1.0

47.4

3.975.7

5.940.0

7.904.1

11.747.1

1.5

3,975.8

9.868.3

12.814.6

15.760.8

21,525.3

2.0

7,904.1

15.760.9

19,689.2

23.617.6

31,303.5

2.5

11,832.5

21,833.4

26,563.9

31.474.3

41,081.7

3.0

15,760.9

27.546.0

33,438.5

.39.331.0

50,860.0

4.0

23,617.6

.39.331.1

47.187.8

55.044.5

70.416.4

Net return was calculated from the gross return minus total annual cost
(7.809.3 USS).

784

Chaitanawisuti et al.

TABLE 8.

Return to capital and management for hatchery production ol'

spotted babylon. B. areolala, juveniles at selected survival rates and

selling prices.

TABLE 9.

Return on in>estnient for hatchery production of spotted babylon,
B. areulata, juveniles at selected survival rates and selling prices.

Survival

(%)

Selli

ing Price (LIS$ per

1,(100 Juveniles)

Selling Price (USS per l.(H)(l .Juveniles)

Survival

9.2

13.8

16.1

18.4

•)■) 9

9.2

13.8

16.1

18.4

22.9

(Vr)

0 5

0 \^

0 06

0 17

0 ""7

0 48

0.5

-1,383.5

580.7

1.562.8

2..544.4

4,466.4

1,0

0.27

0.69

0.91

1.12

1.53

1.0

2,544.9

6.473.2

8.437.5

10.401.6

14.244.6

1.5

0.69

1.33

1.64

1.96

2.58

1.5

6.473.3

12,365.8

15.312.1

18.258.3

24.022.8

2.0

111

1.96

2.38

2.80

3.63

2.0

10,401.6

18,258.4

22.186.7

26.115.1

33.801.0

2.5

1..54

2.59

3.12

3.65

4.68

2.5

14,3.W.0

24,1.50.9

29.061.4

33.971.8

43,579.2

3.(1

1.96

3.22

3.85

4.49

5.73

3.0

18.258.4
26.115.1

30,043.5
41.828.6

35,936.0
49.685.3

41.828.5
57.542.0

53.357.5
72.913.9

4.0

2.80

4.49

5.-34

6.18

7.83

4.0

Return tin investment

was

calculated b\

' di\iil

ini'

return tti cani

tal and

Return to capital and management was calculated tor each level of survival
and selling price by suhtiactmg annual upcrating ci>st (5.31 1.8 US$) frum
gross returns.

production (hatchery) phase ol spotted babylon culture was
7,809.3 US$ (Table 4). Annual ownership and operating costs
accounted for .3 1 .98% and 68.02% of the total annual cost, respec-
tively. The major ownership cost item was depreciation on invest-
ment representing 27.57% of total annual cost. Hired labors was
the highest operating cost item, representing 35.33% of total an-
nual cost. The cost associated with producing juvenile spotted
babylon is expressed as US.$ per 1.000 juveniles (43.5 Thai Baht
is approximately lUSS). The cost of producing 1.200,000 juve-
niles in this hatchery design was estimated at 6.09 US$ per 1.000
juveniles. However, as the total number of juveniles produced per
year decreases, then cost increases. For example, if 426,996 juve-
niles (approximately 0.57( survival) are produced, utilizing the
same level of inputs, the estimated cost of production increases to
18.29 USS per 1,000 juveniles. Estimated total annual cost for
production of juveniles at selected survivals is presented in Table
5. At 1.5% survival in this study reveals the 13.8 U.S.S Baht break-
even price. Thereafter, gross return and net return at these levels
are 17,677.6 USS and 9,868.3 USS, respectively (Tables 6 and 7).
Return to capital and management and return on investment at
these levels are 12.365.8 USS and 1.33. respectively (Tables 8 and
9). Under the basic assumptions in this study (juvenile production
of 1 .2 million/year), a selling price of 13.8 USS per I .OOO juveniles
results in a positive cash flow by year 2 (Table 10).

management by initial capital investment (9,310.5 USS).

DISCUSSION

Based on juvenile production of 1.5% survival and selling price
of 13.8 USS per 1.000 juveniles, the culture system is economi-
cally feasible under the assumptions used. The cost of producing
B. areolata postlarvae according to the procedures and assump-
tions outlined in this study is considerably higher when the sur-
vival is very poor. The stand-alone hatchery operation becomes
profitable at output level of 1.2 million juveniles. Additional re-
search needs to refine the lower limit of profitability and the nature
of marginal costs (i.e., the change in variable costs with changes in
output) within this range of facility sizes. An underlying assump-
tion in this analysis shows that survival rate and market price are
sensitive to farm output. However, potential investors must assess
the impact to local market prices resulting from large production
levels. The analysis assumes a constant market price, which may
not be valid as the production volumes from large-scale operations
are released onto the market. In general, prices are sensitive to
changes in supplies. Prices decrease (increase) as spotted babylon
landing increase (decrease). This relationship is not incorporated
into the preceding analysis, which focuses primarily on cost
changes as output levels vary. Investors in spotted babylon aqua-
culture should be aware of the potential negative effects on market
prices as output levels increase. Costs presented in this study are
based on limited available data. An economic analysis of a pilot
hatchery production for spotted babylon would be commercially

TABLE 10.
Ten-year cash flow of a pilot hatchery production for spotted babylon, B. areolata juveniles.

Variable Cost

Fixed Cost

Investment

Total Annual

Receipt

Net Return

Cumulative

Year

(US$)

(USS)

(USS)

Cost (USS)

(USS)

(US$)

(USS)

Year 1

5.311.8

2.497.5

9,310.5

17.119.8

17.677.6

9.868.3

-7.251.5

Year 2

5.311.8

2.497.5

0

7.809.3

17.677.6

9.868.3

2,616.8

Year 3

5.311.8

2,497.5

0

7.809.3

17.677.6

9.868.3

12.485.1

Year 4

5,311.8

2,497.5

0

7.809.3

17.677.6

9.868.3

22.353.4

Year 5

5,311.8

2.497.5

0

7.809.3

17.677.6

9.868.3

32.221.7

Year 6

5,311.8

2.497.5

0

7.809.3

17,677.6

9.868.3

42.090.0

Year?

5.311.8

2.497.5

0

7.809.3

17.677.6

9.868.3

5 1 .958.3

Years

5,311.8

2.497.5

0

7,809.3

17.677.6

9.868.3

61.826.6

Year 9

5.311.8

2.497.5

0

7.809.3

17,677.6

9.868.3

71.694.9

Year 10

5.311.8

2.497.5

0

7.809.3

17.677.6

9.868.3

81. .563. 2

Based on

an annual

production of 1.2 million juveniles per year and selling price of 13.8 USS per 1,000 juveniles

Economic Analysis of Spotted Babylon Hacthery

785

feasible at current selling price at survival rate of 1 .5%. marginally
feasible. This study serves as a guideline for understanding the
economics of commercial juvenile production. Deviation from the
hatchery specifications and management techniques of this study
will likely result in altered costs. Costs can be lowered consider-
ably by improving growth and survival rate. This economic analy-
sis is intended as a guide and must be modified to reflect individual
situations.

ACKNOWLEDGMENTS

We would like to thank National Research Council of Thailand
(NRCT) for its support of the project and Professor Piamsak Me-
nasveta and Associated Dr. Somkiat Piyatiratitivorakul for their
advice and guidance. Last, we would like to thank Soonthorn
Thepmoon, Siriwan Kathinniai. Mongkol Maklit. and Sailom Tan-
tulvawit for their assistance during the hatchery work.

Chaitanawisuti. N. & A. Kritsanapuntu. 1997. Effects of stocking density
and substrate presence on growth and survival of juvenile spotted bah-
ylon. Babylonia areolata Link. 1807 (Neogastropoda: Buccinidae). ./.
Shellfish Res. 16:429-133.

Chaitanawisuti. N. & .\. Kritsanapuntu. 1999a. Experimental culture of
hatchery-reared juvenile spotted babylon. Babylonia aivolala Link 1807.
(Neogastfopoda: Buccinidae) in Thailand. Asian Fisheiy Sci. 12:77-82.

Chaitanawisuti, N. & A. Kritsanapuntu. 1999b. Growth and production of

REFERENCES

hatchery-reared juvenile spotted babylon. Babylonia areohita Link
1807, cultured to marketable sizes in intensive flow-through and semi-
closed recirculating water system. Aquaculture Research 31:415— H9.

Head, W. D.. A. Zerbi & W. O. Watanabe. 1996. Economic evaluation of
commercial-scale, saltwater pond production of Florida tilapia in Puer-
to Rico. J. World Aijnaciilture Soc. 27:275-289.

Rubmo. M. C. 1992. Economics of red claw (Cherax quadricannatus aqua-
culture). J. Shellfish Res. 11:157-162.

Joiirmil of Shellfish Research. Vol. 21, No. 2, 787-792. 2002.

INCIDENTAL DAMAGE OF BLACKLIP ABALONE (HALIOTIS RUBRA) BY COMMERCIAL

DIVERS IN NEW SOUTH WALES, AUSTRALIA

P. T. GIBSON,' D. G. WORTHINGTON,' C. BLOUNT,' AND N. L. ANDREW' *

^New South Wales Fisheries, Cronulla Fisheries Centre. PO Box 21. Crunulla. NSW, 2230. Australia
and ^National Institute of Water and Atmospheric Research. P.O. Box 14-901, Kilbirnie,
Wellington, New Zealand

ABSTRACT Rales of incidental damage Ui blacklip abalcme (Hciliotis rubra) were invesligated in a retained, commercial catch in
New South Wales, Australia. On average, 10.2% (8.9-1 1.6%, 95% CI) of retained abalone were damaged, but there was no relationship
between the level of experience of divens and the degree or frequency of damage. Most damage was relatively minor, such as small
abrasions to the foot or cuts to the mantle, and wounds healed rapidly in aquana. Major damage, such as large cuts and abrasions to
the foot, occurred in 4.2% (3.6-4.9%) of retained abalone. For individuals with major damage, any mortality was rapid and significantly
greater than for undamaged abalone in aquaria. Rates of growth were also lower for individuals with major damage. An average of
19.1% 117.2-21.1%) of abalone removed from the reef were found to be under the minimum legal size and replaced. E.xperienced
divers removed and replaced significantly fewer abalone under the minimum legal size (11% vs. 23.8% of those handled) than
inexperienced divers. Using the estimated rates described here, approximately 40 tons of abalone are estimated to be damaged by the
fishery each year. Most damaged abalone are retained, heal rapidly in aquaria, and can be exported live, but approximately 3 tons of
damaged abalone (both retained and discarded) may die.

KEY WORDS: abalone, commercial fishing, damage, incidental, mortality

INTRODUCTION

Incidental damage to species not targeted by a fishery can be a
substantial source of mortality (e.g., Broadhurst 2000). Damage to
individuals of species that are targeted but subsequently found to
be inappropriate for market also can be substantia] (e.g.. Blount &
Worthington 20(M ). These types of mortality are most common in
fisheries with nonselective collection methods (e.g., prawn trawls),
or those with uncertainty about the quality or size of the product
(e.g,, sea urchin roe). Despite this, incidental moitality may be an
issue in selective fisheries when a minimum size limit requires the
handling of individuals (to identify their size) before they are
retained. Damage and any consequent mortality to individuals 1 )
above the size limit and retained and 2) below the size limit and
returned can be problematic in fisheries where most of the product
is sent live to markets.

Compared with many other fisheries, commercial tlshing for
abalone in New South Wales (NSW) is relatively benign, with
minimal direct impact on other species or on abalone not being
targeted for collection. In NSW, divers use a blunt, chisel-shaped
iron to remove individual abalone from the reef Abalone are then
quickly measured and, if larger than the size limit (115 mm in shell
length, hereafter referred to as legal size), placed in bags that are
sent to the surface for more controlled measuring. Any individuals
that are removed from the rock, measured, and found to be below
the size limit (hereafter referred to as undersized), either by the
diver in the water or on the boat, are returned to the reef by hand.
Although this process is relatively simple, the action of levering
individuals off the bottom can result in cuts and abrasions to the
foot of the abalone. Similarly, damage can occur during handling
and storage of the catch on the boat before landing at processing
factories.

There has recently been a major change in management of the
NSW abalone fishery. Before 1995, each of the 37 shareholders
was required to harvest the quota they owned, except in excep-
tional circumstances such as illness. This resulted in a group of
shareholders that entered the fishery in the early 1970s and had

*Corresponding author. E-mail: gibsonp@fisheries.nsw.gov.au

collected abalone commercially for over 20 y. The change during
1995 enabled shareholders to nominate a diver to collect abalone
on their behalf and, as a consequence, there was an intlux of
inexperienced divers. This change generated concern within the
industry about the impact of increased rates of damage by inex-
perienced divers on populations of abalone. In addition, over the
same time period, the fishery began to export live almost all aba-
lone that were landed. This has also led to concern within the
industry over the handling of abalone by divers and the rates of
recovery of damaged abalone before export.

Different types of damage are likely to have different effects on
abalone. ranging from decreased growth and reproduction to death.
Even the slightest disturbance, such as tagging, has been known to
reduce growth rates and. in some cases, increase mortality (Mc-
Shane et al. 1986). There is also some evidence from aquaculture
that suggests small disturbances may disrupt reproduction (our
unpublished data). Abalone have no clotting agent in their blood.
so that damage to (he foot (hat severs any of the major arteries is
likely to lead to death (Armstrong etal. 1971 ). Any type of injury may
also inhibit their ability to feed or attach to the reef, leaving them
more susceptible to predation (Pirker 1992). When disturbed, abalone
also clamp down on the reef, making them considerably harder to
remove, which may further increase the likelihocxl of damage.

In this article, we describe the frequency of different types of
damage to abalone by commercial divers in NSW, We estimate
rates of damage in the commercial catch and the number of aba-
lone that are removed from the rock, measured, found to be un-
dersized, and replaced. Finally, to investigate the possible effects
of damage to abalone that are not retained, we estimated the rates
of recovery, growth, and mortality of individuals with different
types of damage when maintained in aquaria. This has implications
for marketing because retained abalone are held in tanks at pro-
cessing plants before export.

MATERIALS AND METHODS

Frequency of Damage by Commercial Divers

To estimate the frequency of different types of damage by
commercial divers, a two-phase sampling design was used. First,

787

788

Gibson et al.

divers" daily catches were liapliuzardly selected for samplint; in
commercial processing factories from August 1996-December
1997. An individual bin (holding 50-100 abalone) from the di\er's
catch was then randomly selected, and the length of all abalone
within measured to the nearest mm and weighed to the nearest 5 g.

Damage was categorized into several types and magnitudes
based on injuries observed in the commercial catch. Categories (in
order of increasing severity) were: no damage (ND), abrasions to
the edge of the foot or mantle (MA), cuts to the edge of the foot
or mantle (MCl, small (<10mm) abrasions to the foot (FAl ), small
(<10 mm) cuts to the foot (FCl ), large (>10 mm) abrasions to the
foot (FA2), large OlO mm) cuts to the foot (FC2), and major
breaks to the shell (BS). These categories were further grouped
into minor damage (MA. MC. FAl) and major damage (FCl. FA2,
FC2, BS). based on their likely effects on abalone. For the analyses
presented here, indi\iduals with more than one type of damage
were categorized to the most severe grouping. Confidence limits
were calculated using the binomial distribution, and a generalized
logit model was used to relate the size of abalone to rates of minor
and major damage (see Richards et al. 1994).

Although the sampling design described above provided de-
tailed information on the frequency of damage, a second sampling
design was used to provide less detailed but more comprehensive
information. In this design, all abalone from all divers' daily catch
supplied to one commercial processing factory were assessed for
damage over a four month period (July to August 1999). Abalone
were assessed as either damaged, not damaged, or dead. In total
68.900 abalone from 15 divers on 207 diver-days were assessed in
this design, compared with only 9.732 in the first design. Although
the second sampling design is more comprehensi\e than the first in
terms of the absolute number of abalone sampled, it covers only a
proportion of the year and only one major processing factory.

To estimate the rates of damage to abalone in the nonretained
catch, information was collected on the frequency of removing,
measuring, and replacing undersized abalone. Commercial divers
were supplied with a logbook to record the number of undersized
abalone removed and replaced and the number of legal-sized aba-
lone retained in their first bag (divers retain harvested abalone in
rope catch bags) of the day. Six commercial divers completed this
logbook for a period of 15-30 days each between April-December
1999.

Recovery. Growth, and Mortality of Damaged Abalone

To investigate the effects of damage on individual abalone, two
experiments were conducted in aquaria at a commercial processing
factory. Experiment 1 began on 31 October 1996 and included 242
damaged abalone selected from the commercial catch. Replicate
individuals from each damage category were placed in one of three
randomly chosen aquaria. The number of replicate individuals
within a damage category was related to the frequency of the
category in the commercial catch supplied to the factory on that
day (Table I ). Every two weeks, all individuals were briefly re-
moved from the aquaria and any damage reassessed. A BS treat-
ment was not included because individuals with this type of major
damage were often dead before landing or were kniiwn to die
rapidly thereafter.

In experiment 2, the effects of damage on growth were exam-
ined and the damage categories with the highest mortality in ex-
periment 1 were further investigated. On 9 April 1997, at least 50
abalone from each of four damage categories (ND, FC 1 , FA2, and

TABLE 1.

Number of abalont within each treatment of different types of

damage, and within each tank for both the first and

second experiments.

Damage Category

Tank

ND MA MC FAl FCl FA2 FC2 Total

Experiment I

1 16

2 4

3 21

5 9 12 10 21 7 80

6 14 15 9 25 7 80
8 14 12 10 10 7 82

Totiil 41 19 37 39 29 56 21 242
Experiment 2

1 17 — — — 17 17 19 70

2 17 _ _ _ 16 16 19 68

3 17 — — — 17 17 18 69
Total 5! — — — 50 50 56 207

Damage calegcines in order of increasing severity, are no damage (NDl.
abrasions to the edge of the loot or mantle (MA), cuts to the edge of the
foot or mantle (MC). small abrasions to the foot <10 mm (FAl ). small cuts
to the foot <I0 mm (FCl ), large abrasions to the foot >1() mm (FA2) and
large cuts to the foot >10 mm (FC2).

FC2) were selected at the factory from the commercial catch and
placed in one of three randomly chosen aquaria (Table 1). Indi-
viduals were not disturbed throughout the six weeks of the experi-
ment.

All abalone used in both experiments were initially measured
(i.e.. maximum shell length, maximum shell width, and wet
weight), and a small plastic tag was attached to the shell with glue.
Each experiment lasted for six weeks, and the animals were fed
fresh drift algae as required. The tanks were checked daily for dead
abalone, which were removed immediately. In experiment 1. aba-
lone were disturbed every two weeks to investigate recovery of
their wounds. This disturbance may have affected growth, so in
experiment 2. abalone were not disturbed and weighed at the end
of the experiment to investigate growth. Results from both experi-
inents were combined to investigate mortality.

RESULTS

Frequency of Damage by Commercial Divers

In total. 9.732 abalone from 29 divers on 123 days were ex-
amined for damage. On average. 10.2% (8.9-1 l.O'/r. 95% CD of
abalone retained by commercial divers were damaged. There was
significant variation among divers in the rate of damage to abalone
(nest of highest vs. lowest. P < 0.05). Total rates of damage varied
among divers from 1-17% of abalone retained, with little relation-
ship to the experience of divers (/?" < 0. 1. P > 0.05, Fig. 1). To
some extent, this was cau.sed by large variation in rates of damage
among days for individual divers, with daily rates of total damage
ranging from 1-30% of abalone retained, and an average coeffi-
cient of variation among days of 67%.

Most damage was relatively minor, occurring in 6.0% of aba-
lone retained (5.2-6.9%, 95% CI), but some divers had signifi-
cantly greater rates of major damage than others (; lest of highest
vs. lowest, P < 0.05, Fig. I ). For example, rates of minor damage
varied among divers from 0-13%, and rates of major damage
ranged from 0-12%. Major damage occurred in 4.2% (3.6—1.9%,
95% CI) of abalone retained. Again, there was no relationship
between the rates of different types of damage and the experience

Incidental Damage of Blacklip Abalone

789

Major damage
Minor damage

7 18 I'J :o 21 22 23 24 25 .

Diver (rank)
Decreasing expenence *

Figure 1. Number of abalone \iilh major and minor damage as a
percentage of all abalone sampled in the commercial catch of 29 divers.
Error is 95% CI on total damage.

of divers (both R- <0.\.P> 0.05). MA was the most common type
of damage (3.7%) whereas FA2 ( 1 .8%) was the most common type
of major damage (Fig. 2). Rates of different types of damage
varied greatly among divers and days. For example, rates of FC2
ranged among days from 0-8% and FA2 ranged from 0-16%.
There was little relationship between the rates of different types of
damage among days (all R~ < 0.07. P > 0.05). but rates of damage
were related to the size of abalone. Large abalone were more likely
to be damaged than small ones (Fig. 3). For e.xample. individuals
of 115 mm were predicted to have rates of minor and major dam-
age of 6.9% and 2.4%, whereas individuals of 140 mm were pre-
dicted to have rates of 9.1% and 4.7%. respectively.

Minor damage Major damage

Damage type

Figure 2. Number of abalone with different types of damage as a
percentage (+95% CI) of all abalone sampled in the commercial catch.
Damage categories were abrasions to the edge of the foot or mantle
(MA), cuts to the edge of the fool or mantle (MC). small (<10 mm)
abrasions to the foot (FAII, small (<I0 mm) cuts to the foot (FCI).
large (>10 mm) abrasions to the foot (FA2), large (>lt) mm) cuts to the
foot (FC2), and major breaks to the shell (BS).

Minor damage

Major damage

800

150

1--

"l-n

J

l^m^^r^^

100

110

150

120 IM 140

Lenglh (mm)

Figure 3. Top, number of abalone with major and minor damage as a
percentage of all abalone sampled. Bottom, number of abalone
sampled in each size class from the commercial catch in NSW. Per-
centage damage is shown for those size classes with more than 150
individuals. A generalized logit model was used to fit the solid line for
size classes with more than 150 individuals and a dotted line for those
size classes with less.

The second sampling design provided a significantly greater
estimate (f test, P < 0.05) of the total rate of damage at 14.1%
(12.6-15.8%, 95% CI). This included 0.7% (0.6-0.8%. 95% CI) of
abalone that were dead at landing. Rates of total damage by indi-
vidual divers ranged from 7-34% and were related to the size of
the daily catch {R- = 0.03. P < 0.05). For example, at an average
catch of 140 kg per day, predicted rates of damage were 12%,
which was 4.5%' lower than at a catch of 40 kg per day. Despite
these differences, there was large variation in rates of damage for
all catches, with a standard error of 9.4%'.

On average, 19.1% (17.2-21.1%, 95% CI) of abalone removed
from the reef were replaced. Rates of replacement varied among
divers from 10-31%. with the two most experienced divers having
significantly lower rates than others (i.e., 1 1.0% vs. 23.8%, / test,
P < 0.05). There was large variation in rates of removal and re-
placement for individual divers, with daily rates ranging from
0-34% for the experienced divers and from 3-55% for the inex-
perienced, with an average coefficient of variation among days of
51%.

Recovery, Growth, and Mortality of Damaged Abalone

Recovery of wounds of abalone occurred rapidly, and at a
similar rate among treatments. After 6 wk, there was no visible
wound in 35.8% (30-42.0%. 95% CI) of previously damaged aba-
lone, varying among treatments from 32-54% (Fig. 4). In addition,
most remaining damaged abalone were recovering, so that after 6
wk only 21.3% (15.8-27.2%, 95% CI) of all abalone had failed to
show any visible recovery. Abalone within the ND treatment re-
mained undamaged throughout the experiment.

790

Gibson et al.

Li

Ll

L

Figure 4. Number of abalone with different types of damage in four
categories of wound progression over a 6-\vk period, as a percentage
(+95% CI) of the original number damaged. Damage categories were
abrasions to the edge of the foot or mantle ( MA ), cuts to the edge of the
fool or mantle <MCl, small (<l(l nmil abrasions to the foot (FAl I, small
(<10 mm) cuts to the foot iFCI), large OlO mm) abrasions to the foot
(FA2), and large OlO mm) cuts to the foot (FC2).

Rates of growth of damaged abalone were significantly differ-
ent among treatments (r test, FC2 vs. ND, P < 0.05). Individuals
within the ND treatments increased their weight by 0.8% over the
6 wk of the experiment (Fig. 5). Within all other treatments, the
average rate of growth was negative. Growth of individuals within
the FC2 treatment was significantly less than ND (Fig. 5). The
remaining treatments were not significantly different from ND {t
tests, FA2 vs. ND, FCl vs. ND, P > 0.05), but followed a consis-
tent gradient of greater weight loss with increasing injury.

In experiment 1, rates of moilality of damaged abalone were
significantly different among treatments after 6 wk in aquaria (Fig.
4). FC2 and FA2 caused the highest mortality of 19% (4.8-38.1%.
95% CI) and 12.5% (5.4-21.4%, 95%. CI), respectively. The re-
maining treatments had much lower rates of mortality ranging
from 0-5.1%'. One individual within the ND treatment died (i.e.,
2.4%), and 88% of all the mortality occurred in the first two weeks
of the experiment. In experiment 2, results were similar, with
generally lower rates of mortality. The highest mortality again
occurred for FC2 with 14.3% (5.4-23.2%, 95% CI), whereas mor-
tality in the other treatments ranged from 2-6%. One individual
within the ND treatment died (i.e., 2%), and 79% of the mortality
occurred in the first 2 wk of the experiment. When rates from both
experiments were combined (Fig. 6), FC2 treatments caused the
highest mortality averaging 15.6%' (7.8-24.7%, 95% CI) followed
by 8.5% for FA2 treatments (3.8-14.2%, 95% CI) and FC! with
5.1% (1.3-10.1%, 95% CI). There was significantly greater mor-

3 1

2 -

'

1 ■

(^

0 •

• 1 -

c

op

-2 ■

.

-3 -

-4 -

ND

FC2

FCl FA2

Damage type

Figure 5. Change in weight as a percentage of original weight (+95%
CI) for abalone with different tvpes of damage after 6 weeks in ex-
perimental aquaria. Damage categories are no damage (ND), small
(<I(» mm) cuts to the foot (FCl), large OlO mm) abrasions to the foot
(F'A2), and large (>10 mm) cuts to the foot (FC2).

tality in the major damage treatments than ND ix' = 5.25. 1 df.
P < 0.05). Most of this mortality was attributable to FC2 treat-
ments, which were significantly greater than ND treatments (x" =
9.84. 1 df. P < 0.01 ) with all other comparisons not significant.

DISCUSSION

Frequency of Damage by Commercial Divers

Cominercial divers damage a significant proportion of the aba-
lone they retain in NSW. Most of the wounds are minor and heal
quickly, but over 4% have major injuries that may affect their
growth and survival (Fig. 2). Although rates of major damage were
variable among divers and days, there was little apparent relation-
ship to the experience of divers. Abalone size and size of the daily
catch did explain some of the variation in rates of damage. The

Damage type

Figure 6. Mortality of abalone with different types of damage as a
percentage (+95 '7f CD of the original number damaged after 6 wk in
an experimental aquaria. Damage categories are no damage (ND),
small (<10 mm) cuts to the foot (FCl), large OlO mm) abrasions to the
foot (F.\2). and large OlO mm) cuts to the foot (FC2).

Incidental Damage of Blacklip Abalone

791

rates of damage were greater for larger abalone and small daily
catches, but this effect was small compared with variation among
divers and among days for individual divers. Local wind and swell
conditions are known to affect catch rates (Worthington et al.
1 998) and are likely to influence rates of damage to abalone due to
the difficulty of handling during harvesting and transport in strong
winds and large swells.

The rate of damage in the commercial catch varied among
divers from 1-17%, and averaged 10.2% (Fig. 1). Although the
second sampling design provided a greater rate of damage
(14.1%), it represented only one major processing factory over a
small portion of the year. Rates of damage in NSW were consid-
erably lower than those found for other abalone fisheries. Pirker
(1992) found rates of damage between 44-78% in the commercial
fishery for Haliotis iris in New Zealand, whereas Bruge et al.
(1975) found rates above 90% in the recreational fishery for H.
rufescens in California. Several differences among the fisheries
may explain these differences. For example, in NSW a limited
number of commercial divers use surface-supplied air and blunt
irons to remove abalone from the reef. Perhaps most importantly,
the NSW fishery exports most of the catch live and divers are
encouraged by beach price to minimize the damage to their catch.
Since this study was completed, further improvements in the han-
dling of abalone by commercial divers have been made so that
current rates of damage may be even lower. These include recir-
culating seawater-holding tanks on board fishing vessels, divers
being encouraged by beach price to handle their catch appropri-
ately, and an improved knowledge of handling techniques through-
out the industry.

In contrast to the frequency of damage to retained abalone, the
rate of removal and replacement of undersized abalone was related
to the experience of divers. Experienced divers removed and re-
placed fewer undersized abalone. For a given catch of abalone, this
would reduce the rate of damage caused by their removal. The
lower rate of removal and replacement by experienced divers
could be caused by a variety of factors. For example, more expe-
rienced divers may be able to identify undersized individuals or
areas where there is little need to remove and measure individuals.
Alternatively, less-experienced divers generally have lower catch
rates and may be forced to work in times and places more expe-
rienced divers can avoid. Some populations are dominated by in-
dividuals who are clo.se to legal size (Andrew et al. 1997) and
fishing in these areas would require more measuring. Indeed, in
some areas few, if any, individuals reach legal size (Worthington
et al. 1995). and this can lead to inexperienced divers measuring
large numbers of abalone to identify any individuals above the size
limit. Damage to undersized individuals at these sites could be a
substantial source of mortality (see also Tegner et al. 1989).

Recovery. Growth, and Mortality of Damaged Abalone

Rates of mortality and growth largely depended on the type of
injury sustained. Damage to the foot that results in severing of any
of the major arteries is likely to lead to death (Armstrong et al.
1971 ). Minor injuries can be controlled and repaired with the aid
of muscle contractions (Armstrong et al. 1971 ). Within the aquaria.
major damage resulted in mortality rates of up to 19% and reduced
body weight (Figs. 5 and 6). Mortality rates and growth reduction
may be much greater in the wild because of increased vulnerability
to infection and predators (Bruge et al. 1975, Pirker 1992). For
similar reasons, reductions in reproductive ability, such as reab-

sorption of eggs (observed when abalone are disturbed in aqua-
culture), may also be more intense in the wild.

Within aquaria, most mortality occurred within the first two
weeks. Recovery of damaged abalone was rapid in aquaria, with
most individuals completely recovered or recovering after six
weeks (Fig. 4). These results confirm the usefulness of retaining
damaged abalone within aquaria until their wounds have recovered
and they are appropriate for live export. Most importantly, the
rapid time for mortality or recovery is consistent with potential
tanking times in the industry within NSW.

The estimated rates of damage and mortality can be used to
estimate the total weight of abalone being damaged and killed each
year by the fishery. During 2000, approximately 1 million abalo-
nes were retained and landed by the commercial fishery. With the
estimated rate of removal and replacement of undersized abalone
from this study (i.e., 19.1%), this suggests almost 1.2 million aba-
lone were exposed to potential damage by being removed from the
rock. At the observed rate of damage in the commercial catch (i.e.,
10.2%), almost 120,000 individuals or approximately 40 tons may
be damaged by the commercial fishery each year. Of these dam-
aged abalone, approximately 8 tons are undersized and replaced on
the reef, and the fishery retains 32 tons. Most of the damaged
indi\ iduals probably recover rapidly but, even at the low rates of
mortality likely in aquaria, the fishery may kill approximately 3
tons of abalone each year (i.e.. 0.81% of those removed from the
reef, estimated from rates of damage and mortality). Of this, ap-
proximately 500 kg are undersized abalone that are replaced, sug-
gesting such mortality has a relatively minor impact on the pro-
ductivity of the population. Furthermore, although many-damaged
abalones that are replaced may survive, they are still likely to
suffer complications, including reduced growth and lower repro-
duction.

Damage in the recreational and illegal abalone fisheries in
NSW may also have important effects. For example, illegal fishers
do not observe the minimum length limit and kill many undersized
abalone. Many recreational fishers also handle and retain under-
sized abalone (unpublished data) with the potential for high rates
of damage because they are not permitted to use scuba and gen-
erally have little experience removing abalone. Recreational divers
often use unconventional tools to remove abalone, which may
cause high rates of damage. During 1997, the recreational catch
was estimated at 52 tons (unpublished data), when the commercial
catch was over 330 tons. As a result, total rates of damage may be
similar between the two sectors.

Early in the history of the commercial fishery for abalone in
NSW there was no minimum legal size. In 1972, a minimum legal
size was introduced at 100 mm, but most abalone were well above
this size and almost all individuals removed from the rock were
retained. With the progression of size limit upwards to the current
115 mm minimum legal size, and the decline in the size-structure
of the population, more removal, measurement, and replacement of
under-size abalone has occurred. Current management measures,
such as temporal closures, attempt to minimize the disturbance and
damage to abalone, particularly during reproductive season. Beach
price incentives have also been developed by the industry to en-
courage the landing of undamaged individuals and to create an
awareness of the damage to the resource caused by injuring aba-
lone that are not retained. Incidental damage of undersized indi-
viduals in the NSW abalone fishery is small when compared with
some other abalone fisheries, but could still be a significant source

792

Gibson et al.

of mortality. This i^ obviously iindesirahle in an already hea\ily
exploited fishery.

ACKNOWLEDGMENTS

The authors thank Rowan Chick. Penny Brett. Nokome Bent-
ley. Greg Otton. Ross Werner. John Vairy. and the divers who

participated with the logbook for their ongoing assistance. We also
thank Southern Ocean Seafoods. NSW Abalone. and Australian
Shellfish Enterprises for providing us with access to their factories
and their abalone. We also thank Steve Kennelly and Doug Ferrell
for reviewing the manuscript. The commercial abalone divers of
NSW provided funding for this work.

LITERATURE CITED

Andrew. N. L.. D. G. Wurthington & P. A. Brett. 1997. Size-structure and
growth of individuals suggest high exploitation rates in the fishery for
blacklip abalone. Hulious nihra in New South Wales. Australia. MoU.
Res. 18:27.S-2S7.

Armstrong, D. A.. J. L. Armstrong. S. M. Krassner & G. B. Pauley. 1971.
Experimental wound repair in the black abalone. Huliotis cracheiddii.
./. Invert. Path. 17:216-227.

Blount, C. & D. G. Worthington. 2001. Identifynig nidividuals with high
quality roe for the sea urchm Centrosteplninus rodgersii. in NSW.
Australia. Fish. Res.

Broadhurst. M. 2000. Modifications to reduce bycalch in prawn trawls: a
review and framework tor development. Rev. Fish Biol. Fislieries 10:
27-60.

Bruge. R.. S. Schultz & M. Odemar. 197.'i. Draft report on recent abalone
re.search in California with recommendations for management. Califor-
nia: Resources Agency. Department of Fish and Game. 48 pp.

McShane. P. E.. M. G. Smith & K. H. H. Beinssen. 1986, Growth and
morphomeu-y in abalone {Huliotis rubra Leach) Irom Victoria. Aus. ./.
Mar. Freslm-at. Res. .^9:161-166.

Pirker. J. G. 1992. Growth, shell-ring deposition and mortality of Paua
(Haliotis iris Martyn) in the Kaikoura region. Masters thesis. Univer-
sity of Canterbury. New Zealand.

Richards. L. J.. J. T. Schnute & J. Fargo. 1994. Application of a general-
ized logit model to condition data for trawl-caught Pacific halibut.
Hippoglossiis steiiolepi.s. Can. J. Fish. Aqiiat. Sci. ."i 1:357-364.

Tegner. M. J.. P. A. Breen & C. E. Lennert. 1989. Population biology of
red abalones. Haliotis rufescens. in southern California and manage-
ment of the red and pink. Haliotis corrugata, abalone fisheries. US
Fish. Bull. 87:31.3-339.

Worthington. D. G.. N. L. Andrew & N. Bentley. 1998. Improved indices
of catch rate in the fishery for blacklip abalone, Haliotis rubra, in
NSW, Australia. Fish. Res. 36:87-97.

Worthington. D. G.. N. L. Andrew & G. Hamer. 1995. Covariation be-
tween growth and morphology suggests alternative size limits for the
blacklip abalone. Haliotis rubra, in NSW. .Australia. US Fish. Bull.
93:551-561.

Joiirihil ofSln-llthh Research. Vol. 21. No. 2, 793-798. 2002.

THE EFFECT OF NONNUTRITIVE FILLERS ON THE DIGESTIBILITY OF A
MANUFACTURED ABALONE DIET

MEEGAN E. VANDEPEER,'* PATRICK W. HONE.^ JON N. HAVENHAND,' AND
ROBERT J. VAN BARNEVELD^

'South Australian Research and Development Institute. PO Bo.x 120. Henley Beach. South Australia
5022: 'Fisheries Research and Development Corporation. PO Box 222. Deukin West. Australian Capital
Territory 2600: ^Flinders University. GPO Box 2100. Adelaide. South Australia 5001: ^Barneveld
Nutrition Pty. Ltd.. 19-27 Coonan Rd. South Maclean. Queensland. Australia 4280

ABSTRACT In this study the effects on nutrient digestibility of adding nonnutntive fillers (kaolin, bentonite. sand and diatomaceous
earth) to a research diet for greenlip abalone. Haliolis laevigata Donovan, were investigated. The addition of kaolin significantly
improved the gross energy digestibility of the diet with the greatest improvement in gross energy digestibility occurring at the highest
level of inclusion investigated (20%). Possible explanations for the positive effect of kaolin on nutrient digestion in abalone include
assisting with the breakdown of diets through an abrasive/grinding effect, increasing gut transit time, decreasing digesta viscosity or
through supplementation of minerals that may be deficient.

KEY WORDS: abalone. digestibility, kaolin, bentonite. sand, diatomaceous earth

INTRODUCTION

Silicates, more commonly known as bentonite, kaolin, zeolites
and other clays, have been used in poultry and swine nutrition for
many years (Dias et al. 1998). The reason for their inclusion is due
to their binding and lubricating property in the production of pel-
leted feeds. There is also suggestion that some clay products may
have direct beneficial effects on animal performance, however,
results from studies aimed at establishing this have produced
mixed results. Experiments with pigs, chickens and fish have
shown responses ranging from increases in growth, feed efficiency
and nutrient utilization (Kurnick & Reid 1960, Onagi 1966, Kondo
& Wagai 1968, Quinsberry 1968, Han et al. 1975, Han et al. 1976,
Smith et al. 1980) to negative or no responses (Reinitz 1983,
Reinitz 1984. Dias et al. 1998).

Because kaolin contributes no protein or energy, it is regularly
used as a tiller in research diets for abalone at the South Australian
Research and Development Institute's Aquatic Sciences Center.
Since the addition of silicates, including kaolin, have been reported
to improve nutrient utilization in other animals, it is important that
its effects on nutrient digestion in abalone be investigated. The aim
of this study is to assess whether four, nonnutritive, fillers (beach
sand, diatomaceous earth, kaolin and sodium bentonite) affect the
digestibility of a research diet for greenlip abalone, Haliotis lae-
vigata.

MATERIALS AND METHODS

Diets

The basal diet used in all experiments was developed by the
South Australian Research and Development Institute's Aquatic
Sciences Center. The exact diet formulation is confidential; how-
ever, semolina constitutes the largest percentage of the diet fol-
lowed by soy flour, which is the main source of protein. Chromic
oxide was included in the diets at 0.5*7? by dry weight at the
expense of semolina, as an indigestible marker for use in calcu-
lating the apparent protein and energy digestibility. All diets were
initially hand mixed and then mixed in a spiral action dough mixer

*Corresponding author: E-mail: vandepeer.meegan@saugov.sa.gov.au

( 'Impastrice', Hill Equipment and Refrigeration. Adelaide. South
Australia). The mixture was then fed through a commercial pasta
machine (La Prestigiosa mediuin. IPA. Vicenza, Italy) where it
was made into 300 mm long strips using a die with slots 18 x 1.5
mm. The strips were dried on mesh trays overnight in a forced
draft oven at 55°C.

Experiment I — The Effect of Different \onnulriti\e Fillers on the
Protein and Energy Digestibility of a Manufactured .Abalone Diet

Six diets were fomiulated in total, including the basal diet
(Table 1). Four of the diets consisted of the basal diet with 5% of
the semolina substituted for one of the following nonnutritive fill-
ers: kaolin, bentonite. diatomaceous earth, and beach sand. The
fifth diet contained 5% of pre-gelatinized waxy maize starch
(Wades BOllC, Goodman Fielder Mills. NSW. Australia). The
beach sand was obtained from West Beach. South Australia and
was autoclaved at 121°C for 20 min before inclusion in the diet.
The size of the sand grains ranged from around 192-346 fjim. Each
dietary treatment was randomly allocated to one of 18 digestibility
tanks to provide three replicate fecal samples per diet.

Experiment 2 — The Effect of Kaolin Inclusion Level on the Protein
and Energy Digeslihilily of a Manufactured Abalone Diet

Six diets were formulated in total including the basal diet and
the basal diet with 5% pre-gelatinized waxy maize as in experi-
ment 1 (Table 2). The other four diets contained increasing levels
of kaolin (5%. 107r. 15%. and 20%) again at the expense of
semolina. The six diets were randomly allocated to one of 18
digestibility tanks to provide three replicate fecal samples per diet.

Abalone and Feeding

Juvenile greenlip abalone (shell length 40-60 mm) were used
in the experiments. The abalone had been obtained from a com-
mercial hatchery and raised on manufactured abalone feed. The
abalone were preconditioned for one week on the test diet assigned
to their tank. During both the pre-conditioning and experiment
periods the animals were fed to excess every day at approximately
1700 h.

793

794

Vandepef.r et al.

TABLE 1.
Composition, proximate analysis and gross energy content (M,I/kg) of experimental diets from experiment 1 (g/kg, air dry basis).

Basal

SC,

5<7r

5%

5%

5%

Diet

Starch

Bentonite

D. Earth*

Sand

Kaolin

Basal diel

995.0

945.0

945.0

945.0

945.0

945.0

Starch

0

50.0

0

0

0

0

Bentonite

0

0

50.0

0

0

0

Diat(.)maceous earth

0

0

0

50.0

0

0

Sand

0

0

0

0

50.0

0

Kaolin

0

0

0

0

0

50.0

Chromic oxide

5.0

5.0

5.0

5.0

5.0

5.0

Gross energy (MJ/kg)

17.79

17.77

17.03

16.93

16.98

16.90

Crude protein (N x 6.25)

311.9

311.9

312.5

312.8

3 1 1 .9

311.3

Dry matter

919.2

916.4

923,4

927. S

926.3

922.4

* diatomaceoiis earth.

Tanks and Collection System

Conical shaped digestibility tanks were used. Abalone were
housed in 20-L buckets (approximately 80-100 per bucket) that
fitted inside the tanks. All the buckets were fitted with plastic mesh
bottoms (1.3 cm x 1.3 cm mesh) allowing containment of the
abalone while permitting feces to drop into the collection tube at
the base of the tank. Three 25 cm lengths of PVC pipe (8 cm in
diameter) were placed in the buckets as shelters for the abalone.
Attached to the bottom of each digestibility tank was a screw-on
collection tube (1 i cm long. 1.5 mm diameter). Tanks were on a
flow-through water system at a rate of about 2 L/min. The seawater
was filtered to 30 jj-rn by primary sand filters, then to 10 p.m by
secondary composite sand filters before entering the tanks. Aera-
tion was supplied at 0.5 L/min to each tank at all times by an air
stone. Water temperature and lighting were controlled during the
experiment with temperature maintained at 18.0°C ± 1.0 and a
light regimen of 12 h light; 12 h dark. Salinity was 35-36%f
throughout the experiment.

Fecal Collection

Feces were collected by settlement every day until 5-6 g of
feces (dry weight) was collected for each replicate sample. This
took approximately 2 wk for each replicate. On each day of fecal
collection the buckets containing the abalone were removed and
the digestibility tanks were drained of water and all fittings were
cleaned of feces and uneaten feed. Following cleaning, the tanks
were refilled and the buckets replaced. Collection tubes were fitted
by 0900 h. A small foam container was placed underneath each
tube and filled with ice to keep the collecting feces cold and thus
reduce its degradation by microbes. The feces were collected from
the lubes at about 1630 h by gently pouring the contents onto a 1
mm diameter mesh. The mesh was then placed into a petri dish and
frozen at -30°C. The following day the frozen fecal sample was
scraped off the mesh, pooled into a composite sample, and stored
in the freezer until required for analysis. Prior to analysis the
samples were freeze-dried and ground with a mortar and pestle.

Chemical Analyses

Dry matter was determined by drying samples at IOO°C over-
night until a constant weight was achieved. Gross energy was
determined using a Parr 1281 bomb calorimeter. Crude protein was
determined bv the combustion method using a LECO® CN-2000

Carbon and Nitrogen Analyser (Royal Australian Chemical Insti-
tute 1999). Chromic oxide was determined using atomic absorp-
tion spectroscopy based on a modification of the methods de-
scribed by Hillebrand et al. (1953). The modified methodology
involved preliminary ignition of the sample at 500"C to remove
organic material and the dissolution of the sample in hydrochloric
acid instead of sulphuric acid (M. Frith, personal communication.
University of Tastnania, Launceston, Australia).

Digestibility Determination

The apparent digestibilities of nutrients in the diets were cal-
culated using the following formula (Hardy 1997):

Apparent digestibility :

C/y,.^.,,, X Nutrient j,^.,

where Cr is chromium content and Nutrient is nutrient or energy
content of the diet.

Statistical Analysis

The data were analyzed by use of general linear model and the
treatment means were compared by least significant difference.
Prior to analysis, data were analyzed using a univariate procedure
and normal plots to establish that the data were in fact normally
distributed, which was the case. The presence of outliers was as-
sessed using the RANK procedure in SAS (SAS Institute Inc..
1988) and normal scores computed from the ranks following Blom
(1958). As no outliers were detected using the RANK procedure,
all data were used in the calculation of digestibility estimates for
all parameters, respectively.

RESULTS

Experiment 1—Tlie Effect of Different Nonnutritive Fillers on the
Protein and Energy Digestibility of a Manufactured Abalone Diet

Although very close, no significant difference was found
among the six diets in their apparent protein digestibility for aba-
lone at the 0.05 significance level (F^,,,, = 2.76: P = 0.08()4i
(Table 3). However, the diets were significantly different at the
0.10 significances level. This was entirely due to the 57r bentonite
diet having significantly lower apparent protein digestibility than
all the other diets. A significant difference was observed in the
apparent dry matter digestibility (F5 ,o = 6.93; P = 0.0048) and

The Effect of Fillers in Abalone Diets

795

TABLE 2.
Composition, proximate analysis and gross energy content (MJ/kg) of experimental diets from experiment 2 (g/kg, air dry basis)

Basal
Diet

5%
Starch

5%
Kaolin

10%
Kaolin

15%
Kaolin

20%
Kaolin

Basal diet

Starch

Kaolin

Chromic oxide

Gross energy (MJ/kg)

Crude protein (N x 6.25)

Dry matter

995.0

945.0

945.0

895.0

845.0

695.0

0

50.0

0

0

0

0

0

0

50.0

100.0

150.0

200.0

5.0

5.0

5.0

5.0

5.0

5.0

17.65

17.74

16.87

15.97

15.11

14.21

.^15..^

308. 1

310.3

302.2

297.2

291.3

914.7

920.6

924.9

928.0

9.16.7

940.7

apparent gross energy digestibility of the diets (F^ m = 3.29. P =
0.0516) (Table 3). The diet containing 5% starch had significantly
higher apparent dry matter digestibihty than all the other diets
(Table 3). No significant differences in dry matter digestibility
occurred among the diets with fillers and the control diet (Table 3).
The diet with 5% starch had significantly higher apparent gross
energy digestibility than all the other diets, excluding the kaolin
diet (Table 3). There was no significant difference in apparent
gross energy digestibility among the control, bentonite. diatoma-
ceous earth and sand diets. Similarly, there was no significant
difference in apparent gross energy digestibility among the kaolin,
bentonite. diatomaceous earth and sand diets, but the kaolin diet
did have significantly higher apparent gross energy digestibility
than the control diet (Table 3).

Experiment 2 — The Effect of Kaolin Inclusion Level on the Protein
and Energy Digestibility of a Manufactured Abalone Diet

The digestibility coefficients for the control diet and that with
5% starch were comparable with those from experiment 1 (Table
3 and Table 4). No significant difference in protein digestibility
occurred among the six diets in experiment 2 (F, ,„ = 1.41. P =
0.3017) but there were significant differences among the diets in
apparent gross energy (Fs j,, = 18.99. P = 0.0001 ) and dry matter

digestibility (F5

8.56. P = 0.0022) (Table 4). The diet with

5% starch had significantly higher apparent dry matter digestibility
than all the other diets (Table 4). The diet with 10% kaolin had

significantly higher dry matter digestibility than the control diet,
but not the other kaolin diets. Apparent gross energy digestibility
significantly increased with increasing level of kaolin in the diet
(Table 4). The diet with 20% kaolin had significantly higher gross
energy digestibility than all other diets (Table 4).

DISCUSSION

Substitution of semolina with kaolin in a manufactured diet for
juvenile greenlip abalone increased the apparent digestibility of its
gross energy. The gross energy digestibility of the manufactured
diet significantly increased with increasing inclusion level of ka-
olin (Table 4). Unlike kaolin, the addition of other inert fillers
(sand, bentonite and diatomaceous earth) did not significantly im-
prove abalone's apparent gross energy digestibility of the diet
when included at 5% (Table 3). It is possible that these fillers may
improve the apparent gross energy digestibility if included in diets
at higher levels.

The improved digestibility/growth rates observed in other ani-
mals fed diets supplemented with inert fillers such as kaolin, may
have resulted, singly or in combination, from their assisting with
the breakdown of diets through an abrasive/grinding effect, in-
creasing gut transit time and through supplementation of minerals
that may be deficient. Any of these mechanisms could also explain
why kaolin significantly improved the gross energy digestibility of
a commercial diet for juvenile abalone. Two other possible mecha-

TABLE 3.
Apparent fecal digestibility coefTicients of protein, gross energy and dry matter of the 6 diets from experiment 1.

Statistics

Basal Diet

5% Starch

5<7f Bentonite

5% D. Eartht

5% Sand

5% Kaolin

SEM

0.708 ±0.01069

0.665 ±0.01287

0.699 ± 0.00543

0.703+11.00742

0,713 ±0.01 144

Prolein 0.717 ±0 01303
Gross

energy 0.490^ ± 0.00589 0.560" ± 0.00841 0.5091^ ± 0.02258 OSOI*^ ± 0.00945 0.515'^ ±0.00665 0.540"" ± 0.00901

DMD 0.362" ± 0.00840 0.434" ± 0.03165 0.331' ± 0.fW935 0.327-' ± 0.01 303 0.34S' ± 0,01041

NS

0.370" ±0.00614

0.01137

0.01435 0.0452
0.01479 0.0466

* P = 0.05.

**P< 0.001.

t diatomaceous earth.

NS, nol significant (P > 0.05).

SEM, standard error of the mean.

DMD, dry matter digestibility.

L.SD. least significant difference.

Diets in a row with different superscripts differ significantly (t,, 05, ,,

Data are mean ± SE, n = 3.

2.23).

796

Vandepeer et al.

TABLE 4.
Apparent fecal digestibility coefficients of protein, gross energy and dry matter of the 6 diets from experiment 2.

Basal
Ditl

5% Stercli

Protein

0.687 ± 0.00542

0.687 ± 0.00846

Gross energy

0.455'' ± 0.00897

0.549'^ ± 0.007484

DMD

(I..U7' ±0.0I.'!20

0.426° ±0.005 15

5% Kaolin

10% Kaolin

ISri Kaolin

20 "^f Kaolin

SEM

LSD

0.685 ±0.00612
0.505' ± 0.02084
0.362'"-± 0.01828

0.696 ± 0.00495
0.553'' ±0.01 155
0..376''± 11,01 148

0.688 ±0.0071.?
0.55 1 "±0.00788
O.UI'" ±0.01225

0.707 ± 0.00532
0.605^ ±0.01 1 12
11.358'"' ±0,01002

NS

0.00692

0.01164 0.0367

0111169 0,0369

**/><O.OI.

*** p < 0.001.

NS. not significant IP > 0.05).

SEM. standard error of the mean.

DMD. dry matter digestibility,

LSD. least significant difference.

Diets in a row with different superscripts differ significantly (Im,,,!

Data are mean ± SE.

2.23).

nisms are its reducing digesta viscosity and osmotic pressure in the
lumen.

Rediulioii in Osmotic Pressure in the Lumen

The increase in apparent gross energy digestibility of diets with
the increased level of kaolin could be related to the unusual oli-
gosaccharides in soyflour, the second largest component of the diet
in this study. Pedersen (1989) suggested that the post-weaning
diarrhea found in piglets fed soy-based diets might not be due
solely to an allergic reaction elicited by soy antigens, but to the
soy's oligosaccharides. The accumulation of these components in
the intestine not only provides a substrate for the growth of unde-
sirable bacteria, but also could lead to an "osmotic" diarrhea that
occurs when body fluid is drawn into the lumen of the gut to
counter a raised osmotic pressure (Pedersen 19S9). Soyflour may
possibly produce the same response in the gut of abalone. The
positive effect kaolin had might be related to its ability to coun-
teract this osmotic diarrhea caused by soyflour and thus increase
the digestibility of dietary nutrients. Evidence for this is that the
addition of zeolites (alumino-silicates) to piglet diets has been
found to reduce diarrhea. Inclusion of zeolite to the diet of piglets
severely affected with scours (prolonged dianhea in livestock)
markedly reversed the progress of this disease within a few days
(Morita 1967). Similarly, the incidence of scours in young swine
fed a diet supplemented with 5% clinoptilolite was significantly
reduced (England 1975). Zeolites are characterized by their ability
to lose and gain water reversibly and by a very effective ion
exchange capacity (Mumpton & Fishman 1977. White & Ohlrogge
1974). It is these properties of zeolites (ion exchange and revers-
ible binding of water) that could have reduced diarrhea in piglets
when added to their diet. Kaolin, also an alumino-silicate, could
have had the same effect in the gut of abalone resulting in the
increased gross energy digestibility of the diet.

Abrasive or Grinding Effect

Kaolin may perform a siinikir function to sand and grit that
abalone ingest while feeding on their natural diet, assisting in
grinding up and breakdown of food. Insoluble grit given to chick-
ens remains in their gi/zard and assists in grinding, crushing and
breaking up food particles (Bruce 1976). Sill is believed to have a
similar effect in the style sac of mussels (Murken 1976). Sand and
grit, which may have a grinding effect in the gut. is taken in by
wild abalone when they feed on their natural diet algae. In the

wild, abalone consume algae by either catching it as it drifts past
or by grazing it off rocks. When they graze rocks, they also take in
sand and other sediments. The fine sand and grit can be seen in
their intestine when dissected. A study on the composition tif food
in the stomachs of wild H. laevigata. H. rubra and H. nwi (Shep-
herd 1973) revealed algae, and other browsed matter, including
sand grains, small gastropods, bryozoa and detrital matter. The
stomachs of H. laevigata and H. scalaris also contained quartz
sand (Shepherd & Cannon 1988). Kaolin contains approximately
46.3% silica (Reis 1908). Like sand, silica may have an abrasive/
grinding action in the gut of abalone. However, the mechanism
that kaolin improves digestion in abalone through its grinding
effect in the abalone's gizzard implies that the addition of sand,
bentonite and diatoinaceous earth would be expected to increase
their digestibility. Therefore, it does not seem likely that a grinding
effect of kaolin caused the increase in digestibility observed in this
present study.

Supplementation of Minerals

It is possible kaolin is not nonnutritive but is actually supplying
essential minerals that abalone require but that are deficient in
manufactured diets. Deshimaru and Kuroki (1974), using a semi-
purified diet, found that mineral rich diets (as high as 19.5% ash)
produced the best growth of Penaeiis japoniciis. Similarly,
Wheeler and Oltjen (1977) reported faster daily weight gains, feed
conversion efficiency and carcass quality in finishing steers fed a
corn and hay ration cttntaining 8% protein and 3.5% cement kiln
dust to replace trace minerals, than in control animals receiving an
otherwise similar diet containing 1 2% protein plus trace minerals.
They speculated that the cement kiln dust may have provided
hitherto unknown but necessary trace minerals. Also, increasing
levels of bentonite in a high roughage ration for steers reduced the
retention of dietary calcium but increased the retention of phos-
phorous (Martin et al. 1969). Thus kaolin may have provided
minerals essential for certain enzyme systems and biochemical
functions involved in digestion in abalone but lacking in the diet
used in this study.

Alteration of Gut Transport Time

It is possible that kaolin increases digestion in abalone by al-
tering gut transport lime. Increased gut transport time can increase
digestibility, because the food is exposed for a longer time to
digestive enzymes. The improved caloric efficiency by the addi-

The Effect of Fillers in Abalone Diets

797

tion of kaolin to poultry diets as observed by Osterhaut (1967), was
due possibly to a slowing down of the rate of feed passage through
the intestinal tract (Quinsberry 1968). Kurniek and Reid (1960), in
their study of the effect of the addition of bentonite to 3 diets
differing in energy levels for Leghorn cockerels, observed a sig-
nificant increase in growth rate with the feeding of 2.5% bentonite
to the low energy level diet. For each diet, the feeding of bentonite
in the diet slightly delayed the passage of feed through the diges-
tive tract. Similarly, by testing the effect of inclusion of silica,
cellulose and chabamin at 10% and 20% in diets for European
seabass juveniles Dias et al. (1998) observed that in fish fed the
control diet, the totality of the feces was expelled after 31 h.
whereas in those fed with 20% bulk incorporated diets, fecal eges-
tion continued over 35 h. By contrast, however. Grove et al. ( 1978)
reported that the gastric evacuation time in rainbow trout was
reduced from 15 h to 10 h when the energy content of the feed
pellet was reduced by 50% by dilution with kaolin.

In the wild, herbivores, such as abalone, eat macrophytes,
which are low in digestibility and possess high levels of indigest-
ible materials that pass rapidly thi'ough the gut (Wee 1992). Their
relatively long guts increase retention time and allow enzymes to
work on the ingested material to optimize nutrient extraction and
absorption. Wild abalone take appro.ximately 24 h to digest pre-
ferred species of algae and species less digestible may remain
identifiable in abalone guts for more than 48 h (Foale & Day 1992.
Day & Cook 1995). The addition of indigestible kaolin to the diet
may have affected transit time in a manner similar to that of poorly
digestible algal species that remain in the gut for a longer period of
time, thereby increasing exposure to digestive enzymes. The di-
gesta transit times of the diets in this study were not measured and
thus cannot be compared, however, an increase in transit time
caused by the addition of kaolin can not be ruled out as a possible
mechanism for the increase in energy digestibility of the diet.

Reduction in Diet Viscosity

Kaolin may have increased the gross energy digestibility of the
commercial diet by reducing its viscosity and hence increasing
access to it by digestive enzymes. The basal diet used in this study
consisted mainly of semolina and soyflour. It is well known, par-
ticularly for poultry, that soluble polysaccharide (fiber), such as
arabinoxylans in wheat (semolina) and (J-glucans in barley and
oats elicit negative effects on digestion through increasing intes-

tinal viscosity (Annison 1990. Bedford et al. 1991, Choct & An-
nison 1992, Annison 1993, Choct et al. 1996, Dusel et al. 1997).
High gut viscosity decreases the rate of diffusion of substrates and
digestive enzymes and hinders their effective interaction at the
mucosal surface (Choct 1997). The viscous polysaccharides prob-
ably complex directly with digestive enzymes and reduce their
activity (Ikeda & Kusano 1983). As with poultry, non-starch
polysaccharides in wheat may also elicit negative effects on di-
gestion in abalone through increasing intestinal viscosity. There-
fore, the significant improvement in digestion by the addition of
kaolin may be because it decreases intestinal viscosity.

If kaolin did increase the digestibility in abalone by decreasing
the diet's viscosity, the fact that other fillers used (bentonite. di-
atomaceous earth and sand) had no effect on its digestibility may
be due to their physical/chemical properties. The contradictory
results of the effects of nonnutritive fillers incorporation on feed
evacuation time in fish are. in some cases, related to the different
physico-chemical properties of the various bulk agents tested (Dias
et al. 1998). The properties, such as ion binding or water holding
capacity, have a strong influence on solubility, gelling and viscos-
ity of food during its passage through the intestinal tract. The
phenomenon of gelation caused by polysaccharides is because they
are hydrophylic molecules and consequently have the ability to
hold water. Kaolin is hydrophilic. disperses in water readily, is
non-expanding, has low viscosity, low sorptivity, and a low sur-
face charge (Murray 1993). Thus, it may reduce viscosity caused
by soluble polysaccharides in diets by binding with water and
reducing the polysaccharide's gelation ability. The same did not
occur with the other fillers and this may be due to their different
chemical properties. For example, bentonite, albeit hydrophilic,
has high viscosity, high sorptivity, is expanding, and has a high
surface charge (Murray 1993). The effect of nonnutritive fillers
seems related to their physico-chemical properties and properties
of other ingredients in the diet. Thus, the positive effect of kaolin
on digestibility may have been related to the basal ingredients in
the diets and it may not have a positive effect on diets with dif-
ferent basal ingredients.

ACKNOWLEDGMENTS

The authors are grateful to Murray Frith for chromic oxide
analysis and Debra Partington for help with .statistical analysis.
This research was supported by financial grants from the Fisheries
Research and Development Corporation.

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Jimnwl of Shellfish Research. Vol. 21, No. 2, 799-803. 2002.

THE DIGESTIBILITY OF WHOLE AND DEHULLED LUPINS (LUPINUS ANGUSTIFOUUS) FED
TO JUVENILE GREENLIP ABALONE, HAUOTIS LAEVIGATA

MEEGAN E. VANDEPEER,' PATRICK W. HONE," JON N. HAVENHAND,^ AND
ROBERT J. VAN BARNEVELD^

^South Australian Research and Development Institute. PO Box 120. Henley Beach. South Australia
5022: -Fisheries Research and Development Corporati(m. PO Box 222. Deakiii West. Australian Capital
Territoiy 2600: -Flinders University. GPO Box 2100. Adelaide. South Australia 5001: ^Barneveld
Nutrition Ptv. Ltd.. 19-27 Coonaii Road. South Maclean. Queensland. Australia 4280

ABSTRACT The apparent digestihility of protein, amino acids, and gross energy from whole and dehulled lupms {l.iipimis an^iis-
lifoliiis) and defatted soyflour was investigated for greenhp ahalone, Haliotis laevificiia Donovan. Protein digestibility of all three
feedstuffs was high with a coefficient of 0.91 obtained for soytloiir and whole lupins and a coefficient of 0.92 obtained for dehulled
lupins. Gross energy digestibility was much more variable with the energy from soyflour being significantly more digestible (coef-
ficient of 0.87) than that from dehulled lupins (coefficient of 0.83). which was significantly more digestible than the energy from whole
lupins (coefficient of 0.50). The significantly lower energy digestibility of the whole lupins compared with the dehulled lupins could
possibly be due to abalone's poor ability to digest cellulose, which is the major component of the hull of L aiif;iislifi>liiis.

KEY WORDS: ahalone, digestibility, lupins. Luphiiis antiit.sufnliiis. Halioiis liicvigiilii

INTRODUCTION

hull. To investigate this the digestibility of both whole and de-
hulled L aiif^iistifoliiis for abalone was determined.

In Australia, lupins, in particular the species Lupinus angusti-
foliiis. are used as a source of dietary protein and energy for both
pigs and poultry. L. angustifdlius has protein levels ranging from
27 1 .9 to 372.3 g/kg and a gross energy content of 1 7.9-1 8.6 MJ/kg
(Petterson et al. 1997). Lupins are an attractive feedstuff for use in
animal diets, as selective breeding programs have produced vari-
eties of lupins with low concentrations of anti-nutritive factors
such as alkaloids (Annison et al. 1996).

Successful results have been obtained on incorporation of L.
angustifolius in manufactured diets for a range of aquaculture spe-
cies including juvenile prawns, silver perch, snapper, carp, gilt-
head seabream and rainbow trout (Jenkins et al. 1994. Viola 1988.
Robaina et al. 1995, Gomes et al. 1995, Smith et al. 1998, Allan et
al. 1998). Jenkins et al. ( 1994) found no significant differences in
growth rates of snapper when fed balanced diets containing either
20% soybean meal, 15% L. angustifolius seed meal plus 13%
soybean or 28% L. angustifolius seed meal. Similarly, excellent
results with L. angustifolius have been found for carp. Growth of
carp fed a diet containing 30% lupins exceeded that of a control
diet by 25% while carp fed a diet containing 45% lupins grew at
the same rate as those on the control diet (Viola et al. 1988).
Robaina et al. (1995) found no significant difference in weight
gain, protein efficiency ratio and feed efficiency between gilthead
seabream (Sparus aurata) fed a fishmeal based control diet and a
control diet with 30% of the fishmeal protein replaced by L an-
gustifolius as a source of protein. Positive results have also been
reported for prawns with no significant differences in daily growth
rate, feed intake, feed conversion rate, protein conversion effi-
ciency or apparent protein utilization of P. monodon when fed
diets containing either dehulled L. angustifolius or deffated soy-
bean meal as the main source of protein (Sudaryono et al. 1999).

This study was undertaken to determine the apparent digest-
ibility of protein, amino acids and gross energy from the lupin,
Lupinus angustifolius, for greenlip abalone, Haliotis laevigata. A
potential problem that could affect abalone's ability to digest en-
ergy from L. angustifolius is the large proportion of cellulose in its

MATERIALS AND METHODS

Diets

Two lupin diets, one containing whole, and the other containing
dehulled L. angustifolius. were formulated (Table 1). A diet con-
taining defatted soyflour was formulated as a control diet for com-
parative purposes ("Bakers Nutrisoy" brand; Archer Daniels Mid-
land Company, Decatur, Illinois). Each feedstuff was the sole
source of protein in the diet and each diet was formulated to have
a crude protein content of 160 g/kg. The lupins were ground in a
hammer mill and then in a ball mill before inclusion in the diets.
Equal amounts of vitamins and ininerals. as described by Uki et al.
(1985). Jack Mackerel oil (Triabunna Fish Oils, Triabunna, Tas-
mania), and pre-gelatinized maize starch were added to each diet.
Kaolin was included as filler and chromic oxide was included as an
indigestible marker (0.5%) for use in digestibility calculations.
Shipton and Britz (2001 ) found that chrotnic oxide was a suitable
marker in protein digestibility studies on Haliotis midae as it was
inert, was not absorbed, did not interfere with the digestive pro-
cess, and moved through the intestine at a similar rate to the
protein. Proximate analyses of the experimental diets and of each
feedstuff were determined (Table 2 and Table 3).

