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PROCEEDINGS
OF THE

Marine Biological Laborat-jfy"
LIBRARY

JUL 81973

WoMli Nolo, ivtui.

NATIONAL SHELLFISHERIES ASSOCIATION

OFFICIAL PUBLICATION OF THE NATIONAL SHELLFISHERIES

ASSOCIATION; AN ANNUAL JOURNAL DEVOTED TO

SHELLFISHERY BIOLOGY

VOLUME 63

Published for the National Shellfisheries Association, Inc. by
Economy Printing Co., Inc., Easton, Maryland

JUNE 1973

THIS VOLUME IS DEDICATED TO
THE MEMORY OF
DR. TAKEO IMAI

The inquiry, knowledge and belief of truth is
the sovereign good of human nature.

-Bacon

PROCEEDINGS

OF THE

NATIONAL

SHELLFISHERIES

ASSOCIATION

Volume 63 - June 1973
CONTENTS

Dedication to Dr. Takeo Imai ii

List of Abstracts by Author of Technical Papers Presented at the 1972 NSA Convention v

Abstracts:

NSA Convention 1

NSA Pacific Coast Section 7

Jose A. Carreon

Ecomorphism and Soft Animal Growth of

Crassostrea iredalei (Faustino) 12

Ramesh C. Dwivedy

A Study of Chemo-Receptors on Labial Palps of the

American Oyster Using Microelectrodes 20

Frederick C. Kopfler and Jack Mayer

Concentration of Five Trace Metals in the Waters and

Oysters {Crassostrea virginica) of Mobile Bay, Alabama 27

Darryl J. Christensen

Prey Preference of Stylochus ellipticus in Chesapeake Bay 35

Haskell S. Tubiash, Sara V. Otto and Rudolph Hugh

Cardiac Edema Associated with Vibrio anguillarum

in the American Oyster 39

Frederick G. Kern, L. Cecelia Sullivan and Michio Takata

Labyrinthomyxa-\\ke Organisms Associated with Mass

Mortalities of Oysters, Crassostrea virginica, from Hawaii 43

Richard J. Marasco

An Appraisal of the Alternative Earning Power of the

Maryland Oystermen 47

Edwin Rhodes and Warren S. Landers

Growth of Oyster Larvae, Crassostrea virginica, of Various

Sizes in Different Concentrations of the Chrysophyte,

Isochrysis galbana 53

John R. Maclnnes and Frederick P. Thurberg

A New Technique for Measuring the Oxygen Consumption of

Larvae of the American Oyster, Crassostrea virginica 60

Judith S. Baab, Gerald L. Hamm, Kenneth C. Haines, Arthur Chu and Oswald A. Roels

Shellfish Mariculture in an Artificial Upwelling System 63

♦ William Duggan

Growth and Survival of the Bay Scallop, Argopecten irradians,

at Various Locations in the Water Column and at Various Densities 68

^Thomas J. Costello, J. Harold Hudson, John R. Dupuy and Samuel Rivkin

Larval Culture of the Calico Scallop, Argopecten gibbus 72

Guy C. Powell, Brian Shafford and Michael Jones

Reproductive Biology of Young Adult King Crabs, Paralithodes

camtschatica (Tilesius) at Kodiak, Alaska 77

Rodner R. Winget, Donald Mauer and Leon Anderson

The Feasibility of Qosed System Mariculture:

Preliminary Experiments with Crab Molting 88

iii

Lynn Goodwin

Effects of Salinity and Temperature on Embryos of the

Geoduck Clam (Panope generosa Gould) 93

Sara V. Otto

Hermaphroditism in Two Species of Pelecypod MoUusks 96

H. Dickson Hoese

Abundance of the Low Salinity Clam, Rangia cuneata, in

Southwestern Louisiana 99

John M. Flowers

Pattern of Distribution of the Surf Clam (Spisula solidissima) in the Point Judith,

Rhode Island Harbor of Refuge 107

Association Affairs 113

LIST OF ABSTRACTS BY AUTHOR OF TECHNICAL PAPERS
PRESENTED AT THE 1972 NSA CONVENTION

Edwin Cake

Larval Cestode Infections in Several Edible Bivalve Mollusks from the Vicinity

of St. Teresa, Florida 1

Melbourne R. Carriker

Discovery of Duck System in Accessory Boring Organ of Urosalpinx

cinerea follyensis by Scanning Electron Microscopy 1

Russell J. Down

The Materials, Methods and Politics of Off-Bottom High Density

Oyster Farming in Cape May County, New Jersey 1

Ramesh C. Dwivedy

Design of an Experimental Self-Supporting, Closed Cycle Oyster

Culture System 2

Susan E. Ford

Recent Trends in the Epizootiology of Minchinia nelsoni (MSX)

in Delaware Bay 2

Warren S. Landers

Early Development in the Ocean Quahog, Arctica islandica (L.) 3

Vance P. Lipovsky and Kenneth K. Chew

Laboratory Control of Pacific Oyster Mortality by Manipulation

of Temperature and Nutrient Concentration 3

Joseph G. Loesch and Dexter S. Haven

Preliminary Estimates of Growth Functions and the Size-Age Relationship

for the Hard Clam, Mercenaria mercenaria, in the York River, Virginia 3

Carol Moore and Albert F. Eble

Cytology and Cytochemistry of Amebocytes of Mercenaria mercenaria 4

Lawrence A. Olsen

Comparative Functional Morphology of Feeding Mechanisms in Rangia

cuneata (Gray) and Polymesoda caroliniana (Bosc) 4

A. Dean Parsons

Prey selection in the Oyster Leech, Stylochus ellipticus 4

Edwin H. Powell

A Potential Use of the Waste Heat Byproducts of a Steam Turbine

Electric Generating Plant 5

Jon Rittgers

Surf Clams and Society: A Rationale for Sound Management 5

George A. Valiulis and Harold H. Haskin

Resistance of Crassostrea virginica to Minchinia nelsoni

and Labyrinthomyxa marina 6

ABSTRACTS OF THE NSA PACIFIC COAST SECTION

Nancy J. Ellifrit, Marvin S. Yoshinaka and Donald W. Coon

Some Observations of Clam Distribution at Four Sites on Hood

Canal, Washington 7

Gary G. Gibson and Dennis S. Lund

A Pilot Economic Study of Oyster Raft Culture in Yaquina Bay,

Oregon 7

R. B. Herrmann

Clam Distribution and Abundance in Grays Harbor as Related to

Environmental Factors 7

Victor L. Loosanoff

Cultivation of Green Mussel in New Zealand 8

Dennis S. Lund

Feeding Studies with Pacific Oyster Larvae 8

David Miyauchi, George Kudo and Max Patashnik

Test for Flavor Differences in Pacific Oysters Related to

Differences in Growing Areas or Methods of Culture 8

David Miyauchi, Max Patashnik and George Kudo

Fish Protein Used to Bind Pieces of Minced Geoduck 9

Richard A. Neve

A Chemical Assay for Paralytic Shellfish Poisoning 9

Russell G. Porter

Preliminary Report on Growth Rate and Reproductive Cycle

of the Soft-Shell Clam at Skagit Bay, Washington 9

A. J. Scholz

Preliminary Evaluation of Oyster Seed Holding-Trays 10

D. W. Smith and N. Bourne

Larval Development of the Piddock, Zirphaea pilsbryi

Lowe 10

Douglas R. Squire

The Japanese Oyster Drill, Ocenebra japonica Dunker,

in Netarts Bay, Oregon 10

Frieda B. Taub, Kathleen Ballard and Fred Palmer

Production of Shellfish Feed by Continuous Algal Culture 10

Christopher Weller and Kenneth Chew

Experiments in Oyster Raft Culture at Clam Bay, Washington 11

R. E. Westley

A Partial Review of Problems and Prospects of the Pacific

Coast Oyster Industry 11

ABSTRACTS OF TECHNICAL PAPERS PRESENTED
AT THE 1972 NSA CONVENTION

LARVAL CESTODE INFECTIONS IN

SEVERAL EDIBLE BIVALVE MOLLUSKS

FROM THE VICINITY OF

ST. TERESA, FLORIDA

EdVin Cake

Department of Oceanography

Florida State University

Tallahassee, Florida

Twenty-five specimens of three edible bivalves,
Atlantic-Bay Scallops, Argopecten irradians concen-
tricus (Say), Sunray Venus Clams, Macrocallista
nimbosa (Lightfoot), and Atlantic Surf Clams,
Spisula solidissima raueneli (Conrad) collected from
the vicinity of St. Teresa Beach, Florida, were
examined for larval cestode parasites. Phyllobothriid
plerocercoids of the genus Echeneibothrium
(Beneden) were found free in the stomach and
digestive diverticula of A. irradians and S. solidis-
sima. One immature phyllobothriid of the genus
Rhodobothrium (Linton) was recovered from a
capsule in the mantle cavity of one M. nimbosa.
Encysted lecanicephalid metacestodes of the genus
Polypocephalus (Braun) were found in the visceral
masses of A. irradians, and of the genus Tylo-
cephalum (Linton) in the visceral masses of all three
species and in the foot musculature of the two clam
species. Encysted plerocercoids of the trypanor-
hynch, Parachristianella dimegacantha (Kruse), were
found in the intestine walls of all three bivalve species
and in the foot musculature of the two clam species.
All five cestode genera encountered have elasmo-
branchs as final hosts and are not known to be harm-
ful to man. Quantitative data are presented on the
cestode larvae from each species and some
cestode-load and host-size relationships are discussed.
Bivalve hosts of the same five cestodes are reported
incidentally from a related, unpublished study of
marine mollusks in the same area.

DISCOVERY OF DUCT SYSTEM IN

ACCESSORY BORING ORGAN OF

UROSALPINX CINEREA FOLLYENSIS

BY SCANNING ELECTRON MICROSCOPY

Melbourne R. Carriker

Systematics - Ecology Programs

Marine Biological Laboratory

Woods Hole, Massachusetts

Accessory boring organs (ABO) of adult snails
were excised, fixed slowly with agitation in increasing
concentrations of glutaraldehyde, and prepared for
examination in the scanning electron microscope by a
freeze dry technique developed by T. Otaka and
S. Honjo (SEM, 1972/11, Proc. Workshop Biol.
Specimen Prep. Techn. SEM, III. Res. Inst., p.
359-363).

Examination of the exterior of ABOs' with the
scanning electron microscope revealed for the first
time a large number of ducts which open conspicu-
ously at the surface among the microvilli. Each duct,
when dilated, was edged by a conspicuous flange. The
ducts were traced into the interior of the ABO in
fracture sections of the gland. Earlier studies with the
transmission electron microscope (Nylen, Provenza,
and Carriker, Amer. Zool. 9: 935-965) revealed
star-shaped dilations among the groups of secretory
cells. It is suggested these dilations may be a part of
the duct system. The function of the ducts is still
unclear.

THE MATERIALS, METHODS AND POLITICS

OF OFF-BOTTOM HIGH DENSITY OYSTER

FARMING IN CAPE MAY COUNTY,

NEW JERSEY

Russell J. Down

P. O. Box 156

Cape May Court House

New Jersey

In 1966, Minchina nelsoni (MSX) - resistant oyster

ABSTRACTS

seed on surf clam shell obtained from the Delaware
Bay side of the Cape May peninsula was transferred
to a natural shell bed in Holmes Creek, a tributary of
the Great Sound, on the ocean side of the peninsula.
All live oysters had disappeared from formerly abun-
dant natural live beds in this area - possibly as a result
of decades of pesticide effect upon larval stages. In
1967-68, two 1,000 x 50 ft lagoons were dredged in
a tidal marsh at the entrance of Holmes Creek, and
the growth of oysters in these lagoons, both in racks
and on vertically suspended punched surf clam shell,
was monitored. During 1968-72, a reusable cultch
assembly of scrap tire beads strung in stacks was
devised and a method for growing oysters using
this material was tested and patented. Growth to
market size of oysters on shell or tire beads was
found to require two years from time of set.

In 1969, the local county government, with ap-
proval from the State Health Department, erected a
sewage treatment plant and outfall pipe within one
tidal cycle of this operation thereby causing it to be
condemned for shellfish harvesting.

During 1970-71, 10% of the rafting of oysters
originally planned for 1969-70 was completed, and
the actual amount and potential loss due to con-
demnation was documented. These oysters are be-
ing maintained suspended from rafts. Documented
actual production from 1970 set equals 95 bu,
plus projected production from approximately
150,000 one year old oysters of the 1971 set
equals 450 bu or a total of 545 bu. Potential
yield per two year period from full rafting in the
two lagoons equals 545 x 10, or 5,450 bu.

DESIGN OF AN EXPERIMENTAL

SELF-SUPPORTING, CLOSED CYCLE

OYSTER CULTURE SYSTEM

Ramesh C. Dwivedy

University of Delaware

College of Agriculture Sciences

Newark, Delaware

The present study describes a unique system that
has been designed, built and is under experimentation
to grow oysters in closed cycle under controlled
environment. The system essentially consists of two
oyster growing tanks with one common biologi-
cal-mechanical filter, charcoal-fiber filters, a bank of
UV lights, water treatment system with ozone and
algal culture system. Importance and use of ozonating
recycling water in such a system are discussed. The
system is unique in two respects. First, the oyster
culture system is coupled with an algal culture system

so that a regulated amount of algae is fed to oysters
and the algal culture tanks are refilled with sterilized
sea-water from the ozone treatment tank. Second, the
complete system has been automated with the help of
electrical timers, pumps, solenoid valves, ball valves,
etc. Only usual maintenance is required.

RECENT TRENDS IN THE EPIZOOTIOLOGY

OF MINCHINIA NELSONI (MSX) IN

DELAWARE BAY'

Susan E. Ford

Oyster Research Laboratory

N. J. Agricultural Experiment Station

Rutgers University

New Brunswick, New Jersey

Activity of the oyster pathogen Minchinia nelsoni
(MSX) has fluctuated markedly in lower Delaware
Bay since it was first recorded there in 1957. Intense
disease pressure and heavy mortalities associated with
the onset of the epizootic in the late 1950's had
dropped to almost negligible levels by the early
1960's. In 1963, MSX activity began to rise and from
1964 through 1967, disease levels were as high as
those recorded during the first years of the epizootic.
A downward trend began in 1968 and, except for a
moderate resurgence in 1970, has continued. Disease
levels resulting from the 1971 infection period were
lower than at any time since the early 1960's. Wide-
spread, heavy mortalities of the type experienced
during the onset of the epizootic have not been re-
peated despite periods of high MSX activity, although
populations of susceptible seed oysters continue to
experience heavy losses when transplanted to epizoo-
tic areas. Additional evidence of resistance has been
soen in an increased tendency to maintain infections
at low, non-lethal levels. This has been noted in all
oysters, but is particularly evident in oyster stocks
native to the lower Bay, which have been exposed to
heavy selective pressure for 15 years.

Two annual peaks in MSX prevalence levels, of
approximately equal height, were seen in oysters in
lower Delaware Bay during the high activity years of
1964-67. These occurred during the winter and late
spring, were often in the 70 - 90% range, and were the
result of early summer and late summer-fall infective
periods respectively. Winter peaks just prior to and
after the years of high disease activity rarely exceeded
50%.; the spring peak was even more abbreviated and
occasionally not seen, indicating an infective period
restricted mainly to early summer.

Monitoring of upper Bay seed beds, whose normal
mid-tide salinities range from 10 - 16 %o , indicates

PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

that MSX in low salinity areas has followed the same
fluctuations as in higher salinity regions. The high
activity years of the mid-1960's coincided with a
severe drought when salinities throughout the Bay hit
peaks 2 - 5°oo above normal, and when the dura-
tion of above average salinities was lengthy and coin-
cided with the infective and immediate post-infective
period. In the middle of the drought, MSX extended
as far up bay as the upper-most of the productive seed
beds where it had not been since the first years of the
epizootic. At the same time, salinities on the lower
seed beds had become high enough to permit MSX
activity comparable to that in the lower Bay. When
salinities are normal, disease levels on the seed beds
are light to non-existent.

Not only was salinity implicated in heightened
disease activity during a period of drought, but high
salinity areas of the lower Bay continually sustain the
highest levels of MSX. Nevertheless, salinity does not
explain all the phases of MSX activity recorded dur-
ing the past 15 years, particularly the early part of
the epizootic when high disease activity coincided
with normal salinity. To explain these fluctuations it
will be necessary to look for factors other than salini-
ty and in addition to resistance of the oyster popula-
tion.

' Supported under PL 88-309 contract 3-3-R-7 with
the National Marine Fisheries Service.

EARLY DEVELOPMENT IN THE OCEAN
QUAHOG, ARCTICA ISLANDICA (L.)

Warren S. Landers

U. S. Department of Commerce

National Oceanic and Atmospheric Administration

National Marine Fisheries Service

Middle Atlantic Coastal Fisheries Center

Laboratory for Experimental Biology

Milford, Connecticut

The normal spawning season of ocean quahogs
in southern New England waters is late summer.
Attempts to ripen mahogany clams out of season
in the laboratory produced limited success. Clams
obtained from the field in late fall and subjected
to a water temperature of 10°C and ample algal
food for 10 weeks failed to ripen. However, clams
obtained from the fishery in late winter and sub-
jected to the same regimen ripened in about 5
weeks.

Ripe clams could not be induced to spawn by
rapidly increased temperature, rapidly decreased
temperature or a sperm suspension. A few untreated,
stripped eggs were found to be fertilizable by stripped

sperm; however, fertilization and the percent develop-
ment of stripped eggs to normal larvae were signifi-
cantly increased when the eggs were exposed to dilute
ammonium hydroxide before fertilization was at-
tempted.

The earliest, fully developed, straight-hinge lar-
vae are about 110 /u long and 80 jj. wide and have
an unusually long hinge line. Metamorphosis takes
place when the larvae are approximately 200 iJi
long. Larvae were reared to metamorphosis at 10°C in
about 60 days.

LABORATORY CONTROL OF PACIFIC OYSTER
MORTALITY BY MANIPULATION OF
TEMPERATURE AND NUTRIENT
CONCENTRATION

Vance P. Lipovsky and Kenneth K. Chew

College of Fisheries

University of Washington

Seattle, Washington

Temperature and nutrient were found to be criti-
cal environmental factors in abating or initiating a
laboratory mortality of adult Pacific oysters. A signi-
ficant mortality did not occur until the temperature
of the seawater was 18 C and above. Prior condition-
ing of oysters at temperatures below 18°C resulted in
a lowered mortality rate. Enrichment of the seawater
with a nutrient medium increased the rate of death.
Ultraviolet light treatment of the seawater reduced
the mortality to the level of the control oysters. The
research gives support to the contention that a micro-
organism is responsible for the mortality.

PRELIMINARY ESTIMATES OF GROWTH

FUNCTIONS AND THE SIZE-AGE

RELATIONSHIP FOR THE HARD CLAM,

MERCENARIA MERCENARIA, IN THE

YORK RIVER, VIRGINIA

Joseph G. Loesch and Dexter S. Haven

Virginia Institute of Marine Science
Gloucester Point, Virginia

Two groups of hard clams ranging from the small-
est size practical for individual marking through the
larger sizes (approximately 30 - 90 mm in length)
were measured, code-marked and planted in similar
natural substrates at two locations in the York River.
Both groups have been harvested, remeasured and
planted annually, and growth functions determined
from length increments.

ABSTRACTS

CYTOLOGY AND CYTOCHEMISTRY OF
AMEBOCYTES OF MERCENARIA MERCENARIA

Carol Moore and Albert F. Eble

Trenton State College

Department of Biology

Trenton, New Jersey

Amebocytes of Mercenaria mercenaria were classi-
fied into cell types by a variety of microscopical and
cytochemical procedures. Three different amebocyte
types were identified: a small (28^) motile granulo-
cyte, a large (45^1) non-motile granulocyte and an
agranulocyte (5m)- The small granulocyte comprised
61% of the total cell population; it had four distinct
types of granules in the cytoplasm. The large granulo-
cyte made up 37% of the cell population; this granu-
locyte possessed the same four types of granules but
contained approximately one-third the number found
in the smaller granulocyte. The agranulocyte had no
visible granules with only a thin peripheral rim of
cytoplasm surrounding the nucleus. The four types of
granules observed in granulocytes in decreasing order
of abundance were; (1) a large (LS^i) blunt type, (2)
a small (0.7m) dot-like type, (3) a large (Iju) sperical
refractile type and (4) a rod-shaped type approxi-
mately 2jL( in length.

The nucleus of all cell types appeared morphologi-
cally similar having uniformly dispersed chromatin
and a rim of chromatin lining the nuclear membrane.
Supravital studies with Janus Green B showed a
preferential uptake by the large, blunt granules. With-
in 10 min the dye had been converted to the red-re-
duction product, diethyl safrnin. When neutral red
was applied supravitally, both the large blunt granules
and small dot-like granules took up the dye. The
color changed from red to yellow in about one-half
hour.

Studies with esterases indicated a strong non-speci-
fic esterase in the small granulocyte. Acid phospha-
tase and NADH dehydrogenase cytochemical studies
are presently under investigation.

COMPARATIVE FUNCTIONAL MORPHOLOGY

OF FEEDING MECHANISMS IN

RANGIA CUNEATA (GRAY) AND

POLYMESODA CAROLINIAN A (BOSC)

Lawrence A. Olsen

Florida State University

Department of Oceanography

Tallahassee, Florida

Preliminary investigations of the functional

morphology of feeding in two estuarine clams are
discussed and compared. The clams, Rangia cuneata
(Gray) and Polymesoda caroliniana (Bosc), were taken
from the same location in the Ochlockonee River
estuary, south of Tallahassee, Florida, on the north-
west Florida Gulf coast. Both clams are morphologi-
cally filter feeders. R. cuneata is shown to have typi-
cal mactrid gill and palp morphology and ciliary cur-
rents similar to other mactrids. P. caroliniana, a
corbiculid, is in the same general gill-palp articulation
category as R. cuneata. Ciliary current pathways on
the gills, visceral mass and mantle in each species are
similar. P. caroliniana, however, appears to possess
more ridge currents on the palps than R. cuneata. The
P. caroliniana palps are also free dorsally, whereas
those of R. cuneata are attached by most of their
dorsal margins, thus limiting their movement.

R. cuneata does not possess a fourth pallial aper-
ture as do some other members of the Matridae for
the ejection of pseudofeces. The orientation of the
waste canal and the siphonal membrane, however, is
such that pseudofeces are extruded through the in-
halent siphon upon quick closure of the valves. P.
caroliniana has no waste canal, but pseudofecal
material is ejected in a manner similar to that in R.
cuneata.

Qualitative samples of stomach and gut contents
of both clams from the same habitats are similar in
composition, including many diatoms, algae and
much unidentifiable material.

PREY SELECTION IN THE OYSTER LEECH,
STYLOCHUS ELLIPTICUS^

A. Dean Parsons

Oyster Research Laboratory

N. J. Agricultural Experiment Station

Rutgers University

New Brunswick, New Jersey

Several different prey preferences have been
previously reported for Stylochus ellipticus. In the
present study, it was determined that preferences
differed from worm to worm and were influenced
by previous diet history. Stylochus when offered
several prey showed the greatest preference for
those on which the worms had been feeding when
collected.

Diet patterns in adult Stylochus were very rigid
and mature worms could not be induced to feed on
certain prey although starved for as long as 30 - 40
days. Evidence was collected indicating selection pat-
terns may be established very eariy in the life cycle of
Stylochus.

PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

An experiment was conducted for six weeks to
determine if prey density could be correlated with
food preferences shown in laboratory feeding experi-
ments. A small population of Stylochus found at the
mouth of Dias Creek in Delaware Bay was selected
for the study. Each week 20 - 30 worms were collect-
ed and estimates were made of the density of four
prey found with the worms: Mya arenaria, Modiolus
demissus, Nassarius obsoletus, and Odostomia impres-
sa. Worms brought to the laboratory were isolated
individually in small aquaria and all four prey species
given as food. Feeding rates were determined on
each prey and an analysis of variance performed.

There was a significant difference between the
feeding rates of the worms on each of the prey for all
six weeks. Preference for Mya was high initially but
decreased sharply. Initial preference for Nassarius was
low but increased as the preference for Mya de-
creased. Modiolus and Odostomia were the least pre-
ferred of the prey. A relationship was noted between
preference and density of the prey. As density of Mya
decreased, the preference decreased. Similarly, an
increase in the density of Nassarius was accompanied
by an increase in preference. Densities of Modiolus
and Odostomia remained low throughout the
six-week period, as did the preferences for these prey.

It is suggested that planktonic Stylochus may be
able to establish wherever suitable prey exist and that
food selection patterns will be determined by the
density of those prey. Several interesting questions
are posed: (1) Can planktonic worms delay meta-
morphosis if suitable prey are not found; (2) Is it
possible that food preferences are established even
earlier than suggested, when both predator and prey
are in the plankton?

' Supported under PL 88-309 contract 3-3-R-3 with
the National Marine Fisheries Service.

A POTENTIAL USE OF THE WASTE

HEAT BYPRODUCTS OF A STEAM

TURBINE ELECTRIC GENERATING PLANT

Edwin H. Powell

Windmill Point Oyster Company

Urbanna, Virginia

and

Potomac Electric Power Company

Washington, D. C.

The basic concept under investigation was that
warm water in the electric generating plant canal
could be beneficially used to provide small cultch-free
oyster spat with normal growth in advance of the
regular season. As applied to the temperatures avail-

able at the generating plant on the Potomac River,
seed oysters could be cultured in the canal beginning
in early March for later planting on conventional
oyster beds in the river during mid-April. This proce-
dure could give up to three months early growth
advantage thus allowing possible harvest the following
November.

A full annual cycle test, utilizing over a thousand
hatchery produced cultch-free seed oysters, (divided
into 14 groups and counted and measured at appro-
priate intervals) showed no significant difference in
survival of the groups located in the canal, the
Potomac River and the Rappahannock River.

SURF CLAMS AND SOCIETY:
A RATIONALE FOR SOUND

MANAGEMENT

Jon Rittgers

U. S. Department of Commerce

National Oceanic and Atmospheric Administration

National Marine Fisheries Service

Gloucester, Massachusetts

Biologists identify four phases in the historical
development of a commerical fishery: 1) the early
period when landings are low; 2) the developmental
period when landings are growing rapidly; 3) the peak
period when landings reach a high level and are main-
tained for a period of time; and 4) the period of de-
cline when landings are falling due to reduction in
stocks brought about by various causes which may or
may not include overfishing. Economists identify
three stages of production which are similar to the
four biological phases of development: Stage I, when
physical returns to investment in the firm (or indus-
try) are increasing at an increasing rate; Stage II,
when returns to investment are increasing but at a
decreasing rate; and Stage III, when further invest-
ment will bring about a reduction in total output.

Some evidence suggests that the surf clam industry
is reaching (and perhaps is well into) the third biologi-
cal phase of development. The exact stage of econom-
ic development is not cleariy defined, but it would
appear that Stage III has not been reached. Thus the
surf clam industry is in the enviable position of not
having to reduce levels of employment and capital
investments to ensure continuing vitality of the indus-
try. It need only cope with the common property
institution which will, if left unaltered, inevitably
lead to operating biologically and economically in the
final (and least desirable) stages of development.

To overcome the problems inherent in the com-
mon property institution, immediate steps should be

ABSTRACTS

taken to develop a system of property rights designed
to delineate resource tenure (ownership rights) and to
ensure high levels of certainty in tenancy (use rights).
State and Federal Government assistance may be
required to solve the problems which will arise in
developing such a system.

RESISTANCE OF CRASSOSTREA VIRGINICA
TO MINCHINIA NELSONI AND
LABYRINTHOMYXA MARINA^

George A. Valiulis and Harold H. Haskin

Oyster Research Laboratory

N. J. Agricultural Experiment Station

Rutgers University

New Brunswick, New Jersey

I*revious reports from this laboratory have indi-
cated an innate resistance to mortality in certain
stocks of Crassostrea uirginica due to Minchinia net-
soni (MSX). Whether there is also a resistance in some
stocks of C. uirginica to Labyrinthomyxa marina, and
whether this resistance (if present) can be correlated
with that for MSX. was the objective of this experi-
ment.

Four laboratory-reared stocks of C. uirginica of
known resistance to MSX were injected with 7 graded
dosages of L. marina cells ranging from 10 to 100,000
cells/oyster. The source of infective inoculum was the
minced, infected tissue of dead oysters collected from
the field. Approximately 2,200 oysters were kept in
aquaria in aerated, running sea water maintained at
28 - 30°C and about 20 %„ salinity for a test period
of 105 days.

The control groups of oysters (uninjected, and
injected with uninfected oyster tissue mince) never
showed infection with L. marina, as determined by
fluid thioglycollate culture and sectioning. Of the
oysters that died in the experimental groups 316
were examined for L. marina and 89'^f of these were
found to be infected; of these 83% had heavy or very
heavy systemic infections. Examination of oysters

still living at the termination of the experiment
showed a trend of progressively higher incidence and
weighted incidence of L. marina in each group receiv-
ing a higher dose regardless of stock.

Sections of the initial live samples and of live
samples at the termination of the experiment showed
that there was a 3 - 20% incidence of light MSX in-
fection which was random and had no correlation
with the dosage of L. marina injected. Mortality due
to L. marina in each of the experimental groups of
oysters was obtained by subtracting the highest con-
trol mortalities of the same stocks from the total
mortalities of the experimental groups of the same
stocks.

Comparisons of final percent cumulative mortal-
ities of the four stocks indicated no distinct differ-
ences in resistance at higher doses of L. marina (500 -
100,000 cells/oyster). However, at lower doses (10 -
100 cells/oyster) one stock showed a consistently
greater susceptibility (2.4 - 11.8 times greater) than
the other three stocks.

Comparisons of the resistances of these stocks of
oysters to L. marina under low dose, laboratory con-
ditions with the resistances of these same stocks to
MSX under field conditions showed that: (1) The two
stocks that were most resistant to MSX were also
resistant to L. marina; (2) The stock most susceptible
to MSX was also the one most susceptible to L.
marina; (3) The stock moderately susceptible to MSX
was resistant to L. marina.

From the results of this experiment and from
other field experiments (the results of which are not
reported here) the trend seems to be emerging that
stocks of oysters most susceptible to MSX are also
the most susceptible to L. marina, those most re-
sistant to MSX are also resistant to L. marina; how-
ever, those stocks which are moderately resistant (or
susceptible) to MSX may or may not be resistant to
L. marina.

' Supported under PL 88-309 contract 3-3-R-3 with
the National Marine Fisheries Service.

PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

NSA PACIFIC COAST SECTION

SOME OBSERVATIONS OF CLAM

DISTRIBUTION AT FOUR SITES

ON HOOD CANAL, WASHINGTON

Nancy J. Ellifrit, Marvin S. Yoshinaka and
Donald W. Coon

U. S. Department of the Interior

Bureau of Sport Fisheries and Wildlife

Portland, Oregon

Personnel of the Bureau of Sport Fisheries and
Wildlife, Division of River Basin Studies, conducted a
study of intertidal shellfish populations at 4 sites on
Hood Canal in March and April, 1972. The purpose
of the study was to determine whether bulkheads and
attendant fill -n the upper intertidal levels have an
effect upon shellfish.

Samples were collected along 4 transects perpen-
dicular to the shoreline at each site. Two transects
were located in front of a bulkhead and 2 on an
adjacent natural beach. Sampling stations were
located at 10 ft intervals on the transects. A sample
of substrate V4 m^ and approximately 8 in deep
taken at each station was sorted through 1 in and V4
in mesh screens, and all clams were saved for classi-
fication and measurement.

At 3 of the sites more than tviice as many clams
were found on natural beaches than on bulkheaded
beaches. There was significant difference between
bulkheaded and natural beaches at 2 sites in numbers
of Japanese littleneck clams, Venerupis japonica,
found in the upper intertidal area. There was also a
trend toward differences in size and distribution.
Clams inhabiting lower intertidal levels did not seem
to be affected by bulkheads.

Several hypotheses for the differences were pro-
posed. The most probable explanation is the change
in current patterns associated with bulkheads which
result in less favorable conditions for settling and
survival of clam larvae. These conditions also may
cause a reduction in availability of nutrients and
food.

A PILOT ECONOMIC STUDY OF

OYSTER RAFT CULTURE IN

YAQUINA BAY, OREGON

Gary G. Gibson and Dennis S. Lund

Fish Commission of Oregon and

Newport Oyster Co.

Newport, Oregon

An oyster raft 12 x 20-feet was anchored in
Yaquina Bay in June 1971. One hundred and

forty-six strings of unbroken Japanese oyster seed
were suspended from the raft. Labor and material
costs were recorded.

Costs of concrete anchors, piling, boom logs and
equipment for harvesting on a larger scale were deter-
mined and added to the actual costs of construction,
stringing and planting. The combination of these
actual and estimated expenses amount to $1.12/6 ft
string. Other expenses such as transportation, tools,
insurance, rent, attorney and accountant fees and
administration were estimated to be $ .25/6 ft string
(based on an average annual producuion of 80,000
strings per year).

Production from the raft after 6 months was 16.7
bu of cocktail-sized oysters (100/pint). The oysters
brought $20/bu in the shell or $334. Potentially, a
raft will support 204 6 ft strings or 34 bu worth
$680 or $3.33 per string.

A gross profit of $3.33/string, minus $1.37/string
for expenses, equals $1.96 net profit per string or
$400 per raft per year.

CLAM DISTRIBUTION AND ABUNDANCE

IN GRAYS HARBOR AS RELATED

TO ENVIRONMENTAL FACTORS

R. B. Herrmann

Weyerhaeuser Company
Longview, Washington

From 1967 - 69 surveys were conducted in Grays
Harbor to determine the distribution and abundance
of native and introduced clams. Nine species includ-
ing 4 softshell-type and 5 hardshell-type clams occur
in the bay. The softshell clams, especially Mya
arenaria, are the most numerous and have the widest
distribution, occurring from within a mile of the bay
mouth eastward to the mouth of the Chehalis River
at Hoquiam. The hardshell-type clams occur mostly
in the western portion of the bay. Clinocardium nut-
talli has the widest distribution in this group, occur-
ring from the mouth of the bay east to Johns River
and Neds Rock.

Clam distributions are discussed in relation to
seasonal levels of salinity and pulp mill effluents.
Persistent conditions of lov/ salinities throughout
most of the bay in winter are thought more import-
ant in limiting the colonization of greater portions of
the bay by hardshell clams. Summer pulp effluent
levels have had little apparent effect on the coloniza-
tion of softshell type clams in the eastern bay areas,
where highest levels occur.

ABSTRACTS

Densities of softshell type clams, excepting
Cryptomya californica, were independent of substrate
organic levels between 0.5% and 3.0% and moisture
content between 10% and 50%. Densities of Mya
arenaria were greater on coarser substrates while finer
substrates had greater densities of Macoma natsuta,
however. Clinocardium nuttalli and Vernerupis
japonica colonized substrates with lower organic
levels, 0.5% - 1.5%, and a particle size similar to that
colonized by Mya.

CULTIVATION OF GREEN MUSSEL
IN NEW ZEALAND

Victor L. Loosanoff

Pacific Marine Station

University of the Pacific

Greenbrae, California

The article describes various methods of cultiva-
tion of the New Zealand green mussel, Pema can-
aliculus, which at present is grown on a relatively
small scale in New Zealand, but the farming of
which seems to offer many promising possibilities.

This mussel, which is native to both the North and
South Islands of New Zealand, lives in water in en-
vironments closely resembling those of the Pacific
Northwest and northern California shores.

Methods of cultivation, rate of growth under dif-
ferent conditions and other aspects of biology, ecolo-
gy and cultivation of these bivalves were discussed.

FEEDING STUDIES WITH
PACIFIC OYSTER LARVAE

Dennis S. Lund

Department of Fisheries and Wildlife

Oregon State University

Marine Science Center

Newport, Oregon

Growth and setting of Pacific oyster larvae fed
Isochrysis galbana were compared with larvae fed
brewers yeast and 4 dry artificial diets prepared by
Dr. Samuel Myers of the Louisiana State University
Food Science Department. The dry diets most effect-
ive in promoting oyster growth were composed of
single-cell protein (yeast), fish meal and solubles, soy-
bean meal, whey, rice bran and vitamins. The com-
ponents of the dry rations were bound with starch or
alginate and dried to form particles of low solu-
bility in seawater.

When 20,000 cells/ml of Isochrysis was fed as a
supplement with the dry diets FDSC 1102-71 P. W.

Flake and TC 1119-71 2 A for the last 10 days
prior to metamorphosis, larvae set as well as those
fed 80,000 cells/ml of Isochrysis. However, in the
absence of the supplemental cilgae, larvae failed to
grow or set well. Optimum feeding level of the
dry rations appears to be 1-2 mg/1 fed once per
day. Concentrations of 4 mg/1 and above greatly
reduced setting of larvae.

Brewers yeast produced erratic results when fed to
larvae for 10 days prior to setting. As in the case of
the dry rations, 10,000-20,000 cells/ml of supple-"
mental algae in addition to the yeast was neces-
sary for larval growth. When fed brewers yeast at
50,000 cells/ml immediately before setting, how-
ever, larvae set much more densely than when fed
50,000 cells/ml of algae.

Larvae of less than 140 ii occasionally grew very
well on the dry rations, but more often growth
was considerably less and mortality significantly
higher than in cultures fed Isochrysis. Brewers
yeast was never a suitable food for larvae of less
than 140 m-

Larvae fed 100,000 cells/ml of Isochrysis for
3-5 days prior to setting set at least 3 times more
densely than larvae fed 50,000 cells/ml, and 10
times more densely than larvae fed 25,000
cells/ml.

TEST FOR FLAVOR DIFFERENCES IN
PACIFIC OYSTERS RELATED TO
DIFFERENCES IN GROWING AREAS
OR METHODS OF CULTURE

David Miyauchi, George Kudo and
Max Patashnik

U. S. Department of Commerce

National Oceanic and Atmospheric Adminstration

Pacific Fishery Products Technology Center

National Marine Fisheries Service

Seattle, Washington

It is a common opinion that Pacific oysters raised
in Hood Canal have a milder flavor than those raised
in other Washington waters, such as Southern Puget
Sound, and that oysters raised "off bottom" have a
milder flavor than those raised "on bottom." During
the Winter of 1971 and the Spring of 1972, the
Pacific Fishery Products Technology Center at Seattle
in cooperation with the Washington State Depart-
ment of Fisheries conducted sensory tests to compare
the flavor of oysters grown near Quilcene in Hood
Canal and in Southern Puget Sound. We also com-
pared the flavor of oysters grown on the bottom and
those grown off the bottom suspended from floats. In

PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

triangle tests, our experienced panel was able to
detect differences in flavor between "on bottom"
oysters raised in Hood Canal and those raised in
Southern Puget Sound. Based on the ability of the
panel members to reproduce their results, the
difference in flavor between these oysters was
statistically significant but equivocal from a prac-
tical point of view. The panel could not distinguish
flavor differences between "off bottom" oysters
grown in Quilcene and in Southern Puget Sound.