Diet Allocations

The three diets were randomly allocated to 4 of 12 digestibility
tanks to provide four replicate fecal samples per diet based on a
completely randomized design.

.Xhalnne Feeding and Feeal Collection

Abalone and Feeding

Juvenile greenlip abalone (shell length 40-60 mm, 70 g wet
weight) were used in the experiment. The abalone had been ob-
tained from a commercial hatchery and raised on manufactured
abalone feed. The abalone were preconditioned for one week on

799

800

Vandepeer et al.

TABLE L
Composition of experimental diets (g/kg, air dry basis).

TABLE 2.

Proximate analysis, total amino acid content (g/k;;. air dry basis),
and gross energy (M.j/kg, air dry basis! of experimental diets.

Whole Dehulled
Lupin Lupin

500.0

421.1
100.0 100.0
369.4 448.4

Soyflour

.Soytlour' 33.S.4

Lupin (whole)"

Lupin (dehulled)-

Pre-gelatinised starch' 100.0

Kaolm .';34.0

Diet

Soyflour
Diet

Whole
Lupin Diet

Dehulled
Lupin Diet

Crude protein (N x 6.25)
Gross energy (MJ/kg)
Dry matter
Ash

167.5

8.28
968.1
539.4

169.8

9.88
950.0
373.8

155.0

10.45
974.6
462.2

Jack Mackerel oil 20.0
Mineral premix"* 2.0
Vitamin preniix'' 3.0
Ascorbic acid 0.5
DL- -tocopheryl acetate 0. 1

20.0 20.0
2.0 2.0
3.0 3.0
0.5 0.5
0.1 0,1

Fiber extract
Crude

Acid detergent
Neutral detergent
Ether extract (bp. 40-60°C)

25.0
427.6
719.3

12.4

34.3
270.0
46').0

38.1

55.3
387.8
618.6

38.6

Chromic oxide 5.0

5.0 5(1

Anuno acids
Aspartic acid
Glutamic acid
Serine
Glycine

11.60

26.10

7.70

6.75

11.50

32.50

8.50

7.00

9.30

23.40

6.60

6.05

' Defatted soyflour (Baker's Nutrisoy; ADM).

" Liipiiuis cingiistifolius cv. Gungunu.

^ Pre-gelatinized waxy maize starch (Wades BOl IC, Inpak Foods. South

Australia).

"* Vitamin and mineral preniixcs as described by Uki el al. ( 1985).

Histidine
Arginine

3.95
9.60

4.00
16,0(1

4.15
13,00

Threonine

6.50

6,00

4.95

the test diet assigned to their tank. During

both the preconditioning

Alanine

7.05

5.50

5.10

and experimental periods the animals were fed to excess every day
at approximately 1700 h.

Proline

Tyrosine

Valine

14.0
5.70
8.30

7.50
6.50
5.50

10.55
5.70
5.65

Tanks and Collection System

Methionine
Cystine

1.25
3.0(1

2.50
2.00

1.05
1,65

Conical shaped digestibility tanks were used. Abalone were

Isoleucine

7.75

6.50

5.85

housed in 20-L buckets (approximately 80-100 per bucket) that
fitted inside the tanks. All the buckets were fitted with plastic mesh
bottoms ( L3 X 1.5 cm mesh) allowinc containment of the abalone

Leucine

Phcnylanaline

Lysine

13.45
8.85
8.20

12.00
6.50

7.50

9.80
6.05
4.80

while permitting feces to drop into the collection tube at the base
of the tank. Three 25-cm lengths of PVC pipe (8 cm in diameter)
were placed in the buckets as shelters for the abalone. Attached to
the bottom of each digestibility tank was a screw-on collection
tube ( 1 1 cm long, 15 mm dianieter). Tanks were on a flow-through
water system at a rate of about 2 L/min. The seawater was tillered
to 30 p.m by primary sand filters, then to 10 jjim by secondary
composite sand filters before entering the tanks. Aeration was
supplied at 0.5 L/min to each tank at all times by an air stone.
Water temperature and lighting were controlled during the experi-
ment with temperature maintained at I8.0°C ± 1.0 and a light
regimen of 12 h light: 12 h dark regimen. Salinity was 35~36%f
throughout the experiment

Fecal Collection

Fecal collection occurred every day until 6-10 g (dry weight)
of feces was collected for each replicate sample. This took 22 days
in total. On each day of fecal collection the buckets containing the
abalone were removed and the digestibility tanks were drained of
water and all fittings were cleaned of feces and uneaten feed.
Abalone were out of water for about 1 to 2 min. Following clean-
ing, the tanks were refilled and the buckets replaced. Collection
tubes were fitted by 0900 h A small foam container was placed
underneath each tube and filled with ice to keep the collecting
feces cold and thus reduce degradation by microbial action. The
feces were collected from the tubes at about 1630 h by gently
pouring the contents onto a 1 mm mesh. The mesh was then placed
into a petri dish and frozen at -30°C. The following day the frozen
fecal sample was scraped off the mesh, pooled into a composite

sample, and replaced into the freezer until required for analysis.
Prior to analysis the samples were freeze-dried and ground with a
mortar and pestle.

Chemical Analyses

Chemical analysis for proximates (i.e.. dry matter, ash. ether
extract, neutral-detergent fiber, acid-detergent fiber, and crude fi-
ber) were undertaken using the methods of the Association of
Official Analytical Chemists (1984). Gross energy was determined
using a Parr 1 28 1 bomb calorimeter. Proteins were analyzed by the
combustion method using a LECO® CN-2000 Carbon and Nitro-
gen Analyser (Royal Australian Chemical Institute 1999).

All amino acids were determined by the Water's PICOTAG
amino acid analysis method (Sarwaret al. 1988) using pre-column
phenylisothiocyanate (PITC) derivilization and liquid chromatog-
raphy was used for the determination of all amino acids. Protein
sources were hydrolyzed for 22 h in duplicate with 6 N HCL at
1 10°C for the determination of all amino acids, except for methi-
onine and cystine. Hydrolysates for the determination of methio-
nine as methionine sulfone and cystine as cysteic acid were pre-
pared by performic acid oxidation of the protein prior to hydrolysis
using 6 N HCL. Alpha-aminobutyric acid was used as an internal
standard.

Chromic oxide was determined using atomic absorption spec-
troscopy based on a modification of Hillebrand et al.'s (1953)
method. The modified method involved preliminary ignition of the
sample at 500°C to remove organic material and the dissolution of
the sample in hydrochloric acid instead of sulphuric acid (M. Frith,

The Digestibility of Lupins for Abalone

801

TABLE 3.

Proximate analysis and total amino acid content (};/kg. air dry

basis), and gross energy content (MJ/kg, air-dry basis) of soytlour,

whole and dehulled lupins used in experimental diets.

Feedstuff

Soyflour

Whole
Lupin

Dehulled
Lupin

Crude prolein (N x 6.25) 454.0 338.6 380.0

Gross energy (MJ/kg) 17.33 17.74 18.28

Dry matter 876.5 894.5 891.9

Ash 58.8 25.0 25.3
Fibre extract

Crude 30.5 76.9 93.9

Acid detergent 79.9 132.8 108.6

Neutral detergent 94.6 186.3 157.3

Ether extract (bp. 40-60°C) 7.5 63.5 64.4
Amino acids

Asparticacid 61.40 32.85 41.00

Glutamic acid 88.10 75.85 82.15

Serine 26.15 18.00 18.45

Glycine 20.80 14.85 14.95

Histidine 12.70 7.60 9.85

Arginlne 35.65 37.65 37.60

Threonine 18.(15 12.15 13.05

Alamne 19.10 11.60 11.65

Proline 20.65 21.15 23.95

Tyrosine 17.95 13.55 12.35

Valine 24.45 12.50 13.45

Methionine 6.10 2.35 2.00

Cystme 6.70 3.50 2.00

Isoleucine 23.10 13.20 14.30

Leucine 36.90 21.40 22.75

Phenylanaline 24.95 13.45 14.05

Lysine 30.85 14.60 15.35

personal communication. University of Tasmania. Launceston,
Australia).

Digestibility Determination

The apparent digestibilities of nutrients in the diets were cal-
culated using the following formula (Hardy 1997):

Apparent digestibility = 1

Cr.iu'i ^ l^'i"''eM/„c.

Cr,,

'jeces ^ Nutrient j,^,,/

where O" is chniniium content and Nutrient is nutrient or energy
content of the diet.

The digestibility of gross energy for each ingredient was cal-
culated by subtracting the amount of digestible energy contributed
from the oil, sodium alginate and pre-gelatini/ed starch in each
diet.

Statistical A nalysis

The data were analyzed by a general linear model and the
treatment means were cotnpared by least significant difference.
Prior to analysis, data were analyzed using a univariate procedure
and normal plots to establish that the data were in fact normally
distributed, as was the case. The presence of outliers was assessed
using the RANK procedure in SAS (SAS Instittite Inc.. 1988) and
normal scores computed from the ranks following Blom (1958).

As no outliers were detected using the RANK procedure, all data
were used in the calculation of digestibility estimates for all pa-
rameters, respectively.

RESULTS

A significant difference was found among the lupins and soy-
llour in abalone's digestibility of protein (F-,,, = 17.22, P =
0.0033). gross energy (F^^, = 1340.96, P = 0.0001), arginine
(F, 7.24. P = 0.0251). proline (F,„ = 10.87, P = O.OIOI).

methionine (F, ,

9.16. P = 0.0150) and isoleucine (F,,

18.94, P = 0.0026) (Table 4).

Dehulled lupins had significantly higher apparent gross energy
digestibility for abalone than whole lupins, but had significantly
lower gross energy digestibility for abalone than soyflour (Table
4). The protein fnmi dehulled lupins was significantly more di-
gestible than that from either whole lupins or soyflour (Table 4).

DISCUSSION

Dehulling significantly improves abalone's digestibility of en-
ergy from L. anf^Ksrifolius. The low gross energy digestibility of
whole L. in}i;iislitoliiis seeds and the significant increase in gross
energy digestibility of dehulled compared with whole L. angiisti-
folius has also been observed for other aquacultured species. For
example, the gross energy digestibility increased from 45-74% for
juvenile P. monodon (Smith et al. 1998) and from 59.4-74% for
silver perch {Bidxanus Indyamis) (Allan et al. 1998) by use of
dehulled as against whole L. angustifoUus lupins.

The abalone's significantly lower digestibility of energy from
whole compared with dehulled L. angustifoUus indicates it has a
poor capacity to digest the cellulose in lupins. Cellulose constitutes
approximately 57.3-58.4% of the hull of L angustifoUus (Evans &
Cheung, 1993) and thus is likely to be cause of abalone's poor
gross energy digestibility of whole L. angustifoUus. Indeed, Uki et
al. (1985) demonstrated that the growth rate of Haliotis discus
hannai decreased as the cellulose content of the diet increased
from 0-20% and concluded that abalone must have a poor capacity
for digesting it.

Some may expect that abalone should be able to digest cellu-
lose, given that it forms the structural basis of many algae, their
natural diet (McCandless 1985) and cellulase activity has been
documented for a number of Haliotis spp. (Gianfreda et al. 1979,
Elyakova et al. 1981, Boyen et al. 1990, Gomez-Pinchetti & Gar-
cia-Reina 1993). A complication in interpretation of those results
is that despite many studies, the origin of the cellulose activity in
abalone is inconclusive. This is because most studies have not
identified whether the cellulase was of microbial origin. Erasmus
( 1997) was the first to demonstrate that abalone possess a cellulase
by detecting the presence of carboxymethylcellulase in gnotobiotic
H. midae. Erasmus (1997) suggested that this cellulase was most
likely to have been poly-fi-glucanase, which hydrolyzes car-
boxymethylcellulose. Whether abalone pos.sess the other two cel-
lulases required to hydrolyze cellulose remains to be confirmed.
The substrate carboxymethylcellulose does not require a true cel-
lulase for hydrolysis. Erasmus et al. ( 1997) hypothesized that bac-
teria possibly secrete a true cellulase to completely degrade cellu-
lose and abalone only partially hydrolyze the substrate. This hy-
pothesis was supported by Enriquez et al. (2000) who examined
the in vitro digestion of pure cellulose (alphacel) using stomach
extracts from Haliotis fulgens and reported that bacteria play an
important role in cellulose digestion in abalone, as stomach ex-

802

Vandepeer et al.

TABLE 4.

Apparent faecal digestibility coefficients of protein, gross energy and amino acids in soyflour, whole and dehulled L. anumtifoliiis lupins fed

to juvenile greenlip abalone I//. laevif;ala). Data are mean ± SK, ii = 4.

Statistics

Whole
Lupin

Dehulled
Lupin

Feedstuff

Soytlour

P

SEM

LSD

Protein

0.904' ±0.00151

0.909" ± 0.00267

0.919" ±0.00222

**

0.00199

0.0069

Gross energy

0.870'' ± 0.00293

0.499*" ± 0.00502

0.83 r± 0.00838

***

0.00556

0.0192

Amino acids

Aspartic acid

0.980 ±0.00153

0.949 ± 0.03647

0.978 ±0.00130

NS

0.02078

Glutamic acid

0.981 ±0.00146

0.982 ±0.00168

0.979 ±0.00124

NS

0.00157

Serine

0.938 ± 0.00233

0.935 ± 0.00229

0.938 ± 0.00335

NS

0.00228

Glycine

0.919 ±0.00573

0.924 ± 0.00306

0.923 ± 0.00924

NS

0.00591

Histidine

0.914 ±0.00702

0.918 ±0.00448

0.929 ± 0.00607

NS

0.00655

Arginine

0.947" ± 0.00425

0.962" ±0.00142

0.962" ± 0.00230

*

0.00306

0.0106

Threonine

0.880 ±0.00716

0.895 ± 0.00360

0.884 ± 0.00666

NS

0.00472

Alanine

0.902 + 0.00561

0.889 ± 0.00250

0.893 ± 0.00363

NS

0.00361

Proline

0.947" ± 0.00549

0.9 16" ±0.00927

0.945" ± 0.00450

*

0.00525

0.0182

Tyrosine

0.885 ± 0.00397

0.908 ±0.00153

0.907 ±0.01080

NS

0.00723

Valine

0.873 ±0.00170

0.857 ± 0.00367

0.869 ± 0.00808

NS

0.00447

Methionine

0.815" ±0.01077

0.907" ± 0.00474

0.828" ± 0.02372

*

0.01647

0.0150

Cystine

0.972 ±0.01379

0.975 ± 0.00688

0.969 ±0.01379

NS

0.00344

Isoleucine

0.693" ± 0.00664

0.660" ± 0.00225

0.596" ± 0.00664

**

0.01 130

0.039 1

Leucine

0.881 ±0.00716

0.888 + 0.00174

0.887 ±0.00716

NS

0.00455

Phenylanaline

0.870 ±0.01009

0.875 ± 0.00206

0.870 ±0.01010

NS

0.00577

Lysine

0.927 ± 0.00895

0.920 ± 0.00634

0.913 + 0.00895

NS

0.00624

NS, not .significant.

* P < 0.05

** P < 0.01

***/>< 0.001

SEM. standard error of the mean

LSD, least significant difference

Values in a row with different superscripts differ significantly (t,)(

2.45).

tracts showed significantly decreased cellulolytic activity in the
presence of antibiotics.

A factor to consider when evaluating abalone's ability to digest
cellulose based on previous studies done to determine cellulase
activity, is that many have estimated cellulase activity by exam-
ining the production of glucose from abalone stomach homoge-
nates incubated with a-cellulose. This type of cellulose is a powder
prepared by treating de waxed cotton (ethanol extracted) for 8 h
with 1% boiling sodium hydroxide solution (Whistler & Smart
1953). Thus, it is probably much easier for the abalone's own
endogenous enzymes and gut bacterial enzymes to digest then
cellulose present naturally in algae or in land based plants. Evi-
dence for this is that cellulose that has been treated with alkali to
increase the proportion of amorphous cellulose is more rapidly
decoiTiposed than untreated cellulose (Whistler & Smart 1953). In
addition bacterial attack of cellulose is inhibited by the presence of
lignin (also a component of lupin hulls). Thus bacteria that readily
hydrolyze isolated cellulose may have little, if any. effect on wood
(Whistler & Smart 1953). In addition, algae contain only low
levels of cellulose. In divisions Phaeophyta. Rhodiiphyta and much
of the Chlorophyta. cellulose is a minor coinponent of the struc-
tural polysaccharides. In fact, in Phaeophytes and Rhodophytes.
the amount of a-cellulose ranges from 1 to 8% of thallus dry
weight whereas the proportion is often 30% in terrestrial plants
(Kloareg & Quatrano 1988). Thus it is possible that although aba-
lone possess cellulase it does not necessarily mean that they are
able to effectively hydrolyze the cellulose present in terrestrial

plants, thereby resulting in the lower gross energy digestibility of
the whole compared with dehulled lupins in this study.

Abalone must be able to digest the major energy reserves in the
cotyledon of lupins (lipids and cell wall non-starch polysaccha-
rides (Evans 1994) relatively well, as indicated by the significant
increase in gross energy digestibility of the lupins after dehulling.
The lipid content of the cotyledon is relatively low and comprises
approximately 85 g/kg by dry weight (Evans & Cheung 1993). The
predominant monosaccharides in the cotyledon non-starch
polysaccharides are galactose (67%), arabinose (12%) and uronic
acid ( 10%) residues (Evans & Cheung 1993), and are the constitu-
ent sugars of the reserve pectic substances. Pectin is also one of the
principal polysaccharides in cell walls of algae (Chapman & Chap-
man 1973). and so it would not be surprising if abalone were able
to digest it.

The finding of comparable digestibility of lupins and soyflour
indicates that there is potential lor use of lupins in manufactured
feeds for abalone. Since lupins are considerably cheaper than de-
fatted soyflour. the replacement of soyflour with lupins in diets
would result in a reduction in the cost of manufactured feeds.

ACKNOWLEDGMENTS

The authors thank Murray Frith for chromic oxide analysis,
Tom Lam for amino acid analysis and Debra Pailington for help
with statistical analysis. This research was supported by financial
grants from the Fisheries Research and Development Corporation.

The Digestibility of Lupins for Abalone

803

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Journal ofShellthh Research. Vol. 21, No. 2. 805-809. 2002.

LARVAL ESCAPE THROUGH ABALONE CULTURE EFFLUENT SYSTEMS: AN ANALYSIS

OF THE RISK

C. D. HAWKINS'* AND J. B. JONES"

^ Depuriment of AiiricuUitre. Western Australia. PO Box 16. Moora, WA 6510, Australia: 'Department of
Fisheries Western Australia. Research Division. P.O. Bo.\ 20, North Beach. WA 6020. Australia

ABSTRACT Expansion of atialone culture in Western Australia has resulted in the acquisition of juvenile stock (Haliotis laevigala)
from interstate, panicularly South Australia. This movement has raised concerns about the possibility of compromising the genetic
integrity of domestic wild stocks of abalone with e.xotic genetic material. Such a compromise could arise from the escape of larvae from
a production system in the event of a spawning event during the growing period. The use of a fine mesh filter may reduce this risk
but is considered impractical because of frequent blockage. A risk model was constructed, which interfaces with an abalone growth
model, to predict spawning events and the likelihood of viable larvae surviving in the open sea until they mature and spawn again. The
model predicts that the probability of a spawning event leading to larvae escaping, maturing and spawning again in the wild is in the
order of 3.7 x 10"'' per production cycle (about 3 y), and if such an event occurred, around 10 abalone would reach maturity, or about
1 7 in the absence of a settling pond for effluent water before discharge. Sensitivity of the model to input variables and implications
for further research are discussed.

KEY WORDS: abalone culture, larval escape, risk analysis

INTRODUCTION

Abalone {Haliotis spp.) are highly prized edible marine gastro-
pod mollusks. Their culture is increasing around the coastline of
Australia with national production anticipated in the order of 485
tons in 2000-2001 (Hone & Fleming 1997). Increasing world
prices for abalone have increased the viability of abalone aquacul-
ture. with the majority of Australian aquaculture production being
in South Australia. Victoria, and Tasmania. The industry is com-
parable to most intensive animal production systems, with a degree
of specialization occurring. In particular, the potential exists for
the production of large quantities of juvenile abalone for sale to
grower units enabling growers to focus on a single aspect of pro-
duction by purchasing stock from a specialist producer.

Production occurs in both sea- and land-based systems, with a
trend toward land-based systems. Land-based systems tend to be
operated close to the ocean, with sea-water supplied from the
ocean, and effluent returned some distance from the inlet system.
Australian abalone farms vary in size and layout. The profitability
of such farms with either a 100 or 200 ton annual production was
modeled by Weston et al. (2001 ).

Development of the industry in Western Australia is expanding,
although only a small number of hatcheries have been established
to date. To facilitate industry development, there has been pressure
on Government to permit the introduction of juvenile stock from
other states in Australia. Movement of any aquaculture stock into
Western Australia is subject to an environmental assessment pro-
cess. When assessing the movement of juvenile green-lip abalone
(Haliotis laevigata) from South Australia to Western Australia, a
concern was expressed that the genetic integrity of the Western
Australian stocks could be compromised. During the growing pe-
riod of some three years, it was hypothesized that a spawning event
could occur in the growing tanks, resulting in discharge of viable
juvenile abalone into the wild. Although the species of abalone in
Western Australia is the same as that around other parts of the
coastline, biotypes may vary (Brown 1991. Elliot et al. 2001 ) and
an exotic or hybrid biotype may establish in the wild following a
spawning event (Gaffney et al. 1996).

*Corresponding author. E-mail; chawkins@agric.wa.gov.au

This proposed risk to the genetic integrity of wild abalone has
been the subject of unpublished consultancies, and there is evi-
dence that in greenlip abalone. stocks are genetically differenti-
ated, that differences are detectable over small distances, and that
interaction between different stocks is limited. More recent data
published by Hancock (2000) show only small differences among
Haliotis roei populations in Western Australia and, on this basis,
genetic zones should no longer be applied for broodstock collec-
tion with this species. For black lip abalone (H. rubra), genetic
differences seem to be even smaller than for green lip. with data on
other species being inadequate for reliable conclusions to be made
(Westaway & Norriss 1997). However, the potential for introduc-
ing new genetic material into existing stocks, and the possibility of
reducing genetic diversity remain of concern to environmentalists
and the abalone aquaculture industry, and management of this
perceived risk is appropriate. Recommended risk management
strategies include the use of progeny derived from a sufficiently
large number of parent stock to ensure adequate genetic diversity;
obtaining broodstock from local populations; and locating the sea
culture facilities at a prescribed distance from wild populations of
abalone. In addition, circumstances may require that effluent tank
water be discharged over sand or filtered to minimize the ability of
escaped larvae to settle on a suitable substrate. CuiTcnt policy in
this state dictates the use of a settlement pond or sediment filter for
land-based farms.

Other risk reduction measures have been suggested, including
the culture of polyploid (specifically triploidi organisms and har-
vesting before sexual maturity is reached. However, these options
are not viable using present technology, because polyploidy is
difficult to guarantee, and sexual maturity may occur some con-
siderable time before harvestable size is reached in some species.

One management strategy to minimize the risk of larval escape
from abalone culture ventures is the use of an effluent filter to trap
larvae should a spawning event occur. In practice, given the vol-
ume of water involved, the use of a fine-mesh (100 |jim) filter on
the discharge outlet from abalone farms poses significant difficul-
ties because of rapid clogging with particulate matter and restric-
tion of water flow.

It is the intent of this risk assessment to quantify the probability
of escape of larvae from a land-based abalone culture system using

805

806

Hawkins and Jones

flow-through seawater in the absence of an effluent filter. The
assessment relates to the risks of a spawning event, which is af-
fected by the volume of water used, but independent of the number
of abalone in the farm and their density.

METHOD

A quantitative risk model (Vose 2000) was constructed using
Microsoft® Excel, with the Palisade® @Risk add-in. However,
because of the changing risks as.sociated with the growth and de-
velopment of abalone during the growing period, relevant parts of
the growth and production system were also modeled. The model
tracks abalone growth and associated aquaculture events daily.

Abalone production is based on a grow-out raceway fed by
seawater, the flow rate of which increases as the abalone grow.
Initially, appro.ximately 0.2 megaliters (ML) of water per day is
used, increasing to 40 ML per day for the 2 mo before harvest. A
linear relationship between size and water flow is assumed: Flow
Rate = Age (days) * 0.04 ML/day to day 1010, then 40 ML
thereafter.

Indications from Purdom ( 1 980) are that growth in fish is linear
under optimal conditions, restrained by food, temperature, and
sexual maturity. Social interaction has an effect in free swimming
fish, but it is anticipated that such interaction is minimal in aba-
lone. Hahn (1989) also showed that growth in length is relatively
linear for abalone over a wide size range. Sexual maturity in green-
lip abalone is generally after reaching 90 mm, so the constraints of
feed and temperature remain the major ones, although precocious
maturation occurs in abalone farms. Under farmed conditions, feed
should be supplied ad libitum and temperatures maintained at or
around optimum. Thus, a linear growth rate between 0.2 and 70
mm, over about 1,000 days is used. The growth expression is;

Size (mm) = Age (days) * 0.07.

Considerable attrition is noted in farmed abalone, with survival
to maturity being very low. Large losses before settlement have
been noted. Shepherd et al. (2000) observed .settlement rates be-
tween 0.02-1.2% for H. rubra, and 0.1-6.5% for H. laevigata in
experimental stock enhancement trials in South Australia. At the
request of the Department of Fisheries, two linear death rates were
used. In this situation, mortalities reach 93% by the time abalone
are 5 mm (approximately day 1 27 of the model ) and a further 50%
mortality by the time abalone reach 88 mm. Simple linear regres-
sion lines were used for this purpose.

Abalone are harvested at approximately 70 mm in length, some
20 mm short of the anticipated size for sexual maturity. However,
abalone have been reported to spawn when only 25 mm in length.
Synchronous spawning may occur in abalone, although Hahn
(1989) reports that spawning is poorly synchronized in some spe-
cies. Egg production is from 0.5 x 10'' to .3 x 10" per abalone, with
larger shellfish producing more eggs. The relationship between
size and spawn production is considered linear. Throughout this
model, assumptions are made that favor the production of larvae
and their escape, so as to avoid underestimating the risk. Thus,
abalone as small as 15 mm are permitted to spawn in the model.
The quantity of spawn (eggs) produced is calculated as:

Spawn (millions) = length in mm * 0.023809524
+ 0.142857143.

Discharge water from the grow-out tanks is fed into a settling
pond, which is believed to remove up to 80% of solid material.
Although more accurate figures would be desirable, this percent-

age is an upper limit, with the minimum and means probably being
30 and 65%. There is a negative correlation between the flow rate
of seawater through the farm and the efficiency of the settling
pond: Proportion settling in the pond is: flow (ML) * (-0.0125628)
+ 0.8. The effectiveness of the settling pond was evaluated by
running the model with zero settling.

From the settling pond, effluent is discharged through a pipe-
line to a subsurface outlet in the sea. This outlet is located at the
maximum practical distance from the seawater inlet for the facility.
Discharge is required to be over sand, to minimize the chance of
any abalone larvae that escape of finding a suitable (rocky) habitat
on which to settle. However, the effect of discharge over sand is
not directly modeled, rather a probability that such larvae will
survive and settle is used. Water from the subsea discharge pipe is
expected to plume rather than disperse evenly into the sea. thereby
maintaining a higher level of suspended particles for some distance
from the outlet.

The model uses a random number generator to determine if a
spawning event occurs during a growing cycle. It is assumed that
spawning is an unexpected event that may occur once before har-
vest. The likelihood of spawning is set as a probability, using a
beta-pert distribution. This type of distribution has been chosen,
because it offers the capacity to specify minimum, maximum, and
most likely values, and the curve is such that the extremes are not
favored, and the bulk of the values generated cluster around the
most likely value. The shape of the beta-pert curve is one that can
be grasped intuitively, appearing as a bell-shaped curve, with or
without a skew. The values for the distribution are: Minimum:
0.001; Most Likely 0.005; Maximum 0.0095. That is, there is a 1
in 200 chance of a spawning event occurring, but this may be as
low as 1 in 1,000, or as high as (approximately) 1 in 100. These
data have been provided from production experience. Although
spawning is most likely to occur with larger abalone in the third
year of the growing period, the model allows spawning on any day
once abalone reach 15 mm. (This may seem very low, but gonad
maturation occurs very early for some species; e.g., Haliutis roei)
The day on which the spawning event occurs is randomly set
between the time abalone reach 15 mm, to the end of the growing
period.

When a spawning event occurs, a proportion of abalone present
in the grow-out tank is able to spawn as females. This is also
represented as a beta-pert distribution, with the following values:
Minimum 0.05; Most Likely 0.1; Maximum 0.3. Proportions cal-
culated in this manner were subsequently used to determine the
number of individuals reaching the next stage, as a binomial prob-
ability.

Although a number of environmental variables interact at the
time of spawning to influence fertilization, and the window of
opportunity for fertilization may be short, is assumed that all
spawn are fertilized — the worst case scenario. (In practice, any lag
between males and females spawning in high flow rate tanks will
impede fertilization.)

There is always a concern that "something drastic could go
wrong" — the unexpected, rare event that throws the whole system
into disarray, such as a meteor crashing directly into the abalone
farm. In this model, the possibility of a rare event is incorporated
in the form of a total washout of all particulate matter from the
grow-out tanks; that is, whatever fertilized spawn is present gets
washed straight out to sea without the benefit of a settling tank.
The likelihood of such a rare event is a beta-pert distribution with
the following parameters: Minimum 0.0000001; most likely

Abalone Larval Escape from Culture: A Risk Analysis

807

0.000001; Maximum 0.000005. That is. the event is most hkely to
occur with a 1 in a milhon probabihty. but could be as high as 5
in a million, or as low as 1 in ten million. This parameter is
included simply to allow for the occurrence of the most extreme
adverse event.

Any fertilized eggs (larvae) that escape the system are subject
to several events: settlement, survival through the first year, and
survival to sexual maturity. Although biologic events, these are
treated as probability distributions as follows. Survival to settle-
ment (Shepherd et ai. 2000) as a beta-pert distribution: Minimum
0.0002; Most likely value 0.02; Maximum 0.08. Survival for the
first year — a uniform distribution between 8 and 16%. Survival
from the first year to spawning as a beta-pert distribution: Mini-
mum 0.1; most likely value 0.2; Maximum 0.5. (It should be noted
that Shepherd et al. (2000) modeled survival of larvae released
directly onto a reef. They also found that survival depended on the
density of abalone already on the reeL Our model assumes that any
larva escaping from the farm will be carried to a reef area as if it
was being released for reseeding. Clearly, this is a worst case
scenario.) An abalone reaching maturity and then spawning is
considered the end point of the model.

Outputs from the model are:

• total fertilized spawn (larvae) discharged:

• number of abalone surviving in the wild to spawn; and

• probability of a spawning event in aquaculture. and subsequent
survival to spawn in the wild.

That IS. if a spawning event occurs, and there is no filter, how
many larvae escape, how many survive to spawn again, and what
probability can be associated with such events occurring?

The model was run for 1.000 iterations, each iteration repre-
senting a full grow-out cycle (approximately 3 y). A random num-
ber seed is generated internally by (S'Risk, and this is allowed to
vary between runs of the model. Consequently, results vary in
detail between runs, but not in order of magnitude. Computation-
ally, the model is a matrix of 17 variables calculated each day for
1,100 days (i.e., 18,700 individual variables determined on each
iteration of the model), of which some 9,000 are generated as
©Risk statistical distributions. This matrix is treated as an Excel
lookup table, and outputs are computed by accumulating looked up
values over the total number of iterations. Hence, outputs are not
single values, but aggregations of data that are presented as means
with upper and lower 95% confidence intervals, where appropri-
ate. Limitations imposed on the model by capacity of ©Risk to
handle large numbers restricts maximum sizes used in some cal-
culations. This was managed by performing such calculations on a
per megaliter of water basis. For example, the use of binomial
sampling is limited to population sizes of less than or equal to
32,767 — a number far smaller than the expected number of spawn
produced by abalone. For the calculation of outputs, the total vol-
ume of water is taken into account.

Where appropriate, sensitivity of output variables to the inputs
was analyzed using the facility available in @Risk. Suitable input
variables are day of spawning, proportion of abalone spawning,
and probability of a spawning event, the main drivers of the model.
Other variables are not suited to this process, because they are
calculated independently each day of the growing cycle (about
1,000 days), and consequently, each daily probability contributes
to a very small component of the final output.

The modeled abalone farm is stocked with five million juvenile
abalone, of two millimeter (2 mm) length.

TABLE I.
Outputs from risk model, with a settling pond.

Output

Mean
Value

Lower 5%

Confidence

Interval

Upper 95%

Confidence

Interval

Total spawn discharged
Abalone surviving to

13,184
10

3.920
0

26.80(1
40

spawn again
Probability of spawning
event and survival to
spawn in the wild

3.7 X 10""

6.1 X I0-'

9.5 X 10-"

RESULTS

Details of Output Variables Are Presented in Table I.

The number of spawn discharged is variable. However, the
mean value is under 20,000, with an upper confidence limit of the
order of 30,000^0,000. Of these, very few survive, with around
10, but fewer than 100, expected to reach sexual maturity and
subsequently spawn again. The likelihood that there will be a
spawning event in the grow-out tank, and larvae will escape, de-
veloping to sexual maturity, is very low. in the order of 4 in a
million, but may be as high as 1 in 100,000. These results include
the possibility of a rare, but catastrophic event, as described pre-
viously.

The effect of the settling tank, although detectable, was small.
The effect of removing the settling tank from the model is given in
Table 2. Output graphs for the data in Table 2 follow (Figs. 1-3).

The impact of a "rare event" is small. Because the rare event
operates in the same manner as removing the settling pond, the
occurrence of a rare event, if it happened at the time of spawning,
would be to increase the number of larvae escaping and reaching
maturity. Because of the very low probability of a rare event, it
does not affect the very low probability that larvae would escape
and establish in the wild.

Sensitivity Analysis

As indicated in Tables I and 2. the model demonstrates that the
presence of a settling pond reduces the quantity of larvae escaping
if a spawning event occurs. However, because the effectiveness of
the settling pond is not perfect, and its effectiveness decreases in
efficiency as flow rates increase, the settling pond does not greatly
affect the probability of larval escape.

TABLE 2.
Outputs from risk model, without a settling pond.

Output

Mean
Value

Lower 5%

Confidence

Interval

Upper 95%

Confidence

Interval

Total spawn discharged
Abalone surviving to

20,796
17

8,382
0

38,520
80

spawn again
Probability of spawning
event and survival to
spawn in the wild

4.0 X 10-"

6.5 X lO"'

9.1 X 10-"

808

Hawkins and Jones

5- CD

CO
>

4,500^

4.000

3.500--

3,000

2.500

2 000

1.500--

1.000

0 500

0.000

leojp-

140-

riitean=:3 972:.f.9E-0e

17,5 35 52 5

Spawn discharged (thousands)

5%

5%

:

8 38 38 52

Figure 1. Distribution I frequency! histogram for total spawn dis-
charged. Horizontal bar indicates percentiles.

The built-in facility of @Risk to detenniiie the correlation of
input variables to output variables indicates a correlation between
total spawn discharged, and the input variables day of spawning (r
= 0.8) and proportion of abalone spawning (r = 0.41). Survival
of escaped larvae to spawn again subsequently is correlated to day
of spawning (r = 0.2) and the proportion of abalone in the grow-
ing tank spawning (r = 0.16). Probability of a spawning event in
the growing tank, and subsequent survival to spawn again in the
wild, is also correlated to the day of spawning (r = 0.40) and
probability of a spawning event (r = 0.,^l.

CONCLUSIONS

The model constructed and described here is a hybrid of risk
and biologic models. Biologic components (growth of abalone.
deaths, water flows) drive the development of abalone in culture,
and risk components drive the events of interest; that is. spawning.
settling, escaping, and maturing. Where available, published data
have been used to determine probabilities. However, the relative
newness of the abalone culture industry is such that much data is
either lacking, or dependent on limited production experience.

In regard to the initial question of the importance of a filter to
prevent the escape of larvae should a spawning event occur, the
incorporation of a filter would seem to be a low utility procedure.
The likelihood of a spawning event leading to establishment of
abalone in the wild is very small (in the order of 10" ''-10*'^). The
consequences of a spawning event would seem to be likewise

0 060-'^

17

[

0.050-

>, 0 040-
u

c

1 0,030-

0)

r«te3r,

■-MAP.7 1
-? '

0 020-

0.010-

0.000-

lj:2 r, — 1 — 1

Number surviving

120

5%

:

80

Figure 2. Distribution (frequency) histogram of abalone surviving to
spawn in the wild. Horizontal bar indicates percentiles.

V C

Is

TO
>

120-

1 00-

60
40-

Pt otaabilitv ot spawning and survival to spawn again
Values in 1 0"-6

I

5%

Figure .1. Distribution (frequency) histogram of the probability of a
spawning event and survival of abalone to spawn in the wild. Hori-
zontal bar indicates percentiles.

small, with very few abalone surviving to spawn again. This result
is in accord with practical experience. Most attempts to outplant
hatchery-reared juvenile abalone on reefs have failed (Burton &
Tegner 2000). It should be remembered that the data used in this
model are considered pessimistic; that is, overestimates of the
input values, and the outputs can likewise be considered pessimis-
tic. It will require additional research to quantify the variables of
concern better. It may also be of benefit to investigate improving
the efficiency of settling ponds, to further reduce the numbers of
abalone escaping should spawning occur.

The nature of the model, using a large matrix as a lookup table
tends to mask the effects of input variables. This occurs because an
input variable may be refen-ed to once in a chain of "events" over
a large i-ange (in this case, 3 y, or about 1,000 day-events). The
model itself has some 9,000 input variables, which refer back
either directly or by way of another variable, to the biologic and
stochastic variables that provide the basic inputs for the models. It
is difficult to determine relationships between inputs and outputs
under these circumstances. However, for the primary driving vari-
ables, day of spawning, probability of a spawning event, and the
proportion of abalone spawning, positive correlations with output
variables were observed. In view of the nature of the model, such
correlations strongly suggest close relationships between the out-
puts and the input parameters, despite traditional statistical think-
ing on the nature of correlations.

These correlations suggest that it would be beneficial to collate
observations associated with spawning events in cultured abalone
and to determine what range of triggers influence such an event,
with the goal of manipulating these triggers to control or prevent
spawning in culture.

Based on the results of this modeling exercise, the source of
broodstock for land-based farms is of little importance in terms of
genetic impact on wild populations. However, the model does not
address disease risks and fine filtration (100 micron absolute) has
been applied to systems in Western Australia to reduce disease
risks associated with use of stock from interstate.

Similariy. settlement ponds did not greatly influence the risk of
larval escape, but this is not their primary purpose. Settlement
ponds are used primarily to reduce nutrient release into the pristine
coastal areas usually selected as abalone farming intake locations.

ACKNOWLEDGMENTS

The authors acknowledge the helpful technical advice provided
either directly by Dr. Greg Maguire of Fisheries Western Australia
or through him from his numerous industry contacts.

Abalone Larval Escape from Culture: A Risk Analysis

809

LITERATURE CITED

Brown. L. D. 1991. Genetic variation and population structure in the black-
lip abalone Haliotis rubra. Austral J. Mar. Freshwater Res. 42:77-90.

Burton, R. S. & M. J. Tegner. 2000. Enhancement of red abalone Haliotis
rufescens stocks at San Miguel Island: reassessing a success story. Mar.
Ecol. Prog. Series. 202:303-308.

Gaffney. P. M., V. P. Rubin. D. Hedgecock. D. A. Powers. G. Morris & L.
Hereford. 1996. Genetic effects of artificial propagation: signals from
wild and hatchery populations of red abalone in California, .-^i/uacul-
ture. 143:257-266.

Elliot, N., N. Conod. G. Maguire. B. Evans & J. Bartlett. 2001 . Preliniuiary
genetic comparison of Western Australian and Tasmanian greenlip aba-
lone. Proceedings .Annual Abalone Aquaculture Workshop July:200l .

Hahn. K. O.. editor. 1989. Handbook of culture of abalone and other
marine gastropods. Boca Raton. FL: CRC Press Inc. 348 pp.

Hancock. B. 2000. Genetic subdivision of Roe's abalone. Haliotis roei
Grey (MoUusca: Gastropoda), in South Western .Australia. Mar. Fresh-
water Res. 51:679-687.

Hone. P. & A. Fleming. 1997. Abalone. In: K. W. Hyde, editor. The new
rural industries. A handbook for farmers and investors. Canberra: Rural
Industries Research and Development Corporation, pp. 83-90.

Purdom, C. E. 1980. Growth in fishes. In: T. L. J. Lawrence, editor.
Growth in animals. London: Butterworths. pp. 273-285.

Shepherd. S. A.. P. A. Preece & R. W. G. White. 2000. Tired nature's
sweet restorer? Ecology of abalone [Haliotis spp.) stock enhancement
in Australia. In; A. Campbell, editor. Workshop on rebuildmg abalone
stocks in British Columbia. Ed. A. Campbell. Canadian. Special Pub-
lication. Fisheries Aquatic Science 130:84-97.

Vose. D. 2000. Risk analysis. A quantitative guide. 2nd ed. Chichester:
John Wiley & Sons Ltd.

Westaway, C. & J. Norriss. 1997. Abalone aquaculture In Western Aus-
tralia: dscussion paper and draft policy guidelines. Fisheries Manage-
ment Paper No. 109, Fisheries Western Australia. 24 pp. (See also the
subsequent 1999 version: Abalone Aquaculture in Western Australia:
Policy Guideline. Fisheries Management Paper 133, Fisheries Western
Australia, Perth. 10 pp.)

Weston. L.. S. Hardcastle & L. Davies. 2001. Profitability of selected
aquaculture species. Chapter 3. Abalone. Australian Bureau of Agri-
cultural and Resource Economics (ABARE) GPO Box 1563. Canberra,
ACT, Australia. 2601. research report 01.3, pp. 16-27.

Journal of Shellfish Research. Vol. 21, No. 2. 81 1-815. 2002.

ISOLATION AND CHARACTERIZATION OF MICROSATELLITE LOCI IN THE PACIFIC

ABALONE, HALIOTIS DISCUS HANNAI

QI LI,' CHOULJI PARK/ AND AKIHIRO KIJIMA^*

' Fisheries College. Ocean University of Qingduo. Qiugdao 266003. China: 'Education and Research
Center of Marine Bio-resources. Graduate School of Agricultural Science. Tohoku University. Onagawa.
Oshika 986-2242. Japan

ABSTRACT Four microsatellite loci, designated HdhLUl. Hdh7S. Hdhl76I, and Hdhl457. were isolated from the Pacific abalone.
Haliotis discus hannai, using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected
genomic library. Primers designed to amplify via polymerase chain reaction the microsatellite loci were used to screen 30 individuals
from a natural Pacific abalone population in Onagawa Bay. Miyagi Prefecture. Japan. The four microsatellite loci were all polymorphic,
with an average of 14.3 alleles per locus (range 7-20). The mean ob.served and expected heterozygosities were 0.48 (range 0.30-0.97)
and 0.79 (range 0.60-0.92). respectively. Significant deviations from Hardy-Weinberg expectations were observed at three loci as a
result of homozygote excess. The expected heterozygosity values were considerably higher than those previously found for allozymes
(range 0.101-0.125), suggesting that these microsatellite loci should provide useful markers for studies of trait mapping, kinship, and
population genetics.

KEY WORDS: microsatellite loci. Pacific abalone, Haliotis discus hannai, genetic vanahility

INTRODUCTION

The Pacific abalone. Haliotis discus hannai, is distributed
along the coastal waters of East Asia, where it is one of the most
valuable and popular fisheries resources. Although cultured seeds
of the Pacific abalone were produced 20 years ago. the develop-
ment of abalone culture has long been hampered by the problems
of low growth rate and mass mortality during seed production
(Hara & Sekino 2001). To resolve these problems and improve
animal breeds for aquaculture production, many genetic studies on
growth-related traits and temperature tolerance have been per-
formed in the Pacific abalone (Wilkins et al. 1980. Okumura et al.
1981. Hara 1990. Kobayashi et al. 1991. 1992, Kobayashi & Fujio
1994, 1996, Furutono et al. 1995, Kijima et al. 1995, Kawahara et
al. 1997. 1999). Allozyme analysis suggested the presence of ho-
mozygote excess and inbreeding depression (Kijima et al. 2002);
however, genetic control of the target traits in H. discus hannai
remains unclear. To tlnd genetic markers associated with loci that
control economically important traits to assist in selective breeding
programs, the development of molecular markers is needed. For
this purpose, allozymes are not appropriate because there are too
few of them and they are not sufficiently variable (Kijima et al.
1992).

Microsatellites are tandemly repeated arrays of short nucleotide
motifs found in all prokaryotic and eukaryotic genomes analyzed
to date (Zane et al. 2002). Because they are evenly dispersed
throughout genomes, usually characterized by high length poly-
morphism, and generally inherited in a Mendelian fashion, micro-
satellite markers have been widely used for genomic mapping,
linkage analysis, pedigree analysis, and population genetics of bio-
logic resources (Schlotterer et al. 1991. Knapik et al. 1998. Hol-
land 2001).

The traditional approach to obtain microsatellites is to create a
size-selected genomic library in a plasmid or phage vector and
then screen clones using oligonucleotide probes containing differ-
ent repeat motifs. For microsatellite repeats that are less abundant
in the genome, it is difficult to isolate them using the method.

*Corresponding author. E-mail: a-kijima@mail. cc.lohoku.ac.jp

Recently, different approaches have been developed to enrich for
microsatellites (Zane et al. 2002). One method is based on selec-
tive hybridization, a protocol that is commonly used in enrichment
procedures (Ostranderet al. 1992, Kandpal et al. 1994, Kijas et al.
1994, Refseth et al. 1997, Zane et al. 2002). In the present study,
we report the first isolation of microsatellite loci in H. discus
hannai by magnetic bead hybridization selection and assess poly-
morphism at microsatellite loci in individuals from a natural popu-
lation.

MATERIALS AND METHODS

DNA Extraction from Abalone

For constructing a genomic DNA library, high-molecular
weight DNA was extracted from a live Pacific abalone from Ona-
gawa Bay, Miyagi Prefecture, Japan. The foot muscle was re-
moved from the abalone and ground with dry ice in a mortar.
About 100 mg of the tissue was digested overnight at 37''C in 0.7
niL of lysis buffer (6 M urea. 10 mM Tris-HCl. 125 mM NaCI, 1%
SDS, 10 mM EDTA, pH 7.5) and 35 |xL of proteinase K (20
mg/mL). The reaction mixture was extracted with phenol xhloro-
form (1:1). precipitated with isopropanol, and dissolved in TE ( 1 0
mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA was treated with
DNase-free RNase (20 p.g/mL) at 37°C for 1 h, and purified again
using a phenol/chloroform extraction.

Digestion of Genomic DNA, Size Fractionation, and Ligation
of Adapters

Extracted DNA was digested with HaelU, Dral. and Hindi
using 100 U each for 50 p-g of abalone DNA. The digested DNA
(5 (xg) was electrophoresed on a 2.5% NuSieve GTG agarose gel
(EMC Bioproducts), and fragments of 400-800 bp were excised
and purified using a Qiagen column (QIAquick Gel Extraction Kit.
QIAGEN). The fragments ( 1 p.g) were ligated with 200 pmol of an
EcoR\-Notl-BamHl adapter (TaKaRa) using a DNA Ligation Kit
(TaKaRa), then collected by ethanol precipitation, and resus-
pended in 20 p.L of H^O.

811

812

Ll ET AL.

Magnetic Isolation of Tarin't Sequences and Adapter Polymerase
Chain Reaction iPCR)

One 0.6-niL tube of the Streptavidin MagneSphere® Paramag-
netic particles (Promega) was washed according to the manufac-
turer's instructions, resuspended in 300 p,L of 5x SSC ( 1 x SSC =
150 mM NaCl. 15 inM sodium citrate), and mixed with 300 pmol
of biotinylated oligoprobe: 5'-(CA)|,GCTTGA-biotin. The six-
base noncomplementary region at the 3' end was used to prevent
the probe, copurified with target DNA during magnetic isolation,
from acting as primers in subsequent adapter PCR (Gardner et al.
1999). The beads and probe were incubated for 15 minutes at room
temperature, then washed three times in 5x SSC and resuspended
in 100 |jiL of hybridization solution (0.5 M NaCl. 47f polyethylene
glycol 8000) at 56°C (Gardner et al.l999). The fractioned DNA
(20 |jlL) was mixed with 80 |j.L of the hybridization solution,
denatured by incubating at 95°C for 5 min, added quickly to the
beads, and incubated at 56°C for 20 min. The beads were then
washed four times at room temperature in 200 ixL 2x SSC fol-
lowed by four times at 30°C in 200 piL 1 x SSC. Immobilized DNA
fragments were eluted from the beads in 50 |xL 0.15 M NaOH at
room temperature for 20 min. The beads were then removed, and
the supernatant was neutralized by the addition of 5.5 |a,L lOx TE,
3.25 JJ.L 1.25 M acetic acid. DNA was purified by a Qiagen col-
umn (QIAquick PCR purification kit. QIAGEN) and eluted in 50
|jiL of TE buffer. PCR amplification was performed in 50 |jlL
volumes containing 5 jxL of the released DNA, 1 .25 U of Ampli-
Taq Gold (Perkin Elmer), 5 |JiL of GeneAmp lOx PCR buffer
(Perkin Elmer), 0.2 mM dNTP mix, 1.5 niM MgCK. and 0.6 jxM
of the adaptor sequence primer, 5'-CGGCGGCCGCGGATCC-3'.
Reactions were denatured at 95°C for 1 1 min before 35 cycles of
94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, followed by
a 5 min, 72°C final extension. PCR products were purified using a
QIAquick PCR purification kit (QIAGEN).

Cloning of the PCR-Amplified DNA Fragments

The purified PCR products were digested with Null, inserted
into the yVofl site of the pBIuescript II SK(-t-) vector (Stratagene),
and the recombinant plasmid vector was transformed into XLl-
Blue MRF' supercompetent cells (Stratagene) following the manu-
facturer's protocol. Recombinant clones were selected by plating
on LB media containing ampicillin (100p.g/mL), IPTGdOO (jlL of
10 mM stock solution) and X-Gal ( 100 |jlL of 27c stock solution).

PCR Screening of Microsatellite-Containing Clones

A small portion of a white colony was transferred to 10 (xL of
10 mM Tris-HCI (pH 8.5) with a toothpick, incubated at 95°C for
10 min, and then used as template (1 p-L) in the PCR with two
vector primers (T3 and T7) and the nonbiotin-labeled (CA),,
primer (Gardner et al. 1999). Each 10-[xL reaction mixture con-
tained 0.25 U of AmpliTaq Gold (Perkin Elmer), Ix the supplied
buffer (Perkin Elmer), 0.2 mM dNTP mix, 1.5 mM MgCK, and 0.2
(i-M of each pnmer. Screening amplifications were pert'ormed as
follows: 1 1 min at 95''C followed by 35 cycles of I min at 94"C.
1 min at 57°C, and 1 min at 72=C, followed by 5 min at 72°C. PCR
products were electrophoresed on 1 .5% agarose gels. Inserts pro-
ducing two or more bands were considered likely to contain a
microsatellite locus. Positive plasmid DNAs were purified using
Qiaprep spin columns (QIAGEN), and then double sequenced on
a Shimadzu DSQ-2000L DNA sequencer (Shimadzu) using Ther-
moSequenase cycle sequencing kit (Amersham) in combination
with the universal forward (5'-CGCCAGGGTTTTCCCAGT-
CACGAC-3') and reverse (5'-GAGCGGATAACAATTTCACA-
CAGG-3') primers, respectively. PCR primers for each microsat-
ellite locus were designed using the OLIGO software package
(National Biosciences Inc.).

Assessment of Polymorphism in Microsatellite Loci

Primer pairs were tested on a random sample of 30 Pacific
abalones from Onagawa Bay. Abalone DNA was extracted by the
method as described above. PCRs were performed in lO-p-L vol-
umes containing 0.25 U of AmpliTaq Gold (Perkin Elmer), Ix
PCR buffer, 0.2 niM dNTP mix. 1.5 mM MgCK, 1 piM forward
FITC-labeled primer and reverse primer, and about 100 ng tem-
plate DNA. The PCR conditions for all loci were 1 1 min at 95°C
followed by 35 cycles of 1 min at 94 C, 1 min at the annealing
temperature listed in Table 1, and 1 min at 72°C, with a final
extension of 5 min at 72-'C. Amplification products were resolved
via 6% denaturing polyacrylamide gel on a Shimadzu DSQ-2000L
DNA sequencer equipped with DSQ-SA program (Shimadzu), and
a pBIuescript II SK(-(-) sequencing reaction was co-electrophoresed
as a size marker.

Statistical Analysis

Number of alleles per locus, expected and observed heterozy-
gosities, and x^ tests of deviations from Hardy-Weinberg expec-

TABLE I.
Characterization of four polymorphic microsatellite loci in the Pacific abalone, Haliotis discus hannai.

Locus

Repeat Motif

Primers (5'-3')

.\nnealing Size No. of GenBank

Temperature IC) (bp) Alleles H,, H^ Ace, No.

HdbLU! (CGCA)j(CA)|s

TTCTGAGATGAGACGCACCAC

TTGGCAGCAGGCGTCGTGT
Hdh7R (CACCD^CACTTlCACCT), GTCGAAACTAGCACAAACATCT

GATCACCGGTCACATCATAC
Hdhl76l (CA),TA(CA),. ..(CCACA),3 GTCCAACACAACCAACTCCTC

CCTGAGCATGTTTGTGGATAAC
Hdhl457 (CGCCA),,(CTCCA)„. .. CACCTGTGTTTCGTACCCAC

(CTCCA),5. . .(CTCCA),, GGGACAATTCCAAGTAGATGC

H„, observed heterozygosity; H^, expected heterozygosity.
*P<0.01.

62 272-,^62 20 0.97 0.92 AB084076

54 177-332 7 0.33* 0.60 AB084077

64 405-596 18 0.30* 0.92 AB084()78

62 481-601 12 0.33* 0.71 AB084079

Isolation of Microsatellites in H. discus hannai

813

tations (HWE) were calculated using GENEPOP 3.1 software
(Raymond & Rousset 1995).

RESULTS

Isolation of Microsalellites in H. discus hannai

An enriched library of 350 white colonies was screened using
the PCR-based technique, and 84 clones were identified by the
presence of two or more bands on the agarose gel. Sequencing the
84 clones gave 46 loci containing microsatellites arrays with a
minimum of five repeats, primarily (CA)„, but some in combina-
tion with other tetra- or pentanucleotide array motifs. Primer pairs
were designed for 10 of the 46 sequences with long, uninterrupted
repeats and adequate unique regions flanking the microsatellite
array. Of the 10 primer sets developed, four loci were successfully
amplified with polymorphisms (Table I).

Genetic variability of Microsatellites in H. discus hannai

Figure I shows the alleles of microsatellite loci Hdhl32l.
Hdh78. Hdhl76L and HdhI457 identified in six Pacific abalone
individuals. The presence of minor peaks below the major ampli-
fication product ("stutter") was observed. At locus Hdhl761. the
peaks of longer alleles tended to be smaller than those of shorter
alleles. Primer sequences, repeat motif, annealing temperature,
number of alleles, amplified product size range, the observed (Hq)
and expected (H,.) heterozygosities for the four microsatellite loci

252 262 272 282 292 302 312 322 332 342 352 362 372 382 392
A]kles (bp)

162 172 182 192 202 212 222 232 242 252 262 272 282 292 302 312 322 332 342 352
Alleles (bp)

410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570
Alleles (bp)

460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620
Alleles (bp)

Figure I, Alleles of microsatellite loci Hdhlill. Hdli78. Hdhl76l. and
HdhI457 identiPied in six Pacific abalone individuals from a natural
population in .Miyagi Prefecture.

are summarized in Table I . The four microsatellite loci were all
highly polymorphic, whereas the degree of variability was differ-
ent at each locus. Hdhl321 had the highest number of alleles (20).
whereas number of alleles at Hdh78. Hdhl761. and Hdhl457 was
7. 18, and 12, respectively. The expected heterozygosity ranged
from 0.60 at Hdh78 to 0.92 at both Hdhl.Ul and Hdhl761. Sig-
nificant deviations from HWE for the observed heterozygosities
were observed in the Hdh78. Hdhl76l. and Hdhl457 (P < 0.01).
Allele frequencies of the four microsatellites for samples from
Miyagi Prefecture are shown in Table 2. At loci Hdh78 and
HdhI457. the frequencies of major alleles 267 and 563 were 0.617
and 0.500. respectively. At loci HdhlJ21 and Hdhl76l. allele
frequencies were overall low. with the most common alleles being
0.150 and 0.167. respectively.

DISCUSSION

The enrichment efficiency seen here (13.1%) is similar al-
though lower than that reported by Gardner et al. (21%, 1999). By
the colony hybridization method, the percentage of positive clones
containing microsatellite repeats was 0.52-0.66'7f in the European
flat oyster (Naciri et al. 1995). 0.1% in the quagga mussel (Wilson
et al. 1999), and an average of 1.96% in molluscs (Zane et al.
2002). Compared with traditional methodologies, the enrichment
procedure using the magnetic bead hybridization selection is more
efficient.

The presence of the microsatellite stutter bands seen at all four
microsatellite loci is a typical artifact of PCR amplication of mi-
crosatellite loci (Johansson et al. 1992). This phenomenon is due to
slipped strand mispairing during PCR (Weber 1990). In this study,
although the variability observed in the microsatellite loci (average
14.3 allele per locus) is possibly underestimated because of small
sample size (30 individuals), it was still much higher than that of
allozymes in the Pacific abalone population (Kijima et al. 1992).
They surveyed 18 allozyme loci using 445 Pacific abalone indi-
viduals in six groups from coastal waters of Japan, and found the
average number of alleles per locus in allozymes was 2.01 (range
1.89-2.17). The average expected heterozygosity in total popula-
tions was 0.116 (range 0.101-0.125). The high level of length
variation found here is similar to that found in H. discus discus and
other abalone species (Huang & Hanna 1998. Kirby et al. 1998.
Miller et al. 2001. Sekino & Hara 2001).

Significant deviations from HWE occurred for the observed
genotype frequencies at loci Hdh78. Hdhl761. and Hdhl457 be-
cause of homozygote excess. The departure from HWE with an
excess of homozygotes may be the result of one or more of the
following reasons. (1) Large allele "dropout" artifacts in the PCR
amplification process: At locus Hdhl76I. we observed that large
allele bands tended to be less intense than small alleles. In hetero-
zygous individuals, preferential amplification of a smaller allele
over a larger allele would result in the mis-scoring of heterozy-
gotes for homozygotes even though larger alleles may indeed exist.
The problem of large allele dropout during PCR has been well
documented for humans (Day et al. 1996), Minke whales (Van
Pijien et al. 1995), and chinook salmon (Banks et al. 1999). (2)
Small sample size: As microsatellite DNA has a rapid mutation
rate, resulting large number of alleles, a large sample size is
needed for accurate reflection of genotypic frequencies (Ruzzante
1998). This sample size was suggested to be at least 50 individuals
per population for microsatellite loci studies. (3) Presence of null
alleles: Null alleles of microsatellite regions, which occasionally

814

Li et al.

TABLE 2.
Allele frequencies of four microsatellite loci of Pacific abalone, H. discus hiiiinai, from Miyagi Prefecture.

Allele

Hdhl32l

Allele

Hdh78

Allele

Hdh 1 76]

Allele

Hdhl457

272

0.017

177

0, 1 33

405

0.067

4X1

0.033

290

0.150

182

0.017

408

0.017

493

0.017

292

0.033

187

0.067

418

0.167

509

0.017

294

0.033

202

0.033

426

0.050

518

0.033

300

0.117

262

0.083

433

0.067

548

0.017

304

0.150

267

0.617

442

0.033

563

0.500

306

0.017

332

0.050

448

0.167

568

0.033

310

O.I 00

450

0.017

578

0.167

312

0.017

478

0.017

586

0.117

316

0.067

483

0.017

591

0.017

320

0.017

526

0.067

596

0.017

322

0.017

537

0.033

601

0.033

324

0.05O

544

0.067

328

0.10(1

553

0.033

336

0.033

570

0.033

340

0.017

576

0.033

346

0.017

591

0.083

348

0.017

596

0.033

356

0.017

362

0,017

fail to yield an amplification product. Jiiay arise tlirough mutations
such as point mutations in the primer annealing site (Callen et al.
1993. Pemberton et al. 1995). Kijima et al. (1997) have found null
alleles at allozyme loci in the Pacific abalone by mating experi-
ment. If null alleles are present but not accounted for. the resulting
.scoring error of heterozygotes for homozygotes can create an ap-
parent excess of homozygotes in population studies (Jones et al.
1998). (4) Inbreeding effects: Extensive heterozygote deficiency
has also been reported at allozyme loci in natural populations of
the Pacific abalone (Fujino 1978, Fujio et al. 1986). Hara and
Kikuchi (1992) showed an excess of homozygotes in all natural
populations of H. discus harmed collected from nine different sam-
pling sites, suggesting extensive inbreeding. The excessive ho-
mozygotes for microsatellite loci were also described for H. rubra.
H. discus discus, and H. kamtschatkana populations (Huang et al.
2000. Miller et al. 2001. Sekino & Hara 2001). (5) Presence of size
homoplasy: Size homoplasy is the co-occurrence of alleles that are
identical in state (PCR products of the same size) without being

identical by descent (Ardien et al. 1999). Mistaking homoplasy for
homology can lead to underestimation of the genetic divergence
within and among population (Taylor et al. 1999). Microsatellite
allele size homoplasy has been confirmed in various animal spe-
cies (Estoup et al. 1995. Viard et al. 1998. Ardren et al. 1999.
Taylor et al. 1999); however, was not reported for abalones. Fur-
ther studies of natural populations and controlled crosses of H.
discus hannai will help to clarify this deviation from HWE.

The high variability of the microsatellite markers identified in
this study will make them excellent tools for paternity testing,
population studies, and the linkage analysis of genes related to
traits with economic significance in the Pacific abalone.

ACKNOWLEDGMENTS

The study was supported by grants from the Japan Society for
the Promotion of Science (No. P00335). Agriculture. Forestry and
Fisheries Research Council of Japan, and National Natural Science
Foundation of China (No. 30170735).

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Journal of Shellfish Re-iearch. Vol. 21. No. 2, XI 7-824. 2002.

TRANSMISSION OF WITHERING SYNDROME IN BLACK ABALONE, HALIOTIS

CRACHERODII LEACH

CAROLYN S. FRIEDMAN.' - * WENDY BIGGS.' JEFFREY D. SHIELDS,' AND
RONALD P. HEDRICK-

Califonua Department of Fish and Game and 'Department of Medicine and Epidemiology, University'
of California. Bodega Marine Laboratoiy. P.O. Bo.x 247. Bodega Bay. California 94923: Virginia
Institute of Marine Science. The College of William and Maiy. Gloucester Point. Virginia 23U62

ABSTRACT Withering syndrome (WS) has been associated with catastrophic declines in black abalone populations in southern and
central California. In an effon to identify the etiological agent of WS and to characterize the progression of this disease, we initiated
a transmission study in which abalone from Ano Nuevo Island, a location free of WS. shared aquaria with animals from Vandenberg
Airforce Base, a location where WS is epizootic. The mean incubation period of WS (time to develop overt signs of the disease) was
245 days with a mean time to death after development of clinical signs of 42 days. Median time to death was 41 wk in the
experimentally exposed Ano Nuevo Island abalone and 16 wk in the positive control Vandenberg abalone. Cumulative mortality was
significantly different between the negative control (unexposed) Ano Nuevo Island abalone (25% mortality) and both the exposed Ano
Nuevo Island abalone (85% mortality; P = 0.0001) and the positive control Vandenberg abalone (100% mortality; P = 0.0001). In
addition, significant differences in prevalences of a recently described Rickettsiales-like procaryote (RLP). "Candidatus Xenohaliotis
califomiensis." were observed between negative control animals (no RLPs) and those with WS (both the experimentally exposed Ano
Nuevo Island and Vandenberg abalone were infected with RLPs: P < 0.001). All abalone infected with the RLP had signs of WS,
including decreased condition indices, foot muscle atrophy, and digestive gland degeneration {P < 0.05). No correlation between
intensity of RLP infection and degree of WS was observed (P > 0.05), suggesting a complex relationship between the RLP and clinical
disease in black abalone. Despite this, these data in conjunction with a lack of observation of any other significant pathogens in the
abalone provides evidence that the RLP infecting abalone {"Cundidtitus Xenohaliotis califomiensis") is the etiological agent of WS.

KEY WORDS: withering syndrome, black abalone, Huliulis. rickettsiales, "CcJ/d/n/ii/i/.v .Xenohaliotis califomiensis"

INTRODUCTION

Withering syndrome (WS) has been associated with cata-
strophic declines in black abalone populations in southern and
central California (Haakeret al. 1992; Steinbeck et al. 1992, Fried-
man and Haaker unpublished data). Initial studies identified a pre-
viously undescribed coccidian parasite. Margolisiella ( = Pseudok-
lossia) haVwtis (Friedman 1991; Friedman et al. 1995, Desser &
Bower 1997). in black abalone with WS that was subsequently
determined to be nonpathogenic as evidenced by field and labo-
ratory studies (Friedman et al. 1993. 1997). VanBlaricom et al.
(1993) documented WS on San Nicolas Island in April of 1992.
These researchers a observed Rickettsiales-like procaryote (RLP)
that was recently described as a new taxon and has been given the
provisional status of "Candidatus Xenohaliotis califomiensis"
(Friedman et al. 2000). Two of six abalone with clinical WS har-
bored RLPs. whereas apparently healthy animals were devoid of
the RLP. The authors indicated that Rickettsiales-like bacteria
were commonly observed in marine invertebrates and that the
pathogenicity of these organisms was unknown. Gardner et al.
(1995) also observed RLPs in association with WS in black aba-
lone from San Nicolas and San Clemente Islands in southern Cali-
fornia. Healthy abalone from Ano Nuevo Island in central Cali-
fornia were not infected with RLPs. suggesting an association
between the RLP and WS. Friedman et al. (1997) examined the
association between the RLP. degeneration of the digestive gland,
and mortality in a laboratory study. No clear associations between
intensity of RLP infection and either condition of the digestive
gland or time to mortality were observed. Recently. Moore et al.

*Corresponding author. School of Aquatic and Fishery Sciences.
University of Washington, Seattle, WA 98195. E-mail: carolynf@
u. Washington. edu

(2000) observed a significant relationship betv\een the intensity of
RLP infection and degree of WS in cultured red abalone. These
conflicting data indicate further examination of the role of the RLP
in WS is warranted. This study was designed to examine the trans-
missibility of WS and to determine the relationship between RLP
infection and WS in black abalone.

MATERIALS AND METHODS

Animals

Healthy black abalone were collected on March 28, 1995 from
Ano Nuevo Island, where WS had never been observed. Black
abalone with WS were collected from Vandenberg Airt'orce Base
(Vandenberg) and Cayucos on April 24. 1995. Abalone were trans-
ported to the Pathology Quarantine Facility at the Bodega Marine
Laboratory, where they were placed in an 88-L aquaria and re-
ceived ambient (8-10°C). flow-through, full-strength seawater.
Macrocystis pyrifera was collected from Bodega Bay and was
surface sterilized by soaking in a tamed iodine solution (Prepo-
dyne: Westagro. Kansas City, MO) for 15 min followed by a
freshwater rinse. Animals were fed M. pyrifera twice per week. All
abalone were lagged and the following data were collected: maxi-
mum length, foot length and total volume (TV), and total weight
(TW). Animals were bled from the pallial sinus with a tuberculin
syringe and a 26-gauge. 0.5-inch needle and the density, cell-type,
and condition of circulating hemocytes was determined using a
hemocytometer. Visual condition of the abalone was assessed ac-
cording to the following scale: (3): healthy abalone with a foot and
viscera that fills the entire shell volume; (2): visible mantle retrac-
tion and moderate atrophy of the foot muscle; and ( 1 ); severe
atrophy of the foot muscle.

817

Friedman et al.

Histology

Selected tissues were placed in Invertebrate Davidson's solu-
tion (Shaw & Battle 1957) tor 24 h and processed for routine
paraffin histology. Deparaffini/ed 5-|j.iti sections were stained
with hematoxylin and eosin (Luna 1968) and viewed by light
microscopy. The intensity of RLP infection was quantified using
the following logarithmic scale at 200x magnification: (0): no
bacterial foci; (1): 1-10 foci per field; (2): I 1-100 foci per field;
and (3): >100 foci per field (Friedinan et al. 1997). Infection in-
tensity was quantified in both the postesophagus (PE) and diges-
tive gland (DG). and an overall infection intensity was calculated
by summing the intensity in the PE and DG (range of 0-6 possible)
(Moore et al. 2000). Intensities were examined according to tissue
type to determine whether the location of infection was correlated
with animal health. Unless otherwise specified, the term RLP in-
fection refers to overall infection intensity. Condition of the di-
gestive gland and foot muscle were assessed using the ( 1 )-( 3 )
scales of Friedman et al. (1997), in which normal was scored as
(3), moderate (up to 30%) alteration from normal was scored as
(2), and tissue that was severely (>30%) altered was scored as ( 1 ).
Three specific morphologic changes that characterized observed
alterations in digestive gland architecture were individually scored
according to the following (1H3) scale: (1) normal architecture;
(2) moderate (up to 25%) degeneration (characterized by an in-
crease in connective tissue between digestive tubules, the primary
tissues responsible for secretion of digestive enzymes and nutrient
absorption in abalone) (Voltzow 1994). transport duct metaplasia,
or inflammation; and (3) abundant (>25%) transport duct metapla-
sia, an increase in connective tissues between degenerating tu-
bules, or inflammation.

Transmission Experiment

Groups of 12 abalone from Ano Nuevo Island were randomly
placed in each of two negative control (NC) and two experimental
aquaria (EA). Groups of 12 abalone with WS (EWS) were ran-

domly added to each of the two experimental aquaria and to each
of the two positive control aquaria (PC) (Fig. 1). Animals were
maintained on ambient seawater for the first 3 mo of the s£udy.
During this time temperatures ranged between 8-10°C (X =
9.53X) for the first 4 wk. 11-15^ (X = 12.89"C) for the fol-
lowing 4 wk and 10.5-15''C (X = I2.47°C) for the third month.
After this time, the animals were acclimated over a 2-wk period to
18 ± rC, the temperature at which the abalone were maintained
for the remaining 34 wk of the 46-wk study. Physical measure-
ments and hemocyte counts were assessed approximately every 8
wk over the course of the experiment. All moribund abalone or
mortalities were sampled as above, including shell weight (SW)
and shell volume (SV), and selected tissues (foot, digestive gland.
PE, and kidneys) were processed for histology. The intensity of
RLP infection and condition of the digestive gland and foot muscle
were quantified as described above. The condition of the abalone
was also assessed upon death using the body weight condition
index of Friedman et al. (1997) = [(TW - SWi/TW]. In addition,
the percentage of live tissue volume relative to the entire volume
of the animal was determined = [(TV - SV)/TV].

Statistical Analysis

The Fisher's exact test was used to test the independence of
exposure to WS and mortality; abalone were grouped as exposed
or unexposed and as alive or dead. Chi square contingency table
analysis (X") was used to test independence between exposure to
WS and measured health parameters. Abalone were grouped as
exposed (laboratory or field exposed) and unexposed. The follow-
ing health parameters or responses were grouped as normal (scores
of 3 for animal condition and that of the digestive gland and foot
and 0 for RLP presence), whereas those with signs of WS and RLP
infection were grouped as abnormal. Observed versus expected
frequencies in each category were compared using 2x2 contin-
gency table analyses. The Fisher's Exact test was used when fewer
than five observations were observed in any cells. These analyses

Experimental Design

n=12

n=12

O O I

n=12 n=12

n=12 11=12

n=12

O O 1

Replicate 1

Replicate 2

n=12

(VAFB)'

(EWS & EA)-

(Ano Nuevo Is.)^ }

Animals

Positive Experimental Negative } Treatment

Control Control

Figure 1 Experimental Design. The dark circles represent black abaU.ne «ith WS collected from the field (Vandenberg Airforce Base or
Cayucos (VBCS)). The open circles represent healthy, naive (no exposure to WS before study) black abalone from Ano Nuevo Island. Animals
with WS collected from Vandenberg Airforce Base and Cavucos in the positive control treatments. -Animals from VBCS (EWS) or Ano Nuevo
Island (EA) with and without WS. respectively, in experimental treatments. 'Animals in the negative control treatments without WS that were
collected from Ano Nuevo Island.

Withering S\'ndrome in Black Abalone

819

were also used to test the independence of RLP infection and WS.
Animals were grouped as infected and uninfected and as above for
survival and health parameters. In a separate analysis to further
assess the independence of specific lesions and RLP infection
intensity animals were grouped as low overall infection levels
(0-3) and high overall infection levels (4-6) and the response (e.g.,
metaplasia) was grouped as present or absent. Spearman rank cor-
relation coefficients were calculated and tested for a (linear) rela-
tionship between intensity of RLP infection and condition of the
digestive gland and foot, condition indices, density of circulating
hemocytes, cumulative mortality, and time of exposure. Stepwise
forward and backward regression models were used to predict the
intensity of RLP infection in exposed abalone from the following
variables: condition of the digestive gland and foot, weight con-
dition index, visual condition assessment, and duration of expo-
sure. Multiple logistic regression analysis was used to predict pres-
ence of RLP infection by using combinations of the five indepen-
dent variables listed above.

RESULTS

All abalone from the PC treatment and all except three animals
in the experimental treatment (EWS and EA) that died in this study
had visible signs of WS, including weakness, weight loss, and
visible atrophy of the foot muscle (Fig. 2). The two EA abalone in
the experimental aquaria that died during the first week of the
study lacked visual and histopathological signs of WS. RLP in-
fections, or visible injuries. A third abalone from the experimental
treatment that died during the 21st week of the study was too
decomposed for gross or histologic examination. The six NC aba-
lone that died during the experiment and 1 8 NC survivors sampled
upon termination of the study did not have visible or microscopic
signs of WS (Figs. 3 and 4). Both Vandenberg/Cayucos (PC and
EWS) and Ano Nuevo Island (EA) abalone with clinical WS had
histopathological and hematological signs of this disease, includ-
ing degeneration and inflammation of and/or metaplastic changes
in the digestive gland, depletion of muscle bundles in the foot,
(Figs. 3-5), and the presence of necrotic cells, cellular debris, and
small hemocytes (-4.5 p,m) with a large nucleus to cytoplasmic
ratio within the hemolymph. In addition, all PC and EWS animals
and all except the two EA abalone that died during the first week
of the study were infected with the RLP. whereas none of the NC
animals were infected. Other than the nonpathogenic renal coccid-
ian, Margolisiella { =PseudokIos.sia) luiliotis. no other parasites
were observed in any of the abalone examined in this study. In this
study, the incubation time for clinical WS is detuied as the dura-

tion between initiation of the study and development of gross
clinical signs such as mantle retraction or visible atrophy of the
foot muscle. The mean incubation period for the EA abalone was
245 days (;; = 21) with a range of 154-301 days. The duration
between onset of visible signs of WS and mortality averaged 42
days (n = 21) with a range of 6-1 13 days. The two abalone that
died during the initial week of the study from handling stress and
the single animal that died at 21 wk did not show signs of WS or
were too decomposed for assessment of WS, respectively, and
were not included in these calculations. Cumulative mortality ap-
proached 100% in the PC aquaria, 85% of the EA animals in the
experimental aquaria, and 25% in the NC aquaria (Fig. 6). A
significantly higher proportion of abalone died upon exposure to
WS (22/24) relative to unexposed animals (6/24; P < 0.0001, Fish-
er's exact test). Median time to mortality was significantly differ-
ent between the exposed EA (41 wk) and PC ( 16 wk) abalone {P
< 0.0001, Mann-Whitney test). As only a few NC abalone died
during the study, median time to death was not calculated for this
group.

A significantly higher proportion of abalone exposed to WS
(EA and PC) had reduced condition indices, morphologic changes,
and RLP infections than did unexposed animals (NC). Reduced
condition indices were observed in 18/24 EA and 19/24 PC aba-
lone. whereas only 3/24 NC animals lost condition {P = 0.002 and
P < 0.001. respectively. X' test). Morphologic changes were ob-
served in the digestive gland of 20/21 EA and 17/24 PC abalone,
whereas only 1/24 NC abalone had an abnormal digestive gland
architecture (P < 0.001. X' test). Of these, degeneration was ob-
served in 14/21 EA and 6/10 PC animals, metaplastic changes in
9/21 EA and 4/10 PC abalone, and inflammation in 6/21 EA and
l/IO PC abalone, whereas 1/24 of the unexposed animals only had
mild digestive gland degeneration. Pedal atrophy was observed in
15/21 EA, 17/24 PC, and only 1/24 NC abalone (P = 0.001 and
P < 0.001. respectively, X' test). Infections with "Candidatus Xe-
nohaliotis califomiensis" were observed only in EA (22/21) and
PC (24/24) treatments (P < O.OOI, X- test and P < 0.001, Fisher's
exact test). As above, significantly higher proportions of animals
with RLP infections died and had clinical signs of WS than did
unexposed abalone (/* < 0.001, A"" test).

Spearman rank correlation coefficients for relationships be-
tween intensity of RLP infection of individuals in each WS-
exposed group (EA, EWS, and PC) versus visual condition, con-
dition indices, condition of the foot and digestive gland, and den-
sity of circulating hemocytes were low and ranged between -0.275
and 0.486 for the Ano Nuevo Island animals and -0.0175 and
0.0567 for Vandenberg animals. Except for metaplasia and overall

Figure 2. Black abalone with and without WS. \. Healthy animals from .\no Nuevo Island from a negative control treatment. B, \n (EA)
abalone from Ano Nuevo Island that contracted WS from infected black abalone in an experimental treatment.

820

Friedman et al.

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Figure 3. Microscopic anatomy of the foot muscle of black abalone with and without \V S. The toot muscle of an uninfected animals are illustrated
in A ( Vandenberg abalone) and B ( Ano Nuevo Island abalone). Note thai dense bundles of muscle libers comprise most the loot. The pedal muscle
atrophy of an abalone that contracted \VS in the field (C) is also observed in those that acquired W'S in this laboratory study (D). Note the severe
reduction in muscle fibers and increase in visible connective tissue in affected individuals. Hematoxylin and eosin, bar = 15(1 pm.

RLP burden in the EA abalone {P < 0.03), all coefficients were
nonsignificant (P > 0.05). This relationship was also mirrored in
X' analysis in which a higher proportion (8/9) of EA abalone with
high overall RLP burdens (scores of 4-6) had metaplastic changes
as the sole or partial response to RLP infections than did those with
low (scores of 0-3) infections ( 1/7. P < 0.01 ). Several of the PC
abalone were too necrotic to assess specific lesions in the digestive
gland, a tissue that degrades more quickly than other tissues
(Friedman, personal observation), and resulted in small samples
sizes for this specific analysis. Intensity of RLP infection in labo-
ratory EA abalone was predicted from the duration of e.xposure
(time) with weight condition index, visual condition, condition of
the foot and digestive gland, and time as independent variables in
the model (P = 0.0156. Forward and Backward stepwise regres-
sions). No prediction of presence of RLP infection could be made
using Multiple logisitic regression analyses using all possible com-
binations of the five independent variables used in this study (P >
0.500). We did observe a significant correlation between hemocyte
numbers and weight condition index of the EA abalone (P =
0.0469), PC abalone (P = 0.0016) and NC abalone (P = 0.0015).
Correlation coefficients, however, were low to moderate and
ranged between 0.2668-0.5612.

DISCUSSION

The present study describes the transmission of WS from black
abalone with WS to previously healthy black abalone held in the
same aquaria. The similarity in physical, histopathological, and
hematological characteristics of WS between black abalone ex-
posed to WS in the laboratory and field, combined with a lack of
these signs in the negative control animals, confirmed that the
experimental abalone contracted WS in this stud\ (Haaker et al.
1992. VanBlaricom et al. 1993, Gardner et al. 1995. Shields et al.
1996, Friedman et al. 1997). These data also suggest that WS is
directly transmissible between sympatric abalone by cohabitation.

WS is a chronic, slow-progressing malady in which clinical
signs appear in the final stages of the disease. The presence of
advanced microscopic morphologic changes throughout the pedal
muscle and digestive gland of affected abalone supports this con-
clusion (Figs. 2-5). Our data also suggests a long incubation pe-
riod for WS (-35 wk) followed rapidly by mortality (-42 days)
under the conditions used in this study. As shown in Figure 6, once
the animals developed clinical WS, the slopes of the mortality
curves from the experimentally (EA) and field-exposed (PC) ani-
mals were very similar. However, median survival times between

Withering Syndrome in Black Abalone

821

Figure 4. Microscopic anatomy of tlie digestive gland of black abalone with and without WS. The digestive gland of unexposed abalone from
both Vandenberg (Al and Ano Nuevo Island (D) is composed primarily of terminal tubules and little transport/absorptive duct epithelia. whereas
those of abalone that contracted VVS in the field (B, Cl and laboratory (E, F) show a reduction in numbers of terminal tubules and an increase
in connective tissue and transport/absorptive duct epithelia. The digestive gland of some abalone with WS is characterized by an atrophy and
loss of terminal tubules (B, E), while other individuals respond to RLP infection (arrow heads) with a transport/absorptive duct metaplasia
(arrows; C, F). Hematoxylin and eosin, bar = 150 fim.

these two groups were quite different (41 wk for Ano Nuevo and
16 wk for Vandenberg/Cayucos animals) and may be due to a
variety of factors. The Ano Nuevo Island EA abalone was unin-
fected before initiation of the study, whereas the Vandenberg and

Cayucos PC abalone were in varying stages of WS. In addition,
differences in susceptibility may e.xist between abalone from these
geographically distant locations. In an earlier study in which
asymptomatic but previously exposed black abalone were col-

822

Friedman et al.

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a:

Body Mass Condition Index
Weight/Length^ Ratio
Visual Condition

Experimental Pos Control Neg. Control

Treatment

Foot Muscle
Digestive Gland
Connective Tissue
Transport Duct Metaplasia
Inflammation

Experimental

Pos Control

Treatment

Neg Control

^^H Postesophagus
I I Digestive Gland
^^M Pooled Intensity

Experimental Pos. Control Neg. Control

Treatment
Figure 5. Microscopic morphologic changes of black abalone that acquired WS in the Held (Pos. Control! or in the laboratory (Experimental)
relative to unexposed (Neg. Control) animals. A, Animal condition; B. relative condition of the foot muscle and disestive gland. C. RLP intensity
of infection. F.ach bar represents the mean of 12 ahalone in each of two replicate treatments :

: standard error (SK).

lecteiJ from Vandenberg and held at 18"C, the initiation of mor-
tality at 1 5 wk was similar to that observed in the PC and EWS
abalone in the cuirent study and also supports a long incubation
period for WS (Friedman & Fan 1998).

The observation of RLPs in the EA Ano Nuevo Island animals
and not in the NC Ano Nuevo Island animals (Fig. 5) suggests that
this bacterium, like WS, is horizontally transmitted by cohabitation
and is the etiological agent of this disease. This is further supported

by a lack of observation of any pathogens besides "Caiulidatus
Xenohaliotis californiensis" in any abalone examined in this study.
Transmission of this RLP is thought to be via a water-bome/fecal-
oral route because of the presence of bacterial foci in the digestive
epithelium and the observation of both intact and lysed RLP foci
in lumina of the digestive tract (unpublished observations). Mor-
talities of the European Saint-Jacques scallop, Pecten maximus,
have been associated vvith a branchial RLP infection (Le Gall et al.

Withering Syndrome in Black Abalone

823

Black Abalone Cohabitation Study
Survivorship Curves

15 20 25 30

Time (Weeks)

Negative Control
Positive Control
■ Experimentally Exposed

Figure 6. Survivorship curve of abalone in the cohabitation study. The
closed circles represent the negative control animals, the open circles
represent positive control animals, and the closed triangles represent
the (EA) Ano Nuevo Island animals in the experimental treatment.

1988. 1991). Transmission of this scallop-pathogenic RLP via
horizontal, water-borne transmission has also been documenleii
(Le Gall et al. 1991). Field and laboratory studies suggested that
transmission of the scallop RLP occurred between -5-28 wk of
exposure (Le Gall et al. 1991). Additional field studies reported
heavy RLP infections in scallops during the winter months fol-
lowed by mortalities in the spring (Le Gall et al. 1991 ). suggesting
a relatively long incubation period for the scallop rickettsial dis-
ease as we have observed for the RLP-induced WS in this study.
The relationship between the RLP and WS in black abalone is
complex as evidenced by higher proportions of mortality and clini-
cal WS in groups of abalone either exposed to WS-affected ani-
mals or infected with the RLP. With one exception, a lack of
significant correlation existed between intensity of RLP infection
and WS in both the experimental and PC animals coupled with a
lack of ability to predict intensity of RLP based on gross or his-
tologic signs that characterize WS (regression models). The single
significant positive correlation between overall intensity of RLP
infection and degree of metaplasia in the EA abalone that re-
sponded, in part, with this morphologic change suggests that sus-
tained high RLP burdens may lead to metaplasia in black abalone
as has been observed in red abalone (Moore et al. 2(!)00). This
relationship was not observed in the small number (\0) of PC
abalone in which the presence or absence of metaplasia was quan-
tified; the small sample size may account for an inability to detect
a relationship. However, when the overall DG condition (alteration
from normal, including all three specific morphologic changes)
was assessed in the EA and PC. these relationships were not ob-
served. This lack of correlation between RLP infection and DG
overall condition in black abalone may relate to the host response
to RLP infection (primarily degeneration of digestive tubules)
(Figs. 4 and 5) combined with the high turnover rate of the target
tissue (digestive epithelia) infected by the WS-bacterium relative

lo the bacterium's growth rate. Significant correlations between
intensity of RLP infection and degree of WS have recently been
observed in both field and laboratory studies using wild and cul-
tured red abalone (Moore et al. 2000. Friedman, unpublished ob-
servation). The authors also suggested that differences in correla-
tions between intensity of RLP infection and disease in red and
black abalone might relate to species differences in host response
to infections. Red abalone respond to the RLP infections predomi-
nantly by a metaplastic change in which digestive gland tubules
are replaced by transport duct epithelium (Moore et al. 2000).
Black abalone respond to RLP infection by a combination of di-
gestive tubule degeneration and. to a lesser extent, transport duct
metaplasia (Gardner et al. 1995. Friedman et al. 1997) (Figs. 4 and
5). Both of these tissue changes result in a loss of key functional
tissue in the digestive gland, the terminal tubules (Voltzow 1994),
which may lead to starvation and account for the utilization of foot
muscle as an energy source followed by death as observed in
abalone with WS (Friedman unpublished data.). As the RLP in-
fects transport duct epithelia and not terminal digestive tubules,
this may result in an increase in RLP intensity of infection in red
and not in black abalone as the infections progress and clinical
disease develops. Figure 5, however, does illustrate alterations in
condition indices and changes in the condition of the foot and
digestive gland only in abalone with RLP infections. This provides
further evidence that the RLP is the etiological agent of WS. RLPs
have been associated with atrophy and degenerative changes in
other invertebrate species (Min & Benzer 1997).

Infections with RLPs have been reported in a variety of mol-
luscs and crustaceans, including the sea scallop, Placopecten ma-
i;ellaiuciis Gmelin (Gulka & Chang 1984al. the blue mussel, Myti-
his ediiUs Linne (Gulka & Chang 1984b), the manila clam. Tapes
japonica Adams and Reeve and the Japanese scallop, Patinopecten
vessoensis (Elston 1986), the European flat oyster, Ostrea edulis
Linntf (Friedman et al. 1989), the black abalone. Haliotis crach-
eroclii Leach (VanBlaricom et al. 1993). and the penaeid shrimp.
Penaeiis inaiginiiliis Randall (Brock et al. 1986). These infections
varied greatly in tissue specificity (nonspecific to highly specific),
length of incubation period, and pathogenicity, ranging from no
apparent harmful effects to lethal effects in the host (Gulka &
Chang 1984a. Brock et al. 1986, Frelier et al. 1993. Gardner et al.
1995, Bower et al. 1996). In addition, the pathogenicity of a spe-
cific RLP has been shown to vary between host species (Brock et
al. 1986). As in these studies, which document that RLPs are
pathogenic for marine invertebrates, our data provides evidence
that "Caiulidatiis Xenohaliotis californiensis," the recently identi-
fied RLP observed in abalone in California, is the etiological agent
of WS. Future studies that examine the interaction between host
gastrointestinal cells and the RLP may provide insight into the
cellular physiology of the host and the physiology and disease
mechanisms of the bacterium.

ACKNOWLEDGMENTS

We appreciate the editorial comments of James D. Moore. This
work was supported, in part, by the National Sea Grant College
and the Saltonstall-Kennedy Programs of the National Oceanic and
Atmospheric Administration, U.S. Department of Commerce un-
der grant numbers NA36RG0537, Project No. R/F-153 (through
the California Sea Grant College Program) and NA76FD0046,
respectively. Additional support was provided by the California

824

Friedman et al.

Slate Resources Agency, California Department of Fish and Game and do not necessarily reflect the views of NOAA or any of its
and the Aquaculture and Fisheries Program, University of Call- subagencies. The U.S. Government is authorized to reproduce and
fornia, Davis. The views expressed herein are those of the authors distribute this work for governmental purposes.

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HISTOPATHOLOGICAL EVALUATION OF THE YELLOW ABALONE HALIOTIS CORRUGATA
AND THE BLUE ABALONE HALIOTIS FULGENS FROM BAJA CALIFORNIA, MEXICO

MA. DEL CARMEN ALVAREZ TINAJERO,' JORGE CACERES-MARTINEZ,'* AND
JOSE GUADALUPE GONZALES AVILES'

^ Laboratorio de Patologia de Mohtscos del Departamento de Aaiiciilliini. Ceiitro de liiveslii;acidn
Cientifica y de Ediicacidii Superior de Ensenada, Apdo. Postal 2732. 22S00 Ensenuda. Bcija Ccdifornia.
Mexico: 'Sociedad Cooperativa de Produccidn Pesquera. Pescadores Nucionales de Ahuldn. S. C. de R.
L. Av. Ryerson 1 17. 22820 Ensenada. Baja California. Mexico

ABSTRACT The yellow abalone Halioris corrugala and the blue abalone Haliotis ful,^eiu are caught in Baja California. Emerging
diseases are affecting the fishery of abalone in several countries around the world. To determine the health status of the yellow and
the blue abalone in Isia de Cedros and Islas San Benito in Baja California. Mexico, a histopathological survey of commercial stocks
of these species was conducted. The results showed the presence of bacterial foci in epithelial cells of the digestive tract resembling
to the intracellular bacterium "CiuuliJulus Xenohaliotis califoniiensis." considered to be the etiological agent of the Withering
Syndrome (WS). The prevalence was higher in the blue abalone than in the yellow abalone. 100% and 63'7r. respectively; and their
presence was not correlated with the external signs of WS. These bacteria were found in WS symptomatic and nonsymptomatic
abalone. Protozoans and copepods were found between the branchial filaments, with a maximum prevalence of 79% and 37%.
respectively; gregarines were also found with a prevalence of 29%. With the exception of the intracellular bacterium, the other
organisms seem to he innocuous for the studied abalone.

KEY WORDS: abalone. Hulions fiili-ens. Halialis corrugatci. histopathology. Rickettsia, withering syndrome

INTRODUCTION

Abalone production in Baja California (Mexico) was 365 met-
ric tons in 1998. reaching a value of about 36 million US dollars.
The abalone fishery has been one of the main sources of economic
resources in the Peninsula of Baja California. Cunently. there are
more than 1.300 direct employees involved in this activity (Ponce
et al. 1998). Two species comprise 97% of this fishery, the blue
abalone Haliotis fulgens and the yellow abalone Haliotis corrii-
gata. Other abalone species included in this production are the
black abalone Haliotis cniclieiodii. the red abalone Haliotis nife-
scens. and the white abalone Haliotis soreiiscni (Ramade et al.
1998). During the last few years, an important decrease in the
production has been recorded, which has been related to overfish-
ing, inefficient application of management regulations, and envi-
ronmental fiuctuatii)ns that have favored the development of some
diseases (Haaker et al. 1992. Vanblaricom et al. 1993, Olivas-
Valdez & Caceres-Marti'nez 2002). In 1984, dramatic mortalities
of California black abalone were recorded after the occurrence of
El Nifio. Lately, it has been suggested that the agent responsible
for those mortalities was an intracellular bacteria from the order
Rickettsiales (Gardner et al. 1995), tentatively named "Candidatus
Xenohaliotis califomiensis" (Friedman et al. 2000, Moore et al.
2000, Friedman et al. 2002). External signs in the abalone include
reduction of the foot muscle in relation to shell size, loss of ad-
herence capacity, and death. Because of the foot muscle appear-
ance, these symptoms are called withering syndrome (WS)
(Haaker et al. 1992). It has been suggested that very high tem-
peratures may increase the mortality of abalone affected by the WS
(Friedman et al. 1997). Other parasites that have been recorded in
abalone species are the protozoan Margolisiella ( = Pseudoklns-
sia) haliotis, which infect the kidney and was first associated with
the WS (Friedman et al. 1995). and the eukaryotic protist Laby-

*Corresponding author. E-mail: jcaceres(a'cicese.mx

riiitliiiloides haliotidis. which is pathogenic to juvenile abalone of
H. kamtschatkana and H. rufescens (Bower 1987). Metazoan para-
sites such as the nematode Echinocephalus pseudoimcinatus have
been detected in H. cnrnigata. H. fulgens. and H. cracherodii
(Milleman 1963). Damage to the host by these parasites varies and
some of them cause severe mortalities (Bower et al. 1994).

Studies of the parasites and symbionts in abalone from Mexico
are scarce. These studies revealed a trematode belonging to the
family Opecolidae in H. fulgens (Romero 1996), a boring clam in
H. fulgens and H. corrugata (Alvarez-Tinajero et al. 2001 ). and the
parasitic load of H. rufescens from a culture facility (Caceres-
Marti'nez & Tinoco-Orta 2001). The present study was conducted
as part of a health evaluation of a commercial catch of H. fulgens
and H. corrugata from Isla de Cedros and Islas San Benito. Baja
California.

MATERIALS AND METHODS

In the first sampling (November 1997), 19 individuals of H.
corrugata and 16 individuals of H. fulgens were obtained from
Punta Norte, north of Isla de Cedros; in March 1 998. 26 H. fulgens
were studied from Islas San Benito; finally, in June 1998. 21 H.
corrugata and 14 H. fulgens were obtained from San Agusti'n.
south of Isla de Cedros (Fig. 1). Abalone species were obtained
from commercial catches (mean size of 145 mm ± 1.73 SD) during
commercial season when abalones are not in the reproductive
stage. The external appearance of the abalone was recorded from
a visual examination using a modification of the WS scale of
Friedman et al. (1997) as: (0) no external symptoms, (1) from I to
lOVr reduction of the foot muscle in relation to the shell, (2) from
II to 20% reduction of the foot muscle. (3) from 21 to 30%
reduction, and finally (4) from 31 to 50% reduction of the foot
muscle.

The visceral mass of the abalone was fixed whole in the David-
. son's fixative (Shaw & Battle 1957) for at least 24 h. Seven trans-
verse sections that contained portions of the digestive tract (pos-

825

826

Carmen Alvarez Tinajero et al.

San Agustin

RESULTS

Figure 1. Map showing the sampling sites in Isla de Cedros and Islas
San Benito, Baja California, Mexico.

terior esophagus included), kidney, gonad, muscle, epipode, and
gills were processed for histology. Sections of 5 |xm were stained
with iron hematoxylin and eosin (Gray 1934). The rickettsia-like
prokaryotes (RLPs) infection intensity was estimated considering
the number of bacterial foci in each histologic preparation at 200x
magnification, which were enumerated using a modification ot the
scale of Friedman et al. (1997): (0+) no RLP. (1 + ) 1 to 10 RLP.
(2+) 11 to 100 RLP. (3+) 101 to 1000 RLP. and (4+) >1000.
Moreover, following the criteria of Friedman et al. (1997). the
appearance of the digestive gland was marked as ( 1) normal tissues
(see Antonio et al. 2000). (2) moderate tissue degeneration, and (3)
severe tissue degeneration. Protozoa in the digestive tract and gill
branchiae were counted in each histologic preparation. Prevalence
of the other organisms was estimated as (number of infested aba-
lone/number of abalone examined) x 100. The Mann-Whitney U
Test was used to compare differences in the intensity of organisms
per abalone species and the Spearman rank order coirelation test
was used to determine the relationship between degeneration of the
digestive gland, intensity of RLP. and external signs of WS (Zar
1984).

RLPs

The RLPs were found in H. fidgens and H. cornigata in the
three study sites. These bacteria infected the epithelial cells of the
digestive tract, including the posterior esophagus, stomach, diges-
tive diverticula, and intestine (Fig. 2a and b). The RLPs formed
colonies inside a large vacuole in the cytoplasm of the infected
cells. These colonies varied in shape from round to elongated and
their size ranged from 17.5 x 10.8 [xm to 24.3 x 10.2 |xm. The host
cell nucleus was displaced to the limit of the cytoplasm, the host
cell became hypertrophied. and in a heavy infection there was a
metaplasia of the epithelium within the digestive gland. Some host
cells were disrupted and bacterial colonies were expelled to the
lumen of the digestive tract (Fig. 2a).

Table 1 shows the RLP intensity of infection in H. fulgens and
H. cornigala per studied site. The yellow abalone from Punta
Norte showed a RLP prevalence of 63.2%; of these, 52.6% had a
severe infection (3-i- to 4+). including a moderate degeneration (2)
of the digestive gland in 41% of the animals, whereas the remain-
ing 52.6% of the abalone did not show signs of degeneration of the
digestive gland. Blue abalone from the same area showed a RLP
prevalence of 43.7%. with severe infection (3+ to 4+) in 31.3% of
the animals. Only 12.4% of the infected abalone showed a mod-
erate degeneration of the digestive gland (2). The results showed a
higher grade of infection in yellow abalone than blue abalone. but
this was not significant statically (U Mann-Whitney. P = 0.33).
There was no significant correlation between the degree of degen-
eration in the digestive gland and the intensity of infection by RLP
in the yellow and blue abalone species (Spearman rank order cor-
relation ;■ = -0.19. P = 0.93 and r = 0.34. P = 0.18. respec-
tively). This correlation was neither significant between the exter-
nal signs and degeneration degree of the digestive gland (Spear-
man rank order correlation r = 0.36. P = 0.12 and /• = -0.23,
P = 0.37).

The blue abalone from Islas San Benito showed a RLP preva-
lence of 96.2%. and the infection was severe (3-i- to 4+) in 92.4%
of infected abalone. In 56.2% of these abalone the digestive gland
was normal whereas the remaining 43.8% showed a moderate

Figure 2. (a) RLP in the intestinal epithelium. An RLP-infected area is shown where the epithelium of the post-esophagus has lost its structure
by hypertrophy of infected cells and rupture (lA). The epithelium in front is in normal omdilion (HEl. There is mucus or cellular debris (M)
in the intestinal lumen. Scale bar = 40 fim. (bl RLP in digestive diverticula. The hypertrophy of infected cells is compressing the neighbor cells.
Scale bar = 20 pm.

Evaluation of Haliotis corrugata and H. fulgens

121

TABLE 1.

Intensity and pre>alente of RLP in H. corrugata and H. fulgens
from different localities studied.

TABLE 2.
External signs scale of the WS in H. corrugata and H. fulgens.

Punta Norte

Islas San

San Agusti'n

Punta Norte

San Agusti'n

H

H

H

H

H.

H.

Islas San

H.

H.

Scale

corrugata

fulgens

Benito H.

corrugata

fulgens

corrugata

fulgens

(<7r)

Benito H.

fulgens ( % )

corrugata

(%)

fulgens

VVS

(%)

{Vc)

fulgens ( % )

(%)

{%)

Scale

0

1

89.4
5.3

75.0
18.8

88.5
0

47,6
4.8

71 6

0

36.8

56.3

3.8

38.1

0

7,1

1

5.3

6.2

0

4.8

21.4

-)

5.3

6.2

7.7

28.6

0

2

5.3

6.2

3.8

42.8

21.4

3

0

0

3.8

19.0

14.2

3

42.1

18.8

46.2

14.3

35.8

4

0

0

0

0

7.1

4

10.5
63.2

12.5

43.7

46.2
96.2

0
61.9

21.4
100

/;

19

16

26

21

14

Prevalence

//

19

16

26

21

14

degeneration (2). There was no significant correlation between the
presence of RLP and degeneration of the digestive gland (Spear-
man rank order correlation /• = 0.34, P = 0.08) or between WS
external signs and the degeneration degree of the digestive gland
(Spearman rank order correlation r = 0.33. P = 0.08).

The yellow abalone from San Agusti'n showed a RLP preva-
lence of 61.9% and the infection intensity was medium (2+) in
42.8% of the organisms. In relation to the degeneration of the
digestive gland. 57.1% of the infected organisms had a moderate
degeneration (2), 14.28% had a severe degeneration (3), and
28.6% had a normal digestive gland. There was no significant
correlation between the RLP intensity and degeneration degree of
the digestive gland (Spearman rank order correlation r = -0.03. P
= 0.86). However, a sign correlation was observed between the
external signs of WS and the degree of digestive gland degenera-
tion (Spearman rank order correlation r = 0.75. P < 0.001). The
blue abalone from the same area showed a RLP prevalence of
100%, with severe infections (3-i- to 4-i-) in 57.2% of these abalone.
Of the severely infected abalone, 28.5% showed a moderate de-
generation of the digestive gland, 14.28% showed severe degen-
eration, and 57.14% showed a normal appearance of the digestive
gland. In both species, the correlation between the RLP intensity
and degeneration of the digestive gland was not significant (Spear-
man rank order correlation ;■ = 0.08. P = 0.75), but it was sig-
nificant between the WS external signs and the degeneration of the
digestive gland (Spearman rank order correlation /■ = 58.36, P <
O.OOI). The infection prevalence from San Agusti'n was signifi-
cantly higher in the blue abalone (U Mann-Whitney P < 0.01 ) hut
not from Punta Norte (Table I).

Table 2 shows the percentage of both abalone .species with
external signs of the WS. At the microscopic level, all these or-
ganisms showed some tissue disorders, such as an increase in
connective tissue between digestive tubules; lack of gonadal matu-
ration and in a few occasions duct metaplasia or intlammation. The
foot muscle showed a reduction in muscle fibers and loss of
muscle bundle orientation, increase in connective tissue and infil-
tration by hemocytes. There was no correlation between the WS
external signs and the presence of RLP in tissues (Table 3); in
some individuals with WS external signs, infection by RLP was
not detected (Fig. 3).

Ciliated Protozoan

A ciliated protozoan (Fig. 4a) was found in the mantle cavity
and gill filaments of the yellow and blue abalone. The unidentified

ciliates measured 26.5 |jim x 12.7 \x.m. No histologic disorders or
lesions were associated with these organisms. Prevalences were
low in both species with 10.57f of the yellow and 6.3% of the blue
abalones from Punta Norte (Table 4. Mann Whitney U, P = 0.66).
Prevalences were higher at the other two locations where over 40%
of the blue abalone from Islas San Benito and over 70% of both
yellow and blue abalones from San Agusti'n were infested (Table
4. Mann Whitney U. P = 0.25).

Copepods

Copepods were found among the filaments of the gill where
some compression and infiltration of hemocytes in the tissue was
detected (Fig. 4b). The mean size of the copepods was 200 (xm x
50 |j,m. Copepod prevalence in the yellow abalone from Punta
Norte was 36.5%, there were no copepods in blue abalone from
this locality. Copepod prevalence in blue abalone from Islas San
Benito was 34.6%. The yellow abalone from San Agusti'n showed
a prevalence of 19.0%, in the blue abalone only one copepod was
recorded. There were no significant differences of copepod inten-
sity between both abalone species (pooled data, U Mann-Whitney
P = 0.34).

Gregarines

Low prevalences of trophozoites of a gregarine protozoan (Fig.
4c) were observed in the branchial epithelium, esophagus epithe-
lium and kidney of the blue abalone (Table 4). The organisms
measured 22 jxm x 14(jLm and no host reaction against this proto-
zoan was observed. Prevalences ranged between sites from 0 to a
high of 28.7% (Table 4). The limited number of data precluded the
use of a statistical test for comparison.

TABLE 3.

Spearman rank correlation between the WS external scale and the
abundance of RLP in H. corrugata and H. fulgens.

Punta Norte

Islas San Benito
H. fulgens

San Agusti'n

H. H.

corrugata fulgens

H. H.

corrugata fulgens

R

P value

0.05 0.06
0.82 0.79

0.18
0.37

-0.09 -0.008
0.68 0.97

828

Carmen Alvarez Tinajero et al.

O Abalone without WS

■ Abalone without WS and with RLP

■ Abalone with WS

□ Abalone with WS and RLP

H. commala H. fulaens _H. I'uluens ii. corru^ata H. fulgens

Punta Norte Islas San Benito San Agustin

Figure 3. Percentage of the WS symptomatic and nonsymptomatic abalone and the intensity of RI>P infection lor Haliotis corriigata and Haliotis
fulgeiis.

«

'-* G

Figure 4. (al filiated protozoan (I'l bet»een branchial gill lilamenls. Scale bar = 20 Mm. (bl Copepod between gill lllaments (arrow). Note the
intlltration of hemocytes in lilamenls surrounding the copepod (IN). Scale bar = 40 urn. (c) (Jregarinc protozoan ((i) in the right kidney of H.
fulgens. Scale bar = 15 jim.

Evaluation of Haliotis corrugata and H. fulgens

829

TABLE 4.
Other organisms in abalone H. corrugata y H. fulgens per locality.

Locality

Ciliated proto/oan
Measured: 26.5 x 12.7 |xni
Location: Mantle cavity and gil
Punta Norte

filaments

Islas San Benito
San Agusti'n

Copepods

Measured; 200 x 50 jjim
Location: Filaments of gill
Punta Norte

Islas San Benito
San Agusti'n

Gregarines

Measured: 22 x 14 \km
Location: Branchial, esophagus, and kidney
Punta Norte

Islas San Benito
San Agusti'n

Abalone
Species

H. corrugata
H. fulgens
H. fulgens
H. corrugata
H. fulgens

H. corrugata
H. fulgens
H. fulgens
H. corrugata
H. fulgens

Mean

H, corrugata

O.I

H. fulgens

0.2

H. fulgens

4.6

H. corrugata

62.7

H. fulgens

11.1

1.0

0.5
0.3
0.1

0.06

(1.07

0.4

Prevalence

10.5

42.3
71.4
78.5

36.5

34.6
19.0
7.1

6.2
3.8

28.5

Max.
Intensity

2

4

28

594

346

DISCUSSION

Caceres-Marti'nez and Tinoco-Oila (2001) and Culver and Ri-
chards (1992) mentioned that the WS was observed in the black
(Haliotis cracherodii) green (H. fulgens) and red {H. rufescens)
abalone from Baja California. Mexico; however, this is the first
record of RLP infecting the tissues of H. fulgens and H. corrugata.
The histologic appearance, shape, size, and targeted cells are simi-
lar to that of the intracellular bacterium "Catulidatus Xenohaliotis
califomiensis" causing the WS in H. cracherodii and H. rufescens
in California. (Friedman et al. 2000). Further characterization is
needed to confirm this hypothesis, therefore molecular methods for
RLP detection are necessary (Andree et al. 2000. Antonio et al.
2000).

The absence of a relationship between the intensity of RLP
infection with the external signs of WS and the degeneration de-
gree of the digestive gland in both abalone species was previously
observed in studies on WS in black abalone (Friedman et al. 1997,
Friedman et al. 2002). Moreover, Caceres-Marti'nez and Tinoco-
Orta (2001) found changes of the digestive gland (metaplasia) in
red abalone infected by RLP but also in noninfected organisms.
Only in San Agusti'n was the relationship between the WS external
signs and the degeneration of the digestive gland significant. Fried-
man et al. (1997) and Friedman et al. (2002) showed that the WS
disease agent requires a long incubation period before the externa!
symptoms become apparent. This could help us to explain this
contradictory result. Another possibility is the existence of sub-
species or strains of RLP, some pathogenic and other nonpatho-
genic. Moreover, the external signs of the WS are not exclusive of
this disease, other diseases or starvation conditions may result as
external symptoms similar to those of the WS (Milleman 1963,
Dixon et al. 1991); also, differences in susceptibility between aba-

lone species and individuals are possible. Friedman et al. (2002)
noted "studies that examine the interaction between host gastroin-
testinal cells and the RLP may provide insight into the cellular
physiology of the host and the physiology and disease mechanisms
of the bacterium."

Vanblaricom et al. (1993) also found a suctorian protozoan in
gill squashes from the black abalone H. cracherodii. both in
healthy specimens and in organisms affected by the WS. Ciliated
protozoa are common in marine invertebrates, and most of them
are considered as commensals (Lauckner 1983). Our histopatho-
logical results did not show any host response to the protozoa, and
the low numbers suggested a lack of potential for a health problem.

To our knowledge, this is the first record of a copepod associ-
ated with abalone species. Copepoda and mollusk associations
have been widely studied, Mytilicola intestiiudis and P.^eiidomyi-
cola spinosus are two of the most studied copepods associated with
bivalve mollusks. These copepods inay produce severe damage to
the epithelial cells of the digestive tract, they may produce encap-
sulations in the connective tissue of the digestive gland, and they
may produce obstruction and rupture of reproductive follicles (Sin-
dermann 1990. Olivas-Valdez & Caceres-Marti'nez 2002). In this
study, no evidence of copepods inside of the abalone digestive
tract was found, but some inflammation in the gills of abalone was
observed in some cases. Their prevalence suggests a symbiotic
association but more studies are needed for determining a kind of
interaction between the copepods and the host.

In the blue abalone, we recorded the presence of gregarines in
the renal tissue and no histologic evidence of reaction of the host
against the protozoan was observed. Some authors (Vanblaricom
et al. 1993) found similar protozoa in the black abalone. Haliotis
cracherodii from California and from Baja California, Mexico. In

830

Carmen Alvarez Tinajero et al.

both studies, no evidence of pathogenic activity was found. A
similar gregarine infection has been observed in mussels, oysters
and other marine bivalves, in some cases the infection has been
associated with a focal, benign inflammatory response, without
significant health effects (Bower et al. 1994).

AKNOWLEDGMENTS

The authors thank Rafael Muiio/ Gallardo and Amado Castro
from the Cooperative Pescadores Nacionales de Abulon for an
agreement with CICESE for the financial support of this work.

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abalone Haliotis cracherodii Leach, 1814, populations at San Nicolas
Island, California. J. Shellfish Res 12:185-188.

Zar. J. 1984. Biostatistical analysis, 2nd ed. Englewood Cliffs. NJ: Pren-
tice-Hall. 178 pp.

Journal of Shellfish Research. Vol, 21. No. 2, 831-834. 2002.

EFFECT OF EL NINO 1997-98 ON THE SNAIL ASTRAEA UNDOSA (WOOD) POPULATION
ALONG THE BAJA CALIFORNIA WESTERN COAST

MARIA GEORGINA GLUYAS MILLAN,*' CASIMIRO QUINONEZ VELAZQUEZ," AND
JESUS TALAVERA MAYA'

'in.stituta Nacional de La Pesca-SAGARPA, Centra Regional de Investigacion Pesquera-La Paz,
Carretera a Pichilingue Km 1 s/n. C.P. 23020, La Paz. B.C.S., Mexico: 'Centra Interdisciplinario de,
Ciencias Marinas-bistitiito Politecnico Nacional. Apdo, Postal 592. C.P. 23000, La Paz, B.C.S.. Mexico.

ABSTRACT The effect of the 1997-98 El Niiio on the snail Astraea undosa population along the Baja California western coast on
biomass changes in June 1997 and July 1998 was evaluated. In 1998. dead snails >40 mm basal diameter were collected and their basal
diameter measured. Individual weight and density (snail,s/10 nr) decreased significantly in 1998 from those registered in 1997, with
an average 47% decrease in biomass. We discuss the possible causes of those changes as a function of the disappearance of the
Macrocystis pyrifera forest and of an e.xtended period of positive anomalies of the sea surface temperature that were present previous
to July 1998.

KEY WORDS: El Nifio 1997-98, snail, Aslraea undosa. biomass, Baja California.

INTRODUCTION

The 1997-98 El Nifio has been considered the most intense of
the twentieth century (Kerr 1998, McPhaden 1999). It was de-
tected along the Baja California western coast through positive
anomalies of the SST >3°C. In July 1998. we observed dying as
well as dead snails Aslraea undosa (Wood) in the imertidal zone
and in fishing banks. No kelp forests M. pyrifera (Agardh) were
found in Bahia Tortugas from October 1997 to April 1998 (Ladah
et al. 1999). This species together with Eisenia arborea (Aresch)
are the food source and habitat of A. undosa. A. turlxviica (Dall),
and other species of herbivores of commercial interest such as the
abalone Haliotis fulgens (Philippi) and H. ci>rrus>atu (Wood)
(Guzman del Proo et al. 1991 ).

In July 1998 we monitored the A. undosa populations located
on the Baja California peninsula western coast to evaluate the
effect of the 1997-98 El Niiio. This region carries the highest
abundance of A. undosa. where >80% of its catch is obtained
(Gluyas-Millan et al. 2000). The size structure of live and dead
snails, average size and weight, density and biomass of July 1998.
were contrasted with those obtained from the same fishing banks
in June 1997. We assumed that the sampled snails in July 1998
reflect the effect of the 1997-98 El Nino.

MATERIALS AND METHODS

The study area is located between Punta Eugenia (27°5rN-
115'04'W) and Bahi'a Asuncion (27°08'N-1 14'^I7'W) (Fig. 1).
The area was divided into three zones and three fishing banks were
selected out of each one (Table 1 ). We believe the selected geo-
graphical area was adequate because it is the main area of abun-
dance of A. undosa in its distribution range along the Baja Cali-
fornia western coast, and also because of the accessibility of the
sampling places, and because this is where the main snail fishing
banks are located. Monitoring was done in June 1997 and in July
1998 in the same places. As an environmental indicator of the 1997
to 1998 El Nino conditions we used the SST monthly anomalies of
1996 to 1998 from the deviations of the monthly average of the
ten-year period before 1997 for the Bahi'a Tortugas area. The SST

values were extracted from NOAA COADS records (Comprehen-
sive Ocean and Atmospheric Data Set) for the Bahi'a Tortugas area.

The comers of the polygons that comprise the area fishing
banks were estimated using a GPS receiver (Gamiin model 45XL)
(Table 1 ). The monitoring in June 1997 and July 1998 consisted of
conducting up to 10 dives at each fishing bank (a, b, c), using a
Hooka diving equipment at depths between 5 and 20 m. In each
dive, all snails inside a 10-m"-sample unit (SU) were taken and the
basal diameter (BD) of each snail was measured, grouping them in
intervals of 5 mm to obtain the size frequency distribution. A
subsample was obtained by randomly selecting up to five snails of
each size interval and measurements were made of the BD of the
shell (±1 mm) and the weight (±1 g) of the adductor muscle to
estimate the biomass.

The area of each fishing bank was transformed to SUs. Snail
density for each fishing bank was stratified according to the snail
number frequency distribution in each SU (Cochran 1978). The
number of density intervals (DI) was determined with the Sturges
rule (Daniel 1984).

An estimate of the population total abundance of snail was
obtained according to the estimators of the random stratified sam-
pling technique (Schaeffer et al. 1987, Conquest et al. 1996):

Average population estimate was

A^

E ^. y,

Total abundance population estimate was

Nv,

S ^. ■^'

Where N = total number of SU, N, = SU number in the fishing
bank,, L = number of density intervals, / = density interval av-
erage.

From the total population abundance estimation, the biomass
was calculated by size interval with the following relationship:

BIT ={aJ^)* ^N,y, * <i>r

*Corresponding author. E-mail: gmillan@balandra.uabcs.m,\

where BIT = size interval biomass. a and p = weight-size rela-
tionship parameters, (Xf= i N, y,) = biomass in snail numbers, 0,

8.31

832

Gluyas Millan et al.

Punta Eugenia

\ r

^

,.\-

^

\ \

A

\__^

Bahia"~- ^

^\^^

)\K^

"27°43' ^

"Tortugas

^

'-J

~-T~

\^^

•v^— -^

^

Ic ^\

\

\\\% c

lla ^

^_^^ Bahia

San\ ] \ ""

^^^ristobal "^^''^^^ |

lib N.

\
\

PACIFIC

lie.

Isia San^

OCEAN

L

iii^ •

-^ Roque

27°13'

lllbSN/ ^

lllc Bahia

114°5r
1

114°28' Asuncion
1

Figure I. Baja Calitornia Hestern coasC and liicalion of sampling sites.
The names of tlie sampling locations are in Table I.

= relative frequency accumuluted by size interval from total snails
measured.

The total biomass for all the size intervals was estimated by:

Bt

^BIT

To determine the empty shells of snails from individuals that died
in 1998, and that did not come from the commercial catch of that
year or earlier, observations of presence-absence of fauna and flora
epibiotics were made inside the shell. Additionally, we know thai
the snails are not killed in the fishing banks, and furthermore the
snail-fishing season was suspended in 1998. except in Bahia Tor-
tugas and Isla Natividad. The latter is 7.5 km NW off Punta Eu-
genia (Fig. 1).

RESULTS

In July 1998 we observed that A. undosa snails at depths of 5
to 20 ni were dying. In addition empty shells were found in the
intertidal zone, and few M. pyrifera stalks between Punta Eugenia
and Bahia Asuncion. July 1998 was preceded by a period of in-
tense warming, the SST rose to 26°C (Fig. 2A). SST positive
anomalies >3°C persisted from September 1997 to January 1998
(Fig. 2B) and the M. pyrifera forests disappeared in Bahia Tortu-
gas. The June 1997 monitoring was not preceded by conditions of
intense warming (Fig. 2A. B). and the snails showed normal con-
ditions, the appearance of the adductor muscle was not tlabby as in
July 1998; dead snails were not observed, and the M. pyrifeni
forests were present.

Dead snails in the inlcilida! area in July 1998 showed remains
of viscera, a flabby adductor muscle, the opercula fixed to the
muscle, and absent epibiotic fauna and flora in the shell internal
surface. This indicates that these snails were not the product of
fishing and that they had recently died.

In each sampling zone in July 1998. the range and the size
frequency distribution of the living as well as of the dead snails
were similar (P > 0.05. KS-test) (Fig. 3): indicating the 1 997-98 El
Nifio affected proportionally those snails whose BD was >40 mm.
This result is reinforced when contrasting the size frequency dis-

TABLE I.

Astraea undosa fishing hanks by zone on the Baja California
western coast. See Figure 1 for geographic location.

References above the Fishing

Fishing Banks

Zone

Banks Location

Dimensions (nr)

1 a

La Banderita-Punta Quebrada

.118.(10(1

1 b

Punta Priela-La Cantina

318.000

Ic

Los Morros-Clam Bay

318.000

lla

Cabo Pruneda-Cerro del Calvillo

104.000

II b

El Muerto-Punta Prieta-Salado

124.000

lie

San Pablo

136000

III a

Punta .San Pablo-Puerto San
Pablo

51.000

lllh

Vuelta del Cerro-Reef-Crucecita

63.000

IIU

Isla .San Roque

75,000

tribulion of 1997 and 1998 (Fig. 3). which showed no significant
differences (P > 0.05. KS-test 1. This suggests that the effect of El
Nino 1997 to 1998 was not selective for snails >40 mm BD.

In 1997 and 1998 the mean BD of the live snails by zone was
not significantly different (P > 0.05. r-test) (Fig. 4). This confirms
the previous results that indicate an effect on the population with-
out size selectivity. The mean individual weight, density, and bio-
mass were significantly greater in 1997 than in 1998 (Fig. 4) {P <
0.05. /-test). This shows a negative effect on individual weight and
density of the snail and in consequence the 1998 biomass de-
creased. The magnitude of the biomass was variable among the
zones because of the different area sizes (Table 1 ). The biomass
decreased 47% on average in 1998 as compared with that regis-
tered in 1997. Because of this decrease, the commercial catch
season was suspended in 1998 in most of the study area.

Observations made in June 1998 of the A. undosa stomach
contents indicated important proportions of silt and crustose cor-

_ 27
O

a; 23

3

re

E
H 15

o

OS

E
o
c
m

Q)

re

c
re

^

■d
O

c
re

^

O

c
re

Q.

<

O

0)
Q.

E

4 n

ft^^^^^^ B

2 -
0-
2-

-3 A^^ ,a ^ ^.-A

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 '

—5

Q. 3
< -^

o ^

O. 3 U

< ^ o ^

re

a. 3

t3

O

1998

1996 1997

Figure 2. i.\) Monthly sea surface temperature (SSI), and (B) SST
anomalies from 1946 to I^VS. Data obtained from NOAA COADS
records of the Bahia Tortugas area.

El Nino Effect on the Astraea undosa Population

833

35
30
25

□ live snail 1998

-^dead snail 1998

■i live snail 1997

Zone I

40 50 60 70 80 90 100 110 120 130 140
Basal diameter (mm)

Figure 3. Size frequency distribution of the snails Astruea undosa from
the Baja Cahfornia western coast in June 1997 and July 1998.

alline algae (Lithothamnium or Lithophylwn). articulated coralline
algae (Bossiella orbigniana [(Manza) Johans], and Coratlina offi-
cianalis [L.]). They were the predominant flora then and are of
temperate and subtropical affinity (Guzman del Pioo et al. 1991 ).
These algae covered a great extension of the rocky surface at all
the levels of the sampling depths. In June 1997 the diet compo-
nents were varied, and we observed in the snail stomachs an im-
portant proportion of M. pyrifem. which was still present in the
area (Raiil Reyes Tisnado. pers. com.). It was not possible, how-
ever, to quantify the stomach contents.

DISCUSSION

Though the 1997-98 El Nifio was detected from summer 1997
in the northern Pacific Ocean (Liu et al. 1998. Connolly & Rough-
garden 1999). its impact on the A. undosa population along the
Baja California western coast was not evident until the summer of
1998, when it became obvious that the biomass of snails had
decreased and dead snails were seen. These events were also re-
ported at La Bocana. 70 km south of Isla .San Roque (Daniel
Aguilar Osuna. pers. com.).

The results and the observations suggest that the observed mor-

^1201

• 1997

g

diameter

00 o

o o

" 1

CD

I/)

CD

^ 60 J

P=0 21

1

o 1998

t

P = 0 45

P=0 32

80
ro 60 -
CT 40
^ 20

0

cT 50
E
o 40

I 30

^ 20

« 10

CD
D

P = 0 02

P=0 03
I 1

0

P<0 01

P=0 01

P<0 01

i

\ \

P<0 01

40 n

■Z: 30

m 20H

CD

E
o

CO

10

\

P<0 01

30

20

10

0 4

\

12n

P<0 01

P<0 01

{

Zone

Zone

Zone

Figure 4. Average and standard deviation of the basal diameter (mm I.
individual weight (g), density (snails/l(l nr), and biomass III by sam-
pling zone of the snail Aslraea undosa from the Baja California west-
ern coast in June 1997 and July I99S.

tality and the diminished biomass that occurred in 1998 were
caused by weakness and starvation because of the absence of M.
pyrifem, and a possible long-warming period temperature effect
that contributed to the disappearance of kelp forest (Ladah et al.
1999). Nearly all snails >40-mm BD are mature individuals, be-
cause the minimum reproductive size is attained at 58-mm BD
(Belmar-Perez et al. 1991). Those snails would be >2-years-old
according to the BD-age relationship reported by Gluyas-Millan et
al. (2000). The individuals <40 mm-BD are in the cryptic phase
(Gluyas-Millan et al. 1999, Gluyas-Millan et al. 2000) and they
were not observed in the study period.

The biomass decrease of the A. undosa population from 1997 to
1998 was the result of both an increased natural mortality and

834

GLUYAS MlLLAN ET AL.

decreased individual vveiL'hl and can be attributed to two factors: 1 )
The prolonged disappearance of the forests of M. pyrifeni from
October 1997 to April I99S (Ladah et al. 1 999 ) affected the habitat
and the food availability of A. imdosa. This alga is one of the main
food sources of /\. iindosa (Guzman del Proo et al. 1991) and also
provides protection against predators; and 2) The physical factors
associated with 1997-1998 El Nifio had a density-independent
influence because their potential effect in each member population
>40-mm BD is the same regardless of population size (Sissenwine
1984). Therefore, the increase of SST and the change of the pre-
dominant diet coinponents could have affected the mollusk physi-
ology and consequently its growth efficiency.

The decrease of the 1998 snail biomass resulted in an important
economic loss for the fishing sector. Because of the low yields and
the poor quality of the adductor muscle fishing ceased at most of
the fishing banks. These fishing yields decreased from 166 t in
1997 to44 t in 1998.

El Nino negative effects have been documented in the eastern
Pacific Ocean on the productivity, abundance and distribution of
invertebrates, the recruitment failure of some fish, and the devas-
tation of the M. pyrifeni forests (Lenarz et al. 1995, Teagner &
Dayton 1987, Ladah et al. 1999). There are also positive effects in
the recruitment of the balanus Balanus glaruhihi and Chthamahis
spp. along the California coast attributed to an increase of the
transport toward the coast (Connolly & Roughgarden 1999).

For A. iindosa along the Baja California western coast, the
1997-1998 El Nino negatively affected its survival. The effects of
this on the structure and function of the M. pyrifera forest ecosys-
tem where A. imdosa is an important species, cannot be ignored,
even when the con.sequences of these impacts on the structure and
function of the ecosystem are difficult to quantify (Gislason et al.
2000). Moving from population to the ecosystem level increa.ses
complexity and we lack the general rules to help us predict
changes in the abundance of the interacting species (Lawton 1999).
However, the annual evaluations of the A. imdosa biomass from
the National Institute of Fishing (INP-Mexico) will allow the
evaluation of the recovery of .4. imdosa associated with the M.
pyrifera forests.

ACKNOWLEDGMENTS

The authors thank Ramon Hernandez, from CRIP-La Paz. for
his collaboration during the monitoring. We also thank the tech-
nicians and members of the following fishing cooperatives for
their support in the collection of the biologic material: SCPP
•'Bahi'a Tortugas", SCPP "Emancipacion" and SCPP "California
San Ignacio". CQV was supported by COFAA-IPN and CONA-
CYT-Mexico. Thanks to Dr. Ellis Glazier who edited the English-
language text, and two anonymous reviewers for advice and edi-
torial help.

LITERATI)

Belmar-Perez, J.. S. Guzman del Proo & I. Martinez-Morales. 1991. Ma-
durez gonadica y ciclo reprodiictor del caracol panocha {Astraea im-
dosa Wood. 1828; Gasteropoda:Turbinidae) en Bahia Tortugas, B.C.S.
.4/7. Insl. Cieiic. del Mar v Limnol. Univ. Nal. Anton. Mexico 18:169-
187.

Cochran, W. G. 1978. Tecnicas de muesU-eo. CECSA. 7th ed. 507 pp.

Connolly, S. R. & J. Roughgarden. 1999. Increased recruitment of north-
east Pacific barnacles during the 1997 El Nifio. Uiiinol. Oceanoi^r.
44:466-469.

Conquest, L., R. Burr, R. Donnelly. J. Chavarria & V. Gallucci. 1996.
Sampling methods for stock asses.sment for small-scale fisheries in
developing countries pp. 179-225. In: V. Gallucci. S. Saila, D. J.
Gustafson & B. Rothschild, editors. Slock Assessment Quantitative
Methods and Applications for small - scale Fisheries. CRC Press Inc.

Daniel, W. W. 1984. Bioestadi'stica. Base para el analises de las ciencias de
la salud. Editoreal Limusa. 485 pp.

Gislason, H., M. Sinclair, K. Sainsbury, & R. oBoyle. 2000. Symposium
overview: incorporating ecosystem objectives within fisheries manage-
ment. ICES J. Mar Sci. 57:468-475.

Gluyas-Millan. M. G., C. Quiiionez-Velazquez, J. A. MasstV-Rojas & F.
N. Melo-Barrera. 1999. Diferencias en la relacion talla-edad del cara-
col panocha Aslraea imdosa entre dos localidades de Bahia Tortugas.
Baja California Sur, Mexico. Cienc. Mar. 25:91-106.

Gluyas-Millan, M. G.. C. Quiiionez-Veldzquez & J. Talavera-Maya.
2000. Parametros poblacionales del caracol Astraea undosa (WOOD
1828) de la costa occidental de la Peninsula de Baja California. Cienc.
Mar 26:643-658.

Guzman del Proo, S., S. Mille-Pagaza, R. Guadarrama-Granados. S. de La

RE CITED

Campa, J. Carrillo-Laguna. A. Pereira-Corona, J. Belmar-Perez, M. J.
Parra-Alcocer & A. Luque-Guerrero. 1991. La comunidad bentonica
de los bancos de abulon (Haliotis spp. mollusca:gaster6poda) en Bahia
Tortugas. Baja California Sur. Mexico. An. Esc. Nac. Cienc. Biol.
Mexico 36:27-59.

Kerr. R. A. 1998. Models win hig in forecasting El Niiio. Science 280:
522-523.

Ladah, B. L., J. A. Zertuche-Gonzalez & G. Hernandez-Carmona. 1999.
Giant kelp [Macrosislys pyrifera. Phaeophyceae) recruitment near its
southern limit in Baja California after mass disappearance during
ENSO 1997-1998. / Phycol. 35:1106-1112.

Lawton, J. H. 1999. Are there laws in ecology? Oikos 84:177-192.

Lenarz, W. H., D. Ventresca, W. M. Graham, F. B. Schwing & F. Chavez.
1995. Explorations of the El Nifio events and associated biological
population dynamics off Central California. Cal.COFI Rep. 36:106-
118.

Liu, W. T.. W. Tang & H. Hu. 1998. Spaceborne sensors observe El Nino's
effects on ocean and atmosphere in north Pacific. EOS 79:249-252.

McPhaden, M. J. 1999. Climate oscillations-Genesis and evolution of the
1997-98 El Niiio. Science 283:950-954.

Schaeffer, R. L. W. Mendenhall & 1. On. 1987. Elementos de muestreo.
Iberoamerica. Mexico D.F.: Grupo Editorial 321 pp.

Sissenwine, M. P. 1484. Why do fish population vary' In: R. Ed & M.
May, editors. Exploitation of marine communities. Dahlem konteren-
zen Bedin: Spring-Veriag. pp. 59-94.

Teagner, M. J. & P. K. Dayton. 1987. El Niiio effects on southern Cali-
fornia kelp forest communities. Adv. Ecol. Res. 17:243-279.

Journal of Shellfish Research. Vol. 21, No. 2, 835-841. 2002.

REPRODUCTION OF THE SPOTTED PINK SHRIMP, FARFANTEPENAEUS BRASILIENSIS

(DECAPODA: PENAEIDAE)

MARTHA E. SANDOVAL-QLINTERO' AND ADOLFO GRACIA"

^ Centra Regional de Inve.stii;aci(in Pesquera de Puerto Morelos. Instititto Nucional de la Pesca.

Apartado Postal 580. Ccinciin. Quintana Roo. 77501. Me.xico. Fa.x: 99H 871 0076.

insandovaI@ww2.net.in.\

'Institiito de Cieneitis del Mar y Linmologia: UNAM. Apartado Postal 70-305. Me.xico. D. F. 045 JO.