When the flavors of Hood Canal oysters raised
"on bottom" were compared vnth those raised "off
bottom," the panel reported a detectable difference
that was statistically significant but not clear-cut.
The same was true of Southern Puget Sound oysters
raised "on bottom" and "off bottom."

FISH PROTEIN USED TO BIND
PIECES OF MINCED GEODUCK

David Miyauchi, Max Patashnik and
George Kudo

U. S. Department of Commerce

National Oceanic and Atmospheric Administration

National Marine Fisheries Service

Pacific Fishery Products Technology Center

Seattle, Washington

In 1970 when the State of Washington started
leasing subtidal geoduck beds for commerica!
harvesting, our laboratory in cooperation with the
Washington State Department of Fisheries obtained
yield data, palatability scores, and information on
cold-storage characteristics of the various edible
components of the geoduck.

The fledgling geoduck processing industry, which
consists of 4 or 5 small processors, requested our
aid in finding a suitable binder with which to make
marketable patties out of the trimmings from their
prime geoduck steaks. In response to this request,
we prepared frozen blocks of minced geoduck, using
our fish binder made from rockfish flesh and
common food ingredients. Breaded portions pre-
pared from 1/4 and V2 in. thick slices from the blocks
were judged to be an improvement over those now
being prepared commercially. The experimental
samples held together well during deep-fat frying
and pan frying. Samples of experimental blocks
have been given to the various geoduck processors
for their evaluation and modification.

A CHEMICAL ASSAY FOR
PARALYTIC SHELLFISH POISONING

Richard A. Neve

Institute of Marine Science

University of Alaska

Douglas, Alaska

Saxitoxin separated from contaminating similar
substances on Amberlite XE-64 can be coupled with
2,4-dinitrofluorobenzene yielding a brilliant
orange-yellow precipitate. This N-substituted,
2,4-dinitroaniline compound is solubilized in ethyl
alcohol. Spectrophotometric analysis revealed a sin-
gle, shatply spiked peak at 372 millimicrons. The test
was developed on certified toxin provided through
the courtesy of the U. S. Food and Drug Administra-
tion. The test has been carried out on toxic butter
clams from Porpoise Island near Juneau, and on razor
clams from beaches throughout Southeast Alaska,
Prince William Sound, and Unmak Island in the
Aleutians. A positive reaction was also observed using
cultures of Gonyaulux catenella kindly provided by
Dr. Ken Chew and Louisa Norris, Department of
Fisheries, University of Washington. Negative results
were observed on other planktonic species: Prorocen-
trum micans, Ostreopsis menotas, Amphidinium
operculatum and Peridinium trochodium. The latter
species were kindly provided by Dr. Richard Norris,
Department of Botany, University of Washington.

PRELIMINARY REPORT ON GROWTH RATE

AND REPRODUCTIVE CYCLE OF THE

SOFT-SHELL CLAM AT SKAGIT BAY,

WASHINGTON

Russell G. Porter

Washington Cooperative Fishery Unit

University of Washington

Seattle, Washington

Growth rate and the annual reproductive cycle of
the soft-shell clam, Mya arenaria L., are being studied
at the Skagit River delta in Puget Sound, Washington.
A brief explanation of the research and methods is
presented. Sampling began in November, 1970 and
will continue through Spring, 1973. The annual
reproductive cycle during 1971 is described and the
various stages of gonadal development enumerated.
Spawning commences a little later for smaller clams,
but in general lasts from late May through early
September. In 1971 peak spawning occurred at Skagit
Bay during July. A general comparison between the
spawning cycle at Skagit Bay and those from studies

ABSTRACTS

along the east coast from Canada to Maryland is
presented.

PRELIMINARY EVALUATION OF OYSTER
SEED HOLDING-TRAYS

A. J. Scholz

Washington State Department of Fisheries
Brinnon, Washington

Seed oysters (Crassostrea gigas) usually suffer
50-75% mortality within the first year of planting
due to siltation, crowding and predation. Oyster
seed held in trays for 4 months and then planted
had twice the survival as oyster seed initially
planted on the ground (evaluation made at 11
months). The growth of the tray-reared seed was
the same as the ground-reared control seed.

LARVAL DEVELOPMENT OF THE PIDDOCK,
ZIRPHAEA PILSBRYI LOWE

D. W. Smith and N. Bourne

Fisheries Research Board of Canada
Nanaimo, British Columbia

localized on a sandy low-tide island in the tail of
Netarts Bay, was the subject of a general investiga-
tion. The drills are inactive in the winter; sheltered
beneath relict Crassostrea gigas left on the crown of
the island by a defunct commercial operation. Egg
capsules are deposited on the relicts in May and June.
Prey items did not include oysters, but were chiefly
cockles, Clinocardium nuttalli, and less abundant
bivalves. Protoconch juveniles were first observed in
August, 1971; Macoma inconspicua and juvenile C.
nuttalli were their major prey. Data from spat-baited
wire traps furnished a good index of adult
distribution, and indicated a dovmshore postspawning
movement followed by a return to the relicts in the
fall.

Aquarium-held snails fed single-prey diets of
oyster, cockle, and Olivella biplicata for 2 months
were tested for prey preference, along with starved
and naive (field) drills. Statistical comparison (X^
homogeneity) of these data demonstrated prey choice
reflected dietary history (Ingestive Conditioning), and
confirmed that the cockle was the most important
prey item in the field.

Implimented control measures consist of the
construction of oyster shell heaps at strategic points
on the island followed by removal of the shell and
predators in the late fall of 1972.

Larvae of the rough piddock, Zirphaea pilsbryi
Lowe, were cultured at 2 temperatures, 15 and
20 C. The larvae have a characteristic round or
circular shape, a dark band around the margin of
the shell, a purple color near the ventral margin and
a pink umbone region. At 15°C, larvae had a mean
shell length increment of 4.6 Ai/day and settled in
35-40 days; at 20°C the mean shell length
increment was 6.7 ju/day and settlement occured in
25-29 days. Metamorphosis occured when the larvae
had a shell length between 240 to 300 m-

THE JAPANESE OYSTER DRILL,

OCENEBRA JAPONICA DUNKER,

IN NETARTS BAY, OREGON

Douglas R, Squire

Oregon State University

Marine Laboratory

Port Orford, Oregon

A viable population of Ocenebra japonica,

PRODUCTION OF SHELLFISH FEED BY
CONTINUOUS ALGAL CULTURE

Frieda B. Taub, Kathleen Ballard and
Fred Palmer

University of Washington
College of Fisheries
Seattle, Washington

A continuous algal culture apparatus of 32
liters (8 gal.) was developed which was capable of a
sustained daily yield of 2.0 x 10' ' cells consisting of
2-5 g ash free-dry weight oi Monochrysis lutheri. This
is a considerably greater yield than could be realized
from this amount of space or effort, had traditional
batch cultures been used.

The protein content of the cells varied from 7-45%
of dry weight but not in the relatively orderly manner
shown in the one liter continuous culture experi-
ments.

Culture units of this size produce enough cell
material for feeding trials of millions of oyster or

PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

dam larvae, thousands of seed animals, or a few
adults.

Scaling up to full hatchery size represents a further
stage of development.

EXPERIMENTS IN OYSTER RAFT CULTURE
AT CLAM BAY, WASHINGTON

Christopher Weller and Kenneth Chew

University of Washington
College of Fisheries
Seattle, Washington

Oyster raft culture was initiated at Clam Bay on
Central Puget Sound in May, 1971. A smaller experi-
mental operation was also set up at Seabeck Bay.
Preliminary information obtained at these 2 sites is
presented.

Spacings between oyster strings of 20, 30 and 40
cm did not appear to effect differences in growth
through December, 1971 in Clam Bay. There was a
significant growth difference of 1.7 cm in length
between Clam Bay and Seabeck Bay by December.

The mussel, Mytilus edulis, and the barnacle,
Balanus glandula, were the most important competi-
tors with respect to effect upon oysters. Observations
at Clam Bay show that barnacles may undermine the
attachment of oysters to cultch. Mussels were severe
competitors at Seabeck Bay. By April, 1972, fouling
comprised principally of mussels, amounted to 89%
of wet weight per cultch. Many oysters appeared
stunted.

The seastar, Evasterias troschelli, set on oyster
strings in the early summer of 1971 at Clam Bay. By
July of 1972, average radius length was 7.9 cm. There
was a significant difference between spacings in num-
ber of seastars per string. The numbers were 2.0 for
the 20 cm, 0.5 for the 30 cm, and 0.4 for the 40 cm
spacing. Damage to mussels and oysters related direct-
ly to numbers and distribution of seastars. In July,

oyster damage was not yet extensive. At the 20 cm
spacing, 4 percent of the cultch demonstrated
signs of attack upon oysters. Oyster damage at the
other 2 spacings was negligible. Mussels were more
severely affected. There was evidence of predation
on 29, 5 and 4 percent of the cultch at the 20,
30 and 40 cm spacings respectively.

A PARTIAL REVIEW OF PROBLEMS AND

PROSPECTS OF THE PACIFIC COAST

OYSTER INDUSTRY

R. E. Westley

Washington State Department of Fisheries
Brinnon, Washington

Some of the problems facing the Pacific Coast
oyster industry are: obtaining an adequate supply of
seed oysters at a feasible price; offsetting the prob-
lems of adult mass mortality; and culturing around
oyster drills. General improvement in methods of
oyster culture, particularly obtaining better first year
survival of seed, is important. Increased competition
for use of water areas may also cause future prob-
lems.

On the plus side, the vast supply of relatively
unpolluted, nutrient-rich water gives this area a major
advantage. Recent efforts locally to upgrade an oyster
product based on Crassostrea gigas, and the tremen-
dous interest in France for use of C. gigas as a gour-
met oyster would suggest that we should take a
second look at the different possibilities of using C.
gigas.

In review of the problems of oyster production
nationally, it would appear that, while the Pacific
Coast area has problems, these have solutions more
readily available than is the case in other areas of the
country. It would appear that with proper organiza-
tion and effort a substantial increase in oyster pro-
duction could be made in this area.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

ECOMORPHISM AND SOFT ANIMAL GROWTH
OF CRASSOSTREA IREDALEI (FAUSTINO)

Jose A. Carreon

INSTITUTE OF FISHERIES DEVELOPMENT AND RESEARCH

COLLEGE OF FISHERIES, UNIVERSITY OF THE PHILIPPINES

DILIMAN, QUEZON CITY, THE PHILIPPINES

ABSTRACT

Marked ecomorphism in Crassostrea iredalei (Faustino) has been measured and
established for three groups of oysters grown by different methods of culture-stick,
hanging and broadcasting. The indexes of ecomorphism developed by the author are
0.84, 0.87 and 0.76 for the groups mentioned in the above order.

Shell volume, which is greatly affected by ecomorphism, has a curvilinear relation-
ship with the weight of the soft animal. From bimonthly samples taken over a
two-year period the shell volume and dry meat weight ratios were established as
73.52, 69.59 and 58.31 mg/cc for stick, hanging and broadcast grown specimens,
respectively. These values were taken as measures of the physiological well-being of
the soft animal.

INTRODUCTION

In general, oyster culture may be categorized
into bottom and off-bottom techniques. The
methods involved in either case influence the shell
formation rather sharply and hence, the growth of
the animal as a whole. Shaw (1965) and Shaw and
Merrill (1966) observed that suspending oysters off
the bottom helped improve the condition of their
meats and promoted a faster growth rate.

Shells of oysters grown on the bottom in tidal
flats were quite different from those of the same
species cultured off-bottom. This ecomorphic
tendency is particularly noticeable in Crassostrea
iredalei, the most important commercial species in
the Philippines, but not in C. malabonensis
(Faustino) which are commonly found growing in
the same areas.

Shuster (1957) stated that he " . . . believed
that information on the relationship of growth
patterns to environmental factors will give addi-
tional insight into the lives of these mollusks, and
thus, may be of practical value in the management
of shellfish crops." The subject of this paper is in
consonance with Shuster's investigation; namely
the intricate relationships between shell formation
as affected by methods of culture (ecomorphism)' ,
and the growth of the soft animal.

STUDY SITE - BACOOR BAY
Bacoor Bay, approximately 10 km^ in area, is
12 km southwest of Manila North Harbor and
almost directly south of a former U.S. military
naval restricted area at Sangley Point, Cavite
Province, Philippines (Fig. 1).

Specimens were collected in the approximate
center of the bay and within a 0.50 km radius.
This area includes an oyster farm of the Philippine
Fisheries Commission and a few private oyster
beds. Rainfall, solar radiation and tides that occur-
red in the bay area at the time of study are given in
the appendix.

METHODS AND MATERIALS

Live specimens were usually collected at bi-
monthly intervals from December 1969 - October
1971. Collections consisted of 12 samples each of
oysters cultured by stick (S) and broadcast (B)
methods and 10 samples of the hanging (H) oys-
ters. Each sample averaged from about 50 - 100-t-
specimens.

Immediately after collecting, live oysters were
scrubbed clean and classified according to Carreon

'Ecomorph: Infraspecific growth of species in
response to special environment.

ECOMORPHISM AND GROWTH OF OYSTERS

FIG. 1. Map portion of Manila Bay indicating location of Bacoor Bay. Scale 1:30,000 at Lat. 14° 32\ traced
from a map made by the Philippine Coast and Geodetic Survey.

(1969). The oysters were partly opened by care-
fully cutting through the hinge ligament. A syringe
needle was then inserted through the opening and
2 - 3 ml of 10 - 15% formalin solution were in-
jected. After the treated specimens had set over-
night, they were shucked and the soft meat
washed with dilute formalin to remove extraneous
materials. The meats were drained for 2 hr and
then individually weighed in tared paper boats.
Specimens were dried in an oven at 50 - 60°C for
48 hr.

The shell measurements included longer and
shorter axes of the left valve in centimeters (Fig.
2) and the shell volume in cubic centimeters. The
oysters under study were equilateral, therefore, the
longer and shorter axes of both valves were more
or less equal.

Soft modelling clay was used to obtain volume
measurements. Each shell was meticulously loaded
so that the valves fitted together in a normal posi-
tion. The volume of the molded clay was then
measured by displacement.

RESULTS

Ecomorphism in S, B and H Samples

Generally, specimens from S samples were later-
ally concave on the left valve and closely des-
cribed the curve of the cross-section of bamboo
post used for attachment (Fig. 3). The shell was
more or less dorso-ventrally equimorphic, rounded
or blunt at the lip region, and with a moderate to
very deep cavity near the hinge. Shell outline was
less elongate than others, commonly oval to sub-
quadrate.

J.A. CARREON

FIG. 2. Pattern for taking shell measurments to as-
sume near elliptical outlines; A, in the case of
shell with growth axis more or less straight: B, for
shells dorso-ventrally deflected or vice-versa; C, for
shells levo-dextro deflected or vice-versa. Longer
axis = average measurements of 1, 2 and 3; short-
er axis = average of 4. 5 and 6.

In contrast, B shells were regularly elongate,
with older specimens wide, thin and flat at the lip
region or posterior end, rarely deep near the hinge
(Fig. 4). H shells were moderately elongate and
normally subtrigonal to oval in outline. The left
valve was regularly deep at the hinge region. (Fig. 5).

The Index of Ecomorphism

The great individual diversity in the shell forma-
tion of C. iredalei makes it very difficult to
establish the specific shell measurement which
would give the best fitting measure of the degree
of ecomorphism. Of the three dimensional relation-
ships of bivalve shells cited by Galtsoff (1964),
only the shell height-shell area regression measure-
ment seemed to slightly differentiate the very
apparent trend of ecomorphism in each of these
three groups of oysters (Fig. 6). However, the in-
formation gathered did not yield clear-cut values
that would comparatively distinguish one group
from the other. For this reason, the author
decided to use shell volume as a function of
ecomorphism and to correlate observed volume to
relative volume (volume of a sphere) whose surface
area was equivalent to the plane shell area
(assumed) with a configuration presumed nearly
elliptical in outline. This assumed shell area was
calculated from the observed dimensions of the
shell's longer and shorter axes (Fig. 2).

Since a sphere contains the greatest volume

FIG. 3. Ecomorphic group of C. iredalei grown by
the stick method. Shells above are in girdle view
while below they are shown correspondingly in
right valve view.

NOTE: All shells of Figures 3-5 show the weak
edges of right valves slightly trimmed away to
facilitate volume measurements by the use of
modelling clay.

with the least surface area as compared to any
other volumetric configuration, the ratio of ob-
served shell volume to that of an assumed
spherical volume will approach unity as the eco-
morphic shell becomes deeper, and much lower
than unity as the shell becomes more flat and shal-
lower. Also, a shell that is flat and shallow has a
greater surface area in proportion to its actual
volume which in turn is much less than the
volume of a spheroid assuming the same surface
area. Based on this principle the indexes of eco-
morphism of S, H and B specimens collected in
this study were computed (Table 1).

Statistics of Shell Volume - Meat Weight Relation-
ship

All the statistical analyses on the shell volume

ECOMORPHISM AND GROWTH OF OYSTERS

FIG. 4. Ecomorphic group of C. iredalei grown by
the broadcast method. Shells above are in girdle
view while below they are shown correspondingly
in right valve view.

FIG. 5. Ecomorphic group of C. iredalei grown by
the hanging method. Shells above are in girdle
view while below they are shown correspondingly
in right valve view.

TABLE 1. Shell areas, volumes and indexes of ecomorphism of
Crassostrea iredalei grown by three different methods of culture.
(All values tabulated are averages of total collections of each
group.)

Sample
Group

Area
(cm^)

Volume (cm^)
a b

Vo Vs

Index of
Ecomorphism^

Stick

Hanging

Broadcast

25.0040
24.4392
32.5206

9.8653

9.7544

13.3386

11.7426
11.2499
17.4820

0.84
0.87
0.76

Observed shell volume content.

Volume of sphere whose surface area = plane shell area as-
sumed nearly elliptical in outline.

'^The ratio of Vo to Vs.

J.A. CARREON

FIG. 6. A: Scatter diagram of shell area us shell height of three ecomorphic g)-oups of C. iredalei;
B: Logarithmic regression of shell area on shell height of the same data in A.

and soft animal weight relationship were computed
by the IBM System/360 in which the Xs and Ys
represented the volumes (mm^) and weights
(mgm), respectively. The nature of x and y rela-
tions was studied in two regression equations,
namely

1) Rectilinear: Y = a + bX

2) Exponential: Y = aX ; and based on the
IBM output the best line of fit for the regression
of meat weight on shell volume is curvilinear in
each group, the degree of curvilinearity being dif-
ferent in each case (Fig. 7).

A comparative summary of the statistical para-
meters obtained by equations 1 and 2 above is
presented in Table 2 (IBM output). From this
Table, the respective exponential equations may
thus be written as follows;

S Group: Y = -19.43 X^

Log Y = 1.03151 Log X - 1.28847

;i.03151

H Group: Y = -30.43 X^-^'^^'^^

Log Y = 1.07673 Log X - 1.48331

B Group: Y = -61.78 X^-^^^^^

Log Y = 1.13991 Log X - 1.79082

Peters and Van Voohris (1940) recommended
that the correlation coefficient, as computed from
the actual sample, must be invariably shown to

differ from 0 , hence its standard error: 5 r = ^ ,

and probable error: P.E.r = 0.6745 xor must' be
computed for samples of the same size whose true
r = 0. Using these formulas, highly significant
values were obtained as tabulated:

Sample Group

5 r

P.E.r

0.036273

0.024484

0.037087

0.025024

0.036394

0.024552

ECOMORPHISM AND GROWTH OF OYSTERS

i^5i- ■•■•

FIG. 7. The regression of dry meat weight on shell volume of three ecomorphic groups of C. iredalei.

TABLE 2. Comparative IBM statistical results between rectilinear and exponential regressions of
meat weight on shell volume of Crassostrea iredalei (Faustino).

Statistical

parameter

Y = a + bX

Y = aX''

Y = a + bX

Y= aX^

Y = a + bX

Y = ax''

761

■do-

728

-do-

756

-do-

Mean

727.16162

2.74721

683.09058

2.69052

835.50244

2.83453

Std. deviation

513.97534

0.33972

493.68896

0.39975

552.70166

0.28881

Correlation

Xvs Y

0.83110

0.86776

0.85543

0.88861

0.82731

0.87189

Intercept of

Y on X

-77.37354

-1.28847

28.69775

-1.48331

-105.56641

-1.79082

Regression

coefficient

0.08267

1.03151

1.06822

0.07673

0.07319

1.13991

Std. error of

reg. coef.

0.00196

0.02144

0.00153

0.02063

0.00181

0.02332

Std. error

of estimate

281.34497

0.16895

255.86574

0.18347

310.68384

0.14152

Computed

T value

42.15930

48.10270

44.50340

52.20300

40.44020

48.98010

F value

1,777.41

2,313.87

1,980.56

2,725.15

1,635.41

2,390.25

J.A. CARREON

FIG. 8. Graphical comparison on the periodical

condition of three ecomorphic groups of oysters

grown by different methods. Condition of soft

animal expressed in milligmms of dry meat per
milliliter of shell volume.

The significance of the statistical parameters
obtained for exponential regression of meat weight
on shell volume were rather acceptable even at P
> 0.001 when referred to the tables of Fisher and
Yates (1957).

Ecomorphism and the Growth of the Soft Animals
Shell volume is perhaps one of the primary fac-
tors affecting the physiology of the soft animal.
When the valves are tightly closed for several
hours at certain intervals within and/or between
tidal cycles, the animal is protectively sealed with-
in a limited amount of space. How much and to
what extent this affects the soft animal is rather
difficult to discern. The author believes that the
meat weight, as a measure of growth, is greatly
affected by all the biophysical activities of the
animal in producing its shell. In one way or the
other, the growth and general well-being of the
soft animal may be related to some parameters of
the shell, particularly shell volume and variations
in shell form as a result of ecomorphism. Follow-
ing this belief, it may be further stated that eco-
morphic groups within the same species which at-
tain greater volume with least shell surface area
will tend to exhibit better meat growth than those
animals vrith a lower volume and greater shell sur-
face.

As a result of the data thus gathered, and
supported by the statistical parameters shown in
Table 3, an analysis of variance was conducted to
measure the significance of the differences in meat

weights of the three ecomorphic groups. The
variance ratio was, F = 16.6789 which exceeded
the table value of 6.91 when n-^ = 2 and ng = in-
finity, with P > 0.001 (Fisher and Yates, 1957).
From all observations, it appears that in terms
of meat weight, oysters of the S ecomorphic
group were in better condition than those of the
other two groups. Oysters of the broadcast
method were the poorest in weight throughout
most of the study as shown in Figure 8. This
graph is based on the computed well-being of the
soft animal per sampling time expressed in mili-
grams of dry weight per cubic centimeter of shell
volume. The annual values obtained are as follows:

1970 1971

S 70.65 mg/cc 79.19 mg/cc

H 66.74 mg/cc 70.70 mg/cc

B 59.65 mg/cc 54.90 mg/cc

For all the samples gathered throughout the
period, the average shell volume-meat weight ratios
are 73.52 mg/cc, 69.59 mg/cc and 58.31 mg/cc
for S, H and B groups, respectively.

LITERATURE CITED

Carreon, J. A. 1969. The malacology of Philippine
oysters of the genus Crassostrea and a review of
their shell characters. Proc. Natl. Shellfish.
Assoc. 59: 104-115.

Fisher, R. A. and F. Yates. 1957. Statistical
Tables for Biological, Agricultural, and Medical
Research. 5th ed. Oliver and Boyd, Edinburgh,
and Hafner Publ. Co. N. Y. 138 p.

Galtsoff, P. S. 1964. The American oyster
Crassostrea virginica Gmelin. U. S. Fish and
Wildl. Serv. Fish. Bull. 64: 1-480.

Peters, C. C. and W. R. Van Voohris. 1940.
Statistical Procedures and Their Mathematical
Bases. McGraw-Hill Book Co.,N.Y. 516 p.

Shaw, W. N. 1965. Pond culture of oysters - past,
present, and future. Trans. 30th N. Am. Wildl.
Nat. Res. Conf. p. 114-120.

Shaw, W. N. and A. S. Merrill. 1966. Setting and
growth of the American oyster, Crassostrea
virginica, on navigation buoys in the lower
Chesapeake Bay. Proc. Natl. Shellfish. Assoc.
56: 67-72.

Shuster, C. N. Jr. 1957. On the shell of bivalve
moUusks. Proc. Natl. Shellfish. Assoc. 47:
34-42.

ECOMORPHISM AND GROWTH OF OYSTERS

APPENDIX

Major ecological factors in Bacoor Bay.

Rainfall*^

Solar

Tide

(feet)'^

(mm)

Radiation

Ref:MLLW

gm cal/
1970

cm"
1971

1970

1971

Month

1970

1971

Highest

Lowest

Highest ]

jQwest

4.5

4.3

Jan

342

360

4.2

-1.7

3.9

•1.3

Feb

0.5

488

346

3.8

-1.5

3.7

-1.0

Mar

464

415

4.1

-1.1

4.2

-0.6

Apr

474

466

4.6

-0.8

4.5

-0.6

May

215

490

416

5.0

-1.1

4.7

-0.6

Jun

256

368

366

401

5.1

-1.2

4.7

-0.8

Jul

284

296

351

391

4.9

-1.0

4.8

-0.7

Aug

222

135

319

448

4.3

-0.6

4.6

-0.4

Sep

347

132

308

408

4.2

-0.3

4.5

0.0

Oct

277

451

223

321

4.3

-0.3

4.7

-0.2

Nov

390

221

324

4.4

-0.9

4.6

-0.8

Dec

155

244

352

-1.6

-1.2

As recorded in Bacoor, Cavite.

^As recorded by the nearest weather station located at latitude 14°39' north and
longitude 121° 04' east.

^As predicted by the Phillipine Coast and Geoditic Survey with reference to the
station at Manila.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

A STUDY OF CHEMO RECEPTORS ON LABIAL PALPS OF THE AMERICAN OYSTER

USING MICROELECTRODES'

R. C. Dwivedy

AGRICULTURAL ENGINEERING DEPARTMENT

UNIVERSITY OF DELAWARE

NEWARK, DELAWARE

ABSTRACT

Tungsten microelectrodes, insulated except for their tips, were used to pick up
receptor potentials from chemical receptors on labial palps of the American oyster.
A functional criterion was used to determine when a microelectrode had penetrated
a receptor cell. The receptor cell responded with differential sensitivity in response
to four major taste substances. An equation was derived which defined the relation-
ship between taste receptor potential and strength of chemical stimulant.

INTRODUCTION

The two pairs of labial palps of the oyster
which lie at the anterodorsal side of the body
under the mantle hood are joined together into a
single unit which serves primarily for the final
sorting of food particles and for the delivery of
the food to the mouth (Galtsoff, 1964). Research-
ers in the past have shown that an oyster may
reject food which has no value to it (Lotsy, 1895;
Grave, 1916; Loosanoff, 1949; Ukeles, 1970).
They speculated that the labial palps possess
chemical receptors. A previous study by the
author confirmed this speculation by demonstrat-
ing electrophysiologically the existence of chemicjil
sensors on the palps (Dwivedy, 1972)^. The ob-
jective of the present study was to further the
previous study in order to define characteristics of
these chemical sensors.

' Published as Miscellaneous Publication No. 658
with the approval of the Director of the Dela-
ware Agricultural Experiment Station. Publication
No. 2 in the Department of Agricultural En-
gineering.

^Dwivedy, R. C. 1972. Instrumentation and
technique of electrophysiological studies of chem-
oreceptors on labial palps of the American oys-
ter. Paper #72-513, American Society of Agricul-
tural Engineers, St. Joseph, Michigan.

ANATOMY AND HISTOLOGY OF THE PALPS
Anatomy

A detailed study of structures of the labial
palps has been made by Galtsoff (1964). A synop-
sis of his work is quoted in part as follows:

"The four soft flaps which lie at the antero-
dorsal side of the body under the mantle hood are
labial palps (Fig. 1). The two pairs of palps, one
on each side, are joined together into a single
unit. Each pair consists of one external and one
internal palp. The two external palps join together
above the mouth where they form the upper lip;
the two internal palps are united below the mouth
into a lower lip. As a result of this arrangement,
the mouth is an irregularly shaped, narrow, curved
slit. Both lips are arched; the lower one is shorter
and its edge is thicker than that of the upper lip."

Histology

Galtsoff (1964) has also studied the histology
of the labial palps in detail. A synopsis of his
work is quoted in part below:

"Each labial palp consists of a layer of con-
nective tissue covered on both sides by columnar
ciliated epithelium set on a basement membrane.
The epithelium of the smooth surface of the palp
consists of almost cubical cells with relatively large
nuclei and small cilia (Fig. 2). Cell boundaries are
distinct, the cells themselves are crowded and com-
pressed, and there is a very thin and transparent
cuticle on the periphery. In the subepithelial layer

CHEMO-RECEPTORS ON LABIAL PALPS OF OYSTERS

Upper

Lip Mouth

■il ci;ii.Led epithelium cells

inopnilic cell
cous cells

muscle fibers

vesicular cells of
connective CiGSues

FIG. 1. Labial Palps of the American Oyster (Re-
printed from Galtsoff 1964).

FIG. 2. Cross section of the smooth side of labial
palp of the American Oyster (Reprinted from
Galtsoff, 1964).

large eosinophilic cells and mucous ceils are very
abundant. The palps are innervated by the nerve
emerging from the cerebral ganglion and entering
the anterior end of the junction between the
paired lobes."

METHODS

The labial palps and part of the gills of adult
American oysters, Crassostrea virginica, were ex-
posed by drilling or breaking through the anterior
and posterior portions of the flat valve of a spec-
imen; this was done without causing injury to the
underlying tissues. A recording microelectrode was
inserted in one of the labial palps and a reference
electrode was inserted in the gill. Two Narsheage
MM3 micromanipulators were used to hold the
electrodes and to regulate the depth of probing.
An optical microscope with a magnification of
lOOx was used during probing of the electrodes.

Tungsten microelectrodes were manufactured by
using technique described by Hubel (1957). Tung-
sten vnres were electropolished until a final tip
diameter of about In was achieved. The electrodes
were then washed in detergent and were insulated,
except for their tip, with a clear stone-mudge
coating material. The impedance of these elec-
trodes, measured in preparation at about 70° F,
was approximately 75 megohms.

Electrical responses of the labial palps were fed

to a Tektronic Dual Trace Oscilloscope, through a
Model P16 D.C. microelectrode amplifier manu-
factured by the Grass Instrument Co. The
oyster-electrode preparation was housed in a cop-
per Faraday cage to prevent stray electrical pick
up by the electrodes. The recording of electrical
responses of the labial palps upon their chemical
stimulation has been termed as Electropalpusgram,
hereafter referred to as EPG.

The recording set up is shown in Figure 3. The
oyster was probed using the technique described
previously. The electrodes were connected through

al Under Test

FIG. 3. Schematic diagram showing set-up for si-
multaneous recording of EPG and latency period.

R.C. DWIVEDY

the amplifier to the upper beam of the oscillo-
scope. A burette was used to drop liquid stimu-
lants over the oyster palps. The open and unin-
sulated ends of two stiff copper wires were placed
just underneath the burette outlet but above the
oyster palps. These two wires were connected
through a 0.5 volt dry cell to the lower beam of
the oscilloscope. During passage, the chemical drop
completed the open D.C. circuit resulting in a sig-
nal of the oscilloscope just before stimulating the
palps. The time difference between this signal
from D.C. circuit and the onset of electrical re-
sponse from labial palps was the sum of the two
time components, time for the chemical drop to
travel from the D.C. circuit to the palps and the
latent period of electrical response of the palps.
The first component was measured by replacing
the oyster preparation by another similar D.C. cir-
cuit which was substracted from the total time lag
to obtain actual latent period.

It is a standard practice to make functional
identification of the particular type of cell or re-
ceptor that initiates an observed electrical response
to a chemical stimulus. For example, a single
nerve fiber dissected free from the chorda tympani
nerve is assumed to be a taste fiber (and not a
temperature, tactile, pain etc., which are also
found in the same nerve bundle) if it responds to
low or moderate concentrations of appropriate
taste stimuli applied to the surface of the tongue.
Since it was not possible to see taste cells at the
surface of the palps, a similar functional criterion
was established for the purpose of this study. The
palps were traversed until a sudden decrease in
D.C. potential was measured; this indicated that
the electrode had penetrated a cell. If the D.C.
potential did not change upon chemical stimula-
tion, then another location was sought. On the
other hand, if the potential did change upon
stimulation, then the penetrated cell was assumed
to be an active taste cell. The sudden decrease in
D.C. potential upon penetration of the electrode
was considered to be the resting potential of the
cell and depolarization of the cell acted as recep-
tor potential. A large number of attempts were
made usually before such a cell was found.
Furthermore, no appreciable response has ever
been observed when the microelectrode was
penetrated into other parts of the body, such as
gills. To verify that the electrical responses were
not just the result of artifacts of electrode, the
oyster was killed by injecting NaCn into its body
after responses had been recorded. The responses
ceased completely when the oyster was dead.

FIG. 4. A typical electrical response of the chemi-
cal sensors in labial palps of the oyster, referred
to as Electropalpiisgram (EPG). Upper trace is
EPG, lower trace is signal from DC circuit for
latency measurement.

Receptor potential of the chemo-sensor in labial
palp was measured in response to distilled water
which served as a response to solutions with zero
molarity. Larger responses were observed as the
concentration was increased. The strength of the
test solution was increased until the receptor
potential (magnitude of negative wave at the onset
of EPG) ceased to increase. Distilled water rinses
were applied to the labial palp between stimuli.
Taste receptor potentials were plotted against
molar concentrations of solutions.

RESULTS

The recording of a typical electrical response
(EPG) of the chemo-sensors of labial palps is
shown in Figure 4. The sensors respond to chemi-
cal stimulation by a sharp negative wave followed
by a slow positive wave (with respect to the refer-
ence electrode in the gills). The D.C. circuit re-
sponds vifith rather a sharp spike (lower trace in
Fig. 4) upon contact of a chemical drop to the
open ends of wires (Fig. 3). The time that it took
the chemical drop to travel from the ends of the
wires to the labial palps was measured as about 25
milliseconds. This time period was subtracted from
the total time lag between the two signals in
Figure 4 to obtain actual latent period of the
receptors. The usual magnitude of latent periods
was about 50 milliseconds for the several chemi-
cals that were tested in this study.

Four distinct taste submodalities are recognized
in human; sweet, salt, bitter and sour. These sub-

CHEMO-RECEPTORS ON LABIAL PALPS OF OYSTERS

0 0.01 O.OS 0.1 0.15 0.20 0.25 0.3 0.35 0.4 0.45 0.5 0.55
Molarity of Stimulant, H

FIG. 5. Taste receptor potential plotted as a func-
tion of molar concentration of sucrose.

modalities are associated with four major sub-
stances, which in the same order are; sucrose,
sodium chloride, quinine sulfate, and hydrochloric
acid. Pure solutions of these four major substances
were tested in this study. Resulting curves are
given in Figures 5, 6, 7 and 8. Each point in
these curves represents an average value of three
recordings from individual oysters. Variation within
corresponding individual readings was insignificant-
ly small for a given solution. The receptor poten-
tial obtained in response to sodium chloride
diminishes as the concentration of solution is in-
creased until it reaches the saturation point. Con-
versely, the receptor potentials with other test
chemicals increase as the concentration of the
solution is increased up to saturation.

Slopes of the curves represented in Figures 5,
6, 7 and 8 were measured at several points by us-
ing a half-silvered mirror. Perpendiculars to the
curves were drawn by positioning the mirror so
that the portion of the curve reflected in the mir-
ror matched the curve behind the mirror. The
slopes of these curves were plotted against molar-
ity of test solution on semi-logarithm graphs. The

0 0.01 0.05 0.1 0.15 0.20 0.25 0.3 0.35 0.4 0.45 0.5 0.55
Molarity of Stimulant . M

FIG. 7. Taste receptor potential plotted as a func-
tion of molar concentration of quinine sulfate.

resulting plots, as shown in Figures 9, 10, 11 and
12 are straight lines with negative slopes. The mag-
nitude of the slope of the straight line multiplied
by 2.3026 to convert to naperian logarithms is
denoted by K and is shown in each plot.

DISCUSSION

It is reasonable to assume that the magnitude
of negative wave at the onset of EPG (Fig. 4) is a
measure of sensitivity of the sensor cell. On this
basis, it is evident by examination of curves repre-
sented in Figures 5, 6, 7 and 8 that the sensitivity
of the sensor cell differs for chemicals tested in
this study. Differential sensitivity of the receptors
indicates the possibility that an oyster is probably
able to discriminate between different chemicals.
Further studies are required to prove or disprove
this assumption.

The cubical ciliated epithelium cells (Fig. 2) are
the only cells that are probably sensory cells. By
using a histological technique as described by
Bultitude (1958), attempts were made without suc-
cess to localize the electrode tip after recording

a 0 0.01 0.05 0.1 0.15 0.20 0.2S 0.3 0.35 0.4 0.45 0.5 0.55 0.6

^ Molarity of Stimulant, M

FIG. 6. Taste receptor potential plotted as a func-
tion of molar concentration of sodium chloride.

0 O.OOi 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.1

Molarity of Stimulant. M

FIG. 8. Taste receptor potential plotted as a func-
tion of molar concentration of hydrochloric acid.

R.C. DWIVEDY

0 0.01 0.05 0.1 0.15 0.20 0.25 O.J 0.35 0.4 0.&5 0.5 0.55 0.& 0.65

FIG. 9. Relationship between logarithm of slope
values and molar concentration of sucrose.

the responses. The reason for this failure was that
the diameter of the colored spot was about 20^.1
whereas the size of the cells is much smaller.
Moreover, a slight movement of the tip of the
electrode during this experiment caused widespread
marking. For this reason, any definite statement
about the origin of the electrical responses record-
ed from labial palps cannot be made. However,
there is a high probability that these electrical
responses were the result of depolarization of the
membrane of the ciliated epithelium cells (Fig. 2).

Characteristic Equation for Taste Receptors of the
Oyster

It was found that a straight line relationship
exists between the molarity of the chemical stimu-
lant and logarithm of the slopes of the curves of
taste receptor potential versus molarity of stimu-
lant. In other words, log .^ and M ai^e related by

dM
a straight line where P is the receptor potential
and M is the molarity of the stimulant.