Me.xico. Fa.x: 55 5616 2745. grucia@inar.icmyl.whim.mx

ABSTRACT Based on the information from catches in Contoy. QR, Mexico (Fehruary-Decemher 1993). the reproductive cycle of
the spotted pink shrimp. Farjanlepenaeiis hrasiliensis. is described. Population si/e structure shows that fishery exploitation affects
small sizes. Females were more numerous than males, particularly during months of greatest reproductive activity. The period for ripe
females period occurs from February to August, peaking in March and April. Main recruitment offshore follows from this spawning
population. The estimated size at first reproduction averaged 148 mm. According to this estimation. 65-80% of the females have not
been able to reproduce at least once before being caught. We observed a significant relationship (Pearson P = 0.05) between the larger
size shrimps and a proportionally larger number of ripe females with depth from February to August. Based on catch-per-unit-effort
indices (CPUE) of ripe females, we propose the region located between 21''40' to 22"10'N and 86°30' to 86°S0'W. at depths of
approximately 25 to 40 fathoms (33 to 59 m) as the main spawning area for the spotted pink shrimp. F. hrasiliensis in the northeast
region of Contoy. QR. Mexico.

KEY WORDS: spotted pink shrimp, reproductive dynamics, maturity, sex ratio

INTRODUCTION

The spotted pink shrimp, FarfanlepenaeiLS hrasiliensis La-
treille 1817, is a commercially important species in Mexico where
the main fishery is located in the Mexican Caribbean along the
coast of the state of Quintana Roo. This impotlant commercially
area is near the noilhem end of the species geographic distribution
which extends from Florida. USA. to the Rio Grande do Norte in
Brazil (Perez-Farfante 1969. 1988).

In the Mexican Caribbean, F. hrasiliensis is the target of a trawl
fishery. Landings also consist of rock shrimp, Sicyonia breviros-
tris, which is of lesser commercial importance and represents ap-
proximately 55'7r of the total catch. Recorded production of spot-
ted pink shrimp is low compared with the penaeid shrimps har-
vested in the Gulf of Mexico (F. aztecus. F. duorarum and
Litopenaeus setiferus). However, fishery exploitation of the spot-
ted pink shrimp represents an important regional activity account-
ing for more than 45% of the crustacean production of the state
(Anonymous 1995).

The annual landings of spotted pink shrimp have declined since
1984—1987. when maximal production was attained (309-474
Metric tons), falling to less than 200 tons during recent years.
Similar to other tropical penaeid stocks (Graci'a et al. 1997). the
spotted pink shrimp, P. hrasiliensis. has reached its maximal sus-
tainable exploitation levels, or even surpassed them, posing serious
problems for the resource. Despite its regional importance, the
spotted pink shrimp fishery has not been described adequately, and
the available literature about its biology and ecology is scarce.
There have been some previous studies about growth (Arreguin
Sanchez 1981a), gonad development (Sandoval-Quintero and Gra-
cia 1998), general distribution (Porras-Ruiz et al. in prep.) and
some fishery aspects (Arreguin Sanchez 1981b. Soto-Aguirre in
prep.), but information about main population processes is still
lacking.

Basic information on the reproductive biology of this species is
needed to establish adequate management policies to sustain this
resource. This study presents data on the temporal pattern of re-

productive activity and distribution of females of F. hrasiliensis in
different stages of gonadal maturation in relation to depth.

STUDY AREA

The F. hrasiliensis fishery in Quintana Roo. Mexico operates
for most of the year, except for the closed season which occurs
around August and September since 1994. The main fishing loca-
tion is on the continental shelf of the Yucatan Peninsula, northeast
of Contoy Island, between 21=27' to 22°I8'N and 86°34' to
87°0I 'W (Porras-Ruiz et al. in prep.). The marine bottom is sandy,
with large coralline formations that limit the operation of trawlers
to regions locally called "blanquizales" which are free from rocks
and corals. It is reported that F. hrasiliensis is also distributed over
coralline bottoms, hence part of the population is not subjected to
fishing gears (Soto-Aguirre in prep.).

MATERIAL AND METHODS

Spotted pink shrimp were collected from the fishing area of
Contoy from February to December 1993 onboard shrimp trawlers.
The monthly information obtained consisted of sampling coordi-
nates, depth and duration of each of the tows, total length of the
shrimp (TL. measured from the tip of the rostrum to the lip of the
telson. at a 0.5 mm precision), sex, and gonadal stage of females
by visual inspection according to a gonad color key based on a
dissecting analysis (Silva Neto et al. 1982, Sandoval-Quintero and
Gracia 1998). The calculated appropiate minimum size of the
monthly sample taken from the commercial catch was 90 individu-
als (Daniel, 1987). at a confidence level of 95% {P = 0.05). Sex
ratio for each month was analyzed through a x"^ independence test
(Mendez et al. 1984).

Periods of greatest reproductive activity during the year as
demonstrated from the commercial catch were determined from an
analysis of gonadal development of females. Females were
grouped according to maturation stage as non-ripe (I and II) or ripe
(III and IV) to avoid misclassification of gonadal stage determi-
nation. The last two stages indicate proximity of the spawning

835

836

Sandoval-Quintero and Gracia

period, and can only be distinguished precisely by histologic sec-
tioning of the gonads (Sandoval-Quintoro and Gracia 1998).

We used catch-per-unit effort (CPUE) as an index of abun-
dance of these stages to analyze variations in the proportion of
non-ripe and ripe females, estimating it monthly from each tow in
the following manner: [CPUE, ^1 = In /t) {%I.M), where n =
number of females in the sample, t = duration of trawl (hr) and
%I.M = percentage of non-ripe (1) or ripe (M) females, expressed
in terms of total number of females per hour towed.

Size at first maturity was defined as mean total length of fe-
males when 50'7f had reached maturity. The relationship between
size distribution and depth was analyzed through a linear regres-
sion analysis (Zar 1974, Bhattacharya & Johnson 1977). We cal-
culated the correlation coefficient (r) to determine whether there
was a statistical association between the gonad stage and depth for
those months in which the variables were not independent (Everitt
1977). Localities were classified arbitrarily according to whether
they yielded >50'7<- non-ripe or ripe females.

RESULTS

Population Size Structure

Total length for entire study ranged from 35 to 205 mm both
sexes, with females typically reaching greater maximum lengths
than males (Fig. I). Both sexes size distribution was unimodal
during most of the months. When two modes were found they
conesponded to large females and small males groups.

Male and female size distribution varied along the period stud-
ied (Table 1). Usually female modal sizes were greater than 140
mm TL. Female average total length varied from 124 mm (inma-
ture females) to 166 m (ripe females). Male average lengths varied
from 116 to 133 mm TL. Modal frequencies were close to the
average in most of the months. May and September modes suggest
the presence of two cohorts (Fig. 1 ).

Sex Ratio

Sex ratio varied in the months sampled (Table 2). Females
prevailed in the months of March to May and in July. Males
prevailed in February and August and from October to December.
The X" test for independence revealed significant differences
(X'ob. = 276.8; x",ah- ^-^^ = 18.307) in the sex proportions
during the year.

Size of First Reproduction

The size at which 5(y/r of the females are mature was estimated
as 148 mm TL (Fig. 2). However, mature females with a minimum
size of 95 mm were found in February and March, while a mini-
mum size of 105 mm was observed in April. In the other months,
minimum sizes of mature females ranged from 1 10 to 125 mm TL.

Distribution of Females on the Fishing Ground

A significant correlation (Pearson P < 0.05 ) between increasing
mean size of spotted pink shrimp females and increasing depth of
capture (Table 2, Fig. 3) was found from February to August. In
most cases the relationship was positive, except in August when
large females were found at shallower depths.

The degree of ripeness of the females and the depth showed a
significant linear relationship (Pearson P ■£ 0.05 ) from February to
May and during July and August. April. May. July, and February

FEMALES <S> NON RIPE (NR)
^ RIPE (R)

Februao' '"^'^=-^08

R=171

100

50

0

rriTfTfflwTKTWI

<
Q

Z

SO
40 i
0 1

100
50
0 -

100

50

0

100

50

0

May

345

'"rrrfliiTlML.

NR=223
R=137

June

n = 345^^^

NR=106
R=239

July
n = 454

August
n = 370

R=180

=274

100

50

0

100 -

50 .

0 -

200
100
0 -

80 I
40 4

0 L

100

50

0

100
50

0

MALES

February^

March
n = 7

April

May
n = 30.

June
n = 356

July
n = 274

JL

NR=I80
JL R=190

100 August „

L

Cri

30
15
0 —

September
n=187

60

30

0

October
n = 274

NR=89
R=98

NR=193

30 . September

JdM^^^lfh-

R=81

30 November

15 n=110
0 —

-n

NR=37
R=73

December

NR=92

30 -

15 . n= 184

35 70 105 140 175
Total length (mm)

30
15
0

30
15
0

November
n=147

'IdMlL

December

ca

Hk ^

35 70 105 140 175
Total length (mm)

Figure I. Size structure of the spotted pink shrimp, F. brasiliensis,
stock from Februar\ to December 1993.

yielded positive correlations between the degree of ripeness and
depth. The inverse condition was observed in March and August.

During all months, except March, at least one area was found
in which more than 50'/f of the females were ripe. From February
to June ripe females were more widely distributed. In March, no
area had high percentages of ripe females; however, the CPUEs in
some of the areas were among the highest ( 1 7. 20, 28 ripe females/
hr towed) along with those observed in April ( 1 3-20 ripe females/
hr towed).

Sites with larger CPUE for non-ripe females occurred in Feb-
ruary (8-15 and 15-20 non-ripe females/hr towed) and March (35,
45. 52 non-ripe females/hr towed). During other months, these
values ranged from 1-13 non-ripe females/hr towed.

One main area which contained ripe females (more than 50%)
dunng almost all months was delimited (Fig. 4). Based on this we
propose that the main spawning area within the general fishing
ground is located at 2r'4()' to 22°10'N and 86 '30' to 86^50' W. In
subarea B. determined only for April, with depths of 25 to 40
fathoms (approximately 42-67 m). the CPUE was estimated at 13

Reproduction of F. brasiliensis

837

TABLE 1.
Female and male size distribution.

Average
Size

Maximum

Size

Minimum
Size

Mode

Size Range
95%

Observations

Immalure females

129-157

195

35

110-160

S5-195

Ripe females

146-166

205

95

130-170

115-180

Males

116-133

200

35

110-145

80-170

Both sex

124-147

205

35

120-160

80-185

February anJ Mareh mean length 130 and 136 mm (recruits)
May and September, two modes
July. August and October, two modes

to 20 ripe females/hour towetd. Subarea C located between 20 to 35
fathoms depth (approximately 33-59 m) was also present for most
of the year with a CPUE of 2 to 11 ripe females/hr towed.

Reproductive Periods

Ripe females were present throughout the whole year, indicat-
ing that reproduction may be continuous. The highest percentages
were found in the months of April (72.3%), June (69.3'7r). and
November (66.4%) (Fig. 5a). Female percentages registered in
May and July to October were lower (less than 50%) than the
previous months. These differences are also related to the catch of
this species within the year, which reaches a maximum in April
(Soto-Aguirre in prep.). Catches yield a relatively high number of
females in April, suggesting that the greatest reproductive activity
occurs during this month.

If we analyze the reproductive activity using CPUE as an index
of abundance (ripe females/hr towed), the pattern obtained differs
from that of percentages (Fig. 5a and 5b). mainly on the last part
of the year. Ripe females are better represented in March and
April, with a CPUE of 8.3 ripe females/hr towed, indicating that
these months are most important in terms of reproduction. June,
also considered as an important month for reproductive activity in
terms of percentage of mature females, had lower CPUE values of
5.6 females/hr towed. February, May, July, and August, varied
from 4.6 to 4.8 ripe females/hr towed. November registered as
important in terms of percentage of ripe females had one of the
lowest values based of CPUE. From September to December the
number of females decreased from 3.2 to 1 .6 ripe females/hr towed
(Fig. 5c).

TABLE 2.

Coefficient correlations (r) of female pink spotted shrimp (n) size

and depth, and monthlv sex ratio (calculated on the basis of

females! in the Contoj fishing ground.

Month

Sex Ratio
(.Male/Female)

February

0.246*

479

1.17

March

0.414*

1118

0.66

April

0.471

988

0.48

May

0.220*

360

0.83

June

0.185*

345

1.03

July

0.351*

454

0.60

August

-0.197*

370

1.12

September

0.060

187

0.95

October

0.096

274

1.35

November

0.047

110

1.33

December

-0.073

184

1,38

DISCUSSION

Ninety-five percent of the spotted pink shrimp catches con-
sisted of organisms that ranged from SO to 185 mm TL, which
suggests that shrimp are recruited at small sizes about 80 mm TL.
This agrees with the emigrating size (80-100 mm TL) of juveniles
of other penaeid shrimps from the nursery areas in the Gulf of
Mexico (Bielsa et al, 1983, Gracia 1989a, Gracia & Soto 1990.
Gracia 1995). Juveniles off. brasiliensis seem to leave the nursery
areas at 67-84 mm TL according to data recorded in coastal la-
goons near the study area (May 1999).

Sex Ratio

In general, the months with the greatest ratio of males/females
are within the main reproductive period (February to August). In
contrast, the months of October to December, which are not con-
sidered as part of the main reproductive period, had a higher pro-
portion of males (1.33-1.38 per female). Guitarl and Hondares
( 1980) also found a difference in the female/male ratio of F. duo-
rarwn and F. azwciis in the Cainpeche Bank.

Female Spatial Distribution Pattern

Some relationships between size of females and gonad stage
were found; however, no consistent pattern was observed. Several
authors have reported that mature female shrimp migrate toward
deeper regions during the spawning time (Bielsa et al. 1983.
Brusheret al. 1972. Garcia and Le Reste 1986, Gracia 1992). This
may be the case for F. brasiliensis. since the reproduction period
established in this paper (February to August) coincides with a
significant relationship of size and gonad stage with depth.

The best correlation between size of females and depth of
catches was observed during March and April that are the months
of greatest reproductive activity. If we also consider that female
size can be related to maturity, one could expect a similar behavior

' Significant at 5% level.

C/3

s

35 65 95 125 155 185
Total length (mm)

Figure 2. Size of first maturity of F. brasiliensis with the criterion of

50%.

838

Sandoval-Quintero and Gracia

200

150

t/D

w

100

J

<

50

s

tu

0

tlH

tlH

o

100

75

e^

w

50

CQ

?5

S

D

0

Z

• Non Ripe

15 20 25 30 35 40 45

Depth ( fathoms )

Figure 3. Number of ripe and non-ripe females of F.
depth.

brasiliensis vs

for the relation^hip between ripeness and depth. This was found for
April but did not occur in March, when no significative correlation
was observed. This correlates well with the fact that March is a
month of marked recruitment to the fishing ground (Porras-Ruiz et
al. in prep.) which can mask this relationship.

In August, when smaller organisms were found at greater
depths, the relationship of size and depth depicts a negative slope.
This finding is related with the second recruitment period of the
population towards the fishing grounds (Soto-Aguirre in prep.)
during summer. In general terms, the results suggest that there may
be a relationship of size and female gonad stage with depth of the
fishing ground, especially during reproductiiin periods.

Nonetheless, monthly variations of non-ripe and ripe females
ocurrence in the fishing ground suggest an area of recruitment with
small shrimp troughout the year (21°30' to 2I°50'N and 86^30'
and 86"40'W) and a spawning area (21°40' to 22°10'N and 86°30'
to 86''50'W) at depths of 20 to 40 fathoms (approx. 33-67 m)
which can be useful as reference areas for the fishery.

This study suggests that the reproductive period for pink spot-
ted shrimp occurs throughout the whole year, but the highest re-
productive activity in terms of number of ripe females occurs from
February to August, peaking in March and April. According to
Garcia and Le Reste (1986). variation in reproductive activity of
shrimps is typically linked to seasons of the year, with two repro-
ductive peaks. Other authors (Crocos and Kerr 1983. Crocos 1987a
and b, Garcia 1988) have also observed a bimodal pattern in egg

B

STW 86°40' 86°20'

87°00' 86°40' 86°20'

135-1^

iio-iiiJ
Q. Rol)

March 'i

CPUE L
(fem/t-h) i

7

1

R

A
B

c

D
E
F

42
10-15
15-22

7-14

35

52

20
3-10
2-4
2-4
17
28

il

.22''10'

21 "50'

21°30'

87°00' 86°40' 86°20'

87°00' 86°40' 86°20'

August

CPLiH22°10'
^fem/t-hj

2r50'

.21°30'

87°00' 86°40' 86°20'

r,

October U

CPUE I

(fem/l-h)J

7

1

K

A
B
C

12
M
4-7

K

3 1

^-155 L

i 1

L21°50'

2r30'

87°00' 86°40' 86f20'

CPUE L22°10'
(fem/t-h)

2-3 L2I°S0'

L21°30'

2I°30'

21°50'

21°30'

Figure 4. Montlily main distribution spawning area of tiie spotted pinit shrimp F

brasiliensis.

Reproduction of F. brasiuensis

839

<D

13

u
ex

80
60
40
20
0

u

T-)

"1

U

o

kri

-;

D.

O

■id

o

a

W

«

P

H

0-

O

"r.

D.

00 i«

f3 t«J

10 n

8 -

Z 6-

4 -

7 .

0

(b)

250 1
200 -
150 -
100
50

(c)

FMAMJ J ASOND

Figure 5. Monthly variation of ripe percentage (a), ripe female CPUE
(b) and mean length (cl of F. hrasiliensis females in the fishery area of
Contoy, QR, Mexico.

production, as well as abundance of larval stages of various pe-
naeid species (Rothlisberg et al. 1987). Gracia ( 1989a) reports that
the L. seriferus population of the Campeche Sound presents two
reproductive peaks of variable magnitude: the most important one
occurs late spring and early of summer and a second less intensive
period occurs in autumn.

We observed only one reproductive peak during spring for F.
brasiliensis. However, it will be necessary to determine whether
this reproductive pattern is typical of the spotted pink shrimp or
whether it is related to environmental factors or high fishing effort
levels that might have suppressed the second reproductive peak
during the year evidenced only from ripe female percentage.
Spawning becomes unimodal, even for species that reproduce all
year round, when they occur near the temperate limits of its geo-
graphical distribution (Dall et al. 1990). The study area is located
in a tropical latitude, so it could be expected that F. brasiliensis
would fit better to a spawning bimodal pattern.

Protracted spawning period allows a high reproductive poten-

tial for F. brasiliensis and also allows a protracted recruitment
through the year. This behavior agrees with the opportunistic re-
productive strategy proposed for white shrimp L. setiferus by Gra-
cia (1989a). Maintaining a high reproductive potential year-round
could permit pink spotted shrimp to cope with environmental fluc-
tuations, thereby adopting an opportunistic strategy to make good
use of microscale variations, mainly in nursery areas which con-
stitute a critical stage (Gracia 1989a, 1991 ). Unfortunately, in spite
of its importance, no data are available on this stage which can
confirm the presence of postlarvae year round or that can give
details about the behavior of F. brasiliensis in the nursery areas.

On the other hand, shrimp spawning during the main peak
reproductive period seems to have a high importance with respect
to the relative contribution to the fishery recruitment. The main
shrimp recruitment that occurs in February-March arises from fe-
male population left at the end of the fishing season of the previous
year (November-December). This may suggest that the decimated
spawning population at the end of the year is more important for
stock renewal than the large female population of younger small-
sized females present during March and April. This behavior con-
forms to a common pattern of penaeid shrimp spawning in which
some of the individuals of a cohort breed at earlier ages and then
have a massive spawning at about 10-12 mo of age (Crocos 1987a
and b, Gracia 1989a). During this time a large proportion of the
spawning stock is removed by the fishery. Other shrimp species
like P. merguiensis. P. seiiiisitkatus. L. notialis. and L. setiferus
(Rothlisberg et al. 1985, Mathews et al. 1987, Lhome and Garcia
1984, Gracia 1989a) present this type of reproduction where the
main recruitment originates from a minor spawning stock. The
success of these spawnings are related to envinronmental factors
which propitiate postlarvae recruitment to the nursery areas, affect
juvenile survival and subsequent recruitment offshore (Gracia 1991 ).

On the other hand, pink spotted shrimp size caught by the
fishery is reducing, which can also influence the reproductive dy-
namics of the stock. Gracia ( 1989b, 1995) points out that consid-
ering shrimp reproductive strategy, where the recruitment is highly
variable due to environmental conditions, the protection of juve-
nile stages has greater influence on increasing population fecun-
dity than protecting spawning population. For F. brasiliensis fe-
males. 60 to 85% of the catches consist of sizes with a total
maximum length of 140 to 150 mm. The size for the first repro-
duction averages 148 mm TL and the high fishing mortality, sug-
gest that most of the females have no chance of reproducing at
least once before being caught. This may induce recruitment over-

Catch
CPUE

0.30

0.25

V 0.20

0.15

o

0.10 g
0.05 U

n r- 0.00

1980 1985 1990 1995 2000

Figure 6. Catch variations of F. brasiliensis in Contoy, Q. Roo

840

Sandoval-Quintero and Gracia

fishing and lead to serious problems for the recovery of the popu-
lation.

This could be one of the reasons for the decreasing shrimp
production which shows a sustained negative trend since 1980.
Both annual shrimp yield and commercial CPUE have shown a
steady decreasing trend to around 20% of maximum records (Fig.
6). It has been reported that shrimp can support exploitation levels
which can reduce spawning stock in a wide range up to around
20% of virgin biomass without affecting seriously the recruitment
(Gracia 1996). However, it also has been demonstrated that a
depleted spawning stock could reduce recruitment and decrease
yield. Fishery statistics seem to indicate that this is the case and
maximum sustainable yield of the resource has been surpassed
affecting the reproduction rate and hence recovery of the popula-
tion.

A closed season during August-September was implemented to
protect spotted pink shrimp and also rock shrimp S. hrevirosrris.
The fishery regulation was mainly designed to reduce fishing pres-
sure on F. brosiliensis spawning stock. This seems reasonable
because location of the main spawning area, as well as the periods
of highest reproductive activity, coincide with the areas and peri-
ods of greatest fishing activities on F. brasiliensis. which could
impact negatively this critical stage for the population renewal.
However, according to our data, this closure does not fill this

objective, as it does not protect the more important spawning at the
end of the year or the more abundant ripe female population earlier
in the year. Neither does this regulation reduce growth overfishing
as it does not protect the main recruitment period of pink spotted
shrimp. A more effective regulation would be to close the fishery
between November-December to protect the remaining and more
important spawning stock. A measure like this would be more
important to enhance spawning potential if a second closure is
considered in March-April. A combination like this could help to
reduce fishing effort on main spawners and pre-spawners of red
spotted pink shrimp which could result in a higher reproductive
potential.

Information provided in this study could serve as a basis to
design management strategies which can help to improve spawn-
ing stock and shrimp yield. It is clear that a closure oriented to
improve spawning potential of spotted pink shrimp should be di-
rected to the more important spawning stock; however, other com-
binations of fishery regulations can also be done.

ACKNOWLEDGMENTS

This study was done as part of the program "Turtles Excluding
Devices" (DETs. for its acronym in Spanish) implemented by the
National Institute of Fishery. SEPESCA. Technician participation
is greatly appreciated.

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Silva Neto, G. L. & J. F. Cruz. Da & A. C. Araujo. 1982. Proceso produtivo
de pos-Iarvas de caniaroes Penaeideos. EMPARN. Bol. Tec, 1 1 . Brasil.
89 pp.

Zar, T, H, 1974. Biostatistical analysis. Englewood Cliffs. New Jersey:
Prentice Hall, Inc. 718 pp.

Journal of Shellfish Research, Vol. 21, No. 2. 843-849. 2002.

INFLUENCE OF THE REPRODUCTIVE CYCLE ON THE BIOCHEMICAL COMPOSITION OF
DEEP-SEA DECAPOD PARAPENAEVS LONGIROSTRIS (LUCAS, 1846) IN THE PORTUGUESE

SOUTH COAST

R. ROSA* AND M. L. NUNES

Departamento de Inovagao Tecnologica e Valorizagcio dos Produtos da Pesca. IPIMAR Avenida de
Brasilia. 1449-006 Lisbon. Portiii'al

ABSTRACT The reproductive cycle unci biochemical composition of the muscle, ovary, hepatopancreas (HP) of Parapenaeus
longirosiris were studied during a period of one year (October 2000-September 2001 ) in the Portuguese south coast. Gonado-somatic
index (GSI) increased significantly in May and June and during the maturation process, suggesting that spawning may start in late
spnng or summer. Hepato-somatic inde.x (HSl) also increased throughout the ovarian maturation, suggesting that the HP resources are
not depleted. Ovarian lipid levels increased with maturation, but no concomitant decrease occurred with HP lipids. The muscle showed
very low lipid levels and higher percentages of polar lipids than ovary and HP. On the other hand, these two tissues presented higher
proportions of neutral lipids, mainly triacylglycerols (TAG). Because both ovarian and HP cholesterol increased with maturation, the
mobilization of HP cholesterol stores to the build-up of ovarian cholesterol was not clear. Protein and glycogen contents in the muscle,
ovary, and HP did not vary as a function of ovary maturity stage. Among the different tissues analyzed, the glycogen is mainly stored
in the HP and to a lesser extent in the muscle. In both ovary and HP. the major fatty acids were 16:0. 18:l(n-7), 18:l(n-9), 20:5(n-3),
and 22:6(n-3). and significant increases in the levels of monounsaturated fatty acids (MUFA) were observed in the ovary during sexual
maturation, which indicates that these compounds act as the major sources of energy during embryonic and early larval development.

KEY WORDS: biochemical composition, Parapenaeus longir<istris. reproductive cycle

INTRODUCTION

The deep-water rose shrimp Parapenaeus longirosiris (Lucas
1846) is the second most valuable species caught by the Portu-
guese crustacean trawl fleet, operating mainly along the south
coast of Portugal (Caramelo et al, 1996). It has a wide geographi-
cal distribution in the Eastern Atlantic, from the north of Spain to
southern Angola, as well as in the Meditenanean and its adjacent
seas (Perez-Farfante 1982). Its growth, reproduction, morphom-
etry, and fecundity have been studied by Ribeiro-Cascalho and
Arrobas (1983. 1984, 1987) and Ribeiro-Ca.scalho (1987) in the
south Portuguese waters. However, there are no biochemical data
associated with the reproductive biology of P. longirostris.

Knowledge of the biochemistry and metabolism processes that
occur during the reproductive cycle are essential for a complete
understanding of reproduction. Biochemical changes during matu-
ration, moulting, and reproduction in gonads, hepatopancreas. and
muscle have been examined for a number of crustacean species
(Pillay & Nair 1973. Guary et al. 1974. Gehring 1974. Galois
1984, Castille & Lawrence 1989, Mourente & Rodriguez 1991.
Palacios et al, 2000), Many of these studies concentrated on lipid
dynamics. The accumulation and mobilization of these organic
reserves constitutes one of the most significant metabolic events in
the physiology of crustaceans (Teshima et al, 1989). Carbohy-
drates are important in the Krebs cycle, in glycogen storage, in
chitin synthesis, and in formation of steroids and fatty acids (New
1976). Synthesis of several proteins, including peptide hormones,
enzymes, high-density lipoproteins (HDLs). and glycoproteins is
especially important in maturation and reproduction (Yehezkel et
al. 2000). These two biological processes are also regulated by
terpenoid, peptide, and steroid hormones (Quackenbush 1986), and
a particular consideration in steroidogenesis is the metabolism of
the steroid precursor, cholesterol. Crustaceans are not capable of

de novo synthesis of the steroid ring (Van den Oord 1966, Teshima
& Kanazawa 1971). and the dietary intake of cholesterol or mo-
bilization of previously consumed cholesterol reserves is required
to support production of steroid hormones (Middleditch et al.
1980).

The purpose of the present study was to follow the biological
changes (gonad and hepatosomatic indices) and the biochemical
composition of the muscle, ovary, and hepatopancreas (HP) of P.
l<nigirostris over a one-year period and link these to the shrimps'
reproductive cycle.

MATERIAL AND METHODS

Samples

♦Corresponding author. Tel.: +351-21-3027000; Fax: ■I-35 1-2 1-301 5948.
E-mail: rrosa@ipimar.pt

The research was performed over a period of one year, between
October 2000 and September 2001. Monthly samples (30-40
shrimps taken from a commercial trawl vessel catches — Costa Sul)
were collected in Algarve (Portuguese south coast). The fishery of
Parapenaeus longirosiris was mainly between 200 and 300 tn. All
the specimens analyzed (a total of 452) had a carapace length
between 20-28 mm. For each shrimp, the following parameters
were recorded: sex, total weight, gonad and HP weight, and ma-
turity stage (only for fetiiales). The maturity stage was based on
Ribeiro-Cascalho (1987). Females were classified as immature
(SI: ovary thin and transparent or translucid white, very small and
with no signs of granulation); maturing (S2: thick ovary, yellowish
or greenish); and mature (S3: stout ovary, olive green or bluish
green colored). Gonad (GSI — gonad wet weight/ body wet weight
xlOO) and hepatosomatic indices (HSl — hepatopancreas weight/
body wet weight x 100) were only calculated for females. To study
the seasonal biochemical changes in the muscle, the monthly
samples were pooled in triplicate taking sex into account. To elu-
cidate how biochemical composition may be associated with matu-
ration process (namely, oogenesis), females tissues (muscle, ovary,
and HP) were pooled in triplicate taking maturation stage into
account.

843

844

Rosa and Nunes

Proximate Chemical Composition and Lipid Class Analysis

Water, protein, fat, and ash (.iintcnts were determined according
to AOAC procedures ( I99S). Total lipids were extracted tising the
Bligh and Dyer (1959) method. Lipid classes were resolved by
analytical thin-layer chromatography (TLC) on plates coated with
0.25 mm silica gel G and developed with hexane: diethylether:
acetic acid (65:35:1 by volume). The developed plates were
sprayed with 10% phosphomolybdic acid in ethanol. Lipid class
identification was made by comparing with standards (Sigma. St.
Louis, MO). Quantification was performed using a scanner and the
software Quantity One (version 2.4) from PDI, Inc. (New York).

Total Lipid Fatty Acid Analysis

The percentage distribution of fatty acids was based on the
experimental procedure of Lepage and Roy (1986) modified by
Cohen et al. (1988). The fatly acid methyl esters were analyzed in
a Varian 34()0 gas chromatograph. equipped with an autosampler
and fitted with a tlame ionization detector. The separation was
carried out with helium as the carrier gas in a fused silica capillary
column Chrompack CPSil/88 (Middleburg, The Netherlands) (50
m X 0.32 mm inner diameter [id]), programmed from 180-200°C
at 4°C/min. held for 10 inin at 20fJ°C. and heated to 210°C for 14.5
min, with a detector at 250"C. A split injector ( 100: 1 ) at 250°C was
used. Fatty acid methyl esters were identified by comparison of
their retention times with those of Sigma chromatographic stan-
dards. Peak areas were determined using the Varian software
(Sunnyvale. CA).

Cholesterol and Glycogen Analyses

The quantification of cholesterol content was based on the ex-
perimental procedure of Naemnii et al. (1995) modified by Oe-
hlenschliiger (1998). The cholesterol was analyzed in a Hewlett-
Packard 5890 gas chromatograph. The separation was cairied out
with helium as the canier gas in a column HP5 (Wilmington. DE)
(30 m X 0.5 mm id). The temperatures of the oven, injector, and
detector were 280°C, 285°C, and 300°C, respectively. Cholesterol
was identified and quantified by comparing with standards
(Sigma) from which a standard curve was prepared. Glycogen
concentrations were determined according to Viles and Silverman
(1949). Glycogen was measured by the anthrone-reagent method,
and the absorbance read at 620 nm. A calibration curve was pre-
pared using glycogen (Sigma, St. Louis. MO) as a standard.

Statistical Analysis

Data were analyzed using a one-way and a two-way analysis of
variance (ANOVA), after the assumptions had been met (normal-
ity and homogeneity of variances were verified by Kolmogorov-
Smirnov and Bartlett tests, respectively). When data did not meet
the assumptions of ANOVA. the nonparametric ANOVA equiva-
lent (Kruskal-Wallis test) was performed. Whenever significance
was accepted at P < 0.05. the Tukey (parametric) and Dunn (non-
parametric) multiple comparison tests were used (Zar 1996).

RESULTS

Biological Changes

The seasonal (intra-annual) patterns of the GSl and HSl ob-
served in Parapeiuiciis l(iiii;ini.\tris females in = 325) can be seen

ONDJFMAMJJAS
Moolb

a)

niiM^*"!*

L.\ Monlh

Figure 1. Box plots of the monthly distribution of gonadosomatic in-
dex ((;S1) (a) («lth the Indication of the dominant maturity stages
found In each month) and hepatosomatic index (HSl) (b) of I'arap-
enaeiis longirostris females.

in Figure
July.

GSl and HSl are the highest in May, June, and

Proximate Chemical Composition. Cholesterol, and Glycogen Contents
of Muscle, Ovary, and Hepatopancreas

The biochemical composition of the muscle of Parapenaeus
liiiinirostris exhibited seasonal variations in the water, protein, and
lipid contents (Fig. 2). The water content ranged from 12.9-15.1%
in females and 73.6-75.5% in males, revealing significant tempo-
ral variations. The protein content ranged from 20.1-22.3% in
females, and 19.5-21.3% in males. The statistical analysis re-
vealed a significant increase from November/December to April/
May in both genders (Females; F,, ,4 = 4.63, P < 0.05; Males:
F, I -4 = 4.25, P < 0.05). The lipid content ranged from 0.1-0.5%
in females and 0.1-0.4% in males, exhibiting a considerable rise in
the spring. The ash levels varied between 1 .8-2.1 % in females and
1.9-2.3% in males. The cholesterol coiiient in the muscle ranged
from 56.1-72.3 mg/100 g in females and 50.5-67.9 mg/100 g in
males (Fig. 2), exhibiting significant seasonal variations between
winter and summer months (Females: F,, ,4 = 4.55. P < 0.05;
Males: F, 1.24 = 4.09, P< 0.05). The glycogen content varied from
0.8-2.6 mg/100 nig in females and from 1.2-2.8 mg/100 mg in
males (Fig. 2) (Females: F, |.:4 = 4.32. P < 0.05; Males: F, ,24 =
4.25, P < 0.05), being the highest between March and May for both
genders.

The protein content of the muscle, ovary, and HP of the females
as a functiim of ovary maturity stage showed no significant varia-
tions (Table I). Statistical differences were obtained when com-
paring the different tissues; the protein content varied from 20.6-
22.3% in the muscle, 42.1^5.3% in the ovary, and from 8.5-9.7%
in the HP. The cholesterol content increased significantly from SI

Biochemistry and Reproduction of Rose Shrimp

845

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r

Females

T

24 -
23 -

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22 ■

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-1 _ 1

21 .

■ , k

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20 .

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19.

18-

— ( — 1 — 1 — 1 — 1 — 1 — 1 — 1 — p — 1 — 1 —

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- 25

20

1 5

1 0

. 0 5

25
24

? ^^

£ 22

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20
19
18

Males
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30
25

'^ I

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00

1

77 -

-#- Moisture

— Lipid

-

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J T

75

1 1^^

S^

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74 ■

■'-.H^ ' '

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T -L.

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■ '-

72 -

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b-~.

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— 1 — 1 — 1 — 1 — 1 — 1—

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80

70 ..

60

50

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Figure 2. Monthly variations in proximate chemical composition (% wet wt.). cholesterol (nig/lOOg wet wt.), and glycogen (mg/lOOmg wet wt.)
contents in the muscle of Parapenaeus loiigirostris females and males (values are means of three pooled samples ± SD).

to S3 (62.3-73.5 mg/100 g) in the ovary and from S 1 to S3 (150.2-
181.9 mg/100 g) in the HP. In the muscle, this content decreased
significantly from SI to S3 (70.0-56.7%) (Table 1 ). On the other
hand, muscle, ovary, and HP glycogen content did not show sig-
nificant variations throughout the maturation process (Table 1).

Lipid Class Distribution Among Female Tissues

To elucidate how lipids may be associated with maturation and
reproduction processes, lipid content was determined in the
muscle, ovary, and HP of females as a function of ovary maturity

TABLE 1.

Variations in protein (% wet wt.), glycogen (mg/l((0 mg wet wt.), cholesterol (mg/100 g wet wt.), total lipids (nig/lOO mg dry wt.), lipid class
content (% total lipids) in the muscle, ovary, and hepatopancreas of Parapenaeus longirostris females at different stages of

ovarian development.

Muscle

Ovary

Hepatopancreas

Stage 1

Stage 2

Stage 3

Stage 1

Stage 2

Stage 3

Stage 1

Stage 2

Stage 3

Protein

20.6 ± ().7-'

21.2 ±0.5"

22.3 ± 1.0"

42.1 ± 1.5"

45.6 ± 1.6"

45.3 ± 1..^"

8.5 ± 1.2"

8.4 ± 1.0"

9.7 ± 1.0"

Glycogen

1 .9 ± 0.2"

2.1 ±0.2"

2.4 ±0.3""

2.7 ± 0.3"

2.6 ±0.5"

2.5 ± 0.6"

2.6 ± 0.4"

2.7 ±0.3"

2.5 ± 0.3"

Cholesterol

70.0 ±2.1"

68.8 ± 2.8"

56.7 ± 3.2"

62.3 ± 4.5"

64.4 ± 3.7"

73.5 ± 3.4"

150.2 ±6.7"

173.3 ±5.8"

181.9 ±5.3"'

Total lipids

2.9 + 0.3"

3.1 ±0.2"

3.4 ± 0.4"

19.8 ± 1.9"

25.2 ± 1.7"^

29.1 ±2.2"

37.8 ±2.1"

44.6 ± 2.5"*

48.3 ± 2.6"*

Triacylglycerols

8.3 ±3. 1"

8.9 ±2.8"

9.5 ± 1 .5"

40.5 ± 6.5"

49.8 ±5.3"

58.7 ± 4.5"

53.6 ±5.5"

60.9 ± 4.2"

71.1 ±3.5"

Phospholipids

25.1 ±3.5"

25.6 ±3.7"

26.2 ±4.4"

12.5 ±3.6"

9.3 ±4.2"

6.1 ±3.2"

2.8 ± 0.5"

1 .6 ± 0.4"

1.6± 1.1"

Diacylglycerols

6.9 ±0.7"

6.6 ± 0.4"

6.3 ± 0.8"

4.7 ± 1.2"

5.6± 1.1"

6.9 ± 1.5"

3.5 ± 0.3"

2.6 ±0.7"

3.0 ± 0.2"

Monoacylglycerols

2.5 ± 0.2"

2.8 ±0.1"

3.2 ± 0.2"

5.5 ± 0.-5"

4.7 ± 0.4"

4.6 ± 0.4"

1 .8 ± 0.6""

2.4 ±0.2"

0.8 ±0.1"

Free fatty acids

18.1 ± 1.4"

17.0 ±1.6"

15.9 ± 1.1"

7.0 ±0.8"

4.3 ± 0.8"

2.8 ± 1.8"

13.4 ±1.3"'

15.2 ±0.2"

6.6 ± 0.5"

Free cholesterol

29.3 ± 3.3"

28.6 ±3.8"

27.8 ±2.6"

9.7 ± 2.4"

10.8 ± 1.4"

10.2 ± 1.2"

6.5 ± 1.0"

5.1 ± 1.1""

3.7 ± 0.7"

Cholesterol esters

5.7 ± 1.2"

6.0 ± 0.9"

6.3 ± 1.7"

1I.0±2.7"

7.9 ± 1.4"

6.2 ± 3.5"

16.1 ±4.0"

9.9 ± 2.9"

10.8 ±0.9"

Hydrocarbons

4.1 + 1.9"

4.4 ± 1.6"

4.8 ± 1.8"

6.7 ± 1 .7"

7.4 ± 0.5"

4.5 ± 0.5"

2.3 ± 1.4"

2.3 ± 3.5"

2.3 ± 4.9"

Values are the means ± SD of three pooled samples. Different superscript letters within rows repre.sent significant differences (P < 0.05).

846

Rosa and Nunes

stage (Table 1 ). The lipid levels did not vary significantly in the
muscle (from 2.9-3.4 mg/100 mg), but in the other tissues showed
significant variations; namely, between SI and S3 (ovary: 19.8-
29.1 mg/lOO mg; HP: 37.8-+8.3 mg/100 mg).

Different patterns of distribution of the polar and neutral lipids,
(expressed as % total lipids) among the muscle, ovary, and HP can
be found throughout the maturation period (Table 1 ). Neutral lipids
were dominated by triacylglycerols (TAG), which are by far the
largest fraction in the ovary and HP. TAG increased significantly
from stages I to 3 (ovary: 40.5-58.7%: HP; 53.6-71.7%). The free
fatty acids (FFA) percentage decreased during the maturation pro-
cess, and the neutral lipids, diacylglycerols (DAG), and mono-
acylglycerols (MAG) showed no clear trends in the different tis-
sues. Similar trends of variation were shown by the other classes.
In the muscle, the polar lipids attained higher percentages, because
phospholipids (PL) reached about 25% during the maturity pro-
cess. Significant differences in PL percentages were also detected
between the ovary and HP.

Total Fatty Acid Composition of Female Tissues

The fatty acid composition in the muscle of females at different
stages of ovarian development is shown in Table 2 (only the quan-
titatively most important fatty acids are represented in the table).
Saturated fatty acids (SFA) content ranged from 6.9-7.1 mg g' dry
weight. The most predominant was 16:0. attaining 4.6— l.7-mg g"'.
The monounsaturated fatty acids (MUFA) content ranged from
6.8-8.0 mg g~'. Most of this content was present as 18:1. Poly-
unsaturated fatty acids (PUFA) were the major group attaining the
highest values in stage 3 ( 12.2 mg g"' ) because of the increase of
ARA (20:4n-6). EPA (20:5n-3). and DHA (22:6n-3) in that period.
These fatty acids accounted for almost 80% of the PUFA.

The fatty acid composition of the ovary and HP of females in
relation to ovary maturity stage is also presented in Table 2. With
respect to the SFA fraction, there were significant differences
among the developmental stages. A similar trend was obtained in
the most predominant saturated fatty acid ( 16:0). The MUFA frac-

tion increased significantly during oogenesis and represented more
than half of the total fatty acids. The significantly higher values of
the MUFA in the ovary and HP compared to muscle were attrib-
utable to the significantly higher contribution of 18:l(n-9). 18:l(n-
7). 20:l(n-9). and 2():l(n-7). PUFA ranged from 46.1-58.5 mg g^'
in the ovaries and from 72.9-90.0 mg g"' in the HP. The major
PUFA were ARA. EPA. and DHA, and significant differences
were obtained between the tissues analyzed.

DISCUSSION

Oogenesis and primary vitellogenesis usually correspond to the
juvenile and prepubescence phases, which are characterized by a
slow increase in ovary weight. The onset of puberty is distin-
guished by a rapid deposition period, the secondary vitellogenesis
(Adiyodi & Adiyodi 1970. Aiken & Waddy 1980). In this study,
the maturation of P. longirostris ovary, indicated by GSI. seemed
to show this basic phase pattern, with a slow increase up to the S2.
followed by a rapid increase in S3. The temporal trend of GSI
seems to be concordant with the seasonal spawning pattern ob-
served by Ribeiro-Cascalho & Arrobas (1987) in Portuguese wa-
ters, which suggests two peaks of reproduction, one at the end of
spring and another at the beginning of autumn in October.

On the other hand, the variations of HSI throughout the year
and during the maturation process did not seem to corroborate the
general pattern among the decapods; namely, the storage of or-
ganic reserves in the HP and the utilization of these reserves in the
ovarian development (Gibson & Barker 1979. Kyomo 1988) or in
the formation of a new exoskeleton (Adiyodi & Adiyodi 1970).
The fact that both GSI and HSI increased with the ovarian matu-
ration suggests that the HP resources are not depleted and. accord-
ing to Tuck el al. (1997). if resources are mobilized from this
organ, then these resources seem to be compensated hv those
gained from feeding.

The increase in lipid levels in the ovary occurs as a result of the
maturation process. In fact, neutral lipids, particularly TAG. are
the major energy source, and the predominant form of energy

TABLE 2.

Fatty acid composition (mg g ' dry wt.) in the muscle, o>ary, and hcpatopancreas of Parapenaeus longirostris females at different stages of
ovarian de>elopnient ((mly the quantitatively most important fatly acids are represented).

Muscle

Ovary

Hcpatopancreas

Fatty ,\cids

Stage 1

Stage 2

Stage 3

Stage I

Stage 2

Stage 3

Stage 1

Stage 2

Stage 3

14:0

0.2 + ().()■'

0.2 ± 0.0"

0.3 ±0.1"

2.6 + 0.9"

3.2 ± 1 .3"

4.0 ±2,0'"

6.4 ± 1.2'

8.3 ±1.5''

8.7 ± 1.9"

16:0

4.6 ± c.g-"

4.7 ± 0.7"

4.6± 1,1"

24.3 ± 4.2"

32.7 ± 3.7'

38.7 ±5.1'

54.7 ± 4.9''

61.7 ±6.4"'

73.9 ± 7.9'

18:0

1.7 + 0.5"

1.7 ±0.4"

1.4 + 0.8"

5.4 ±1..^"

9.0 ±1.7'

9.5 ±1.9'

9.2 ±2.1'

10.2 ±2.7'

11.6 ±3.0'

X saturated

7.1 ±1.3"

7.2 ±1.5"

6.9 ±1.2"

34.3 ± 5.9"

47.5 + 6.8'

57.4 ± 7.9''

764 ±10.-^'

87.4 ±12.1"

98.2 ± 9.9s

16:l(n-7)

1.5 ±0.8-'

1.6 ±0.9"

1.5 ±0.6"

8.5 ± 2.2"

11.0 ±4.5"

19.5 ±3.6'

28.6 ± 8.9''

33.3 ± 7.6"'

37.6 ± 6.3'

18:l(n-9)

4.3 ±1.6-'

3.6 ± 0.9"

3.6 ±1.2"

44.3 ±8.2"

61.1 ± 10.9'

74.3 ±12.4"'

83.1 ±12.5''

107.8 ±18.1"'

1 16.8 ± 20.6'

18:l(n-7)

1.1+ 0.5"

1.1 ±0.6"

1.3 ±0.4"

12.1 ±3.2"

13.5 ±2.8"

16.0 ±3.3"'

29.0 ± 7.5'

28.4 ±6.0'

26.0 ± 5.4'

20:l(n-9)

0.7 ± 0.3"

0.2 ±0.0"

0.2 ±0.1 '■

8.3 ± 1.5^

10.3 ±2.3'

18.0 ±2.7''

16.9 ±4.2"'

20.1 ±3.4"

21.5 ±5.0"

20:l(n-7)

0.1+0.0"

0.1 ±0.0"

0.1 ±0.0"

1 .5 ± 0.5"

2.2 ± 0.8"

7.7 ± 3.6'

5.1 ± 1.3'

6.1±2.r

6.6 ±2.4'

1 monounsaturated

8.0 ± 1 .9"

6.8 ±1.6"

7.0 ± O.S"

83.0 ±15.6"

109,0 ±25.3"'

140.7 ± 19.8'

169.0 ±23.5'"

204.0 ±18.7"'

224.6 ± 26.6'

18:2(n-6)

0.3 ±0.1'

0.3 ± 0.2"

0.2 ±0.1"

1.5 ±0.3"

1,3 ±0.5"

2.5 ± 0.8"'

3.2 ± 0.9'

4.2 ±2.1'

3.3 ± 1.3'

20:4(n-6)

1.5 ±0.8"

1.5 ±0.6"

1.8 ±0.9"

3.4 ± 0.8"

4.9 ± 1.7"

7.7 ± 3.4'

11.1 ±3.2'^

12.9 ±2.2"

|4,y±4.4"

20:5(n-3)

3.8+1.2"

3.8 ±1.5"

4.0 ±1.0"

1 1 .3 ± 2.8"

14.2 ±4.4"'

16.5 ± 3.0'

23.2 ±4.2"

27.2 ±4.0"'

30. 1 ± 3.9'

22:6(n-3)

4.4 ±1.7"

5.4±2.1"

5.7 ± 1 .9"

24.0 ± 5.8"

30.3 ±7.1'

19.7 ±6.3"

24.5 ± 3.3"

29.5 ± 4.8'

3 1.4 ±5.2'

2. polyunsaturated

10.9 ±2.5"

1 1 .8 + 0.9"

12.2 ±0.9"

46.1 ±15.6"

58.5 ± 12.4"'

51.4± 12.0"

72.9 ±11.7'

87.0 ± 14.5"

90.0 ± 12.6"

Values are the means ± SD of three pooled samples. Different superscript letters within rows represent significant differences (P < 0.05).

Biochemistry and Reproduction of Rose Shrimp

847

storage in the adult, egg. and prefeeding larva (Xu et al. 1994.
Nates & Mckenney 2000), comprising primarily 16:0 and omega-9
family fatty acids (Teshima et al. 1988). PL. DAG. and sterols are
the other main lipid classes found in marine shrimps and are also
associated with the maturation of oocytes (Teshima 1997. Ravid et
al. 1999. Wouters et al. 2001).

The HP is the major lipid storage and processing organ for
postembryonic stages (Voght et al. 1985). but during maturation
the ovary becomes an additional center for lipid metabolism, in-
cluding lipogenesis — TAG synthesis (Teshima et al. 1988). In the
present study, and like some previous studies in decapods (Castille
& Lawrence 1989. Cavalli et al. 2001). the increase in ovarian
lipids is not accompanied by a decrease in HP lipids. Under these
circumstances, the lipid requirements of the developing ovary
seem to be more dependent on the ingestion of dietary lipids than
on HP reserves.

The profile of fatty acids in the ovaries of P. longirostris is a
reflection of the fatty acid requirement of this tissue or of what is
required for transfer to the developing embryos after fertilization.
Alava et al. (1993). Cahu et al. (1994). and Cahu et al. (1995)
demonstrated the benefits of high levels of highly unsaturated fatty
acids in the diet on reproductive parameters and on offspring qual-
ity of penaeid shnmps. In fact, the long-chain fatty acids are nec-
essary for vitellogenesis of crustaceans (Middleditch et al. 1980.
Millamena & Pascual 1990) and ARA and EPA are precursors of
eicosanoids in marine animals (Lawrence et al. 1979. Sargent.
1995). The high values of MUFA in the ovary and HP are con-
sistent with previous findings of Clarke et al. (1990) and Roustaian
et al.( 1999), which indicated that these compounds are the major
sources of energy during embryonic and early larval development.

In the present study, the protein content of the ovary and HP did
not vary significantly. On the other hand, the seasonal (intra-
annual) variation of protein content of the muscle may be linked
with changes in feeding activity. Muscle protein loss during star-
vation has been observed in other deep-sea decapod species (Dall
1981).

Because cholesterol is a precursor of steroid hormones
(Kanazawa & Teshima 1971), the increase of ovarian cholesterol
levels with maturation in the P. longirostris was not surprising.
This increase can also be related to the role of cholesterol as
precursor of ecdysteroids. as these compounds are known to in-
crease during gonadal maturation (Wilder et al. 1991). Similar
trends were observed in HP cholesterol, which differs from the
results obtained by other authors (Adiyodi & Adiyodi 1970, Lau-
tier & Lagarrigue 1988), where the decrease in HP cholesterol
during vitellogenesis suggests that mobilization of HP cholesterol
stores may contribute to the build-up of ovarian cholesterol. The
explanation of our findings could be found in Teshima et al.
(1988). which indicated that cholesterol is sequestered to the ova-
ries from the muscle stores. In fact, in the present study, the muscle
cholesterol content decreased significantly from SI to S3, and the
seasonal variation of the cholesterol content also seems to confirm

the conclusions of Teshima et al. (1988). On the other hand, be-
cause cholesterol stores within the HP and gonads are derived from
the diet (Middleditch et al. 1980). because of the incapacity of de
novo synthesis of the steroid ring, if the steroid resources are
mobilized from the HP to the gonad, then the HP resources seem
to be compensated by those gained from feeding.

Glycogen did not show significant variations throughout the
maturation process, in contrast to what was stated by Kulkami and
Nagabhushanam (1979). Moreover, because carbohydrates have
specific roles in the production of nucleic acids, are precursors of
metabolic intermediates in the production of energy and nones-
sential amino acids, and as a component in ovarian pigments (Har-
rison 1990). they have to be especially important for the matura-
tion process and for embryogenesis. Among the different tissues
analyzed in this study, the glycogen is mainly stored in the HP and
to a lesser extent in the muscle, but according to the studies by
Hagerman et al. (1990) and Baden et al. (1994). on the decapod
Nephiops nonegkus (Linnaeus 1758). the occurrence of glycogen
depletion in the muscles, following hypoxia and starvation, sug-
gests that the muscle contains a particularly important store of
glycogen, because it is more readily accessible when there is a shift
to anaerobic metabolism or when there is decrease in the feeding
activity dunng winter. This can explain the seasonal variation of
glycogen content in the muscle of P. longirostris. because the
lowest values were obtained in the winter. Although there is no
evidence of a decreasing feeding activity during this period of the
year, the diet composition of P. longirostris should vary signifi-
cantly between seasons like the other deep-sea crustaceans species
(Cartes & Sarda 1989, Labropoulou & Kostikas 1999), because
these changes correspond basically to the period of abundance of
the different dietary group in the deep-sea environment (Cartes
1994).

In summary, it is evident that reproductive cycle has profound
effects upon the biochemistry of P. longirostris. Reproduction and
gonadal maturation have large associated energy costs attributable
to the increase in biosynthetic work, which will support the leci-
thotrophic strategy (reliance on egg yolk nutrition) of the embryos
and prefeeding larval stages. Moreover, these processes seem to be
influenced or even synchronized with seasonal feeding activity or
food availability. Despite not being mentioned or discussed in this
study, moult cycle can also have an important effect in the bio-
chemistry and physiology of decapod Crustacea, manifested in
changes in their physiological ecology and behavior. Lack of data
on this matter in P. longirostris indicates that further work will be
necessary to understand better the biochemistry and physiology of
this species.

ACKNOWLEDGMENTS

The Foundation for Science and Technology (FCT) supported
this study through a doctoral grant to the first author. The technical
assistance of Angelino Martins is greatly appreciated. We express
our aratitude to the crew of the trawler Costa Siil.

Adiyodi. K. G. & R. G. Adiyodi. 1970. Endocrine control of reproduction

in decapod Crustacea. Biol. Rev. 46;121-165.
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& B. F. Phillips, editors. The biology and management of lobsters, vol.

1. physiology and behavior. New York: Academic Press, pp. 215-276.

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Journal of Shellfish Reseairh. Vol. 21. No. 2, 851-860. 2002.

A COMPARISON OF GROWTH PERFORMANCE ACROSS THE SQUID GENUS ILLEX
(CEPHALOPODA, OMMASTREPHIDAE) BASED ON MODELLING WEIGHT-AT-LENGTH AND

AGE DATA

SERGIO RAGONESE,'* PATRIZIA JEREB,' AND EARL DAWE"

Usthuio di Ricerche sidle Risorse Marine e I'Ambiente, IRMA-CNR. Via Vaccani 61. 91026 Mazara.
Italy: -Department of Fisheries and Oceans. Science Oceans and Environment Brancli. P.O. Sm 5667.
St. John's. Nfld.. Canada. AlC 5X1

ABSTRACT A new approach to study growth performance in squid is described, based on modeling the ratio of body weight to
mantle length (BW/ML) as a function of age (squid "condition"). This approach has several advantages above traditional size-at-age
modeling, including the concurrent use of two size indices, the fact that it is not constrained by theoretical considerations regarding
the form of growth in absolute size, and that it is also less severely affected by sampling bias. It was tested on three species of the genus
lllex using raw data sets for two species as well as BW/ML values calculated from sizes predicted by models available in the literature
for all three species. Several experimental models were explored, but the specific logistic model was found to be the most suitable,
especially when most of the life cycle was sampled. This model was successfully applied to all species and case studies, thereby
elucidating common features of growth performance throughout the genus llle.x. It provided similar or superior model fits when
compared with corresponding models of length-at-age or weight-at-age. Comparisons within and among species indicated some effects
that have also been suggested from independent length-at-age studies, including a positive effect of temperature on growth perfor-
mance. This new approach also indicates a relationship between growth performance and sexual maturation.

KEY WORDS: squid, lllex. growth performance, age

INTRODUCTION

Three species of short-finned squids of the genus llle.\:
(Teuthoidea. Ommastrephidae) are commercially exploited (Roper
et al. 19981; lllex illecehrosiis (Lesueur 1821 ). /. ari>entinus (Cas-
tellanos 1960), and /. coindetii (Verany 1839). The fisheries they
support have recently gained increasing attention because of con-
cerns regarding potential for overexploitation (Haimovici et al.
1998, O'Dor & Dawe 1998, Sanchez et al. 1998).

Effective squid fisheries management, however, is hampered
by uncertainties concerning the life cycle. As is typical of most
squid, llle.x species show great and unpredictable variability in
growth, maturation, and spawning patterns over very short time
periods (Mangold 1987, Forsythe 1993, O'Dor & Lipinski 1998).
Such great variability in life history parameters largely accounts
for the great variability evident in size-at-size (e.g., weight-at-
length) and size-at-age relationships observed in lllex sp. and the
difficulty in applying length-frequency methods (Caddy 1991,
Jereb & Ragonese 1995) conventionally used for estimating
growth rates in other fisheries resources (Pauly & Morgan 1987).
Recent direct ageing techniques based on squid statolith micro-
structure (Jereb et al. 1991; Jackson 1994), despite the inevitable
existence of ageing bias (Pauly 1998, Gonzalez et al. 2000), indi-
cate very high growth rates, even higher than those observed in fast
growing pelagic fish such as the scomberoids (Longhurst & Pauly
1987, Jarre et al. 1991).

Despite the many studies of the past decade, there is no con-
sensus on which model is the most suitable to describe squid
growth. Among those used for various species and portions of the
life cycle, the simple linear, power, log linear, piece-wise, expo-
nential, seasonally oscillating von Bertalanffy, double exponential
(or Gompertz), and logistic (Jackson 1994) have most frequently
been applied. To date, five models have been used to describe

*Corresponding author. E-mail: ragonese(3irma.pa.cnr.it

absolute growth in either length or weight within the genus lllex
(Table 1).

Until now, size-at-size and size-at-age relationships have been
analyzed separately, despite the strong correlation existing be-
tween body mass and mantle length. Here, these relationships are
analyzed jointly by relating body mass to mantle length and in-
vestigating trends in this ratio with age. The main purpose of our
study was to combine two indices of size (both important factors
in ecology and evolution studies; Peters 1983, La Barbera 1989)
into a new index, which may better indicate variation in growth
performance than either length or weight alone and thereby facili-
tate broad-scale comparison within and among species. Potential
advantages of this approach include that variability surrounding
models of weight-at-length on age may be lower than for more
familiar models of size on age. Weight-at-length on age models
may be simpler, more consistent, and provide better fits than single
size variable on age inodels. They may also better reflect suitabil-
ity of the biotic environment than growth in either length or weight
alone.

The models we developed were applied to three congeneric
squid species {lllex spp.) to test the utility of this approach for
comparison of growth performance patterns.

MATERIALS AND METHODS

The weight-at-length index (WaL) is defined as the ratio of
whole body weight (BW; g) to dorsal mantle length (ML; mm),
i.e.. WaL = BW/ML; it is, therefore, analogous to other more
conventional condition indices, derived from the classic Huxley's
allometric formula y = ax*" (La Barbera 1989) and widely used in
aquaculture and fisheries science (Dawe 1988, Bolger & Connolly
1989, Scott Cone 1989).

Two kinds of size-at-age databases were used (Tables 1 and 2).
The first one consisted of original individual estimates of size (ML
and BW) and age. Such data sets were available for two micro-
cohorts of /. coindetii from the Strait of Sicily (Arkhipkin et al.

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r^

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d

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-t

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II

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II

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II

II

II

II

II

II

II

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II

II

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II

II

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>^

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r*-)

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ri

ri

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nD

ri -^ ri —

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rj ri rj n ri rj ri ri r<^ r*~i

1/1 O m, o _ '/". w"- '/^. O —

OO' — O-"! — — r^, OCOC

^' -J ^ b Cm b a. c

— U -t -.'i fli _3 flj r3

Q i; Q

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ii ii t t>5

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t 1/5

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< S S < S 5

— rir*-, ^ — rir^. -t— ri— 'rir;-, -t_,~|r^, -j-i/-, sO —

Growth Performance Across the Squid Genus Illex

853

TABLE 2.

Ranges of ape. mantle length (ML), and weight-at-length ratios (WaLl for data sets based on individual observations and on predicted size
values for Illex species and populations. For cases A and E, the predicted values were calculated only for the age ranges sampled

(i.e., without extrapolation).

Original 1 individual 1 Observations

Theoretical (mean) Observations

Age Range

MI. Range

WaL

(g/mm)

■Age Range

ML Range

WaL

( g/nim )

Species-Area

Case

Sex

Group

Idaysl

(niml

Imin)

(max)

N

(days)

(mm)

(min)

(max)

N

A cvlmlcni

Al

f

Oct.-Dec.

75-240

41-202

0.073

1.284

189

70-230

33-186

0.054

0.891

17

Central Mediterranean

A2

f

May-July

74-181

52-175

0.102

1.027

144

70-180

31-156

0.089

0.687

12

A.I

ni

Oct.-Dcc.

76-230

45-171

0.078

1.1.54

218

80-230

50-143

0.105

0.866

16

A4

m

May-July

81-191

56-141)

0 1 17

0.857

98

100-180

89-129

0,251

0.720

9

/, coindelii

Bl

r

Oct-March

220-475

115-204

0.407

1.206

IS

W Mediterranean

B2

r

Apr.-Sept.

160-460

92-205

0.266

1.218

21

83

ni

Oct. -March

160-430

68-165

0,191

1.127

19

84

m

Apr.-Sept.

160-370

69-158

0.198

1 .042

15

/. coindt'tii

CI

f

pooled animals

90-450

37-tOl

0.099

4.160

25

Galician (Atlantic)

C2

m

pooled animals

90-390

53-248

0.053

2.773

21

L coindetii

Dl

f

Sierra Leone

120-255

113-192

0.418

0.757

10

NW African Coasts

D2

f

W. Sahara

90-300

84-294

0,282

2.522

15

D3

m

Sierra Leone

105-210

84-135

0,206

0.627

8

D4

ni

W. Sahara

90-255

80-192

0,187

1.823

12

/. illecebrosus

El

r

March

116-247

L54-299

0.383

1.753

32

115-250

166-261

0,488

1.070

10

NW Atlantic

E2

f

April

108-228

159-286

0.396

1.7.34

32

100-235

150-291

0,366

1 ,574

10

E3

f

May

112-195

175-281

0.491

1.697

12

120-210

181-297

0.535

1.869

7

E4

m

March

116-204

152-227

0.409

1.128

48

115-220

169-225

0.488

0.922

8

E5

m

April

115-216

152-246

0.357

1.447

35

115-220

174-237

0.463

1,189

8

E6

m

May

141-200

217-265

0.917

1 620

17

135-210

212-268

0,812

1.749

6

/. araentinus

Fl

r

June

180-360

149-311

0,377

2.206

13

Palagonian Shelf

F2

m

June

1 80-360

172-298

0.589

2.567

13

1998, Arkhipkin et al. 2000; Case A: Tables 1 antJ 2) and three
micro-cohorts of /. illecebrosus from the Northwest Atlantic
(Dawe & Beck 1997; Case E; Tables 1 and 2). /. coindetii samples
were collected during an experimental depth-stratified random bot-
tom trawl survey (stretched mesh size in the cod-end of 31 mm),
whereas /. illecebrosus samples were collected from the New-
foundland inshore jig fishery during July to November 1990.

The second kind of database consisted of size values (ML and
BW) at age predicted by the specific size-at-age models reported
by sex for different hatching groups or populations (pooled over
hatching groups) of Illex spp. in the literature (Tables I and 2).
When only ML-at-age models were available (Case B), the corre-
sponding BW-at-age data were roughly approximated from pub-
lished BW-ML relationships obtained for the same season. There-

00
-0.5

O 1

O A

A l^^^^^o ^

-1.0

A^

^0

J^ AOO

o

-1.5

A CP^^^I

1)8

o

O A1-F
A A3-M

-2.0 -

Al - F com

- - - A3 - M com

-0 9,

— 1

70

90

110

170

190

210

130 150

Age (days)

Figure I. Individual log,, of body weight (BVV) at mantle length (ML) ratio vs. age and computed (com) specified logistic model for Al and Xi
cases, Illex coindetii females (F) and males (M) of the Central Mediterranean (see Table 1 for case specifications).

854

Ragonese et al.

CD
(U

o>

o

0.0

-0.5

-10

-15

-2.0

-2.5

>

Aa Q .^^ °0 A

Ad A^°A

A

_A

O

O A2-F

A A4-M

A2 - F com

- - - A4 - M com

r r

70

90

110

130 150

Age (days)

170

190

210

Figure 2. Individual !»(;,. of hody weigiit (BW) al maiUk- lengtii (Mlj ratio vs. age and computed (com) .specified logistic model for A2 and A4
cases, lllex coindelii females (F) and males (M) of tlie Central Mediterranean (see Table 1 for case specifications).

fore, they have to be considered only as representing the form of
growth.

To explore the most suitable models to apply. WaL-at-age scat-
ter plots were analyzed by points interpolation with a model-fitting
procedure (Systat 1992). A variety of available models were ap-
plied to the untransformed WaL and age data, as well as to data
sets with the dependent variable only and with both variables
transformed (using natural, base e, logarithms). This exploratory
analysis indicated that the relationship of WaL on age (t) was best

described by the power function in some cases (WaL = at*") and
by asymptotic models in other cases. Among the asymptotic mod-
els, the sigmoidal logistic model was preferred (Ratkowsky 1983).
Therefore, the following two models (applied to log trans-
formed data to stabilize the variance and normalize the residuals)
were tested:

the allometric model, hereafter referred to as the log-log model,

which represents the linear transformation of the power model:

TABLE 3.

Estimated coefficients of the log-log and specific logistic models by using individual vveight-at-length (WaL I at-age observations: log =
natural (log,) logarithm; r' = coefficient of determination; MSE = mean squared error; N = sample size.

Case

Sex

Group

Log-log Model (i)

Specific Logistic Model (ii)

Species-Area

log^a

b

r"

MSE

A

B

C

r"

MSE

N

/. coiinli'lii

Al

f

Oct.-Dec.

-13.552

2.549

0.859

0.059

0.1335

0.0314

159.4

0.950

0.054

189

Central Mediterranean

s.e.

0.378

0.076

s.e.

0.0856

0.0021

6.2

A2

f

May-July

s.e.

-11.754
0.493

2.26(1
0.101

0.777

0.055

s.e.

-0.2708
0.0499

0.0508
0.0040

114.4
2.6

0.953

0.041

144

A3

m

Oct.-Dec.

s.e.

-13.134
0.298

2.479
0.060

0.887

0.050

s.e.

-0.0373
0.0515

0.0360
0.0017

143.0
3.5

0.968

0.041

218

A4

m

May-July

s.e.

-13.118
0.547

2.552
0. 1 1 3

0.840

0.057

s.e.

-0.2046
0.0602

0.0527
0.0045

114.4

3.2

0.961

0.041

98

/. illecebrosus

El

f

March

-9.9.34

1 .883

0.685

0.040

Model failure

25

NW Atlantic

s.e.

1.359

0.266

E2

f

April

s.e.

-14.893
1 .542

2.877
0.302

0.777

0.039

s.e.

0.7126
0.2702

0.0335
0.0082

175.3
17.3

0.839

0.037

28

E3

f

May

-13.280

2.618

0.827

0.013

Model failure

11

s.e.

2.052

0.400

E4

m

March

-6.288

1.164

0.5,36

0.024

Model failure

47

s.e.

0.814

0.162

E5

m

April

-7.827

1.488

0.416

0.090

Model failure

35

s.e.

1 .549

0.307

E6

ni

May

-9.L54

1.819

0.590

0.019

Model failure

15

s.e.

2.168

0.420

Growth Performance Across the Squid Genus Illex

855

log^WaL = log^.a + blog^t + e

and;

the specific logistic (Weatherley 1972):

log^WaL = A - log,( 1 + e-'"'^'^') + e'

where log^.a and b are the intercept and the slope parameters re-
spectively, A denotes the asymptote (log^WaL-,), B is the slope, C
is the inflection point (days), t is the age (days), considered as the
independent variable, and e and e' are the error terms.

A possible disadvantage of the specific logistic model is the
apparent lack of a direct biologic interpretation for the asymptotic
parameter (A). However, a direct biologic meaning may be found
for the parameter C, which, when modeling growth in length,
represents the inflection point at the end of the initial exponential
growth phase and the beginning of the logarithmic growth phase.
We investigate the possible relationship of the age at inflection
point (i.e.. the C parameter) with the approximate mean age of all
mature animals of a specific group, as derived from the literature.

Model coefficients were estimated by the ordinary (Type I)
simple least squares method, using both linear (Model i) and non-
linear (Model ii; "Quasi Newton" and "Simplex") iterative proce-
dures. The coefficient of determination (r) and the mean square
errors (MSE) were used as approximate indices of goodness of tit.
The former statistic, in particular, was used to compare results
derived by modeling WaL with those derived by modeling ML and
BW separately (original size models. Table 1 ). Probability levels
for acceptance of significance of the regression coefficients and
the overall regressions were fixed at P < 0.05* (significant) and
P < 0.01** (highly significant). Statistical analysis, model fitting,
and computations were performed using the package Systat
(1992).

RESULTS
Models for Data Sets from Individuals

I. coindetii

The log-log transformation (Model i) was not successful in
achieving linearity between WaL and age for /. coindetii from the
Strait of Sicily, with the exception of females of the October to
December group (Al). However, the scatter-plots of log^. trans-
formed WaL values vs. age according to Model ii (Figs. 1 and 2),
showed an acceptable homogeneity of variance. Male and female
curves overlapped, but the two seasonal groups did not. October to
December specimens (A1-A3) showed lower log^.WaL values for
a given age than did their May to July counterparts (A2-A4),
suggesting a seasonal effect with higher growth performance of
spring-summer group.

Considering cases A and Model i (Table 3). all regressions
were significant, but the regression parameters were rather impre-
cise, as reflected by high standard errors. The Model i. therefore,
did not properly fit the data sets, as already indicated by failure to
achieve linearity. Improvement in both r~ and MSE was achieved
by fitting Model ii (Table 3). For both hatching groups and sexes
convergence was rapid, and various starting values led to very
similar final estimates of regression parameters, regardless of
which non-linear fitting procedure was used. The precision of the
regression parameters was acceptable. Model ii parameter values
were similar between sexes of the May-July group (A2 and A4:
Table 3, Fig. 2), whereas some sex effect was detected in the
October to December group (Al and A3, Fig. 1). In that fall to

winter group, females exhibited higher asymptotic values than
males and a small difference in the slope parameter and inflection
points (Table 3, Fig. 1 ). Overall, /. coindetii specimens hatched in
May to July showed a higher growth performance than their Oc-
tober to December counterparts.

/. illecebrosus

For this species (E cases), parameter estimation was achieved
only using Model i (Table 3), except for the E2 case. The precision
in the estimation of parameter A (Model ii) was very low in that
case, however, as reflected by the ratio of that parameter to its
standard error (0.27/0.71, i.e.. 38% of the variation).

The Model i (log-log regression) coefficients (Table 3) indicate
that the slopes were higher for May and April than for March, and
higher for females than males.

Models for Data Sets of Predicted Values

Results obtained from fitting both models to the data sets of
predicted values (all cases) are presented in Table 4. The consis-
tently higher model fits (reflected by higher r and lower MSE)
than for models fitted to data sets of individuals (Table 3), reflects
the much lower variance in the data sets of values predicted by
other (single size-at-age) models than in raw empirical data sets. A
comparison of specific logistic models among cases is shown for
females (Fig. 3) and males (Fig. 4).

It is interesting to initially compare the A and E cases for which
models were also fitted to the data sets based on individuals.

Case A: /. coindetii

The log^WaL-at-log^.-age plots (Model i) were nearly linear for
the October to December group (cases Al and A3) and more
strongly curvilinear, with an asymptotic trend, for May to July
hatched squid (cases A2 and A4). The log^.WaL-at-age plots
(Model ii) showed a curvilinear trend in October to December
specimens and a clear asymptote in May to July animals.

Application of Model (i) resulted in clear differences in models
between seasonal groups and sexes. Model fits were better (higher
r and lower MSE) and slopes were higher for October to Decem-
ber than for May to July models and also for female than for male
iTiodels (Table 4).

The application of Model ii (Table 4) resulted in rapid conver-
gence and good agreement between empirical and predicted val-
ues. The asymptotic (A) and inflexion (C) parameters were higher
in the October to December group; with the exception of the A
parameter (which showed great variability), males and females
within the each group showed very similar inflection and steepness
parameters (Table 4).

Case E: /. illecebrosus

Linearity was not fully achieved by the log-log transformation
and the application of Model i resulted in different parameter
values among groups (cases El to E6; Table 4); however, the fit
was good (high r and low MSE) with only slight differences
between sexes, except for E3 and E6 cases (higher slopes for
females than males in the May-hatched group).

The log^WaL-at-age plots (Model ii) resulted in very similar
(virtually coincident) curves for both sexes of the March (El, E4)
and May (E3. E6) groups while larger values of log^,WaL-at-age
were observed in females than in males of the April group. (E2,
E5); the slopes consistently increased from March to May for both
sexes, suggesting increase in growth rate throughout the spring.

856

Ragonese et al.

TABLE 4.

Estimated coefficients of the log-log and specific logistic models bv using predicted weight and mantle length to derive weight-at-length
(WaL)-at-age observations: log = natural (log,! logarithm; r" = coefficient of determination: MSE = mean squared error; N = sample size.

Case

Sex

Group

Log-log Model (i(

Specific L

jgistic Model (iil

Species-Area

log,.a

b

/■■

MSE

A

B

C

r'

MSE

N

/. cnindetii

Al

f

Oct.-Dec.

-14.029

2.628

0.964

0.037

0.002

0.038

148.7

0.997

0.004

17

Central Mediterranean

A2

f

May-Julv

-11.912

2.288

0.918

0.048

-0.301

0.054

108.5

0.996

0.003

12

A3

m

Oct.-Dec.

-11.551

2.152

0.953

0.026

-0.065

0.036

140.0

0.998

0.00 1

16

A4

m

May-Julv

-8.650

1.639

0.799

0.03 1

-0.315

0.078

108.4

0.999

0.000

9

/. cointletii

Bl

f

Oct.-March

-8.569

1.418

0.999

0.0001

0.7747

0.0065

443.0

0.999

0.0000

18

W Mediterranean

B2

f

Apr.-Sept.

-8.729

1 .452

0.999

0.0003

0.5215

0.0083

360.0

0.999

0.0002

21

B?

ni

Oct.-March

-10,S25

1.803

1.000

0,0001

0 8'-)73

0.0085

443,0

0.997

O.OOIO

19

B4

m

Apr.-Sept.

-11.680

1.982

1 .000

0.00(.)0

0.4756

0.0119

323.1

0.999

0.0002

15

/. coindetii

CI

r

pooled animals

-13.337

2.405

0.997

0.0041

1 .5600

0.0140

355.9

0.997

0.0037

25

Galician (Atlantic)

c:

m

pooled animals

-15.041

2.689

1.000

0.0000

1 .0460

0.0181

297.0

0.995

0.0083

21

/. coindetii

Dl

f

Sierra Leone

-4.258

0.727

0.941

0.0024

-0.2693

0.0251

110.8

0.994

0.0003

10

NW African Coasts

D2

f

W. Sahara

-9.506

1.845

0.995

0.0020

1.0167

0.0196

198.5

0.999

0.0007

15

D3

m

Sierra Leone

-8.214

1.473

0.889

0.0185

-0.4887

0.0482

118.3

0.994

0.0012

8

D4

m

W. Sahara

-10.972

2.126

0.961

0.0229

0.5799

0.0327

152.9

0.996

0.0026

12

/. ilU'cehrosus

El

f

March

-5.947

1.010

0.999

0.0001

0,3350

0.0131

161.8

0.999

0.0000

10

NW Atlantic

E2

f

April

-8.848

1.705

1.000

OOOOO

0.8022

0.0180

1 89.3

0.999

0.0002

10

E3

f

May

-11.324

2.235

1.000

0.0000

1.2347

0.0205

202.0

0.999

0.0001

7

E4

ni

March

-5.364

0.981

0.999

0.0000

0.2224

0.0139

146.6

0.999

0.0000

8

E5

ni

April

-7.662

1 .454

1.000

0.0000

0.5779

0.0164

178.5

0.999

0.0000

8

E6

m

Mav

-8.728

1.737

1.000

0.0000

1.1411

0.0171

196.4

0.999

0.0000

6

/. an^eutinus

Fl

f

June

-14.200

2.548

1 ,000

0.0000

1.2977

0.0140

333.2

0.999

0.0002

13

Patagonian Shelf

F2

ni

June

- 1 1 .606

2.130

1,000

0.0001

1.6207

0.0113

358.9

0.999

0.0000

13

-3.50

60

90

120

150

240

270

300

330

360

-0-A1-IC
-•-A2-IC
-^B1-IC
-^ B2-IC
-»^C1-IC
-ChDI-IC
-m- D2-IC
-^E1-ll
-♦^ E2-II

E3-II

H-FI-IA

180 210

Age (days)
Figure 3. Log„ of body v»eight (BW) at mantle length (ML) ratio (based on predicted values, symbols) vs. age and computed specified logistic
models (lines) for Illex females (see Table 1 for case specifications).

Growth Performance Across the Squid Genus Illex

857

1.50

-3.50

60

90

120

150

270

300

330

360

A3-IC
A4-IC
B3-IC
-k- B4-IC
-^ C2-IC
-O- D3-IC
-^- D4-IC
^ E4-II
-*- E5-II

E6-II

-^ F2-IA

180 210 240
Age (days)

Figure 4. Log^ of body weight (BW) at mantle length (ML) ratio (based on predicted values, symbols) vs. age and computed specified logistic
models (linesl for Illex males (see Table 1 for case specifications).

Application of Model ii resulted in rapid convergence and sat-
isfactory agreement between predicted and empirical values (Table
4). Both the slopes (B) and asymptote (A) increased with month of
hatching in each sex. Differences in these parameter values be-
tween sexes were slight for all hatching groups.

Case B, C, D, and F: Illex spp.

For these cases, for which no raw data were available, both
models performed quite well (Table 4). but Model i did not achieve
complete linearity in D cases. There were some strong differences
in model parameters between the sexes but none that were con-
sistent among groups and between Models i and ii. All cases for all
three species are compared using Model ii for females (Fig. 3) and
males (Fig. 4). Two patterns emerged: relationships with a slight
curvature (Cases B1-B4; C1-C2; F1-F2) and relationships with a
pronounced curvature and asymptotic trend (all D cases).

Clear differences in model slopes between sexes were evident
in /. coindetii off Sierra Leone (Dl, D3) and in /. argentinus (Fl ,
F2), but in the former the slope was higher for females than males,
whereas the opposite was true in the latter.

A comparison among and within species indicates that B cases
(/. coindetii from the Catalonian Sea; Figs. 3 and 4) are unique.
They are consistently different from the other curves, suggesting
relatively slow and near linear growth, regardless which model is
considered.

The Inflection Point and Maturity

The relationship between age-at-the-inflection point (i.e.. C pa-
rameter; Table 4) and the mean age of mature animals of a specific

group (as directly computed or roughly estimated from the litera-
ture) was examined (Fig. 5). Despite the rough approximation, a
positive and significant linear trend between the two parameters
was evident, suggesting that C values reflect to a certain extent
ages at maturity. The estimated slope ( 1 .7). however, is higher than
the value expected in case of direct proportionality (close to 1 ).
The inflection parameter tended to be relatively lower than (or
equal to) the age a! maturity for groups where almost all the life
stages were represented in the samples (i.e., A1-A4 and D1-D4).
It tended to be relatively higher in cases in which mature speci-
mens were underrepresented in the samples, i.e.. mean age at ma-
turity was probably underestimated (i.e.. C1-C2 and Fl).

DISCUSSION

The most appropriate model for describing growth in cephalo-
pods is currently a subject of strong debate. One argument is that
squid are fundamentally different from fish and so any of the
several available empirical growth models may be appropriate and
applied (Jackson 1994). The alternative argujiient is that most
principles of fish population biology do apply to squid: therefore
the most appropriate growth function to use is the von Bertalanffy
Model (Pauly 1985. Longhurst & Pauly 1987. Pauly 1998).

There is no need of entering into this matter here because an
index of growth performance was iflodeled. not growth in absolute
size itself. Therefore we were justified in embracing, the view of
Ricker ( 1979). that "the only criteria for choosing a growth curve
that have proven valid are goodness of fit and convenience." It is,
however, of considerable interest to compare trends in growth
performance obtained here with trends in growth from studies of
absolute size-at-age.

858

Ragonese et al.

260

300

140 180 220

Mean age (days) of mature specimens

Figure 5. Relationship between age at inflexion point (C parameter of llie specific logistic model) and mean age of mature Illex females (see Table
1 for case specifications). Tlie fitted straight line is significant Ir" = (1.835: df = 9).

Trends in Growth Performance

A single curve has seldom been suitable to describe the whole
life cycle when modeling size-at-uge (Ricker 1979, Jackson 1994);
therefore, a single model is more often a compromise that deals
with different growth phases, each of which could be better de-
scribed independently.

In the case of lUe.x spp. at least three phases of growth can be
identified: an early (larval) exponential phase, a "juvenile"' (power
or log linear) phase and a "mature and post mature" phase. Be-
cause of this, the exponential and the power models are often
defined as "early growth curves" (Peters 1983).

Linear modeling of length-at-age data fits satisfactorily some
restricted portions of the life cycle (Rodhouse & Hatfield 1990.
Gonzalez et al. 1996, Dawe & Beck 1997), but clearly does not
apply to the entire cycle (Dawe & Beck 1997. Gonzalez et al.
1998).

A logistic model was already used quite satisfactorily to rep-
resent growth in length and weight of A1-A4 squids (Arkhipkin et
al. 2000).

However, several different models can fit the same set of data
(Arkhipkin et al. 1998) and in some situations both linear and
curvilinear models could perform quite well, as was recently re-
ported for length-at-age of/, argentinus (Uozumi & Shiba 1993).

In our study of growth performance, the logistic model was
quite suitable for cases in which most of the ontogenetic develop-
ment was represented in the samples (i.e.. individual and predicted
data for cases A and values based on predicted sizes for cases D).
Nevertheless, some imprecision in the estimate of the A (asymp-
totic) parameter was evident, likely as a consequence of the poor

representation in the samples of spent males and females, i.e., a
poor representation of the last portion of the life cycle.

Besides the problem related to sampling the whole life cycle,
poor model fits obtained with raw data may be caused by several
different biases that are related to the different aspects of the
methodology applied, starting with the partitioning of specimens
into groups or "microcohorts" based on periods of hatching (e.g..
month). Statolith increment counts in fact, can be affected by
systematic and measurement errors due to preparation techniques
and reader's interpretation (Jackson 1994. Dawe & Beck 1997.
Arkhipkin et al. 1998. Gonzalez et al. 1998, Gonzalez et al. 2000).
Thus, the hatching month to which any individual is assigned may
be largely a function of ageing error (Gonzalez et al. 2000).

The use of mean values, despite statistical problems in fitting
models, reduces the incidence of these errors thus reflecting in a
better model fit.

Comparisons of absolute growth can also be biased by differ-
ences in the sampling gear used. Although little is known about the
catching capacity of gear such as trawls, traps and jigs, it is most
likely that their selectivity by size do differ, so that none of them
individually would provide a truly representative sample. Our ap-
proach facilitates comparisons when such sampling biases are
present, since different gears are likely to be much more highly
selective for absolute size than for physical condition.

Evaluation of Models

One of the immediate questions to answer, i.e.. "Is there any
advantage to modeling WaL above modeling ML and BW sepa-
rately'?" requires comparisons to be answered. Such comparisons
were possible only for individual /. coindetii (A1-A4 cases) and /.

Growth Performance Across the Squid Genus Illex

859

illecebrosiis {E1-E6) data sets, based on the logistic models re-
ported in Arkhipkin et al. (2000) and the simple linear and log-log
models presented in Dawe and Beck ( 1997).

The r^ for A cases ranged between 0.87-0.91. 0.77-0.89, and
0.95-0.97 for ML-at-age, BW-at-age, and log^WaL-at-age, respec-
tively. Considering the E cases, the r values ranged between
0.37-0.84, 0.37-0.90, and 0.35-0.90 for ML-at-age, log^.BW-at-
log^age, and log^.WaL-at-age, respectively. Results indicate that
similar (E1-E6) or even better (A1-A4) model tits were obtained
by using WaL instead of ML and BW separately, with the ad-
vantage of using only one model to take into consideration two
size indices. A possible explanation for that is that while length
will increase with age under any conditions, weight may increase
or decrease, but does not account for concurrent increases in
length. However, weight-at-length does account for concurrent
length increase, and so it is a better descriptor of growth perfor-
mance.

Another very interesting point is the comparison of results ob-
tained by applying the same models to the same data sets and using
alternatively raw data from individuals and values computed from
predicted lengths and weights. While both Models i and ii (i.e., the
log-log and the specific logistic) do fit consistently well for both /.
coincletii and /. illecebrosiis datasets when using data based on
predicted sizes, when raw individual data were used. Model ii
fitted well /. coindetii datasets (Cases A), but did not fit /. illece-
brosiis datasets. Such differences in Model ii fits are likely the
consequence of the "quality" of the available datasets. Raw indi-
vidual age data in fact, are almost neither "balanced" (i.e.. the
same number of aged specimens for each age level) nor "optimal"
(i.e., number of counts proportional to the variance of the esti-
mates) because statolith processing is very time consuming and
age classes are numerous. In cases of such high variability, few
points could have a great influence on the estimates and the model
may not fit properly. If the samples are larger, however, or if
repeated blind counts are available (see, for example, Arkhipkin et
al. 1998), then a statistical "weight" can be assigned to each point
and a weighed regression performed to improve the fitting quality
(Neter et al. 1985).

Comparisons Within and Among Species

WaL modeling showed some common patterns among species
and groups, with the exception of the /. coindetii population of the
Catalonian Sea. Regardless of the model applied to those excep-
tional cases, the parameters generated were quite different from

those of all the other cases. To a certain extent, such differences
may be related to the indirect method used in the present work to
compute body weight-at-age, but it is also likely that age was
originally overestimated for that group, as a consequence of the
ageing method used (Arkhipkin personal communication: Dawe &
Beck 1997, Gonzalez et al. 2000).

Results obtained for the other groups showed several common
features of growth performance within the genus Illex, which are
consistent with results of other studies (e.g., Dawe & Beck 1997,
Haimo\ici et al. 1998, Arkhipkin et al, 2000). These features in-
clude superior growth performance of /. coindetii and /. illecebro-
siis specimens hatched in warm conditions relative to those
hatched in colder conditions. Also consistent with previous stud-
ies, a superior growth performance in populations inhabiting warm
waters (Strait of Sicily in the Central Mediterranean Sea. West
Sahara, and Sierra Leone in the Eastern Atlantic) relative to that of
con-specitlc or con-generic populations living in colder waters
resulted. Last but not least, the previous conclusion that the Gali-
cian /. coindetii population appears to be more similar to the South
Patagonian stock of /. argentinus than to any other Illex popula-
tions (Gonzalez et al. 1996) was confirmed.

One of the most interesting results of our study is the direct
relationship found between the intlexion parameter C (from appli-
cation of the specific logistic model) and the age-at-maturity. Size-
at-maturity in Illex squid has rarely been objectively defined (e.g.,
as the size at which 50% of the specimens were classified as
mature; cf Jereb & Ragonese 1995). Usually, only ranges or mean
size values of mature animals have been reported, although the
onset of sexual maturation is considered to be a critical point in
cephalopod life cycles (Mangold et al. 1993).

The C parameter may represent a useful maturity objective
index for future studies of changes or trends in sexual maturation.
For example, Pauly (1998) argued that principles of fish dynamics
are also applicable to cephalopods. If this were true, then tempera-
ture would be directly related to growth rate and inversely related
\.o maximum size- and age-at-maturity. Consequently, when com-
paring among populations, a shift of the inflection parameter to
younger ages would be expected from colder to wanner habitats,
related to an improvement in growth performance. This is consis-
tent with results obtained in the present work.

Obviously further comparative studies are required especially
within populations. Independent estimates of size- and age-at-
maturity would be very useful in evaluating if a true relationship
exists between the C parameter from the specific logistic model
and the age at the onset of maturity.

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Jounwl of Shellfish Research. Vol. 21. No. 2. 861-870. 2002.

COMPARISON OF NUTRIENT COMPOSITION OF GONADS AND COELOMIC FLUID OF
GREEN SEA URCHIN STRONGYLOCENTROTUS DROEBACHIENSIS

CHANDRIKA LIYANA-PATHIRANA,' FEREIDOON SHAHIDI,' " * AND ALAN WHITTICK'

^Department of Biology, -Department of Biochemistry. Memorial University of Newfoundland. St.
John's, Newfoundland, AlB 3X9, Canada

ABSTRACT The compositional characteristics of sea urchin gonads and coeloniic lluid from Stnmgylocemnitus droehuchiensis
harvested in the coasts of Newfoundland and thereafter reared in an aquaculture facility and fed on a Laminaria diet for a 3-week
period, were assessed. Evaluations were performed on the basis of proximate composition, lipid class distribution, fatty acid compo-
sition, total and free amino acid composition, and contents of nucleic acids and carotenoids. Noticeable changes existed between
proximate composition of sea urchin gonads and coelomic fluid. Moisture content was 74.7 + 0.04 and 96.5 ± 0.03% in gonads and
coelomic fluid, respectively. Gonads contained very high levels of lipids, proteins, and carbohydrates; whereas, these were present at
very low levels in the coelomic fluid. Major nonpolar lipid classes were triacylglycerols (TAG), free fatty acids (FFA). and sterols (ST)
while dommant polar lipid classes were phosphatidylcholine (PC), phosphatidylethanolamine (PE). sphingomyelin/ lysophosphatidyl-
choline (SM/LPC). and phosphatidylserine / phosphatidylinositol (PS/PlI in both the gonads and the coelomic fluid. Major saturated
fatty acids (SPA) were 14;0 and 16:0; whereas. 20:ln-15 was the main monounsaturated fatty acid (MUFA) present. Furthermore,
20:5n-3 (eicosapentaenoic acid, EPA) was the dominant polyunsaturated fatty acid (PUFA) in the gonads and the coelomic fluid. The
total amino acid (TAA) and free amino acid (FAA) profiles were dominated by glycine. The total FAA content was much higher in
the gonads than in the coelomic fluid. In addition, the total carotenoid content of sea urchin gonads was approximately 6.4 times greater
than that of coelomic fluid. Hence, most of the carotenoids were concentrated in the gonadal tissue. Echininone and fucoxanthin were
the dominant carotenoids in the gonads and the coelomic fluid, respectively. The content of deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) was much higher in the gonad than in the coelomic fluid, thus indicating greater biomass and protein synthetic
activity in the former tissue. The present study demonstrates that sea urchin gonads have much in common with sea urchin coelomic
fluid on a qualitative basis. However, there were marked quantital;ve differences between the two tissues.

KEY WORDS: amino acid composition, carotenoids. fatty acid composition, lipid class distribution, nucleic acids, Slmngyhcen-
trolus droehachiensis

INTRODUCTION

Sea urchins belong to the marine invertebrate phylum Echino-
dermata or spiny-skinned animals. These relatively small echino-
derms have spherical bodies enclosed in a hard shell or "test"
completely covered with numerous sharp spines. Sea urchins are
omnivorous animals that live on the ocean floor, feeding on small
crustaceans, fish offal, but mainly seaweed (Smith 198(5). Thus,
the eating quality of sea urchin gonads is dictated, to a certain
degree, by the quality of kelp consumed. Laminaria kelps are the
preferred source of feed for sea urchins. Kramer and Nordin ( 1979)
reported that the green sea urchin Strongyloceiitniliis droebachien-
sis produces high-quality gonads when the availability of fresh
kelp is adequate. The edible green sea urchin S. droehachiensis is
abundantly distributed in the North Atlantic, Arctic, and North
Pacific Oceans, but this species is currently exploited to a much
lesser extent in the Northwest Atlantic and in the Northeast Pacific,
and Northeast Atlantic (Walker & Lesser 1998). Furthermore, S.
droehachiensis is a target species for the development of commer-
cial echiniculture (Hagen 1996).

The edible portions of the sea urchin body are its reproductive
organs; ovaries, and testes. Gonad yield from sea urchin may vary
with the time and the site of harvest and generally ranges from
8-20% of the total body mass. When sea urchins are processed for
gonads, the initial step is to break the shell and open it so that the
five gonad sacs are exposed. The cracked shells are then allowed
to drain for several minutes to dispose of coelomic fluid. Thus,
during extraction of sea urchin gonads, large amounts of coelomic

fluid are obtained. So far, there are no effective means of using sea
urchin coelomic fluid in a useful manner. Furthennore, no infor-
mation is available on the nutrient composition of sea urchin coe-
lomic fluid. In fact, knowledge of nutrient composition may be
useful to determine whether sea urchin coelomic fluid could serve
as a potential source of a flavoring in fabricated seafood.

The objective of this study was to assess the nutrient compo-
sition of sea urchin coelomic fluid as compared with that of the
gonads. Thus, proximate composition, lipid class distribution, fatty
acid composition, amino acid composition, and contents of carot-
enoids and nucleic acids of gonads and coelomic fluid were de-
termined. This may lead to potential commercial utilization of the
processing byproducts from sea urchins, which would otherwise be
discarded.

MATERIALS AND METHODS

Materials

*Corresponding author. Tel.: (709) 737-8552; Fax: (709) 737-4000;
E-mail: fshahidi@mun.ca

One hundred twenty-five sea urchins were procured from the
Sea Urchin Research Facility (SURF) at Bonavista Bay, New-
foundland and subsequently transported in aquarium coolers to our
laboratory at Memorial University of Newfoundland. Urchins were
captured from the wild (June 2000) and raised in raceways feeding
on a Laminaria diet. Urchins were harvested for analysis after
three weeks of feeding on a purely algal diet. Live urchins were
stored at 4°C before the extraction of tissues. The gonads and
coelomic fluid of sea urchins were separated after breaking the
shell, using a specially devised sea urchin cracking tool. After
extraction, sea urchin gonads were homogenized for 2 min using a
cooled Waring blender (Dynamics Corporation, New Hartford,
CT), and coelomic fluid was used for analysis as it is. In this study.

861

862

Liyana-Pathirana et al.

sea urchin male and female gonads were pooled together for analy-
sis. The tissues (both gonads and coelomic fluid) were flushed with
liquid nitrogen and stored at -20°C until used for further analyses.
All chemicals used were obtained from either Fisher Scientific
(Fair Lawn. NJ) or Sigma Chemical Co. (St. Louis. MO). The
solvents were of ACS-, pesticide-, or HPLC-grade.

Determination of Proximate Composition

Moisture and ash contents of sea urchin tissues were deter-
mined according to the standard AOAC ( 1990) procedures. Crude
protein content was obtained by Kjeldhal method (AOAC 1990).
and total lipids were extracted and quantified by the Bligh and
Dyer ( 1959) procedure. Carbohydrate content of each sample was
determined by difference.

Analysis of Lipid Classes by latrnscan

Instrumentation

The crude lipids obtained from Bligh and Dyer ( 1959) extrac-
tion were chromatographed on silica gel-coated Chromarods - S III
and then analyzed using an latroscan MK-5 (latroscan Laborato-
ries Inc., Tokyo) analyzer equipped with a flame ionization detec-
tor (FID) connected to a computer loaded with TSCAN software
(Scientific Products and Equipment. Concord. ON) for data han-
dling. A hydrogen flow rate of 160 niL per min and an airflow rate
of 2.000 mL per min were used in operating the FID. The scanning
speed of rods was 30 sec per rod.

Preparation of Chromarods

The Chromarods were soaked in concentrated nitric acid over-
night followed by thorough washing with distilled water and ac-
etone. The Chromarods were then impregnated by dipping in a 3%
(w/v) boric acid solution for five minutes to improve separation.
Finally, the cleaned Chromarods were scanned twice to burn any
remaining impurities.

Standards and Calibration

A stock solution of each of the nonpolar lipids; namely, free
fatty acid (FFA; oleic acid), cholesterol ester (CE). cholesterol
(CHOL), monoacylglycerol (MAG; monoolein). diacylglycerol
(DAG; diolein). and triacylglycerol (TAG; triolein), and the polar
lipids; namely, phosphatidylcholine (PC), phosphatidylethanol-
amine (PE). phosphatidylinositol (PI), phosphatidylserine (PS),
lysophosphatidylcholine (LPC). lysophosphatidylethanolamine
(LPE). cardiolipin (CL). and sphingomyelni (SM) was prepared by
dissolving each in a chloroform/methanol (2:1. v/v) solution and
stored at -20°C. A range of dilutions of the stock solution, from
0.1 to 10 |j.g per (xL. was prepared for use as working standards.
Each compound was developed individually and run on the latro-
scan-FID to determine its purity and Rf value. For each compound
peak, area was plotted against a series of known ct)ncentrations to
obtain the calibration curve.

latroscan (TLC-FID) Analysis of Sea Urchin Lipids

The total lipids extracted were dissolved in chloroform/
methanol (2:1, v/v) to obtain a concentration of 1 p.g lipid per mL.
A I ji,L aliquot of sample was spotted on silica gel-coated Chro-
marods - S III and conditioned in a humidity chamber containing
saturated CaCl, for 20 min. The Chromarods were then developed
in two solvent systems. The solvent system hexane/diethyl ether/

acetic acid (80:20:2. v/v/v) was u.sed for separation of nonpolar
lipids (Christie 1982). Following their development, Chromarods
were dried at 1 10°C for three minutes and scanned completely to
reveal nonpolar lipids. For polar lipids, following the same proce-
dure and drying, the Chromarods were scanned partially to a point
just beyond the MAG peak to bum the nonpolar lipids. These
partially scanned Chromarods were developed in a second solvent
system of chloroform/methanol/water (80:35:2, v/v/v) for the sepa-
ration of polar lipid classes (Christie 1982) followed by drying at
110°C for three minutes. Finally, the Chromarods were scanned
completely to reveal polar lipids; the identity of each peak was
determined by comparison with a chromatogram of standards ac-
quired concurrently with the samples. The determination of weight
percentages of individual lipid classes was achieved using the
standard curves obtained for each authentic standard.

Analysis of Fatty Acid Composition of Lipids

Fatty acid composition of lipids was determined using gas
chromatography (GC) as described by Wanasundara and Shahidi
(1997). Fatty acid methyl esters (FAMEs) of total lipids of sea
urchin gonads and coelomic fluid were prepared by transmethyl-
ating approximately 10 to 20 mg of each lipid sample in 2 mL of
freshly prepared transmethylating reagent [6% (v/v) sulfuric acid
in 99.9 moK/f HPLC-grade methanol containing 15 mg of t-
butylhydroquinone (TBHQ)] at 65°C for 15 h in a 6 mL Teflon-
lined screw-capped conical vials. After incubation, the mixture
was cooled, and 1 mL of distilled water was added to it. This was
followed by extracting the FAMEs three times with 1.5 mL pes-
ticide-grade hexane. A few crystals of TBHQ were added to each
sample before extraction with hexane. The hexane layers were
removed and combined in a clean test tube followed by washing
twice with 1.5 mL of distilled water by vortexing. The aqueous
layer was discarded after the first wash, while the hexane layer was
removed and placed in a GC vial following the second wash.
Hexane was evaporated under a stream of nitrogen in a fume hood.
The dried FAMEs were then dissolved in 1 mL of carbon disulfide
and used for GC analysis. FAMEs were separated using a gas
chromatograph (Hewlett-Packard 5890 Series II, Hewlett-Packard.
Mississuaga, ON) equipped with a fused silica capillary column
(SUPELCOWAX-IO. 0.25-mm diameter. 30-m length. 0.25-p,m
film thickness; Supeico Canada Ltd.. Oakville, ON). The sample
was injected into the GC analyzer using a Hewlett-Packard 7673
autoinjector (Hewlett-Packard. Toronto. ON). The temperature of
the oven was programmed at 220°C for 10.25 min followed by
ramping to 240°C at 20°C per min. where it was held for nine
minutes. Helium at a flow rate of 2 mL per min was used as the
carrier gas. The FAMEs were identified by comparing their reten-
tion times with those of authentic standard mixtures (GLC - 461.
Nu-Check-Prep) and literature values (Takagi et al. 1980. Takagi
et al. 1986). The relative content of fatty acids in the sample was
determined using the peak areas of fatty acids.

Carotenoid Pigments

Extraction and Determination of Total and Individual Carotenoids

Carotenoids from each tissue were extracted three times with a
total of 50 mL of acetone for two minutes. The homogenized
samples were centrifuged (lEC Centra MP4 Centrifuge. Interna-
tional Equipment Co.. Needham Heights. MA) at 4000 x g for five
minutes. The supernatant was subsequently filtered through a

Comparison of Nutrient Composition of Sea Urchins

863

Whatman No. 1 filter paper. Carotenoid pigments in acetone were
then transferred to 40 niL of n-hexane in a 250-mL separatory
funnel. One hundred niilhliters of a 0.57c sodium chloride solution
were added to the mixture to maximize the transfer of carotenoids.
The hexane layer was then transferred into a 50-mL volumetric
flask and made up to volume. The absorption spectrum was then
recorded (400-600 nm) using a Spectronic spectrophotometer
(Spectronic Genesis. Toronto. ON I. The total and individual ca-
rotenoid contents were determined by the method of McBeth
(1972). The total content of carotenoids present per 100 g of tissue
was calculated using the following equation.

mg Carotenoid per 100 g tissue = (A x V x 10')/e x W)

where, A = absorbance at \„„,; V = total volume of the sample
(mL); e = molar extinction coefficient, and W = weight of the
tissue (g). Because the crude extracts usually contained a variety of
carotenoids an average coefficient of 2,500 was used in the cal-
culations.

The total pigment extracted was separated into individual ca-
rotenoids by means of thin-layer chromatography (TLC). The
crude carotenoids were separated by preparative TLC on silica gel
G (20 X 20 cm, 230 (jim, Aldrich Chemical Co., Inc., Milwaukee,
WI) using acetone/n-hexane (3:7, v/v) as the developing solvent.

Characterization of Fractions

Cochromatography on TLC provided the ultimate test for iden-
tification when authentic samples were available for comparison
with unknown pigments. The unknown fraction and the authentic
sample were spotted on either side in an equally proportionated
mixture of the two pigments on silica gel G plates (20 x 20 cm. 250
fjLm, Aldrich Chemical Co., Inc., Milwaukee, WI): unknown frac-
tions were considered to be identical to the authentic sample if the
two did not separate upon subsequent development of the plate.
When authentic samples were unavailable, the type of carotenoid
in each fraction was tentatively identified according to its absorp-
tion maximum in n-hexane, ethanol, and chloroform (Goodwin
1955, Krinsky & Goldsmith 1960, Fox & Hopkins 1966, Britton
1995).

Determination of Total Amino Acids

The amino acid composition of sea urchin gonads and coelomic
fluid was determined according to the procedure described by
Blackburn (1968). Samples were lyophili/.ed and then hydrolyzed
for 24 h at 1 10°C with 6M HCl. Hydrochloric acid in the hydro-
lyzate was removed under vacuum, and the dried sample was
reconstituted with a lithium citrate buffer (0.2 M, pH 2.2) for
analysis. The amino acids in the hydrolyzate were separated, iden-
tified and quantified using a Beckman 121 MB amino acid
analyzer (Beckman Instruments Inc., Palo Alto, CA). Sulfur-
containing amino acids were determined by oxidizing the samples
with pertbrmic acid before their hydrolysis in a 6M HCI solution
(Blackburn 1968). Cysteine and methionine were measured as cys-
teic acid and methionine sulphone, respectively. To determine
tryptophan, samples were hydrolyzed in 3M mercaptoethane-
sulfonic acid at 1 10°C for 22 h under nitrogen and then neutralized
with lithium hydroxide and adjusted to pH 2.2 (Penke et al. 1974).

Determination of Free Amino Acids

Samples (10 g) were extracted with 20 mL of a 6% (v/v)
perchloric acid (PCA) solution by homogenization using a Poly-

tron homogenizer (Brinkmann Instruments, Rexdale, ON) at
10,000 rpm for two minutes in an ice bath. The homogenized
samples were then incubated in an ice bath for 30 min. This was
followed by centrifugation (lEC Centra MP4 Centrifuge, Interna-
tional Equipment Co., Needham Heights, MA) at 2,000 x g for 15
min. The residue was re-extracted with another 20 mL of 6% PCA.
The supematants were combined and filtered through a Whatman
No. 4 filter paper. The pH of the filtrate was adjusted to 7.0 using
a 33% KOH (w/v) solution. Precipitates of potassium perchlorate
were removed by centrifugation at 2000 x g for 10 min. The
supernatant was then acidified to pH 2.2 using a 10 M HCI solu-
tion, and the volume of the extract was brought to 30 mL with
distilled water. Three milliliters of lithium citrate buffer (pH 2.2,
0.3M) were added to I mL of the extract, and the resultant solution
was analyzed using a Beckman 121 MB amino acid analyzer
(Beckman Instruments, Inc., Palo Alto, CA) for individual amino
acids.

Determination of Nucleic Acids

The DNA and RNA contstituents of gonads and coelomic fluid
of sea urchins were extracted according to the method of Schmidt
and Thannhauser ( 1945) as modified by Munro and Fleck (1969).
Five grams of each sample were homogenized in 80 mL ice-cold
deionized water using a Polytron homogenizer (Brinkman Instru-
ments, Rexdale, ON) at 10.000 rpm. Five milliliters of the homo-
genate were allowed to stand for ten minutes in ice and then
centrifuged (lEC Centra MP4 Centrifuge, International Equipment
Co., Needham Heights, MA) at 2000 x g for ten minutes. The
residue was subsequently washed with 2.5 mL of ice-cold 0.2 M
PCA and centrifuged at 2,000 x g for ten minutes followed by
digestion of the residue in 4 mL of a 0.3 M KOH for one hour at
37°C in a water bath. The resultant solution was cooled in ice and
mixed with 2.5 mL of 1.2 M PCA and allowed to stand for ten
minutes, which finally resulted in the coagulation of proteins. The
mixture was centrifuged at 2,000 x g for ten minutes, and the
supernatant was recovered (Extract No.l). The precipitate was
then washed twice with 2.5 mL of a 0.2 M PCA solution and
centrifuged at 2,000 x g for five minutes. The supernatant was
combined with extract No. 1 and 10 niL of a 0.6 M PCA were
added to the mixture. This was used for RNA determination after
diluting it up to 100 niL with distilled water. The residue was
dissolved in 17 mL of a 0.3 M KOH solution at 37 C and diluted
to the 50 mL mark in a volumetric flask with distilled water. The
content of DNA in the samples was estimated by determining the
deoxyribose content in the extract using the indole procedure of
Ceriotti (1952), while RNA was determined by recording the ab-
sorbance of the nucleotide extracts at 260 nm using a Hewlett-
Packard diode array spectrophotometer (Hewlett-Packard, Model
8452A, Hewlett-Packard [Canada] Ltd., Mississauga, ON). Protein
interference at this wavelength was eliminated by employing a
correction factor of 0.001 absorbance unit for each I (xg per mL
protein concentration in the extracts. The protein concentration ot
the extracts was measured using the Folin-phenol procedure ot
Lowry et al. (1951). Bovine serum albumin (BSA) was used as a
standard. Calf thymus DNA (containing 82% single stranded
DNA) and calf liver RNA (96% purity) were used as the standards
for DNA and RNA determinations, respectively.

Statistical Analysis

Each experiment was replicated three times and mean values ±
standard deviations reported for each sample. For statistical analy-

864

Liyana-Pathirana et al.

ses, mean values of the experimental data were subjected to one
way analysis of variance (ANOVA) using GraphPAD Instat Ver-
sion 1.0. Significance was determined at 5% probability level.

RESULTS

I'roximale Coiiiposilidii

Proximate composition of sea urchin gonads and coelomic fluid
is shown in Table I . The moisture and ash contents of sea urchin
coelomic fluid were much higher than those of the gonads. On the
other hand, the levels of protein, lipid, and carbohydrate in the
coelomic fluid were much lower than those in the gonads on a
fresh weight basis.

Lipid Class Distrihiilion

The nonpolar and polar lipid classes of gonads and coelomic
fluid of sea urchin S. droebachiensis are shown in Table 2. Major
nonpolar lipid classes were TAG. FFA. and ST; whereas, main
polar lipids classes were PC. PE. SM/LPC. and PS/PI in both
gonads and coelomic fluid. Triucylglycerols constituted the main
energy reserve in both tissues, contributing more than 63% to the
total nonpolar lipids. On the other hand, PC was the dominant
polar lipid, accounting for more than 60% in both gonads and
coelomic fluid. The polar lipid classes SM and LPC as well as PS
and PI did not show a clear chromatographic separation from each
other during latroscan analysis.

Fatty Acid Composition

Fatty acid composition of sea urchin gonads and coelomic fluid
is presented in Table 3. Qualitatively, the fatty acid compositions
were the same in both tissues, while there were significant (P <
0.05) quantitative variations. In both tissues. 14:0 and 16:0 were
the main saturated fatty acids. In addition. 18:0 and 20:0 were
present in considerably high levels. The fatty acid 20: In- 1 5 was
the dominant MUFA in both gonadal and coelomic fluid lipids.
Furthermore, 16:ln-7. l6:ln-9. 18:ln-7, 20:ln-7, 20;ln-9, and 22:
In- II were detected in noticeable amounts. Among PUFA, 20:
5n-3 contributed the highest proportion to the total fatty acid con-
tent in both gonadal and coelomic fluid lipids,

Carotenoid Pigments

The total carotenoid content, on a dry weight basis, of sea
urchin gonads and coelomic fluid was 23.2 ± 0.04 and 3.7 ± 0. 1 mg
per g tissue, respectively. Crude pigments from gonads and coe-

TABLE 1.

Proximate cumposition of sea urchin gonads and coelomic fluid after
feeding urchins on a Laminaria diet for three weeks.

Constituent

Gonads

Coelomic Fluid

Moisture

74.7 (0.04)

96.5(0.031

Ash

2.2 (0.2)

3.0(0.02)

Protein

7.4 (0.2)

0.1 (0.02)

Lipid

4.7(0.1)

0.1 (0.03)

Carbohydrate'

10,6 (0.2)

0.4(0.1)

TABLE 2.

Quantification of non-polar and polar lipids (weight % ) of sea

urchin gonads and coeliiniic fluid alter feeding the urchins on

Laminaria diet for three weeks.

Lipid Cla.sses

Gonad

Coelomic Fluid

Non polar lipids

TAG

66.7(0.8)

56.5 (0.7)

FFA

22.7(1.))

37.4(1.0)

•ST

10.6(1.2)

6.1 (0.8)

MAG

tr

tr

DAG

tr

tr

Polar lipids

PC

65-7(1.4)

68.9 (0.8)

PE

17.3(0.2)

27.3 (0.9)

SM/LPC

8.4 (0.7)

2.6 (0.2)

PS/PI

8.5 (0.6)

1.1 (0.5)

Results are mean values of three replicates (standard deviation). Values in

each row with the same superscript are not different (f > 0.05) from one

another.

' Determined by difference.

Results are mean values of three replicates (standard deviation). Values in
each row with the same superscript are not different (P > 0.05) from one
another. Abbreviations: TAG. triacylglycerol; FFA, free fatty acid; ST,
sterol; MAG, monoacylglycerol; DAG, diacylglycerol: PC, phosphatidyl-
choline; PE, phosphalidylelhanolaniine; SM, sphingomyelin; LPC. lysi-
phosphatidylcholine; PS, phosphatidylserme and PI, phosphatidylinositol;
and tr. irace.

lomic fluid were separated by TLC into eight and seven fractions,
respectively. Crude pigments of both tissues, upon TLC separa-
tion, exhibited two major bands. In gonads, fractions I (Rf = 0.96)
and II (Rf = 0.88) ran close to the solvent front, but they were
adequately separated. For coelomic fluid, fraction I (Rf = 0.94)
ran almost close to the solvent front; whereas, fraction IV (Rf =
0.42) ran well behind. The carotenoid fraction I of both gonads and
coelomic fluid of sea urchin S. droehachiensis was confirmed to be
3-carotene using an authentic ^-carotene sample as established by
cochromatography on silica gel TLC plates. Similarly, gonadal
fractions III. IV. VI. and VII contained astaxanthin ester (Rf =
0.57), zeaxanthin (Rf = 0.51), canthaxanthin (Rf = 0.22), and
free astaxanthin (Rf = 0,1), respectively. Furthermore, fraction II
of gonadal crude pigments corresponded to echininone by means
of absorption maxima in hexane. chloroform, and ethanol (Good-
win 1955. Krinsky & Goldsmith I960. Fox & Hopkins 1966.
Britton 1995). Thus, the observed X^nax values of echininone were
484/460. 466. and 475 nm in hexane. ethanol. and chloroform,
respectively. In coelomic fluid, the pigment in fraction IV corre-
sponded with fucoxanthin based on absorption maxima of 424/
447/474, 423/446/472, and 454/488 nm in hexane. ethanol, and
chloroform, respectively. Other minor carotenoids in the coelomic
fiuid were astaxanthin etser (Rf = 0.58), canthaxanthin (Rf =
0,23), and free astaxanthin (Rf = 0.1), which coiresponded to
fractions III, V, and VI, respectively. The other minor carotenoids
were not analyzed because of their insufficient concentration to
obtain absorption maxima and also lack of authentic .samples.

Amino Acid Composition

The total and free amino acid compositions of sea urchin go-
nads and coelomic fluid are shown in Tables 4 and 5, respectively.
Results so obtained did not show any clear variation on a quali-
tative basis for tissues examined. Thus, the spectrum of amino
acids, both total and free, present was nearly the same for both
gonads and coelomic fluid. Total amino acid profile indicated the

Comparison of Nutrient Composition of Sea Urchins

865

TABLE 3.

Fatly acid composilimi (Ht'i(;lit '7f I of total lipids sea urchin gonads

and coeloniic fluid after feeding the urchins with iMininaria diet for

three weeks.

TABLE 4.

Total amino acid content Img/g protein! of sea urchin gonads and

coelomic fluid after feeding urchins on a Laminaria diet for

three weeks.

Fatty Acid

14:(1

15:0

KrO

IS:()

20:0

14:ln-7

l6:ln-9

l6:ln-7

16:1 11-5

lS:lii-4

lS;ln-7

lS:ln-5

20; In- 15

20:ln-9

20:ln-7

22: In- 11

22:ln-9

16:2n-fi

Hi:4n-h

l(r4n-3

l.S:2n-'-)

lS:2n-6

lS:3n-6

lS:3n-3

lS:4n-.^

20:2A5. 1 1

20:2A5. 13

20:2n-6

20:4n-6

20:3n-3

20:4n-3

20:5n-3

22:5n-6

22:5n-3

22:6n-3

Gonads

Coelomic Fluid

Amino Acid

Gonads

Coelomic Fluid

9.4(0.1)'
0.4(0.02)''
11.1 (O.!)-*
2.2(0.04)''
2.9(0.1)-'
0.8 (0.03)"
4.8(0.1)"'=
1.5(0.1)"
0.3 (0.03)°
1.8(0.1)"
3.6(0.1)''
0.5 (0.02)"
7.5 (0.2)"
4.0(0.03)"
2.2(0.1)"
2.9 (0.04)"
0.4(0.03)"
0.5 (0.03)"
1.9(0.1)"

ND

ND
1.1 (0.1)"
1.4(0.03)"
1.3(0.04)"
3.8(0.03)"
1.8(0.1)"
0.9(0.1)"
1.7(0.1)"
7.0(0.1)°
1.7(0.2)"
1.2(0.2)"
16.3(0.1)"
0.2(0.1)"
0.6(0.1)"
1,4(0.1)"

8.4(0.1)"
0.7 (0.02)"

17.6(0.3)"
2.3(0.1)"
1.2(0.1)"
0.3(0.03)"
1.6(0.1)"
3.4(0.2)"
0.6(0.04)"
2.2(0.1)"
4.2(0.1)"
3.4(0.2)"
5.6(0.1)"
1.2(0.03)"
1.9(0.1)"
1.2(0.1)"
1.1(0.1)"
0.2(0.02)"

ND
2.8(0.1)"
0.5 (0.02)"
0.9(0.1)"
1.3(0.1)"
0.5 (0.04)"
2.2 (0.03)"
1.7(0.1)"
0.7(0,03)"
1.7(0.1)"
9.9(0.1)"
0.9 (0.04)"
0.3(0.02)"

16.5(0.5)"
0.3 (0.03)"
0.7 (0.04)"
0.6(0.1)"

ResuUs are mean values of three replicates (standard deviation). Values in
each row with the same superscript are not significantly different {P > 0.05)
from one another. ND, not detected.

dominatice of glycine in both tissues analyzed. However, on a dry
weight basis, the content of glycine in sea urchin coelomic fluid
was significantly (P < 0.05) higher than that of gonads. Almost all
the essential amino acids were present in both gonads and coelo-
mic fluid of S, droebachiensis. With respect to FAA content of sea
urchin tissues, the total FAA content was much higher in sea
urchin gonads than that in the coelomic fluid. However, in both
tissues glycine was the dominant FAA contributing 57.1 and
56.3% to the total amount in the gonads and coelomic fluid, re-
spectively.

Content of Nucleic Acids

The content of nucleic acids in sea urchin gonads and coelo)nic
fluid, on a dry weight basis, was different. The content of DNA of
sea urchin gonads and coelomic fluid was 3.93 ± 0.1 and 1.02 ±
0.07 jjLg per g tissue, respectively, whereas, corresponding values
for the content of RNA were 2.63 ± 0.06 and 0.49 ± 0.03 p,g per
g tissue, respectively. Thus, the content of DNA was higher than

Alanine

Arginine

Cysteine

Glutamic acid

Glycine

Histidine

Hydro\yproline

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Proline

Serine

Threonine

Tryptophan

Tyrosine

Valine

Total (mg/g protein)

42.5 (0.4)"
80.7(0.5)"

7.2(0.1)"
87.2(0.2)"
118.0(0.7)"
35.5 (0.3)"

3.3 (0,2)"
77.1 (0,1)"
65,9(1.0)"

81.1 (0.9)"
1.2(0.3)"

53.2 (0.9)"
40.2(0.5)"
47.1 (0.1)"
47.8 (0.4)"

1.4(0.1)"

36.3 (0,8)"
78,3 (0,4)"

989,4(2,2)

63.1 (0.5)"

74.2 (0,6)"
5,7 (0,2)"

90.6 (0.6)"
143.6(0.7)"

27.0 (0.8)"
2.6 (0.2)"

60.9 (0.9)"
62.6 (0.3)"

77.1 (0,9)"
0,9(0,1)"

42.1 (0,6)"
48.4(0,1)"

46.5 (0,7)"

47.2 (0,5)"
1,1 (0,2)"

38.6 (0,4)"
75.1 (1.0)"

985.4(1.3)

Results are mean values of three replicates (standard deviation). Values in
each row with the same superscript are not different (P > 0.05) from one
another.

that of RNA in both tissues analyzed. Furthermore, the ratio of
RNA/DNA was 0.7 ± 0. 1 and 0,5 ± 0,03 for sea urchin gonads and
coelomic fluid, respectively,

DISCUSSION

Reproductive Stale of Sea Urchins

For feeding experiments, sea urchins were obtained from the
wild in the month of June, representing the spring season. In gen-
eral, gonad development in sea urchins may include five different
stages of resting, growing, premature, mature, and spawning (de
Jong-Westman et al, 1996). In resting, gonad size is at a minimuiT),
which usually occurs after spawning. S. droi'lnwhiensis has an
annual reproductive cycle with major spawning period in the late
winter or early spring (Keats et al. 1984). Therefore, at this stage,
uichins were presuniably in the state of resting; hence, they have
undergone a large drop in gonad size following spawning.

In general, gonadal yield is strongly affected by the seasonal
reproductive cycle of sea urchins. During spawning, a high pro-
portion of the gonad mass is released as gametes (Thompson
1984). Once spawning occurred, this may exert a significant effect
on the biochemical composition of gonads. Because urchins were
fed on a Laminaria diet after harvesting and consequently subject
to intense feeding, this may have a significant effect on the nutrient
composition of sea urchin tissues. Generally, both food quality and
quantity affect sea urchin growth (Lawrence & Lane 1982), thus
excessive feeding resulting in the accumulation of nutrients in the
tissues.

Proximate Composition

The major nutrients of sea urchin S, ilroehachiensis gonads
were polysaccharides, proteins, and lipids, siniilar to that reported

866

Liyana-Pathirana et al.

TABLE S.

Free amino acid conttnl in/ft. dry weight) of sea urchin gonads and

coelomic fluid after feeding urchins on Laminaria diet for

three weeks.

characteristics of sea urchins' coelomic tliiid. In our study, when
urchins were fed on u Lainimiria diet, gonads contained 74.7 ±
0.04% moisture, which was significantly (f < 0.05) lower than that
of the coelomic tluid.

Gonads

Coelomic Fluid

Amino Acid

Mg/g

%

Mg/g

%

Alanine

2.872 (77 )■■

14

899(17)"

7.5

a-aminoadipic acid

68 IS)-*

0.3

Arginine

180 (3I''

0.9

507(19)"

4.2

Asparagine

13(1)-'

0.1

Aspartic acid

73 (Sr

0.4

80(3)"

0.7

Cystathionine

115(2)"

0.6

76 (.^9)"

0,6

Cysteine

214(12f

1.0

90(7)"

0,7

Glutamic acid

874(12)"

4.2

478(14)"

4,0

Glulamine

647 (34)"

3.1

184(8)"

1,5

Glycine

11.751 (223)-'

57.1

6,771 (4.30)"

56,3

Histidine

100(6)-

0.5

111 (10)"

0,9

Hydro.xyproline

124(11)-

0.6

88(6)"

0,7

Isoleucine

313(11)-'

1.5

175(6)"

1,5

Leucine

370 (23)"

1.8

293(7)"

2.4

Lysine

356 (7)"

1.7

312(11)"

2.6

Methionine

68 (4)"

0.3

134(8)"

1,1

Phenylalanine

164(2)"

0.8

164(12)"

1.4

Proline

140(10)"

0.7

4.909(147)"

2.7

Sarcosine

332(14)

1.6

Serine

316(5)"

1.5

163(10)"

1.4

Threonine

521 (27)"

2.5

189(12)"

1,6

Tryptophan

264(11)"

1.3

140(3)"

1,2

Tyrosine

217(16)"

1.1

212(5)"

1.8

Valine

273(16)"

1.3

272(9)"

2.3

Total ( mg/g )

21 (1,0)"

12.0(0.8)

Results are mean values of three replicates (standard deviation). Values in
each row with the same superscript are not different (P > 0.05) from one
another.

by Fernandez et al. (1995). However, coelomic fluid contained
96.5 ± 0.03% moisture; thus, its contents of lipid, protein, and
polysaccharide were extremely low. On the other hand, the ash
content of sea urchin coelomic tluid was much higher than that of
the gonads. In general, sea urchin gonads are known to contain
high levels of protein. They also have considerably high lipid
levels; whereas, carbohydrate levels are low (McClintock & Pearse
1987). However, in our study, the carbohydrate content was high
and accounted for approximately 10% of the total amount, on a
fresh weight basis. Furthermore, in coelomic fluid, although the
relative proportion of carbohydrate was only 0.4 ± 0.1%. this was
four times more than that of its protein and lipid contents. From
this study, it is apparent that content of lipid, protein and carbo-
hydrate in the coelomic fluid was much less than that of the go-
nads. The urchins in this work were given a diet purely comprised
of Lantinaiia kelp to resemble the urchin's prefened natural diet.
In general, the diet plays a very important role in the compositional
characteristics of these animals (Nishikiori 1989, Fernandez et al.
1995, Agatsuma 1998) and almost all studies have only determined
compositional characteristics of the gonads. Thus. Agatsuma
(1998) showed that a diet of fishmeal increased moisture levels,
and Nishikiori (1989) observed that moi.sture content in the gonads
of 5. niiJus was below 70% when the urchins were fed Laminaria
japonica to satiation. None of these studies reported compositional

Lipid Class Composition

The lipid composition of marine invertebrates is influenced by
several factors, including pattern of feeding, gametogenesis, and
possibly environmental conditions (Jezierska et al. 1982). Wax
esters have been reported to constitute energy reserves in various
marine invertebrates (Sargent 1976). but this was not the case for
sea urchin 5, draelniciucnsis. TAG formed the main energy reserve
in these animals, and their gonads and coelomic fluid were quali-
tatively composed of similar lipid classes. The same nonpolar lipid
class distribution has been observed in S. droelyuchiensis gonads
and coelomic fluid collected form Nova Scotia (Takagi et al.
1980). The nonpolar lipids of gonads and coelomic tluid consisted
mainly of TAG, FFA, and ST. Triacylglycerols are usually con-
sidered to serve as storage lipids in eukaryotic cells (Sul et al.
2000). Thus, sea urchin lipids contained much larger amounts of
storage lipids, principally TAG, which constituted more than 60%
of the total nonpolar lipids of gonads and coelomic tluid.

Although, qualitative composition of nonpolar lipids of gonads
and coelomic fluid of .S'. droehachiensis was similar, relative con-
tent of individual classes differed. Hence, both sea urchin gonads
and coelomic fluid were composed of the same major lipid classes,
both nonpolar and polar, but their relative contents were markedly
different. Thus, relative content of TAG in sea urchin gonads was
much higher than that of coelomic fluid; whereas, that of FFA in
gonads was much less than that of coelomic fluid. It was apparent
in the preliminary experiments that both gonads and coelomic fluid
contained high levels of FFA. Therefore, it was thought that partial
hydrolysis of TAG may lead to an underestimation of TAG con-
tent. However, the impact of this on a nutritional value of the fatty
acids involved is inconsequential. To verify the above fact, fatty
acid content was determined in a set of freshly harvested sea
urchins. The gonads were extracted as quickly as possible at 0°C
immediately after homogenization. It was assumed that hydrolysis
of lipids because of the activity of endogenous enzymes is mini-
mized under these conditions. The FFA content was 15.5 ± 1.7%
of the total nonpolar lipids upon latroscan analysis. Hence, the
high levels of FFA observed for stored sea urchin tissues following
homogenization could be attributed to the hydrolysis of TAG dur-
ing storage of samples at -20°C.

In general, the energy supplied to the animal by the breakdown
of lipid reserves comes primarily from oxidation of fatty acids.
Farkas (1979) has shown that the production of FFA can be in-
duced by stress. Thus, the environmental temperature and diet can
be specified as factors exerting a major impact on the content and
metabolism of fatty acids in animals (Farkas et al. 1978).

There were noticeable differences in the relative content of
sterols in sea urchin tissues. In general, cholesterol level may
depend on dietary level and stage of sexual development (Love
1970), In fact, diet and nutritional status are known to be the main
factors that influence cholesterol levels (Dave et al. 1975). On the
other hand, during gametogenesis, a redistribution of cholesterol
takes place that may lead to high levels of cholesterol in the gonads
(Idler & Tsuyuki 1985). Therefore, all relevant factors must be
considered to explain the content of cholesterol in different tissues.

Comparison of Nutrient Composition of Sea Urchins

867

III the present study, the relative content of ST in the gonads was
significantly (P < 0.03) higher than that in the coelomic fluid.

Vaskovsky and Kostetsky (1969) have performed TLC on polar
lipids of sea urchin 5. iiiuhis and S. intennediiis. The polar lipid
fraction was separated into five components of which PC, PE, and
SM constituted the major polar lipid classes present. Furthermore,
lipid extracts of different organs of the same animal had a similar
qualitative polar lipid composition (Vaskovsky & Kostetsky
1969). In this study, both gonads and coelomic fluid showed quali-
tative similarities in their polar lipid fractions. Thus, PC, PE, SM/
LPC, and PS/PI constituted the polar lipids of S. droebachiensis
gonads and coelomic fluid, and PC was dominant in both tissues,
with a contribution of more than 65'^/c to the total content of polar
lipids. Similarly, Floreto et al. ( 1996) demonstrated that sea urchin
Tiipneiistes gratila fed on a seaweed diet had PC and PE as the
major lipid constituents, and PC contributed a greater proportion
than PE.

Fatty Acid Composition

The fatty acids of total lipids of sea urchin gonads and coelomic
fluid were typically similar to those of other marine species with a
dominance of 16:0 and 20:5n-3 (Wanasundara 1996). Although.
22;6n-3 is a typical fatty acid in marine lipids, it contributed only
1 .4 ± 0. 1 and 0.6 ± 0. 1 % to the total fatty acids in the lipids of sea
urchin gonads and coelomic fluid, respectively. Holland (1978)
reported that the predominance of 20:5n-3 and 22:6n-3 in typical
marine fatty acids is a result of low- temperature adaptation. This
helps in the maintenance of cell membrane fluidity in organisms
living in the cold environments.

Considerable data are available on the fatty acid composition of
sea urchins (Takagi et al. 1980. Kaneniwa and Takagi 1986). The
fatty acid 16:0 was the major SFA in the sea urchin 5. droe-
bachiensis harvested from Herring Cove, Nova Scotia (Takagi et
al. 1980). Fujino et al. (1970) analyzed fatty acid composition of
sea urchins Anthocidaris crassispina. S. piilclwninuis. S. fraii-
ciscanus. S. intermedins, and Echinus esculentus. In all these
samples, 16:0 was the prominent SFA followed by 14:0. The fatty
acid 18:0 was found to occur in considerable amounts. Similarly,
in the present study, the predominant SFA were 16:0 and 14:0 in
the lipids of both gonads and coelomic fluid of S. droebachiensis.

Among MUFA 20: In- 1 5 was present up to 11% in the total
fatty acids of urchins (Takagi et al. 1980). Ackman and Hooper
(1973) reported that such marine animals as periwinkle {Littorine
littorea). moon snail (Liinata triseriata). and sand shrimp
(Crangon septemspinosu.s) contain 20:ln-l5, but at much lower
levels of up to 0.2% of the total fatty acids. However, this has not
been commonly reported as being typical of marine lipids. In our
study, 20:ln-15 was also the major MUFA in both tissues ana-
lyzed. On the other hand, seaweeds, the natural diet of sea urchins,
have not been reported to contain 20:ln-15 (Ackman &
McLachlan 1977); hence, the formation of 20:ln-l5 in sea urchin
tissues may be biosynthetic in origin, because this was not depen-
dent on the diet.

The occurrence of such unusual ."i-olefinic fatty acids as
18:ln-13, 20:ln-15, 20:2A5,11, 20:2A5,13, 20:3A5,1 1,14. and
20:3A5, 11,14,17 has been noticeable in lipids of sea urchins ac-
counting for as much as 6-2 1 % of the fatty acids of total lipids
(Takagi et al. 1980. Kaneniwa & Takagi 1986). In this study, the
amount of 5-olefinic acids found in the lipids of both gonads and

coelomic fluid was in the range of 7-10%. The presence of 5-ole-
finic fatty acids has been reported in 12 species of Echinoidea
collected in Japan (Takagi et al.l986); thus, they serve a common
and characteristic feature of sea urchin lipids.

The amount of eicosapenlaenoic acid (20:5n-3) was quite high
in sea urchin lipids (Takagi et al. 1980). Pohl and Zurheide (1979)
reported that urchins that consumed Laniinaria had a high content
of 16:4n-3, 18:4n-3, 20:4n-6. and 20:3n-3. Similariy. sea urchin S.
droebachiensis in our study consumed Laniinaria for only a three
week period, and their gonadal and coelomic fluid lipids contained
quite high levels of these fatty acids. Thus, the fatty acid profiles
of sea urchin tissues somewhat reflect that of their diets as was also
observed by Floreto et al. (1996). However, certain fatty acids,
such as 16:4n-3, 20:4n-6, 20:5n-3, and 20:ln-l I, which constitute
the major fatty acids of sea urchin tissues, were not detected in
their diets; therefore, suggesting that sea urchins are capable of
synthesizing them from lower fatty acid precursors. Similarly, in
the present study 16:4n-3, 20:ln-l 1, 20:4n-6, and 20:5n-3, among
others, may have been formed by chain elongation of precursors.
In general, the sea urchin fatty acids; namely, 16:4n-3, 20:4n-6,
and 20:5n-3, may possibly confer some structural function and,
hence, are purposely synthesized by the animal (Floreto et al.
1996).