A typical equation of the straight lines shown

in Figures 9, 10, 11 and 12 is

Log ^ + Log C, = -KM
^ dM ^1

where log C-i is a constant equal to the

ordinate-intercept of the straightline plot and K is

another experimentally determined constant (K is

0 0.01 0.05 0.1 0.15 0.20 0.25 0.3 0.35 0.4 0.45
Molarity of Stimulant, M

FIG. 10. Relationship between logarithm of slope
values and molar concentration of sodium chloride.

equal to slope of straight line multiplied by
2.3026 to convert to naperian logarithms). The
above equation can be solved for P as follows:

Log ^ + Log Ci = -KM (1)

dM 1

Log [ (dP) C, ] = -KM (2)

dM ^

i£=e±^ (3)

dM Cj

Where e is the base of naperian logarithm:

i£=Ce-KM (4)

dM
where C =_L
Cl
Integration of the equation (4) results into:

P = C e-KM ^ C„ (5)

K 2

CHEMO-RECEPTORS ON LABIAL PALPS OF OYSTERS

Slope, K = 25.6 H

1 Ll L_

_1 L

_X.

0 0.01 0.05 0.1 0.15 0.20 0.25 0.3 0.35 0.4 0.45
Molarity of Stimulant, M

FIG. 11. Relationship between logarithm of slope
values and molar concentration of quinine sulfate.

When the molarity of the test solution was
zero, i.e., distilled water, then the receptor poten-
tial was experimentally obtained as 2.3 mv. (Figs.
5, 6, 7 and 8).

Therefore C<-

2.3 +-^
K

e-KM)

P = 2.3 +±1(1 - e"'^") (6)

K
Where P is the taste receptor potential in mv, C

and K are experimentally determined constants
and M is molarity of the t«st solution.

Equation (6) governs the relationship between
taste receptor potentials and molarity of solutions
for any chemicals tested in this study except
NaCl. For NaCl, since magnitude of the receptor
potential diminishes as the concentration of solu-
tion goes up until saturation the characteristic

0 0.001 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.1
Molarity of Stimulant, M.

FIG. 12. Relationship between logarithm of slope
values and molar concentration of hydrochloric
acid.

equation would be as follows:
C

P = 2.3

e-KM)

(6a)

For large values of the molarity of solutions M,
the factor -KM becomes very small (K being con-
stant) and can be practically neglected. The equa-
tions (6) and (6a) then reduce to:

P = 2.3 +.^

and
P = 2.3

respectively.

The pattern of diminishing receptor potentials
in response to increasing concentrations of sodium
chloride is compatible to the fact that the oyster
lives in saline water and therefore, its sensory
system may not be aroused when exposed to

R.C. DWIVEDY

changes within the natural environment.

ACKNOWLEDGMENT

The author wishes to thank Professors C. W.
Woodmansee, R. L. Salsbury and C. Epifanio for
their critical review of the manuscript. This re-
search was supported by Sea Grant No. 2-35223
awarded to the University of Delaware by the U. S
Department of Commerce.

LITERATURE CITED

Bultitude, K. H. 1958. A technique for marking
the site of recording with microelectrodes. Q. J.
Microsc. Sci. 99: 61.

Galtsoff, P. S. 1964. The American oyster Crassos-
trea uirginica Gmelin. U. S. Fish and Wildl.

Serv. Fish. Bull. 64: 111-120.

Grave, C. lO^e. The process of feeding in the oys-
ter. Science, 44: 178-181.

Hubel, D. H. 1957. Tungsten microelectrode for
recording from single units. Science, 125:
549-550.

Loosanoff, V. L. 1949. On the food selectivity of
oysters. Science, 110: 122.

Lotsy, J. P. 1895. The food of oyster, clam and
ribbed mussel. Rep. U. S. Comm. Fish and
Fisheries 1893. 19: 375-386.

Ukeles, R. 1970. Nutritional requirements in shell-
fish culture. In K. S. Price and D. L. Maurer
(ed.). Proceedings of the Conference on Arti-
ficial Propagation of Commercially Valuable
Shellfish - Oysters. Univ. Delaware, Newark,
Del. p. 43-64.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

CONCENTRATIONS OF FIVE TRACE METALS IN THE WATERS AND OYSTERS
(CRASSOSTREA VIRGINICA) OF MOBILE BAY, ALABAMA

Frederick C. Kopfler^ and Jack Mayer^

U. S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND MONITORING

GULF COAST WATER SUPPLY RESEARCH LABORATORY

DAUPHIN ISLAND, ALABAMA

ABSTRACT

From January 1968 through June 1969, samples of water and oysters were
collected at monthly intervals from eight locations in Mobile Bay, Alabama. These
samples were analyzed for cadmium, chromium, copper, lead and zinc by atomic
absorption spectrophotometry and the results analyzed statistically.

Oysters from Mobile Bay contained less cadmium, copper and zinc than the
average concentrations reported for Atlantic Coast oysters. The concentration of
chromium was approximately the same while the lead concentration was about two
times that of the average value for Atlantic Coast oysters.

Oysters collected from the western side of the Bay were found to contain a
significantly greater concentration of copper and zinc than oysters collected from
the eastern side. These differences were attributed to differences in river systems
that contribute the fresh water discharge and runoff to opposite sides of the Bay.

Although concentrations of the trace metals investigated were 10^ - 10^ higher in
oysters than the concentrations in the environmental water samples, poor correlation
was observed between the two sets of data.

INTRODUCTION

Marine organisms have the ability to accumulate
trace elements from the environment (Vinogradov.
1953). Hiltner and Wichmann (1919) demonstrated
that metallic wastes in industrial effluents could be
responsible for abnormally high concentrations of
copper and zinc in oysters. Hunter and Harrison
(1928) reported detecting lead and arsenic in oys-
ters growing in industrially polluted waters. The
potential danger to public health that could arise
from the consumption of shellfish contaminated
with heavy metals was discussed at the National
Shellfish Sanitation Workshop held in Washington,
D. C. in 1961 (McFarren, Campbell and Engle,
1961). Concern continued to grow as the coastal

'Current Address: Water Supply Research
Laboratory, National Environmental Research
Center, U. S. Environmental Protection Agency,
4676 Columbia Parkway, Cincinnati, Ohio*

waters became more heavily industrialized, and the
U. S. Public Health Service initiated a program to
provide information on the relationship of trace
metal levels in the environmental waters and the
levels in oysters.

The purposes of this study, performed at the
Gulf Coast Water Hygiene Laboratory^, were (a)
to provide data to serve as background concentra-
tions of cadmium, chromium, copper, lead and
zinc in oysters in Mobile Bay for future reference;
(b) to determine if the trace metal concentrations
in samples bf shellfish growing waters could be
correlated wCth the trace metal concentrations in
oysters and (c) to determine the variations that
can occur In trace metal concentrations in both
oyster and water samples from different localities
in a relatively small area such as Mobile Bay.

^Former name of the Gulf Coast Water Supply
Research Laboratory, Dauphin Island, Alabama.

F.C. KOPFLER AND J. MAYER

FIG. 1. Location of sampling sites in Mobile Bay, Alabama.

FIELD SAMPLING PROCEDURES

Monthly collection of oyster and water samples
began during January of 1968 from the eight loca-
tions in Mobile Bay shown in Figure 1. Both oys-
ter and water samples were collected through
January 1969. From February 1969 through June
1969 when sampling ended, only oyster samples

were collected. Ten or 12 oysters, collected by
dredging, were used as a sample from each loca-
tion. Since stratification in the relatively shallow
waters over the oyster reefs in Mobile Bay is not
pronounced (Austin, 1954; McPhearson, 1970), the
water samples from each location were collected
by submerging a one gallon polyethylene bottle
below the surface.

TRACE METALS IN WATER AND OYSTERS

LABORATORY PROCEDURES

All laboratory glassware was washed in a deter-
gent solution and rinsed in tap water followed by
rinsing in deionized water. The glassware was then
rinsed in dilute nitric acid (1:4) and finally rinsed,
three times in glass-distilled water.

A Perkin Elmer Model 303 atomic absorption
spectrophotometer^, with instrument settings
recommended by the manufacturer, was used to
determine the metal concentrations in the prepared
samples. The samples were prepared for analysis as
described below.
Oyster Samples

The shells of the oysters were scrubbed with a
stiff brush under running tap water to remove
mud. The oysters were then shucked, and the
pooled meats were drained and homogenized for
three minutes at high speed in a Sorvall Omni-Mix-
er. Duplicate 10 - 20-gram aliquots of each homo-
genate were weighed to the nearest 0.01 g into
300 ml tall form beakers. Twenty ml of concen-
trated reagent grade nitric acid was added to each

^Mention of commercial products does not
necessarily imply endorsement by the U. S. Gov-
ernment.

beaker. To prevent foaming, two drops of a
dilute aqueous suspension of Dow Antifoam C were
added to each sample. The beakers were covered
with watch glasses and the contents heated to
boiling on a hot plate. Gentle boiling was con-
tinued until the tissue had been completely digest-
ed, about 4-6 hr. Each digested sample was
filtered through glass wool into a 50-ml volu-
metric flask and diluted to volume with distilled
water.

Preliminary studies indicated that the solids
content of the prepared samples did not interfere
with the analysis and also that the iron content
was not of sufficient magnitude to interfere with
the determination of chromium as described by
Giammarise (1966). Recoveries of the five ele-
ments from fortified samples ranged from
95-103%.
Water Samples

A 2-liter water sample was filtered through a
0.45/n membrane filter. The sample was placed in
a 3-liter beaker and concentrated to 200 ml by
gentle boiling under a stream of clean, dry air.
The concentrated sample was adjusted to pH 3
with HCl and transferred to a separatory funnel.
One ml of 2% aqueous solution of ammonium
pyrrolidine dithiocarbamate was added, and the

TABLE 1. Summary of cadmium concentrations in oyster and water samples from Mobile
Bay.

Sampling

Cadmium concentrations

Total
Samples

Number

Location

Range

Median

Mean ± S.E.

Quantifiable

Oyster samples (mg/kg wet weight)

0.07-1.61

1.00

1.04±0.07

0.05-1.16

0.65

0.60±0.08

118

0.03-1.16

0.68

0.70±0.07

119

<0.05-1.20

0.50

0.65±0.10

0.20-1.30

0.47

0.53±0.08

0.10-0.80

0.51

0.52±0.08

104

<0.05-0.60

0.45

0.49±0.04

112

0.05-0.75

0.49

0.46±0.04

Water samples (|Ug/l)

<0.1-1.4

0.6

—

<0.1-1.5

0.4

—

118

<0.1-1.1

0.3

119

<0.1-9.1

0.6

—

<0.1-1.8

0.5

<0.1-1.3

0.5

104

<0.1-1.2

0.6

112

<0.1-1.4

0.5

F.C. KOPFLER AND J. MAYER

TABLE 2. Summary of chromium concentrations in oyster and water samples from Mobile
Bay.

Sjunpling

Chromium concentrations

Total

Number

Location

Range

Median

Mean ± S.E.

Samples

Quantifiable

Oyster samples (mg/kg wet weight)

0.70-3.30

0.28

0.52±0.20

<0.10-0.63

0.24

0.27±0.04

118

<0.10-0.83

0.25

0.33±0.06

119

<0.10-0.65

0.23

0.30±0.05

0.12-0.70

0.34

0.34±0.04

0.12-0.80

0.37

0.38±0.04

104

<0.10-1.00

0.28

0.38±0.06

112

<0.10-0.58

0.26

0.29±0.03

Water samples {figll)

<0.1-0.8

<0.1

<0.1-1.4

<0.1

118

<0.1-2.3

<0.1

119

<0.1-1.0

<0.1

<0.1-3.7

0.2

<0.1-2.6

0.2

104

<0.1-2.9

0.2

112

<0.1-2.9

0.8

funnel was shaken and then allowed to stand for
several minutes to chelate the metal ions. The
chelated metals were then extracted with methyl
isobutyl ketone (MIBK). The MIBK fraction con-
taining the metal chelates was placed in a 50 ml
beaker and carefully evaporated to dryness. The
residue was taken up in 10 ml concentrated nitric
acid and heated until the solution was clear. The
sample was then made to an appropriate volume
for analysis (10-25 ml) with distilled water.

Recoveries for cadmium, copper, lead and zinc
from fortified estuarine water were found to range
from 91-100%. Results of the sample analyses
were not corrected for recovery.

RESULTS AND DISCUSSION

The results of the analyses of the water and
oyster samples are summarized in Tables 1 through
5. The values for the median and mean concentra-
tions in oysters are in fair agreement. Since the
concentrations of the elements were below detect-
able quantities in many of the water samples and
occasional samples contained extremely high con-
centrations, the median and mean concentrations
were quite different; the median values were be-
lieved to reflect more accurately the conditions
over an extended period of time.

The data obtained were statistically analyzed in
the following manner. Analysis of variance was
used to compare concentrations of each of the
metals in oyster samples from the eight stations.
The relationship of each metal concentration in
the oyster samples to the overlying water at eight
sampling stations were determined by calculating
correlation coefficients. Results of these analyses
of the data were compared for statistical signifi-
cance at the 5% probability level.

Water Samples

The concentration of each metal in the water
samples varied highly from month to month, and
no seasonal trends were readily observable. The
ranges of concentration (^fg/l) of the metals in all
water samples were: cadmium, <0.1 - 9.1; chro-
mium, <0.1 - 3.7; copper, <0.1 - 15.0; lead, <0.3
- 29.4; zinc, <0.1 - 25.0 (Tables 1-5). Many of
the water samples contained concentrations of
metals too low to quantify, notably chromium
with 45% of the samples indeterminate.

When the data for cadmium were examined no
obvious differences were apparent among the con-
centrations in the water samples from the eight
stations. Similar observations were made for cop-
per, lead and zinc. However, tvdce as many sam-

TRACE METALS IN WATER AND OYSTERS 31

TABLE 3. Summary of copper concentrations in oyster and water samples from Mobile
Bay.

Sampling
Location

Copper concentrations

Total

Number

Range

Median

Mean ± S.E.

Samples

Quantifiable

Oyster

samples (mg/kg wet weight)

27.0-78.3

37.8

43.2±4.2

13.0-57.6

21.0

24.113.0

118

10.8-36.7

15.2

20.0±2.3

119

10.1-54.1

15.4

22.7±4.1

3.7-17.5

9.0

9.6±1.1

5.7-17.8

10.0

10.7±0.8

104

5.0-20.0

10.8

10.9±0.9

112

5.0-33.0

13.0

15.2±1.7

Water sampl

es (A/g/1)

0.1-13.0

1.0

<0.1-15.0

1.0

118

<0.1-6.0

1.2

119

<0.1-7.0

1.7

<0.1-7.2

1.7

0.2-3.0

1.8

104

0.5-8.0

1.4

112

<0.1-7.1

1.8

TABLE 4. Summary of lead concentrations in oyster and water samples from Mobile
Bay.

Sampling

Lead concentrations

Total

Niirnhpr

Location

Range

Median

Mean ± S.E.

Samples

Quantifiable

Oyster samples (mg/kg wet weight)

0.17-1.59

0.76

0.8010.11

<0.10-1.79

0.70

0.8310.12

118

<0.10-1.22

0.70

0.7210.09

119

<0.10-1.50

0.67

0.8410.12

0.13-1.60

0.88

0.8510.11

<0.10-1.50

0.80

0.92+0.10

104

<0.10-1.70

0.78

0.8610.10

112

0.17-1.75

0.68

0.7710.11

Water samples (iJtgll)

<0.3-11.8

0.5

<0.'3- 7.8

1.0

118

<0.3-10.2

1.2

("g/1)

119

<0.3- 7.2

2.0

<0.3-16.4

2.7

<0.3-13.2

2.0

104

<0.3-14.0

2.2

112

<0.3-29.4

3.0

F.C. KOPFLER AND J. MAYER

TABLE 5. Summary of zinc concentrations in oyster and water samples from Mobile Bay.

Zinc

concentrations

Total

Number

Sampling

Location

Range

Median

Mean ± S.E.

Samples

Quantifiable

Oyster samples (mg/kg wet weight)

925-3800

1980

22001194

350- 911

603

611+ 38.2

118

250- 702

478

496± 33.8

119

235- 900

478

497+ 46.8

140- 600

319

350± 29.1

238- 529

350

366± 29.2

104

200- 540

371

364± 24.5

112

140- 678

412

436± 33.8

Water samples (^g/l)

<0.1-17.0

2.3

0.1- 9.8

2.6

118

<0.1- 7.7

2.8

119

0.2-25.0

2.2

<0.1-21.2

2.4

0.6-12.0

2.5

104

0.3- 9.1

2.5

112

<0.1-11.2

3.6

pies from eastern stations contained detectable
concentrations of chromium and the median con-
centrations were substantially higher at those sta-
tions (Table 2).

Oyster Samples

The average concentrations of the trace metals
in all oyster samples were compared with concen-
trations in oysters from the Atlantic Coast (Table
6). The most pronounced differences were that the
Atlantic Coast oysters contained approximately
five fold more cadmium and copper and twice as
much zinc. The chromium content was about the
same, and the Mobile Bay oysters contained al-
most twice as much lead.

The data for the metal concentration of the
oyster samples were subjected to statistical analysis
to determine if the oyster populations at the vari-
ous stations were homogeneous with respect to
each element. No significant difference (P>0.05)
was found among the stations with respect to
chromium and lead concentrations and none for
cadmium concentrations, except those from station
50 which were significantly higher. Oysters from
station 50 also contained significantly more copper
and zinc than those from the other stations.

Copper and zinc concentrations in oysters from
the other stations followed a common pattern.

The concentrations of each of these metals in oys-
ters from stations 83, 92 and 104 on the eastern
side of the Bay were not significantly different
(P>0.05) and the concentrations of each metal in
oysters from stations 89, 118 and 119 on the
western side of the Bay were not significantly dif-
ferent (P>0.05). The concentrations from stations
89, 118 and 119 were, however, significantly high-
er than those in oysters from 83, 92 and 104
(P<0.05). Oysters from station 112 also contained
significantly (P<0.05) more copper and zinc than
those from the other eastern stations.

Hugget, Bender and Sloan (In Press) reported
that as the freshwater source of an estuary is ap-
proached the oysters contain increasing amounts of
copper and zinc. This may be responsible for the
copper and zinc levels in oysters from station 50
being significantly higher than the levels in oysters
from stations 89, 118 and 119 in lower western
Mobile Bay. Although the levels of these two ele-
ments in oysters from the latter stations were sig-
nificantly higher than those in oysters from the
lower eastern section (stations 83, 92 and 102), it
has been calculated from the data of McPhearson
(1970) that the salinities in those two areas of the
Bay are not significantly different.

Austin (1954) has shown that because of the
prevailing circulation in Mobile Bay, oysters at

TRACE METALS IN WATER AND OYSTERS

TABLE 6. A comparison of trace metal concentration in Mobile Bay oysters
with levels reported for Atlantic Coast Oysters.

Concentration (mg/kg wet weight)

Element

Mobile Bay

Atlantic Coast

Cadmium

Chromium

Copper

Lead

Zinc

0.62
0.35
19.5
0.82
665

3.10

0.40

91.50

0.47

1482

^Average values of all samples in this study.

''Pringle, Hissong, Katz and Mulawka (1968): Oysters from Maine to North Carolina.

stations 83, 92 and 102 would receive the dis-
charge and runoff from Fish River and Bon
Secour River whereas those on the western side
would receive water from the river system at the
head of the Bay. The differences observed in the
zinc and copper levels in these two groups of oys-
ters is most certainly influenced by differences in
the copper and zinc concentrations in the different
rivers flowing into Mobile Bay. Whether the signi-
ficantly higher copper and zinc burdens in the
oysters from the western side represent industrial
pollution or naturally higher copper and zinc con-
tent in the river system is not known.

COMPARISON OF SAMPLES

When the data were arrayed so that the concen-
trations of each metal in companion oyster and
water samples from each station could be com-
pared, little correlation existed. The interdepen-
dence of the two sets of data was further ex-
amined by calculating the correlation coefficients
for each metal in oyster and water samples when
both contained a quantifiable concentration of the
element. Correlation coefficients for the chromium
data were not calculated since the method used to
determine chromium in water detects only hexa-
valent chromium (Midget and Fishman, 1967), and
the total chromium concentration was determined
in the oysters; thus correlation would not be ex-
pected.

The correlation between the concentration of
copper or cadmium in oysters and the concentra-
tion of these elements in the water samples was
not significant at the 5% probability level at any
station, and correlation between the zinc concen-
trations of oyster and water samples existed only
at station 50. Correlation at the 5% probability

level existed between lead concentrations in the
oyster and in the water samples at five of the
eight stations. Correlation was observed at the
three southernmost stations (89, 118 and 119) on
the western side of the Bay and at stations 83
and 104 on the eastern shore.

Shuster and Pringle (1969) exposed oysters to
various levels of lead, cadmium, chromium, copper
and zinc under controlled conditions. They re-
ported that the rate of accumulation of each
metal occurred in three phases and that an ap-
proximate doubling of metal concentration oc-
curred in the tissue upon doubling the concentra-
tion of the metal in the water. Since their data
indicate that lead is concentrated in a manner
similar to the other metals, the reasons for cor-
relation only between oyster and water lead con-
centrations observed in this study are not ap-
parent. The poor degree of correlation observed
here agrees with the findings of Ikuta (1958) who
demonstrated the difficulty of correlating levels of
copper and zinc in the Pacific oyster with the
levels in the environmental waters. The mechanism
of trace element concentration by shellfish is not
well understood. The poor degree of correlation
observed between trace metal concentrations of
companion oyster and filtered water samples sug-
gests that such concentration may occur through
particulate ingestion of suspended material from
seawater or ingestion of elements via their precon-
centration in algae or other food material as
proposed by McFarren, et al. (1961) and Brooks
and Rumsby (1965).

Since trace metal concentrations in estuarine
waters will fluctuate with the tidal stages, amount
of fresh water runoff and variations in discharges
containing trace elements, the metal levels in the

F.C. KOPFLER AND J. MAYER

shellfish, regardless of the mechanism of concentra-
tion, reflect differences in the long-term levels of
the trace metals in the water better than the data
obtained by direct analysis of water samples them-
selves.

LITERATURE CITED

Austin, G. B. 1954. On the circulation and tidal
flushing of Mobile Bay, Alabama, Part I. Tex.
A & M Coll. Res. Found. Proj. 24, Tech. Rep.
12, 28 p.

Brooks, R. R. and M. G. Rumsby. 1965. The bio-
geochemistry of trace element uptake by some
New Zealand bivalves. Limnol. Oceanogr. 10:
521-527.

Giammarise, A. 1966. The use of ammonium
chloride in analyses of chromium samples con-
taining iron. Atomic Absorption Newsletter 5:
113-114.

Hiltner, R. S. and H. J. Wichmann. 1919. Zinc in
oysters. J. Biol. Chem. 38: 205-221.

Hugget, R. J., M. E. Bender and H. D. Sloan.
1973. Utilizing metal concentration relationships
in the eastern oyster (Crassostrea virginica) to
detect heavy metal pollution. Proc. 7th Natl.
Shellfish Sanit. Workshop. (In Press)

Hunter, A. C. and C. W. Harrison. 1928. Bacteri-
ology and chemistry of oysters, with special
references to regulatory control of production.

handling, and shipment. U. S. D. A. Tech. Bull.
No. 64, p. 77.

Ikuta, K. 1958. Studies on accumulation of heavy
metals in aquatic organisms - II. On accumula-
tion of copper and zinc in oysters. Bull. Jap.
Soc. Sci. Fish. 34: 112-116. (In Japanese,
English summary)

McFarren, E. F., J. E. Campbell and J. B. Engle.
1961. The occurrence of copper and zinc in
shellfish. Proc. Shellfish Sanit. Workshop, p.
229-234.

McPhearson, R. M., Jr. 1970. The hydrography of
Mobile Bay and Mississippi Sound, Alabama. J.
Mar. Sci. Alabama 1(2): 1-83.

Midget, M. R. and M. F. Fishman. 1967. Determi-
nation of total chromium in fresh waters by
atomic absorption. Atomic Absorption Newslet-
ter 6: 128-131.

Pringle, B. H., D. E. Hissong, E. L. Katz and S. T.
Mulawka. 1968. Trace metal accumulation by
estuarine mollusks. J. Sanit. Eng. Div. Proc.
Am. Soc. Civ. Eng. 94: 455-475.

Shuster, C. N., Jr. and B. H. Pringle. 1969. Trace
metal accumulation by the American eastern
oyster, Crassostrea virginica. Proc. Natl. Shell-
fish. Assoc. 59: 91-103.

Vinogradov, A. D. 1953. The elementary chemical
composition of marine organisms. Mem. Sears
Found. Mar. Res., No. II. New Haven, Conn.,
647 p.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

PREY PREFERENCE OF STYLOCHUS ELLIPTICUS
IN CHESAPEAKE BAY

Darryl J. Christensen

U. S. DEPARTMENT OF COMMERCE

NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION

NATIONAL MARINE FISHERIES SERVICE

MIDDLE ATLANTIC COASTAL FISHERIES CENTER

OXFORD, MARYLAND

ABSTRACT

Flatworms, Stylochus ellipticus, collected from two locations in Chesapeake Bay and
randomly offered oysters and barnacles exhibited similar feeding behavior, preying on
both species. However, when the flatworms were segregated, based on known prey at the
time of collection, they exhibited marked prey preference. These experiments support
the hypothesis of "ingestiue conditioning" in S. ellipticus as proposed by other investi-
gators.

INTRODUCTION

Several investigators have documented predation
by Stylochus ellipticus and other poly clad flatworms
on various marine organisms. These accounts have
been reviewed by Hopkins (1949, 1950), Provenzano
(1961), Landers and Rhodes (1970) and Christensen
(1971). S. ellipticus has been described as either an
oyster predator, barnacle predator or both. Extensive
predation on raft-caught seed oysters at the Oxford
Laboratory prompted an investigation of the feeding
habits of S. ellipticus. Flatworms with known feeding
habits from two areas in Chesapeake Bay were of-
fered various combinations of oysters, barnacles or
both, and their predatory activity was monitored.

METHODS AND MATERIALS

Flatworms, barnacles and oysters were collected
during the spring and summer of 1969 from oyster
shells suspended from rafts in the Tred Avon River
and Harris Geek, two streams located on the Eastern
Shore of Chesapeake Bay. aiells or shell fragments
bearing oysters, barnacles or both were cleaned of
other fouling organisms, flatworms, debris and barna-
cles or oysters in excess of the numbers chosen for
the experiment. The barnacles and oysters were held
in laboratory tanks provided with running seawater at
ambient river temperature for several days to detect
mortality due to handling, and then placed in con-

tainers to condition them to room temperature.
Shells bearing known numbers of barnacles or oysters
were then placed in appropriate containers with room
conditioned worms. Controls containing only prey
were included in all experiments.

Experiments were conducted in containers com-
patible in size to the predators and prey being used.
Glass petri dishes filled with water were used in ex-
periments involving very small flatworms measuring
1.0-2.0 mm in length; glass finger bowls holding 250
ml of water were used with flatworms from 2.0-4.0
mm; and glass beakers holding 900 ml of water were
used with worms larger than 4.0 mm. The water was
changed five times a week in the petri dishes and
bowls and twice a week in the beakers. Only the
beakers were aerated.

In all experiments, Tred Avon River water was
used. During the period of these experiments, the sa-
linity varied from a low of 11.8 %o on 23 July 1969
to a high of 14.9 %c on 28 October 1969. According
to Landers and Rhodes (1970), a salinity difference
from 7.5 9cc to 27-28 % has no affect on initiation
or rate of predation of S. ellipticus on oysters.

Room temperatures during the experiment ranged
from 20-22°C. Landers and Rhodes (1970) found
that at temperatures from 10 - 22°C there was no
difference in time of initiation or rate of predation of
S. ellipticus on oysters or barnacles.

The water used for the first three sets of experi-

D.J. CHRISTENSEN

ments involving very small worms and barnacles was
centrifuged and autoclave-sterilized to prevent intro-
duction of larval worms or barnacles. Water used in
the beakers was only centrifuged, since the chance of
mistaking a recently set individual from a larger one
used in the experiment was negligible.

The worms and their prey were counted at varying
intervals depending on the length of the experiment.
In experiments involving large numbers of small bar-
nacles, the shells were marked off into grids with the
number of barnacles per grid section recorded to fa-
cilitate future counting. When prey mortality exceed-
ed 50%, they were replaced with the original number
of new individuals. The same worms were used during
an experiment. An exact count of worms was made
with a dissecting microscope at the termination of
each experiment or when it was necessary to replace
the original prey stock.

Predation rate, in each given series of experiments,
is expressed as the number of oysters or barnacles
killed per worm per week.

RESULTS

Predation on Barnacles

The first three series of experiments involved only
worms and barnacles collected from the Tred Avon
River. Ropes and bags bearing oyster shells were sus-
pended from a raft in late April 1969. Initial setting
of barnacles, Balanus sp., began during the first week
in May and by the end of May a density of approxi-
mately 600 individuals per 100 cm^ was observed.
Setting of S. ellipticus also began during the first
week of May and approximately 40 worms per 100
cm^ accumulated on the shells by the end of May. No
oyster setting occurred during this period; therefore.

all flatworms used for the first three series of experi-
ments had never eaten any oysters.

In the first series of experiments with very small
worms (1.0-2.0 mm) and barnacles, predation rates in
five separate glass petri dishes were observed to be
.16, .16, 1.01, 1.40 and 2.50 barnacles killed per
worm per week. None of the barnacles in either of
two controls died during the two-week experimental
period. In a second series of experiments conducted
in eight glass finger bowls, predation rates were .70,
.80, 1.00, 1.25, 2.00, 2.10, 2.10 and 2.40 barnacles
killed per worm per week. None of the barnacles in
the four controls died during the two-week experi-
mental period. In the third series of experiments con-
ducted in seven glass beakers, predation rates were
.95, .95, 1.11, 1.15, 1.25, 1.75 and 2.00 barnacles
killed per worm per week. Mortality in the seven con-
trols was negligible. The mean predation rate for the
three series of experiments was 1.34 barnacles killed
per worm per week.

Predation by Unselected Worms

Flatworms were collected from two different
areas. Tred Avon River specimens were obtained from
the same source as those used in the previous three
experiments. Harris Creek specimens were obtained in
a similar manner from shells suspended from a raft on
26 June. Both oysters and barnacles were setting at
the time the shells were suspended. Two weeks after
suspension the shells contained an average of 61 oys-
ter spat, 26 barnacles and 3 flatworms. The results of
the prey-preference experiment series with worms
from the two areas are presented in Table 1. Mortal-
ity was negligible in the 12 control experiments used
in these experiments.

A comparison between Tred Avon River worms

TABLE 1. Results of feeding experiments using flatworms from two different areas in Chesapeake Bay.

Oyster Mortality

Barnacle Mortality

Range of

Mean

Range of

Mean

Number of

Predation

Experiment

Replicates

Rate

Tred Avon

River Worms

Oysters

.00-.90

.37

Barnacles

1.15-1.60

1.38

Both

.00-.50

.19

.40-.70

.54

Harris Creek

Worms

Oysters

.00-.90

.45

Both

.14-.27

.21

.50-.86

.68

PREY PREFERENCE OF STYLOCHUS ELLIPTICUS

TABLE 2. Results of feeding experiments using wdrms selected on the basis of prey utilization at
pme of collection.

Oyster

Mortality

Barnacle

Mortality

Range of

Mean

Range of

Mean

Number of

Predation

Experiment

Replicates

Rate

Oyster Worms

Oysters

.40-.80

.55

Barnacles

.08-.21

.14

Both

.22-.29

.25

.03-.04

.04

Barnacle Worms

Barnacles

.07-.21

.15

Oysters

Both

.00-.14

.07

and Harris Creek worms indicates that they have very
similar predatory activities. Regardless of the worm
source, the mean predation rate on barnacles was
about three times the mean predation rate on oysters
if both were offered. If only oysters were offered, re-
gardless of the worm source, the mean predation rate
was double that on oysters in experiments where
both prey species were available. When both oysters
and barnacles were offered to Harris Creek worms,
they preyed on oysters in all five experiments. Tred
Avon River worms, however, preyed on oysters in
only three of five experiments. The mean predation
rates on oysters were essentially the same regardless
of worm source. When only barnacles were offered to
Tred Avon River worms, the mean predation rate
(1.38) was consistent with that found in the previous
experiments (1.34).

Prey Preference With Selected Worms

In other experiments involving prey choice, worms
taken from the same sources as in previous experi-
ments were segregated on the basis of prey utilization
at the time of collection; i.e. flatworms found feeding
on oysters were separated from those found feeding
on barnacles. Flatworms found in empty oyster or
barnacle boxes or on the substrate were not used.

The results of these experiments are presented in
Table 2. In no experiment did known barnacle eating
worms prey on oysters, even wh«n an alternate food
source was not offered. Known oyster eating worms
preyed on oysters and barnacles in all experiments.
However, predation by oyster eating worms on oys-
ters was 3.5 times greater than on barnacles, whether
they were offered together or separately. Barnacle
predation rate (.14-. 15) was considerably less than in

previous experiments. However, this phenomenon is
probably due to the size of the barnacles used which
were relatively larger in proportion to the worms than
those used in prior experiments. Negligible mortality
occurred in the 10 controls.

DISCUSSION

These experiments lend further support to the
"ingestive conditioning" hypothesis offered by Wood
(1968) and later supported by Landers and Rhodes
(1970). When the predatory activities of S. ellipticus
from seven different sources were compared. Landers
and Rhodes (1970) found that worms from six
sources preyed on either barnacles or oysters but not
on both, while worms from only one source attacked
both prey. In the two instances where worms preyed
on oysters alone, the worms were obtained from
raft-caught suspended seed. In the one case where
both oysters and barnacles were preyed on, the
worms were obtained from a recently planted oyster
seed bed (Landers, personal communication). It is
possible that worms obtained from the recently plant-
ed bed may have included individuals which had
previously fed on barnacles and then moved into the
oyster plant area.

Worms collected from rafts in the Tred Avon River
and Harris Creek included individuals which had ac-
cess to either prey species, as both oysters and barna-
cles were present on the rafts at the time worms were
collected for prey preference experiments. This might
account for the similarity in predatory activity of
worms from both rivers (Table 1); that is, their preda-
tion on both oysters and barnacles. However, when

D.J. CHRISTENSEN

worms were segregated based on prey at the time of
collection rather than by source, prey selection was
different (Table 2) and suggested "ingestive condi-
tioning." Known barnacle-eating worms appeared to
have established a preference for barnacles. Known
oyster-eating worms had not established the same
preference and fed on barnacles when oysters were
not available, yet only rarely did oyster-eating worms
feed on barnacles when oysters were available.

It is interesting to note that, in all cases of high
oyster mortalities caused by S. ellipticus, the oysters
were crowded either as raft-suspended seed or dense
bottom beds. Although Webster and Medford (1961)
saw S. ellipticus in fresh spat boxes in Chesapeake
Bay and suggested the worms killed oysters, no ex-
tensive mortalities on natural oyster bars caused by S.
ellipticus have actually been observed. If oysters are
the usual prey species, observations of naturally oc-
curring mortalities of these important commercial
bivalves should have been reported. In the experi-
ments described here, the worms all had opportunity
to feed at an earlier stage on either oysters or barna-
cles. Some worms became conditioned to the lack of
oysters but not the lack of barnacles. It would ap-
pear, therefore, that barnacles are the preferred prey
of S. ellipticus under most conditions. However, if
barnacles are not available, as under certain aqua-
culture situations, S. ellipticus may become condi-
tioned to feed heavily, and perhaps exclusively, on
oysters.

LITERATURE CITED

Christensen, D. J. 1971. Early development and
chromosome number of the polyclad flatworm
Euplana gracilis. Trans. Am. Microsc. Soc. 90:
457-463.

Hopkins, S. H. 1949. Preliminary survey of the litera-
ture of Stylochus and other flatworms associated
with oysters. Texas A & M Res. Found. Proj. 9,
1-16. (Mimeograph)

Hopkins, S. H. 1950. Addendum to "Prehminary
survey of the literature on Stylochus and other
flatworms associated with oysters." Texas A & M
Res. Found. Proj. 9, 1-4. (Mimeograph)

Landers, W. S. and E. W. Rhodes, Jr. 1970. Some
factors influencing predation by the flatworm,
Stylochus ellipticus (Girard), on oysters. Chesa-
peake Sci. 11: 55-60.

Provenzano, A. J., Jr. 1961. Effects of the flatworm
Stylochus ellipticus (Girard) on oyster spat in two
salt water ponds in Massachusetts. Proc. Natl.
Shellfish. Assoc. 50: 83-88.

Webster, J. R. and R. Z. Medford. 1961. Flatworm
distribution and associated oyster mortality in
Chesapeake Bay. Proc. Natl. Shellfish. Assoc. 50:
89-95.

Wood, L. 1968. Physiological and ecological aspects
of prey selection by the marine gastropod Uro-
salpinx cinerea (Prosobranchia: Muricidae).
Malacologia, 6: 267-320.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

CARDIAC EDEMA ASSOCIATED WITH VIBRIO ANGUILLARUM IN
THE AMERICAN OYSTER

Haskell S. Tubiash, Sara V. Otto and Rudolph Hugh

NATIONAL MARINE FISHERIES SERVICE

MIDDLE ATLANTIC COASTAL FISHERIES CENTER

OXFORD, MARYLAND

MARYLAND DEPARTMENT OF NATURAL RESOURCES

ANNAPOLIS, MARYLAND

AND

GEORGE WASHINGTON UNIVERSITY

SCHOOL OF MEDICINE

DEPARTMENT OF MICROBIOLOGY

WASHINGTON, D.C.

ABSTRACT

During a survey for parasite distribution in Chesapeake Bay oysters (Crassostrea
virginica), sporadic cases of greatly enlarged and edematous pericardia were noted.
Prevalence of the edematous syndrome, which we have termed "cardiac vibriosis",
was estimated at 0.04%. Examination of aspetically aspirated pericardial fluid showed
heavy concentrations of gram-negative motile rods which proved morphologically and
culturally compatible with Vibrio anguillarum, an organism implicated in diseases of
fishes and larval bivalve mollusks. Except for pericardial enlargement, the animals
appeared to be grossly and histologically normal. Attempts to reproduce the
pericardial edema experimentally by injection of V. anguillarum proved unsuccessful.