Carotenoid Pigments

In the sea urchin .S'. dnfcbacliiensis. carotenoids were mainly
concentrated in the gonadal tissue. Hence, the total content of
carotenoids in the gonads was about 6.3 times more than that of the
coelomic fluid. However, the content of carotenoids in different
tissues may vary with the reproductive stage of urchins. Hence,
during gametogenesis most of the carotenoids in other tissues may
be transferred into gonads, consequently increasing their carot-
enoid content (Griffiths & Perrott 1976).

Echininone and fucoxanthin were characterized as the major
carotenoids present in the gonads and the coelomic fluid, respec-
tively. In addition, (J-carotene was identified in both tissues.
Echininone was found to be the main pigment with a lesser amount
of P-carotene in the gonads of S. piirpiiraliis (Griffiths 1966), S.
droebachiensis (Griffiths & PeiTOtt 1976) and Tripnenstes gratila
(Shina et al. 1978). Tsushima et al. (1995) found that (J-echininone
and p-carotene were the major carotenoids in the gonads of 1 9 out
of 20 sea urchin species examined. Meanwhile, the major carot-
enoids of brown algae, the natural preferred diet of sea urchins,
consist of p-carotene, violaxanthin, and fucoxanthin (Matsuno &
Hirao 1989). Furthermore, there is bioconversion of p-carotene to
P-echininone via P-isocryptoxanthin in sea urchins; which takes
place mainly in the gut wall, and the resultant p-echininone is
incorporated into the gonads (Tsushima et al. 1993). Kawakami et
al. ( 1998) showed that fucoxanthin, the major carotenoid in brown
algae, did not accumulate in the gonads. In fact, in the present
study on 5. droebachien.sis. fucoxanthin did not occur in the go-
nads. On the contrary, coelomic fluid had fucoxanthin as its major
carotenoid.

Amino Acid Composition

Although marine invertebrates characteristically contain a high
intracellular concentration of FAA, the composition of the FAA
pool may vary among species (Gilles 1979). In the present study,
glycine was the dominant amino acid in both TAA and FAA

868

Liyana-Pathirana et al.

profiles in both sea urchin gonads and cocloniic fluid. Komata el
al. (1962) reported that glycine was dominant ni the gonads ot" sea
urchin S. piilclwnimiis. and its content ranged from 35—41% of
total FAA. Lee and Haard ( 1982) reported that glycine constituted
18-60% of the FAA in the gonads of sea urchin S. droebachiensis.
The gonads and coelomic fluid of sea urchin .S'. droebachiensis in
this study contained 1 1.9-14.6% glycine in the TAA profile, re-
spectively. However, glycine was not the dominant amino acid in
the gonads of the sea urchin Paracentrotus lividiis. although it
contributed a considerable amount to the TAA pool (Cruz-Garcia
et al. 2000). Other than glycine, alanine, arginine. glutamic acid,
lysine, and methionine are considered important for taste, even
though some of them were present in small quantities (Lee &
Haard 1982). These amino acids were present in considerable
amounts in both gonads and coelomic fluid of sea urchins in this
study.

It has been found that different combinations of taste-active
components (substances that influence the taste of any food) as
well as their relative amounts are of paramount importance in
producing the characteristic flavor of each seafood (Puke 1994). In
general, glutamine and glycine, which were present in higher
amounts in the gonads than coelomic fluid, are known to be taste-
active in sea urchins and other seafoods, regardless of their quan-
tity. Sea urchin gonads seemed to be sweeter when little or no
glutamine was present, and alanine was found in considerably high
levels. Alanine is a taste-active component in sea urchin tissues,
contributing noticeably to both TAA and FAA contents. Further-
more, valine and methionine are known to be taste-active only in
sea urchins; whereas, arginine was also taste-active in sea urchins
because of its high content (Fuke 1994). Both methionine and
arginine were present at a higher proportion in the coelomic fluid
than in the gonads. Similarly, the contents of aspartic acid, histi-
dine. and especially proline were much higher in the coelomic
fluid than those in the gonads. Thus, amino acids play a major role
in the taste of sea urchin gonads. In our study, various amino acids
contributed differently to both the TAA and FAA of sea urchin
gonads and coelonnc fluid.

Contents of Nucleic Acids

In general, quantitative analysis of nucleic acid provides a rela-
tively simple means of estimating recent growth rate of sea ur-
chins. The processes of cellular growth and division require the
synthesis of nucleic acids and proteins. The fact that RNA is a
precursor to protein synthesis led to its use as an indicator of
growth rate (Church & Robertson 1966). The primary function of
RNA involves protein synthesis; whereas. DNA is the primary

carrier of genetic information. Because the majority of cellular
DNA is chromosomal, the quantity of DNA per cell is quasicon-
stant in soinatic tissues; the tissue DNA concentration reflects cell
numbers (Sulkin et al. 1975; Bulow 1987). Therefore. DNA con-
tent has usually been used as an index of cell numbers or biomass
(Regnault & Luquet 1974). In this study, the DNA content in the
gonads was approximately four times higher than that in the coe-
lomic fluid. Although the gonad is a tissue with a higher biomass
as compared with coelomic tluid, the latter contains mostly coe-
lomic fluid with a lower biomass. On the other hand, the RNA/
DNA ratio has been used as an estimate of growth for a variety of
invertebrates (Sulkin et al. 1975). Thus, the RNA/DNA ratio is an
index of protein synthetic activity per cell and reflects the protein
synthesizing capacity for estimating recent //; situ protein increa,se
(Bulow 1987, Hovenkamp & Witte 1991 ). In fact, correlation be-
tween RNA concentration or RNA/DNA ratio and growth rate has
been observed for a wide variety of organisms (Sutcliffe 1970).
Furthermore, the gonadal RNA content was about 5.4 times higher
than that in the coelomic tluid, thus demonstrating higher protein
synthetic activity in the gonads. In general, gonad is the site of
gametogenesis, which involves much protein synthesis. Further-
more, the RNA/DNA ratio was much lower in coelomic tluid than
that in gonads, indicating greater protein synthetic activity per cell
in the gonads. This is an indication that gonad is a tissue with
greater //; sitit protein growth as compared with coelomic fluid.

CONCLUSIONS

The pi'esent study demonstrated that sea urchin gonadal and
coelomic tluid tissues had many common compositional charac-
teristics. Most of the parameters analyzed did not show qualitative
diffeiences; whereas, there were quantitative differences. In fact,
gonads of sea urchins are a site of nutrient storage in addition to
being the reproductive organs. The accumulation of nutrient re-
serves contributes to the growth and development of the commer-
cially important sea urchin gonads. Although .sea urchin coelomic
fluid has not yet been exploited commercially, evaluation of its
composition may lead to its potential use as a flavoring source.

ACKNOWLEDGMENTS

The author (C. L.-P.) gratefully acknowledges the assistance of
the Canadian International Development Agency (CIDA) through
a Mai'ine Science Scholarship. Thanks are also extended to Mr.
Keith Collins at the Sea Urchin Research Facility (SURF) at
Bonavista Bay, Newfoundland for providing sea uichin samples
for the study.

Acknian. R. G. & S. N. HcmpL-i, 1973. Non-methylene interrupted fatty
acids in lipids of shallow water marine invertebrates: a comparison of
two mollusks {Littorina liltorea and Limatia triseriata) with the sand
shrimp {Cnmgitn .scpli'iiispiiin^ii.s). Copiii. Bim hem PItysiol. 4<iB:l.'i.3-
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Ackman. R. G. & J. McLachlan. 1977. Fatty acids in some Nova Scotian
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Journal of Shellfish Research. Vol. 21. No. 2, 871-873. 2002.

THE DEVELOPMENT OF A POSITIVE NON-INFECTIOUS CONTROL FOR THE DETECTION

OF PERKINSUS USING THE RAY TEST

B. R. MOORE, S. N. KLEEMAN, AND R. J. G. LESTER*

Department of Microbiology and Parasitology, The University of Queensland. Brisbane. Australia 4072

ABSTRACT To establish a noncontagious control for the Ray thioglycollate test for the detection of Perkinsiis in moUusks we
evaluated nonviable stages of P. olseni for enlargement of hypnospores and blue/black iodine stain. Trophozoites made nonviable with
formalin, irradiation or colchicine failed to swell in thioglycollate. They remained small and did not differentially stain in iodine.
Trophozoites that had already developed into hypnospores in thioglycollate were rendered inactive by freezing, ethanol or formalin
immersion. They retained their iodinophilic properties and thus could provide a partial control for the Ray Test.

KEY WORDS: Perkinsiis. abalone. fluid thioglycollate medium

INTRODUCTION

Members of the genus Perkinsiis are protozoan parasites found
exclusively in mollusks. Perkinsiis parasites have long been rec-
ognized as a cause of mortality in commercially important mol-
lusks (Ray & Chandler 1955). Perkinsiis mariinis has been dem-
onstrated to have profound effects on the oyster Crassostrea vir-
ginica. leading to reduced growth, reduced fecundity, and
increased mortality (Menzel & Hopkins 1955, Mackin 1962). In
Australia, P. olseni infections result in deep abscesses and soft
yellow pustules in commercially important abalone. HuUotis rubra
and H. laevigata. This is of great concern to the endemic abalone
industry as infected individuals are unacceptable for processing
(Lester & Davis 1981). Transmission appears to occur through
zoospores that develop via hypnospores (Goggin et al. 1989). Hyp-
nospores can be found within the abscesses (Goggin & Lester
1995) and may also be produced by culturing trophozoites in fluid
thioglycollate medium (FTM) (Ray 1952). Other species oi Per-
kinsiis produce hypnospores though the role of zoospores in trans-
mission between mollusks is not clear (Perkins 1996).

Diagnosis of Perkinsus infections is commonly accomplished
by the FTM assay (Ray 1952). Trophozoites in infected tissue
develop into usually much larger hypnospores that stain blue-black
with Lugol's iodine (Ray 1952). The reaction with iodine relies on
the development of hypnospores; trophozoites remain brown (Ray
1952, Stein & Mackin 1957). Prior to the development of this
technique, diagnosis relied on fresh tissue smears or histologic
sections. These methods are labor intensive, use expensive mate-
rials, rely on a high degree of expertise, and lack sensitivity to
detect low levels of infection (Ray 1954).

Infected hosts frequently show no overt sign of disease, and as
such, infected tissue can easily reach processing plants undetected
(Goggin et al. 1990). Here they may be examined for the presence
of Perkinsus with the FTM assay, however, due to inexperience in
recognizing the parasite or through errors in the formulation of the
culture media, investigators may fail to detect infections.

The development of positive reference material to assist Per-
kinsus diagnosis in fisheries laboratories would be an invaluable
tool to help minimize misidentifications and false negatives. The
material needs to be noncontagious to prevent cross-infection into
local mollusk populations during shipping or in disposal after use,
while maintaining the key attributes of the FTM assay: enlarge-

*Corresponding author. E-mail: R.Lester@mailbox.uq.edu.au

ment of hypnospores in FTM and uptake of the iodine stain (Ray
1952).

Fisher and Oliver (1996) stated that dead trophozoites of P.
mariniis fail to enlarge in FTM. We sought to make P. olseni
material nonviable to see if swelling would still occur. We also
sought to determine whether hypnospores, following enlargement
in FTM, could be rendered inactive while retaining their iodino-
philic properties.

MATERIALS AND METHODS

Trophozoite Inactivation

Pieces of infected mantle tissue from three blood cockles, Ana-
dara trapezia, infected with P. olseni (Murrell et al. 2002) were
exposed to either dilute formalin, irradiation, and colchicine to
attenuate the viability of hypnospores while retaining the charac-
teristics of the Ray Test. Several tests were run on tissues from one
animal to enable the results from different dose levels to be com-
pared. Tissue from animal 1 was immersed in one of four dilute
formalin solutions |1:4 x 10', 1:4 x lO^*; 1:4 x lO'; 1:4 x 10"
formalin:seawater (approximately 349;r)]. For a period of 30 min-
utes tissue from animal 2 was treated with gamma irradiation. The
self-contained gamma radiation source of "'Co had a dose rate of
670 Gy h"'. Duplicate wet tissue samples were placed in glass
petri dishes and irradiated to a maximum absorbed dose rate of 600
Gy in increments of 200 Gy. Variations in absorbed dose were
minimized by placing thin tissue samples within a uniform portion
of the radiated field. Two tissue samples from animal 3 were
placed in FTM to which was added colchicine, at one of two
concentrations: 10'"* M and 10"* M for six hours, after which they
were rinsed in seawater.

After treatment all tissues were placed in FTM, supplemented
with 200 mg Chloromycetin and 200 units of mycostatin to reduce
fungal and bacterial contamination (Ray 1966), and incubated at
25°C for five days. A second pair of samples from animal 3 was
left in the colchicine-supplemented FTM for the full period of
incubation (5 days).

Following incubation, a portion of infected tissue was exam-
ined and hypnospore abundance counted, using a compound mi-
croscope at x40 magnification in five fields of view. To facilitate
easy enumeration, part of the tissue sample was stained with iodine
prior to counting. Hypnospores were teased out of the unstained
tissue and transferred to a glass petri dish containing seawater. The
seawater in the dish was replaced twice daily. Hypnospores that
adhered to the dish were allowed to develop; a process that typi-

871

872

MOORR ET AL.

TABLE 1.

Erfet'ts of formalin, irradiation and cokhicini' triatment iin trophozoite enlargement, resulting hypnospore viability, and iodinophilia.

Swelling Evident

No.

Enlarged

Duration of

After FTM

Hy

pnospores

'i Hypnospores

Presence of

Treatment

Treatment

Culture <V/N|

Present (5 Fields 4(lx)

\lal)le

Iodinophilia tV/N)

Control

—

Y

4S(1

s()<;;

Y

1:4 X 10' formalin:seawater

1 h

N

0

-

~

1:4 X lO'* formalin:seawater

1 h

N

0

-

~

1:4 X 10^ formalin:seawater

1 h

N

0

-

-

1:4 X 10" fornialin:seawater

1 h

Y

510

90%

Y

Control (no treatment)

—

Y

520

90%

Y

Gamma 200 Gy

—

Y

400

100%

Y

Gamma 400 Gy

—

Y

30

85.7%

Y

Gamma hOO Gy

—

Y

4

100%

Y

Control

—

Y

90

100%

Y

10^-" M Colchicine

6h

Y

80

83.3%

Y

10"" M Colchicine

6h

Y

70

100%

Y

K)--' M Colchicine

entire

incubation

Y

170

93.3%

Y

10"" M Colchicine

entire

incubation

Y

200

100%

Y

cally took 1-5 days. Parasites were inspected under a dissecting
microscope and were deemed viable only if cell division occurred
within five days. To assess whether treated ceils retained their
iodinophilic properties irrespective of viability, individual hypno-
spores were isolated from the treated tissue and transferred to a
separate petri dish, where they were stained with 3-4 drops of
LugoFs iodine. Iodinophilia was based qualitatively on the uptake
of stain by the hypnospore and was assessed approximately three
minutes after application (Quick 1972). Control tissues from all
three mollusks were placed directly into FTM and incubated for
the equivalent period of time to confirm that they were infected
with Peikinsiis.

Hypnospore Inactivation

To obtain hypnospores. pieces of mantle, foot, digestive gland,
and gill from A. trapezia, from Wynnum. Queensland and from H.

rubra and H. Inevii^aui collected from South Australia, were in-
cubated in 20 ml FTM at room temperature (approximately 24°C)
for 4-6 days. The medium was supplemented with 200 mg Chlo-
romycetin and 200 units of mycostatin (Ray 1966). The incubated
tissues containing resulting hypnospores were then subjected to
various treatments: freezing at -20°C; immersion in 10% formalin;
and immersion in 70% ethanol. The effectiveness of each treat-
ment on hypnospore viability and iodinophilia was assessed after
24, 48. and 72 h. The viability of hypnospores prior to treatment
was confirmed by viability testing of a random subset of enlarged
cells.

RESULTS

Trophozoite Inactivation

In all treatments, trophozoites that developed into hypnospores
in FTM were capable of further development and were evidently

TABLE 2.
Effects of \arious treatments on hypnospore viability and iodinophilia.

Host

Treatment

Duration of
1 reatment

% Hypnospores
Viable

Presence of
Iodinophilia (V/N)

A.

trapezia

H.

rubra

H.

laevigata

A.

trapezia

A.

trapezia

A.

trapezia

H.

rubra

H.

laeviaala

A.

trapezia

A.

trapezia

A.

trapezia

H.

rubra

H.

laevigata

A.

trapezia

A.

trapezia

A.

trapezia

H.

rubra

H.

laevigata

Control
Control
Control
10% formalin
10% formalin
10%. formalin
10% formalin
10% formalin
70% ethanol
70% ethanol
70% ethanol
70% ethanol
70% ethanol
Freezing (-20°C)
Freezing (-20°C)
Freezing (-20°C)
Freezing (-20''C)
Freezing (-20°C)

24 h
48 h

72 h
72 h
72 h
24 h
4S h
72 h
72 h
72 h
24 h
4S h
72 h
72 h
72 h

80%

80%

100%

0%

0%

0%

0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%

Y
Y
Y

Y
Y
Y

Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y

Controlled Diagnosis of Perkinsus

873

viable (Table 1 ). Treatment had little effect on the parasitic stages
that had enlarged. No hypnospores were evident in tissues exposed
to tornialin concentrations of 1:4 x 10\ 1:4 x 10"*; 1:4 x 10''.
Tissues exposed to 1:4 x l(f formalin:seawater provided similar
hypnospore numbers to those of the control (Table 1 ). Tropho-
zoites exposed to gamma irradiation showed a progressive decline
in hypnospore numbers with increasing radiation exposure (200
Gy, 400 Gy, and 600 Gy ) (Table 1 ). Colchicine treatment had little
effect on hypnospore enlargement and viability, irrespective of
treatment concentration. The majority of hypnospores retained
from incubation appeared viable, with motile zoospores evident
after five days examination (Table 1).

Hypnospore inactivation

All treatments killed hypnospores within 24 h of treatment.
Nevertheless, all the dead hypnospores exhibited clear iodino-
philia. staining a deep blue on application of Lugol's iodine (Ta-
ble 2).

DISCUSSION

The negative coiTelation between treatment intensity (or dura-
tion) and hypnospore abundance, particularly for the irradiated
tissue, followed by the successful development of recovered hyp-
nospores indicate that nonviable trophozoite stages fail to enlarge
in FTM. This is in agreement with Fisher and Oliver (1996). who
stated that enlargement of P. marimis trophozoites in FTM only
occurs if the parasite is viable. Formalin treatment at concentra-
tions >1:4 x lO'' formalin:seawater appeared to prevent hypno-
spore enlargement, with no hypnospores recovered from these con-
centrations. The extremely weak formalin (1:4 x 10" formalin:
seawater) appeared to have little effect on trophozoite viability.
with treated tissues exhibiting a similar enlarged hypnospore abun-
dance to that of the control. Similarly, colchicine concentrations of

10""" M or 10"" M had little effect on parasite viability. Colchicine
has been demonstrated to be a potent microtubule inhibitor at such
concentrations (Wiest et al. 1993). thus preventing cell division.
The fact that parasites treated with colchicine remained viable and
underwent division once out of the treatment is consistent with
microscopic observations that no cell division occurs during tro-
phozoite differentiation to hypnospores and cell enlargement.
Swelling of Perkinsus cells in FTM had been suggested to occur as
a result of the direct uptake of the media (Ray 1932). although the
mechanisms of this process are not yet described.

As nonviable trophozoites failed to enlarge in FTM. and thus
failed to become iodinophilic, we tested whether hypnospore
stages could be killed and still retain their iodinophilic priiperties.
All treatments tested killed hypnospores. The sensitivity of hyp-
nospores to low temperature is in accordance with Chu and Greene
(1989). who ob.served that hypnospores of P. marimis exhibited
1009f mortality at 0°C for 24 h. Goggin et al. (1990) observed
motile zoospores from cultured tissues previously chilled at 4"C.
0°C and frozen at -20°C. showing that trophozoites are more
tolerant to low temperatures than hypnospores.

In all cases, nonviable hypnospores were iodinophilic. Thus,
such tissue could be transported risk-free to processuig plants to
serve as a positive reference for sample comparison. Although this
is not a control for FTM development, it still supplies users with
a positive control that is safe, noncontagious, and simple to use.

ACKNOWLEDGMENTS

Mr. Tavis Anderson. Department of Microbiology and Parasi-
tology. University of Queensland, assisted with experimentation
and viability testing, and Dr. David Hunter, Department of Chem-
istry, University of Queensland, guided the in'adiation experi-
ments. Financial support from the Fisheries Research and Devel-
opment Corporation (2000/151) is gratefully acknowledged.

Chu, F. E. & K. H. Greene. 1989. Effect of temperature and salinity on in
vino culture of the oyster pathogen Perl<insiis marimis ( Apiconiplexa:
Perkinsea). / Invert. PallntL 53:260-268.

Fisher, W. S. & L. M. Oliver. 1996. A whole oyster procedure for diagnosis
of Perliinsus marimis disease using Ray's fluid thioglycollate culture
medium. J. Shellfish Res. 15:109-117.

Goggin, C. L. & R. J. G. Lester. 1995. Perliinsus. a protistan parasite of
abalone in Australia: a review. Mar. Freshwater Res. 46:639-646.

Goggin, C. L., K. B. Sewell & R. J. G. Lester. 1989. Cross infection
experiments with Australian Perkinsus species. Dis. Aq. Org 7:55-59.

Goggin. C. L.. K. B. Sewell & R. J. G. Lester. 1990. Tolerances of Per-
kinsus spp. (Protozoa, Apicomplexa) to temperature, chlorine and sa-
linity. J. Shellfish Res 9:145-148.

Lester, R. J. G. & G. H. G. Davis. 1981. A new Perkiii.-.ii.\ species (Api-
complexa. Perkinsea) from the ahalone Huliinis rubra. ,/. Invertehr.
ftir/(()/ 37:181-187.

Mackin, J. G. 1962. Oyster disease caused by Dermocystidiiim mariimin
and other microorganisms in Louisiana. Phi. Inst. Mar, Sci. Univ. Tex.
7:132-229.

Menzel, R. W. & S. H. Hopkins. 1955. Effects of two parasites on the
growth of oysters. Proc. Natl. Shellfish Assoc. 45:184-186.

Murrell, A., S. N. Kleeman, S. C. Barker & R. J. G. Lester. 2002. Syn-
onymy of Perkinsus otseni Lester & Davis, 198) and Perkinsus atlan-
ticus Azevedo, 1989 and an update on the phylogenetic position of the
genus Perkinsus. Bull. Europ. Ass. Fish Pathol. 22:258-265.

LITERATURE CITED

Perkins, F. O. 1996. The structure of Perkinsus marimis (Mackin, Owen
and Collier, 1950) Levine, 1978 with comments on taxonomy and
phylogeny of Perkinsus spp. J. Shellfish Res 15:67-87.

Quick, J. A. Jr. 1972. Fluid thioglycollate medium assay of Labyrin-
thomyxa parasites in oysters. Florida Department of Natural Resources.
Leaflet Series Volume 6, Part 4, No. 3. 1 1 pp.

Ray, S. M. 1952. A culture technique for the diagnosis of Infection with
Dermocvstidium marimim Mackin. Owen, and Collier in oysters. Sci.
116:360-361.

Ray, S.M. 1954. Biological studies oi Dermocvsiuhum marinum. a fungus
parasite of oysters. Rice Inst. Pamph, Special Issue, November, 1954.
113 pp.

Ray, S. M. & A. C. Chandler. 1955. Dermocysiiiliuiii munnum a parasite
of oysters. Expl. Purasit, 4:172-200.

Ray, S. M. 1966. Effects of various antibiotics on the fungus Dermocys-
tidiiim marinum in thioglycollate cultures of oyster tissues. J. Imerlebr
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Stein, J. E. & J. G. Mackin. 1957. An evaluation of the culture method used
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Wiesi, P. M.. J. H. Johnson & T. P. Flanlgan. 1993. Microtubule inhibitors
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Joiinnil of Shellfish Research. Vol. 21, No. 2, 875-881. 2002.

EVALUATION OF SUBSTITUTE DIETS FOR LIVE ALGAE IN THE CAPTIVE MAINTENANCE

OF ADULT AND SUBADULT UNIONIDAE

S. J. NICHOLS'* AND D. GARLING^

^USGS-GLSC, 1451 Green Road. Ann Arbor. Michigan 48105: -Department of Fisheries and Wildlife,
217 Natural Resonrces. Michigan State University, East Lansing. Michigan 48824

ABSTRACT Ten nonlive algal diets were evaluated as potential hroodstock diets for adult and subadult unionids. These diets varied
significantly in their ability to support growth, reproduction and survival. Growth, increase in glycogen stores, and limited glochidial
formation were seen in most unionid species on two of the diets. However, long-term survival (>3 y) remained problematic, and the
cause of mortality in these animals could not be determined. While two of the diets tested are potentially useful for supplemental
feeding of adult unionids to increase glycogen levels during quarantine, or during short-term captive maintenance in the laboratory,
none can be recommended without reservation for long-term maintenance because of the lack of survival after three years during this
study.

KEY WORDS: unionidae diets

INTRODUCTION

Nearly 70% of the freshwater mussels (Unionacea) in North
America are currently facing extinction (Williams et al. 1993).
Conservation efforts have focused on relocation of endangered
populations, and aquaculture of recently transformed larvae. Cap-
tive maintenance of endangered animals is a common technique
used to enhance and preserve species-at-risk. however unionids
have proved difficult to maintain in captivity or to relocate into
new habitats (Cope & Waller 1995). Most aquaculture efforts have
concentrated on developing live algal diets that will support the
growth and survival of larvae (<l year of age). A tri-algal diet has
recently been produced that appears to support survival of a few
species for about one year after larval transformation (see Gatenby
et al. 1994. Gatenby et al. 1996. Gatenby et a!. 1997). Adult
unionids have rarely been kept alive for more than three years even
in hatchery ponds or raceways. One problem in maintaining adult
unionids is the lack of information regarding actual nutritional
requirements. Recent studies have indicated that while live algae
may be supplying certain key nutrients, detritus and bacteria are an
important part of unionid diets (Nichols & Garling 2000).

Marine aquaculturists have e.xperienced similar difficulties in
long-term feeding of adult oysters and clams, and usually rely on
natural food supplies found in offshore grow-out areas. Hatchery
production of live algae is often used to rear seed marine bivalves
to planting size (1-2 mm), but is rarely used as a sole food source
for adult animals due to the difficulties and costs associated with
maintaining sufficient year-round supplies and lack of knowledge
of long-term nutritional needs (Knauer & Southgate 1999. Heas-
man et al. 2000). In recent years, attempts have been made to
replace live algae with artificial diets to reduce the need for ex-
pensive algal production (Coutteau & Sorgeloos 1993). These ar-
tificial feeds have been successful, but this success varies with
both feed type and farm operator. The objective of our study is to
determine if similar types of non-live algal feeds could be devel-
oped to support adult unionid survival, growth, and reproduction
under captive conditions.

MATERIALS AND METHODS

We tested commercial and laboratory-prepared (experimental)
feeds from 1994 to 1998 on eight unionid species Amhlema plicaia

*Corresponding author. E-mail: SJerrine_Nichols@usgs.gov

(Say, 1817). Cycloiiai.s titberculata (Rafinesque. 1820), Lampsilis
fasciola (Rafinesque, 1820), Lampsilis ventricosa (Say, 1817).
Lampsilis siliquoidea (Barnes, 1823), Leptodea fragilis
(Rafinesque. 1820). Pyganodon grandis (Say. 1829). and Qiia-
diiihi quadrula (Rafinesque. 1820). The size and age of the ani-
mals varied and both adult and subadult animals were tested. Adult
animals in our experiments were at least five years of age accord-
ing to external annuli. Subadults animals were less than three years
but younger than one year based on external annuli. Shell mor-
phometries for all animals were measured upon arrival in the labo-
ratory and individual tracking numbers etched onto one of the shell
valves.

Unionids were held at the Great Lakes Science Center in a
fiow-through system of 8-L rectangular aquaria, containing 10 cm
of coarse gravel, with a water replacement rate of 4L/h. Baseline
water quality parameters were: CaCO, of -100 mg/L (EDTA
titrimetric method. APHA 1989); dissolved oxygen 8.0 ppm
(Winkler method. APHA 1989); dissolved ammonia of <0.5 ppm
(phenate method. APHA 1989); and a pH of 7.8 (Fisher Scientific
Accumet pH meter model #AB15). Water quality parameters were
measured weekly and during any die-off of unionids. Water tem-
perature averaged \5°C. and the light regimen was on a 12 light/ 12
dark cycle.

Diet Formulations

We tested 10 diet formulas in this study (Table I). Five were
commercially available (treatments #1-5). and 5 were experimen-
tal mixtures (treatments #6-10). The diets were chosen on avail-
ability and/or prior successful use in culturing marine bivalves or
zebra mussels Dreissena polymorpha (Pallas. 1771).

The five commercial diets were: treatment (TR) #l-dried Clilo-
rella sp. (Earthrise Co., California); TR#2-marine algal paste
Thalassiosira pseudonana (Hust.) (Hasle and Heimdal) (from
Coastal Oyster Inc.); TR#3-Hatchfry Encapsulon (30 |j, size par-
ticles. Argent Co. Washington); TR#4-fish fiake food (tropical,
various retailers), and TR#5-a manipulated yeast diet (Artemia
Reference Center. Ghent Belgium).

The five experimental diets were a combination of bacterial/
ciliate cultures grown in the laboratory, commercially available
invertebrate enrichment feeds, and aniinal feed supplements incor-
porated for protein, dextrose, and other nutrients. The first experi-
mental diet (TR #6) was a microencapsulated feed with food par-
ticles embedded in a gel matrix prepared according to Langdon"s

875

876

Nichols and Garling

TABLE 1.
Diets fed to adult and subadult unioids.

TR#1. TR#2. TR#3. TR#4. TR#6. TR#8. TR#9. TR#1().

Dried Marine Hatchlrv Fish Flake TR#5. Encapsulated TR#7. Bacterial Bacterial Bacterial

Chlorella algal paste encapsulon Food ^ east feed Kj;;; chow slurry #A slurry #B slurry #C.

Adults
Subadults

+

work on marine bivalves (Langdon & Levine 1983, Langdon &
Bolton 1984; Buchal & Langdon 1995). Diet Tr#7. was based on
the analysis of the gross biochemical composition of freshwater
bivalves (Secor et al. 1993). This formula, (called egg chow) was
a mixture of 60% dried powdered chicken egg (ICN Biomedicals
Inc.), 30% powdered dextrin (ICN Biomedicals Inc.), 9% liquid
safflower oil, and 1% vitamin supplement (Rep-Ca! Herptivite.
Los Gates, California) mixed and finely ground prior to feeding.

The other three experimental diets were formulated from bac-
terial/ciliate sluiTy based on Stuart's (1982) work on the marine
bivalve Aidacomya ater (Molina, 1782). These were prepared by
soaking finely ground vegetation in water for three days to en-
courage the growth of bacteria and ciliates and the breakdown of
cellulose. Stuart used kelp as a base; we used freshwater marsh
grass {Phalaris spp.). This basic bacterial slurry formula is TR#8
(bacterial/slurry #A). Treatment #9 (bacterial slurry #B) was a
mixture of 50% TR#9 and 50% dried Chlorella spp. Treatment #10
(bacterial slurry #C) was a mixture of 30% TR#8, 30% dried
Chlorella. 10% Rich Advanced (a liquid mixture of lipids and
algal growth enhancers from Sanders Corp. Ogden, Utah) and 107f
Sanders Black Gold (a flake similar in composition to Rich .Ad-
vanced).

The ration level of all diets was maintained at 5-8 mg diet dry
weight/L of aquarium water for at least 15 h out of the day. This
ration was based on the average total organic particulate matter
values found in the Huron River near a large free-living union id
bed as described in Nichols and Garling (2000).

Measuring Success of Diet Formulations

Growth and survival were the critical criteria for assessing diet
success for subadult unionids; reproduction (glochidia formation)
and survival were the criteria used for adult unionids. Changes in
maximum shell length were also recorded, but not further statis-
tically tested. There was a wide variation in age of adult animals
used and even a nutritionally ideal diet, older animals could not be
expected to show the same potential for shell growth as younger
adults. In tests using adult unionids. 10 randomly selected indi-
viduals from each of the following six species for six species — A.
plicata (si/.e range measured across longest anterior/posterior shell
plane 45-110 mm), L. siliijitoidea (56-119 mm), L. veiUricosu
(71-98 mm), L. fragilis (61-167 mm), Pyganodon grandis (78-
149 mm), and Quadnda quadrula (48-61 mm) — were assigned to
all ten diet treatments. Fewer C. tuberculata (51-77 mm) and L.
fascinla (36-45 mml were available (15 individuals each) thus,
only 5 adults from these two species were randomly assigned to
three diets (TR#7, TR#9 & TR#10).

The number of subadult unionids available varied by species;
A. plicata in = 75), C. tuberculata {it = 15), Lfasciola in = 2),
L. ventricosa in = 10), L. siliquoidea in = 2), L. fragilis in =

100), P. grandis in = 100), and Q. quadrula in = 50). Within
each species, ages and sizes of animals were similar to minimize
variability. Due to the unequal sample size, the number of indi-
viduals and number of diets tested were limited. Two diets (TR#7
& TR#10) were tested for a period of 280 days. The distribution of
mussels in these treatments was as follows; in TR#7 A. plicata in
= 27). C. tuberculata in = 7), L. fasciola (n = \).L. ventricosa
in = 5). L. siliquoidea in = 1). L fragilis in = 50), P. graiulis
in = 50), and Q. quadrula in = 18); in TR#8 and TR#9, A.
plicata in = 5) and Q. quadrula in = 3). The remaining subadults
were fed TR#I().

Adult unionids were measured monthly (maximum shell length
to the nearest mm) and survival checked daily. Reproductive ef-
forts were monitored by the development of glochidia in the mar-
supium, mantle lure behavior, and glochidial release in the test
aquaria. Marsupia were examined by gently prying open the ani-
mal and visually examining the gills for obvious swelling on a
monthly basis. Each individual subadult unionid was measured
every two weeks (maximum shell length to the nearest mm) and
survival checked daily. Autopsies were performed on any animals
that died and tissues dissected and examined for flukes, fungus, or
bacteria, or gross structural changes in appearance.

Changes in glycogen content were measured in an additional
group of animals from January to December 1998. Only one spe-
cies, P. grandis. was readily available and used for the test.
Twenty adult P. grandis were placed on TR#7 and TR#10 (10
animals each diet) to determine the glycogen status of all soft
tissues as a measure of fitness. Glycogen content based on wet soft
tissue weights was obtained using the homogenized tissue and the
phenol-sulfuric acid method used by Haag et al. (1993) to assess
clam fitness and reported as mg/g wet tissue. This was a whole
body analysis, using 3 randomly selected animals from each of the
two diets at the beginning of the (January 2) and at the end (De-
cember 28) of the 1997 test year. All ten animals were from the
same age class based on external age lines (10 y old) and had a
shell length of 135-140 mm.

Two of the nonproprietary diets were analyzed for biochemical
content. The diets, TR#7 and TR#10 were prepared in the labora-
tory and then whole samples frozen to -40°C. Analyses were
performed on an HPLC using standard techniques as cited in As-
sociation of Official Analytical Chemists (AOAC 1995). Protein
(total), lipids (cholesterols, phytosterols, and total lipids), and car-
bohydrates (total) and fiber content were measured.

The statistical relationship between differences in growth rates,
survival rates and glycogen concentrations of adult and subadult
unionids on the various diets was tested. Differences in growth
rates between subadults of within each species over time, on dif-
ferent diets, were analyzed using a linear regression of the monthly
measurements of all individuals. Since the test group within each
species was identical in number and similar in length and age, no

Unionid Diets

877

data transformations were performed. Analysis of covariance
(ANCOVA) in a sequential analysis of the slopes was used to
determine growth differences between species in the same diet
treatment. Growth statistics were based on changes in length, not
in length at T = 0. Percent survival data was analyzed using a x"
test. Treatments without survivors were not included in any of the
analyses. Results were considered significantly different at the P <
0.05 level.

RESULTS

Adults

Of the ten diets tested on adult unionids. none can be recom-
mended without reservation. At least two. however, do show po-
tential for use in long-term captive maintenance. Initially, all of the
ten diet formulations were cleared from the water column, and
directly ingested (based on fecal production) by all species of
unionids. However, two of the diets caused apparent stress in the
animals and failed to support growth or survival for more than 30
days: six diets supported growth and survival for at least one year;
three diets supported glochidia formation, and; one supported
growth, reproduction, and survival but the animals died after the
third year (Table 2). All diet formulations caused problems with
water quality and some mortality was more directly related to rapid
changes in water quality than to diet.

Diet TR#2 caused all adult animals tested to extrude extensive
mucous strands outside of the shell that proved detrimental to
water quality. Within 24 h after feeding long strands of mucus
were drifting through the water column, the water was cloudy, and
ammonia levels spiked from <1 to 8 ppm. Twenty-five percent of
the unionids on this feed died within the first month, probably due
to water quality problems; thus this diet was eliminated from the
experiment after M days.

A second commercial diet, TR#3, was dropped from the tests
after eight months although this commercial rotifer-replacement
feed was initially very successful. All adult unionid species fed
well on it, and growth was seen in the first three months on the diet
in some species. Five of the ten adult Q. quadntki grew an average

of 2 mm in the first three months, with one individual adding 4 mm
of shell in that time period. Two of the ten adult L. siliquoidea
grew 2 mm each and seven of the ten Leplodea fragilis grew an
average of 3 mm each in the first three months. No growth was
seen in any animal after this period or in other species during the
test. After the fifth month of testing, when we began using feed
from a different batch, the unionids refused to eat the new feed.
The feed was not ingested (no fecal matter produced). We reor-
dered the feed to see if the problem was batch related, but the
mussels did not feed on the next batch either. Since the unionids
continued to refuse this diet, it was eliminated from the experiment
after an additional sixty days.

Of the three remaining commercial diets. TR#4 and TR#5
could not support survival of any adult unionid species for > 1 3 mo
(Table 3 and Table 4). None of the unionids showed any shell
growth while on these diets. Percent survival and growth did occur
on the last commercial feed TR#1. Individuals of all species sur-
vived at least 15 mo and two species, P. grandis and L. fragilis.
showed shell deposition during the first four months, but not af-
terwards. Four out of 10 adult P. grandis and 3 out of 10 adult L
fragilis grew 1-2 mm over the first four months on these dried
green algae, but shell growth then ceased. Mortality rates of 100%
occurred within 13 mo.

Four of the five experimental diets (TR #7-10) were successful
in supporting initial limited growth and survival, but long-term
survival (>3.5 y) was still problematic (Table 2 and Table 4). The
exception was the encapsulated feed, TR#6. This encapsulated
feed was ingested by the unionids, but within 12 h of feeding all
the animals were gaping and non-responsive to touch on soft body
pans. We dropped this feed from the diet tests after 30 days and
60% mortality in all species.

The best diet for supporting survival, growth and reproduction
of adults of most species, at least up to year 3. was the high-protein
egg chow (TR#7). The exception was L fasciola, all of who died
regardless of diet treatment. At the beginning of year 3. the sur-
vival rates of the adult unionids feeding on TR#7 were; A plicata
81%. C. mberculata 80%, L. ventricosa 72%, L. siliquoidea 65%,
Leptodea fragilis 64%, P. grandis 71%, and Q. qiiadnila 69%.

TABLE 2.
Test response of adult and subadult unionids to treatment diets.

Survived

Showed

Injjested but

at least

Survived

Survived

Survived

shell

Initiated

Ingested

caused stress

6 months

~\ year

-2 years

-3 years

growth

glochidia

TR#1. Dried Chlorellu

+

+

-f

+

TR#2. Marine algal paste

+

+ (1)

TR#3. Hatchfry encapsulon

+

-y

+

TR#4. Fish Flake Food

+

+

+

TR#5. Yeast

-I-

+

TR#6. Encapsulated feed

+

-Kl)

TR#7. Egg chow

-1-

+

+

+

-H

+

+ (5)

TR#8. Bacterial slurry #A

+

-K2)

+

+

(4)

+

TR#9. Bacterial slurry #B

+

+

-h

(4)

(4)

+

TR#10. Bacterial slurry #C

+

+

-H

(3)

(3)

-1-

-1-

( 1 ). Stopped testing after one month

(2). Kills Pyganodon grandis almost immediately and Leptodea fragilis within a couple a weeks

(3). Tested only for one year

(4). Tested only two years.

(5). 20% of females died after glochidial relea,se

878

Nichols and Garling

TABLE 3.

Percent survival of adult unionid at 12 months on treatment diets. N = 10 for each species, each diet, except for C. luberculata and

L. fasciola where N = 5/diet tested.

TR#I.

TR#3.

TR#8.

TR#9.

TR#1(I.

Dried

Hatchfrv

TR#4.

TR#5.

TR#7.

Bacterial

Bacterial

Bacterial

Unfed

Chlorella

Encapsulon

Fish flake food

Yeast

Egg show.

slurry #A

slurry #B

slurry #A

A. plicala

45%

100%

51%

25%

100%

100%

100%

100%

100%

C. tuhenulata

*

*

*

*

*

100%

*

100%>

100%

L fascinla

*

*

*

*

*

0%

*

0%

0%.

L venlricosa

21%

100%

14%

5%

2%

100%

3%

100%

100%

L siliqouidea

15%

100%

11%

10%

1%

100%

4%

100%

100%

L fragilis

12%

100%

6%

0%.

0%

100%

0%>

0%

100%

P. grandis

32%

100%c

37%

0%

0%.

100%

0%

0%

100%.

Q. qiuidruki

0%

100%

54%

0%

79%

100%

100%

100%

100%

Indicates diet not fed to that species

Twenty-one percent of the P. grandis and 15% of the L fragilis
feiTiales formed glochidia during year 2, on this diet. However,
during the third year adult unionids began to die. and by the
beginning of year four all had perished. The body weight and
glycogen levels (discussed below) of these animals were high.
indicating that starvation was not a factor. At times soft tissue
growth was so rapid that the mussels could not completely close
their shells. Autopsies showed no signs of parasitism or other
disease factor, but all of these animals had greatly enlarged kid-
neys. The proximate analysis indicates that TR#7 is a high protein
(~65%)/low carbohydrate (22%)/high lipid (13%) feed that is natu-
rally high in cholesterol (78%- of total lipid), but contains no phy-
tosterols.

The series of bacterial/ciliate slurries. TR#s 8-10, differed in
their ability to support adult unionid growth and survival. Treat-
ment #8 proved an acceptable feed for species such as A. plicala
and Q. qiiadnila. but killed all of the P. grandis within a day or
two of the initial feeding and 99%* of the Linnpsilis species within
a few weeks. Most of the other species died within the 13-month
period. On the other hand, Amblema plicata and Q. quadrnia
adults survived and grew well on this diet during the two years i.i'i
testing. The greatest increase in shell growth was seen in A. pli-
cata. The average increase was 6 mm; maximum was 1 1 mm and
minimum 3 mm over the 48-nio period. The amount of growth in
Q. (jiiadrnla was about half that seen in the A. plicara adults. No
reproductive effort was seen in any species (Table 4).

TR#9 did not improve survival and growth when compared
with TR#8. Neither adult P. grandis nor L. fragilis could tolerate
this feed, but once again, 82% of the A. plicata and 77% of the Q.
qiiadmla survived for 2 y. Amblema plicata grew more than Q.
qiiadnda, averaging 9 mm. with a maximum of 17 mm. and a
minimum 4 mm over the 48-mo period. The amount of growth in
Q. quadrnia averaged 9 mm, with a maximum of 12, and a mini-
mum of 2. No reproductive effort was seen in any species
(Table 4).

Treatment #10, which combined the basic bacterial/ciliate
slurry, dried algae, with various micronutrients and lipids proved
acceptable to adults of all species. Pyganodcm grandis and L.
fragilis did well on this diet, as did all the other unionid species
tested. Survival of the adults of all species was 100% after one
year, with the exception of L. fasciola. which we were not suc-
cessful at handling regardless of what they were fed or handled.
Note that this diet was only tested for a one-year period and long-

term data is not available. TR#IO is a low protein (-8%), low lipid
(5%), high carbohydrate (87%) feed. Cholesterol comprises 75%
of total lipid, algal sterols at 20% and miscellaneous lipids at 5%.
Pyganodon grandis fed TR#7 and TR #10 showed a consistent
increase in glycogen levels from January 1998 to December 1998.
The glycogen levels of animals on TR#IO rose from an average of
7.2 ± 1.9 mg/g in January 1998. to an average of 9.7 ± 2.4 mg/g
by December 1998 but this increase was not significantly different
(f-test. n = 10. P = 0.059). During the same period, animals on
the TR#7 showed a statistically significant increase {P < 0.05) in
glycogen from an average of 7.6 ± 1.1 mg/g to 1 1.2 ± 0.5 mg/g
(/-test. H = 10. P = 0.036). Glycogen concentrations of P. ^ra«rf(.5
that had been feeding on TR#7 for at least 36 mo (36-45 mo) were
significantly higher than P. grandis that had been on TR#7 for only
12 mo, averaging 14.1 ± 2.6 mg/g as compared with the 1 1.2 ± 0.5
mg/g (?-test. n = 20. P = 0.042). Initial glycogen concentrations
are not available for the animals kept on TR#7 for the 36-mo
period.

Siihadiills

The growth rates of subadult uniomds led on TR#7 and
onTR#IO differed more by species than by diet. Pyganadon gran-
dis and L. fragilis grew significantly faster on TR#7 and on the
TR#IO than did the other species over a 350-day period, with P.
grandis showing an average increase in shell length of 8.7 mm and
L. fragilis. 8.1 mm. There was no significant difference in growth
rates between these species on either diet. The thick-shelled spe-
cies {A. plicata. C. tuherculata. and Q. quadrnia) grew signifi-
cantly less, averaging 6 mm. Lxnnpsilis siliqnoidea and L. ventri-
cosa grew even less, averaging only 3 mm during the test period.
Survival during the test period was 100% for all species except L.
fa.uiola. Mortality rates for L fasciola were 100%. The growth
equations, r", and P values are presented in Figure I . Significant
differences in growth rates are as follows, with species sharing a
line not significantly different (Arcsine transformed data. AN-
COVA. P < 0.05): A = A. plicata. C = C. tuherculata. L = L.
fragilis. P = P. grandis. Q = Q. quadrula. E = TR#7, B =
TR#10.

Within diet

LB PB AB CB QB LE PE AE CE QE
Between diets

AB AE CB CE LB LE PB PE QB QE

Unionid Diets

879

TABLE 4.

Adult unionid % survival at 24 montlis on various diets. N = 10 for each species, eacli diet, except for C. luberailata and L. fasciola where
N = 5/diet tested. The bacterial slurry #C was not tested for longer than one year.

TR#1.

TR#3.

TR#8.

TR#9.

Dried

Hatchfrv

TR#4.

TR#5.

TR#7.

Bacterial

Bacterial

Unfed

CMorella

Encapsulon

Fish flake food

Yeast

Egg chow

slurry #A

slurry #B

A. plicala

0%

0%

0%

0%

0%

87%

78%

82%

C. tuberculata

*

*

*

*

*

89%

*

0%

L. fasciola

*

*

*

*

*

0%

*

0%

L. ventricosa

0%

0%

0%

0%

0%

78%

0%

0%

L siliqouidea

0%

0%

0%

0%

0%

71%

0%

0%

L. fragilis

0%

0%

0%

0%

0%

70%

0%

0%

P. grandis

0%

0%

0%

0%

0%

72%

0%

0%

Q. quadnila

0%

0%

0%

0%

0%

75%

80%

77%

* indicates diet not fed to that species.

Water Quality

Water quality was difficult to maintain in the test chambers,
particularly when feeding the bacterial slurries. There were 22
episodes of water quality problems during the four years of tests.
During these events, dissolved oxygen levels would plummet to <1
ppm and ammonia levels rise to >3 ppm often in less than 12 h.
Mortality was seen in adult mussels of all ten species being fed
TR#2 and TR#6 during these events (25% and 60% respectively
within 24 h). None of the other test animals died during these
events, but gaping and lack of response to touch on soft tissue body
parts were noted. No other diet treatments lead to mortality events
affecting all ten species of unionids within 1 2 h of feeding. Rapid

mortality seen in TR#8 and 9 were limited to two species (P.
grandis and L. fragilis). not all ten.

DISCUSSION

One problem in evaluating these diets is that none of the union-
ids survived for longer than 3.5 y. although a couple of feeds
supported growth and short-term survival (<3.5 y). Our data in-
dicate that unionids are capable of feeding on a wide variety of
materials, and can survive and grow for months on non-live algae
diets. Long-term survival as would be needed for broodstock inain-
tenance remains problematic. Other than survival, the criteria we
selected for measuring diet success, such as growth, reproduction

TR#7-Egg Chow

■« — Pyganodon
Leptodea
— — Amblema
--K- Quadrula

« — Cyclonaias

TO T2 T4 T6 T8 TIO

Time
Figure 1. Comparison of growth equations of various unionid subadults fed the diet TR#7 for 350 days. Ambelma plicata y = 3.33677Ln(x) -

0.7147 R- = 0.9095; Cyclonais tuberculata y = 3.8l23I.n(x) - 0.6746 R" = 0.935: Leptodea fragilis y = 5.7222Ln(x)
grandis y = 5.5916Ln(x) - 0.8214 R" = 0.9602: Quadrula quadrula y = 3.1152Ln(.\) - 0.6959 R" = 0.9112.

1.0147 R- = 0.941; Pygaiidoii

880

Nichols and Garlfng

and glycogen concentrutinn were not capuble ol predicting the
gradual die-off of ail test animals after 36 mo. Additional criteria
relating to physiologic mcchanisins and underlying digestive ca-
pabilities are needed so that changes in diet and environmental
conditions can be implemented before mortality occurs.

The two diets that were most effective in our tests. TR#7 and
TR#1(), differed substantially in protein, lipid, carbohydrate, and
phytosterol composition, but did not differ in their ability to sup-
port subadult unionid growth or adult survival. TR#10 is a low
protein (-8%), low lipid (5%), high carbohydrate (87%) feed with
added algal sterols. In contrast, TR#7 is a high protein (-65%), low
carbohydrate (22% I. higher lipid ( 13%) feed that is naturally high
in cholesterol, but contains no phytosterols. Our hypothesis is that
TR#10 is the closest in nutritive content and physical structure to
the food resources used by wild unionids in shallow temperate
rivers (see Nichols & Garling 2000). This diet proved acceptable
to all species tested in our study, supported significantly higher
growth rates in subadults and kept 100% of the test animals alive
for the entire year of the study. However, while promising results
were obtained, the problem in whole-heartedly recommending this
diet is that it was tested for only one year. It is possible that this
diet, like TR#7 cannot support long-term (>3 y) survival.

Up until year three of our study, we thought TR#7 was a
successful diet formula. It was easy to make in the laboratory, was
readily ingested by all species, and supported growth, survival,
glycogen storage, and limited reproduction, at least until year
three. The question remains unanswered as to why long-term sur-
vival was not supported. One problem might relate to protein
levels. This TR#7 is a high protein feed (-65%- protein), and high
protein may not be a dietary requirement of the age class of mus-

sels used in this study. Subadults grew as well on the low protein
(8%) levels found in TR#IO as they did on the high protein TR#7
(Fig. I and Fig. 2). Our work on a unionid community in a small
temperate river indicate that they preferentially use a lower protein
food (the <28 jx fine particulate organic matter.-6.7% protein) as
their main food source (Nichols & Garling 2000).

Feeding a high-protein feed such as the TR#7 as a sole diet at
the amount used in this study may have overfed the animals.
Glycogen levels in animals fed on egg chow were far higher than
those recorded by Naimo et al. (1998), Naimo and Monroe ( 1999),
and Patterson et al. (1999) for newly caught unionids. However,
food quantity may not have been the only problem. Feeding ex-
cessive dietary protein to vertebrates that normally use low protein
feeds will support rapid growth and reproduction initially, but can
cause high mortality due to excess nitrogen excretion causing kid-
ney failure after a few years and alter mineral tnetabolism. This
supposition is not a direct cause-and-effect relationship in union-
ids. While all the dying unionids on this feed did show greatly
enlarged kidneys, unionid kidneys do not excrete excess nitrogen.
as do vertebrate kidneys; the gills perform that function. However,
the primary function of unionid kidneys is to control ionic balance,
including mineral balance, of bodily fluids. Theoretically, excess
protein can affect ionic balance due to alteration of blood pH
through excess amine production and stress the metabolic balance
of the unionid; we could not determine cause and effect from our
study. We cannot recommend the long-term use of high protein
feeds for unionids. However, short-term or supplemental feeding
at a lower ration than used in this study might be acceptable to
prevent glycogen loss during quarantine as reported by Patterson et
al. (1999).

TR#10-Bacterial Slurry C

12 1

Time

Figure 2. Comparison of growth equations of various unionid subadults fed diet TRSKI over 350 days. Amhelma plicala y = 3.9346I,n(\l - 0.4899
R= = 0.9649: Cychnais liiherculala y = 4.l877I.n(\( - 0.3063 R" = 0.9909; l.iplodea fragilis y = 5.5873Ln)x) - 0.7751 R" = 0.9608: Pygandon grandis
y = 5.77851>n(x) - 0.903 R" = 0.9426: Qiiadnihi qiHidnila \ = 3.l988I,n(\l - 0.5076 R" = 0.9533.

Unionid Diets

U

There is certainly the possibility that unionid mortality after
three years in captivity relates more to en\ ironment than nutrition.
Even aquaculture efforts that feed their unionids live algae report
incidents of poor water quality that at times does not lead to
immediate mortality (Gatenby et al. 1994, Gatenby et al. 1996).
However, such events may produce sublethal stress that over a
period of time and after a number of incidents may eventually kill
the adult unionids. One of the greatest problems in using the types
of feeds we tested is that of maintaining water quality, especially
with bacterial/ciliate slurries (Tr#s 8-10).

There are a nuinber of other environmental factors that may
produce sublethal stress, including altered tlow. light, and tem-
perature regimes. Such environmental factors need further study
before we can establish captive management protocols for the
various unionid species, as there will be variability in environmen-

tal tolerances. Survival was to some degree species-specific. L
fasciola could not be kept alive under any type of environmental
condition, even though we were able to keep other Lampsilis spe-
cies alive for several years.

Unionids can survive, grow, and even reproduce for about
3-3.5 y on non-live algal diets, but long-term survival remains
problematic. Health monitoring criteria using glycogen concentra-
tions, shell growth rates, reproduction, and survival did not provide
enough warning to prevent mortality. Additional criteria to judge
success or failure of captive management protocols, other than
death, need to be developed. These types of non-live algal diets may
function for supplemental feeding, but at this time, maintaining
adult unionid populations in captivity will require access to natural
foods and water supplies from water systems that support native
unionid fauna to increase the likelihood of long-term survival.

LITERATURE CITED

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the Examination of Water and Wastewater. In: M. Franson. editor.
Washington DC: APHA.

Association of Official Analytical Chemists (AOAC). 1995. Official meth-
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Buchal. M. & C. Langdon. 1995. Lipid spray beads for the delivery of
water-solable materials to marine bivalves. Annual meeting of the Na-
tional Shellfisheries Association. Pacific Coast Section and Pacific
Coast Oyster Growers Assoc. 14:227.

Cope. W. & D. Waller. 1995. Evaluation of freshwater mussel relocation
as a conservation and management strategy. Rei>ul. Rivers Res. Mnna^.
11:147-156.

Coutteau. P. & P. Sorgeloos. 1993. Substitute diets for live algae in the
intensive rearing of bivalve moUusks- a state of the art repon. World
Aquae. 24:45-52.

Gatenby. C. R. Neves & B. Parker. 1994. Development of a diet for
rearing early juvenile freshwater pearly mussels. J. Shellfish Res. 13:
289.

Gatenby, C, R. Neves & B. Parker. 1996. Influence of sediment and algal
food on cultured juvenile freshwater mussels. / North. Am. Benthol.
Sac. 15:597-609.

Gatenby. C. B. Parker & R. Neves. 1997. Growth and survival of juvenile
rainbow mussels. Villosa iris (Lea. 1829) (Bivalvia: Unionidae). reared
on algal diets and sediment. Am. Malaeol. Bull. 14:57-66.

Haag. W., D. Berg. D. Garton & J. Fams. 1993. Reduced survival and
fitness in native bivalves in response to fouling by the introduced zebra
mussel (Dreissena polymorpha) in western Lake Erie. Can. J. Fish.
Aquat. Sci. 50:13-19.

Heasman. M.. J. Diemar. W. O'Connor. T. Sushames & L. Foulkes. 2000.
Development of extended shelf-life microalgae concentrate diets har-
vested by centrifugation for bivalve molluscs a summary. Aquacul-
lure Res. 31:637-659.

Knauer. J. & P. Southgate. 1999. A review of Nutritional Requirements of
bivalves and the development of alternative and artificial diets for
bivalve aquaculture. Rev. Fish. Sci. 7:241-280.

Langdon. C. & E. Bolton. 1984. A microparticulate diet for a suspension-
feeding bivalve mollu.sc. Crassoslrea virginica (Gmelin). / Exp. Ma-
rine Biol. Ecol 82:239-258.

Langdon. C. & D. Levine. 1983. Technological innovations in the devel-
opment of microparticulate feeds for marine suspension feeders. Pro-
c. Oceans Conf 1983. Effective use of the sea: an update. San Francis-
co, pp. 1005-1008.

Naimo. T.. E. Damschen. R. Rada & E. Monroe. 1998. Nonlethal e\alu-
ation of the physiological health of unionid mussels: Methods for bi-
opsy and glycogen analysis. J. North Am. Benthol. Soc. 17:121-128.

Naimo. T. & E. Monroe. 1999. Variation in glycogen concentrations within
mantle and foot tissue in Amblema plicatu plicata: Implications for
tissue biopsy sampling. Am. Malaeol. Bull. 15:51-56.

Nichols. S. & D. Gariing. 2000. Food-weh dynamics and trophic -level
interactions in a multispecies community of freshwater unionids. Can.
J. Zool. 78:871-882.

Patterson. M.. B. Parker & R. Neves. 1999. Glycogen concentration in the
mantle tissue of freshwater mussels (Bivalvia: Unionidae) during star-
vation and controlled feeding. Am. Malaeol. Bull. 15:47-50.

Secor. C. E. Mills. J. Harshbarger. T. Kuntz. W. Gutemann & D. Lisk.
1993. Bioaccumulation of toxicants, element and nutrient composition,
and soft tissue histology of zebra mussels {Dreissena polymorpha)
from New York state waters. Chemosphere 26:1559-1575.

Stuart. V. 1982. Absorbed ration, respiratory costs and resultant scope for
growth in the mussel Aiilaeomya ater (Molina) fed on a diet of kelp
detritus of different ages. Mar. Biol. Lett. 3:289-306.

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Conservation status of freshwater mussels of the United States and
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Joimwl of Shellfish Research. Vol. 21. No. 2. 883. 2002.

ABSTRACTS OF TECHNICAL PAPERS

Presented at The 4th Annual

INTERNATIONAL CONFERENCE

ON

SHELLFISH RESTORATION

Hilton Head Island. South Carolina
November 15-18. 2002

883

Shellfish, Restoration. Hilton Head Island. SC Abslracls, December 2002 885

CONTENTS

S. A. Allen. Jr.

Brood stock sources for hatchery-based stock enhancement of oyster reefs: Essential questions and recommendations. . 889
T. D. Alphiii and M. H. Posey

Ecological function of oysters in southeastern North Carolina 889

D. Bishop

Simplification of shellfish restoration methods 889

M. Bowen, K. Groves, C. Heinig, and A. Frick

Expanding and sustaining shellfisheries of Casco Bay 889

M. F. Frana, E. A. Venso, K. Brohawn. W. Beatty, M. Ellwanger, R. McKay, B. Evans, and M. Phipps-Dickerson

DNA fingerprinting of nonpoint source Escherichia coli contamination in a Chesapeake Bay watershed 890

D. W. Brown

A national strategy for coastal habitat restoration 890

R. J. Bruckner and R. L. Takacs

Beyond the project: Values of community-based habitat restoration 890

R. I). Brunbaugh. W. J. Goldsborough. L. A. Sorabella, and J. A. Wesson

Oyster bloodstock enhancement in Virginia and application of a new monitoring technique 891

D. Bushek, R. F. Dame. D. M. Allen, A. J. Lewitus, E. T. Koepfler. and D. Edwards

Nutrient cycling in intertidal creeks along the southeast US: Are oysters in control? 891

G. W. Calvo. L. M. Ragone Calvo, and E. M. Burreson

Disease resistance in a selectively bred Cntssostrea virginica strain 891

D. Cheney. R. Elston. B. MacDonald, A. Kinnan, A. Suhrbier, G. Cherr. C. Friedman, F. Griffin, A. Hamdoun, J.
Mitchell, L. Righetti. and L. Burnett

Summer mortality of the Pacific oyster. Crassostrea gigas: Influences of culture methods, site conditions, and stock

selection °"'-

F.-L. E. Chu. V. G. Encomia, S. Stickler. S. Allen, and J. La Peyre

Physiological condition and defense-related activities among easleni oyster populations 892

P. Comar. L. Kracker, P. Bauersfeld, and M. Meaburn

A unified information system for shellfish restoration 892

M. Davidson. L. Hoist. H. Bokuniewicz, C. Smith, and K. Tetrault

Effects of pearl net stocking density on survival, growth, and gonadal maturation of bay scallops 893

R. A. Elston, R. M. Estes, A. Gee. R. P. Herwig. K. Kinnan, and S. Rensel

Probiotic approach to enhance health of hatchery produced shellfish seed 893

E. Fahy

Management by size limit of the Whelk Biicciinim wuhuwn Fishery in the south west Irish Sea 893

L. D. Coen and A. Fischer

Managing the future of South Carolina's oysters: An experimental approach evaluating current harvesting practices

and boat wake impacts 894

P. M. Gaffney. K. S. Reece, and J. C. Pierce

Genomic approaches to marker development and mapping in the Eastern oyster. Crassostrea virginica 894

W. J. Goldsborough. R. D. Brumbaugh. D. W. Meritt, and J. A. Wesson

Community-based oyster restoration: Case studies from Chesapeake Bay 894

M. Gomez-Chiarri and P. Munoz

Molecular immune responses of the Eastern oyster to the parasite Perkinsus marimts 895

G. C. Cherr, C. S. Friedman. F. J. Griffin. A. Hamdoun. J. Mitchell. L. Righetti. D. P. Cheney. R. A. Elston, and B.
McDonald

Factors affecting the stress response in oysters on the west coast: Implications for summer mortality 895

N. H. Hadley and L. D. Coen

Community-based oyster habitat restoration and enhancement in South Carolina 895

J. M. Harding and R. Mann

Essential or just opportunistic fish habitat' Utilization of restored complex shellfish habitat by fish species 896

E. E. Hofmann, J. M. Klinck, E. N. Powell, S. E. Ford. S. Jordan, and E. Burreson

Effects of climate variability on the prevalence and intensity of Dermo and MSX diseases in Eastern oyster

populations 896

886 Abstracts, December 2002 Shelllish. Restoration. Hilton Head Island, SC

C. Smith, S. Dumais. L. K. Hoist, and M. Davidson

Field trial of a Bay scallop [Argopecten inadians) spawner sanctuary 896

A. C. I be and P. O. Abohweyere

Restoring critical habitats in the Niger Delta for shellfish production 896

M. Janowicz

Stakeholder and concensus - How do we make these elements work for effective action? 897

S. J. Jordan, K. N. Greenhawk, C. B. McCollough, and M. L. Homer

Oyster biomass and abundance in niirthern Chesapeake Bay: Trends in relationship lo harvest, recruitment, parasitic

diseases, and environmental variation 897

C. J. Judy and E. Campbell

Public and private oyster restoration in Maryland's Chesapeake Bay 897

P. Kamerinans and A. C. Sinaal

Mussel culture and cockle fisheries in The Netherlands: Finding a balance between economy and ecology 898

R. J. Diigas, W. R. Keithly, M. Bourgeois, P. Meier, D. Lavergne, and A. Diagne

An economic analysis of public ground oyster reef restoration in central Louisiana damaged by hurricane Andrew 898

A'. E. Landrum, K. M. St. Pe, B. Ache, and F. Kopfler

The rerouting of stormwater discharges for wetlands enhancement, levee protection, and oyster habitat protection and

restoration 899

K. E. Landrum

The shellfish challenge initiative: A cooperative success story in the Barataria-Terrebonne National Estuary 899

T. Landry, M. Ouellette, and P. Cormier

Oyster population restoration in Caraquet, N.B.; Phase 1. population assessment 899

A'. L. LeBlanc

Community-based initiatives for improving water quality in Southwestern New Brunswick. Canada — an update on

success 899

A. T. Leggett, R. Brumbaugh, W. Goldsborough, and A. McDonald

Application of commercial-scale oyster aquaculture to reef restoration 899

M. Luckenbach, F. O'Beirn, P. Ross, J. Nestlerode, and L. Sorabella

Dollars and sense of oyster restoration: An examination of nitrogen removal by a restored oyster reef 900

R. B. Luftig and W. Pelon

Reduction in the Vibrio vulnificus load of oysters by a novel short-term combination biodepuration treatment 900

R. Mann and J. M. Harding

Coming soon to a restoration site near you: The invading predatory oriental gastropod Rapana venosa 900

R. Mann

Up close and personal: A suggested quantitative approach to broodstock enhancement on shellfish restoration sites .... 901

D. W. Webster and D. W. Meritt

Linking public and private partners for restoration aquaculture in Maryland's seaside bays 901

D. W. Meritt and S. Tobash

Production of disease-free oyster seed using shallow water nurseries in the Mid-Chesapeake Bay 901

K. A. Nelson and G. L Scott

A multifactorial approach for describing the relationship between the classification of shellfish harvesting waters and

adjacent land use in Murrells and North Inlet, South Carolina 901

J. A. Nestlerode, M. W. Luckenbach, P. G. Ross, and F. X. O'Beirn

Influence of oyster reef structure on fish assemblages: Does the placement of artificial substrate enhance fish

populations? 902

M. Norman

Shellfish restoration in Ireland: The need for novel partnerships 902

M. Ouellette, M. Hardy, T. Landry, N. G. MacNair, and A. Boglien

Reproductive biology of the northern quahaug. Mercenaria mercciuiiia. in Prince Edward Island, Canada 902

K. T. Paynter, Jr.

Considerations for oyster restoration in Maryland: Disease genetics, density, reproduction, and habitat creation 903

Shellfish. Restoration. Hilton Head Island, SC Abstracls. December 2002 887

P. J. Pazo

Culture techniques applied to wild bivalve beds in Galicia. NW Spain 903

A. L Pereira. F. A. Ruano. L. Chicharo. and D. Matias

The influence of environmental factors in Japanese oysters health condition cultivated in the South of Portugal 903

P. Perina and D. Perina

The "EEEOHM" (Easttlelds" Environment Enhancing Oyster Holding Module) 903

E. A. Bochenek, E. N. Powell, J. M. Klinck, and E. E. Hojmann

A biochemically-based model of the growth and development of Crassostrea gigas larvae 904

J. M. Klinck, E. N. Powell. J. N. Kraeuter, and S. E. Ford

A fisheries model for managing the oyster fishery during times of disease 904

J. Power and D. B. Walker

Shellfish data management and reporting system (SDMRS) 904

S. P. Powers, C. H. Peterson, and J. H. Grabowski

Restoring oyster reefs for fish: Estimating enhanced secondary production of restored oyster reefs 905

B. RaLonde

Restoring the littleneck clam resource for Native American subsistence use in the Prince William Sound, Alaska 905

A. M. Reece, W. L. Ribeiro, K. L. Hudson, and S. A. Allen, Jr.

Development of Crassostrea virginica microsatellite markers for a genetic linkage map and genetic monitoring of

restoration projects 905

D. B. Rouse, R. A. Wallace, and F. S. Rikard

Oyster reef restoration research in Mobile Bay. Alabama 905

D. C. Sherwood

Transplanting broodstock oysters, Crassostrea virginica. onto reconstructed oyster reefs to increase spat recruitment

in the Piankatank River 906

S. E. Shumway

Harmful algal blooms and shellfish restoration: Can they co-exist? 906

L. A. Sorabella, M. W. Luckenbach, and F. X. O 'Beirn

A comparison of two oyster {Crassostrea virginica) strains for productivity and suitability for use in oyster reef

restoration efforts 906

S. M. Tobasfi and D. W. Meritt

Oyster restoration and the University of Maryland: Interactions between research, industry, and the public 907

A. A. Volety, M. Savarese, and S. G. Tolley

Effects of watershed alterations on oyster populations in Southwest Florida estuaries: An ecological approach 907

J. A. Wesson and L. B. McKay

The Virginia Oyster Heritage Program 907

J. J. Whitten

Enhancing and sustaining North Shore shellfishing through aquaculture 908

A. Willner

Community-based oyster restoration in an urban estuary: Developing an oyster culture and reef restoration program in

the Hudson-Raritan Estuary 908

Shellfish, Restoration. Hilton Head Island. SC

Abstracls. December 2002 889

BROOD STOCK SOURCES FOR HATCHERY-BASED
STOCK ENHANCEMENT OF OYSTER REEFS: ESSEN-
TIAL QUESTIONS AND RECOMMENDATIONS. S. K.
Allen, Jr.. Aquaculture Genetics and Breeding Technology Cen-
ter, Virginia Institute of Marine Science, College of William And
Mary, Gloucester Point, VA 23062.

Oyster populations are subdivided into genetically distinct units
with major divisions occurring over large geographic scale be-
cause of larval dispersal. Populations resist local adaptation be-
cause of population mixing through migration. However, over the
course of the last 50 years, populations of Chesapeake Bay oysters
may have lost alleles for disease resistance from the combination
of disease mortality followed by heavy harvesting of survivors.
Artificial breeding can increase the frequency of disease-resistant
alleles, and several varieties of disease-resistant oysters are avail-
able as brood stock today. Some of these strains have been used to
populate reefs and are likely to survive, grow, and breed on the
reefs. Reproduction of the disease-resistant strains will produce
disease-resistant spat over surrounding areas. There may also be
natural stocks of oysters that resist disease, such as those from the
Gulf of Mexico, where Dermo historically occurs. However, using
"artificial" or genetically distmct oysters from the hatchery for reef
restoration could also entail some risk to natural genetic diversity.
For example, artificially selected populations have (by definition)
reduced genetic variation over wild stocks. Interbreeding of the
two may alter wild populations. At present, the risk (or benefit) of
using alternative stocks is unknown. The results and recommen-
dations from a workshop on genetic considerations for hatchery-
based reef restoration will be presented.

ECOLOGICAL FUNCTION OF OYSTERS IN SOUTH-
EASTERN NORTH CAROLINA. T. D. Alphin and M. H. Po-
sey, Center For Marine Science, UNC-Wilmington, 1 Marvin
Moss Lane, Wilmington, NC 28409.

Oysters serve a variety of functions within the estuarine sys-
tems of Southeastern North Carolina. A number of juvenile tlsh
and decapods use oyster habitat for refuge/forage during some
portion of their lives. Here, we present results from several studies
evaluating the use of oyster habitat by juvenile fish and decapods
compared with alternate habitats. An abundance of fish and deca-
pods were examined in isolated and mixed oyster habitats using
Breder traps in small tidal creek estuarine systems whereas net
sampling was used to compare faunal abundances in oyster reefs
along larger spatial scales in the presence and absence of sea grass
beds. In both cases, abundance in oyster reefs were compared with
vegetated or unvegetated marsh edge habitats. On the larger scale,
use of oyster reefs was also compared with abundance patterns
within seagrass beds. The mixed results of these studies indicate
that the importance of oyster reefs as a refuge/forage habitat varies
seasonally within a given system as well as among small estuarine
systems based on the presence of alternate habitats. We also

present preliminary information on more indirect effects of oysters
as modifiers of water quality using transplant/removal studies.

SIMPLIFICATION OF SHELLFISH RESTORATION
METHODS. D. Bishop, Fukui North America, P.O. Box 119, 523
Island View Drive, Golden Lake, Ontario, Canada.

As the shellfish aquaculture industry continues to grow, meth-
ods of husbandry to reduce labor, increase yield, and produce
higher quality products are in a constant evolution. Based on the
simple fact that smart people learn from their mistakes and really
smart people learn from other's mistakes, there is a lot that can be
learned from the aquaculture industry to transfer to restoration
projects. Although all the answers for husbandry are not in place,
many dynamics and protocols positively affect restoration projects
by moving them forward at a faster pace. Attitudes towards labor
efficiency using different equipment ideas and management tech-
niques will be discussed. This will give attendees references that,
used as-is or with slight modification, could benefit their efforts
significantly. An interactive audiovisual presentation with ex-
amples of methods from around the world used today will enhance
the presentation.

EXPANDING AND SUSTAINING SHELLFISHERIES OF
CASCO BAY. M. Bowen,' K. Groves,- C. Heinig,' and A.
Frick,'' 'Normandeau Associates, Normandeau Associates Inc.,
251 Main Street. Yarmouth. ME 04096; "Casco Bay Estuary Proj-
ect, University of Southern Maine, Law School Building, Portland.
ME 04104; 'Mer Assessment Corporation, 14 Industrial Parkway,
Brunswick. ME 04011; and ■*Albert Frick Associates. Inc.. 95a
County Road, Gorham ME 04038.

One of the missions of the Casco Bay Estuary Project is to
ensure communities around Casco Bay in Maine have a healthily
shellfish harvest that sustains commercial and recreational shell-
fishing for generations to come. A "clam team" of stakeholders,
including the US Environmental Protection Agency, the Friends of
Casco Bay, Maine Department of Marine Resources, individual
cities and towns, and the Maine Department of Environmental
Protection was formed to find the most productive shellfish areas
currently closed to harvest, determine sources of contamination,
and find ways to remediate. A field review of the 57 clam flats —
800 acres — of soft-shell clam habitat that are currently closed to
harvest targeted 22 of these, totaling 370 acres of highly produc-
tive clam flats. Review of water quality data pinpointed sources of
contamination. Many of the flats are closed simply because of the
presence of an overboard discharge system that treats household
waste. The project is currently supporting an intensive effort to
design and construct replacement systems, a collaborative effort
between the towns, state, and individual homeowners. Additional
water sampling efforts are in progress to determine other nonpoint
sources of contamination, including farm runoff, leaking septic
systems, and wildlife. A third element of the project is investigat-

890 Abstracts. December 2002

Shellfish, Restoration, Hilton Head Island, SC

ing the sustainability aspect, investigating the effectiveness of
regulatory options, including licensing, harvest limits and tech-
niques, and conservation closures.

DNA FINGERPRINTING OF NONPOINT SOURCE ES-
CHERICHIA COLl CONTAMINATION IN A CHESA-
PEAKE BAY WATERSHED. M. F. Frana.' E. A. Venso,'
K. Brohawn/ W. Beatty,' M. Elluanger,' R. McKay/
B. Evans,"' and M. Phipps-Dickerson.'' 'Department of Biologi-
cal Sciences, Salisbury State University, 1101 Camden Avenue.
Salisbury, MD 21801; "Environmental Health Science, Salisbury
State University, 1101 Camden Avenue, Salisbury. MD 21801;
'Maryland Department of the Environment, Technical & Regula-
tory Services Administration, 2500 Broening Highway, Baltimore.
MD 21224; and ""Wicomico County Environmental Health Depart-
ment. Seth H. Hurdle Health Center. 108 East Main Street, Salis-
bury, MD 21801.

Fecal coliform contamination has closed shellfish harvesting
areas and public beaches and threatened recreational areas in the
Chesapeake Bay watershed. Bacteriological water quality testing
currently performed in these watersheds does not identify the
sources of contamination. Therefore, no pollution control or miti-
gation efforts have been undertaken, despite the large economic
impact for this area of the Midatlantic. Possible sources include
runoff from crop fields, wildlife, discharge from boats, and runoff
from >1..^00 animal production farms on the Eastern Shore of the
Chesapeake Bay. Although municipal waste water plant effluent
and on-site waste water treatment (septic) systems could contrib-
ute, shoreline surveys conducted by the Shellfish Sanitation Pro-
gram of the Maryland Department of the Environment indicate that
nonpoint sources are responsible for the elevated levels of coliform
bacteria in this watershed. It is understood that these sources would
contribute not only bacteria but also excess nutrients and possibly
other water contaminants that can negatively impact public health
as well as the sensitive plant and animal species that dwell in the
watershed. The methodologies used to determine the specific
sources oi Escherichia colt contamination are described, including
choice of sample locations and environmental variables, sampling
techniques. DNA analysis of strain-specific E. cc^li. and interpre-
tation of the data. Preliminary data are presented, including se-
lected DNA fingerprints and relationships among and between
total conforms and E. coli MPNs and six environmental and water
chemistry variables. Ultimately. Geographic Information Systems
mapping will be used for spatial analysis as a key to the under-
standing needed for pollution control and mitigation.

A NATIONAL STRATEGY FOR COASTAL HABITAT
RESTORATION. D. W. Brown, National Marine Fisheries Ser-
vice. 1313 East West Hwy. Ssmc#.3. Room 15221. Silver Spring.
MD 20910-3282.

Shellfish habitats make up a significant portion of the important
aquatic habitats in our coastal waters that provide the living space

for marine and estuarine fish and shellfish. Unfortunately, in many
areas along our coastline, many habitats, including shellfish habi-
tats, are being destroyed and the natural systems they support are
failing. The National Oceanic and Atmospheric Administration
recently joined with Restore America's Estuaries and the Pew
Charitable Trusts to launch a major partnership initiative to restore
important habitats in our coastal estuaries. A major element of this
initiative is to develop a national strategy for coastal habitat res-
toration, including important shellfish habitats. The purpose of the
strategy is to identify specific habitat problems in each coastal
region and to determine the most viable restoration approaches to
address degraded areas for these regions. The National Strategy
will ( 1 ) actively promote the increased protection of existing habi-
tats; (2) establish specific regional and national restoration goals
and objectives; (3) provide a framework for setting restoration
priorities; (4) identify and integrate the science and new technolo-
gies needed for effective restoration; and (5) energize cooperative
partnerships among private and public stakeholders. This presen-
tation will review ongoing and planned actions by the NGO com-
munity, federal agencies, and the private sector to develop a na-
tional strategy for coastal habitat restoration, including shellfish
habitats, by the fall of 2001.

BEYOND THE PROJECT: VALUES OF COMMUNITY-
BASED HABITAT RESTORATION. R. J. Bruckner' and R.
L. Takacs,- 'NCAA Restoration Center, 1315 East West High-
way, Silver Spring, MD 20910 and "NCAA Chesapeake Bay Of-
fice, 410 Severn Avenue, Annapolis, MD 21403.

The NCAA Community-based Restoration Program (CRP) be-
gan in 1996 to inspire local efforts to conduct meaningful, on-Ihe-
ground restoration of marine, estuarine. and riparian habitat. The
CRP is a systematic effort to catalyze partnerships at the national
and local level to contribute funding, technical assistance, land,
volunteer support, or other in-kind services to help citizens imple-
ment restoration projects that promote stewardship and a conser-
vation ethic for living marine resources. The CRP links funding
and technical expertise to citizen-driven restoration projects and
emphasizes collaborative strategies built around improving NCAA
trust resources and the quality of the communities they sustain.
Oyster restoration projects, although not all explicitly off-limits to
harvesting, have emphasized the habitat benefits of reef restora-
tion, from three-dimensional habitat conducive to spat settlement,
to the benthic organisms that make up the ecological diversity of
oyster reefs themselves, to the fish and openwater communities
that aggregate around hard-bottom reef habitat. In addition to
implementing projects, this innovative funding/partnership source
has provided the mechanism to "field test" new restoration strat-
egies, such as reef design and construction, unique management
approaches like sanctuaries, reserves, and satellite bars, and often
has served as the springboard for larger-scale, river-wide restora-
tion efforts. The availability of technical expertise and matching

Shellfish. Restoration. Hilton Head Island. SC

Abstracts. December 2002 891

funds and the positive results achieved by community-based shell-
fish restoration efforts have catalyzed other federal, state, and local
entities to participate, effectively broadening the partnering and
stewardship opportunities, increasing the areas available for shell-
fish restoration, and leveraging the amount of funds available for
habitat restoration efforts.

OYSTER BROODSTOCK ENHANCEMENT IN VIRGINIA
AND APPLICATION OF A NEW MONITORING TECH-
NIQUE. R. D. Brumbaugh.' W. J. Goldsborough,' L. A. Sora-
bella,' and J. A. Wesson," 'Chesapeake Bay Foundation, 142 W.
York Street. Suite 318. Norfolk. VA 23510 and -Virginia Marine
Resources Commission. P.O. Box 756. 2600 Washington Ave..
Newport News, VA 23607.

The transplanting of both wild and hatchery-produced oysters
onto oyster sanctuary reefs is increasingly frequent as a component
of oyster restoration efforts in the Chesapeake Bay. Since 1996.
oysters have been added to more than a dozen state or privately
managed sanctuary reefs in Virginia in an effort to enhance local-
ized oyster spawning success. Wild oysters, purchased with both
state and private funds, have accounted for approximately 70% of
the total number of oysters added to reefs. Increasingly, however,
the oysters added to reefs are hatchery produced, grown by citizens
and students volunteering through programs such as the Chesa-
peake Bay Foundation's Oyster Corps. To date, more than 800.000
oysters grown by volunteers have been added to Virginia's system
of reefs. Although definitive data are scarce, there appears to be
good empirical evidence that these stocking efforts have enhanced
spat settlement rates on and around sanctuary reefs. Dive surveys
and patent-tong data show substantial increases in localized spat
settlement in tributaries where oysters have been added to reefs in
recent years. To better understand spat settlement dynamics around
the reefs, spat cages, small cages filled with a known volume of
shell, are now being used to monitor oyster settlement around
selected reefs. A strong correlation exists between spat cage data
and diver-surveys on nearby reefs (r = 0.95, P < 0.01). suggesting
that spat cages may be a low-cost means of both involving the
public in restoration and of evaluating results of broodstock en-
hancement and reef restoration projects.

NUTRIENT CYCLING IN INTERTIDAL CREEKS ALONG
THE SOUTHEAST US: ARE OYSTERS IN CONTROL? D.
Bushek,' R. F. Dame," D. M. Allen," A. J. Lewitus.' E. T.
Koepfler,- and D. Edwards,' 'Baruch Marine Field Laboratory.
Baruch Institute for Marine Biology and Coastal Research. Uni-
versity of South Carolina. Georgetown. SC 29442 and "Depart-
ment of Marine Science, Coastal Carolina University. Conway, SC
29528.

Ecologically, oyster reefs provide habitat, filter water, and fa-
cilitate nutrient cycling. We experimentally removed oyster reefs
to examine their role in the structure and function of intertidal

creek ecosystems. Surprisingly, the removal of oyster reefs did not
significantly alter nutrient concentrations, nekton usage, or phy-
toplankton production. Our calculations show that oysters do not
produce enough ammonium to satisfy phytoplankton productivity,
but nekton, water column remineralization, and sediments more
than account for the deficit. These observations were interpreted as
an indication of functional redundancy in the system. Flagellates,
which are preferred over diatoms as food by the oysters, dominated
the phytoplankton during summer when ammonium concentra-
tions were high. Diatoms dominated during the colder months.
Shifts in phytoplankton dominance corresponded to the seasonal
arrival and departure of nekton in the creeks. Because nekton
comprised more than double the biomass of oysters during sum-
mer, fishes and macrocrustaceans may play a greater role in nu-
trient remineralization than has been previously considered. At the
meso-scale ecosystem level, the loss of nutrient remineralization
activities attributable to the removal of oyster reefs was compen-
sated by other components within the system, but phytoplankton
communities changed, apparently in response to changes in graz-
ing. Oysters clearly play important roles, but defining the impor-
tance of shellfish restoration in the management of coastal eco-
systems requires an understanding of the ecosystem science, a
consideration of scale, and the realization that tidal creek systems
exhibit complex responses.

DISEASE RESISTANCE IN A SELECTIVELY BRED CRAS-
SOSTREA VIRGINICA STRAIN. G. W. Calvo, L. M. Ragone
Calvo. and E. M. Burreson, Virginia Institute of Marine Science
(VIMS). College of William and Mary, Gloucester Point, VA
23062.

During 1997 to 1999, DEBY oysters, a VIMS stock that was
selectively bred for four generations at a disease endemic site in
the lower York River, Virginia, were evaluated for survival,
growth, and disease susceptibility in comparison with progeny
from wild Mobjack Bay (MB) and Tangier Sound (TS) brood
stocks. MB and TS stocks are relevant to rehabilitation of Chesa-
peake Bay oysters because the former have been routinely used for
aquaculture and the latter have been recently used for reef resto-
ration because of their putative disease resistance. Oysters (n =
1500 of each group, mean shell height = 15-17 mm) were de-
ployed in floating mesh cages at a low salinity (<15 ppt) site and
a moderate salinity (15-25 ppt) site in the lower Chesapeake Bay,
and at a high salinity (>25 ppt) site on the Atlantic Coast of
Virginia. Twenty-eight months after deployment, cumulative mor-
tality in MB and TS was 84-100%. In contrast, cumulative mor-
tality in DEBY at low, moderate, and high salinity sites was,
respectively, 21, 51. and 36%. By November 1999. mean shell
height in MB and TS at low-, moderate-, and high-salinity sites
was. respectively. 77. 88-90. and 57-59 mm. In comparison, mean
shell height in DEBY was 92. 101. and 72 mm. Although similar
low levels of MSX were observed in all groups, P. marimis infec-

892 Ahsmicis. December 2002

Shelltish, Restoration. Hilton Head Island, SC

tions in MB and TS were more intense than in DEBY throughout
the study. This promising oyster strain has potential to facilitate
commercial aquaculture and reef restoration efforts in Chesapeake
Bay.

SUMMER MORTALITY OF THE PACIFIC OYSTER.
CRASSOSTREA GIGAS: INFLUENCES OF CULTURE
METHODS, SITE CONDITIONS, AND STOCK SELEC-
TION. D. Cheney.' R. Elston.' B. MacDonald.' K. Rinnan,' A.
Suhrbier.' G. Cherr." C. Friedman." F. {Jriffin.- A. Ham-
doun,- J. Mitchell,- L. Righetti," and L. Burnett,' 'Pacific Shell
fish Institute, 120 State Ave NE #142. Olympia, WA 98501 ; "Uni-
versity Of California, Davis. Bodega Marine Laboratory. P.O. Bo,\
247, Bodega Bay, CA 94923; and 'Grice Marine Laboratory, 20.5
Fort Johnson, Charleston, SC 29412.

During the late summer to early fall period. Pacific oy.sters
cultured on the west coast of the United States and elsewhere may
experience high levels of mortality. In the 1960s to 1980s, this
condition was subject to intensive investigation focusing on broad
areas of disease pathology, genetics, physiology, and the environ-
ment. Results of these studies were largely inconclusive or pointed
to a poorly defined etiology. Recent studies in Puget Sound. Wash-
ington and Tomales Bay, California, center on the influence of
multiple stressors and their affects on oyster survival, physiology,
and pathology. The goal of this research is to identify possible
modifications in culture practices, brood stock selection, or grow-
out location to increase survival of Pacific oysters. Field observa-
tions indicate oysters are subject to extreme variations in a number
of parameters during intertidal cycles. An increased rate of oyster
mortality and modified physiological response appear to be
strongly correlated with both elevated temperatures and extended
periods of depressed DO. The DO reductions are sometimes
coupled with heavy macroalgae blooms and high phytoplankton
densities. This and other works indicate oyster summer mortality
rates are also strongly influenced by ploidy and broodstock origin/
stock selection. The.se observations have renewed interest in test-
ing stocks selected for reduced rates of summer mortality and
which retain desirable characteristics of good growth and meat
yield. This research was supported by grant numbers
NA86RG0015 and NA96RG0488 from the National Sea Grant
College Oyster Disease Research Program and matching contribu-
tions from West Coast shellfish farmers.

PHYSIOLOGICAL CONDITION AND DEFENSE-
RELATED ACTIVITIES AMONG EASTERN OYSTER
POPULATIONS. F.-L. E. Chu.' V. G. Encomio,' S. Stickler.'
S. Allen,' and .1. La Peyre." 'Virginia Institute of Marine Science.
College of William and Mary, Gloucester Point. VA 2.^062 and
"Louisiana State University, Baton Rouge, LA 70803-6002.

The goal of our study is to identify oyster slocks that are re-
sistant/tolerant to the disease caused by the parasite, Perkinsus

iiuiniiKs (Dermo). We are comparing the physiological condition
and defense factors of putative "Dermo resistant" and "nonresis-
tant" oysters (Crasssostrea virgiiiica) deployed in the fall of 1999
at two sites in the Chesapeake Bay (Port Kinsale. Yeocomico
River; Regent Point. Rappahannock River), where Dermo disease
is known to occur, but not MSX (disease caused by Haplospo-
ridiiim nelsoni). These oysters are Fl progenies from presumably
genetically distinct oyster populations (three Gulf of Mexico and
three Chesapeake Bay populations, and one hatchery strain) and
represent geographical disparity. Oysters have been sampled
monthly since May 2000. Initial analysis showed that all the stocks
have grown significantly since deployment and the Rappahannock
River Stock has the fastest growth. Tissue dry weights of this slock
increased significantly over lime at both sites. Contents of glyco-
gen, protein, and lipid increased with growth. All stocks sampled
from May to July had low P. mariiuts infection and prevalence.
Mortality was low in all stocks and lower in the Gulf of Mexico
than Chesapeake Bay populations. No significant differences were
noted in levels of plasma protein and lysozyme among stocks.
Currently we are analyzing oysters sampled in August and Sep-
tember. Correlation between growth, physiological, biochemical,
and defense condition and P. mariiuts infection among oyster
stocks will be discussed. This research was funded by the NOAA-
Vir'jinia Sea Grant-Oyster Disease Research Program.

A UNIFIED INFORMATION SYSTEM FOR SHELLFISH
RESTORATION. P. Comar, L. Kracker. P. Bauersfeld, and M.
Meaburn. Center For Coastal Environmental Health and Biomo-
lecular Research. National Ocean Service. NOAA. 219 Fort
Johnson Road. Charleston. SC 29412.

The Shellfish Information Management System (SIMS) is an
intergovernmental data system designed to provide a current cen-
tral source of information on shellfish safety, resource, and habitat
useful to multiple users at local, state, regional, and national levels.
SIMS is being developed as a GlS-enabled, web-accessible rela-
tional database of shellfish harvest water survey, classification,
and resource information. Most of the data in SIMS is provided by
state agencies, and SIMS will allow more extensive access to and
integrative analysis of that information. In 1999. the National
Ocean Service. Center for Coastal Environmental Health and Bio-
molecular Research in Charleston. South Carolina, began partner-
ing with a growing number of coastal states in the design and
applications for SIMS. This spatially enabled. Oracle database is
designed with extensive query functionality, visualization, and
analytical capabilities for a wide range of shellfish safety, water
quality, resource, and restoration concerns. Shellfish restoration is
a new component being developed for incorporation into SIMS so
that trends in restoration can be quantified and visualized. Water
quality, benthic and habitat suitability, shellfish resource, presence
of disease agents, social and economic factors, and other influ-
ences impact shellfish restoration decisions and actions. Discus-

Shellfish. Restoration. Hilton Head Island, SC

Abstracts. December 2002 893

sion will include the rationale and means for collecting and inte-
grating such restoration data layers into SIMS.

EFFECTS OF PEARL NET STOCKING DENSITY ON SUR-
VIVAL, GROWTH. AND GONADAL MATURATION OF
BAY SCALLOPS. M. Davidson,' L. Hoist.' H. Bokuniewicz."
C. Smith,-' and K. Tetrault,-' 'Nysdec, 205 North Belle Mead
Road. East Setauket. NY 1 1733. "Marine Science Research Cen-
ter, Suny Stony Brook. NY 1 1790; and 'Dornell Cooperative Ex-
tension Marine Program, 3059 Sound Avenue, Riverhead. NY
11901.

The stocking densities under which bay scallops are reared can
have long-term effects on survival, growth, and spawning success
that may not be evident while the scallops are in culture. To
investigate the influence of stocking density on scallop production,
hatchery reared bay scallops were stocked in pearl nets at three
different densities during the summer. In the fall, bay scallop sur-
vival and shell heights were recorded. The animals were trans-
ferred to lantern nets and stocked at two different densities,
grouped by their initial densities in the pearl nets, and over win-
tered. Bay scallops raised at high densities exhibited lower survival
and slower growth than those raised at lower densities. Regardless
of density in the lantern nets, growth and survival still showed the
negative effects of initial crowding in the pearl nets. Two-way
analyses of variance revealed significant differences among the
peari net and lantern net treatments in scallop survival and growth.
Gonadal indices show that all the bay scallops, regardless of treat-
ment, spawned at the same time. At the time of spawning there
were no significant effects of density on gonadal index. Bay scal-
lop restoration efforts should ensure that scallops are reared under
conditions that maximize survival and growth.

PROBIOTIC APPROACH TO ENHANCE HEALTH OF
HATCHERY PRODUCED SHELLFISH SEED. R. A. Elston,'
R. M. Estes,^ A. Gee,' R. P. Herwig,^ K. Kinnan,^ and S.
Rensel,' 'Aquatechnics/Pacific Shellfish Institute, P.O. Box 687,
Carisborg, WA 98324; "School of Fisheries, University of Wash-
ington, 3707 Brooklyn Ave. N.E., Seattle, WA 98105-6715; 'De-
partment of Biology, Pacific Lutheran University, Tacoma. WA
98447-0003; "'School of Fisheries, University of Washington, 3707
Brooklyn Ave. N.E., Seattle, WA 98105-6715; ^Aquatechnics/
Pacific Shellfish Institute. P.O. Box 687, Carisborg, WA 98324;
and ^'Department of Biology, Pacific Lutheran University,
Tacoma, WA 98447-0003.

Bacterial diseases of intensively cultured larval and juvenile
shellfish cause significant losses in hatcheries and nurseries. In
addition, chronic bacterial infections are a significant cause of
bivalve seed losses postplanting. From commercial hatchery case
histories, a number of virulent juvenile oyster bacterial pathogens
have been isolated, characterized, and pathogenicity confirmed by

challenge procedures. Prevention and control strategies for bacte-
rial pathogens in hatcheries and nurseries must include routine
sanitation of system surfaces, water filtration, brood stock sanita-
tion, and maintenance of low dissolved organic levels. Antibiotics
have been used in experimental settings but are not routinely used
on production scale systems because of cost as well as risk of
producing resistant strains. A program to select and test probiotic
strains of bacteria, as an alternative to antibiotic use, is underway,
and results to date will be presented. Bacterial pathogens were first
screened by comparing whole cell fatty acid profiles. Based on this
evaluation, most pathogens were consistent or close to the Vibrio
genus, but probiotic candidates represented a variety of bacterial
genera. Selected representative isolates were further characterized
using biochemical criteria and 16s rDNA sequencing. Candidate
probiotic bacteria are first tested in agar plate inhibition tests.
Strains showing inhibition to isolated pathogens are tested for
haemolytic activity and pathogenicity to shellfish seed. Candidates
passing these tests are then tested for inhibition of mortality and
morbidity response in laboratory pathogen challenges. This re-
search was supported in part by Saltonstall-Kennedy program (Na-
tional Marine Fisheries Service, U.S. Department of Commerce)
grant to Pacific Shellfish Institute. Olympia, Washington.

MANAGEMENT BY SIZE LIMIT OF THE WHELK BUC-
CINUM UNDATUM FISHERY IN THE SOUTH WEST
IRISH SEA. E. Fahy, Marine Fisheries Services Division, Marine
Institute, Abbotstown, Castleknock, Dublin 15, Ireland.

Whelk landings in the south west Irish Sea increased from 56
t in 1990 to 6,575 t in 1996 after which they stabilized between
3,600 and 4,600 t annually. At its peak, the fishery supported
approximately 80 vessels but this number has halved since this
time. In 1994, a size limit of 50 mm was introduced for conser-
vation purposes. Age-based assessments of the landings were con-
ducted in 1994, 1996, 1997, and 1999, for which purpose the
fishery, ranging from 52°10' to 53°30', is divided into four sectors.
Landings to the four sectors display biological characteristics that
indicate the occurrence of a number of stocklets rather than a
single stock unit. Compliance with the size limit has been poor.
From 20 to 33% of total landings (by number) in any of the
assessed years have been less than the legal limit. Trends in cpue
have been monitored since 1990. Some fishermen in the center
sectors improved their yield between 1994 and 1998. Whelk have
responded to a reduction in fishing effort since 1996, immediately
after which averaged mortality coefficients (Z) were highest
(0.79); they declined to 0.61 in 1999. The survival of the whelk
fishery in the south west Irish Sea is attributed to the instability of
the market which is dominated by a single customer. South Korea.
A more effective size limit for this fishery would be 68 mm (83
mm in the northern sector), and this is considered unrealistic,
suggesting that alternative management measures will have to be
introduced.

894 Abstracts, December 2002

Shellfish, Restoration, Hilton Head Island, SC

MANAGING THE FUTURE OF SOUTH CAROLINA'S
OYSTERS: AN EXPERIMENTAL APPROACH EVALUAT-
ING CURRENT HARVESTING PRACTICES AND BOAT
WAKE IMPACTS. L. D. Coen and A. Fischer, Marine Re-
sources Research Institute. SCDNR. Charleston. SC 29412.

Oyster reefs provide an important intertidal habitat to the
Southeastern United States. However, harvesting and recreational
boating invariably impact these critical habitats and their associ-
ated functions. In 1998, we began to experimentally evaluate the
direct impacts of four harvesting practices (complete harvest, cull-
in-place. clamming, and rake down) on intertidal oyster resources.
Initially. 26 sites were sampled by quadrat to establish baseline
assessments. Initial mean oyster size (SH) across sites ranged from
23-.^,-' mm. with initial densities ranging from 1.700-7,500 oys-
ters/nr. Then, the above harvesting practices were simulated at
replicated sites, each paired with an adjacent control site. Water
quality (temperature. DO. salinity, chl a) was measured during the
study period. Trays of shell were deployed at each site to evaluate
oyster recruitment and growth. After approximately I year, inore
than LI .^.000 oysters recruited to the 1.^0 deployed trays. This
recruitment, a surrogate for larval supply/habitat quality, and the
baseline assessments are analyzed and discussed. In 1999. we con-
ducted experiments to understand how boat wakes compromise
shell (cultch) deployments for oyster restoration and marsh erosion
control. For this, we deployed stabilized (mesh) and unstabilized
shell treatments, monitoring cultch retention after controlled boat
wakes. In the first pilot experiment, unstabilized treatments lost
3.3.6'^ (7.7 cm) rnore shell than stabilized treatments after expo-
sure to 32-controlled boat passes. In a second experiment, 22.4%
(5.17 cm) more was lost after only 24 passes. Both harvesting
practices and recreational boating wakes can potentially impact the
growth, recruitment, and recovery of intertidal oyster resources.
Additionally, oyster reefs that fringe marshes can serve as mod-
erators of both marsh and bank erosion. Further studies with re-
mote sensing technologies should be employed to monitor the
oyster-marsh interaction.

GENOMIC APPROACHES TO MARKER DEVELOPMENT
AND MAPPING N THE EASTERN OYSTER, CRASSOS-
TREA VIRGINICA. P. M. Gaffney,' K. S. Reece,- and J. C.
Pierce,"' 'College of Marine Studies, Lewes, DE 19958; ^Virginia
Institute of Marine Science. Rt. 1 208, Gloucester Point. VA
23062; and 'University of the Sciences in Philadelphia. 600 S. 43d
St.. Philadelphia, PA 19104.

In response to the dramatic decline in the Atlantic oyster fish-
ery, efforts are underway to expand hatchery production of the
Eastern oyster, for both commercial farming and for replenishment
of disease-challenged natural populations. In particular, there is a
strong demand for genetically improved oyster strains resistant to
two common protozoan parasites, Deniio and MSX. The genetic

improvement process will be enhanced by the development of
molecular markers and a genetic linkage map. To facilitate future
marker development in C. viiginica. we obtained 0.7 MB of ran-
dom genomic sequence data from a small-insert fl kb) pGEM
library. A modest number of significant BLASTX hits may prove
valuable for designing type I markers for comparative mapping
with the Pacific oyster. In addition, we the searched the sequence
database for repetitive sequences. Several satellite DNA sequences
were identified and compared with putative satellite sequences
obtained by traditional cloning methods. Our database yielded use-
ful information on the distribution of microsatellite loci. Dinucle-
otide microsatellites were dominated by the AG motif (669<"). Tri-
nucleotide microsatellites included all possible motifs in appar-
ently equal frequencies. Tetranucleotide microsatellites were more
common than trinucleotides and, unlike the other microsatellite
classes, were frequently associated with repetitive sequences, with
a strong tendency for certain tetranucleotide motifs to be associ-
ated with particular repetitive sequences. This information will be
useful for tetranucleotide microsatellite marker design, as well as
interpretation of linkage mapping data. The repetitive sequence
database will be used as an adjunct for designing new primers, to
reduce the frequency of non-target amplification.

COMMUNITY-BASED OYSTER RESTORATION: CASE
STUDIES FROM CHESAPEAKE BAY. W.J. Goldsborougii,'
R. D. Brumbaugii,' D. W. Meritt,- and J. A. Wesson,' 'Chesa-
peake Bay Foundation, 162 Prince George Street. Annapolis. MD
21401; "University of Maryland, Center For Environmental Sci-
ence, P.O. Box 775, Cambridge, MD 21613; and 'Virginia Marine
Resources Commission, P.O. Box 756, 2600 Washington Avenue,
Newport News. VA 23607.

Public support for oyster restoration in the Chesapeake Bay
region has increased in recent years, largely because of expanded
opportunities for direct citizen involvement in restoration work
The commercial value of oyster restoration is the most easily ap-
preciated aspect of restoration, whereas associated benefits such as
impro\ed fish habitat and water quality are only recently being
more widely recognized. As opportunities for public participation
have expanded, the support for restoration has increasingly been
based on these associated ecosystem benefits, particularly in de-
veloped areas where water quality may preclude commercial or
recreational harvest of bivalves. One of the principal ways that the
public now participates in oyster restoration is by growing hatch-
ery-produced oysters using small-scale aquaculture techniques
(i.e., "oyster gardening") for eventual transplanting onto brood-
stock sanctuary reefs. Analyses of four local examples of citizen
involvement in oyster gardening/restoration in the Chesapeake re-
veal a general pattern of roles and responsibilities for successtui
community-based restoration. Local leadership, sources for shell
and seed, education, technical guidance, amenable government
rules and regulations, media exposure, and funding emerge as key

Shellfish, Restoration, Hilton Head Island, SC

Abstracts, December 2002 895

factors. In the Chesapeake, a partnership approach based on co-
operation between various combinations of citizens, schools, local
businesses, local service organizations, watershed associations,
academic institutions, state and federal agencies, conservation or-
ganizations, and private foundations has successfully addressed
these needs. It is becoming increasingly clear that the groundswell
of public support for restoration resulting from community-based
approaches is a key factor in generating increased public funding
for restoration.

MOLECULAR IMMUNE RESPONSES OF THE EASTERN
OYSTER TO THE PARASITE PERKINSUS MARINUS. M.
Gomez-Chiarri and P. Mufioz, Department of Fisheries, Animal
and Veterinary Science, University of Rhode Island, 127 Wood-
ward Hall. Kingston, RI 02881.

Microbial pathogens and parasites like Peikinsiis marinus and
Haplospnridiwn iielsniu place a large economic burden on oyster
fisheries and aquaculture. Although there has been a steady in-
crea.se in our knowledge on the pathology and epizootiology of the
diseases caused by these parasites, relatively little is known about
the molecular mechanisms involved in the response of oysters to
infection. The goal of this project funded by the ODRP is to
monitor systematically the induced expression of genes involved
in the response oi Crassostreu virginica to infection by the parasite
Perkinsiis marinus. An mRNA differential display technique
coupled with stringent verification assays (reverse Northern blot)
will be used to isolate oyster and parasite sequences expressed in
a differential manner after challenge of oysters with cultured P.
marbuis. Genes will be cloned and sequenced using standard mo-
lecular techniques. The temporal and tissue patterns of expression
of the candidate genes in oysters will be studied using Northern
blot. Preliminary results from the challenge experiments will be
presented at this meeting.

FACTORS AFFECTING THE STRESS RESPONSE IN OYS-
TERS ON THE WEST COAST: IMPLICATIONS FOR SUM-
MER MORTALITY. G. C. Cherr.' C. S. Friedman,' F. J.
Griffin.' A. Hamdoun,' J. Mitchell,' L. Righetti,' D. P.
Cheney,^ R, A, Elston,^ and B. McDonald,^ University of Cali-
fornia. Davis, Bodega Marine Laboratory. P.O. Box 247. Bodega
Bay. CA 94923 and "Pacific Shellfish Institute. 120 State Ave.
N.E. #142. Olympia, WA 98501.

Summer mortality of Crassostrea gigas on the West Coast of
the United States is an unpredictable phenomenon of unknown
etiology but one that is hypothesized to be caused by multiple
stressors. Previous research has identified a dinofiagellate (Gyiu-
nodinium sanguineum). temperature, and low dissolved oxygen as
possible contributors. We have attempted to delineate the indepen-
dent effects of two of these suspected factors, phytoplankton and
temperature, while conducting parallel field studies in California

and Washington to determine the effects of seed stock lineage and
seed planting times. Laboratory challenges confirmed that G. san-
guineum can produce stress/mortality in the absence of other in-
sults. Phytoplankton bloom events have coincided with field mor-
tality; however, the species present were a Pseudonitzchia-Vike
species and Proroccntrum spp.. not G. sanguineum. Previous re-
search on temperature effects showed thai the ability of C. gigas to
tolerate otherwise lethal temperatures occurred after sublethal ther-
mal shock and induction of the heat shock protein 70 (HSP70)
family. This is termed the heat shock response (HSR). We have
examined the abilities of C. gigas from three different habitats
(Toten Inlet, WA; Mud Bay, WA; Tomales Bay. CA) to mount a
HSR and compared this ability with environmental and summer
mortality data. Our current findings suggest that chronic sublethal
environmental stressors such as heat and immersion can induce
HSP70 expression and acquisition of thermal tolerance in C. gigas.
However, these chronically stressed animals exhibit a compro-
mised HSR; they do not tolerate post-heat shock temperatures as
high as nonchronically stressed counterparts. This research was
funded by National Sea Grant College Program Office: Oyster
Disease Research Program.

COMMUNITY-BASED OYSTER HABITAT RESTORA-
TION AND ENHANCEMENT IN SOUTH CAROLINA. N. H.
Hadley and L. D. Coen. Marine Resources Research Institute.
SCDNR. P.O. Box 12559, Charleston. SC 29422.

Oyster reefs provide important habitat for finfish, crabs, and
shrimp; improve water quality; and. when located adjacent to
Spartina marsh, form a natural bulwark to reduce erosion. Oyster
habitats nationwide are threatened by adverse effects of coastal
development. The majority of oysters in South Carolina occur
intertidally. where they may be exposed for as much as 6 h because
of the -2 meter tidal range. This makes them especially vulnerable
to physical disturbances such as boat wakes. Substrates in South
Carolina are typically soft mud and oyster shell provides one of the
few hard surfaces for larval oyster attachment. Oysters readily
recruit to shell placed in areas which otherwise may have no re-
cruitment because of lack of suitable substrate. At sites with ap-
propriate characteristics functional oyster reefs may be established
in 3 to 5 years, with some attributes beginning earlier. This pro-
gram will use community volunteers to establish multiple small-
scale oyster habitats by planting oyster shell and covering it with
stabilizing mesh. We will also develop a volunteer-based moni-
toring program to evaluate restoration success. Community part-
ners with existing volunteer contacts have been enlisted to assist in
this program. An oyster shell-recycling program is being estab-
lished to generate shell for future restoration projects. Schoolchil-
dren will be involved through collaboration with the Charleston
Math and Science Hub to develop classroom and field activities
directly related to oyster habitats. Materials (pamphlets, a website.
CD) will be developed to educate the public about oyster habitats
and shell recycling.

896 Abstracts, December 2002

Shellfish, Restoration, Hilton Head Island, SC

ESSENTIAL OR JUST OPPORTUNISTIC FISH HABITAT?
UTILIZATION OF RESTORED COMPLEX SHELLFISH
HABITAT BY FISH SPECIES. J. M. Harding and R. Mann,

School of Marine Science, Virginia Institute of Marine Science,
College of William and Mary. Gloucester Point. VA 23062.

Shellfish restoration typically creates complex habitat in re-
gions where such habitat is limited or absent. Observations to date
suggest that increasing habitat complexity supports more diverse
representation in other trophic levels. Such observations have been
used to argue for shellfish restoration sites in the wider context of
essential fish habitat. We present temporal and spatial data on fish
utilization of a dine of habitats from a complex, "restored"" site,
through a two-dimensional but spatially complex site, to a mo-
notonous sand bottom, and pose the question as to whether fish
utilization of this cline suggests "essential"" or simply opportunistic
utilization of the varying resource.

EFFECTS OF CLIMATE VARIABILITY ON THE PREVA-
LENCE AND INTENSITY OF DERMO AND MSX DIS-
EASES IN EASTERN OYSTER POPULATIONS. E. E. Hof-
niann.' J. M. Klinck.' E. N. Powell,- S. E. Ford." S. Jordan,'
and E. Burreson,"* 'CCPO, Old Dominion Llniversity, Norfolk,
VA 23529; "Haskin Shellfish Research Laboratory, Rutgers Uni-
versity, Port Norris. NJ 08349; 'Sarbanes Cooperative Oxford
Laboratory. 904 South Morris Street. Oxford, MD 21654; and
■^Virginia Institute of Marine Science. P.O. Box 1346, College Of
William and Mary. Gloucester Point, VA 23062.

With previous funding from the National Sea Grant Oyster
Disease Research Program, we developed numerical models that
simulate the annual cycles of intensity and prevalence of the dis-
ea.ses, Dermo, caused by Perki)isiis mariiiKs. and MSX, caused by
Hapliispordiiim iielsoni. in Eastern oyster (Cmssosiiea viri;inica>
populations in Delaware Bay and the upper Chesapeake Bay. The
host-parasite models consist of models for the growth of the para-
sites, as well as a model for the growth and development of the
oyster. The external forcing for the models is from time series of
temperature, salinity, food supply, and total suspended solids. Our
recent project has focused on combining the two disease models
with the oyster growth model. Simulations w ith the combined two
di.sease-oyster model provide insight into the effect of variability
in environmental conditions in initiating and controlling epizootics
of Dermo and MSX in Chesapeake and Delaware Bays. The com-
bined model also provides a mechanism for investigating possible
interactions between Dermo and MSX diseases that modulate the
level of intensity and overall prevalence of the two diseases in
oyster populations. Initial results suggest that there is only limited
interaction between the two diseases in the host and that environ-
mental conditions are the primary determinant of which disease is
dominant at a given location, as long as the oysters are susceptible
to both disease agents and that the dose of infective particles does
not vary.

FIELD TRIAL OF A BAY SCALLOP (ARGOPECTEN IRRA-
DIANS) SPAWNER SANCTUARY. C. Smith,' S. Dumais,' L.
K. Hoist,- and M. Davidson," 'Cornell Cooperative Extension of
Suffolk County, Marine Program, 3059 Sound Avenue. Riverhead,
NY 11901 and "New York State Department of Environmental
Conser\ation. Division of Fish. Wildlife, and Marine Resources,
205 North Belle Meade Road, East Setauket. NY 1 1733.

The populations of Bay scallop in New York waters have ex-
perienced critical decline over the past two decades, caused in
large part by the occurrence of Brown Tide algal blooms and its
concomitant effects on habitat and shellfish health. Management
efforts in the past have focused heavily on hatchery-produced
stock, which has been free planted into the estuary. Frequently,
follow-up investigations show no juvenile scallops at the release
sites, and the ultimate fate of the seed stock is unknown, except
through extrapolation of harvest data. New York State Department
of Environmental Conservation, working with Cornell Coopera-
tive Extension"s Marine Program, set out to evaluate "spawner
sanctuaries"" as a management method to ensure that reproducing
scallops are present in the system in densities sufficient to increase
the population. Mature scallops (n = 15.000) that had been over
wintered in a creek adjacent to Comelfs hatchery were stocked
into lantern nets at a density of 100 animals per tier and deployed
into Cutchogue Harbor in Peconic Bay. New York. A larval drift
model and diver transects were used to calculate relative contri-
bution of the sanctuaries to the 1999 year class set in Cutchogue
Harbor. Results were further normalized to reflect differences in
gonad weight between wild stock and hatchery-reared scallops.

RESTORING CRITICAL HABITATS IN THE NIGER
DELTA FOR SHELLFISH PRODUCTION. A. C. Ibe' and P.
O. Abohweyere.- 'The Regional Coordination Centre Gef s Large
Marine Ecosystem Project for the Gulf t)f Guinea and "United
Nations Industrial Development Organization, Nigerian Institute
for Oceanography and Marine Research, P.M.B. 12729, Victoria
Island, Lagos, Nigeria.

The Niger Delta is a fan-shaped piece of land located between
504'. 00 and 7040'. 00 longitude and stretching from the Benin
Ri\er in the west to the Bonny River in the east, covering an area
t)f about 16,340 knr. This low lying region, riddled with intricate
water channels through which the river Niger empties into the sea,
consists of three broad ecological zones: freshwater, mangrove,
and the coastal sandridges. The mangrove ecosystems are prime
areas for production, constituting spawning and nursery grounds
for near shore, demersal and pelagic fish species including shell-
fishes. Shellfish of economic importance found in the Niger Delta
include the oyster Cnissastrea gnsar that settles as spat on man-
grove aerial roots at intertidal levels, the periwinkles Tympanoto-
lunts fuscatus and Pachymelia quadriserata. and Penaeid shrimps
that generate over US $195,977.26 from 10,664 MT annually in
foreign exchange. Anthropogenic activities in the form of defor-

Shellfish. Restoration. Hilton Head Island. SC

Abstracts. December 2002 897

estation. sand mining and nourishment, channelization, dredging,
oil and gas exploitation, and rapid urbanization are presently re-
ducing the aerial extent of the mangroves as well as degrading the
mangrove environment. This has an impairing effect on the shell-
fish production potential of critical habitats of the Niger Delta.
This work thus advocates rational use of the mangrove ecosystem
and reforestation of the mangrove swamp where possible such that
one of the goals of the shellfish restoration effort of "restoration or
enhancement of populations of commercially exploited shellfish
depressed by over-harvesting and or reduced environmental qual-
ity" could be achieved in the Niger Delta.

STAKEHOLDER AND CONSENSUS— HOW DO WE
MAKE THESE ELEMENTS WORK FOR EFFECTIVE AC-
TION? M. Janowiez, New Brunswick Department of Environ-
ment and Local Government. 364 Argyle Street. Fredericton. New
Brunswick. E3B 1T9 Canada.

One model for effective stakeholder insohement requires
implementation of a consensus decision-making process. This al-
lows each stakeholder to understand the context within which each
participant is working and requires the development of respect
among the participants. This is an age-old concept but in reality,
few in 21st century North America can easily accept and work
within it. This article will examine why stakeholder in\olvement is
a necessary means to formulate approaches to shellfish restoration
or any other local economic development and planning program. It
will explore the foundations of consensus decision-making includ-
ing examination of concepts of democracy, discussion on Native
North American decision-making processes and other decision-
making models. And. finally, it will identify some methodology
for achieving an effective stakeholder, consensus decision-making
process with the emphasis on developing a common ground of
understanding.

OYSTER BIOMASS AND ABUNDANCE IN NORTHERN
CHESAPEAKE BAY: TRENDS IN RELATIONSHIP TO
HARVEST. RECRUITMENT, PARASITIC DISEASES. AND
ENVIRONMENTAL VARIATION. S. J. Jordan, K. N. Green-
hawk, C. B. McCollough, and M. L. Homer, Maryland Depart-
ment of Natural Resources, Sarbanes Cooperative Oxford Labo-
ratory. 9045 S. Mon-is St.. Oxford. MD 21654.

The Chesapeake Bay Program has committed to a 10-fold in-
crease in the Bay"s oyster population. Oysters are patchily distrib-
uted over about 1,500 km" of the Bay floor. Therefore, it is im-
practical to assess their absolute numbers by direct means. Tradi-
tionally, landings data, with their inherent inaccuracies and biases,
have been the only means of estimating trends in the population.
Maryland's monitoring program records relative numbers and size
distributions of oysters annually at 43 fixed sites. By applying a
length: weight equation to size-frequency data from this fishery-
independent survey, we computed an index of relative biomass that

varied from year to year in response to the relative abundance and
size distribution of the oyster populations. The index reflects in-
terannual variations in recruitment and growth, as well as mortality
caused by the oyster parasites Haplosporidiiiiu iielsoni and Perk-
insiis marinus. An index of market oyster (>72 mm shell height)
biomass had a strong predictive relationship with annual harvests,
but an index of sub-inarket oysters (<72 mm) was not a good
predictor of harvests in subsequent years, probably because of high
and variable rates of natural mortality due to parasitic diseases.
Relative biomass is a sound indicator for measuring progress to-
wards the oyster restoration goal, and has promising applications
in fisheries-related stock assessment.

PUBLIC AND PRIVATE OYSTER RESTORATION IN
MARYLAND'S CHESAPEAKE BAY. C. J. Judy and
E. Campbell, Maryland Department of Natural Resources. Shell-
fish Di\ision. 580 Taylor Avenue. Annapolis. MD 21401.

Oyster restoration is a shared venture between government and
the private sector. Maryland oyster projects through the 1990s
have been a collaboration between state, federal, and private
groups. Projects have focused on the creation of oyster sanctuaries
to protect broodstock and enhance benthic community diversity,
restoration of habitat; and planting of seed oysters, primarily from
hatcheries. In round numbers, the acreages for a cooperative proj-
ect range from a few to over 10 and the number of oysters planted
in a year range from about 10 million to over 50 million total. A
wide range of participants constitutes the private sector: environ-
mental groups, non-profit oyster restoration groups, community
groups, private citizens, watermen and school groups. The number
of projects by such groups has risen dramatically since the eariy
1990s and encompasses types of projects not normally conducted
by State agencies alone. Other projects are more uniquely govern-
mental. The long standing Maryland Department of Natural Re-
source's seed and shell programs plant about 400 acres of seed and
800 acres of shell per year. The number of oysters planted as seed
range between 120 million to over 800 million per year. These
projects mostly produce market oysters, but environmental and
broodstock benefits accrue from such mass plantings. The Feder-
ally funded Reef Program conducted by the State restores oyster
populations in sanctuaries using shell and seed resources. To-
gether, public and private entities are working toward improving
oyster habitat and oyster populations to improve the industry and
the ecological role of oysters.

MUSSEL CULTURE AND COCKLE FISHERIES IN THE
NETHERLANDS: FINDING A BALANCE BETWEEN
ECONOMY AND ECOLOGY. P, Kamermans and A, C.
Smaal, Netheriands Institute For Fisheries Research. Centre for
Shellfish Research. P.O. Box 77. 4400 Ab Yerseke. The Nether-
lands.

In the Netherlands, mussel seed is fi.shed in a coastal sea in the
North of the country (Wadden Sea) and cultured in an estuary the

898 Abstracls. December 2002

Shellfish, Restoration, Hilton Head Island, SC

South (Oosterschelde). Dredging for cockles takes place in the
Wadden Sea, and two estuaries in the South (Oosterschelde and
Western Scheldt). The Wadden Sea and the Oosterschelde are
nature reserves where human activities are possible only when
they do not cause negative effects. In 1993. a policy was formu-
lated to ensure preservation of bird populations and restoration of
mussel banks and seagrass meadows. As a result of this policy,
fishing for mussel seed and cockles is not allowed in areas with a
high potential for the development of mussel banks and seagrass
fields. The location of these areas is based on CIS models. All
vessels are equipped with a black box to control the closed areas.
There is some debate about the closure because fishermen have the
impression that fishing improves the sediment for settling of mus-
sel larvae. Consumption-sized cockles and mussels are also the
prefeiTed prey of oystercalchers and eider ducks. Therefore, the
policy makes use of a quotum system in the cockle fisheries. Each
year, basin-wide surveys take place to determine the total amount
of cockles present. In years when cockle stocks are low an amount
is reserved for the birds. Both fishermen and environmentalist
question the calculated amounts needed by the birds. An overview
of the viewpoints of the interest groups and the role of policy
makers and scientists is given.

AN ECONOMIC ANALYSIS OF PUBLIC GROUND OYS-
TER REEF RESTORATION IN CENTRAL LOUISIANA
DAMAGED BY HURRICANE ANDREW. R. J. Dugas,' W. R.
Keithly," M. Bourgeois/ P. Meier,' D. Lavergne."* and A. Di-
agne,'* 'Louisiana Department of Wildlife and Fisheries, Marine
Fisheries Division, 1600 Canal Street, New Orleans, LA 701 12;
'Louisiana State University. Coastal Fisheries Institute, Wetland
Resources Building, Baton Rouge. LA 70803-7503: 'Louisiana
Department of Wildlife and Fisheries, Marine Fisheries Division,
1600 Canal Street. New Orieans, LA 70112; and ^Louisiana De-
paitment of Wildlife and Fisheries, Socioeconomic Division, P.O.
Box 98000, Baton Rouge. LA 70898-9000.

In August 1992, Hurricane Andrew heavily damaged Louisiana
coastal environments, particularly oyster, Crassostrea virginica.
reef communities. The transport and transfer of tremendous
amounts of sediment and vegetative matter resulted in massive
oyster mortalities and extensive reef damage. The Louisiana De-
partment of Wildlife and Fisheries received $5.\ million of federal
funds for restoration of oyster habitats on both Louisiana public
and private oyster grounds. Of these funds, $3.2 million were used
in Terrebonne Parish, the area most severely impacted. Restoration
efforts were comprised of sweeping buried reefs and depositing
cultch material for oyster reef construction. Some 1,780 acres of
waterbottoms were swept by commercial oyster harvesters with
bag-less oyster dredges. Mined oyster shells/clam, Rangia. shell
mixture were deposited (at a rate of approximately 1 32 cubic yards
per acre) on 306 acres of waterbottoms in 1994 (42,576 cubic
yards) and 553 acres in 1995 (70,902 cubic yards). Economic

benefits associated with a restoration effort of this nature accrue to
both oyster consumers and oyster producers. To consumers, the
benefits reflect a reduction in price paid for the harvested product,
which in tuni translates to an Increase in willingness to pay relative
to what was paid (i.e., consumer surplus). To producers, the ben-
efits reflect an increase In returns to the scarce resource, oyster
population, used in the production process (i.e.. producer surplus).
This study provides an estimate of benefits derived from the res-
toration efforts and compares these benefits to costs. Overall, the
results indicate a favorable benefit to cost ratio.

THE REROUTING OF STORMWATER DISCHARGES
FOR WETLANDS ENHANCEMENT, LEVEE PROTEC-
TION, AND OYSTER HABITAT PROTECTION AND RES-
TORATION. K. E. Landrum,' K. M. St. Pe,' B. Ache,- and F.
Kopfler,'' 'Barataria-Terrebonne National Estuary Program. P.O.
Box 2663, Nicholls State University. Thibodaux, LA 70310; "Bat-
telle, 191 East Broad Street, Suite 315, Athens, GA 30601; and
'Epa/Gulf Of Mexico Program, Stennis Space Center, Building
1103, Room 202, MS 39529-6000.

The Barataria-Terrebonne estuary is losing over 22 square
miles of emergent wetlands each year because of erosion, saltwater
intrusion, and natural and anthropogenically-induced subsidence.
An extensive levee system has successfully halted overbank flood-
ing of the Mississippi River, eliminating sustaining inputs of sedi-
ments and freshwater to the Barataria-Terrebonne estuary. This
situation represents not only the imminent loss of a nationally
significant wetland resource but also threatens a unique culture,
local infrastructure, and the region's significant contribution to the
national economy. Runoff from rural and agricultural areas is col-
lected in a borrow canal inside the back levee and then pumped
into adjacent wetland areas by a series of stormwater pump sta-
tions. Over 250 pump stations currently discharge stormwater.
draining approximately 500,000 acres, in the Barataria-Terrebonne
estuary. These pump discharges are generally directed into large,
human-made canals to ensure that stormwater is quickly evacuated
from the leveed area and they often flow directly to high-salinity
bays through some of Louisiana's prime oyster growing waters.
Redirecting discharges so that they are retained in adjacent wet-
lands may maintain lower local salinities, provide a sediment
source to subsiding wetland areas, and support plant growth, di-
rectly benefiting the degrading wetland systems, especially those
directly seaward of levees that protect property from storm surges
and flooding. Retention of storm water may also produce corollary
water quality benefits, such as nutrient uptake and pathogen die-off
prior to encountering oyster-growing areas. The Barataria-
Terrebonne National Estuary Program is leading an effort to moni-
tor changes at pump station sites in the estuary to demonstrate the
benefits of this unique process.

Shellfish. Restoration. Hihon Head Island, SC

Abstracis, December 2002 899

THE SHELLFISH CHALLENGE INITIATIVE: A COOP-
ERATIVE SUCCESS STORY IN THE BARATARIA-
TERREBONNE NATIONAL ESTUARY. K. E. Landrum.

Barataria-Terrebonne National Estuary Program. P.O. Box 2663.
NichoUs State University, Thibodaux, LA 70310.

The Shellfish Challenge Initiative is an interagency and inter-
state effort undertaken to establish progress on the Environmental
Protection Agency's Gulf of Mexico Program Shellfish Challenge.
With an overall goal of increasing Gulf shellfish beds available for
safe harvest by ten percent, more than 200 experts in shellfish
management, habitat restoration, and pollution control helped de-
velop 32 shellfish restoration strategies targeting 24 watersheds in
the Gulf of Mexico. A watershed implementation initiative was
developed within the Barataria-Terrebonne National Estuary re-
sulting in the identification of 61 oyster restoration opportunities,
including geographically targeted projects to reduce inputs of fecal
coliform bacteria, enhance shellfish habitat, revise shellfish man-
agement procedures, and collect and analyze additional needed
information to better assess project feasibility. The 61 candidate
restoration projects were ranked by members of the Barataria-
Terrebonne National Estuary Program Management Conference,
and detailed implementation plans were developed for the four
selected priority projects. The priority projects included the fol-
lowing: the installation and improved use of marina pumpouts and
dump stations; connecting poorly operating individual wastewater
treatment systems to community level treatment systems; rerouting
stormwater runoff to suitable wetlands; and revising the shellfish
relay system. Implementation of the four projects is underway with
active educational and interacfive workshop components designed
for state and local officials and the general public. Funding allo-
cations by local and state government attest to their involvement
and acceptance of the implementation process and their agreement
to promote active stewardship of an economically important re-
source and conservation principal.

OYSTER POPULATION RESTORATION IN CARAQUET,
N.B.; PHASE I, POPULATION ASSESSMENT. T. Landry.
M. Ouellette, and P. Cormier, Department of Fisheries and
Oceans, GFC. P.O. Box 5030. Moncton N.B., ElA 4Y1 and De-
partment of Agriculture, Fisheries and Aquaculture. 22 Boul.
Saint-Pierre. Caraquet, N.B. EIW 1B6. Canada.

A decrease in the productivity of oysters in Caraquet Bay. N.B.
is generating some interest in restoration projects. The first phase
of this initiative is to conduct a quantitative assessment of the
distribution, abundance and population structure of the natural
beds in this bay. which represents the most northern location with
a sustainable oyster (Crassostrea virginica) population. The results
from 1999 assessment is the fifth of a series of similar exercises
conducted in 1974, 1979,1987, and 1991 but the first to use a
geostatistical approach to data analysis. A comparison between the
two assessment methods reveals that the geostatistical approach is
more accurate and of greater use for the next phase of this project,

w hich will look at identifying and characterizing the suitable oys-
ter habitat of this bay for restoration efforts. The comparison
among the five assessments over the past three decades is showing
that the status of this population is approaching a critical state in
tenns of recruitment and habitat quality and quantity. The resto-
ration of this oyster population is of great socio-economical and
ecological importance to this area.

COMMUNITY-BASED INITIATIVES FOR IMPROVING
WATER QUALITY IN SOUTHWESTERN NEW BRUNS-
WICK, CANADA— AN UPDATE ON SUCCESS. K. L.
LeBlanc, Eastern Chariotte Waterway Inc.. 17 Main Street. St.
George, New Brunswick, E5c 3h9. Canada.

The Southwestern New Brunswick Clam Resource Committee
(CRC) was formed in 1997 to better understand the importance of
the soft-shell clam iMya areiiaiUO fishery and to prevent further
loss of shellfish growing areas due to monitoring cutbacks in
Southwestern New Brunswick (NB). Soft-shell clams are the main
molluscan shellfish harvested in the region. The committee has
over 20 stakeholders that include nonprofit groups, industry and
government and is chaired by Eastern Chariotte Waterways Inc.
(ECW. a nonprofit group). The mandate of the committee is to
preserve the clam fishery in Southwestern NB because of its long-
term socio-economic importance to the region. In 1998. the CRC
developed the Cooperative Bacterial Monitoring Program
(CBMP), which allows industry and community-based contribu-
tions for the maintenance and improvement of the classification of
growing areas. Under this community-based initiative coastal wa-
ter samples are collected over a 1,500-km length of shoreline over
a two-year period. Presently, 67% of the growing areas are avail-
able for the harvest of clams in Southwestern NB during all or
selected times of the year, an increase of 32% over 3 years. How-
ever, it is important to note that clean-up efforts in growing areas
coupled with the CBMP are responsible for the improvements of
water quality that have permitted an increase in soft-shell clam
harvest. The CRC coordinates clean-up efforts through the Fundy
Flats Remediations plan, a program managed under ECW.

APPLICATION OF COMMERCIAL-SCALE OYSTER
AQUACULTURE TO REEF RESTORATION. A. T. Leggett.
R. Brumbaugh, W. Goldsborough, and A. McDonald, Chesa-
peake Bay Foundation. 142 W. York Skeet. Suite 318, Norfolk,
VA 23510.

Oyster reef restoration projects in the Chesapeake Bay increas-
ingly involve the addition of broodstock to enhance localized oys-
ter spawning activity. Since 1996. more than 4 million adult oys-
ters have been transplanted onto sanctuary reefs in Maryland and
Virginia waters. Volunteers and school students have grown and
transplanted a significant number of hatchery-produced oysters, in
collaboration with state management agencies. In an effort to in-
crease the numbers of hatchery-produced oysters being trans-
planted onto sanctuary reefs, the Chesapeake Bay Foundation has

900 Abstracts. December 2002

Shellfish, Restoration, Hilton Head Island. SC

initiated a commercial-scale grow out operation in the lower York
River with a annual production goal of I million adult oysters.
Oysters produced by this program will approximately double the
number of oysters available for transplanting onto sanctuary reefs
in the lower Chesapeake Bay each year. A new cage system was
developed in accordance with existing regulations in Virginia gov-
erning shellfish aquaculture. and was used to maximize the number
of oysters produced over a relatively small acreage of leased oyster
grounds. Data on growth and mortality as well as water quality
parameters are collected as a routine part of the operation. Early
monitoring results showed a 78% increase in size (measured volu-
metrically) of -1-8 mm seed, and a 63% increase in 8-12 mm seed
one week after deployment in mid-July. The oysters produced by
the operation will not only be used to enhance broodstock popu-
lations on sanctuary reefs, but will also be used as "natural capital"
to enticing further public and private investment in oyster resto-
ration.

analysis of Gulf coast oysters, clams and shrimp has shown stimu-
lation of a unique anti-molluscan protein that varies in MW 4 to 22
kd and has anti-V'//)/7V) viiliuficus activity. When the protein and
phage are used together, eradication of Vibrio vidiiificus occurs to
more than 8 logs. Analysis by Edman degradation of the 22-kd
oyster protein revealed a unique N-terminal 16 amino acid frag-
ment, as did analysis of two cyanogen bromide gel purified frag-
ments. The proteins were not detected in Japanese or Olympia
(Washington) oysters (kindly provided by Dr. C. Kaysner. FDA I.
Finally, a new rapid assay to study the effect of temperature and
brief bacterial exposure has been developed, suggesting the pos-
sibility that Vibrio viiliiificiis could undergo a non-culturable state
under certain conditions (K. Johnston, pers. comm.). Our intention
is to isolate the genes expressing the 4- and 22-kd proteins; then
express them in large amounts to use with the specific phage in a
biodepuration procedure. This work was supported by SK Grant
#NA97FD0062 to RBL from NOAA.