INTRODUCTION

Oysters fall prey to many protozoan parasites
and metazoan predators (Sindermann and Rosen-
field, 1968; Farley, 1968). While a bacterial
disease of larval oysters, called bacillary necrosis,
has been described (Tubiash, Chanley and Leifson,
1965) no bacterial diseases of adult Crassostrea
virginica have been reported. In our experience,
adults of this species and other bivalve mollusks
are also refractory to experimental bacterial infec-
tion.

Toward the end of the 1950's a fast-spreading,
highly lethal oyster epizootic of unknown etiology
appeared in productive areas of Delaware Bay,
then rounded the Virginia Capes into the lower
half of Chesapeake Bay (Haskin, Canzonier and
Myhre, 1965; Wood and Andrews, 1962). Since
the center of the fishery was threatened, a
multi-agency, multidisciplined study was launched.

By 1966 the etiology was established as a
haplosporidan parasite, Minchinia nelsoni (Haskin,

Stauber and Mackin, 1966), whose proliferation
was evidently mediated by intrusion of high
salinities, concurrent with an extensive deficiency
of rainfall.

Careful resource management, and more import-
antly, the return of normal rainfall, have contained
the epizootic, but biologists continue to monitor
the prevalence of the disease in enzootic and
disease-free areas of the Chesapeake. For example,
in Maryland this surveillance consists of gross and
histologic examination of 25-50 oysters from 24
locations semi-annually.

In the course of this routine survey a previous-
ly undescribed syndrome was discovered. Out of
more than 10,000 oysters examined during four
years (1967 - 1970), four animals (0.04%) were
found with grossly enlarged hearts and pericardial
chambers (Fig. 1). Affected animals were found
during 1969 and 1970 from the Manokin, St.
Marys and South Rivers, which are Maryland
tributaries of Chesapeake Bay.

H.S. TUBIASH, S.V. OTTO AND R. HUGH

FIG. 1. American oyster, Crassostrea virginica, with greatly-enlarged, fluid-filled pericardial chamber
(arrow). Darkened areas in center of upper shell m.argin mark old and new invasion by Polydora sp.

OBSERVATIONS

Oyster No. 1 - This oyster was collected in
October 1969 slightly upstream from the mouth
of the South River and was one of a large
collection for a disease resistance study. It was
16.5 cm long, had a light infection of Polydora
sp. inside the shell and was judged to be in
"medium" market condition. The heart and car-
diac chamber were greatly enlarged and obviously
gorged with fluid (Fig. 1).

Oyster No. 2 ■ This oyster was collected from
the Manokin River in November 1969 as one of a
routine sample of 25. It was 8 cm long with ripe
gonads, a light invasion of Polydora sp. and was
judged in "medium" condition. The heart was
swollen and the cardiac chamber was enlarged,
containing a jelly-like fluid.

Oyster No. 3 - The animal was collected from
the St. Marys River during March 1970. It was 11
cm long and was judged in "medium" condition.

Mantle recession, usually indicative of pathology or
physiological stress (Farley, 1968) was evident.
The heart and pericardial chamber were both
greatly enlarged and fluid-filled.

Oyster No. 4 - This oyster was collected from
the Manokin River in September 1970. It was 9
cm long and in "watery" or "poor" market
condition. Holes caused by the oyster drill,
Urosalpinx cinerea, were present on the shell and
invasion by Polydora sp. and mantle recession
were seen in the shell interior. The heart was
swollen and the pericardial chamber greatly dis-
tended.

PROCEDURE
Bacteriological

Pericardial fluid was aspirated aseptically from
each animal for microbiological study before the
oysters were processed for histological examina-
tion. Gram stains were prepared from the peri-

CARDIAC EDEMA IN OYSTERS

cardial ha^molymph and blood agar plates were
streaked. Samples (0.2 ml) of the pericardial fluids
were diluted serially in Tryptose-Glucose-Yeast
extract (TOY) broth prepared in seawater (Tubi-
ash, et al., 1965) to estimate bacterial counts and
to isolate the predominant organisms. Incubation
was at 28°C for 48 hr. Blood agar plates were
streaked from the highest dilutions showing
growth, and isolations made of the predominant
organisms. Isolates were initially transferred to
Eosin Methylene Blue agar (EMB), Krumweidie's
Triple Sugar agar slants prepared with 1% NaCl
and Difco MOF fermentation medium with 1%
glucose. Determinative tests were performed as shown
in Table 1.

Histological

After macroscopic examination was completed
and pericardial fluid had been aseptically aspirated
from the affected oysters, they were rinsed in
membrane-filtered seawater to remove as much
mud, sand, shell fragments and detritus as possible.
Two transverse cuts were made just posterior to
the palps to prepare a section about 10 mm thick.
The heart, pericardial cavity and surrounding tissue
were also removed intact, placed in Davidson's
fixative and refrigerated for at least 48 hours. The
tissues were then run through successive changes
of ethyl alcohol and embedded in paraffin. Six
micron sections were cut, stained with Harris
hematoxylin and alcohol-soluble eosin, and mount-
ed in Permount.

RESULTS

The gram stains revealed heavy to moderate
concentrations of small gram-negative rods and on
culture the predominant organisms also proved to be
gram-negative rods. Bacterial concentrations on the
three cardiac haemolymphs successfully cultured
were estimated between 10* and lO'' per ml. The
fourth fluid was lost through contamination. Growth
on Krumweide's Triple Sugar agar with 1% NaCl
showed acid slants and butts, with no gas. MOF
glucose medium showed acid production in the open
and sealed tubes, hence glucose was fermented. The
bacteria failed to grow on EMB. These findings led us
to suspect that we were dealing with strains of a
motile marine vibrio and determinative tests were
performed as listed in Table 1. Isolates were
forwarded to Dr. Riichi Sakazaki at the National
Institute of Health in Tokyo, who confirmed our
identification of Vibrio anguillarum.

V. anguillarum is one of the etiologic agents of
bacillary necrosis in larval bivalve mollusks (Tubi-
ash, Colwell, and Sakazaki, 1970). The three

TABLE 1. Cardiac Vibrio Charge teris tics

Gram-negative polar monotrichous rods +

Acid, no gas produced from:

Glucose (fermentative) +

Sucrose +

Maltose -i-

Trehalose -i-

Mannitol -t-

Lactose -

Inositol -

Citrate Utilization -

Gelatin Liquefaction -i-

Production of:

Indole -I-

Catalase +

Oxidase -i-

Arginine dihydrolase +

Lysine decarboxylase -

Ornithine decarboxylase -

Phenylalanine deaminase -

Growth in:

0% NaCl Broth -

1% NaCl Broth +

6% NaCl Broth -i-

7% NaCl Broth -

cardiac isolates were therefore used to challenge
week-old oyster larvae using methods described
by Tubiash, et al. (1965). Forty-eight hour larval
mortalities averaged 92, 87 and 96% respectively,
while controls exposed to Escherichia coli averaged
only 11%.

Histologic examination revealed no abnormalities
other than the cardiac involvement. Microscopically
the tissues appeared normal.

In October, 1970, 24 oysters were injected
intracaridally, via a small trocar puncture between
the shell valves, with 24-hour vibrios washed from
the surface of TGY agar. The 0.1 ml injection
consisted of a suspension of about 8 x 10^ viable
organisms. The oysters were maintained in flowing
water throughout the vdnter. Five months later, in
March, 1971, they were sacrificed and examined.
Aside from mud blister formation at tlie injection
sites, the animals were in good condition and the
hearts appeared normal. Reproduction of the
cardiac enlargement syndrome was therefore not
achieved.

DISCUSSION

Histologic examination of the four affected
oysters revealed no abnormalities other than the
cardiac involvement, except that the oyster rated

H.S. TUBIASH, S.V. OTTO AND R. HUGH

in "poor" condition was also parasitized by
Nematopsis ostreanim, a gregarine protozoan para-
site and tiie shell had been invaded by Polydora
sp., an annelid blister-forming worm. This combi-
nation of parasitic stress, recovery from a possible
infection with M. nelsoni (as evidenced by mantle
cell recession and pigment cell infiltration) added
to the cardiac infection, could well be responsible
for the poor condition of the animal.

After studies of the oysters exhibiting cardiac
enlargement was completed, 0.2 ml of pericardial
fluid was aspirated and similarly cultured from
each of six aseptically-opened normal oysters. Only
a scattering of colonies appeared on the blood
plates streaked with cardiac fluid from five of the
six oysters, but a count estimated at 150 per ml
was obtained from the fluid of the sixth oyster.
The predominant organisms proved to be indis-
tinguishable from and apparently identical to V.
anguillarum.

Vibrios are knovra to be pathogenic to many
species of finfish and larval bivalve moUusks
(Anderson and Conroy, 1970; Tubiash, et al.,
1965) but we have isolated V'. anguillarum pre-
viously from apparently normal Chesapeake Bay
oyster tissue (Tubiash, et al., 1970). The signifi-
cance of our present finding is moot. Perhaps the
lesson to be releamed is that all potential
pathogens need not necessarily be associated with
overt pathology and that in the present case, the
vibrios are probably opportunists which may be
eliciting a host-response bordering on pathology.

We hesitate to categorize this syndrome as a
"disease", but are designating the condition "car-
diac edema".

LITERATURE CITED
Anderson, J. I. W. and D. A. Conroy. 1970.

Vibrio disease in marine fishes. In S. F.

Snieszko (ed.), A Symposium on Diseases of

Fishes and Shellfishes. Am. Fish. Soc. Spec.

Publ. No. 5, p. 266-272.
Farley, C. A. 1968. Minchinia nelsoni (Haplo-

sporida) disease syndrome in the American

oyster, Crassostrea virginica. J. Protozool. 15:

585-599.
Haskin, H. H., W. J. Canzonier and J. L. Myhre.

1965. The history of "MSX" on Delaware Bay
oyster grounds, 1957-1965. Am. Malacol. Union
Annu. Rep., Bull. No. 32: 20-21. (Abstract).

Haskin, H. H., L. A. Stauber and J. A. Mackin.

1966. Minchinia nelsoni n. sp. (Haplosporida,
Haplosporidiidae): causative agent of the Dela-
ware Bay oyster epizootic. Science 153:
1414-1416.

Sindermann, C. J. and A. Rosenfield. 1968.
Principal diseases of commercially important
marine bivalve Mollusca and Crustacea. U. S.
Fish Wildl. Serv., Fish. Bull. 66: 335-385.

Tubiash, H. S., P. E. Chanley and E. Leifson.
1965. Bacillary necrosis, a disease of larval and
juvenile bivalve mollusks. I. Etiology and epi-
zootiology. J. Bacteriol. 90: 1036-1044.

Tubiash, H. S., R. R. Colwell and R. Sakazaki.
1970. Marine vibrios associated with bacillary
necrosis, a disease of larval and juvenile bivalve
mollusks. J. Bacteriol. 103: 272-273.

Wood, J. L. and J. D. Andrews. 1962. Haplo-
sporidium costale (Sporozoa) associated with a
disease of Virginia oysters. Science 136:
710-711.

Proceedings of the National Shellfisheries Association
Volume 63-- June 1973

LABYRINTHOMYXA-LIKE ORGANISMS ASSOCIATED WITH

MASS MORTALITIES OF OYSTERS,

CRASSOSTREA VIRGINICA, FROM HAWAII

Frederick G. Kern, L. Cecelia Sullivan and Michio Takata

NATIONAL MARINE FISHERIES SERVICE

MIDDLE ATLANTIC COASTAL FISHERIES CENTER

OXFORD, MARYLAND

AND

STATE OF HAWAII, DIVISION OF FISH AND GAME

HONOLULU, HAWAII

ABSTRACT

In July 1972, a massive mortality ravaged oyster stocks (Crassostrea virginica) in
West Loch, Pearl Harbor, Hawaii. Oyster tissues cultured in fluid thioglycollate medi-
um were found to be infected with a fungal parasite similar to Labyrinthomyxa
marina (=Dermocystidium marinum). Examination of histological sections revealed
hypnospore stages which had enlarged, formed presporangia and were believed to be
forming planonts of developing zoospores.

INTRODUCTION

During July 1972, Eastern oysters, Crassostrea
virginica, in West Loch, Pearl Harbor, Hawaii, ex-
perienced a massive mortality of 90-99%, or ap-
proximately 30-34 million oysters. With the pos-
sible exception of barnacles, no other forms of
marine life appear to have been affected. No ap-
parent environmental anomaly was found to be as-
sociated with the mortality. A less intense mor-
tality occurred during the month of June in Mid-
dle Loch, Pearl Harbor. This mortality involved
not only moUusks but crustaceans and polychaetes
as well and could be directly related to low levels
of dissolved oxygen. Oyster tissues from West
Loch and Middle Loch were examined to deter-
mine if the mortality was of biotic etiology. In
our examination of oysters from both areas, a fun-
gus parasite similar to Labyrinthomyxa marina
(Mackin, Owen and Collier, 1950; Mackin and
Ray, 1966) was found.

L. marina has been extensively studied and
documented as a serious oyster pathogen (Mackin
et al., 1950; Mackin, 1952; Ray, 1954; Andrews
and Hewatt, 1957; Sindermann and Rosenfield,
1967; Quick and Mackin, 1971). Its reported range
appears limited to the Atlantic and Gulf coasts of

the United States and Mexico (Quick and Mackin,
1971). Its presence in oysters from Hawaii forms
the basis of this report.

MATERIALS AND METHODS

Fifty oysters (C. virginica) from West Loch and
25 oysters from Middle Loch were examined 2
weeks after the initial report of the West Loch
mortality. Each oyster was coded, opened at the
hinge and examined for gross abnormalities. Rectal
tissues from 25 of the West Loch oysters and 15
of the Middle Loch oysters were cultured in fluid
thioglycollate medium, incubated at room tempera-
ture for 72 hrs, and examined after staining with
Lugol's iodine solution (Ray, 1966).

A cross section of tissue approximately 6 mm
thick was cut from the visceral mass of each oys-
ter, fixed in Davidson's fixative (Shaw and Battle,
1957), dehydrated in ethanol and embedded in
paraffin. Sections were cut at 6fx and stained with
periodic acid Schiff with Weigert's acid iron chlo-
ride hematoxylin as a counterstain (PASH).

In September 1972, in order to determine
whether the fungus parasite was present in other
mollusks, additional samples of 50 Eastern oysters
(C. virginica) from West Loch and 50 each of

F.G. KERN, L.C. SULLIVAN AND M. TAKATA

FIG. 1. Sporocyst of developing hypnospores.
(PASH) lOOOX.

Pacific oysters (C. gigas) and Manila clams (Tapes
philippinarum) from Kaneohe Bay were processed
and examined by both of the methods described
above. Percentages of fungal infections reported
are based on the thioglycoUate technique.

RESULTS AND DISCUSSION

Fifty-two percent of the West Loch oysters and
27% of the Middle Loch oysters were found to be
infected with a fungal parasite similar to L.
marina. All oysters were alive at the time of pro-
cessing and without apparent tissue degeneration,
and, based on gross observations, the physical con-
dition of the infected oysters varied from medium
to poor, depending on the extent of the fungus
infection.

Additional samples of C. virginica taken in Sep-
tember 1972 from West Loch confirmed the
continued presence of the fungus pathogen in 44%
of the oysters surviving the original mortality.
Samples of C. gigas and T. philippinarum taken at
the same time from Kaneohe Bay were found to
be free of this pathogen.

Labyhnthomyxa-Vike organisms have been re-
ported in a wide variety of mollusks (Ray, 1954;
Andrews, 1955; Andrews ana Hewatt, 1957). Ray
(1954) demonstrated fairly rigid host specificity
and was unsuccessful in establishing cross infec-

tions. This possibly is why only C. virginica was
found infected.

Hypnospores observed in thioglycollate-cultured
tissues of Hawaiian oysters were considered com-
parable to those from L. marina infections of Gulf
coast oysters described by Ray (1966). No abnor-
mal increase in size or unusual staining characteris-
tics were observed in any of the stained prepara-
tions. Examination of histological sections revealed
hypnospores measuring 4-17/j, 58% larger than the
3-1 0;U hypnospores described from stained sections
and fresh preparations by Mackin, et al. (1950).
Hypnospores and sporangia possessed PAS-positive
walls. Small hypnospores were generally intra-
cellular; larger hypnospores and sporangia were fre-
quently seen encapsulated by oyster hemocytes.
Sporangia of hypnospores were numerous and
often quite large (Fig. 1).

Enlargement of L. marina spores in thio-
glycoUate media (Ray, 1966) is believed to repre-
sent an exaggerated developmental stage which oc-
curs naturally in degenerating post-mortem oyster
tissue (Mackin, 1962). Ray (1954) reported spore
enlargement on rare occasions in living oyster tis-
sue and believed the large spores to be abortive
sporangial bodies. Perkins and Menzel (1966) be-
lieved that enlargement signaled the formation of

V ,.y
» «

A '*• «

* 1

*•*

C *^'«

FIG. 2. A. Enlarged hypnospores B. Presporangia;
C. First cleavage two cell stage; D. Three-four cell
stage. (PASH) lOOOX.

MASS MORTALITY OF OYSTERS

FIG. 3. Sporangium of presumptive planonts with
suspected areas of discharge tube formation
(arrows). (PASH) lOOOX.

presporangia. They were able to induce sporulation
in L. marina which, through successive bipartition
of the protoplast, resulted in the formation of
motile bi flagellated zoospores. A similar sporula-
tive process has been reported in Labyrinthomyxa
sp. from the clam Macoma balthica (Perkins, 1968;
ValiuHs and Mackin, 1969).

The large spores found in the tissues of the
Hawaiian oysters were accompanied by early divi-
sion stages (Fig. 2) similar to those described by
Perkins and Menzel (1966). Sporangia of what ap-
pear to be developing planonts occasionally had
areas corresponding to the discharge pore and as-
sociated tube (Fig. 3) described from L. marina in
oysters (Perkins and Menzel, 1967) and from
Labyrinthomyxa sp. in M. balthica (Valiulis and
Mackin, 1969). We were unable to determine from
the examination of fixed tissue whether the plan-
onts completed their development and formed
motile biflagellated zoospores.

The similarity of the Hawaiian parasite to L.
marina and the unusually high prevalence of the
parasite suggests that it 'was the etiological agent
responsible for the mortality which occurred in
West Loch and possibly, to a lesser extent, for the
mortality which occurred in Middle Loch.

As an incidental observation, the parasitic ces-
tode Tylocephalum sp. was found in 18% of the

West Loch oysters and in 24% of the Middle Loch
oysters. No histopathology, other than cyst forma-
tion, is associated with Tylocephalum sp. infec-
tions (Sparks, 1963).

LITERATURE CITED

Andrews, J. D. 1955. Notes on fungus parasites of
bivalve moUusks in Chesapeake Bay. Proc. Natl.
Shellfish. Assoc. 45: 157-163.

Andrews, J. D. and W. G. Hewatt. 1957. Oyster
mortality studies in Virginia. II. The fungus
disease caused by Dermocystidium marinum in
oysters of Chesapeake Bay. Ecol. Monogr. 27:
1-25.

Mackin, J. G. 1952. Oyster disease caused by
Dermocystidium marinum and other micro-
organisms in Louisiana. Publ. Inst. Mar. Sci.
Univ. Tex. 7: 132-229.

Mackin, J. G. and S. M. Ray. 1966. The
taxonomic relationships of Dermocystidium
marinum ,Mackin, Owen, and Collier. J.
Invertebr. Pathol. 8: 544-545.

Mackin, J. G., H. M. Owen and A. Collier. 1950.
I*reliminary note on the occurrence of a new
protistan parasite. Dermocystidium marinum, n.
sp. in Crassostrea virginica (Gmelin). Science
111: 328-329.

Perkins, F. O. 1968. Fine structure of zoospores
from Labyrinthomyxa sp. parasitizing the clam
Macoma balthica. Chesapeake Sci. 9: 198-202.

Perkins, F. 0. and R. W. Menzel. 1966. Morpho-
logical and cultural studies of a motile stage in
the life cycle of Dermocystidium marinum.
Proc. Natl. Shellfish. Assoc. 56: 23-30.

Perkins, F. O. and R. W. Menzel. 1967. Ultrastruc-
ture of sporulation in the oyster pathogen Der-
mocystidium marinum, J. Invertebr. Pathol. 9:
205-229.

Quick, J. A., Jr. and J. G. Mackin. 1971. Oyster
parasitism by Labyrinthomyxa marina in
Florida. Fla. Dep. Nat. Resour. Prof. Pap. No.
13, 55 p.

Ray, S. M. 1954. Biological studies of Der-
mocystidium marinum, a fungus parasite of oys-
ters. Rice Institute Pamphlet, Spec. Issue, Nov.
1954, 114 p.

Ray, S. M. 1966. A review of the culture method
for detecting Dermocystidium marinum vnth
suggested modifications and precautions. Proc.
Natl. Shellfish. Assoc. 54: 55-69.

F.G. KERN, L.C. SULLIVAN AND M. TAKATA

Shaw, B. L. and H. L Battle. 1957. The gross and
microscopic anatomy of the digestive tract of
the oyster Crassostrea virginica (Gmelin). Can J.
Zool. 35: 325-347.

Sindermann, C. J. and A. Rosenfield. 1967. Prin-
cipal diseases of commercially important marine
bivalve MoUusca and Crustacea. U. S. Fish
Wildl. Serv., Fish. Bull. 66: 335-385.

Sparks, A. K. 1963. Infection of Crassostrea vir-
ginica (Gmelin) from Hawaii with a larval tape-
worm, Tylocephalum. J. Insect Pathol. 5:
284-288.

Valiulis, G. A. and J. G. Mackin. 1969. Formation
of sporangia and zoospores by Labyrinthomyxa
sp. parasitic in the clam Macoma balthica. J. In-
vertebr. Pathol. 14: 268-270.

Proceedings of the National Shellfisheries Association
Volume Q3 - June 1973

AN APPRAISAL OF THE ALTERNATIVE EARNING POWER
OF THE MARYLAND OYSTERMEN'

R. J. Marasco

AGRICULTURE AND RESOURCE ECONOMICS
UNIVERSITY OF MARYLAND
COLLEGE PARK, MARYLAND

ABSTRACT

Information collected from personal interviews with oystermen is used to determine
labor market participation potential. Data presented indicate that the oystermen living in
two communities located on Maryland's Eastern Shore would have more difficulty
finding employment outside the fishing industry than their counterparts living in two
western shore communities.

INTRODUCTION

For many years, the Chesapeake Bay and its
tributaries have supported one of the United States'
major commercial fisheries, the oyster fishery. It has
been said that the watermen who participate in the
fishery may very well be the last living specimens of
an almost extinct species: the independent, the
individual man (Lang, 1961). In the aftermath of
Hurricane Agnes' destruction, interest has surfaced in
the waterman's job mobility. The objective of this
study was to assess the employment mobility of the
Maryland oysterman.

SCOPE

The investigation was designed to serve as a limited
effort pilot study. Consequently, instead of en-
compassing the entire State of Maryland, four com-
munities. Shady Side, Rock Hall, Crisfield-Smith
Island and Avenue, were selected for analysis. Selec-
tion of these communities was made on the basis of
geographical considerations, the overall number of
licensed fishermen and the importance of the oyster
industry to the local economies.

In the selection process, the existence of some
contrasts in the structure of the local economy and
relative importance of fishing activities were heavily
weighted. Avenue, the smallest of the four com-
munities with a population of 600 people, represents

an isolated economy where farming and fishing are
the predominant activities. Of the cities selected,
Crisfield's economy, while isolated, is the most
diversified. Economic activity in Rock Hall and
Shady Side is centered around the fishing industry
with a limited amount of manufacturing activity
located in each community. Because of its close
proximity to Washington, D. C. and Annapolis,
Maryland, Shady Side offers the greatest number of
job alternatives.^

PROCEDURE

Numerous factors serve to determine the employ-
ability and alternative earning power of an individual.
Of the various factors that influence job mobility,
age, level of education and amount of vocational
training were considered critical. Skills acquired from
part-time, off-season and other miscellaneous job
experience also contribute to a person's mobility. The
current and projected demand for and supply of
individuals with various skills were viewed as im-
portant.

The data required for the investigation were
generated by selecting a stratified random sample of

'This work was supported by the National Marine
Fisheries Service, Contract No. N-043-7-71.

It is difficult to say how representative these four
communities are of the entire oyster fishery. Suf-
ficient information is not readily available to iden-
tify the characteristics of the population of oyster-
men in Maryland and relate them to those of the
sample fishermen in these communities.

R.J, MARASCO

TABLE 1. Age frequency of licensed oystermen.

Location

1-19

19-24

25-29

Age in Years
30-34 35-44

45-54

55-64

65-H

Shady Side

Rock Hall

Crisfield-
Smith Island

Avenue

Percent of Total

133 oystermen. Forty-one oystermen were inter-
viewed in Rock Hall. This represented the largest
number of interviews taken in a single community.
Crisfield-Smith Island, Avenue and Shady Side fol-
lowed with 33, 30 and 29 interviews, respectively.^

Taking into account information collected on
employment, related variables such as skills obtained
either from currently held part-time jobs or alterna-
tive jobs held in the past, level of education, age and
the labor market in the four communities, a labor
mobility table was constructed. Oystermen inter-
viewed were classified as being either "potentially
employable," "potentially trainable," "potentially
hardcore unemployed" or "not in the labor force."

Individuals who had either sufficient educational
training or marketable skills which matched the
demand in the local labor market were classified as
"potentially employable." The category "potentially
trainable" included persons capable of participating
in a training program. Age and level of education
were used to get a first approximation of an individ-
ual's suitability for further training. Oystermen who
had no marketable skills, who fell into the age
bracket, 45-65, and who had completed less than 6
years of education were classified as being potentially
hard-core unemployed. These individuals in all likeli-
hood would find it difficult to make vocational

^The validity of the actual sample sizes was verified
by making comparisons with optimal sample sizes
calculated from the information obtained in the
preliminary interviews.

"it should be borne in mind that the above classifi-
cation is only a preliminary step in identifying the
differences in labor market participation potential.

re-adjustments. The last category, "not in the labor
force," included oystermen who were either over 65
years of age or students."*

RESULTS

Age of the Oystermen Interviewed

The average ages of the interviewees from Shady
Side, Rock Hall, Crisfield-Smith Island, and Avenue
were 41.3, 44.8, 42.2 and 45.8 years, respectively.
Statistical analysis failed to reveal any significant dif-
ferences between the averages. Further summariza-
tion of the survey information revealed that Rock
Hall had the largest number of older oystermen
(Table 1).

Level of Education

Table 2 summarizes the educational data obtained
from the interviews. Of the oystermen interviewed
from Shady Side, 21% had an eighth grade education
or less, whereas in each of the remaining communi-
ties, the percentage of watermen having the same
level of formal schooling was substantially higher.
Specifically, 42% of the oystermen living in Rock
Hall had an eighth grade education or less. The per-
centage of watermen having no more than 8 years of
formal education jumped to 60% in Avenue and 67%
in Crisfield-Smith Island.

The average level of education completed for each
of the communities was: Shady Side - 9.9 years. Rock
Hall - 9.4 years, Crisfield-Smith Island - 7.8 years and
Avenue - 8.3 years. Statistical analyses indicated that
the average level of education for watermen living in
Crisfield-Smith Island was significantly lower than
that completed by oystermen based in Rock Hall and
Shady Side.

Cross-Tabulation of Age Versus Education

Cross-tabulation of the age and level of education
data revealed that 28% of the watermen interviewed

EARNING POWER OF MARYLAND OYSTERMEN
TABLE 2. Level of education.

Location

1-5

Educational level (in years)
7-8 9 10-11

13-15

Shady Side

Rock Hall

Crisfield-
Smith Island

Avenue

Percent of Total

had a high school education (Table 3). The majority
of oystermen in this group(32%) were betvi^een 19-45
years of age. However, 48% of those interviewed had
an eighth grade education or less, and 68% of this
group were 46 years old or older.

Vocational Training^

To delve further into the educational level of the
oystermen interviewed, each was asked whether
he had ever received any vocational training and if so,
had he used it within the past five years. Twen-
ty-three percent of the licensed oystermen inter-
viewed stated that they had received some type of

'Vocational training was defined as schooling re-
ceived either on the job or from a trade school.

vocational training. Only 26% of the oystermen that
responded "yes" to the vocational training question
also stated that they had not used the acquired skills
within the last five years (Table 4). The number of
watermen receiving some vocational training ranged
from none in Crisfield-Smith Island to 12 of the 29
fishermen interviewed in Shady Side.

Part-time and/or Off-season Job Status

Table 5 summarizes how interviewed oystermen
allocated their working time. Of the watermen inter-
viewed, 63 reported that they spent all of their time
fishing. Out of the 63, 38 reported spending all their
time harvesting oysters and crabs. A majority, 29, of
the crabbers/oystermen lived in Crisfield-Smith
Island. Fishermen located in the remaining com-
munities concentrated more on clams and finfish.

TABLE 3. Ages of licensed oystermen versus the level of education of oystermen interviewed.

Age

Years of education

Percent of Total

1-5

7-8

10-11

13-15

1-19

-.„

.—

19-24

—

...

....

—

25-29

....

...

....

30-34

—

...

....

35-44

—

45-54

—

55-64

—

65 +

....

Percent of Total

100

R.J. MARASCO

TABLE 4. Percentage of oystermen who have received voactional training.

Location

Shady Side
Rock Hall
Avenue
Crisfield-Smith Island

If so, have you

Have received

used it within the

vocational training

last five years

42%

69% responded yes

20%

67% responded yes

50% responded yes

0% responded yes

Only 17 out of the 133 oystermen interviewed
reported that they oystered full-time during the
oyster season and held down a full-time job outside
the fishing industry during the off-season. Those
interviewed in this group were concentrated in Shady
Side.

When asked if they held a non-fishing job in addi-
tion to their oystering activities, 49 of the 133 oyster-
men interviewed responded "y^s." Seventy-eight of

those interviewed stated that they did not participate
in other employment activities (Table 6). Of the four
communities, Crisfield-Smith Island had the smallest
number of oystermen who stated that they did hold
either part-time or off-season jobs in addition to their
oystering activities.
Recognized Employment Alternatives

In order to gauge the degree of recognition of
employment alternatives, the oystermen were asked

TABLE 5. Allocation of working time for interviewed oystermen.

Allocation

of Working Time

Shady
Side

Rock
Hall

Crisfield-
Smith Island

Avenue

Total

Finfish-Oyster
full-time

Crab-Oyster
full-time

Finfish-Crab-Oyster
full-time

Crab-Oyster-

outside employment
full-time

Clam-Oyster
full-time

Fin fish-Clam-Oyster
full-time

Oyster with outside
employment in
off-season

Oyster part-time
with outside
employment

Retired

Other

EARNING POWER OF MARYLAND OYSTERMEN

TABLE 6. Response to the question, "Do you hold a non-fishing job in addition
to oystering?"

Crisfield-

Response

Snady Side

Rock Hall

Smith Island

Avenue

Total

Yes

No response

TOTAL

133

what they would do to support their families if the
oyster supply failed due to pollution and/or disease.
The responses •given to this question are summarized
in Table 7. Out of the 133 oystermen interviewed
only 23 (17%) were unsure as to what they would do
if the oyster fishery failed. However, 13 watermen
who were undecided as to what type of work they
would do stated that they would actively seek em-
ployment. Only 6 of the interviewees stated that they

would have to go on welfare.

Labor Market Participation Potential

Taking into account information collected on
employment related variables such as skills obtained,
past job experience, level of education, age, and the
current labor market in the four communities, a labor
mobility table, (Table 8) was constructed. Oystermen
interviewed were classified as being either "poten-

TABLE 7. Types of action that would be taken if there was a failure in the oyster
fishery.

Response

Shady

Rock

Crisfield-

Avenue

Total

Side

Hall

Smith Island

Undecided or No Response

Undecided as to what type

of work, but would try

to find a job

Welfare

Stay on in some other

aspect of watering

Retire or Retired

Construction

Farm

Mechanic

Bricklayer

Painter

Fireman

Management

Electrical Work

Iron Work

Police

Other

R.J. MARASCO

TABLE 8. Labor market participation potential.

Community

Oystermen
Interviewed

Potentially
Employable a

Possibly
Trainable b

Potential

Hard-Core

Unemployed

Not in

Labor

/ Forced

Avenue

25d

Rock Hall

Shady Side

Crisfield-

Smith Island

a Those having marketable skills or sufficient education.

b Those capable of participating in a training program. Age and level of education were
used as a first approximation of trainability.

c Those having no marketable skills between the ages of 45 and 65 who have completed
less than 6 years of school.

d Students and those over 65 years.

tially employable," "potentially trainable," "poten-
tially hard-core unemployed," or "not in the labor
force."

Of the four communities surveyed, Avenue and
Shady Side were found to contain the largest number
that were "potentially employable." The booming
construction industry in these two areas served as one
of the major explanatory factors. The large number
of fishermen in these two communities with employ-
ment either part-time or during the off-season con-
tributed to their mobility. Employment opportunities
in Rock Hall and Crisfield-Smith Island were found to
be limited. Further, the jobs available required educa-
tional training beyond that received by many of the
surveyed watermen. The employability dilemma was
indicative of the possible need for training programs
and possible relocation of Rock Hall and Cris-
field-Smith Island watermen, if a major disaster were
to occur. Results reported in Table 8 indicate that a
large number of the watermen located in these two
communities were potentially trainable.

The relatively large concentration of potential
hard-core interviewees in Rock Hall and Cris-
field-Smith Island reflected the high concentration of
older and less educated watermen located in these
two communities, and the demand for individuals
with high school educations and beyond.

SUMMARY
Information obtained from 133 interviews viith

oystermen located in four Maryland communities
indicated that the labor market participation
potential was significantly higher for the two western
shore communities of Avenue and Shady Side than
for the eastern shore communities of Rock Hall and
Crisfield-Smith Island. Vocational training and out-
side employment coupled with an expanding con-
struction industry were found to contribute greatly
to the potential employability of the interviewed
watermen living in Shady Side. The employability of
Avenue oystermen was found to be aided greatly by
the strong demand for unskilled labor. Potential
vocational readjustment for oystermen living in Rock
Hall and Crisfield-Smith Island was found to be
hampered by the lack of jobs for people with limited
educational and/or vocational training, indicating a
possible need for training programs and potential
relocation if a serious disaster were to occur. The high
concentration of potentially hard-core unemployed
watermen located in Rock Hall and Crisfield-Smith
Island could necessitate additional assistance pro-
grams. Age, limited educational training and addition-
al job experience were factors that led to the classifi-
cation of approximately 20% of the interviewees in
the two eastern shore communities as being "po-
tentially hard-core unemployed."

LITERATURE CITED

Lang, 1961. Follow the Water. J. F. Blair Publishing
Co., Winston-Salem, N. C, 222 p.

Proceedings of the National Shellfisheries Association
Volume 63- June 1973

GROWTH OF OYSTER LARVAE, CRASSOSTREA VIRGINICA,

OF VARIOUS SIZES IN DIFFERENT CONCENTRATIONS

OF THE CHRYSOPHYTE, ISOCHRYSIS GALBANA

Edwin W. Rhodes and Warren S. Landers

NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION

NATIONAL MARINE FISHERIES SERVICE

MIDDLE ATLANTIC COASTAL FISHERIES CENTER

MILFORD, CONNECTICUT

ABSTRACT

Oyster larvae I'Crassostrea virginicaj of seven different size groups were fed
different concentrations of Isochrysis galbana. The optimum concentration of I. gal-
bana for each size group was determined by measuring the increase in mean length
of larvae during the 48 hr test period. The optimum concentration increased with
increasing larval size and ranged from 2.5 /i/ of packed cells per liter of larval cul-
ture for larvae 74 fi long to 32.5 id of packed cells per liter of larval culture for lar-
vae averaging 246 ti in length. It was found to be more efficient to increase the
Isochrysis concentration as the larvae grew than to feed the larvae at constant rates.

INTRODUCTION

There is much current interest in the culture of
oyster larvae by large privately owned hatcheries
and other organizations raising oyster larvae for
their research programs. One of the most impor-
tant factors in bringing large numbers of oyster
larvae successfully to metamorphosis is the type
and amount of food used during the rearing pro-
cedure.

Most studies of the food requirements of oyster
larvae have been concerned primarily with the rela-
tive growth achieved with particular micro-
organisms. Cole (1937) was the first to demon-
strate that pure cultures of naked flagellates could
be used to produce significant growth of Ostrea
edulis larvae under laboratory conditions. Bruce,
Knight and Parke (1940) cultured six species of
flagellated algae and found two, Isochrysis galbana
and Pyraminomonas grossi, that were good foods
for O. edulis larvae. Walne (1963) reported that /.
galbana, among other algal species, was an accept-
able food for O. edulis larvae. Davis (1950, 1953)
tested a number of potential foods for Crassostrea
virginica larvae and found that five flagellated
species and Chlorella sp. were utilized. Later, 10
genera of microorganisms were tested by Davis and

Guillard (1958) and they found that the chryso-
phytes, /. galbana and Monochrysis lutheri, were
of approximately equal value and the best single
foods for C. virginica larvae.

Some information is available on the quantita-
tive aspects of feeding shellfish larvae. Loosanoff,
Davis and Chanley (1953, 1955) studied the larvae
of Mercenaria mercenaria and reported that heavy
concentrations of Chlorella sp. killed larvae, that
larval growth was abnormally slow when an insuf-
ficient amount of food was present, and that the
optimum larval growth over a 12-day period occur-
red at concentrations of 50,000 large (8 p.) or
400,000 small (4 p) Chlorella sp. cells/ml. Davis
and Guillard (1958) found the optimum concentra-
tions of /. galbana and M. lutheri for M. mer-\
cenaria larvae to be 200,000 and 250,000 cells/ml, 1!
respectively, with little difference in growth occur- '''
ring over a wide range of concentrations.

Bayne (1965) reported that Mytilus edulis
larvae exhibit a general increase in growth rate
with increasing /. galbana concentrations up to
100,000 cells/ml, the highest cell concentration
tested. Bayne's data also showed that the grazing
rate and the number of cells caught per larva in
24 hr increased with an increase in larval size.

E. RHODES AND W.S. LANDERS

Walne (1956, 1963, 1965, 1966) investigated
the quantitative aspects of feeding O. edulis larvae.
Walne (1965) reported a rapid increase in assimila-
tion of radioactively labeled /. galbana as food
concentrations increased until at 50,000 cells/ml
about 70% of the maximum assimilation is
obtained. He further showed that at cell densities
over 100,000/ml the increase in assimilation is
slight for substantial increases in cell density.
Walne also performed experiments which indicated
that, as larval sizes increase from about 170 - 260
H, the numbers of cells assimilated by a larva in
24 hr increase from 6,000 - 15,000. At larval den-
sities of 1.0 - 1.5/ml Walne (1966) reported that
it was necessary to add food to cultures more fre-
quently than every 24 hr to maintain cell concen-
trations high enough for optimum growth of the
grazing larvae.

Davis and Guillard (1958) reported some in-
formation on the relatonship between algal concen-
tration and the growth of C. virginica larvae.
These workers fed five different concentrations of
/. galbana and M. lutheri to oyster larvae. They
found that a concentration of 250,000 M. lutheri
cells/ml was optimum at each sampling in a
14-day experiment. With /. galbana young larvae
grew best at 100,000 cells/ml, whereas older larvae
grew fastest at 400,000 cells/ml. The data of Davis
and Guillard, however, do not reveal the quantity
of /. galbana to feed to larvae of specific sizes to
obtain maximum growth.

Ukeles and Sweeney (1969) also reported some
food concentration data for C. virginica larvae.
They fed ^^C-labeled M. lutheri to straight-hinge
C. virginica larvae and found that retention is
most efficient at a food concentration of about
200,000 cells/ml or 13,000 cells/larva. At these
food concentrations approximately 150 - 250 M.
lutheri cells were taken up and retained per larva
in 24 hr. No data are reported for older larvae.

In the present work the concentrations of /.
galbana necessary to effect maximum grovrth of C.
virginica larvae of various sizes are reported, and
some comparisons are made between feeding at
constant rates and feeding on a graduated schedule
according to larval size.

METHODS

Algal Culture

I. galbana was chosen for this study because it
has been found to be one of the best foods for C.
virginica larvae (Davis and Guillard, 1958) and
because similar studies have been performed using

this species with O. edulis larvae (Walne, 1956,
1963, 1965, 1966). The Isochrysis used in these
experiments was grown in semicontinuous unialgal
cultures (not bacteria free) in a heat-sterilized, en-
riched seawater medium following the methods
described by Ukeles (1971). The Isochrysis re-
quired was harvested daily and the density of the
culture determined by centrifuging a 10-ml sample
in a Hopkins tube for 15 min at 1,000 g. The re-
sulting packed cell volumes were used to deter-
mine the appropriate quantities of algal suspension
to feed to the larval cultures. The food concen-
trations reported, therefore, are expressed as
microliters of packed cells per liter of larval cul-
ture.

Feeding Concentration Experiments

All of the feeding concentration experiments
were short-term, acutely measured tests, molded
after the methods of Walne (1965). For each
series of experiments a stock population of oyster
larvae, consisting of the pooled progeny from a
number of Long Island Sound parents, was reared
according to the methods of Loosanoff and Davis
(1963). The stock populations were reared at 28°C
in 15-liter polyethylene containers containing fil-
tered seawater to which 100 ppm sodium sulfa-
methazine (Sulmet, American Cyanamid Co.)' had
been added, and were fed exclusively on a diet of
Isochrysis. To obtain larvae of a uniform size for
an individual experiment and to make the results
more applicable to commercial hatcheries where
larvae are separated and grown by size, the entire
stock population was screened through a series of
nylon mesh screens and the desired size group
selected. The nylon screens used had square open-
ings of 54, 75, 100, 135, 151, 180 and 216 /i.
Mesh size refers to the screen in this series which
retained larvae after a 3 min seawater rinse.
Straight-hinge larvae were not screened for size,
but were rinsed on a 36 ^i nylon screen before use
in the tests.

In the first series of experiments eight groups
of C. virginica larvae in four basic size categories
were tested in duplicate 1-liter cultures to which
Isochrysis concentrations of 0, 2.5, 5.0, 10.0, 20.0
and 40.0 /J/1 were added daily. The cultures of
about 15,000 larvae each were maintained in
Pyrex glass beakers at 28°C in filtered and ultra-
violet-treated seawater to which 100 ppm of

' Trade names mentioned in this paper do not
imply endorsement by the National Marine
Fisheries Service.

GROWTH OF OYSTER LARVAE

TABLE 1. Average optimum Isochrysis concentra-
tions (iil/l) for maximum growth of oyster larvae of
various sizes in 48 hr.

Average Initial Initial Larval Average Optimum
Larval Size (^i) Mesh Size Feeding Concentration

74.4

S-H

2.5

80.1

5.6

107.0

75.

13.8

139.8

100

17.5

170.0

135

22.5

200.4

151

32.5

246.4

180

32.5

sodium sulfamethazine had been added. The
experiments were sampled and terminated at 48
hr. Growfth data consisted of 100 larval measure-
ments for each sample. Larval lengths were
measured to the nearest 5 jd with an ocular micro-
meter.

Using the results from the first series of experi-
ments, series of seven Isochrysis concentrations
were selected for testing each of seven larval size
groups. Each size group was tested in two 48 hr
experiments, and duplicate 1 -liter cultures were
used at each of the seven concentrations of food
tested in each experiment. Experimental methods
were identical to those above. In the two experi-
ments involving larvae larger than 237 /j in initial
length a clean oyster shell was added to each
beaker to provide a suitable substrate for larvae
that might attain a size sufficient for meta-
morphosis.

Reding Schedule Experiments

After the feeding concentration experiments had
revealed the optimum feeding rate for the seven
sizes of larvae, two experiments were performed in
which the growth of oyster larvae using a gradu-
ated feeding schedule and several constant feeding
rates were compared. The average optimum food
concentrations, which comprised the graduated
feeding schedule, are indicated in Table 1. The
constant feeding rates were 0, 2.5, 5.0, 10.0, 20.0
and 40.0 /J/1. The tests were set up with
straight-hinge larvae and were terminated when
eyed larvae were observed. Larvae in all treatments
were screened from the cultures and resuspended
in clean seawater every two days. Larvae in one
set of cultures in the first feeding schedule experi-
ment were separated by size every two days, set
up in beakers according to mesh size at a density
of 15 larvae/ml, and the beakers fed at the con-
centrations of Isochrysis indicated in Table 1. All
other methods and materials were identical to
those above.

RESULTS AND DISCUSSION

Food Concentrations and Larval Growth

The results of the first series of experiments are
presented in Table 2. These data show generally
the concentrations of Isochrysis necessary for good
growth of oyster larvae of various sizes. However,
in the first and seventh experiments reported in
Table 2 the maximum growth was achieved in the
lowest and highest food concentrations tested,
respectively, making it necessary to expand the
ranges of concentrations used in later tests. Be-
cause our initial food concentrations were vddely
spaced, we also wanted to test some intermediate
concentrations. Therefore, for the remainder of the

TABLE 2. The average growth increments (n) of oyster larvae of various sizes after
being fed different concentrations of Isochrysis in Experimental Series 1.

Food

Initial

Larval

Length (ii)

Concentration

(Ml/1)

77.1

77.7

104.1

104.2

139.4

145.8

200.6

204.2

40.0

3.3

10.1

17.1

22.0

5.9

31.9

30.9

8.4

20.0

4.1

13.1

19.2

33.4

3.4

41.0

19.9

27.0

10.0

7.9

14.9

19.5

38.4

11.8

20.9

8.0

14.6

5.0

11.7

18.8

15.1

28.8

9.0

7.1

3.8

4.9

2.5

13.8

19.3

9.1

16.1

4.7

1.4

0.0

4.2

Unfed

3.9

4.6

2.7

5.5

0.0

1.2

E. RHODES AND W.S. LANDERS

TABLE 3. The average growth increments (n) of oyster larvae of various sizes
after being fed different concentrations of Isochrysis in Experimental Series
2.

Food

Initial Larval Length (/n)

Concentration

(Ml/1)

74.2

80.1

104.6

137.1

168.1

200.4

255.0

60.0

9.7

50.0

17.1

45.0

38.1

16.8

40.0

38.1

11.9

35.0

20.5

35.6

16.3

30.0

27.6

38.1

17.6

25.0

43.1

30.6

37.7

11.1

20.0

41.9

42.9

34.2

26.7

17.5

43.8

41.9

33.6

28.0

15.0

17.0

45.1

43.6

33.2

12.5

13.2

41.7

39.3

27.5

10.0

11.7

14.7

39.0

25.8

7.5

12.3

17.7

34.7

15.5

5.0

15.0

14.2

18.6

3.8

15.6

12.3

2.5

14.6

12.7

1.2

7.5

0.6

5.6

Unfed

2.2

0.0

2.3

3.4

0.0

food concentration experiments reported here we
used the data in Table 2 to select a series of food
concentrations to be tested against larvae of
specific sizes.

Tables 3 and 4 present the results of the sec-
ond and third series of experiments. In Figure 1
the Isochrysis concentrations which produced the
most rapid growth of larvae of various sizes are
plotted against initial larval length. Duncan's multi-
ple range tests (Steel and Torrie, 1960) were
performed to determine in each experiment which
grovrth increments were not significantly different
from the maximum increment obtained (95% con-
fidence level), and these are indicated as vertical
lines in Figure 1. The average optimum Isochrysis
concentrations for larvae in the seven size groups
tested are presented in Table 1.

These feeding experiments indicate that, as oys-
ter larvae grow, their food requirements increase
substantially. A 13-fold increase in Isochrysis con-
centrations was found necessary to support maxi-
mum growth of the larvae over the range of sizes
tested. Straight-hinge larvae 74 n in length grew

• SESrES 2

■

o SERIES 3

•

50 100 150 200 250

INITIAL MEAN LENGTH IN MICRONS

FIG. i. The optimum Isochrysis concentrations for
C. virginica larvae of various initial mean lengths.
Points indicate concentrations in which the
greatest growth increment was obtained in the raw
data. Vertical lines indicate concentrations in
which growth increments were not statistically dif-
ferent from those obtained in the concentration
producing the greatest growth increment.

GROWTH OF OYSTER LARVAE

TABLE 4. The average growth increments (id) of oyster larvae of various sizes
after being fed different concentrations of Isochrysis in Experimental Series
3.

Food

Initial Larval Length (/j)

Concentration

(Ml/1)

74.7

80.1

109.4

142.4

172.0

200.4

237.7

60.0

0.0

50.0

0.0

45.0

20.7

0.5

40.0

22.9

0.0

35.0

20.4

25.3

4.5

30.0

20.4

22.4

0.0

25.0

36.8

23.3

21.6

1.3

26.0

22.6

38.1

22.6

15.1

17.5

24.7

37.7

20.2

11.2

15.0

17.3

26.2

28.1

18.8

12.5

18.8

27.4

23.6

14.1

10.0

6.7

20.6

20.5

19.1

7.5

7.9

19.8

18.6

11.0

5.0

9.4

14.0

14.8

3.8

9.5

21.6

2.5

8.5

17.7

1.2

10.3

0.6

8.5

Unfed

3.7

4.7

1.7

0.0

fastest at an average concentration of 2.5 /l(1/1,
while larvae 200 /j in length required an average
Isochrysis concentration of 32.5 /jl/1 for maximum
growth.

Larvae longer than 237 jd grew slower than the
other groups tested (Tables 3 and 4). There are no
data in the literature to suggest that oyster larvae
grow more slowly as they approach metamorphosis
in the presence of a substrate suitable for setting.
We suspect that this slow growth was due to a
toxic substance associated with the Isochrysis cul-
tures used for these tests since the larvae in the
fed beakers failed to swim actively, while the lar-
vae in the unfed beakers did swim actively.

The experiments of Davis and Guillard (1958),
although not specifically designed to reveal opti-
mum feeding concentrations for larvae of various
sizes, did indicate a general increase in food re-
quirements as oyster larvae grow. The data of
these workers showed the optimum Isochrysis con-
centration for 75 Id larvae to be 10)L(1/1, whereas
140 Id larvae grew best at 40 jUl/1. These con-
centrations are somewhat higher than those found
to be optimum in the present study. For the most
part, larvae in the present study grew faster and at
lower concentrations of food than did those of

Davis and Guillard. The lower temperatures (21 -
23° C) used by Davis and Guillard and differences
in the quality of the Isochrysis cultures used may
account for this discrepancy.

Guillard (1958) considered the data of Davis
and Guillard (1958) and the levels of food
organisms encountered by shellfish larvae in natur-
al situations and suggested that an algal concen-
tration of 10 lAH be used as a guide in feeding
oyster larvae at densities of 3 - 15/ml. The data
from the present study show that this concentra-
tion of cells is less than optimum for larvae over
100 Id long.

The studies of Ukeles and Sweeney (1969)
showed that about 150 - 250 M. lutheri cells/larva
were taken up and retained by straight-hinge larvae
in 24 hr at a concentration of 13,000 celis/larva,
the most efficient feeding concentration. Assuming
that 100,000 Isochrysis cells/ml is equal to 10 ^1
of packed cells/liter (Davis and Guillard, 1958), in
our study only about 1,600 Isochrysis cells were
available to each straight-hinge larva in a 24-hr
period at the concentrations that produced the
most rapid growth. Although we made tio final al-
gal counts, significant clearing of the cultures was
observed and most of the those cells available

E. RHODES AND W.S. LANDERS

were probably utilized. The slower growth of
straight-hinge larvae which we obtained at higher
food concentrations (Table 2) shows that under
the conditions in the present experiments the low-
er feeding rate is superior.

Walne (1956, 1963, 1965, 1966) provided much
information on the feeding behavior of O. edulis
larvae. Because this species is larviparous and
releases larvae averaging 170 n in length, no com-
parisons of food requirements are possible for
small larvae, but some can be made for larger
ones. Walne (1965) reported that O. edulis lar\'ae
averaging 219 /J in length catch an average of
24,000 Isochrysis cells in 24 hr. The growth data
in the present study indicate maximum growth of
similar size C. virginica larvae at a concentration
of about 20,000 cells/larva in 24 hr. Walne (1965)
also reported that, as O. edulis larvae grow from
about 170 - 260 /j in the planktonic phase, the as-
similation of Isochrysis cells increases 2.5 fold.
The data presented here indicate an approximate
doubling of optimum cell concentrations for C.
virginica larvae of similar sizes.

One of the prime considerations in evaluating a
feeding schedule is the number of larvae that can
be reared per unit volume. The present experi-
ments indicate that acceptable growth can be
achieved with proper feeding concentrations over a
wide range of larval sizes at a density of 15 lar-
vae/ml. Walne (1965) obtained rapid growth at a
density of 140 O. edulis larvae/1 and a cell con-
centration of 123,000/ml; but to get similar
growth at a larval concentration of 5,000/1 the
Isochrysis concentration had to be tripled. In the
first case the small number of larvae grazing did
not significantly reduce the Isochrysis concentra-
tion, while at the higher larval density food be-
came a limiting factor. Davis (1953) observed an
inverse relationship between larval density and
growth at various Chlorella sp. concentrations for
C. virginica larvae. Loosanoff et al. (1955) who
fed various amounts of Chlorella sp. to clam lar-
vae, M. mercenaria, concluded that an increase in
larval densities beyond a certain limit cannot be
compensated for by a proportionate increase in
the quantity of food. The limits in this situation
appear to result from the mechanical interference
with feeding at high algal concentrations
(Loosanoff et al., 1955), the possible occurrence
of toxins produced by the algal cells or present in
the algal suspension from some other source, and
the accumulation of inhibiting quantities of meta-
bolic wastes from the larvae at high densities.

At the larval density of 15 /ml used in the

UNFED

y 1 r

I"'"'- ./.^//M

GROWTH INCREMENTS
W?L 2ND TO 6TH DAY

F 1 6TH TO lOTM DAY

I I lOTH TO I2TH DAY

10 Oyl/I

r;-;/ .;,...,/;;.

200).(//

t rwM -■<■■■

v-:>^-sv-'>" , 1

40 0|. f/l

I ".'4 ,

, 1

GRADUATED SCHEDULE

GRADUATED SCHEDULE (SCREEN SEPARATED LARVAE)

IV^v.-».-^.v------/--/-^.^il..

^\r\ 1 1-

75 100 125 150 175 200 225 250

MEAN LENGTH IN MICRONS

FIG. 2. The growth of C. virginica larvae on dif-
ferent feeding schedules.
Experiment 1.

present study the Isochrysis concentrations in the
larval cultures were substantially reduced by graz-
ing in the 24 hr between feedings. The ideal feed-
ing situation should probably include provisions
for continuous feeding so that an optimum con-
centration of cells would be present in the culture
vessel at all times.

Feeding Schedules and Larval Growth

The results of the first feeding schedule experi-
ment are presented in Figure 2. From 65 - 90% of
the original larval population were alive in the dif-
ferent treatments on the twelfth day. A Duncan's
multiple range test of the data from the final

UNFED

mmuy/:'A 1

S.O/il/l

mm::iM

JgiJ 1

io.Om'/i

mm^Mifimmmmi 1

20.0 m '/'

mm/t:mmmm-siMmm

40 0/i///

y/m , . ) 1

GRADUATED

SCHEDULE

y////M .:: ! 1

GROWTH INCREMENTS
rM 2ND TO 6TH DAY

[ 1 6TH TO lOTH DAY

I I lOTH TO I4TH DAY

A^H h

H 1 1 H

75 100 125 150 175 200 225 250

MEAN LENGTH IN MICRONS

FIG. 3. The growth of C. virginica larvae on dif-
ferent feeding schedules. Experiment 2.

GROWTH OF OYSTER LARVAE

sampling date showed all treatment means to be
significantly different from each other (95% confi-
dence level), except between the means repre-
senting 20 /jl/l and the graduated feeding schedule
for screened larvae. The best growth occurred at
40 jul/l, but the cultures fed according to the
graduated feeding schedule produced larvae only
5% smaller and required only 46% of the total
Isochrysis used to feed the 40 /il/1 cultures. The
larvae that were screen-separated into size groups,
adjusted to 15/ml, and fed according to size, were
fed 56% of the food required to maintain the lar-
vae in the fastest growing treatment.

The growth data from the second feeding sche-
dule experiment are presented in Figure 3. All
treatment means for the final sample are statis-
tically different from each other (Duncan's multi-
ple range test, 95% confidence level), except those
representing 10 ^1/' and 40 lAjl. On the 14 day of
the experiment 70% of the original larval popula-
tion were alive in the best two treatments. The
larvae at 20 /il/1 were 4% larger than those fed ac-
cording to the graduated feeding schedule, but to
effect this increase 63% more Isochrysis was re-
quired. These results show that using constant
feeding rates Isochrysis concentrations of 20 to 40
/j1/1 are required to effect maximum growth rates
of C. virginica larvae at densities of 10 - 15/ml.
Similar high rates of growth can be achieved by
starting at much lower feeding rates and then in-
creasing the Isochrysis concentration as the larvae
grow. This latter method requires a smaller volume
of algae than the constant concentration method
and could yield significant savings to organizations
rearing substantial numbers of oyster larvae.

ACKNOWLEDGMENTS

We thank Dr. Ravenna Ukeles, who provided
the phytoplankton; Mr. Bruce Collins, who per-
formed some of the larval measurements; Mr. John
Maclnnes, for the statistical treatment of the data
and Mr. Harry C. Davis, for his review of the
manuscript.

LITERATURE CITED

Bayne, B. L. 1965. Growth and the delay of
metamorphosis of the larvae of Mytilus edulis
(L.). Ophelia 2: 1-47.

Bruce, J. R., M. Knight and M. W. Parke. 1940.
The rearing of oyster larvae on the algal diet. J.
Mar. Biol. Assoc. U. K. 24: 337-374.

Cole, H. A. 1937. Experiments in the breeding of
oysters (Ostrea edulis) in tanks, with special
reference to the food of the larva and spat.

Fish. Invest., Minist. Agric. Fish Food Ser. 2

15: 1-28.
Davis, H. C. 1950. On food requirements of larvae

of Ostrea virginica. Anat. Rec. 108: 132-133.
Davis, H. C. 1953. On food and feeding of larvae of

the American Oyster, C. virginica. Biol. Bull. 104:

334-350.
Davis, H. C. and R. R. Guillard. 1958. Relative

value of ten genera of micro-organisms as food

for oyster and clam larvae. U. S. Fish Wildl.

Serv. Fish. Bull. 58: 293-304.
Guillard, R. R. 1958. Some factors in the use of

nannoplankton cultures as food for larval and

juvenile bivalves. Proc. Natl. Shellfish. Assoc.

48: 134-14?.
Loosanoff, V. L. and H. C. Davis. 1963. Rearing

of bivalve mollusks. Adv. Mar. Biol. 1: 1-136.
Loosanoff, V. L., C. Davis and P. E. Chanley. 1953.

Behavior of clam larvae in different concentrations

of food organisms. Anat. Rec. 117: 586-587.

Loosanoff, V. L., H. C. Davis and P. E. Chanley.
1955. Food requirements of some bivalve lar-
vae. Proc. Natl. Shellfish. Assoc. 45: 66-83.

Steel, R. G. D. and J. H. Torrie. 1960. Principles
and Procedures of Statistics. McGraw-Hill, New
York.

Ukeles, R. 1970. Nutritional requirements in shell-
fish culture. In K. S. Price and D. L. Maurer
(ed.). Proceedings of the Conference on Artifi-
cial Propagation of Commercially Valuable
Shellfish - Oysters. Univ. Delaware, Newark,
Del. p. 43-64.

Ukeles, R. and B. M. Sweeney. 1969. Influence of
dinoflagellate trichocysts and other factors on
the feeding of Crassostrea virginica larvae on
Monochrysis lutheri. Limnol. Oceanogr. 14:
403-410.

Walne, P. R. 1956. Experimental rearing of the
larvae of Ostrea edulis L. in the laboratory.
Fish. Inves., Minist. Agric. Fish. Food Ser. 2
20(9): 1-23.

Walne, P. R. 1963. Observations on the food value
of seven species of edgae to the larvae of Ostrea
edulis L. I. Feeding experiments. J. Mar. Biol.
Assoc. U. K. 43: 767-784.

Walne, P. R. 1965. Observations on the influence
of food supply and temperature on the feeding
and growth of the larvae of Ostrea edulis L.
Fish. Invest., Minist. Agric. Fish. Food Ser. 2
24(1): 1-45.

Walne, P. R. 1966. Experiments in the large-scale
culture of the larvae of Ostrea edulis L. Fish.
Invest., Minist. Agric. Fish. Food Ser. 2 25(4):
1-53.

Proceedings of the National Shell fisheries Association
Volume 63 - June 1973

A NEW TECHNIQUE FOR MEASURING THE OXYGEN CONSUMPTION
OF LARVAE OF THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA

John R. Maclnnes and Frederick P. Thurberg

NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION

NATIONAL MARINE FISHERIES SERVICE

MILFORD, CONNECTICUT

ABSTRACT

The oxygen consumption rates of larvae of the American oyster, Crassostrea
virginica, were determined using an all-glass differential microrespirometer. Oxygen
uptake was found to increase logarithmetically as the larvae grew in size. This rate
ranged from less than 0.2 iil O^/hr/lOOO larvae for individuals 60 n in length to 20
Ijj 02/hr/lOOO larvae for individuals 200 ji in length.

INTRODUCTION

The American oyster, Crassostrea virginica, is an
estuarine bivalve that is naturally exposed to a
wide range of environmental conditions. The de-
velopment of techniques for rearing bivalve mol-
lusks (Loosanoff and Davis, 1963) has prompted a
number of studies on the effects of environmental
changes (temperature, salinity, pH, industrial pollu-
tion) on the survival of bivalve embryos and larvae
(Woelke, 1967; Calabrese and Davis, 1970; Cala-
brese, Collier, Nelson and Maclnnes, 1973). Oxy-
gen consumption is a parameter often used as an
indicator of sublethal environmental stress on the
metabolism of the organism studied. The small size
and low respiration rate of bivalve larvae, however,
have made accurate determinations of larval oxy-
gen consumption extremely difficult. Conventional
respirometers, such as the Warburg apparatus, are
not sufficiently sensitive to determine a precise
oxygen uptake rate. Vemberg and Costlow (1966),
using a differential microrespirometer developed by
Grunbaum, Siegel, Schulz and Kirk (1955), were
able to measure the oxygen consumption of fid-
dler crab larvae (Uca, various species). Recently,
Sastry and McCarthy (1972)' were able to meas-
ure oxygen consumption rates of larvae of two
brachyuran crabs. Cancer irroratus and C. borealis,
using the same type of microrespirometer.

The present study was designed to evaluate the
use of a similar microrespirometer in measuring
normal oxygen consumption rates of oyster larvae
during the period of development from several

hours after fertilization to metamorphosis two or
three weeks later. This evaluation may prove valu-
able in future studies using oxygen consumption
of bivalve larvae as an indicator of stress induced
by abnormal environmental conditions.

MATERIALS AND METHODS

Oyster eggs were obtained following the pro-
cedure described by Loosanoff and Davis (1963).
Adult oysters were induced to spawn by thermal
stimulation and by addition of sperm stripped
from a sacrificed male. The eggs were collected
from more than one female to insure a hetero-
geneous sample. The number of fertilized eggs per
unit volume was determined by microscopic exam-
ination of a subsample and approximately 500,000
eggs were then transferred to a 15 liter container
maintained in a water bath at 26°C. The larvae
were reared in natural seawater (salinity-25%o )
that had been circulated through 15 ^ and 1 11
Orion filters, an ultra-violet light sterilization unit
and an activated charcoal filter. The water was
changed every other day, and the larvae were fed
laboratory grown phytoplankton cultures of Iso-
chrysis galbana and Monochrysis lutheri. A sample

' Sastry, A. N. and J. F. McCarthy. 1972. Oxygen con-
sumption of larval stages of two sympatric species of
brachyuran crabs. Cancer irroratus and C. borealis.
In Abstracts of Papers Submitted for the 35th An-
nual Meeting, American Society of Limnology and
Oceanography, Inc., Tallahassee, Florida.

MEASURING OXYGEN CONSUMPTION IN OYSTER LARVAE

of larvae was taken prior to each oxygen con-
sumption experiment and the mean length of 50
larvae was determined. The all-glass microrespiro-
meter used in this study was essentially that des-
cribed by Grunbaum et al. (1955) and consisted
of a capillary with a 0.3 mm bore and two 5 ml
flasks; a respiration flask containing oyster larvae
and a control or compensation vessel containing
seawater but no organisms. Glass loops were
formed on each end of the capillary to hold a car-
bon dioxide absorbent; a filter paper disc soaked
with 1% KOH and tied in place. Several hundred
larvae, in 2.0 ml seawater, were placed in each res-
piration flask and then immersed in a constant
temperature (26 C) water bath. The amount of
oxygen consumed by the larvae was determined by
measuring the movement of the red distilled kero-
sene indicator in the capillary bore. The change in
oxygen is given by K times h where h is the dis-
tance covered by the indicator and the proport-
ionality factor is derived from the equation:

273 /P-Pw\ /Vg + l\

K =

in which:

,Po

,Vgi

T = absolute temperature

P = atmospheric pressure (mm Hg)

P^ = vapor pressure of water at T (mm Hg)

P = standard atmospheric pressure (mm Hg)

A = cross section of area of capillary bore

Vg = volume of respiration chamber

Vg' = volume of compensation chamber

Readings were recorded every 15 min during ex-
periments of at least 2 hr in duration. The res-
piratory rate was calculated as microliters of oxy-
gen consumed per hour per thousand larvae.

RESULTS AND DISCUSSION

The results of this study are presented in Fig-
ure 1. Since the relationship between mean length
(ii) of oyster larvae and the oxygen consumption
rate is curvilinear, it is best expressed logarith-
metically by the equation:

10 (log Y) = -4.556 + 2.941 (log X)

where X = mean length of larvae sampled on

the day of the experiment

and Y = consumption rate of (p\ OQ/hr/lOOO

larvae)

The high correlation coefficient of the relationship
(r - 0.875) indicates a rise in oxygen consumption
rate as the larvae grew in size. This rate increased
from less than 0.2 tx\ OQ/hr/lOOO larvae for indivi-
duals 60 li in length to 20Ad Og/hr/lOOO larvae

20 0 -

10 0 -

1 0 -

'/ IO(logY)--4 556

2 94l(logX)

/ r-0e75

60 0 100,0 200 0

LENGTH (>i)

FIG. 1. Oxygen consumption rates of American
oyster larvae (/ul 02/hr/lOOO larvae) vs size
(length in ji) at 26°C and 25 %o salinity.

for individuals that had grown to 200 n in length.
Other workers have noted similar increases in oxy-
gen consumption of developing oyster eggs (Black,
1962; Cleland, 1950), but little information is
available on the normal oxygen consumption of
60-200 /i larvae.

This study has demonstrated the suitability of
this microrespirometer in measuring oxygen con-
sumption of larval bivalves. The small size of this
instrument allows great sensitivity and performs
well if operated under constant temperature condi-
tions. The results, however, are valid only under
the conditions described in this study and may
not represent the actual respiratory rates in the
environment or in a hatchery situation. The rela-
tive values, however, are valuable and should prove
useful in future studies of the effects of environ-
mental changes on bivalve larvae.

LITERATURE CITED

Black, E. 1962. Respiration, electron-transport en-
zymes, and Krebs-cycle enzymes in early devel-
opmental stages of the oyster, Crassostrea vir-
ginica. Biol. Bull. 123: 58-70.

Calabre^, A. and H. C. Davis. 1970. Tolerances
and requirements of embryos and larvae of bi-
valve molluscs. Helgol. Wiss. Meeresunters. 20:
553-564.

J.R. MACINNES AND F.P. THURBERG

Calabrese A., R. S. Collier, D. A. Nelson and J.
R. Maclnnes. 1973. The toxicity of heavy
metals to embryos of the American oyster,
Crassostrea virginica. Mar. Biol. (Berlin) 18:
162-166.

Cleland, K. W. 1950. Respiration and cell division
in developing oyster eggs. Proc. Limn. Soc. N.
S. W. 75: 282-295.

Grunbaum, B. W., B. V. Siegel, A. R. Schulz and
P. L. Kirk. 1955. Determination of oxygen up-
take by tissue growth in all-glass differential
microrespirometer. Mikrochim. Acta 6:
1069-1075.

Loosanoff, V. L. and H. C. Davis. 1963. Rearing
of bivalve mollusks. Adv. Mar. Biol. 1: 1-136.

Vernberg, F. J. and J. D. Costlow, Jr. 1966.
Studies on the physiological variation between
tropical and temperate-zone fiddler crabs of the
genus Uca. IV. Oxygen consumption of larvae
and young crabs reared in the laboratory. Phys-
iol. Zool. 39: 36-52.

Woelke, C. E. 1967. Measurement of water quality
with the Pacific oyster embryo bioassay. In
Water Quality Criteria. Spec. Tech. Publ. No.
416. American Society for Testing Materials,
Phila., Pa. p. 112-120.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

SHELLFISH MARICULTURE IN AN ARTIFICIAL UPWELLING SYSTEM'

Judith S. Baab, Gerald L. Hamm, Kenneth C. Haines, Arthur Chu

and Oswald A. Roels

LAMONT-DOHERTY GEOLOGICAL OBSERVATORY

COLUMBIA UNIVERSITY AND CITY UNIVERSITY INSTITUTE OF OCEANOGRAPHY

NEW YORK, NEW YORK AND PALISADES, NEW YORK

ABSTRACT

A ri^ariculture system was established on St. Croix, U. S. Virgin Islands, using
"artificial upwelling" to obtain deep water, rich in nutrients necessary for plant life.
Water from 870 m depth in the sea was pumped into 45,000-liter pools in which
species of three diatoms, Bellerochea sp., Chaetoceros simplex and Thalassiosira
pseudonana ('=Cyclotella nanaj were grown to feed shellfish. The St. Croix site was
chosen because the ocean reaches a depth of 1000 meters approximately 1.6 km
offshore.

Initially, 100,000 juvenile Crassostrea virgimca(Gmelin)and 100,000 juvenile Mer-
cenaria mercenaria Linne, from Long Island Sound were put into the system. The
oysters grew very well during the first few months but died rapidly thereafter. While
experiments indicated that clam survival and growth were good, they ceased feeding
for 24-48 hr after handling. Clams in sediment grew faster than those in wire trays.

Later introductions of F^ hybrid (or racial cross) clams (male M. mercenaria X
female M. campechiensis Say), grew far more rapidly than M. mercenaria and ap-
peared very well suited to the conditions of our system.

Comparative growth studies of C. virginica, C. gigaa (Thunberg) and Ostrea edulis
Linne are underway.

INTRODUCTION

Deep ocean water is cold and rich in nitrates,
phosphates, silicates and other dissolved nutrients
necessary for plant life. In our artificial upwelling
system on the north shore of St. Croix, water
from 870 m is pumped into 45,000-liter concrete
onshore pools where planktonic algae are grown as
food for shellfish in a controlled food chain
(Roels, Van Hemelrijck, Gerard and Worzel, 1971).

The accumulation of nutrients in the deep
water results from photosynthesis occurring in the
euphotic zone which is limited to a depth of ap-

'This work was supported by Sea Grant 1-36119
from the U.S. Department of Commerce. La-
mont-Doherty Geological Observatory Contribu-
tion No. 1919; City University Institute of
Oceanography Contribution No. 11.

proximately 100 m. Unicellular phytoplankton
converts solar energy, carbon dioxide, water, ni-
trate, phosphate and minor mineral elements into
protoplasm for their cells, the first link in the
food chain. As a result of this photosynthetic pro-
cess in the upper layer of the seas, carbon, nitro-
gen and phosphorus are extracted from solution
thereby depleting the surface waters of these nutri-
ents.

Particulate organic material resulting from dead
and disintegrating phytoplankton and the excreta
from zooplankters which have grazed on the
phytoplankton, sinks through the water column.
Bacterial and chemical activity eventually oxidizes
the organic matter to inorganic nitrate, phosphate
and silicate, etc., resulting in the high dissolved
nutrient content of deep water. Table 1 compares
some parameters for surface water and water from
870 m depth pumped up through our deep-water

J.S. BAAB, G.L. HAMM, K.C. HAINES, A. CHU AND O.A. ROELS
TABLE 1. Comparison of properties of deep and surface water.

In Situ
Temperature Salinity
°C "Z""

'NOc

^PO^- ^Si04-

Surface
Water

26-29

35.83

2.3

0.1

Deep
Water

34.87

32.5±0.7 2.15+0.09 26.5±0.5

^Values in microequivalents/liter.

pipe in St. Croix.

The cold temperature of deep ocean water can
be used for a wide variety of cooling applications
and for sea tliermal power production by the
"Claude" process (Claude, 1930) in areas where
the temperature differential between the surface
and the deep water is great enough. Some of the
possible cooling applications are air conditioning,
ice making, cooling for electrical power generating
plants and desalination plants (avoiding thermal
and brine pollution) and condensing atmospheric
moisture for fresh-water production. The discharge
water from these cooling systems would be a valu-
able resource for mariculture since its nutrient
content, essential for algal growth, is much higher
than that of surface water.

The advantages of using deep water over in-
shore or estuarine surface waters are: (1) its rela-
tive sterility, i.e., lack of human disease-producing
organisms, shellfish parasites, predators and fouling
organisms; (2) its negligible content of oxygen-con-
suming dissolved organic matter and suspended
sediment, especially pesticides and other man-made
pollutants; and (3) the constancy of its chemical
and thermal characteristics.

Progressive closing of shellfish beds due to: (1)
increased coliform counts in near-shore waters; (2)
mass shellfish mortality resulting from low salinity
caused by the 1972 hurricane "Agnes", floods in
the Chesapeake Bay System; (3) the September
1972 disastrous red tide in New England and (4)
the destruction of shellfish beds due to a variety
of pollutants, could all be avoided by use of this
mariculture system.

This paper reports results of growth experi-
ments with juvenile oysters (C. virginica, C. gigas
and O. edulis) and clams (M. mercenaria and M.
mercenaria X M. campechiensis F-, hybrids) in our
system.

MATERIALS AND METHODS

Deep ocean water was pumped from 870 m
depth into 1.2 m deep pools of 45,000 liter capa-
city. The pools were inoculated with planktonic
diatoms Bellerochea sp. clone STX-114, Chaeto-
ceros simplex clone STX-105 or Thalassiosira
pseudonana clone 3H. Details of the algal mass
culture system will be reported in a later publica-
tion. The algae grown in the pools to a concentra-
tion of 10"* - lO' cells ml'' were pumped contin-
uously at metered rates to a series of epoxy-coat-
ed plywood 750 liter shellfish tanks measuring 2.4
X 0.6 X 0.6 m (Fig. 1).

Water temperature in these tanks varied be-
tween 22° and 29° C. Cell concentrations in the
water entering and leaving the shellfish tanks were
monitored twice daily by counting in a Spiers-Le-
vy eosinophil counter under 200-power phase il-
lumination. Flow rates of the phytoplankton sus-
pension to the tanks were based on growth rates
of shellfish in each tank. Flow-rates to the differ-
ent tanks were set according to the following
formula:

weight gain of the shellfish
in that tank per 24 hr

weight gain of the shellfish
in all the tanks per 24 hr

total flow rate of phytoplankton
suspension available

The shellfish were grown in wire trays (0.6 x 0.5
X 0.1 m) stacked in the tanks. Effluent from the
tanks was used as a nutrient source to grow car-
rageenan-producing seaweed. Effluent from the sea-
weed tanks was filtered through sand to avoid pol-
lution and introduction of new species in the nat-
ural environment.

MARICULTURE IN ARTIFICIAL UPWELLINGS

Seaweed tanks

3 4

r-r-r~f

^s.

pump

Pool 2

Pool

Shellfish tanks

FIG. 1. The flow of deep water and phytoplankton suspension through the mariculture system of St. Croix.

Hatchery-reared shellfish were used in all
growth experiments. The first populations of
100,000 juvenile C. virginica and 100,000 juvenile
M. mercenaria were kindly supplied by Long Is
land Oyster Farms, Inc., in December, 1970
Juvenile oysters (50,000 each of C. virginica, C.
gigas and O. edulis) obtained from Pacific Maricul
ture. Inc., and F-. hybrid clams supplied by Dr. R
Winston Menzel, were used in later growth studies
Wet weight, linear dimensions and stacked and dis
placement volumes were measured regularly. Wet
weight was determined by placing the shellfish in
a pre-weighed wet net bag, draining for one min-
ute and weighing on a Chatillon autopsy scale.
Linear growth was determined by measuring ex-
treme width, length and/or thickness with calipers.
Stacked and displacement volumes were deter-
mined by immersing the shellfish in a graduated
cylinder filled to the «brim with seawater and
measuring the volume of seawater displaced from
the cylinder (displacement volume) and the volume
occupied by the shellfish in the cylinder (stacked
volume).

The effect that cleaning the animals had on the
filtering efficiencies (per cent cells removed from

the incoming phytoplankton suspension by the
animals) was determined. Comparable groups of M.
mercenaria were cleaned at one, two or three-week
intervals and their filtering efficiencies were com-
pared. The growth of clams in sediment (grain size
less than 0.821 mm) was compared to that of
clams kept in wire trays.