DOLLARS AND SENSE OF OYSTER RESTORATION: AN
EXAMINATION OF NITROGEN REMOVAL BY A RE-
STORED OYSTER REEF. M. Luckenbach.' F. O'Beirn,' P.

Ross,' J. Nestlerode.- and L. Sorabella." 'Virginia Institute of
Marine Science, College of William and Mary. Wachapreague.
VA 23480 and "Virginia Institute of Marine Science. College of
William And Mary. Gloucester Point. VA 23062.

Arguments for the conservation and restoration of oyster reefs,
often at the expense of fisheries exploitation, include water quality
benefits derived from feeding activities of the oysters and reef-
associated fauna. Yet, there has been limited basis for directly
evaluating the water quality improvements associated with con-
servation or restoration of oyster reefs and for comparing those
benefits to the economic value derived from oyster fishery pro-
duction. Using data from oyster populations developing on experi-
mental reefs near the mouth of Chesapeake Bay. we model the
nitrogen uptake and release attributable to the oysters and develop
nitrogen budgets for the reefs on an area-normali/ed basis. We
then explore the potential effects of fisheries exploitation of these
reefs by modeling the harvest of market-sized oysters and exam-
ining the effects on nitrogen removal. Finally, we consider the
economic returns from oyster harvesting in relation to the costs
associated with alternative nitrogen removal. The results give con-
text to water quality benefits to be derived from oyster reef sanc-
tuaries and should help to guide fisheries management decisions
related to balancing conservation and exploitation.

REDUCTION IN THE VIBRIO VULNIFICUS LOAD OF
OYSTERS BY A NOVEL SHORT-TERM COMBINATION
BIODEPURATION TREATMENT. R. B. Luftig and W.
Pelon, Department of Microbiology, LSU Health Sciences Center.
New Orleans. LA 701 12-1393.

Based upon an improved method. Vibrio vulnificus phage can
be maintained and stored at high titer. Further, mass spectroscopic

COMING SOON TO A RESTORATION SITE NEAR YOU:
THE INVADING. PREDATORS ORIENTAL GASTROPOD
RAPANA VENOSA. R. Mann and J. M. Harding. School of
Marine Science. Virginia Institute of Marine Science. College of
William and Mary. Gloucester Point. VA 23062.

Rupana venosa Valenciennes 1846 ( Neogastropoda. formerly
Muricidae. currently Thaididae) is a predatory gastropod native to
the Sea of Japan. Yellow Sea. East China Sea. Bohai Sea. and
Taiwan. The species has been introduced to the Black Sea. Adri-
atic Sea. and Aegean Sea. where it is generally considered to be
responsible for decimation of local commercially valuable mollusc
species. It was first reported in the Chesapeake Bay in 1998. Bal-
last water transport of larval stages from the eastern Meditcrtanean
or Black Sea is the suspected vector of introduction. To date over
1200 specimens of adult Rapaini have been collected from Hamp-
ton Roads and a limited region of the Southern Chesapeake Bay.
Population demographics, records of Rapana egg cases in the field
and our ability to culture early life history stages at prevailing
temperature and salinity strongly suggest active breeding in this
receptor location. Temperature and salinity tolerance data for Ra-
panii suggest that it can both invade the higher salinity regions of
most East Coast estuaries and survive on exposed shorelines from
Cape Cod, MA to Charleston. SC. Dispersal is facilitated by pe-
lagic development, and may be exacerbated by ballast water trans-
port of larval stages originating in Hampton Roads. Hard substrate
habitat, typical of many curtent shellfish restoration efforts, ap-
pears optimal for post settlement stages, but larger adults may
invade soft sediments. Predation has been demonstrated on a range
of commercially valuable shellfish species including Mercenaria
incrceiiaria. Crassostrca viraiuica. Myii urcnaria and Mytiliis edu-
lis.

Shellfish. Restoration. HiUon Head Island. SC

Abslracts. December 2002 901

UP CLOSE AND PERSONAL: A SUGGESTED QUANTITA-
TIVE APPROACH TO BROODSTOCK ENHANCEMENT
ON SHELLFISH RESTORATION SITES. R. Mann, School of
Manne Science. Virginia Institute of Marine Science. College of
William and Mary. Gloucester Point. VA 2.^062.

Shellfish broodstock are typically added to restored habitat to
facilitate rapid recruitment by aggregating spawning adults and
thus increasing fertilization efficiency. Although this is conceptu-
ally attractive there exist few data on which to build quantitative
guidelines to optimize the practice. For example, published size
versus fecundity relationships for oysters are based on data that has
both methodological and size limitations. Similarly, fertilization
models are based on sea urchin studies from flow regimes that are
arguably quite different from shellfish restoration sites. A quanti-
tative approach is proposed wherein a variety of size-fecundity and
fertilization models are proposed for examination in building
guidelines to optimize both size and density of placement of shell-
fish used in broodstock enhancement. The biological and eco-
nomic aspects of these alternatives are compared.

LINKING PUBLIC AND PRIVATE PARTNERS FOR RES-
TORATION AQUACULTURE IN MARYLAND'S SEASIDE
BAYS. D. W. Webster' and D. W. Meritt,^ 'University of Mary-
land. Wye Research & Education Center. PC Box 169. Queen-
stown MD 21658 and "Shellfish Aquaculture Specialist, Univer-
sity of Maryland Center for Environmental Science. Horn Point
Lab. Po Box 775, Cambridge, MD 21613.

Traditional harvest of the bard clam (Menenaria ineicenaria)
in Maryland's seaside bays utilizes the hydraulic escalator dredge.
This method has raised concern among environmental groups due
to its perceived impact upon eel grass [Zostera spp.) in shallow
estuarine waters. Meanwhile, an increase in demand for hard clams
and strong wholesale prices have caused many clammers who
normally harvest softshell clams (Mya arenaria) in the Chesa-
peake Bay to shift harvest to the seaside bays, placing increasing
pressure on that area. Hard clam aquaculture is well known and
may provide an alternative to harvesters, who will likely come
under increasingly restrictive regulations. Differences in culture
methods for the clam have evolved in many states to take advan-
tage of local conditions. In Maryland, the clam aquaculture indus-
try has only recently begun, with few persons currently investing
in the technology to produce these animals through husbandry.
During 1998, investigations were begun with cooperation from
university and private companies to develop seed production and
evaluate grow out techniques. The Maryland Industrial Partnership
(MIPS) program has funded development of a hard clam nursery/
grow out operation. University of Maryland Sea Grant Program
(UMSG) funded a survey of the hard clam disease QPX to assess
background levels of this potential problem. University of Mary-
land Cooperative Extension (UMCE) funded an extension project
to assess grow out techniques, as well as conduct outreach educa-

tional programs designed to bring the technology to those who can
use it. These cooperative studies are described as well as future
directions for the project.

PRODUCTION OF DISEASE-FREE OYSTER SEED USING
SHALLOW WATER NURSERIES IN THE MID-
CHESAPEAKE BAY. D. W. Meritt and S. Tobash, University
of Maryland, Center for Environmental Science. Horn Point Labo-
ratory, Po Box 775, Cambridge. MD 2161.^.

Recently in Maryland, there has been an emphasis on the pro-
duction of disease-free oyster spat for use in oyster restoration.
Spat produced using traditional methods utilize sites where oyster
parasites are common and are typically infected at the time of relay
to the grow-out site. As part of the Action Plan for Oyster Recov-
ery in Maryland, the Maryland Oyster Roundtable established oys-
ter recovery zones in several major tributaries of Chesapeake Bay
into which only disease-free oysters can be introduced. Given the
problem with producing disease-free oyster .seed using natural
methods, hatcheries have been employed for disease-free seed pro-
duction. Since 1994, the University of Maryland's Horn Point
hatchery has produced over 90 broods of spat using in-water nurs-
ery systems. Dermo, the disease caused by the parasite Perkinsus
marinus, is of greatest concern in these systems due to low salini-
ties. Only one brood of spat has tested positive for Dermo since
1994. Based on trials conducted over the past six years, we have
demonstrated that it is not only possible but also likely that unin-
fected oyster seed can be produced using hatcheries and shallow-
water nursery systems. Disease-free seed are being used to test the
idea that by prohibiting the movement of parasites into upstream
portions of the oyster producing rivers, dermo will be naturally
purged from oyster populations in those regions. Early data sug-
gest that there is some validity to this concept.

A MULTIFACTORIAL APPROACH FOR DESCRIBING
THE RELATIONSHIP BETWEEN THE CLASSIFICATION
OF SHELLFISH HARVESTING WATERS AND ADJA-
CENT LAND USE IN MURRELLS AND NORTH INLET,
SOUTH CAROLINA. K. A. Nelson and G. I. Scott. National
Ocean Service. 218 Fort Johnson Road. Charieston. SC 29412.

Urbanization poses a particular threat to the coastal areas of the
southeastern United State, where the lands surrounding the wet-
lands are still relatively undeveloped compared with other regions.
Fecal coliforms. including Escherichia coli. are important indica-
tors of public health since human and/or animal feces may come in
contact with and contaminate drinking water supplies or filter-
feeding shellfish. The measurement of the concentration of fecal
coliforms is the current criterion for deciding when and if shellfish
harvesting should be approved. Predictive models that would cor-
relate information on land use change and development would be
useful so that downgrades in water quality can be predicted before

902 Ahslrculs. December 2002

Shellfish. Restoration. Hilton Head Island. SC

they occur. The approach used for this study involved an historical
comparison of land use change and fecal coliforni bacterial den-
sities on Murrells Inlet (Ml) (urbanized site) and North Inlet (Nl)
(pristine site). Both MI and NI are bar-built estuaries are located on
the northern coast of South Carolina near Myrtle Beach. The mi-
crobiological and water quality data used in this research covers
the period of 1967-1995 and the following parameters were uti-
lized: date of sampling, most probable number of fecal coliforni
bacteria, salinity, rainfall, and water temperature. The regressions
models utilized the above parameters and a change in trend term
that accounted for both instantaneous and gradual changes in water
quality that may arise from a particular intervention. For MI, the
1980 intervention consisted of both the construction of a jetty and
the conversion from septic tanks to a main sewer line. For NI, the
1973 intervention was the construction of Baruch Laboratory. For
MI, the intervention, controlling for other parameters, was found to
be significant at the alpha = 0.05 level. This means that there was
a significant decrease in the increasing trend of bacteria for MI and
that the conversion to the sewage line had a beneficial effect on
water quality and probably dominated the jetty effect. For Nl, the
laboratory construction had no impact on water quality so back-
ground natural sources of bacteria probably dominated the small
increase from human sources. These findings indicate that the use
of Intervention Analysis may provide coastal managers with an
effective process to evaluate landscape changes on bacterial water
quality in shellfish harvesting areas.

INFLUENCE OF OYSTER REEF STRUCTURE ON FISH
ASSEMBLAGES: DOES THE PLACEMENT OF ARTIFI-
CIAL SUBSTRATE ENHANCE FISH POPULATIONS? J. A.
Nestlerode.' M. W. Luckenbach,- P. G. Ross,^ and F. X.
O'Beirn," 'Virginia Institute of Marine Science, College of Wil-
liam & Mary, Gloucester Point, VA 2.3062, "Virginia Institute of
Marine Science, College of William & Mary, Wachapreague, VA
23480.

The objectives of oyster reef habitat restoration within Chesa-
peake Bay are not only the enhancement of oyster stocks but also
to restore the physical structure and ecological function of these
systems. We revisit the artificial reef fisheries attraction vs. pro-
duction issue by comparing transient nekton community structure
among reef structures constructed of different substrates. The sub-
strate materials (oyster shell, surf clam shell, and pelletized coal
ash) used in this study offer the opportunity to examine how habi-
tats with various degrees of structural complexity contribute to
differences in habitat u.se in terms of microhabitat availability,
predation risk, and diversity and abundance of prey. Results indi-
cate that oyster shell reefs, which have best supported the devel-
opment of an oyster population, offer the highest degree of struc-
tural complexity and support a more diverse community of both
resident and transient nekton. Furthermore, increased availability
of nesting sites (empty articulated oyster shells) and a high abun-

dance of benthic prey items support the thesis of increased fish
production rather than simply attracting t"ish to the reef structure
from nearby habitats. The patterns observed here provide evidence
that proper reef architecture and subsequent reef community de-
velopment lead to increa.sed finfish production and should give
context to the importance of substrate selection in similar restora-
tion activities.

SHELLFISH RESTORATION IN IRELAND: THE NEED
FOR NOVEL PARTNERSHIPS. M. Norman, Taighde Mara
Teoranta (Marine Research Limited), Cama Co.. Galway Ireland.
Historically shellfish restoration projects in Ireland have been
undertaken for a singular reason and driven by a single user group.
Frequently, the goal has been increased commercial production of
shellfish, and the restoration has been pursued by a grouping of
fishermen or aquaculturists. The.se projects have a narrowly de-
fined objective, profitability, and frequently "fail" when they do
not achieve this. However, some recent restoration attempts have
been performed by partnerships. These projects are driven by mul-
tiple user groups and thus have a range of goals. It is postulated
that this partnership approach has more chance of success as the
partners support each other in "staying the course" and as the goals
are broader, makinc success more achievable.

REPRODUCTIVE BIOLOGY OF THE NORTHERN QUA-
HAUG, MERCENARIA MERCENARIA, IN PRINCE ED-
WARD ISLAND, CANADA. M. Ouellette, M. Hardy, T. Lan-
dry, N. G. MacNair, and A. Boghen, Department of Fisheries and
Oceans, Science Branch, Gulf Fisheries Centre, P.O. Box 5030,
Moncton. New Brunswick, EIC 9B6; Department De Biologie,
Universite De Moncton, Moncton. Nouveau-Brunswick, ElA 3E9;
and Department of Fisheries and Tourism, PC Box 2000, Char-
lottetown. Prince Edward Island. CIA 7N8. Canada.

The northern quahaug, Mercenuria mercenaria. is an important
species for both the commercial and recreational fisheries as well
as for aquaculture purposes in Prince Edward Island. The manage-
ment strategy of the quahaug resource is largely based on the
minimum legal size of 50 mm. At the same time, there is a growing
concern regarding the sustainability of the clam industry and
hence, an evolving interest in stock enhancement. Effective brood-
stock management, however, requires basic information about the
animal's reproductive biology. Sexual maturity, ovocyte size, go-
nado-somatic ratios, and time of spawning were established for
quahaugs sampled from two sites in West River, PEL Histological
methods and physiological condition indices are used to determine
of the spawning activities. Findings revealed that the minimum
size at sexual maturity was 25 mm and 30 mm (shell length) for
males and females respectively. Furthermore, there was a positive
correlation between ovocyte size and shell length. Seasonal varia-
tion coincided with spawning predictions based on conventional

Shellfish. Restoration. Hilton Head Island. SC

Ahsuactx. December 2002

903

physiological condition indices. As well, the gonado-somatic con-
trihiition increased as a function of length. Both histological and
condition index data support the likelihood that a major spawn
occurs in mid-June. The study provides useful information on the
reproductive biology of M. meixenaria and could contribute to-
wards a reassessment of existing management and grow-out strat-
egies. Establishment of reproductive sanctuaries is also being in-
vestigated as a method to increase the annual recruitment success
in this study bay.

CONSIDERATIONS FOR OYSTER RESTORATION IN
MARYLAND: DISEASE, GENETICS, DENSITY. REPRO-
DUCTION, AND HABITAT CREATION. K. R. Paynter. .Jr..

Department of Biology. University of .Maryland. College Park.
MD 20742.

Over the last 2 years, we have conducted numerous experi-
ments and monitored several State and Federally funded restora-
tion projects in the Maryland portion of Chesapeake Bay. A sum-
mary of the results of these activities will be presented. Field
experiments have revealed that oyster seed cohorts from different
broodstocks appear to have differing resistances to disease. Video-
graphic observations from the field have shown that high-density
oyster plantings result in significant community enhancement
leading to diverse benthic ecosystems. Laboratory studies have
shown that benthic fishes such as gobies and blennies prefer natu-
ral clumps of oyster shell compared to equal volumes of loose
oyster shell. In addition, other laboratory studies have shown that
eggs introduced into the water column more than a few centimeters
from introduced sperm will have little chance of becoming fertil-
ized. However, other studies have shown that high densities of
oysters (>400/m") may result in deleterious effects on oysters
themselves. Many aspects of oysters and their ecosystem must be
considered when planning restoration projects. Those projects
seeking to restore ecological function should bear in mind the
complex relationships between oysters, the habitat they create as
biogenic reef builders, and the water column in which they reside.

CULTURE TECHNIQUES APPLIED TO WILD BIVALVE
BEDS IN GALICIA, NW SPAIN. P. J. Pazo, Delegacion Ter
ritorial Conselleria De Pesca CI Palma 4. 36202 Vigo Spain.

The region of Galicia is located in NW Spain. It has a coastline
of 1.195 km. Galicia is the first producer of molluscs in Spain,
taking advantage of natural oceanographic conditions: a seasonal
upwelling and existence of positive estuary bays (Rias). Molluscs
have been exploited in Galicia since prehistoric times. The present
shellfishing situation is developed in two ways: the gathering of
molluscs on foot, raking the substratum for macroinfaunal bivalves
in the intertidal belt. The other type of shellfishing exploits the
subtidal molluscs beds and involves the use of small boats. In order
to maintain and enhance bivalve production in intertidal wild beds.

a series of culture techniques are applied, acting on the bivalve
population (lowering high densities, enlarging area beds, sowing
and repopulating new areas), fighting against predators and com-
petitors (starfishes, drilling gastropods), removing green algae of
the bed surface to avoid deleterious effects in young bivalves,
changing substrate granulometry by adding coarse sand to areas
with mud and silt condition, and by other means. To act on the
recruitment problems of two very valued species: butterfish clam
{Riulitapes decussata) and European flat oyster (Ostrea edulis), a
plan was established by the Fisheries Department of Galician Re-
gional Government: "Plan Galicia". This Plan began in 1997 and
was aimed to gradually transform traditional intertidal shellfish
gathering into a professional activity by enhancing both the inter-
tidal wild molluscan beds and the social organization of the mainly
female population of shellfish gatherers. This Plan is presently
developing and relevant achievements are being achieved, mainly
in the social area.

THE INFLUENCE OF ENVIRONMENTAL FACTORS IN
.JAPANESE OYSTERS HEALTH CONDITION CULTI-
VATED IN THE SOUTH OF PORTUGAL. A. L. Pereira,' F.
A. Ruano.' L. Chicharo." and D. Matias.' 'IPIMAR, Reseaich
Institute for Fisheries and Sea, Av. De Brasilia 1449-006 Lisbon,
Portugal; "UCTRA. Algarve University, Campus De Gambelas,
8000-062 Faro, Portugal: and 'CRIPsul, South Research Center of
Ipimar, Av. 5 De Outubro, 8500 Olhao, Portugal.

The effect of environmental parameters as well as the organic
contamination, heavy metals and tribulytlin (TBT) upon the de-
velopment of diseases in cultivated Japanese oysters Crassostrea
gigas (Thunberg, 1793) was studied over 6 months. The results
were also related with the condition index, growth and mortality
rates. The study was performed in two different sites on a coastal
lagoon in the South of Portugal. One site ("Elisatnar") is located in
a clean area whereas the other (Olhao). due to its proximity to
urban areas and to an important fishing harbour, is exposed to
higher contamination levels. In Olhao. nosological examinations
showed the higher levels of lesions and the greatest incidence of
parasites. Two ciliates. Ancistniin sp. and Tiichodina sp.. were the
most abundant at the two areas. The intensity of the infections and
the lesional picture observed at the two sites, didn't seem to affect
significantly the condition, growth and mortality of the studied
animals. However, the differences in the infection intensity regis-
tered in both sites could be an indicator of a lack of defensive
response from the individuals submitted to more intense stressful
conditions.

THE "EEEOHM" (EASTFIELDS' ENVIRONMENT EN-
HANCING OYSTER HOLDING MODULE). P. Perina and D.
Perina, Eastfields Farms, Box 275, Mathews, VA 23109.

The "EEEOHM" (Eastfields' Environment Enhancing Oyster
Holding Module) was originally developed to be used commer-

904 Abstracts. December 2002

Shellfish. Restoration. Hihon Head Island. SC

cially, but the system is extremely versatile and is adaptable to a
small garden size operation. The emphasis in developing the
EEEOHM was on "Keep It Simple. Keep It Cheap." The
EEEOHM module consists of three ADPI square-sided oyster
cages (also called bags). Each cage has attached four 2-liter soda
bottles for flotation. The cages are strung together with a 13-foot
piece of crab pot rope or clothes line running laterally through their
centers. The modules can also be separated into single floats as the
needs of the grower dictate. The reason for using just three cages
per module is simply "ease of handling". Whether employing the
system from a dock or a skiff, it's a lot easier to detach and lift just
three cages at a time than to struggle with maybe 40 or more cages
all attached to a single rope. No heavy lifting. The EEEOHM can
be floated under or along side of a dock, tied between posts, or
deployed in rows secured to two parallel ropes anchored to the
bottom at each end. The latter usually requires the use of a skiff.
During the last 14 years, we at Eastfields have tried many ways of
growing oysters. We've found the EEEOHM to be one of the most
efficient and cost effective systems of off bottom culture. The
ADPI cages last for many years, and the soda bottles are free. We
at Eastfields Farms are proud to have developed the EEEOHM and
would appreciate the opportunity to answer any questions concern-
ing this environment enhancing system.

A BIOCHEMICALLY-BASED MODEL OF THE GROWTH
AND DEVELOPMENT OF CRASSOSTREA GIGAS LAR-
VAE. E. A. Bochenek,' E. N. Powell,' J. M. Klinck." and E. E.
Hofmann,' 'Haskin Shellfish Research Laboratory. Rutgers Uni-
versity. Port Norris, NJ 08349 and "CCPO. Old Dominion Uni-
versity. Norfolk, VA 23529.

A biochemically-based model was developed to simulate the
growth, development, and metamorphosis of larvae of the Pacific
oyster. Crassostrea iiii;(is. This model, which is the first of its type,
defines larvae in terms of their gross biochemical compositiim;
protein, neutral lipid, polar lipid, carbohydrate, and ash content.
The model includes parameterizations for larval filtration, inges-
tion, and respiration, which determine growth rate, and processes
controlling larval mortality and metamorphosis. Changes in the
initial ratios of protein, carbohydrate, neutral lipid, and polar lipid
occur as the larva grows and in response to the biochemical com-
position of available food. The model results show increased larval
survival when low protein food sources are available. High-protein
food sources result in insufficient carbohydrate and neutral lipid to
cover metabolic costs and to permit successful metamorphosis.
The result is lar\ ae that are unable to successfully complete meta-
morphosis. Thus, food quality as well as food quantity appear to be
primary controls on the ability of Crassostrea gigas larvae to reach
the body condition needed for metamorphosis. Other simulations
show that initial egg size (lipid content) controls the ability of the
larva to sustain itself until it reaches a size where it can effectively
filter and assimilate food. Large eggs produce larvae that are more

able to withstand food-poor environments, suggesting that egg size
variability may account for the range of larval sizes at which
metamorphosis is attempted.

A FISHERIES MODEL FOR MANAGING THE OYSTER
FISHERY DURING TIMES OF DISEASE. J. M. Klinck.' E.

N. Powell.- J. N. Kraeuter,- and S. E. Ford," 'CCPO. Old Do-
minion University, Norfolk, Virginia 23529 and "Haskin Shellfish
Research Laboratory, Rutgers University, Port Norris, NJ 08349.
In Delaware Bay. market-size oysters have been produced by
moving oysters from the seedbeds to planted grounds in higher
salinity, where oysters increased in size and meat condition. In
1989. the oyster disease Dermo became active on the planted
grounds and lower .seedbeds. The increase in disease reduced the
desirability (loss of shell resource from seed areas) and profitabil-
ity (high mortality) for moving oysters into higher salinity areas.
As such, oyster production has focused on two production
schemes: ( 1 ) direct harvest of oysters produced on the lower seed-
beds, essentially a wild fishery and (2) the transplantation of oys-
ters from the mid-estuary seed beds to the lower estuary seedbeds.
Transplanted oysters are then harvested after the meat condition
improves. One of the consequences of these approaches is the need
to estimate the allowable production from the seedbeds each year,
which is equivalent to setting a yearly quota. It seems clear that
present oyster populations are below pre-disease levels, and that
continued high disease levels will prevent recovery to pre-disease
levels. The presentation describes a model developed for the man-
agement of fished oyster populations that lie over a salinity gra-
dient and for which disease mortality is a controlling influence. We
will present a review of the Delaware Bay stock assessment for
1998 and 1999. We will then describe a model developed to ad-
dress management issues when B,„„ and K are not appropriate
options and apply it to Delaware Bay oyster populations.

SHELLFISH DATA MANAGEMENT AND REPORTING
SYSTEM (SDMRS). J. Power' and D. B. Walker,^ 'Environ-
ment Canada, 224 West Esplanade. North Vancouver. British Co-
lumbia. V7M 3H7, Canada, and "E-Carta Services, 419 N 18th
Ave. E, Duluth. MN 55812-1352.

The objective of this work was to design a user- friendly inter-
face to digital watershed maps and hydrographic charts, thematic
layers such as clam harvesting areas, farms, shellfish leases, and
closures, and databases relating to pollution sources, water quality
sampling, shellfish growing areas, locations of marine mammals
and seabirds, fish farms and weather, for Canada's West Coast.
Such an interface will allow scientists in the shellfish program of
Environment Canada, Pacific and Yukon Region to readily gen-
erate data reports including maps detailing shellfish closures and
shoreline assessments. The Shellfish Data Management and Re-
porting System (SDMRS) is an ArcView CIS application linked to

Shellfish. Restoration. Hilton Head Island. SC

Abstracts, December 2002 905

an Access database, which requires no special knowledge of SQL
queries of databases or GIS. From ArcView the user chooses the
sector to be mapped and the additional area around the sector to
display map information from drop down menus. The system then
generates the appropriate base map by clipping out the underlying
hvdrographic charts and/or watershed maps. Then the user selects
the themes and data sets to add to the map. again by choosing from
drop down menus. The system adds the themes, uses ODBC to
query the database and retrieve the appropriate data sets, converts
them to the proper projection, and adds them to the map. When
completed the map is then exported to the report document.

RESTORING OYSTER REEFS FOR FISH: ESTIMATING
ENHANCED SECONDARY PRODUCTION OF RESTORED
OYSTER REEFS. S. P. Powers. C. H. Peterson, and J. H.
Grabowski, Institute of Marine Sciences. University of North
Carolina at Chapel Hill. 34.^1 Arendell Street. Morehead City, NC
28557.

The recognition that oyster reefs provide an important resource,
but also provide habitat for a variety of other species, has encour-
aged restoration of oyster reefs as a method to enhance production
of finfish and shellfish within estuaries. Here, we describe an
approach for determining the augmented secondary production of
bottom areas that were sand/mud flats and restored to oyster reefs.
First, through a review of published and unpublished studies, we
compared densities of animals on oyster reefs to nearby areas
without biogenic structure. We divided those species that showed
enhanced densities on reefs into three groups: ( I ) species that
recruited exclusively to reefs, (2) species that had higher recruit-
ment to reefs, but still recruited and used non-structured habitats,
and (3) species that showed higher aggregations around reefs, but
were not limited by reef habitat based on diet and life-history
analyses. For this first group, all production is attributed to the
reef. Because some proportion of the enhanced density of species
belonging to this second group would probably have recruited to
other habitat, the production attributed to the reef is adjusted by a
coefficient of reef-habitat exclusivity (CRE) that we developed
using diet analysis and life history information. For the final group
of animals, the reef only receives credit for that fraction of growth
that is enhanced by the presence of the reef, this determination is
made through the application of a CRE. Applying this approach to
proposed oyster reef restoration in Tampa Bay, FL, we estimated
the augmented secondary production of 10 m" of oyster reef to be
2.57 kg y-'.

RESTORING THE LITTLENECK CLAM RESOURCE FOR
NATIVE AMERICAN SUBSISTENCE USE IN THE
PRINCE WILLIAM SOUND, ALASKA. R. RaLonde, Univer-
sity of Alaska, School of Fisheries and Ocean Sciences, Anchor-
age, AK 99508-4140.

Natural and man caused disasters decimated the littleneck clam
(Protnthaca stammea) populations on the intertidal beaches of

Pruice William Sound. Alaska. Subsequently. Native American
Villages have been unable to harvest clams to meet their subsis-
tence needs. Since 1995. the Quteckak Native Corporation has
been actively pursuing restoration of the clam populations by de-
veloping seed production technology, conducting site selection
studies, and managing growout trials. The initial success of their
restoration efforts now enables communities to harvest clams from
the restored beaches and broaden the program. This presentation
will describe the research and restoration results of the project and
the human impact of the restored resource on the Native Villages
of Prince William Sound. Alaska.

DEVELOPMENT OF CRASSOSTREA VIRGINICA MICRO-
SATELLITE MARKERS FOR A GENETIC LINKAGE MAP
AND GENETIC MONITORING OF RESTORATION PRO-
JECTS. K. M. Reece, VV. L. Ribeiro. K. L. Hudson, and S. K.
Allen Jr., Virginia Institute of Marine Science, The College of
William and Mary. Gloucester Point, VA 23062.

Dermo and MSX have had significant impacts on natural popu-
lations of the eastern oyster Crassostrea vir}>inica and have been a
detriment to oyster aquaculture development. A potential solution
to this problem is genetically improved disease-resistant strains ot
C. virginica that can grow to market size despite disease challenge.
Traditional selective breeding programs have resulted in strains of
oysters that are being assessed for disease resistance. One means of
accelerating selective breeding programs is to identify genetic
markers associated with traits such as disease resistance or growth
rate. A goal of this project is to develop genetic markers for con-
structing a linkage map and to identify markers associated with
disease resistance to use in marker-assisted selection programs. In
addition, markers developed in the genomics project are being
surveyed for use in genetic monitoring of reef restoration projects.
To assess relative genetic contributions of wild and planted stocks
to restored reefs; markers are being identified that are able to
genetically distinguish selected strains and nattiral populations in
Chesapeake Bay. C. virginica microsatellite markers are being
developed in an ODRP funded genome mapping project. Several
di-, tri-, and tetranucleotide repeat sequences have been identified.
Primers for use in the polymerase chain reaction have been de-
signed to anneal to regions flanking 39 microsatellites and ampli-
fication reactions for 21 loci have been optimized. Fl individuals
from four reference families have been screened at twelve micro-
satellite loci for generating a genetic linkage map. Microsatellite
allelic profiles of selected strains and natural populations are being
examined.

OYSTER REEF RESTORATION RESEARCH IN MOBILE
BAY, ALABAMA. D. B. Rouse,' R. K. Wallace,- and F. S.
Rikard,^ 'Department of Fisheries and Allied Aquacultures, Au-
burn University. Auburn. AL 36849 and "Mobile, AL 36615.

Oyster reef restoration in Mobile Bay has consisted primarily
of shell planting on active reefs in the lower sections of the bay.

906 Ahstmcts. December 2002

Shellfish, Restoration, Hilton Head Island, SC

Efforts are now underway to restore reefs in the mid-bay area.
Studies are being conducted to determine why these reefs are no
longer productive and what should be done to restore them to a
productive state. Bottom surveys were performed to quantify
cultch availability. Sediment traps were deployed to determine
rates of sediment accretion and spat collectors were used to evalu-
ate natural oyster set. Spat were deployed on the bottom and on
platforms 20 cm and 40 cm above bottom. Data loggers were
deployed on bottom and 40 cm above bottom to measure tempera-
ture, salinity and oxygen concentration. Surveys revealed hard
bottoms but little exposed cultch on non-productive reefs. Sedi-
mentation was high and consisted mainly of silt with more than
10% organic matter. Single peak oyster sets occurred in the fall.
Oysters at the three experimental levels grew to approximately 60
mm in the first year. During the second year, total mortality was
observed at all three levels when oxygen levels dropped to 0 mg/L
for 3 consecutive days. Similar periodic low oxygen events may be
occurring at the study site and on other relic reefs that will hinder
their successful restoration. Water quality studies suggest that
cultch mounding will be necessary to elevate oysters above anoxic
bottom conditions.

TRANSPLANTING BROODSTOCK OYSTERS, CRASSOS-
TREA VIRGINICA, ONTO RECONSTRUCTED OYSTER
REEFS TO INCREASE SPAT RECRUITMENT IN THE
PIANKATANK RIVER. D. C. Sherwood, Sandston. VA 2,^150.
Virginia oyster reef restoration in the form of three-
dimensional structures began in the Piankatank River. Virginia in
1993. From 1993 to 1998. fifteen artificial reefs were built in
tributaries of the Chesapeake Bay. In December 1996. reproduc-
tively active broodstock oyster from Tangier and Pocomoke
Sounds were transplanted to the Shell Bar reef in the Great
Wicomico River. Virginia. Surveys in the fall of 1997 indicated a
high spat recruitment both on the reefs and nearby oyster bars.
With this success, broodstock oysters from Tangier and Pocomoke
Sounds were transplanted in December 1997 to two of the four
reconstructed reefs in the Piankatank River. Since total quantity as
well as density of broodstock was believed to be limiting factors
for recruitment in this river, adding stock was expected to raise
spat recruitment. Spat recruitment data was collected via dive sur-
veys on the reefs, and via dredge and patent long surveys on the
natural oyster bars. The recruitment of spat to both reefs and bars
was significantly higher in 1998 (P < 0.001 ) than in the previous
four years and a positive interaction {P < O.OO.'i) was seen between
the reefs and the year 1998. Based on these data, stock enhance-
ments in the Piankatank River successfully improved recruitment
and suggest oyster restoration may be facilitated in other areas of
the Chesapeake Bay by strategic enhancement ol spawning stocks.

HARMFUL ALGAL BLOOMS AND SHELLFISH RESTO-
RATION: CAN THEY CO-EXIST? S. E. Shumwav. Depart

ment of Marine Sciences. University of Connecticut, 1080 Shen-
necossett Road, Groton, CT 06340

Habitat quality is an obvious and important consideration for
all shellfish restoration efforts including aquaculture and reseed-
ing. Harmful algal blooms (HABs). worldwide threats to habitat
quality, are naturally occurring phenomena and their number and
frequency are increasing. These blooms impact ecosystem integ-
rity, species interactions, aquatic animal health, population growth,
hinnan health, economy, industry, and ecology and often pose a
threat to wild and cultured shellfish populations. While algal spe-
cies that impact human health receive the most attention, there are
numerous HABs that cause the destruction and demise of shellfish
beds and aquaculture operations. These HABs can also dictate the
successful citing of restoration efforts. This presentation will re-
view our knowledge of harmful algal-shellfish interactions world-
wide including threats associated with the presence of recently
identified problem species, e.g., Pfwsleria spp. and Helerocapsa.
and discuss ways in which shellfish restoration efforts may be
undertaken successfully in the face of these imposing threats. Miti-
gation of these HABs has become an important focus for coastal
research. Data will be presented on recent studies on the use of
clay as a means of alleviating the impacts of HABs. Careful man-
agement-science interaction, not eradication, remains the only safe
and functional means of mitigation currently available.

A COMPARISON OF TWO OYSTER {CRASSOSTREA VIR-
GINICA ) STRAINS FOR PRODUCTIVITY AND SUITABIL-
ITY FOR USE IN OYSTER REEF Rt;STORATION EF-
FORTS. L. A. Sorabella,' M. W. Luckenbach," and F. X.
O'Beirn," Virginia Institute of Marine Science. College of Will-
iam and Mary. P.O. Box 1346. Gloucester Point. VA 23062. Vir-
ginia Institute of Marine Science, College of William and Mary.
P.O. Box 350, Wachapreague, VA 23480.

Over the past ,5 years, eastern oyster (Cnisso.strt'u virgiiuca)
restoration efforts in Virginia have focused on constructing reef
structures to act as sanctuaries. Increasingly, shell plants are
stocked with hatchery-produced brood stock oysters that spawn
and increase recruitment to the reefs. This involves rearing hatch-
ery-produced seed oysters in floating containers and out-planting
them onto reefs when they reach an appropriate size (20-30 mm).
To assist with the labor involved in this process, we recruited
citizen and student oyster-gardeners who raised the bulk of the
oysters for transplant onto the sanctuaries. Two strains were hatch-
ery-reared for transplantation onto these reefs: CROSBreed oysters
and wild-caught oysters from the lower Chesapeake Bay. The
CROSBreed strain has been selectively bred since 1962 for resis-
tance to the parasite Haplosporiduium nelsoni that causes MSX.
and has more recently been bred for resistance to Perkinsus mari-
nus. that causes Dermo. The second strain were large wild-caught

Shellfish. Restoration. Hilton Head Island. SC

Abstracts. December 2002 907

oysters collected from the Lynnhaven River (Chesapeake Bay,
VA) where the oysters were surviving under presumed high pres-
sure from both parasites. The objective of this research is to evalu-
ate the performance of the CROSBreed stock and wild stock oys-
ters deployed on sanctuary reefs in the Lafayette River (Chesa-
peake Bay, VA). Evaluation compares the two strains based on
female fecundity, growth, survival, and incidence of Perkinsus
marinus and Haplosporiduium nelsoni infection measurements. As
large-scale restoration projects proceed, it will be fundamentally
important to assess which stock is most appropriate for use in the
oyster restoration effort.

OY.STER RESTORATION AND THE UNIVERSITY OF
MARYLAND: INTERACTIONS BETWEEN RESEARCH,
INDUSTRY, AND THE PUBLIC. S. M. Tobash and D. W.
Meritt, University of Maryland. Center for Environmental Sci-
ence, Horn Point Laboratory. PC Bo.\ 775. Cambridge. MD 21613.
Maryland oyster populations have been declining for many
years. The LIniversity of Maryland is committed in its involvement
to assist in restoration programs, placing an emphasis on the eco-
logical contribution that oysters provide to the overall health of the
Chesapeake Bay. Research efforts are focused on diseases, genet-
ics, culturing techniques and basic oyster biology and ecology. The
efforts of the Maryland Oyster Recovery Partnership and other
concerned groups, along with increasing public involvement has
enabled the oyster hatchery at the University of Maryland Center
for Environinental Science Horn Point Laboratory to increase pro-
duction of disease-free hatchery seed. These seed oysters are then
used in a variety of restoration, education, and outreach activities
statewide. Educational programs are targeted to a wide audience,
ranging from nonprofit organizations of school-aged children to
commercial watermen. Outreach projects promote the involvement
of concerned citizens like the Chesapeake Bay Foundation's Oys-
ter Gardening Program. Cooperation between commercial fisher-
men, management agencies, and concerned groups and combina-
tion of resources are key to a successful restoration program. The
University of Maryland represents an important link in this pro-
cess. The ongoing commitment of UMCES Horn Point Laboratory
together with cooperative programs will continue to strengthen the
future of the Chesapeake Bay oyster restoration effort.

EFEECTS OF WATERSHED ALTERATIONS ON OYSTER
POPULATIONS IN SOUTHWEST FLORIDA ESTUARIES:
AN ECOLOGICAL APPROACH. A. K. Volety, M. Savarese,
and S. G. Tolley, College of Arts and Science. Florida Gulf Coast
University. 10501 Fgcu Boulevard South, Fort Myers. FL 33965.
Southwest Florida is one of the country's fastest growing re-
gions. Consequently, watersheds are heavily managed to accom-
modate development. Studies on the effects of altered watershed in
this region involving valued ecosystem components, like oysters.

are lacking, but clearly necessary. Using the oyster, Crassostrea
virfiiuica. as an indicator species, we are investigating ecosystem-
wide health effects of watershed management practices in altered
(Faka-Union. Henderson Creek, and Caloosahatchee River) and
pristine (Blackwater River) estuaries. Measurements of oyster spa-
tial distribution, condition index, spat recruitment, energy reserves,
and disease prevalence of Perkinsus imiriiuis are underway using
a "spatial homologue approach" (among-estuary comparisons at
hydrologically and geoniorphically similar locations along the sa-
linity gradient). Preliminary results indicate that in summer
months, depending on the location, mean prevalence of P. iiuinmis
infection in oysters varied between 33-73%, whereas the mean
condition index varied between 2.4-4.7. The distribution of reefs,
regions of maximum living density, and maximum oyster produc-
tivity are shifted seaward in altered relative to pristine systems
when water management practices are supplying excessive fresh-
water to estuaries. These populations, however, exhibit lower P.
iiHiriiiKs prevalence because of the greater freshwater influence.
This project represents the first study of watershed alteration on
oysters in Southwest Florida and will help provide target environ-
mental conditions for restoration efforts.

THE VIRGINIA OYSTER HERITAGE PROGRAM. J. A.

Wesson' and L. B. McKay," 'Virginia Marine Resources Com-
mission. 2600 Washington Ave.. 3rd Floor. Newport News. VA
23607-0756 and "Virginia Department of Environmental Quality,
629 E. Main St., Richmond, VA 23219.

The Virginia Oyster Heritage Program (VOHP) is a bold ini-
tiative to capitalize on recent advances and consensus on strategies
for oyster restoration. The VOHP seeks to re.store oyster popula-
tions and oyster reef habitat, and to. thereby, improve ecological
function, water quality and the oyster industry in Virginia's portion
of Chesapeake Bay and its seaside bays. Initiated in 1999 by the
Department of Environmental Quality and Marine Resources
Commission, the VOHP is a partnership of Federal. State, and
private entities. Phase One focuses on the Lower Rappahannock
River, with a goal of rebuilding 8 to 10, three-diinensional, oyster
sanctuary reefs and restoration of more than 200 acres of oyster
beds for direct harvest. The combination of funding from all
sources totaled more than ,$1,500,000 in the first year, resulting in
six reefs constructed, and 85 acres of harvest area restored. The
Lower Rappahannock River, closed to harvest for six years, is an
area of high salinity with consistent exposure to oyster diseases.
Annual monitoring during this closure demonstrated that a signifi-
cant proportion of the native oysters survived and grew, and that
spatset became dependable. We believe that the lack of cultch has
limited the increase in oyster populations and that enough natural
broodstock has accunuilated to colonize these restored areas. With
proper management of sanctuary and harvest areas, we also believe
that a sustainable fishery can be established while simultaneously

908 Abstracts. December 2002

Shellfish. Restoralion. Hilton Heud Island. SC

increasing the standing stock of oysters. We anticipate comparable ties for shellfishemien as well as other marine-based job opportu-
funding in 2001. and the expansion of this model into other areas. nities.

ENHANCING AND SUSTAINING NORTH SHORE SHELL-
FISHING THROUGH AQUACULTURE. J. J. Whitten, Mer

rimack Valley Planning Commission. 160 Main .Street, Haverhill.
MA 01830.

Soft-shell clamming has long been a vital commercial fishery
on the North Shore of Massachusetts. Earlier in the century, the
Merrimack River Estuary alone produced up to 100.000 bushels of
soft-shell clams per year ( among the highest of shellfish harvesting
communities on the East Coast). During the past decade, the fish-
ery has seen significant declines and wide fluctuations in produc-
tivity. Landings have plummeted as much as 809r along the North
Shore. The dramatic downturn in this once prominent industry is
due to a combination of factors such us: natural "boom-and-bust"
reproduction cycles, predation from non-native species such as the
green crab, over harvesting, as well as regulatory closures of pre-
viously undocumented contaminated areas. The wide, unpredict-
able fluctuations in bed producti\ ity make it impossible to ensure
a sustainable and predictable harvest. The seeding of Massachu-
setts' North Shore shellfish beds with hatchery-reared soft-shell
clams offers the potential to moderate this cyclical and declining
nature of productivity. Efforts to improve shellfishing through res-
toration/enhancement efforts in other states ha\e demonstrated
several methods by which a more consistent and sustainable har-
vest can be achieved. This presentation examines regional efforts
conducted by the Merrimack Valley Planning Commission
(MVPC) in collaboration with the coastal communities of Glouc-
ester. Ipswich and Rowley Massachusetts to enhance and sustain
soft-shell clam (M\a arenana) populations. Ultimately a more
sustainable harvest provides more secure employment opportuni-

COMMUNITY-BASED OYSTER RESTORATION IN AN
URBAN ESTUARY: DEVELOPING AN OYSTER CUL-
TURE AND REEF RESTORATION PROGRAM IN THE
HUDSON-RARITAN ESTUARY. A. Willner. NY/NJ Bay

keeper. Building 18, Sandy Hook, Highlands. NJ 07732.

Goals, progress, and challenges to development of a restoration
program led by a nonprofit organization in an urban estuary will be
discussed. The Hudson-Raritan Estuary supports a sparse oyster
population that has collapsed since the beginning of the 1900s.
Major natural oyster beds were exhausted by commercial shell-
fishing in the eariy I800"s. since the closure of the industry in the
1920s the population has continued to decline to the current state
of small groupings of individuals located in disparate reaches of
the system. Prompted by the success of oyster restoration in other
estuaries, NY/NJ Baykeeper, with guidance from a scientific ad-
visory board, has begun to investigate the feasibility of restoring
reefs to provide habitat for larval settlement and population recov-
ery. Spat surveys conducted in 1998 demonstrating minimal re-
cruitment, lead to the 1999 experiment of placing a large amount
of shell on the historic footprint of an oyster population in New
York Harbor to provide a greater area to increase probability of
recruitment. Preliminary results demonstrate the potential for reef
construction to increase oyster recruitment. To stock the reef in an
effort to further improve recruitment success, a community-based
oyster-culturing program has recently been established involving
schools, marinas, civic groups and families. Oyster restoration in
estuaries with pollution concerns such as this provide the oppor-
tunity to investigate factors affecting population recruitment and
maintenance as well as challenges to public education and out-
reach in urban areas.

THE NATIONAL SHELLFISHERIES ASSOCIATION

The National Shellfisheries Association (NSA) is an international organization of scientists, manage-
ment officials and members of industry that is deeply concerned and dedicated to the formulation of
ideas and promotion of knowledge pertinent to the biology, ecology, production, economics and man-
asement of shellfish resources. The Association has a membership of more than 1000 from ail parts of
the USA. Canada and 18 other nations; the Association strongly encourages graduate students' mem-
bership and participation.

WHAT DOES IT DO?

— Sponsors an annual scientific conference.

— Publishes the peer-reviewed Joimuil of Shellfish Research.

— Produces a Quarterly Newsletter.

— Interacts with other associations and industry.

WHAT CAN IT DO FOR YOU?

— You will meet kindred scientists, managers and industr>' officials at annual meetings.

— You will get peer review through presentation of papers at the annual meeting.

— If you are young, you will benefit from the experience of your elders.

— If you are an elder, you will be rejuvenated by the fresh ideas of youth.

— If you are a student, you will make useful contacts for your job search.

— If you are a potential employer, you will meet promising young people.

— You will receive a scientific journal containing important research articles.

You will receive a Quarterly Newsletter providing information on the Association and its activities, a

book review section, information on other societies and their meetings, a job placement section, etc.

HOW TO JOIN

—Fill out and mail a copy of the application blank below. The dues are 65 US $ per year ($35 for students)
and that includes the Journal and the Newsletter!

NATIONAL SHELLFISHERIES ASSOCIATION-APPLICATION FOR MEMBERSHIP

(NEW MEMBERS ONLY)

Name: __^_ For the calendar year: Date:

Mailing address: -^

Institutional affiliation, if any: .
Shellfishery interests:

Regular or student membership:

Student members only — advisor's signature REQUIRED:

Make checks {MUST be drawn on a US bank), international postal money orders or VISA for $65 ($35 for
smdents with advisor's signature) payable to the National Shellfisheries Association and send to Nancy Lewis,
Bookkeeper, PO Box 350. V.I. M.S. Eastern Shore Lab. Wachapreague. VA 23480. USA.

INFORMATION FOR CONTRIBUTORS TO THE
JOURNAL OF SHELLFISH RESEARCH

Original articles dealing with all aspects of shellfish re-
search will be considered for publication. Manuscripts will be
judged by the editors or other competent reviewers, or both, on
the basis of originality, content, merit, clarity of presentation.
and interpretations. Each article should be carefully prepared in
the style followed in prior issues of the Journal of Shellfish
Research before submission to the Editor. Papers published or
to be published in other journals are not acceptable.

Title, Short Title. Key Words, Abstract: The title of the
paper should be kept as short as possible. Please include a
"short running title" of not more than 48 characters including
spaces, and key words. Each manuscript must be accompanied
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the research reported. The abstract will be published at the
beginning of the article. No separate summary should be in-
cluded.

Text: Manuscripts must be typed double-spaced throughout
on one side of the paper, leaving ample margins, with the pages
numbered consecutively. Scientific names of species should be
underlined or in italics and, when first mentioned in the text,
should be followed by the authority. Common and scientific
names of organisms should be in accordance with American
Fisheries Society Special Publications 16 and 17; Common and
Scientific Names of Aquatic Invertebrates from the United
States and Canada: Mollusks and CSNAIUSC: Decapod Crus-
taceans, or relevant publications for other geographic regions.

Abbreviations, Style, Numbers: Authors should follow the
style recommended by the sixth edition ( 1994) of the Council
of Biology Editors [CBEJ Style Manual, distributed by the
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Tables: Tables, numbered in Arabic, should be on separate
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Literature Cited: References should be listed alphabeti-
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should be those recommended in the American Standard for
Periodical Title Abbreviations, available through the American
National Standard Institute, 1430 Broadway, New York. NY
1 00 1 8. For appropriate citation format, see examples below;
Journal:

Watts, R. J., M. S. Johnson & R. Black. 1990. Effects of re-
cruitment on genetic patchiness in the urchin Echinonietra
mathaei in Western Australia. Mar. Biol. 105:145-151.
Book:

Claudi, R. & G. L. Mackie. 1994. Practical manual for Zebra
Mussel monitoring and control. Boca Raton. FL; CRC Press.
227 pp.

Chapter in Edited Book:

Davio, S. R., J. F. Hewetson & J. E. Beheler. 1985. Progress
toward the development of monoclonal antibodies to saxitoxin;
antigen preparation and antibody detection. In: D. M. Ander-
son, A. W. White & D. G. Baden, editors. Toxic dinoflagel-
lates. Amsterdam; Elsevier, pp. 343-348.

Page Charges: Authors or their institutions will be charged
$100.00 per printed page. All page charges are subject to
change without notice. A handling fee of $50 will be charged
for all manuscripts accepted for publication.

Proofs: Page proofs are sent to the corresponding author
and must be corrected and returned within seven days. Alter-
ations other than corrections of printer's errors may be charged
to the author(s).

Reprints: Reprints of published papers are available at cost
to the authors. Information regarding ordering reprints will be
available from The Sheridan Press at the time of printing.

Cover Photographs: Appropriate photographs may be sub-
mitted for consideration for use on the cover of the Journal of
Shellfish Research. Black and while photographs and color
illustrations will be considered.

Corresponding: An original and two copies of each manu-
script submitted for publication consideration should be sent to
the Editor, Dr. Sandra E. Shumway, Department of Marine
Sciences, University of Connecticut, 1080 Shennecossett Rd.,
Groton, CT 06340. E-mail; sandra.shiunw ay@uconn.edu or
sandrashumway@hotmail.com

Membership information may be obtained from the Editor
or the Treasurer using the form in the Jounud. Institutional
subscribers should send requests to: Jounud of Shellfish Re-
.search. P.O. Box 465. Hanover, PA 17331.

Sergio Ragonese, Patrizia Jereb. and Earl Dawe

A comparison of growth performance across the squid genus Ille\ (Cephalopoda, ommastrephidae) based on

modelHng weight-at-length and age data 85 1

Chandrika Liyana-Pathirana, Fereidoon Shahidi, and Alan Wliittick

Comparison of nutrient composition of gonads and coelomic fluid of green sea urchin

Strongylocentroliis drnebachiensis °6 1

B. R. Moore, S. N. Kleeman, and R. J. G. Lester

The development of a positive non-infectious control for the detection of Perkiiisii\ using the Ray test 87 1

S. J. Nichols and D. Garling

Evaluation of substitute diets for live algae in the captive maintenance of adult and subadult freshwater unionidae 875

Abstracts of technical papers presented at the 4th International Conference on Shellfish Restoration. Hilton Head Island.

South Carolina. November 1 5- 1 8. 2000 883

COVER PHOTO: Red crab {Gennii qmnquc dens) are found in deep water off of the coast of North America. These
crabs were caught 140 miles south of New England aboard the Duimoiut Girl. Photo by Richard W. Dionne. Jr. with
special thanks to the Sakonnet Times.

The Journal of Shellfish Research is indexed in the following: Science Citation Index*. Sci Search®. Research Alert^. Current
Contents«/Agriculture. Biology and Environmental Sciences. Biological Abstracts. Chemical Abstracts. Nutrition Abstracts. Current
Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review. Environmental Periodicals Bibliography.
Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts.

Patrick Rice, Sammy M. Ray. Sherry D. Painter, and Gregg T. \'agle

An intrinsic membrane protein in oyster sperm stimulates spawning behaviors in Crassostrea virtiiiiica: implications

tor aquacultiire 715

Qiaoxiang Dong, Benoit Eiideline, Standish K. Allen, Jr., and Terrence R. Tiersch

Factors affecting sperm motility of tetraploid Pacific oysters 719

Amy D. Nickens, Jerome F. La Peyre, Eric S. Wagner, and Terrence R. Tiersch

An impro\ ed procedure to count Perkinsiis imiriiuis in Eastern oyster hemolymph 725

Stephen J. Jordan. Kelly N. Greenhawk. Carol B. McCollough. Jessica Vanisko, and Mark L. Homer

Oyster biomass. abundance, and harvest in northern Chesapeake Bay: trends and forecasts 733

Andrew G. Jeffs. B. J. Dunphy, and R. M. G. Wells

Experimental effects of water temperature on the gametogenic development of broodstock in the oyster,

Oslreci cliilensis 743

Raymond E. Grizzle. Jamie R. Adams, and Linda J. Walters

Historical changes in intertidal oyster iCnissosrrea vir^inica) reefs in a Florida lagoon potentially related to

boating activities 749

Sonia Rodriguez-Astiidillo. Marcial Villalejo-Fuerte. Federico Garcia-Dominguez. and Rafael Guerrero-Caballero

Biochemical composition of Spi'iulyliis Iciicacaiithus Broderip, 1833 (Bivalbvia: Spondylidae) and its relationship

with the reproductiv e cycle at Isla Dan/ante. Gulf of Calitbrnia. Mexico 757

Ana Bratos. Jaksa Bolotin. Melita Peharda. and Jakica Sjire

Seasonal distribution of the oyster Osrrea cJitlis (Linnaeus. 1758) larvae in the Bay of Mali Ston. .Adriatic Sea 763

Alan J. Power. Ellie Covington. Todd Recicar. Randal L. Walker, and Nelson Filer

Observations on the egg capsules and hatchlmgs of the knobbed whelk. Biisyccm carica (Gmelin. 1791 ) in

coastal Georgia 769

Dario Savini. Juliana M. Harding, and Roger Mann

Rapa whelk Rcijuiini veiuisa (Valenciennes. 1846) predation rates on hard clams Menenaria meneiuiria

(Linnaeus. 1 758) 777

N. Chaitanawisuti. S. Kritsanapuntu. and Y. Natsukari

Economic analysis of a pilot commercial hatchery-based operation for spotted babylon. Biihylmiia areolata Link

1 807. juveniles in Thailand 781

P. T. Gibson. D. G. Worthington. C. Blount, and N. L. Andrew

Incidental damage of blacklip abalone (Halintis nibnt) by commercial divers in New South Wales, Australia 787

Meegan E. Vandepeer, Patrick W. Hone, Jon N. Havenhand, and Robert J. Van Barneveld

The effect of nonnutritive fillers on the digestibility of a manufactured abalone diet 793

Meegan E. Vandepeer, Patrick W. Hone, Jon N. Havenhand, and Robert J. Van Barneveld

The digestibility of whole and dehulled lupms [Liiphnis niii^KstifhUus) fed to juvenile greenlip abalone,

Haliotis liievifiata 799

C. D. Hawkins and J. B. Jones

Lar\ al escape through abalone culture eftluent systems: an analysis of the risk 805

Qi Li, Choulji Park, and Akihiro Kijima

Isolation and characteriz-ation of microsatelhte loci in the Pacific abalone. Huluilis discus luiiiiuii 811

Carolyn S. Friedman, Wendy Biggs, Jeffrey D. Shields, and Ronald P. Hedrick

Transmission of withering syndrome in black abalone. Haliolis ciculicnHlii leach 817

Ma. Del Carmen Alvarez Tinajero, Jorge Cdceres-Mariinez, and Jose Guadalupe Gonzales Aviles

Histopathological evaluation of the yellow abalone Haliaris comtgahi and the blue abalone Haliotis fidgens from

Baja California. Mexico 825

Maria Georgina Gluyas Milldn, Casimiro Quinonez Velazquez, and Jesiis Talavera Maya

Effect of El Nino 1 997-98 on the snail Astniea iiiiddsa (Wood) population along the Baja California western coast — 83 1
Martha E. Sandoval Quintero and Adolfo Gracia

Reproduction of the spotted pink shrimp. Faifuntepenaeus brasiliensis ( Decapoda: Penaeidae) 835

R. Rosa and M. L. Nunes

Influence of the reproductive cycle on the biochemical composition of deep-sea decapod Parapenaeus longirostris

(Lucas, 1 846) in the Portuguese .south coast 843

CONTENTS CONTINUED ON PREVIOUS PAGE

Sizhong Wang, Peter F. Duncan, Wayne Knibb, and Bernard M. Degnan

Byssal attachment of Amusimn hallolt (Bernardi. 1861 ) (Bivahia: Pectinidae) spat 563

Maite Narvarte and Marina Kroeck

Intraspecific variation in the reproductive cycle of the tehuelche scallop Aeqiiipectcii tchuclchiis (Pelecypoda.

Pectinidae). in San Mati'as Gulf. Patagonia. Argentina 571

G. Roman, M. J. Campos, J. Cano, C. P. Acosta, Pilar Iglesias, and Oscar Garcia

Reproductive and reserve storage cycles in Aequipeclen opercularis (L.. 1758) in Galicia. NW Spain 577

Gonzalo Gajardo, Margarita Parraguez. and Nelson Colihueque

Karyotype analysis and chromosome banding of the Chilean-Peruvian scallop Argopecten pitrpunitiis

(Lamarck. 1819) 585

Mohsin U. Patwary, Akelia Waiichope, Timothy W. Short, and Edward J. Catapane

Molecular cloning and characterization of a fructose- 1.6-biphosphale aldolase cDNA from the deep-sea scallop

Placopecteii iiicigellaiiiciis 591

Rosali'o Maldonado-Amparo and Ana M. Ibarra

Comparative analysis of oocyte type frequencies in diploid and triploid catarina scallop (Argopecten ventricosiis) as
indicators of meiotic failure 597

Martin LaFrance, Helga Guderley, and Georges Cliche

Low temperature, but not air exposure slows the recuperation of juvenile scallops. Placopecten mageUanicus. from
exhausting escape responses 605

Jose L. Riieda and Aad C. Smaal

Selecti\e ingestion of pelagic versus henlhic algae by the cockle Ceraslodenna ediilc (Linne. 1758) 619

Donald Brown, Bernardita Campos, and H.-Jiirg Urban

Reproductive cycle of the bivalve clams Semele solida (Gray. 1828) (Semelidae) and Gari soliJa (Gray. 1828)
(Psammobiidae) from Chile 627

A. L. Heck, Jr., L. D. Coen, and D. M. Wilson

Growth of northern \Meixenaria lucirciiaria (L.)] and southern \M. amipechiensis (Gmelin)] quahogs; influence of
seagrasses and latitude 635

C Tirado, C. Salas, and J. I. Lopez

Reproduction of Calllsia cluone L., 1758 (Bivalvia: Veneridae) in the littoral of Malaga (southern Spain) 643

S. Novoa, D. Martinez, J- Ojea, P. SoudanI, J.-F. Samain, J. Moat, and J.-L. Rodriguez

Ingestion, digestion, and assimilation of gelatin-acacia microcapsules incorporating deuterium-labeled arachidonic acid

by larvae of the clam Venerupis piilltislrn 649

William S. Arnold, Dan C. Marelli, Melanie Parker, Philip Hoffman, Marc Frischer, and John Scarpa

Enhancing hard clam {Meixeiuuia spp.) population density in the Indian River Lagoon. Florida: a comparison of

strategies to maintain the commercial fishery 659

Stefan Aki Ragnarsson and Gudriin G. Thorarinsdottir

Abundance of ocean quahog. Arclica isUindua. assessed by underwater photography and a hydraulic dredge 673

Stefano Peruzzi and Ximing Guo

Tetraploid induction by meiosis inhibition with cytochalasin B in the dwarf surfclam. Mtdiiiia lateralis Say: effects

of temperature 677

Alexandra Leitdo, Raqiiel Chaves, Sara Santos, Pierre Boudry, Henrique Guedes-Pinto, and Catherine Thiriot-Quievreiix
Cytogenetic study of Oslrea coiiclniphllii (Mollusca: Bivalvia) and comparative karyological analysis
within ostreinae 685

Eric N. Powell, Kathryn A. Ashton-Alcox, Joseph A. Dobarro, Meagan Cummings, and Sarah E. Banta

The inherent efficiency of oyster dredges in survey mode 691

E. Kenchington, C. J. Bird, J. Osborne, and M. Reith

Novel repeat elements in the nuclear ribosomal RNA operon of the flat oysters Oslrea edulis C. Linnaeus. 1758 and

O. aiigasi Sowerby. 1871 697

Maria Eugenia Valdez-Ramirez, Anne Donval, and Marcel Le Pennec

Ultrastructural and histochemical criteria for determining normality in mature oocytes of the Pacific oyster

Crassostrea gigas 707

2(28 057

CONTENTS CONTINUED ON PREVIOUS PAGE

JOURNAL OF SHELLFISH RESEARCH
Vol. 21, No. 2 December 2002

CONTENTS

IN MEMORIAM

Harold Haley Haskin (141 5-2002 ) 441

Eugene Burreson

Honored Life Member: Jay Donald Andrews 445

Kenneth Chew

Honored Lite Member: Neil F. Bourne 447

Loren D. Coen and M. Yvonne Bobo

Honored Life Member: Vietor G. Bunell 449

Richard A. Lutz

Honored Life Member: Herbert Hidu 45 1

George R. Abbe

Honored Life Member: Sandra E. Shumway 453

F. H. Mackintosh and E. A. Smith

Evakialion of Mist Alert '^' rapid test kits for the detection of paralytic and amnesic shellfish poisoning toxins

in shellfish 455

Ana Maria Gayoso, Stacie Dover, Steve Morion, Mark Busman, Peter Moeller, Vanesa K. Fulco, and Lucie Maranda

Diarrhetic shellfish poisoning associated with Pronicenlruiii limn (Dinophyceae) in Patagonian Gulfs (Argentina) 461

Graham C. Fletcher, Brenda E. Hay, and Margaret F. Scott

Reducing neurotoxic shellfish poison (NSP) in Pacific oysters iCrassostrea ,?i,?rt.s) to levels below 20 mouse

units • 1 00 g-' 465

F. M. Harper, E. A. Hatfield, and R. J. Thompson

Recirculation of dinoflagellate cysts by the mussel, Mxlihis cdiilis L.. at an aquaculture site contaminated by

Alexandrium fundyense { Lebour) Balech 47 1

Rodrigo Caspar Soria, Marcela Susana Pascual, and Victor Hugo Fernandez Cartes

Reproductive cycle of the cholga paleta. Atriini seminuda Lamarck. 1819 (Bivalvia: Pinnidae) from northern

Patagonia, Argentina 479

Qin-Feng Gao, Ka-Kei Mak, and Siu-Gin Cheung

The physiological ecology of black-ribbed mussels, Septifer virgatus (Wiegmann) (Bivalvia: Mytilidae) on a

sub-tropical wave-exposed shore in Hong Kong 489

Nestor Fernando Ciocco and Ana Maria Gayoso

Microalgal food of the ribbed mussel Aiiliuomra atra (Molina, 1782) in Golfo Nuevo (Patagonia, Argentina) 497

John Brake, Jeffrey Davidson, and Jonathan Davis

Triploid induction of Mylihis editlis using 6-dimethylanimopurine 503

Pauline Kamermans and Aad C. Smaal

Mussel culture and cockle fisheries in The Netherlands: finding a balance between economy and ecology 509

Dirk A. Campbell and Maeve S. Kelly

Settlement of Poimiloceros triqiieter (L.) in two Scottish lochs, and factors determining its abundance on mussels

grown in suspended culture 519

Per Dolmer

Mussel dredging: impact on epifauna in Limfjorden, Denmark 529

Katherine A. Ross, John P. Thorpe, Trevor A. Norton, and Andrew R. Brand

Fouling in scallop cultivation: help or hindrance? 539

Laura Schejter, Claudia S. Bremec, Rut Akselman, Daniel Hernandez, and Eduardo D. Spivak

Annual feeding cycle of the Patagonian scallop Zygoclilamys patagoiuca (King and Brodenp, 1832) in Reclutas bed

(39S-55 "W), Argentine Sea 549

Karin B. iMhrmann, Andrew R. Brand, and Stephen W. Feist

Comparison of the parasites and pathogens present in a cultivated and in a wild population of scallops (Argopecten

piirptiiaHis Lamarck. 1819) in Tongoy Bay, Chile 557

CONTENTS CONTINUED ON INSIDE BACK COVER

MBL WHill 1 IKRARY

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