The growth of the F^ hybrid clams was com-

■'"

fio '

1£LE£J

*^'

—

,»

>*■

/''

—

-• —

tost

CO V

rginr

-^7

— »»

****

«♦)!

11^

FIG. 2. Growth of C. virginica and M. mercenaria
ouer an 18-month period.

J.S. BAAB, G.L. HAMM, K.C. HAINES, A. CHU AND O.A. ROELS

FIG. 3. Phytoplankton filtering efficiencies of M.
mercenaria as a function of frequency of cleaning
and cell density.

pared with that of the northern clam, M. mercen-
aria.

The growth rates of three different species of
juvenile oysters (C. uirginica, C. gigas and O. edu-
lis) were compared.

RESULTS AND DISCUSSION

The first populations of C. virginica and M.
mercenaria were directly influenced by difficulties
encountered in the early stages of our mariculture
system. Figure 2 illustrates the growth of both
populations. It is clear from the growth curves
that, compared to C. virginica, M. mercenaria grew
fairly well in the system. C. virginica grew well
initially but were subject to high mortality rates
afterward. The definite cause of this mortality is
unknown. A possible cause of death may have
been the high temperatures combined with high
salinities. E. Mandelli (personal communication)
found that salinities above 35 %c were lethal to
juvenile and adult C. virginica at 28-32°C.

Initially, the M. mercenaria were cleaned and
measured and their tanks scrubbed weekly. The

A
/

sediment

' ^^^^ wire trays

0 20 40 60 8

time in days

FIG. 4. Increase in weight over 64 days of M.
mercenaria grown in sediment or in wire trays.

filtering efficiencies of the clams dropped off se-
verely for 24-48 hrs after cleaning. This decrease
in filtering did not appear to be correlated with
changes in phytoplankton density flowing into the
shellfish tanks (Fig. 3).

To determine whether this frequent cleaning of
tanks and shellfish slowed down the growth of M.
mercenaria, we monitored populations of clams in
three tanks and varied the interval between clean-
ings. There were no differences between growth
rates and filtering efficiencies of the clams in the
tanks cleaned weekly and every two weeks. The
clams in the tank cleaned every three weeks fil-
tered cells more efficiently and had a higher
growth rate (Table 2).

Clams growing in wire trays required frequent
cleaning to remove fouling organisms: such as
bryozoans (Bowerbankia gracilis Leidy) and epi-

TABLE 2. Comparison of growth rates and filtering efficiencies of three populations of M.
Mercenaria cleaned and measured weekly, every two weeks or every three weeks.

Frequency of tank cleaning

Average cell filtering
efficiency for 29 days

% increase in
weight in 29 days

Weekly

Every two weeks

Every three weeks

34.4%
44.9%
57.8%

4.8%
5.2%
9.5%

MARICULTURE IN ARTIFICIAL UPWELLINGS

phytes (Enteromorpha spp.). By allowing the-
clams to bury in sediment we avoided fouling and
eliminated the need for cleaning, thus improving
their growth rate (Fig. 4). However, cleaning of
the tanks at 4-6 week intervals was continued and
scheduled to coincide with routine measurements
of the shellfish.

When phytoplankton production had been
stabilized, experiments were undertaken to find
the most suitable shellfish species for our system.
Juvenile F-. hybrid clams grew five times faster
than the M. mercenaria, as shown in Figure 5.
These results confirm the potential use of Mercen-
aria hybrids in mariculture systems (Menzel, 1971).
Comparative growth studies of the second series of
juvenile oyster? (C. virginica, C. gigas and O. edu-
lis) indicate that the survival and growth rate of
O. edulis were far better than of C. virginica and
C. gigas. (Fig. 6).

F, h

brjd

(

•

M. mere

enaria

^^ A"^

time in days

FIG. 5. Comparison of the growth of Fj hybrids
of (fM. mercenaria x o M. campechiensis with M.
mercenaria. (The average length of the clams at
the beginning of the experiment was 34 mm).

100
80
60

10
8

- 6

S 4

D Ostreg edulis

O Crossoslreo virginica

^ CfQssoslreo gigos

FIG. 6. Growth of 3 species of oysters: 0. edulis,
C. virginica and C. gigas ('s/se of oysters at begin-
ning of experiment was 2 to 3 mm).

Thus, we feel that this system offers a unique
opportunity to optimize shellfish growth under
managed conditions with controlled phytoplankton
and water flow, low fluctuations in temperature
and salinity and absence of predators. An eco-
nomic and engineering study (unpublished) of clam
production based on present small-scale results and
extrapolated to a commercial scale indicates high
profit potential for "artificial upwelling" maricul-
ture.

ACKNOWLEDGMENTS

We wish to thank the following for their assist-
ance with various aspects of this study: L. Aust,
M. Bishop, C. Carson, L. Fick, W. Green, M. Lom-
bard, P. McDonald, W. Tobias and L. van Hemel-
rijck.

LITERATURE CITED

Claude, G. 1930. Power from the tropical seas.
Mech. Eng. 52: 1039-1044.

Menzel, R. W. 1971. Possibilities of molluscan cul-
tivation in the Caribbean. FAO, Fish. Res. Div.,
Fish. Rep. 71: 183-200.

Roels, O. A., L. Van Hemelrijck, R. D. Gerard
and J. L. Worzel. 1971. Cold, nutrient-rich
water: the most abundant resource of the deep
sea. CNEXO Colloque International sur I'Ex-
ploitation des Oceans, Bordeaux, France, March
9-12, 1971, Th. IV, Sect. G2-02, 21 p.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

GROWTH AND SURVIVAL OF THE BAY SCALLOP, ARGOPECTEN IRRADIANS,
AT VARIOUS LOCATIONS IN THE WATER COLUMN AND AT VARIOUS

DENSITIES

William P. Duggan

VIRGINIA INSTITUTE OF MARINE SCIENCE
WACHAPREAGUE, VIRGINIA

ABSTRACT

Two experiments were carried out in 1971 using bay scallops spawned in August
and September of 1970. Four groups of 150 scallops were held at the surface, one
meter below the surface, two meters off the bottom, and one meter off the bottom,
to find the effects of depth on growth and survival. Scallops were held in surface
enclosures at four different densities (100, 75, 50 and 25/ft^ ) to find out the
effects of crowding.

In the depth experiment growth was approximately equal throughout the water
column. Mortality decreased with increasing depth with the exception of those held
at one meter off the bottom.

In the density experiment growth up to a height of 27.0 - 28.0 mm (1.1 in) was
approximately equal at all densities. Above this size, growth decreased with
increasing density. Mortality was low and about equal at all densities for the first
two months but increased with increasing density during the last two months.

INTRODUCTION

The bay scallop is a likely species for maricul-
ture. It grows rapidly, has a high market value,
can be readily conditioned and induced to spawn
and its larvae are amenable to mariculture (Wells,
1927; Loosanoff and Davis, 1963; Sastry, 1965;
Castagna and Duggan, 1971).

The Virginia Institute of Marine Science Eastern
Shore Laboratory began investigating the possi-
bility of culturing the bay scallop in 1968. Work
completed up to 1971 established the biological
feasibility of culturing this species from egg to
market size. The purpose of the present study is
to show how growth and survival are affected by
the location of the scallops in the water column
and by the density at which the scallops are held.

This work is a result of research sponsored by
NOAA Office of Sea Grant, Department of
Commerce under grant number NG572. The U. S.
Government is authorized to produce and distri-
bute reprints for government purposes not with-
standing any copyright notation that may appear
hereon.

DESCRIPTION OF AREA

Experiments were carried out in Finney Creek
in front of the Virginia Institute of Marine Science
Eastern Shore Laboratory. Tidal amplitude is 1.2 -
1.5 m (3.6 - 4.9 ft). Water depth is 5-6 m at high
tide. Temperatures ranged from 17.2 - 28.7°C and
salinities from 20.8 - 31.6 °oo during the experi-
mental period. The bottom is soft mud. Tidal
currents average approximately 30.0 cm/sec
throughout the entire water column (Joseph and
Van Engle, 1967).'

MATERIALS AND METHODS

Juvenile scallops used in these experiments were
spawned in the laboratory from brood stock in
late summer of 1970 and tray reared in Finney
Creek until May and June 1971 when the

'Joseph E., and W. A. Van Engle. 1967. Nursery
ground study. Bureau of Comm. Fish., Comm.
Fish. Res. & Devel. Act. Funded under 88-309,
Suppl. Rep. Vol. e. (Unpublished manuscript).

GROWTH AND SURVIVAL OF BAY SCALLOPS

SURFACE

FIG. 1. Enclosures held at the surface, one meter below the surface, and one and two meters off
the bottom.

experiments began.

Enclosures used in each experiment were con-
structed of % in pine covered top and bottom
with plastic screen (mesh opening 7.0 mm). Those
used in the depth experiment measured 64.0 x
55.5 X 15.0 cm while those in the density
experiment measured 122.0 x 56.5 x 15.0 cm.
The surface enclosures used in the density experi-
ments had 14.5 x 1.9 cm boards added on each
side for stabilizing wings. Enclosures held at the
surface were tied to stakes and maintained at the
surface by their own buoyancy. Enclosures held
below the surface were either suspended from
surface floats or secured to poles at the appro-
priate depth (Fig. 1).

Experiments were run in duplicate. Mean
growth measurements and mortality counts were
averaged from duplicate enclosures at two-week
intervals and enclosures were cleaned of the mud
and fouling organisms that had accumulated during
that period.

All enclosures were held in a line parallel to
the tidal flow. All measurements of scallops refer
to the height or distance from the hinge to ventral
edge.

RESULTS

Depth Experiment: Growth and Mortality

This experiment ran from 10 June, 1971 - 7
October, 1971. One hundred and fifty scallops

with a mean size of 14.4 mm had been placed in
each enclosure held at the surface, 1 m below the
surface and 1 and 2 m above the bottom (Fig. 1).

At the end of this experiment scallops averaged
44.7 mm at the surface, 44.6 mm at 1 m below
the surface, 47.0 mm at 2 m above the bottom
and 42.7 mm at 1 m above the bottom, indicating
approximately equal growth at all depths (Fig. 2).

With the exception of those scallops held at 1
m above the bottom, total percent mortality
decreased with increasing depth: 16.5% at the

TEMPERATURE

SURFACE
-ONE METER BELOW SURFACE
-TWO METERS ABOVE BOTTOM
-ONE METER ABOVE BOTTOM

A
19 7 1

FIG. 2. Growth data for scallops held at various
locations in the water column (surface, one meter
below the surface, one and two meters above the
bottom.)

W. DUGGAN

30-1

25-

H 20

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o

2 15

Ll.

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CO

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W

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O
CD

U
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CD
<

IT
hi

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DEPTH

FIG. 3. Total percent mortality of scallops held at
the four experimental depths.

surface, 8.0% at 1 m below the surface and 4.0%
at 2 m above the bottom. Mortality at 1 m above ;
the bottom was 29.0% (Fig. 3).

Density Experiment: Growth and Mortality

This experiment ran from 12 May, 1971 - 20
September, 1971. Initial densities of 100, 75, 50
and 25/ft^ were tested.

Figure 4 indicates approximately equal growth
at all densities until the scallops reached 27.0 -
28.0 mm. Above this size growth decreased with
increased density.

Figure 5 indicates low mortality at all densities
during the first two months and increased mortal-
ity during the last two months with higher
densities having the greater mortalities. Mortalities
began to increase when the scallops were about
37.0, 39.0, 43.5 and 46.2 mm at densities of 100,
75, 50 and 25/ft^ respectively.

Total mortality at the end of the experiment
averaged 35.0, 16.0, 6.2 and 3.2% at densities of
100, 75, 50 and 25/ft^ respectively (Table I).

DISCUSSION
Although scallops grew and survived best at a

50-1

40 -

X 30H

TEMPERATURE

100/ ft'

75/ ft'

50/ ft'

25/ft'

r 30

I-

10 £

£
UJ

-1 r

1 1

J A

197 1

-1 r-

FIG. 4. Growth data for scallops held at the four
experimental densities (100, 75, 50 and 25/ft^).

density of 25/ft^, the data suggests that densities
as high as 60-65/ft^ could be used (Table I).
Control of factors mentioned below would prob-
ably allow scallops to grow and survive equally
well throughout the water column.

Those factors which affected growth and sur-
vival in both experiments were: (1) heavy fouling
of the screen meshes vnth hydroids, mud and/or
algae resulting in poor water circulation; and (2)
mechanical disturbance of enclosures due to boat
wakes, wave action and/or tidal currents. The
effect of these factors seemed to depend on the
location of the enclosures in the water column,
the density at which the scallops were held, the
size of the scallops and stability of the enclosure.

In the density experiment fouling and mechani-

FIG. 5. Average monthly mortality of scallops
held at the four experimental densities (100, 75,
50 and 25/ft^).

GROWTH AND SURVIVAL OF BAY SCALLOPS

TABLE 1. Initial densities, total percent mortality
and final densities of scallops in density experi-
ment.

Initial Densities
scallops/ft^

100
75
50
25

Total Percent
Mortality

35.0
16.0

6.2

3.2

Final Densities
scallops/ ft^

65
63

47
24

cal disturbance were common to all enclosures.
Decreased circulation due to fouling probably
resulted in a greater competition for food which
became more intense as the scallops increased in
size. This was particularly true at the higher
densities indicated by the decreased growth rate
and increased mortality. Mechanical disturbance of
the enclosures probably disturbed the scallops
feeding behavior. Occasionally scallops were
washed to one end of the enclosure causing some
smothering of the scallops. Again this was more
intense at the higher densities.

In the depth experiment the high mortality of
scallops at one meter off the bottom is believed
due to heavier accumulations of silt or mud in
these enclosures than in the enclosures just above
them. Scallops were frequently found buried.

Fouling was common to all the enclosures in this
experiment and undoubtedly had an adverse affect
on growth and survival.

The decrease in mortality from the surface to
two meters off the bottom (Fig. 3) is believed due
to the decreased effects of wave action and other
surface turbulences vvith increased depth. The
relatively stationary position in which the en-
closures at two meters off the bottom were held
helped reduce disturbances at this depth and
probably accounts for the slightly higher mean size
and percent survival attained by the scallops held
here. The effects of the mechanical disturbance of
the enclosures in this experiment were similar to
those described for the density experiment.

LITERATURE QTED

Castagna, M. and W. Duggan. 1971. Rearing the
bay scallop, Aequipecten irradians. Proc. Natl.
Shellfish. Assoc. 61: 80-85.

Loosanoff, V. L. and H. C. Davis. 1963. Rearing
of bivalve mollusks. Advan. Mar. Biol. 1: 1-136.

Sastry, A. N. 1965. The development and external
morphology of pelagic larval and post-larval
stages of the bay scallop, Aequipecten irradians
concentricus Say, reared in the laboratory. Bull.
Mar. Sci. 15: 417-435.

Wells, W. F. 1927. Report of experimental shell-
fish station. N. Y. State Conserv. Dep., 16th
Annu. Rep. p. 113-130.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

LARVAL CULTURE OF THE CALICO SCALLOP, ARGOPECTEN GIBBUS ' ' '

T. J. Costello, J. Harold Hudson, John L. Dupuy and Samuel Rivkin

NATIONAL MARINE FISHERIES SERVICE

SOUTHEAST FISHERIES CENTER

MIAMI, FLORIDA

AND

VIRGINIA INSTITUTE OF MARINE SCIENCE

GLOUCESTER POINT, VIRGINIA

ABSTRACT

Mature calico scallops, Argopecten gibbus, collected from the grounds off Cape
Kennedy, Florida, were induced to spawn in the laboratory. Fertilized eggs were reared to
postlaruae in sea water of 23° C ± 2.0° C at a salinity of 35 %o . The external mor-
phology of eggs and developing larval stages are described.

INTRODUCTION

The calico scallop, Argopecten gibbus (Linne),
(Fig. 1)^ is a commercially valuable shellfish which
supports a developing fishery off the southeastern
coast of the United States and in the Gulf of Mexico.
Large concentrations of this benthic marine pele-
cypod occur on the continental shelf in the area of
Cape Kennedy, Florida, in depths from 9-74 m
(Drummond, 1969). Concentrations also occur south
of Cape Hatteras off North Carolina in depths from
ca. 13 m (Bullis and Thompson, 1965) to at least 94
m (Cummins, Rivers and Struhsaker, 1962). The
general distribution of this organism is given by Allen
and Costello (1972).

The National Marine Fisheries Service (NMFS)

'Contribution No. 225, Southeast Fisheries Center,
National Marine Fisheries Service, NOAA, Miami,
FL 33149.

^Contribution No. 478, Virginia Institute of Marine
Science, Gloucester Point, VA 23062.

Two terms are used in this paper to define shell
dimensions. They are: (1) Length (L), a straight line
measurement of the greatest distance between the
anterior and the posterior shell margins; (2) Width
(W), a straight line measurement of the greatest
distance between the umbo and the ventral shell
margin. Several authors use the term "height" for
the dimension we define as width.

initiated a life history study of calico scallops in
1969. A portion of the study was concerned with the
early life history of this mollusk. The purposes of this
paper are: (1) to present illustrations of the gross
morphology and time sequence of larval development
so these stages may be readily identified in plankton
samples, and (2) to make available procedures for the
mass culturing of this species.

Previous works on larval development of moUusks
of the genus Argopecten " are by Belding (1910),
Outsell (1930) and Sastry (1965). These papers deal
with a closely related species, the bay scallop, Argo-
pecten irradians.

MATERIALS AND METHODS

Techniques to induce spawning and rear moUuscan
larvae suggested by Loosanoff and Davis (1963) were
modified at the Virginia Institute of Marine Science
(VIMS) in rearing calico scallop larvae. Mature calico
scallops (shell width 55 - 65 mm) were collected by
otter trawl from the grounds off Cape Kennedy,
Florida. They were transported to the NMFS Labora-
tory in Miami, Florida, in insulated containers of
aerated sea water maintained at 20 - 23° C. At the
laboratory, scallops were held on water tables and/or
troughs of running sea water. Subsequently, a portion

Waller (1969) rejected the generic name Aequipec-
ten and suggested Argopecten, the name currently in
use.

LARVAL CULTURE OF SCALLOPS

FIG. 1. The right valve of a mature cahco scallop,
Argopecten gibbus - shell width 56 mm.

of these mature scallops was air-shipped to VIMS at
Gloucester Point, Virginia, where spawning and larval
rearing to setting were accomplished.

All culture techniques and most of the mor-
phology were described from specimens, photomicro-
graphs and information obtained from induced
spawning and larval rearing at VIMS.

Induction of Spawning

Ovarian color is a reliable index of sexual maturity
in calico scallops (Miller, Hudson, Allen and Costello,
1972).^ Before we attempted to induce spawning,
scallops were selected that showed orange-red
("ripe") ovarian color. The ovarian color was easily
observed as the scallops gaped in the troughs of run-
ning sea water. Preliminary observations indicated
that induced spawning in ripe calico scallops is easily
achieved. We induced spawning several times in less
than one hour by raising the water temperature from
ca. 20 - 25° C. To trigger spawning, in addition to
raising the water temperature, it was occasionally
necessary to strip gametes from one mature calico
scallop specimen and, with a pipette, introduce them
gently into the water containing gaping scallops.

Calico scallops are hermaphrodites. Sperm cells are

^Miller, G. C, J. H. Hudson, D. M. Allen, and T. J.
Costello. 1972. Ovarian color changes in calico scal-
lops, Argopecten gibbus. Unpublished manuscript
filed at the National Marine Fisheries Service, South-
east Fisheries Center, Miami Laboratory, Miami, Fla.

usually extruded first when spawning is induced in
the laboratory. After sperm cells have been dis-
charged for 30 min to an hour, discharge of eggs
begins. Once spawning begins, it may continue for
several hours.

When techniques to induce spawning were estab-
lished, 10 ripe scallops were selected. Their shells
were carefully scrubbed to remove a variety of en-
crusting invertebrates which are frequently affixed to
the outer shell (Wells, Wells and Gray, 1964). If these
fouling organisms, e.g., the serpulid polychaete,
Pomatoceros caeruleus, are not removed, they may
spawn when spawning is induced in the scallops and
contaminate the larval culture.

After cleaning, the scallops were placed, one to a
dish, in 3"x5"x9" Pyrex glass containers, each %
filled with filtered 20 C sea water at a salinity of
32.1 /oo . The containers were then placed on a water
table. A black cloth was placed between the glass con-
tainers and the table top to aid in observing when
spawn was first extruded. Temperatures in the dishes
containing scallops were raised from 20 - 25 C
by flowing warm tap water around them. In two of
the dishes, sperm cells stripped from another mature
calico scallop were introduced with a pipette. The
scallops in these two dishes began to spawn 78 min
after the water temperature reached 25°C. Six addi-
tional scallops spawned at various intervals in the
next hour.

When the water in each dish became clouded
(opaque) with suspended sperm, the scallop was re-
moved and placed in a clean dish of 25 C filtered sea
water. This procedure was continued until the scallop
began to discharge only eggs. The scallop was then
placed in a clean dish of 25° C filtered sea water
where it was kept until spawning was completed.
Dishes containing mixed sperm and eggs were dis-
carded.

Since the eight scallops induced to spawn began
extruding sperm and then eggs at various times over
ca. a 2-hr period, we had available, simultaneously,
dishes containing freshly spawned, unmixed suspen-
sions of sperm cells, and freshly spawned, unmixed
suspensions of eggs. A light suspension of sperm (35
cc) was added to each of the dishes containing eggs,
and the mixtures were gently agitated. Following
fertilization, the eggs were washed in a stainless steel
screen (152 iJ. openings) to remove debris that ac-
companies spawning. We followed the washing pro-
cedure described by Loosanoff and Davis (1963).

After the fertilized eggs were washed, they were
added to a container of filtered sea water and the
number of eggs per unit of sea water was determined
with a Sedgwick-Rafter cell. A sufficient quantity of

T.J. COSTELLO, J.H. HUDSON, J.R. DUPUY AND S. RIVKIN

the washed egg suspension was added to a 20-liter
container* of filtered sea water to provide 25
eggs/ml. This concentration was reduced to ca. 10
larvae/ml at the straight-hinge stage.

Temperature in the culture was maintained at 23
C ± 2.0° C throughout larval development. To simu-
late conditions in the calico scallop's natural offshore
spawning area, salinity was adjusted to 35 %o imme-
diately after fertilization and held at this concentra-
tion. The culture was not aerated, and no illumina-
tion was provided. Water was changed every other
day by straining the entire 20 liters through a stain-
less steel screen. A screen with mesh openings of 50/i
was used initially; larger openings were used as the
larvae increased in size. Larvae retained on the screens
were returned to clean 20-liter containers of filtered
sea water. Following the first two water changes, 0.2
cc of "twin biotic" (a mixture of streptomycin and
penicillin) was added per liter of culture to retard
bacterial growth. Feeding of the larvae w£is initiated
30 hr after fertilization. Unialgal cultures of Mono-
chrysis lutheri were fed in quantities sufficient to
provide, initially, concentrations of ca. 60,000
cells/ml. As the larvae grew, adjustments to concen-
trations of food were made to quantities where
observations showed complete utilization.

EMBRYONIC DEVELOPMENT

Embryonic development of A. gibbus is similar to
that described by Sastry (1965) for A. irradians. A
detailed study of early cleavage was not made; there-
fore, the times that are reported for early embryonic
development are approximations based on the most

* Plastic garbage can

cir

FIG. 2. Argopecten gibbus eggs ca. 35 min after
spawning. Note irregular shape of most eggs.

FIG. 3. Embryonic development of Argopecten
gibbus: a) unfertilized eggs; b & c) zygotes 40-60 min
after fertilization showing polar bodies; d) cell divi-
sion ca. 100 min after fertilization; e) a ciliated
trochophore 24 hr after fertilization.

typical stage represented in the culture samples ob-
served. Developing zygotes from a single spawning
showed considerable disparity in rates of develop-
ment during the first 24 - 36 hr. Newly spawned eggs
of A. gibbus were asymmetrical (Fig. 2), though
observations of A. irradians eggs observed after
spawning also appeared similarly asymmetrical.

Unfertilized eggs, measured with an ocular micro-
meter, averaged 60/u in diameter (Fig. 3a). Approxi-
mately 40 min after fertilization, two polar bodies
formed as the zygote gradually modified to form a
polar lobe (Figs. 3b and 3c). In most cultures discern-
ible cleavage began 70 min after fertilization. As in
the embryonic development of many other molluscs,
unequal blastomeres were noted in all early cleavages,
and micromeres proceeded with more rapid division
than macromeres during the first 8 hr of develop-
ment. Figure 3d depicts typical cell division 100 min
after fertilization. Active ciliated trochophores were
observed 24 hr after fertilization (Fig. 3e).

Shell secretion began during the early trochophore
stage. The shell gradually enveloped the body and an
active straight-hinge veliger was formed before the
larvae were 48 hr old.

Larval Culture

Under our laboratory conditions, the larval period
of the calico scallop was 16 days. Figure 4 is a com-
posite made from photomicrographs taken every 24
hr. The larvae, items B through J in Figure 4, repre-

LARVAL CULTURE OF SCALLOPS

70 H

80 M

I 70 W

B
90 L

I 80 W

69 W

c Z)

112 L X I 00 W

H
2 I 0 L X I 90 W

120 L X I 08 W

250 L X 208 W

140 L X I 30 W

250 L X 208 W

FIG. 4. Composite photomicrograph of larval Argo-
pecten gibbus. Age in days: a) 1 ; b) 2; c) 4; d) 5; e) 7;
f) 9; g) 11; h) 13; i) 15; j) 16. Length x width dimen-
sions are given in microns.

sent the average sizes for each time stage obtained by
measurement of 25 larvae from several photomicro-
graphs of each 24-hr period. The early straight-hinge
larvae appeared to be chopped off at one point along
the hinge line. The umbo appeared at about 140 fi,
rounded and poorly defined. It remained incon-
spicuous throughout larval development. Figures 5
and 6 show typical morphological features in the lat-
ter stages of larval development and just prior to set-
ting. Chanley and Andrews (1971) made effective use
of hinge line shapes in describing 23 species of bivalve
larvae. The hinge line shape of the calico scallop lar-
vae (Fig. 7) is distinctive but very similar to A.
irradians. The toothed area is comprised of three
taxodont teeth at each end of the hinge line. The
central hinge area is undifferentiated. Other identify-
ing characteristics of calico scallop larvae are their

FIG. 5. Photomicrograph of live 12-day-old larvae of
Argopecten gibbus length 21 0 ^i.

pale color and development of an inconspicuous eye-
spot when the larvae reach a length of ca. 250 fi.

SUMMARY

Calico scallops, A. gibbus, have been induced to
spawn in the laboratory and the larvae have been
reared to setting. Development, on the basis of ex-
ternal morphology, is quite similar to that recorded
for a closely related form, A. irradians (Sastry, 1965).

FIG. 6. Photomicrograph of live 16-day-old larvae
with foot extended, showing anatomical relationship
and early structure of gill and foot.

T.J. COSTELLO, J.H. HUDSON, J.R. DUPUY AND S. RIVKIN

FIG. 7. Dorsal view of hinge of the larval Argopecten
gibbus.

The major difference is that A. gibbus has a much
larger pediveliger or newly set larvae which ranges in
length from 235-270 jj. The difference is significant
when compared to the bay scallop, A. irradians,
which sets at a length of from 170-190 y.

ACKNOWLEDGMENTS

This work was supported in part by the NOAA
Office of Sea Grant, Department of Commerce, under
Grant No. 1-36032. We express our sincere thanks to
Dr. Kenneth Chew, Mr. Robert Work, and Mr.
William Shaw for their very helpful editorial sug-
gestions.

LITERATURE CITED

Allen, D. M. and T. J. Costello. 1972. The calico scal-
lop, Argopecten gibbus. NOAA Tech. Rep. NMFS
SSRF-656, 19 p.

Belding, D. L. 1910. A report upon the scallop fish-

« ery of Massachusetts, including the habits, life
history oi Pecten irradians, its rate of growth, and
other facts of economic value. Wright and Potter
Printing Co., Boston, 150 p.

Bullis, H. R., Jr. and J. R. Thompson. 1965. Col-
lections by the exploratory fishing vessels Oregon,
Silver Bay, Combat, and Pelican made during
1956-1960 in the southwestern North Atlantic. U.
S. Fish Wildl. Serv., Spec. Sci. Rep. Fish. 510, 130
P-

Chanley, P. and J. D. Andrews. 1971. Aids for identi-
fication of bivalve larvae of Virginia. Malacologia,
11: 45-119.

Cummins, R., Jr., J. B. Rivers and P. J. Struhsaker.
1962. Exploratory fishing off the coast of North
Carolina, September 1959 - July 1960. Commer.
Fish. Rev. 24(1): 1-9.

Drummond, S. B. 1969. Explorations for calico scal-
lop, Pecten gibbus, in the area off Cape Kennedy,
Florida, 1960-66. Fish. Ind. Res. 5: 85-101.

Outsell, J. S. 1930. Natural history of the bay scallop.
Bull. U. S. Bur. Fish. 46: 569-632.

Loosanoff, V. L. and H. C. Davis. 1963. Rearing of
bivalve mollusks. Adv. Mar. Biol. 1: 1-136.

Sastry, A. N. 1965. The development and external
morphology of pelagic larval and post-larval stages
of the bay scallop, Aequipecten irradians concen-
tricus Say, reared in the laboratory. Bull. Mar. Sci.
15: 417-435.

Waller, T. R. 1969. The evaluation of the Argopecten
gibbus stock (Mollusca: Bivalvia), with emphasis
on the Tertiary and Quaternary species of the
eastern North America. Paleontol. Soc. Mem. 3,
125 p. Also J. Paleont. 43 (5, Suppl.)

Wells, H. W., M. J. Wells and I. E. Gray. 1964. The
calico scallop community in North Carolina. Bull.
Mar. Sci. 14: 561-593.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

REPRODUCTIVE BIOLOGY OF YOUNG ADULT KING CRABS
PARALITHODES CAMTSCHATICA (TILESIUS) AT KODIAK, ALASKA

Guy C. Powell, Brian Shafford and Michael Jones

ALASKA DEPARTMENT OF FISH AND GAME

DIVISION OF COMMERCIAL FISHERIES

KODIAK, ALASKA

ABSTRACT

King crab mating was studied in the natural environment while simultaneously
conducting mating experiments in undersea pens in an adjacent location. Pubescent
females, 86-119 mm in carapace length, began mating 14 February, 1971, a month
earlier than adults. The majority of females were mature at a length of 111 mm.
The smallest adult was 96 mm. In nature pubescent females averaging 99 mm in
carapace length mated with males averaging 142 mm in length. The smaller and
more abundant males (90-109 mm) molted at the same time pubescent females were
molting, and mated with experimental females when placed in undersea pens. Males
mated with females larger than themselves but appeared to be incapable of mating
during the 10-day interval bracketing the male molt.

Average growth of pubescent females is similar to that of juveniles and 3 mm
more than that of small adult females. Males were found to attain sexual maturity
at a smaller size than females.

INTRODUCTION

King crab harvest in tlie Kodialt Island area in-
creased slowly through 1958 when 5 million
pounds were landed. Annual production increased
rapidly after 1958, peaking in 1966 at 91 million
pounds (Powell and Gray, 1969)'. Since 1966
average catch per effort has declined steadily and
in 1971 landings were down to 12 million pounds.
This was the lowest in the last 12 years.

Kodiak's fishing grounds have yielded 438 mil-
lion pounds of male king crabs since 1950. Female
king crabs have always been protected by regula-
tion and the intense fishing pressure on males
greater than 7 in. in shell width (6V2 in. prior to
1963) has caused marked changes in the composi-
tion of the brood stocks. Tagging studies in the

'Powell, G. C. and P. L. Gray, 1969. A study of
the king crab fishery (Paralithodes camtschatica,
Tilesius) within Kodiak Island Management Unit,
Alaska, with emphasis upon recent catch
statistics, 1960-1968. Typewritten manuscript.
140 pp.

upper Gulf of Alaska have shown king crabs to
have a longevity of about 14 years and attain legal
size in 7 or 8 years (Powell, 1967). Male crabs
are, therefore, susceptible to harvest for as many
as 8 years. As early as 1960, annual fishing mor-
tality was a minimum of 33% in areas of fleet
concentration (Powell, 1964) and average size and
proportion of anexuviant males in the stocks de-
clined as a result of fishing (Nickerson, Ossiander
and Powell, 1966).

Trawl fishing studies during the 1962 mating
season, vhen sexes are congregated, revealed four
times as many females as males (Gray and Powell,
1966). During 1967 trawling, 11 times more fe-
males were captured than males (McMuUen, 1967).

Scuba surveys of natural mating areas during
1963 and 1964 revealed that all males grasping fe-
males were larger than 119 mm and were just one
molt away from commercial size (Powell and
Nickerson, 1965). Further, that the male mates
with the female within hours after she molts. In
1970, mating studies illustrated that recently
molted males, just under legal size, could mate as
many as 13 successive times but that mating

G.C. POWELL, B. SHAFFORD AND M. JONES

ability decreased after the sixth mating (Powell,
James, and Hurd, 1972)^

The present investigation was initiated to study
the adequacy of the current 7-inch size limit in
providing adequate protection to male king crab
brood stocks (legal carapace width of 178 mm
converts to a carapace length of 145 m m)^. Males
were believed to attain sexual maturity at the
same or at a larger size than females because
growth of the latter decreases markedly at sexual
maturity. Quantitative data for sizes of females at
maturity were lacking but it had been learned that
adult females were as small as 96 mm and that
many females attained adulthood at the length of
108 mm (Powell, 1958). Determining proportions
of various size king crabs which are sexually
mature became vital to insure proper management
of the resource. The primary objective of the
1971 mating study, therefore, was to obtain
qualitative data regarding the reproductive ability
of small male and female king crabs in the size
range 90-109 mm.

METHODS

Pubescent'' crabs were located in Middle Bay
(Fig. 1) by fishing with pots and then sub-
sequently monitored for several months until mat-
ing was completed. Simultaneously, specimens were
obtained for examination and for controlled
studies in undersea pens. The controlled study al-
lowed determinations of mating ability of in-
dividual small males in the absence of competition
from larger males.

Exploratory pot fishing

Continuous fishing for 4 months with 18 pots
enabled us to locate and study a school of small
crabs before, during and after the molting and

^Powell, G.C, K. E. James and C. L. Hurd, 1972.
Ability of male king crabs (Paralithodes
camtschatica, Tilesiusj to mate repeatedly,
Kodiak, Alaska, 1973. In preparation, Fish. Bull.

^Carapace length is used exclusively throughout
the manuscript because it is the standard
measurement used by researchers (see page 13 of
Powell, 1967).

''For convenience in writing, the authors are using
the term pubescent to refer not only to the fe-
male about to mate for the first time but also to
that same female soon after molting and mating.
In this way we can separate pubescent crabs that
have just become adults from those that have
been adults previously.

FIG. 1. Location of crab school in Middle Bay
and site of undersea pens.

mating season. Scuba diving was employed to
supplement pot fishing and to capture grasping
pairs from natural mating areas. Scuba diving also
confirmed the continued presence of the crabs
during the molt when they could not be captured
by pot fishing.

Seven different vessels ranging in length from
30 - 90 ft were used at various times throughout
the study. Two pot sizes were used: 6-foot square
by 3-foot high, and 4-foot square by 2-foot high.
Stretched mesh was 2% in. on all pots to insure
retention of small crabs. Pots were lifted at our
convenience and as weather permitted.

A random sample of the catch from each pot
was measured and studied to determine com-
position by size, sex, shell-age and ovigerousness.
Partial clutches of eggs, matted abdominal setae
and stage of molt were also recorded.

Crabs of the size and condition needed for the
controlled study and for dissections were kept
alive and brought back to Kodiak for these pur-
poses. Crabs were handled carefully to avoid in-
jury.

KING CRAB REPRODUCTIVE BIOLOGY

Dissections

A separate group of small male and female
crabs were dissected every 5 or 6 days so that the
newly developing exoskeletons could be examined
to predict the advent of the molting season. Ab-
dominal cavities of various sized juvenile females
were opened and oviducts examined to determine
the presence or absence of internal eggs. A length
frequency distribution was prepared showing the
proportions of the various size females which were
pubescent. Microscopic examination of repro-
ductive tracts of seven males was also undertaken.
Knowledge obtained from dissections was used to
help determine size of experimental crabs for the
controlled study, and beginning date for the study.

Controlled study

Experimental crabs were housed in four under-
sea pens which were each subdivided into four
separate compartments (total of 16), each a 4-foot
cube, 64 cubic feet in size. Undersea pens were
made of steel bars welded together and covered
with small mesh web. Pens were bottomless en-
abling crabs to dig into the substrate as they
would do in nature thus creating as near natural
conditions as possible. Since pens could not be
lifted to the surface, they were tended daily
throughout the study by divers. Activities of ex-
perimental crabs were recorded underwater on
bakelite slates. Pens were placed in 35 feet of
water at Near Island Basin. This area is adjacent
to the natural mating grounds and is one-half mile
east of the City of Kodiak (Fig. 1). Several
Dungeness pots situated alongside the pens served
as temporary crab storage facilities.

New-shell males, 90-109 mm, were used in the
controlled study (old-shell males this size are rare).
One male and several females were placed into
each of the 16 compartments. As soon as one of
the females molted and mated (ovulation occurred)
both she and her partner were removed from the
compartment and placed in separate storage. A
new male and female were added to the compart-
ment shortly thereafter. As many individual mat-
ings as possible were arranged during the early
mating season before mating of small females end-
ed. Mated females were kept in storage from 8 -
14 days, depending upon water temperatures, until
eggs had time to deVelop to at least the
8-blastomere stage. After eggs had adequate time
to cleave, several hundred were collected from
numerous locations among the egg mass and pre-
served in Bouin's solution. A sample of 100 eggs
was taken from the Bouin's solution and examined
microscopically for final determinations as to

whether or not mating had been successful.

In cases where ovulation did not occur during
the first 4 days after female molting, the male was
considered to have had adequate opportunity to
mate, and was removed as a failure. Additional
males were introduced and if ovulation still did
not occur the female was dissected to determine if
she was pubescent or still juvenile. If internal eggs
were absent; i.e. the female was juvenile, males
were given another opportunity to mate and were
not considered unsuccessful in their initial attempt.

An interval of 8 or more days after molting
was adequate for shell hardening. Both males and
females were measured after an interval of this
magnitude to determine growth increment and
were subsequently released as soon as mating had
been proven successful.

Bottom water temperatures were collected at
the pen site using Ryan thermographs (Model F,
fast response, waterproof). Divers also recorded
temperatures with hand-held mercury thermome-
ters.

Experimental animals

Experimental animals used in the undersea pen
study were all tagged with permanent loop tags so
that individual crabs could be readily identified.
Details on tagging procedure are presented by
Gray (1965). Crabs were fed sea urchins, shrimp
and fish every 5 or 6 days.

Graspee"^ females were preferred because of
their impending molt and because males were at-
tracted to them; however, some non-graspees, i.e.;
females not ready to mate, were used successfully
during intervals when graspees were scarce. Prac-
tically all of the experimental females were
pubescent except for those molting and mating in
late April. The majority of the graspees were cap-
tured from natural mating areas by scuba divers,
three were captured in pots as were all of the
non-graspees and males.

RESULTS AND DISCUSSION

Mating Season of Pubescent Females and Location
of Mating

Exploratory pot fishing and scuba diving dis-
closed a large school of both juvenile and pubes-
cent crabs in the vicinity of Viesoki Island, Middle
Bay, 7 miles south of the city of Kodiak, Alaska

^Graspee refers to a female crab being grasped by
a male. Adult females become attractive to males
prior to the molt and are grasped by a male and
held until molting and copulation are completed.

G.C. POWELL, B. SHAFFORD AND M. JONES

TABLE 1. Size of mating crabs in the 35 grasping pairs which contained
pubescent females, captured from natural mating areas, Kodiak Island,
Alaska, 1971.

Carapace
length (mm) ^

1.
1

C/2

c
0

•a

1)
N

c c

OX)

c
'S.

Carapace
length (mm) ^

0. a.

2 2
0 0

Cm
0

C
0

£,

■a

c
■5.

2
0

<u
0.

2
0

13
S

-9

104

145

119

145

123

104

146

106

123

104

147

103

124

147

103

129

148

101

135

102

148

136

113

148

106

137

149

138

107

150

138

100

152

104

139

106

154

140

102

155

140

155

143

159

143

108

160

145

111

177

145

3,479

4,967

Graspees are the females and graspers are the males.

(Fig. 1). Crabs were abundant in depths ranging
from 7 - 16 fathoms and generally remained with-
in this area throughout the study period, Decem-
ber - March.

The 65 pubescent females that were captured
from this school and placed in undersea pens
molted and mated during the period 14 February
- 19 April the majority (85%) doing so prior to
21 March. Scuba divers swimming in the vicinity
of Viesoki Island on 26, 27 February and 7 March
observed thousands of shed exoskeletons lying on
the bottom, confirming that molting of juvenile

and pubescent crabs was well underway. Grasping
pairs were also more abundant at this time than
on previous dives. Molting was further implicated
by the disinterest in feeding evident by the sharp
decline in average catch per pot lift (from 60/pot
to 6) which occurred 22 February and continued
through 7 March.

No pubescent graspees were captured after 13
March. Continued searth effort disclosed adult
females being grasped, indicating that pubescent
females had mated first. Molting of adult females
in undersea pens complemented observations in the

KING CRAB REPRODUCTIVE BIOLOGY

CARAPACE LENGTH

FIG. 2. Size distribution of 14,635 king crabs cap-
tured in Middle Bay, 17 December, 1970 - 31
March, 1971, Kodiak Island, Alaska.

natural environment. Adult females began molting
and mating 23 April in the same general depth
and locality as the pubescent females before them.
Size of Males Grasping Pubescent Females in
Natural Mating Areas

The majority (65%) of graspee females captured
from natural mating areas were pubescent (35 of
54) primarily because we stopped searching for
mating crabs shortly after adults began molting.
Average length of males grasping pubescent females
was 142 mm, 19 were legal size (54%). Average
size of females was 99 mm (Table 1). Males
averaged 42 mm larger than their female mates.
The only male smaller than his partner was 85
mm, next smallest male was 119 mm. The major-
ity of males (89%) ranged from 123-159 mm with
the largest male 177 mm.

Exploratory fishing and diving revealed that
males of the size which were grasping in the
natural environment (123 mm and larger) were
relatively scarce, and yet it was this scarce group
of males that was mating rather than the smaller
males (63-100 mm) which were abundant (Fig. 2).

A size relationship between mates opposite to
that occurring naturally was created in the con-
trolled undersea pen study because we wanted to
test mating ability of small males. There, every
male except one, was smaller than his female part-
ner. Pubescent females averaged 14 mm larger than
their mates but in spite of the difference, males
mated successfully. Had the thousands of small
males less than 100 mm participated in mating in
nature, they would have been found grasping fe-
males to a greater extent than they were. The
relative absence of crabs ranging in length from

102-116 mm is unexplained, but may represent
the void between two age classes.

Between 1963 and 1971, 3,402 grasping pairs
have been captured from natural mating areas.
Only 6 males were smaller than 110 mm (Powell,
Rothschild and Buss, 1972)*.

Small males may mate in the absence of large
males or when a surplus of pubescent females
exists. Since the majority of small newshell males,
90-109 mm, used in the undersea pen study mated
successfully, the absence of this size from grasping
pairs captured in natural mating areas suggests that
they are not aggressive and do not compete with
larger males.

During the last 6 days of fishing, 1,035
new-shell females were captured, 41 were ovigerous
and 994 were non-ovigerous. Non-ovigerous females
ranged from 59-106 mm while the ovigerous fe-
males ranged from 96-122 mm. The 994
non-ovigerous females were considered juveniles
rather than unmated pubescents because most of
them were too small to be adults; partial clutches
and unfertilized eggs commonly found among
unmated females were completely absent from
those of adult size, and none of the ovaries exam-
ined contained developed eggs. A total of 1,673
males ranging in length from 47-186 mm were
captured during the same period and in the same
area indicating the presence of an adequate num-
ber of males to service all pubescent females. Un-
mated females commonly extrude and carry in-
fertile eggs while females that mate with males
that have mated repeatedly have partial clutches of
eggs. Neither partial clutches nor infertile eggs
were observed.

Molt Increment of Females and Males

Average growth increment of pubescent female
crabs was greater than that of either small or large
adult females. The average growth of 59 pubes-
cent, 11 small adult, and 2 large adult females was
11, 7, and 4 mm respectively (Table 2). Average
growth of 3 juvenile females was similar to that of
pubescent females (11 mm). Another estimate of
the growth of pubescent females was obtained by
comparing the size difference between the smallest
pubescent and smallest adult female (Fig. 3).
Growth estimated in this way was 10 mm, the

'^Powell, G. C, B. J. Rothschild, and J. Buss.
1972. A Study of King Crab (Paralithodes camt-
schatica, Tilesius) Brood Stocks, Kodiak Island,
Alaska, 1963-1971. 30 pp. Typewritten manu-
script.

G.C. POWELL, B. SHAFFORD AND M. JONES

TABLE 2. Growth increment per molt of experimental female and male king crabs used in
undersea pen study, 1971. ^

Carapace

No. of

Increment

No. of

Increment

No.

Increment

length

pubescent
females

per

molt

adult
females

per

molt

of
males

per

molt

(mm)

range average

72-77

78-83

84-89

10-16

90-95

10-17

96-101

8-12

13-18

102-107

5-12

4-9

12-16

108-113

6-14

4-8

114-119

6-14

120-125

126-131

132-137

138-143

*Three juvenile females 96, 108 and 109 increased 7, 14 and 11 mm respectively.

same as the average for those which actually molt-
ed. Average increment for males of comparable
size was greater than that for either pubescent or
adult females. The 29 males which molted in
undersea pens averaged 14 mm. This data com-
pares favorably with and complements that pre-
sented by Powell (1967).

Size at Which Females Attain Sexual Maturity
Small females without externally developing

^ PUBESCENT Od JUVENILE,
^^ WITHOUT EXTERNAL EGGS

lOUlts, with external eggs

Smo Bs) oubescenr lafimm)

_il

.rteffl

IB^

CARAPACE LENGTH (MM

eggs possess silky incubatory setae and are either
juvenile or pubescent^. Dissection is necessary to
make the determination; oviducts full of eggs re-
veal that females are pubescent.

During exploratory fishing in Middle Bay, 8,439
female king crabs were captured, measured and
examined for presence or absence of external eggs.
Juvenile and pubescent females combined totaled
4,856, adults numbered 3,583. The smallest adult
female captured was 96 mm in carapace length.

Dissections of 180 pre-molt females including
juveniles, pubescents and adults revealed that all
females less than 86 mm were juvenile, i.e., had
empty oviducts; and that all pubescent and adult
females had ripe eggs in their oviducts (Fig. 4).

Absence of both external and internal eggs in-
dicates females are juvenile and will remain as
such for at least another year, being incapable of
ovulating in the ensuing mating season. Figure 4
shows that pubescent females ranged from 84-119
mm but that at the size 111-113 mm the majority
were adults. It is interesting to note that the
majority of females 90-92 mm were juveniles and,
that the majority of females 108-110 mm were

FIG. 3. Size distribution of juvenile and adult fe-
males in a sample of 8,439 king crabs, 17
December, 1970 - 31 March, 1971, Middle Bay,
Kodiak Island, Alaska.

'An exception to this statement could exist if a
small adult female were not mated. Unmated
small females are uncommon.

KING CRAB REPRODUCTIVE BIOLOGY

CaRAPaCE LENGTH (mm)

FIG. 4. Size at sexual maturity of female king
crabs, December 1970, Middle Bay, Kodiak Island,
Alaska (10 crabs in each size group).

pubescent and would become 118-120 mm
through molting before having their first oppor-
tunity to mate.

Size at Which Males Attain Sexual Maturity

At present, no easy method to determine
maturity of male crabs exists. An attempt to
determine maturity by microscopic observation of
reproductive tracts from 49 males, 71-160 mm
carapace length, revealed no apparent difference
except size of the tract. All tracts were similar in
color and convolution and all contained sperma-
tophores. Non-motile spermatozoa similar to those
illustrated by Marukawa (1933) were found within
spermatophores of two crabs checked, 79 and 132
mm in carapace length. Smaller crabs probably
should have been included in the examination to
insure the inclusion of juveniles. More research of
male reproductive tracts is needed.

Placing males in pens with ripe females proved
to be a direct, practical and dependable approach
for determining sexual maturity of male crabs.

Experimental new-shell males within the
100-109 mm size class were the first group to be
placed with females in the undersea pens. Each of
the 18 males within this class mated successfully.
Egg clutches on all females were large and no at-
tempt was made to differentiate between their
relative sizes. Four failed to mate their first op-
portunity, but their failure may be attributed to

the fact that their females had molted 10-17 days
earlier. Many females attempting to mate 10 days
after molting are unsuccessful (McMullen, 1969;
Kurata, 1961)*. When introduced to females which
had just molted, males mated quickly at the
second opportunity.

Each of the males in the 100-109 mm size class
except one was smaller than his female mate.
Males averaged 11 mm smaller, two males success-
fully mated females 38 and 40 mm larger than
themselves (Table 3). Males which failed to mate
for reasons other than their own inability, such as
a male with a juvenile female, are omitted from
the comparison since no useful purpose would be
served by including them. One such male is be-
lieved to have failed because of his impending
molt.

Witnessing the success of the 100-109 mm
males, the smaller second group, 90-99 mm, was
subsequently incorporated into the experiment. Of
the 18 within this class, 16 mated successfully and
two failed. It is believed that the two were im-
mature. One of the 16 failed on his first oppor-
tunity but succeeded when given another try
(Table 3). Each of the 16 males was smaller than
their mates, averaging 14 mm less. One male
mated with a female 26 mm larger than himself.
All egg clutches were large and no attempt was
made to differentiate between their relative sizes.
The proportion of males mating successfully in
this group was .875 with a standard error of .08.

In supplemental experiments, 6 new-shell males
from 84-89 mm in size were tested for mating
ability. Three were successful and three failed.
Two possibly were immature, and the third is be-
lieved to have failed because of an impending
molt. The two males that were considered
juveniles were 20 and 23 mm smaller than their
female partners. The authors would like to em-
phasize that the experimental males were neces-
sarily smaller than the females because adult fe-
males have never been found smaller than 96 mm
and small adult females are scarce.

In other supplemental experiments, 10 mature
males larger than 109 mm had opportunities to
mate. The only ones which failed were those pre-
paring to molt or completing their molt.

Males attain sexual maturity a year or two
before females and at a smaller size. These smaller

Kurata, H. 1961. King crab investigations in the
eastern Bering Sea in 1961. International North
Pacific Fish. Comm. Prelim. Transl. Doc. 481. 6
P-

84 G.C. POWELL, B. SHAFFORD AND M. JONES

TABLE 3. Size relationships of mating partners within the 38 mating pairs tested experimentally.^

Size

of new-shell

males with

in the two

experimental groups (capapace

length (mm))

90-99

100-109'

Male

Carapace length (mm)

Size

Male

Carapace len

gth (mm)

Size

File

tag #

Male

Female

difference

(mm) tag -

Male

Female'^

difference
(mm)

849

116

851

100

109

841

109

830

100

112

829

105

863

100

108

824

115

871^^

101

141

842

101

871

101

112

826

108

855

109

860

101

114

856
850^

113

845

101

118

113

866

103

118

840

113

866

103

107

854

112

867"^

103

116

857

112

766

104

665

109

862^

104

142

770

104

864

105

110

847

108

827

106

116

846

100

108

859

106

114

825^

111

869

106

111

839^

111

731

107

104

-3

870

108

112

861

108

112

865^

109

115

Pairs in which the male failed to mate for reasons other than immaturity are omitted.

Males 871 and 866 mated 2 females within a 3-day period of time.

'^Sizes given are those for new-shell females after molting.

Indicates those males which failed to mate at their first opportunity; they are listed with the female with which
they mated.

^Indicates those males which failed to mate.

males have growth rates equal to or greater than
the pubescent females. The average size difference
between partners was equal to one year's growth.
The size difference between a 90 mm experi-
mental male, such as those which mated in the
undersea pens, and a male just smaller than legal
size (144) is 55 mm. The smaller 90 mm male
would have to molt four times to attain commer-
cial size, taking 4 years, illustrating that some
males may have five opportunities to mate before
becoming available to the commercial fishery; the
last opportunity to mate occurs just before the
fishing season opens but after the crab has molted

to legal size. Caution is recommended, however,
because 90-109 mm males which mated under con-
trolled experimental conditions seldom are found
mating in nature, suggesting that behavior in
undersea pens may be limited in its applicability
to conditions existing in nature.

Grasping as an Indicator of Mating Ability

Grasping activity of small males (100-109 mm)
used in the 1971 pen study was compared with
that of 24 large males (138-193 mm) used in a
1970 pen study. Only males with graspee females
are included in the comparison.

KING CRAB REPRODUCTIVE BIOLOGY

Small males were observed by divers to be
grasping their female partners 56% of the time as
compared to 80% for large males. Small males
grasped their female partner only 34% of the time
when total time together is used in the calculation
rather than just the last 6 days prior to copula-
tion. Regardless of how the 2 groups were com-
pared, smaller males consistently grasped less than
larger ones. No relationship between grasping and
the time of day that observations were made was
apparent. No diving was conducted after dark.

All males in the comparison mated successfully.
Less grasping or lack of aggression might lead to
reduced mating in nature by small males not only
because they would often be coexisting with large
males, but also because interruptions in grasping
activity could allow females to escape. In the
undersea pens, females were unable to leave the
proximity of the males even if not grasped.

Data suggest that only adult males grasp fe-
males, but data also reveal that small adult males
do not grasp to the extent that large males do,
and therefore lack of grasping cannot be inter-
preted as a sign of immaturity.

Of the 7 males which failed to mate, (those
marked d & e in Table 3), 56% (4 crabs) were
never seen grasping during a combined observation
total of 23 days. The three unsuccessful males in
supplemental studies were seen grasping 10 of 21
days observed or 48% of the time. The relative
amount of time each male was seen grasping was
similar to that of the successful males.

Males have not been known to grasp juvenile
females. In the present pen study the only three
females that were not grasped were found upon
dissection to be juveniles. These three females
were vrith their male partners for a combined total
of 98 days, and were observed for a total of 70
days.
Interval of Time for Copulation and Ovulation

Copulation and the deposition of sperm on the
female's gonopores can occur only after the female
molts, and precedes ovulation. It was hypothesized
that longer intervals of elapsed time for comple-
tion of copulation and ovulation might be in-
dicative of reduced male mating ability, especially
since females ovulate soon after sperm deposition
has occurred. Total days elapsed time for comple-
tion of copulation and ovulation, recorded during
the 1970 and 1971 mating studies, were com-
pared.

Small males during 1971 copulated almost as
quickly as the larger males which mated in 1970.
In 1970, all 74 females had been mated within

two days after molting. In most of these matings,
copulation and ovulation probably occurred within
24 hr after molting. In all 10 cases where females
were examined within 24 hr after molting, eggs
were present indicating that mating had occurred.

In 1971, 45 of 47 females were mated within
two days after molting, only two required more
than two days. Observations were made more fre-
quently than during 1970 and several females
examined 24 hr after molting still had not ovula-
ted, however, colder temperatures during 1971
may have retarded ovulation.

The two males which required more than two
days for mating were not typical individuals. One
was the smallest male that mated during the study
(85 mm length) and the other was the first
new-shell male captured after the molt in Middle
Bay.

Apparently, the 10 - day interval of time
bracketing the male molt is a period during which
the male is limited in his ability to mate. Some
males may be limited for a greater period of time.
Numerous males had opportunities to mate just
before and after their molt but the closest to the
molt that any male mated was five and six days
respectively (Table 4). One of the males that
mated seven days after molting actually molted
the same day as his female partner and chose not
to mate wath her until the seventh day afterwards.
Some of the males that failed to mate could have
been juveniles but it is not likely that many were.
Effects of Temperature on Mating

Bottom water temperatures of Near Island Basin
in 1971 were 3-6°F lower than during the same
period in 1970. Temperatures in 1971 were ap-
proximately 35° F on 14 February when the first
female molted and mating began. Temperatures
gradually declined for the next month and were
approximately 31.5°F on 10 March. Most of the
pubescent females in the pens were molting and
mating while water temperatures were declining.
Small crabs in nearby Middle Bay were molting at
the same time and probably under the similar
temperature conditions. Temperatures increased
after 10 March and 1 April were back to 35°F.
Adults mating later in April were mating during
rising temperatures. Data suggest that molting and
mating are not closely regulated by declining or
rising temperatures of this magnitude.

Maximum daily fluctuations of 2°F were as-
sociated with "spring" tides. Temperature would
rise quickly, remain level for 6 hr, and then de-
cline to the original temperature. Cold air
temperatures (0-20° F) cooling exposed inter-tidal

areas during low tides probably had a marked ef-
fect in reducing sea water temperatures each time
the tide would rise.

Colder temperatures slowed egg development in

G.C. POWELL, B. SHAFFORD AND M. JONES

1971. McMullen (1970) reported the 8-blastomere
stage was attained 5-7 days after fertilization at
temperatures of 37-40° F. During this study, the
8-blastomere stage was attained 10-14 days after

TABLE 4. Effect of Male Molting Upon Mating Ability.

Premolt Male Crabs

No. of Males

Z .5 [I. 2 S

Comments - how soon males
mated after having the
opportunity^ (first com-
ments refer to the males
which failed to mate)

7
9

14 -
Tot. 4

Had opportunity for 1 day.

Both had opportunity for

3 days.
Had opportunity with 2

females for 5 days.

Both mated right away.
Male mated right away.

Male mated right away.

Post molt Male Crabs

No. of Males

oil

-o

%-i

4->

Comments - how soon
males mated after having
the opportunity^ (first
comments refer to the
males which failed to
mate)

Postmolt Male Crabs (Cont.)

Had opportunity for 3
days.

10 1

11 1

12 1

13 -

16 -

17 1
20 -
22 1

27 ■

Tot. 10

One had opportunity for
2 days, other had oppor-
tunity for 4 days.

Had opportunity for 5
days.

Male mated right away.

Had opportunity for 7 days.

One mated right away, the

other had opportunity for
7 days.''
One male had opportunity

for 8 days, the other mated

right away.
Male had opportunity for 8

days, the other mated right

away.
Male had opportunity for 8

days.
Male mated right away.

Male mated on 4th day.

One male had opportunity for

6 days.
Mated on the 8th day.

Male had opportunity for
3 days, the other mated
right away.

Male mated on the 3rd day.

Each day the male is with the new-shell female constitutes one opportunity.

This male molted the same day as the female.

KING CRAB REPRODUCTIVE BIOLOGY
fertilization at temperatures of 32-35° F.

SUMMARY

Male king crabs attain sexual maturity at a
smaller size and at a younger age than females.
The crucial question is whether or not these small
mature males are functioning as brood stock.
Pubescent males and females are congregated in
schools along with juveniles of similar age and size
with each group molting prior to adults in late
February and March. The approximate 10 - day
interval bracketing a male's molt is a period during
which males are incapable of mating, therefore
many pubescent males are unavailable for mating
with pubescent females. This partially accounts for
larger males, many of which molt a month later
and/or are anexuviants, being available to mate
with pubescent females. Larger adult females segre-
gate in separate schools located in similar depth
but molt and mate in April and May. Since small
males mated with females considerably larger than
themselves in the pen studies, it is likely that
some would also mate in the natural environment
if they had the opportunity. The degree to which
they attempt to mate and their ability to compete
remains unknown.

The 7-inch size limit appears to protect most
brood stock males from commercial harvest for
two or more years, especially when used simul-
taneously with a quota and closed season, but in-
tense harvest on some grounds, if allowed to per-
sist, may still create undesirable sex ratios. A few
young males probably have four seasons to mate
before attaining legal size, and many at least three
seasons. Past intensive commercial harvests (prior
to quotas and extended closures) in locations
where schools of older males and females were
segregated from those of younger crabs, particu-
larly off-shore areas, has resulted in the occurrence
of unmated females as high as 30% (Powell, 1969;
Powell and Davis, 1969)'. Harvest of legal-size
males must be regulated in areas inhabitated only
by older crabs if full particiaption by females is to
be obtained. Abundance of unmated females is
much less in shoreward areas where undersize
males are abundant.

'Powell, G. C. and R. A. Davis, 1969. Further
Contributions to King Crab Paralithodes camt-
schatica (Tilesius) reproduction. Typewritten
manuscript 105 pp.

LITERATURE CITED

Gray, G. W., Jr. 1965. Tags for marking king

crabs. Progr. Fish-Cult. 27: 221-227.
Gray, G.W., Jr. and G. C. Powell. 1966. Sex ratios
and distribution of spawning king crabs in
Alitak Bay, Kodiak Island, Alaska (Decapoda
Anomura, Lithodidae). Crustaceana 10:
303-309.

Marukawa, H. 1933. Biological and fishery research
on the Japanese king crab Paralithodes camt-
schatica (Tilesius). J. Imp. Fish. Exp. Stn.,
Tokyo 4 (37): 1-152. (In Japanese with English
abstract)

McMullen, J. C. 1967. King Crab Paralithodes
camtschaticp (Tilesius) offshore breeding study
on Marmot Flats, Kodiak Island, spring of
1967. Alaska Dep. Fish. Game Inf. Leafl. No.
112, 12 p.

McMullen, J. C. 1969. Effects of delayed mating
on the reproduction of king crab, Paralithodes
camtschatica. J. Fish. Res. Board Can. 26:
2737-2740.

McMullen, J. C. 1970. Aspects of eariy develop-
ment and attachment of fertilized king crab
eggs. Alaska Dep. Fish Game Inf. Leaflet No.
140, 12 p.

Nickerson, R. B., F. J. Ossiander and G. C.

Powell. 1966. Change in size-class structure of
populations of Kodiak Island commercial male
king crabs due to fishing. J. Fish. Res. Board
Can. 23: 729-736.

Powell, G. C. 1958. King crab research. Annu.
Rep. Alaska Fish Game Comm. and Alaska
Dep. Fish Game No. 10, p. 34-44.

Powell, G. C. 1964. Fishing mortality and move-
ments of adult male king crabs, Paralithodes
camtschatica (Tilesius) released seaward from
Kodiak Island, Alaska, Trans. Am. Fish. Soc.
93: 295-300.

Powell, G. C. 1967. Growth of king crabs in the
vicinity of Kodiak Island, Alaska, Alaska, Dep.
Fish Game, Kodiak Res. Center, Kodiak,
Alaska, Inf. Leafl. No. 92, 106 p.

Powell, G. C. 1969. Some aspects of king crab
biology. Proc. Am. Fish. Soc, West Div. Meet.,
Jackson Hole, Wyo., p. 142-143.

Powell, G. C. and R. B. Nickerson. 1965. Repro-
duction in king crabs, Paralithodes camtschatica
(Tilesius). J. Fish. Res. Board Can. 22:
101-111.

Wallace, M. M., C. J. Pertuit and A. R. Hvatum.
1949. Contribution to the biology of the king
crab Paralithodes camtschatica (Tilesius). U. S.
Fish Wildl. Serv., Fish. Leafl. 340, 50 p.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

THE FEASIBILITY OF CLOSED SYSTEM MARICULTURE:
PRELIMINARY EXPERIMENTS WITH CRAB MOLTING'

Rodner R. Winget'^ , Don Maurer and Leon Anderson

FIELD STATION

COLLEGE OF MARINE STUDIES

UNIVERSITY OF DELAWARE

LEWES, DELAWARE

ABSTRACT

A recirculation system for inducing shedding in the blue crab, Callinectes sapidus
Rathbun, and preliminary experiments on crab molting are described. The most
important result was the inducement of out of season molting (January-March) in
the Delaware Bay area. It appeared that temperature was u key factor in promoting
out of season molting. Regardless of its present limitations, year round crab molting
and growth may be feasible in a closed recirculation system.

INTRODUCTION

Since 1968 tnis laboratory has been engaged in
research directed toward developing facilities for a
completely enclosed, environmentally controlled
pilot shellfish hatchery (Harman and Maurer, 1971;
Price and Maurer, 1971; Maurer, 1972). An important
by-product of the research was consideration of new
species for inclusion in closed system mariculture.
The blue crab, Callinectes sapidus Rathbun, was one
species which was studied. To accomplish this
research it was necessary to design and construct a
recirculation system.

One objective of this paper is to report results
of an experiment concerning growth and molting
in the closed system. Factors such as temperature,
salinity, photophase, nutrition, season, privacy,
moisture and hormone concentrations influence
molting in reptantian decapods. The purpose of
the experiment was to determine which factors are
necessary for inducing molt in blue crabs out of
season, in this case January through March. Shed-
ding of blue crabs in Delaware waters under natu-
ral conditions normally occurs from April -

' College of Marine Studies Publication No.

2-81-103
^Present address: Zoology Department, University

of Minnesota, Minneapolis, Minnesota

November. By developing methods to molt blue
crabs in the winter the soft shell industry can be
pursued throughout the year. In addition, by in-
ducing molting the year round, faster growth rates
are realized and marliet size crabs can be obtained
much quicker.

MATERIALS

Closed System Design

The recirculating seawater system is housed in a
1.52 X 2.74 m insulated room and consists of a
reservoir, filtering unit, pump, eight trays, lights,
an air cooling unit and two timers (Fig. 1). The
volume of the entire seawater system is 1,135
liters. The reservoir conveniently holds 680 liters
with dimensions of 1.82 x 1.23 x 0.33 m. The
trays (Fig. 2-1) which are 2.74 x 0.36 x 0.17 m
are divided into 26 cubicles. Dividers are made
from asbestos boards in which slots are cut in the
center and cross pieces such that they interlock
forming cubicles (0.15 m on all sides). Qearance
of 3.2 cm is provided on the bottom of all cross
pieces to reduce accumulation of waste material.
Water also flows through holes drilled in the cross
pieces at the water line. Water depth in the trays
is 6 cm.

The filtering unit is 0.46 x 1.22 x 0.46 m and
is divided into three compartments (Fig. 3). The
pump draws its water from the middle compart-

EXPERIMENTS WITH CRAB MOLTING IN CLOSED SYSTEMS

AIR COOLER

ITTMER

11 \r

1 1 1 1

FIG. 1. Schematic of recirculating seawater system.

3-3). After salinity and temperature are adjusted
the rubber stopper is pulled and the water fills the
filtering unit. The pump 20 gpm (Fig. 3-4), is
turned on and the water flows to the individual
trays. At each tray there is a valve that controls
the rate of flow (Fig. 2-3). The water then fills
the tray to the desired depth determined by the
length of stand pipe inserted in the drain hole
(Fig. 2-4). When the water level reaches the top of
the stand pipe it flows into the return line via
flexible pipe (Fig. 2-5) and is returned to the fil-
tering unit. At that point the cycle is completed.
The stand pipes are pulled out, and the drain
valve is opened to flush the system.

Water was changed every two weeks during the
molting experiments. Temperature and pH remained
stable and salinity increased about 2 %o in the same
period. The holding capacity of this system is 200
crabs. The entire system including construction of
two insulated rooms cost $3,500.

ment (0.46 x 0.30 m) through a 2.81 cm PVC
pipe in which a foot valve is installed for conven-
ience in priming (Fig. 3-1). A float switch is also
installed to protect the pump in case of line ob-
struction or breakage. The lateral compartments
are 0.46 x 0.46 m, each with four 2.54 cm PVC
pipes spaced equally 1.27 cm from bottom. Each
pipe (Fig. 3-2) leads to the middle compartment.
Small slits are cut into these pipes allowing water
to flow into the pipe and then into the center
compartment. The lateral compartments are half
filled with crushed clam shells, approximately 1.22
cm in diameter, which serve as filtering and buf-
fering agents. All wooden components are made
from 1.91 cm marine plywood coated with fiber-
glass.

Each tray is equipped with a light fixture con-
sisting of a F-72 cool white fluorescent bulb en-
closed in a moisture proof plastic cover (Fig. 2-2).
Lights for each bank of trays are controlled
separately by a time clock which allows simultane-
ous testing of two photoperiods. To avoid inter-
ference from other light sources, opaque curtains
are installed in front of each bank. Heat from the
lights is modified by a Tecumseh cooling compres-
sor, model No. C2516 MTK, which enables a con-
stant temperature to be maintained.

Water is pumped to a head tank and remains in
it several days to facilitate sedimentation. From
there the water is pumped through a heat ex-
changer into the reservoir which is closed from the
rest of the system by a rubber stopper inserted in
a 30 cm long, 3.81 cm PVC connecting pipe (Fig.

METHODS
Crabs were collected from the field in late

MARINE PLYWOOD

FROM WATER PUMP

FIG. 2. Schematic of crab tray holding facilities for
recirculating seawater system.

R.R. WINGET, D. MAUER AND L. ANDERSON

FUDW TO TRAYS

FIG. 3. Diagram of filtering unit for seawater recirculating system.

December 1970 (3-6° C). They were placed in
ambient, still, aerated sea water in the laboratory.
The water was gradually raised to 20-22 C over a
period of two days. After acclimation each crab
was weighed and measured and placed into a com-
partment (Fig. 2-1) within the recirculation sys-
tem. Throughout the experiment the temperature
and salinity were 25° C and 25 o/oo respectively.
Crabs were fed silversides, Menidia menidia Linne,
five days a week. Based on Aiken's (1969) re-
search concerning molting in crayfish, two photo-
phases were established. Thirty-four crabs were
exposed to a 16 hr day and thirty were exposed
to an 8 hr day. The dates crabs entered the sys-
tem and molted or died were recorded together
with growth determinations (weight, length,
width). All measurements were recorded during the
C4 molt stage (Drach, 1939). The experiments
were terminated in late March.

RESULTS AND DISCUSSION
For the 16 hr and 8 hr photophases, 20 of 34

crabs (59%) and 16 of 30 crabs (53%) molted
respectively. The average time to first molt was
27.3 days (16 hr photophase) and 28.6 days (8 hr
photophase). Crabs in the 16 hr phase grew (aver-
age increase in width - 19.3%, length - 22.3% and
weight - 104%) slightly more than crabs in the 8
hr phase (average increase in width - 17.4%, length
- 19.1% and weight - 83.1%). The Mann-Whitney
Test, a non parametric statistic (Conover, 1971),
indicated no significant difference between photo-
phases ■ (P > 0.05) in width, length, weight and
days to the first molt. Suvivorship in the 16 and
8 hr photophase was 68% and 70% respectively. In
addition, 5 crabs in the 16 hr photophase molted
a second time; on the average the second molt
occurred 23 days after first molt. There was essen-
tially no difference in length between the first
molt (average increase of 22.3%) and second molt
(average, 22.6%), a slight increase in average width
from 19.3 to 24.6%, and a reduction in average
weight from 104 to 94.6%. Initiation and com-
pletion of a complete molt cycle in a closed sys-

EXPERIMENTS WITH CRAB MOLTING IN CLOSED SYSTEMS

tern was an encouraging result.

The most important result was the inducement
of out of season molting. It appears that tempera-
ture is a key factor in promoting out of season
molting. Experiments in progress also confirm this
(Epifanio, personal communication). Field survey
data show that blue crabs in a local thermal ef-
fluent (Island Creek, Indian River Bay, Delaware)
were molting in January. The water temperatures
in the effluent may be 7-8° C higher than ambient
sea water. No statistics on the frequency of shed-
ders per month are recorded for Delaware, but
molting of this species does not normally occur in
winter waters of the Delaware Bay region. In
other laboratory experiments out of season molt-
ing has been induced in blue crabs from Virginia
waters (Haefner, personal communication). Tem-
peratures ranging from 18 - 25° C were used in
these experiments. Haefner (1971) found the inci-
dence of mortality among peeler crabs higher in
recirculated water (55%) than in new seawater
(38%) and highest (65%) in artificial seawater.

Based on the present experiments the effect of
photophase on molting is statistically insignificant.
However, it would be premature to discount the
biological significance of photophase on molting
particularly in view of the small number of crabs
used in the experiments and initial mechanical
problems with the recirculation system. Research
on other reptantian decapods has demonstrated
that photoperiods affect molting and breeding
(Little, 1968; Aiken, 1969). Refinements in this
system together with larger numbers of crabs
under combinations of temperature, photophas^,
nutrition and seasonality must be pursued to
determine optimum relationships. For example,
water purity may be improved by filtration
through a 5 /J (AFCO) filter bag or by chlorina-
tion. After this, the water is dechlorinated, ozo-
nized or passed through an ultraviolet radiation
treatment to kill bacteria and viruses. Sanders and
Fryer (1972) recommended combinations of the
above procedures to control fish pathogens in
hatcheries.

Regardless of its present limitations, this re-
circulation system has some advantages. The estu-
arine waters of the Delaware Bay region are ex-
tremely turbid (Secchi readings less than 0.5 m)
which makes laboratory work difficult. It is im-
perative to have particulate free water in con-
trolled laboratory experiments not only for water
quality control but to prevent clogging water
passages through the partitions, making isolation of
individual crabs possible. Past work in the labora-

tory has shown that heavy mortality of crabs is
caused by cannibalism. Since this was a molting
study the effect of cannibalism became even
more serious during intermolt stages. Isolation was
important because it provided privacy eliminating
fighting and cannibalism. Without controlling silta-
tion and cannabilism these experiments could not
have been conducted. Fouling of seawater systems
in Delaware waters can also be a serious problem
particularly during the summer. With improved
filtration the present system would essentially
avoid this problem. In research on disease of blue
crabs Cook (1972) faced similar problems and was
obliged to design and construct a recirculation
system to hold large numbers of crabs in a
healthy environment. His crabs were held over
several months in the system. In the present work
water was changed every two weeks. The senior
author found that water quality in closed systems
can reduce ingestion rates on the horseshoe crab,
Limulus polyphemus Linne, in a month (un-
published data). This demonstrates that water
quality in closed systems for mariculture must be
improved. Our work together with Haefner's
(1971) and Cook's (1972) leads us to believe that
year round crab molting and growth is definitely
feasible in a closed recirculation system.

ACKNOWLEDGMENTS

The research was supported in part by P. L.
88-309, the NOAA Sea Grant Program, and the
Delaware Department of Natural Resources and
Environmental Control. We would like to thank
Dr. Charies Epifanio and Dr. Paul Haefner for
reading the manuscript and providing constructive
criticism.

LITERATURE CITED

Aiken, D. E. 1969. Photoperiod, endocrinology
and the crustacean molt cycle. Science 164:
149-155.

Cook, D. W. 1972. A circulating sea water system
for experimental studies with crabs. Prog.
Fish-Cult. 34: 61-62.

Conover, W. J. 1971. Practical Nonparametric Sta-
tistics. John Wiley & Sons Inc. New York, 461

P-
Drach, P. 1939. Rue et cycle d'lnternue chez les

crustaceas decapodes. Ann. Inst. Oceanogr.

(Paris) N. S. 19: 103-391.
Haefner, P. A. 1971. An approach to shedding

blue crabs Callinectes sapidus in a recirculated

seawater system. Am. Zool. 11: 658. (Abstract).

R.R. WINGET, D. MAUER AND L. ANDERSON

Harman, O. R. and D. Maurer. 1971. Environ-
mental considerations for shellfish production.
Am. Soc. Agric. Eng., Abstract p. 11-26.

Little, G. 1968. Induced winter breeding and larval
development in the shrimp, Palaemonetes pugio
Holthius (Caridea, Palaemonidae). Crustaceana,
Supplement 2, Studies on Decapod Larval
Development, p. 19-26.

Maurer, D. 1972. The development of closed sys-
tem oyster culture. Am. Malacol. Union. Bull.

for 1971, 37th Annu. Meet., p. 18-20.

Price, K. S., Jr. and D. Maurer (ed.) 1971. Pro-
ceedings of the Conference on Artificial Propa-
gation of Commercially Valuable Shellfish -
Oysters. Univ. Del. Publ., Coll. Mar. Stud.
Newark, Del., 212 p.

Sanders, J. E., J. L. Fryer, D. A. Leith and K. D.
Moore. 1972. Control of the infectious proto-
zoan Ceratomyxa shasta by treating hatchery
water supplies. Prog. Fish Cult. 34: 13-17.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

EFFECTS OF SALINITY AND TEMPERATURE ON EMBRYOS OF THE
GEODUCK CLAM {PANOPE GENEROSA GOULD)'

Lynn Goodwin

WASfflNGTON DEPARTMENT OF FISHERIES

SHELLFISH LABORATORY - POINT WHITNEY

BRINNON, WASHINGTON

ABSTRACT

Combined effects of salinity and temperature on embryos of geoducks were
ex'amined. Results indicate narrow salinity and temperature limits for geoduck em-
bryos. For optimum development to the straight-hinge larval stage salinities must
remain between 27.5 and 32.5 °oo , and temperatures between 6 and 16° C. Environ-
mental requirements delineated by these experiments agree with the natural distri-
bution of adult geoducks.

INTRODUCTION

Recent findings by the Washington State De-
partment of Fisheries of large populations of geo-
ducks in the subtidal zones of Puget Sound have
led to a commercial fishery for these large clams.
This new fishery is restricted to divers who harvest
geoducks with small hand held water nozzles.
Landings from the first year's fishing exceeded
400,000 lb. Annual yield could increase consider-
ably because estimated standing crops are well
over 100 million lb (Goodwin, 1973)* i

The increased interest in this species requires
detailed ecological information upon which man-
agement decisions can be based. Objectives of this
study on the effects of salinity and temperature
on embryonic^ stages are to supply some of this
needed information. The work was conducted at
the Washington State Department of Fisheries
Shellfish Laboratory located at Point Whitney on
Hood Canal, Washington.

' The work reported here was partially financed by
the National Marine Fisheries Service, Fisheries
Research and Development Act, PL 88-309.

^Goodwin, C. L. 1973.' Subtidal geoducks of Puget
Sound, Washington, Technical Report, Wash.
State Dept. of Fish., in preparation.

^Fertilized egg to straight-hinge stage = embryo;
straight-hinge stage to setting size = larvae.

METHODS

The bifactorial approach of testing two environ-
mental parameters simultaneously in many differ-
ent combinations was used in this study (Brenko
and Calabrese, 1969). The general methods report-
ed in this paper were developed by Woelke
(1968)" in oyster embryo bioassays. Spawnings
were induced by thermal stimulation and normally
occurred at temperatures between 12-14 C. Fertil-
ized eggs (30,000/liter) were held at different
temperature and salinity ranges in one-liter poly-
ethylene beakers. After allowing the embryos to
develop into straight-hinge larvae, samples of about
250 larvae from the cultures were preserved and
later counted to determine the number which
developed normally. The percentage of embryos
which developed normally to the straight-hinge
stage was used as a measure of the stress of the
culture medium.

Salinities were determined by a hydrometer. Ac-
curacy of this method was within ± 1.0 %o as
verified by chemical titration. Culture temperatures
were maintained in water baths within ± 1 C of the
designated temperatures.

A preliminary test was conducted on the rate

'' Woelke, C. E. 1968. Development and validation
of a field bioassay method with Pacific oyster,
Crassostrea gigas, embryo. Ph. D. Thesis, Univ. of
Wash., Coll. Fish., 140 p.

L. GOODWIN

of embryonic development at different temper-
atures so that the bifactorial cultures could be
incubated the proper amount of time. This
allowed embryos to reach the straight-hinge stage
at the various temperatures tested.

Salinities in the first bifactorial experiment were
prepared by mixing seawater (Dabob Bay, Puget
Sound) and Spencer Creek water (a small unpollut-
ed stream near the laboratory) for salinities below
30 %= , and seawater and Rila Marine Mix (synthe-
tic seawater compound, Rila Products, Teaneck, N.
J.) for salinities above 30 %o . Control cultures of
seawater (29.1 %<, ) and mixtures of Spencer Creek
water and Rila Marine Mix (29.8 %•> ) were also
prepared.

Because of the low percentage of larvae which
developed normally in the Spencer Creek-Rila
Marine Mix controls, the experiment was repeated.
For this experiment fresh water from another
nearby stream (Jackson Creek) was mixed with
seawater (Dabob Bay) for salinities less than 30
%o, and seawater mixed with highly saline sea-
water, concentrated by freezing, for the salinities
above 30 %o . Frozen seawater controls were pre-
pared by freezing seawater then thawing it; main-
taining the original salinity to assess the effects of
freezing on water quality. In the latter experiment
the 6°C cultures were omitted and 16°C cultures
added to refine the upper temperature threshold.
Salinities below 22.5 °oo and above 35 °oo were
omitted.

RESULTS AND DISCUSSION

Results of Table 1 illustrate the marked effect
of temperature on the rate of development of geo-
duck embryos. At 6°C, 132 hr were required be-
fore the maximum number of straight-hinge larvae
was present, whereas, at 18°C the maximum num-
ber was present as early as 36 hr after fertiliza-
tion. Numbers of straight-hinge larvae in the 6 and

TABLE 2. The combined effects of temperature and
salinity on geoduck embryos; percentage of normal
straight-hinge larvae (each figure represents the
mean of triplicate cultures).

Salinity

Temperature (°C)

( %°)

20.0

22.5

25.0

27.5

30.0

32.5

35.0

■ 18-

37.5

40.0

Marine

mix and

Spencer

Creek (29

1.8 %. )

Seawater

Control

(29.1 %o )

1 -

18°C cultures were lower than in 10 and 14 C
cultures indicating that the former temperatures
are outside the optimum temperatures for geoduck
embryos.

The combined effects of temperature and salin-
ity are shown in Tables 2 and 3. The dotted line
encloses salinities, 27.5 - 32.5 %= , and tempera-
tures 6 - 14°C, at which 70% or more of the em-
bryos developed normally to the straight-hinge
stage. Temperatures of 18°C or above are clearly

TABLE 1. Effect of temperature on rate of development of geoduck embryos; percentage of embryos
which developed to the straight-hinge stage (each figure represents the mean of duplicate cultures).

Temperature

Age in hr at termination

(°C)

24 28 32

40 44

52 60 66 72 78 90

118

132

141

196

220

6
10
14
18

.- - 0
1 0 3

0
27

2 75
25 13

86
26

4 11 57 81 90
94 94 - - - -

60
94

64
92

EFFECTS OF SALINITY AND TEMPERATURE ON GEODUCK EMBRYOS

TABLE 3. The combined effects of temperature
and salinity on geoduck embryos; percentage of
normal straight-hinge larvae (each figure represents
the mean of triplicate cultures).

Salinity

Temperature ( C)

22.5

25.0

27.5

75 ■

30.0

32.5

35.0

Salt water

concentrate

and Jackson

Creek

(30 %= )

Frozen

seawater

control

(30 %o )

Seawater

control

(29.1 %o )

detrimental to geoduck embryos and 6 C appears
to be the lower temperature threshold. Survival
and normal development were low at salinities be-
low 25 %o and above 32.5 %o regardless of

temperature.

Kesults ot the second bifactorial experiment

were similar to the first. The percentage of normal
larvae was slightly higher at 14°C compared to
16° C in comparable salinities which indicates that
16°C is the upper tolerance limit for geoduck em-
bryos.

The experiments suggest that geoduck embryos
have relatively narrow salinity and temperature
limits. For satisfactory percentages (70% or above)
of embryos to develop into straight-hinge larvae,
salinities must remain between 27.5 and 32.5 %<>
and temperatures between 6 and 16 C. Salinity
limits are comparable with two previous experi-
ments conducted on the effects of salinity on geo-

duck embryos held at a constant 14 C. Salinity
and temperature limits for geoduck embryos are
narrower than those of the coot clam, Mulinia
lateralis (Calabrese, 1969). For the development of
a satisfactory percentage of coot clam embryos,
the salinity must remain between 20 and 30 %o ,
and the temperature from 12.5 • 27.5°C.

The low percentage of normal development of
embryos in the controls with Rila Marine Mix and
freshwater of the first experiment and those with
concentrated salt water (freezing method) mixed
with freshwater of the second experiment reduces,
somewhat, the reliability of the results. These
control cultures were slightly toxic to geoduck
embryos. This may have artificially narrowed the
tolerance limits established by the experiments.
The freezing and thawing of seawater apparently
lowered the water quality as shown by the lower
percentage of normals in the frozen seawater
controls of the second bifactorial experiment.
Calabrese (1969) and Brenko and Calabrese (1969)
did not include controls needed for a comparison
of my results. My preliminary experiments indicate
that controls other than those prepared from
unaltered seawater are needed to properly interpret
the results of these types of experiments.

Salinity tolerance limits suggest that the geo-
duck is an estuarine animal which cannot tolerate
salinities found in the open ocean or to prolonged
exposures of water less than 25.0 %» . Temperature
requirements show that they prefer cold water and
would not be expected to be found in areas where
water temperatures are above 16°C during their spring
and early summer spawning season. These require-
ments agree with the known distribution of geo-
ducks in the State of Washington (Goodwin,
1973)^ Tolerance limits of larvae and older stages
of geoducks are probably wider than those of em-
bryos. Larval stages in some of my earlier feeding
experiments and adults held in the laboratory have
survived prolonged temperatures of 18 C and
short-term exposures of 20 C.

LITERATURE CITED

Brenko, M. H. and A. Calabrese. 1969. The com-
bined effects of salinity and temperature on lar-
vae of the mussel Mytilus edulis. Mar. Biol.
(Berl.) 4: 224-226.

Calabrese, A. 1969. Individual and combined ef-
fects of salinity and temperature on embryos
and larvae of the coot clam, Mulinia lateralis
(Say). Biol. Bull. (Woods Hole) 137: 417-428.

Proceedings of the National Shellfisheries Association
Volume 63 - June 1973

HERMAPHRODITISM IN TWO SPECIES OF PELECYPOD MOLLUSKS

Sara V. Otto

MARYLAND DEPARTMENT OF NATURAL RESOURCES

FISHERIES ADMINISTRATION

ANNAPOLIS, MARYLAND

ABSTRACT

Five additional hermaphroditic soft shell clams (Mya arenaria) were found in samples
from several beds in Chesapeake Bay in 1971. With these new findings, a total of ten
hermaphrodites, 7 bilateral and 3 mixed, have been found among 1,311 specimens exam-
ined. These are the only known cases of soft clam hermaphroditism ever reported for
Chesapeake Bay. One bilaterally hermaphroditic hard clam (Mercenaria mercenaria) was
found from 546 examined in Chincoteague Bay. This is the first known case of herma-
phroditism in hard clams in Chincoteague Bay. A total of 520 hard clams were also exam-
ined from Chesapeake Bay, but no hermaphrodites were found.

INTRODUCTION

Otto (1972) reported the discovery of five herma-
phroditic soft shell clams (M. arenaria) collected from
various areas of Chesapeake Bay. These clams were
considered as "accidental functional ambisexual" by
the classification of Coe (1943). Four of the clams
were bilaterally hermaphroditic and the fifth was of
the mixed type wherein the alveoli contained both
male and female gametes. To my knowledge this was
the first report of hermaphroditism in Mya collected
from Chesapeake Bay tributaries. Since the publica-
tion of the earlier report, five more hermaphroditic
Mya and one hard clam (M. mercenaria) have been
found. All the Mya v/ere collected from Chesapeake
Bay tributaries, while the single Mercenaria was part
of a sample collected from Chincoteague Bay. Table 1
details the results of macroscopic and microscopic
examinations. The hard clam specimen will be dis-
cussed separately.

The specimens were from samples collected regu-
larly in a project (#3-131-R under P.L.88-309) under-
taken by the State of Maryland, Department of
Natural Resources. This project consists of the col-
lection and examination of mollusks (Crassostrea
virginica, M. arenaria, M. mercenaria, Tagelus sp., and
others) to determine parasite prevalences, distribu-

tions and pathological conditions. Mya were collected
during 1971 only. Collection of Mercenaria is a con-
tinuing part of the project.

HISTOLOGICAL METHODS

All specimens in our samples are processed with
the same methods. Samples are individually coded
when received. The animals are scrubbed, measured
and examined macroscopically before and after open-
ing. They are graded according to condition: Fat,
Medium or Watery. A transverse section 10 mm thick
is taken from each animeil through the visceral mass,
gonad, gills and kidney. The tissues are placed in
Davidson's fixative for at least 48 hr, dehydrated in
successive changes of ethanol and xylene and embed-
ded in paraffin. Sections 6u thick are permanently
stained with Harris hematoxylin-eosin for examina-
tion.

HERMAPHRODITISM IN M. ARENARIA

Results of Examinations

Table 1 details pertinent information related to
samples where hermaphroditic Mya were found. In all
cases the clams were in developmental phase. The
total number of Mya examined in the project was
1,311. The percent prevalence of the 10 herma-

HERMAPHRODITISM IN CLAMS 97

TABLE 1. Summary of data related to samples where hermaphroditic Mya were found in Chesapeake Bay.

Sample

% Sex ratio

Av. Shell

% Hermaphroditic

Number in

Salinity

Temp.

Site

Date-1971

Female: Male

Length (cm)

Bilateral

Mixed

Sample

°C

Chester River

20 May

7.0

G.76

17.41

Potomac River

11 Jun

7.0

3.00

23.22

Corsica River

30 Jun

6.0

7.80

27.46

Corsica River

2 Sep

6.0

7.46

26.00

Chester River

2 Sep

6.5

10.23

25.49

Chester River

16 Sep

6.5

9.70

26.42

Corsica River

4 Oct

6.5

9.89

21.82

Eastern Bay

4 Oct

6.0

13.80

22.64

phroditic clams found was 0.76% (Bilateral type:
0.53%; mixed type: 0.23%).

HERMAPHRODITISM IN M. MERCENARIA

This hermaphroditic clam was found in a sample
of 20 clams collected from Chincoteague Bay on 26
May 1972. Up to and including this sample of clams,
a total of 1,066 were examined. The incidence of this
condition in the hard clam samples was 0.094%. A
total of 546 clams were collected from this area
(0.18% incidence). The other clams were collected
from various areas in Chesapeake Bay. A review of
the hterature indicates that this condition is rare in
M. mercenaria. Loosanoff (1936) stated that he
found only two cases of functional hermaphroditism
among several hundred adult clams. He also stated
that there was strong evidence of protandry in young,
immature clams.

Results of Macroscopic Examination

The average size of the clams in the sample was 8.5
cm, while the hermaphrodite was 8.0 cm. The range
of physical conditions was as follows: Fat - 3 (15%);
Medium plus - 7 (35%); Medium - 9 (45%); and
Medium minus - 1 (5%). The hermaphrodite was
Medium minus. No abnormalities were noted in this
clam.

Results of Microscopic Examination

The following sex ratio was noted: 5 males, 14
females, and the single hermaphrodite. The herma-
phrodite and the rest of the sample was free of para-
sites.

The clam was bilaterally hermaphroditic. Both
sperm and eggs appeared to be of mature size; the
development being parallel (Fig. 1). The physical
condition of the clam, in addition to the sexual
anomaly, appeared to be slightly abnormal in that

phagocytic infiltration was heavy throughout the
animal; an indication of physiological stress.

DISCUSSION

The reason or cause for the relatively high level of
hermaphroditism in the Mya samples can be only
speculated upon at this time. That the condition is
rare, according to the literature, cannot be disputed.
The discovery of ten such endowed animals in a small
number of areas (5) in Chesapeake Bay would remove
this condition from that category; at least in Chesa-
peake Bay. That environmental conditions or stresses
on these Mya may be one of the causative agents is
very possible. Since Mya here are near the southern
limit of their geographical distribution, any change,
however slight, in their environment probably affects

Wk ■■ ■ ••■■■:■

*>» '

W-' ■ ■ ■•'

■-

♦

«j^jr^i^^ ■;•,.*

•

• •

•

••1 .'Kt. •».••'•• V;.

V »

A •:-•> • • . .

* »

..U- *. . .

•ijvl^'. ^^'f- ♦• •

♦ , ,' '

FIG. 1. Bilaterally hermaphroditic gonads of M.
mercenaria. Sperm are small, dark-staining bodies.
(About 430X).

S.V. OTTO

them greatly. It could be that gonad development is
also affected by subtle changes that are not reflected
in the Bay's hardier species such as the oyster (C.
virginica).

As far as concerns the Mercenaria case, this clam,
moreso than the Mya, deserves the classification of
Coe (1943) as an "accidental functional ambisexual"
(italics mine). Loosanoff (1936) noted the presence
of small ovocytes along the walls of adult male alveoli
and stated that "this may be the potentiality of
changing sex even in the adult condition." In this
case, as with Mya, environmental conditions, may, in
part, effect this phenomenon.

AKNOWLEDGMENTS

I wish to thank Mrs. Janet B. Hammed, project
#3-131-R, Fisheries Administration, State of Mary-

land, for the histological processing of the material;
Dr. Aaron Rosenfield, Mr. William N. Shaw, and Mr.
John W. Ropes, National Marine Fisheries Service,
Biological Laboratory, Oxford, Maryland, for their
advice and review of the paper; Mr. Frederick G.
Kern, N.M.F.S., Biological Laboratory, Oxford,
Maryland, for the photomicrography in this paper;
and Mr. Frank Hamons and Mr. Frank Nelson, F.A.,
State of Maryland, for the collection of the samples.

LITERATURE CITED

Coe, W. R. 1943. Sexual differentiation in mollusks.

I. Pelecypods. Quart. Rev. Biol. 18: 154-164.
Loosanoff, V. L. 1936. Sexual phases in the quohog.

Science. 83: 287-288.
Otto, S. V. 1972. Hermaphroditism in the soft clam

(Mya arenaria). Proc. Nat. Shellfish Assoc. 62:

47-49.

Proceedings of the National Sfiellfisheries Association
Volume 63 - June 1973

ABUNDANCE OF THE LOW SALINITY CLAM, RANGIA CUNEATA
IN SOUTHWESTERN LOUISIANA

H. Dickson Hoese

DEPARTMENT OF BIOLOGY

UNIVERSITY OF SOUTHWESTERN LOUISIANA

LAFAYETTE, LOUISIANA

ABSTRACT

The low salinity clam Rangia cuneata, was found to be very common in oli-
gohaline waters of southwestern Louisiana, discontinuously distributed across a band
over 100 miles long and 10 miles wide, occurring in tidal creeks, lakes and bays
from the shoreline to at least 4 m in depth. It was replaced in the intertidal zone
by Polymesoda caroliniana, in saltier, deeper waters by Tagelus plebius and Macoma
mitchelli, and in fresh water by unionids. It was inexplicably absent or rare in many
areas, showing no correlation with total sediment carbon, except for being very rare
in very highly organic sediments rich in plant detritus.

Populations were usually composed of uniformly sized animals ranging from
means of 28 mm in Grand Lake to 57 mm in one tidal creek in Vermilion Bay.
Large populations of juveniles were rare although recently metamorphosed juveniles
were sometimes taken. It is estimated that southwestern Louisiana has a minimum
standing crop of between 24 and 48 billion clams based in part on an average of
11.1 clams/m^ found over the whole study area.

INTRODUCTION

Although perhaps as common in their habitat as
oysters are in their's, the moderate sized Louisiana
road clam or rangia, Rangia cuneata Gray, long
utilized by prehistoric man for food (Mclntire,
1958), has received little interest until very recent-
ly. Indian mounds composed largely of rangia
provide part of the basis of an extensive mudshell
industry, which in 1966-67 (2 years) removed
nearly 9V2 million cubic yards of shell. Louisiana is
the only state with large enough fossil populations
to support such an industry, cdthough rangia is
now being considered in much of its range as a
possible source of food. However, suspected slow
growth rates (Fairbanks, 1963; Wolfe and Pet-
teway, 1968; Gooch, 1971' ) may render this clam
less amenable to harvest than oysters, which reach
market size very rapidly in Louisiana (Hopkins,

'Gooch, D. M. A study of Rangia cuneata Gray in
Vermilion Bay, Louisiana. M. S. thesis, USL: 50
pp.

Mackin and Menzel, 1953).

Nevertheless, southwestern Louisiana probably
contains more R. cuneata than any other compar-
able area of the world, except perhaps Lake
Ponchartrain, and the animal is undoubtedly of
enormous significance to the ecology of the area.
To this end, this study was devoted to deter-
mining the distribution and abundance of R.
cuneata and associated moUusks from about the
Atchafalaya River mouth to near but not including
Sabine Lake (Fig. 1).

METHODS

Qams were collected in deep water with an
angle iron frame dredge 85 x 20 x 93 cm long,
pulled behind either a 40 ft or 18 ft boat at
about 3 kn for 3 min at each station. The bag
was constructed out of 1 in stretched mesh which
retained clams as small as 25 mm, with a few
down to 15 mm. Shallow waters (less than 2 m)
were sampled with two random square meter
frames throvra from a small boat. Clams were then
removed from the quadrat by diving. Juvenile

100

H.D. HOESE

ATCHAFALArA
RIVER

FIG. 1. Map of study area showing estimated concentrations of clams. 1=0 clams, 2 = less than
lO/rn^, 3 - over 10/m^. For more details, see Figure 2. White areas not sampled.

clams were collected with a 2 or 5 m long
cylinder of fiberglass or plexiglass with diameters
of 56 or 63 mm. Two cores were taken at each
station, sieved and examined for small mollusks,
but large amounts of plant fiber at some stations
undoubtedly obscured some of the clams. At each
station salinity was measured by a Beckman RS5-3
conductivity meter, ph by meter or Hach color
kit, oxygen with YSI model 54 m and tempera-
ture by thermistor.

Ninety-three shallow water stations were spaced
three nautical miles apart around major water
bodies, with some sampling elsewhere. Thirty-nine
deep water stations were laid out in a grid sep-
arated by three nautical miles. Some areas could
not be sampled due to shallow water and other
problems.

Sediment samples were collected along with
juvenile clams. Organic matter was measured by
loss on ignition and is expressed in percent total
carbon, including a small amount of carbonate car-
bon.

DESCRIPTION OF AREA

The area of study includes a very old reworked
delta of the Mississippi, now known as the chenier
plain region (Russell and Howe, 1935; Van Lopik,
1955). Cheniers are low, sandy intrusions above an
otherwise flat marshland composed of several
species of fresh and brackish water plants, with
true salt marsh plants rare (O'Neil, 1949;
Chabreck, 1970^). Degradation of these plants
with other allochthonous sources results in high
concentrations of plant detritus or peat mixed in
with clays and silts. In addition mud is being

added continually from the rivers and is reworked
with the detritus (Coleman, 1966).

From Sabine Lake to Vermilion Bay (Fig. 1)
the marsh is nearly continuous except for numer-
ous tidal creeks and ponds and the estuaries of
two rivers, the Calcasieu and the Mermentau.
Grand Lake, associated with the latter and White
Lake, with no apparent river system, are oblong
ovate "lakes" roughly parallel with the shoreline.
These lakes are isolated on all three sides from
salt water by control structures completed in
1951. From Vermilion Bay to the Atchafalaya
River mouth there is a system of shallow bays
(2-3 m) separated from the Gulf of Mexico by
marsh on the western end (Marsh Island) and dead
oyster reefs on the eastern end (Point au Fer).
These reefs have been killed by the increasing flow
of the Atchafalaya, which has been capturing
much of the Mississippi River flow (Gunter, 1952;
Thompson, 1955) and now is building its own del-
ta in Atchafalaya Bay (Shlemon, 1971).

HYDROGRAPHY

Except for Vermilion Bay there is relatively
little hydrographic data on the area, although the
mouth of the Atchafalaya River has attracted
some interest due to the increased flow. Salinities
there have been very low, usually within the range
of fresh water through Atchafalaya Bay into West
Cote Blanche Bay. Salinities increased to an aver-

Xhabreck, R. H. 1970. Marsh zones and
vegetative types in the Louisiana coastal marshes.
Ph.D dissertation, LSU. 113 pp.

ABUNDANCE OF RANGIA CUNEATA

101

VERMILION BAY
WEST COT_E BLANCHE BAY

10
N A MIL eS

FIG. 2. Area of highest concentrations. Shoreline concentrations in nos./m^. 1 = less than 1. 2 = 1 -10. 3
= more than 10. Offshore are lines of equal density. Numbers are clams caught per 3 minute dredge haul.
(To estimate numbers/ m^ divide by 6 )

age of 3.7 %„ in Vermilion Bay. Although! this is
about the same as reported by Dugas (1970)^ for
1969, it is 2-3 %o lower than that observed in
1963-64 (Fontenot, 1967").

Westward through the marsh salinities decrease
to near fresh water in Grand and White Lakes.
Data given by Gunter and Shell (1958) showed
similar salinities for this area although they noted
some as high as 2.7 %<, . Calcasieu Lake has been
reported to be somewhat saltier (Kellogg, 1905)
and the highest salinities (15.5-26.0 %» ) in the
study were found there. Probably the Lake Charles
Ship Channel has caused an increeise in the average
salinity of the Lake.

DISTRIBUTION AND ABUNDANCE
Rangia was not continuously distributed across

^ Dugas, R. J. 1970. An ecological study of
Vermilion Bay. 1968-69. M. S. thesis, USL:107
pp.

"Fontenot, B. J. 1967. Seasonal relative abundance
and distribution of postlarval white and brown
shrimp in Vermilion and Cote Blanche Bay. M.S.
thesis, USL: 77 pp.

southwestern Louisiana. It was absent in much of
the shallow water of Atchafalaya Bay, at Terrapin
Reef between Vermilion and West Cote Blanche
Bays, White Lake, Calcasieu Lake and most of the
northern marsh area between Calcasieu and Sabine
Lakes. Its center of abundance lies in western Ver-
milion Bay (the area studied by Gooch, 1971'),
central and eastern West Cote Blanche Bay, with
lesser concentrations in parts of Grand Lake, cen-
tral East Cote Blanche Bay and western Atcha-
falaya Bay (Fig. 2, Tables 1 and 2). In Vermilion
Bay clams appeared equally abundant along the
shoreline and in deep water. However, in West and
East Cote Blanche Bays clams were scarce along
much of the shoreline while reaching high densities
in deeper water.

The highest density of clams found in shallow
water in a single sample was 238/m^ in Vermilion
Bay. Doubtlessly higher densities could be found
by further searching since Gooch (1971)' reported
concentrations up to 756/m^. Nevertheless, our
data indicates an average concentration in shallow
water of 11.1/m^ with highest numbers in Ver-
milion Bay to none found in White and Calcasieu
Lakes (Table 1). In core samples covering 1.5 m^,
an average of 14/m^ was taken for clams over 10

102

H.D. HOESE

TABLE 1. Average numbers/m'
several Louisiana bays.

at shallow water stations in

Highest

No.

Avg.

concentration Stations Abundance

Atcliafalaya Bay

6.1

East Cote Blanciie

Bay

7.0

West Cote Blanche Bay

130

8.5

Vermilion Bay

238

26.6

White Lake

Grand Lake

116

16.9

Calcasieu Lake

Miscellaneous

11.8

Total (all stations)

11.1

Lowest sample in all bays was 0.

mm while for clams under 10 mm the rate was
28/m^. The number of clams taken by core in
shallow and deep water were exactly the same
(0.08/core). While this does not constitute proof
that deep and shallow water samples are compar-
able there are no data refuting this hypothesis.
Various estimates of abundance based on our data,
based on weights given by Hopkins (1970) and
based on the acreages given by Chabreck (1971)'^
and Ferret, et al. (1971) are shown on Table 3.

Accepting the slow growth rate of rangia as sug-
gested by previous workers (Fairbanks, 1963;
Wolfe and Petteway, 1968; Gooch, 1971'), it

^Chabreck, R. H. 1971. Ponds and lakes of the
Louisiana coastal marshes and their value to fish
and wildlife. 25th Ann. Conf. S. E. Assoc. Game
and Rsh Comm. (mimeo. 19 pp.).

might be prudent to harvest no more than 5% of
the population annually until more information is
gathered about the actual deep water concentra-
tions, the effect of harvesting, recruitment, pos-
sible culture methods and the importance of the
clam to the ecology of the bays. This should still
give a potential annual harvest of about 2 billion
clams, at a wet meat weight of 22 million pounds
(45.5 million kg.).

Regardless of the precise figure, rangia popula-
tions between Sabine Lake and Atchafalaya Bay
must number in the tens of billions, with total
weights in the billions of pounds (85% is shell
weight). Based on our recommendations a few
billion rangia could be harvested each year. How-
ever, current harvest is about 8-9 billion pounds of
shell a year, which exceeds the replacement
amount by a factor greater than 18, assuming the
whole Louisiana coast is producing the same
amount of rangia as the western part.

TABLE 2. Average numbers/3 min haul at deep water stations by
dredge.

Highest No.

concentration Lowest Stations Avg.

Atchafalaya

233

East Cote Blanche

352

West Cote Blanche

1458

Vermilion

273

White Lake

Grand Lake

190

Total (all stations)

143.7

83.4

37.8

53.5

95.0

60.0

ABUNDANCE OF RANGIA CUNEATA

103

TABLE 3. Total amounts of Rangia cuneata in southwest Louisiana study area
based on various means of estimation.

Total Study Area (Chabreck, 1971^)

Total from

Shallow water avg.

(11.1/m^)

Tube samples (14.0/m^)

Number
(in millions)

38,457
48,504

Shell Weight Wet Meat Weight
(lbs. in millions) (lbs. in millions)

1,864
2,350

390

491

By Bay System (Perret et al., 1971)

Bay

Number

(in millions)

Atchafalaya

3,325

East Cote Blanche

2,332

West Cote Blanche

3,092

Vermilion

13,091

Grand

2,170

Total of above

24,011

Total based on total

acreage

32,332

Most of the clams lie in the area from Ver-
milion to western Atchafalaya Bay (Fig. 2). Within
this area, which covers about 330 thousand acres
(133.5 thousand hectares), there is an estimated
standing crop of 23 billion clams.

Several workers have suggested that rangia abun-
dance might be correlated with sediment type or
amount of organic matter. Figure 3 shows organic
matter concentrations versus rangia abundance. Ex-
cept for the scarcity of rangia in very highly or-
ganic sediments (over 10%) there seem to be no
correlations. These highly organic sediments are
predominately broken down plant detritus. In
these areas rangia may have difficulty in becoming
stabilized. Rangia does occur in sediments high in
plant detritus where small pockets of detritus col-
lect in swales in hard packed clay. In this clay the
clam burrows with difficulty. Therefore, the cor-
relation may only represent problems of maintain-
ing stability where loose plant detritus exceeds the
normal burying depth o'f the clam.

SIZE

The majority of clams collected exceeded 34
mm. At only 19 of 55 shallow water stations were
smaller clams found and at 6 of these no clam
was under 30 mm. Only at Grand Lake was there
an abundance of small clams, over 10/m^ (Fig. 4),

and the majority of these were between 20 and
30 mm. However, numerous clams over 35 mm
were also present at some stations in the Lake and
at one station in the northeastern part they
averaged 48 mm, or about as large as that found
anywhere in the study area.

At deep stations the majority of clams were
within the 30-42 mm range although some smaller
clams were often found. The mean size for clams
at the deep water stations ranged from 30-52 mm
which was closely comparable to those found in
shallow water. The majority of clams over 50 mm
were taken in numbers under 10/m^, the only ex-
ceptions being at one station in Grand Lake and
two in Atchafalaya Bay. The largest clam taken
was in a tidal creek off Vermilion Bay; it mea-
sured 75 mm. Gooch (1971)' reported a record
86 mm clam from the area and the average size of
some populations was over 75 mm. While large
rangia seem most common in tidal creeks where
the water remains practically fresh, there is no
obvious correlation of size with environmental
factors.

An example of the most common length-fre-
quencies are shown in Figure 4. Means of rangia
populations in excess of 10/m^ (outside of Grand
Lake) ranged from 38-52 mm. Only 14% of the
clams were over 48 mm and 40% of them were

104

H.D. HOESE

100

•

% TOTAL CARBON-

10'

20"

FIG. 3. Comparison of number of clams/m^
shallow water with amount of sediment carbon.

between 40 and 44 mm. In contrast, at Grand
Lake only 2 populations were above 37 mm (both
at 48 mm) and 84% of the clams were between
22 and 31 mm.

Samples containing juveniles below 10 mm were
rare. Collections were made at all times of the
year, and occasionally coincided with the time
that veligers were metamorphosing. For example,
collections made between 24 March and 21 April
1970 in Vermilion and West Cote Blanche Bays
coincide with the t'me of setting previously
reported by Fairbanks (1963). A total of 27 small
juveniles was taken, mostly from areas where less
than 5 adults were taken per drag. While this may
suggest that rangia larvae only settle in areas
where clams are scarce, large populations of small
clams are often found very close to, although not
intermixed, with adults.

One accidental capture of very small clams may
provide some insight into settlement of larvae. A
large uncounted group of young less than 1 mm

GRAND

LAKE

WEST COTE BLA^^CHE
BAY

•^

:;:::;:;:;:;;;;;U

L^:;;;;;;;:;;:;;;x;::

--.•\

30 35

LENGTH

40 45

FIG. 4. Comparison of size distribution (in 2 mm
intervals) of rangia from one station on north
shore of Grand Lake and one on north shore of
West Cote Blanche Bay.

in length was accidently snagged with a small
hydroid colony caught on the end of the oxygen
probe in West Cote Blanche Bay. The clams had
apparently clamped onto the colony by the shell
margins.

ASSOCIATED SPECIES

Rangia apparently has no infaunal competitors
in southwestern Louisiana estuaries. Occasionally
we found the marsh clam, Polymesoda caroliniana,
the small low salinity tellinid, Macoma mitchelli
and unidentified unionids among the rangia pop-
ulations.

P. caroliniana lives in the intertidal zone buried
in mud in Spartina patens-Sagittaria lancifolia type
marshes and sometimes reaches fair abundance'
there. Young clams (1-4 mm) were also found in-
tertidally on Mud Point above mean sea level. Live
P. caroliniana were common in intertidal burrows
and loose clams were found scattered all the way
to adjacent subtidal areas, where they are un-
doubtedly inadvertently transported. In an adjacent
tidal creek rangia were abundant; however, none
were found above mean low water. It appears,
therefore, that these two species do not mix.
Harry (1942) reported P. caroliniana among roots
of marsh grasses in Barataria Bay, Louisiana, and
Andrews and Cook (1951) describe their range and
habitat in Virginia.

Macoma mitchelli was found only at the saltier
and deep water stations in southern Vermilion
Bay close to southwest Pass. Here they barely
overlap rangia populations in the western part of
the bay.

ABUNDANCE OF RANGIA CUNEATA

105

Closely associated forms seem largely limited to
the two tiny gastropods, Littordina sphinctostoma
and Vioscalba louisianae (Gooch, 1971)^ These
two species live among the rangia, but their mode
of life is unknown.

Oysters, Crassostrea virginica, and hooked mus-
sels, Brachidontes recurvus, occur predominately
seaward of rangia, although both occasionally set
and survive for a short period of time in areas
where rangia are found. Other than these animals
and several demersal fishes and crustaceans
(Norden, 1966; Ferret, 1967) only two other mol-
lusks were found. The gastropod Nereitina re-
clivata, is common in the lower intertidal zone
and on stfme of the higher oyster reefs. They feed
on green and blue-green algae, and occasionally
overlap with rangia. The mussel. Modiolus de-
missus, occurs rarely in the marshes; only two
records are knovm. Other than a rare chironomid
larva or polychaete, there was no other macro-
scopic animal associated infaunally with rangia.

Only R. cuneata (Gray) was found; living speci-
mens of R. flexuosa (Conrad) seem to be very
rare, and have been reported in Louisiana by
Harry (1942), Behre (1950), and Gooch (1971)\
Although recent R. flexuosa seem rare, many
shells were found in old assemblages.

DISCUSSION

The distribution of rangia in Louisiana clearly
follows the lower salinity waters that range from
0.5 - 9.0 %o . This zone is perhaps best called oli-
gohaline, although the term does not fit the salin-
ity limits given by other authors. However, rangia
clearly occupies this lower zone where there is
some salt water intrusions. Other infaunal pelecy-
pods, both fresh-water and marine, are absent. The
absence of rangia along the eastern Atchafalaya
Bay shoreline may be explained by the possible
lack of salt water intrusion. This came about in
the past two decades with increasing river flow.
However, we were unable to sample the open
waters of the Bay because of recent shoaling in
the central and eastern part (Shlemon, 1971). It is
possible that rangia would be in these waters.

The same may be true of White Lake which,
like Grand Lake, has been isolated since 1951 by
control structures to prevent seasonal salt water in-
trusion. Rangia were abundant in White Lake in
1952 (Gunter and Shell, 1958), but very few were
taken there by Gooch. (1970) as late as 1969.
Howe, Russel and McGuirt (1935) reported that in
1934 Grand Lake was too saline for rangia. Today
Grand Lake has considerable numbers of rangia in-

cluding populations of small individuals (below
30mm). These must have set after the control
structure was built. Penaeid shrimp are still found
in White Lake indicating that there may be some
salt water intrusion, especially since the opening of
Freshwater Bayou to the Gulf of Mexico. How-
ever, no rangia were found in this lake.

The lack of rangia in Calcasieu Lake can be
explained by the higher salinities, probably in-
creased by the ship channel. Instead, Tagelus
plebius is a common infaunal moUusk. Large
numbers of recent rangia shells on the bay bottom
attest to its presence within historic times. Rangia
was reported to be extremely common in upper
Calcasieu Lake by Kellogg (1905). We failed to
find any although no samples were taken in the
center of the bay, in the river above the bay or in
Lake Charles.

Although rangia does not penetrate the mod-
erate salinity area of estuaries, it is not clear what
factors are limiting. It tolerates moderate salinities
(O'Heeron, 1966)* and occurs in small numbers off
Marsh Island in the Gulf of Mexico where salin-
ities often reach 20 oo. O'Heeron (1966)'' sug-
gested predation by Thais, but this is an epifaunal
feeder. Two drilled rangia were found in lower
Vermilion Bay, but based on the bevel of the hole
they were apparently drilled by Polynices. Poly-
nices is a common predator of infaunal pelecy-
pods. Other possible predators discussed by Gooch
(1971)' do not seem to be segregated by salinity.
There is no evidence of competition with other
pelecypods at the seaward edge or no changes in
bottom types which might explain the lack of ran-
gia.

One of the most intriguing findings made
during this study was the uniform size of popula-
tions and the apparent slow growth (Gooch,
1971)'. Another interesting observation was the
lack of clams, young or old, in many apparently
suitable areas suggesting that recruitment is rare.
One explanation is the possible need for degrading
plant detritus on which rangia might first attach
to before burying into the sediments. The lack of
plant detritus or other suitable materials at time
of setting may contribute to setting failure.
However, other hypotheses need to be investigated
and studies on spawning, larval abundance, settle-
ment and recruitment should be done.

The great abundance of rangia in southwestern

^O'Heeron, M. K., Jr. 1966. Some ecological
aspects of the distribution of Rangia cuneata
Gray. M.S. thesis, Texas A & M Univ. 55 pp.

106

H.D. HOESE

Louisiana is undoubtedly related to the great
width of the upper part of the estuarine zone. Its
width in the Vermilion-Cote Blanche area is about
12 miles and the length exceeds 100 miles. Rangia
appears well adapted to the very organic, turbid
waters and reduced sediments that typify this area.
The importance of the clam to the area seems to
be as follows:

(1) Important converter of detritus to animal
matter and reservoir for many nutrients, especially
CaCOg-,

(2) Fills a niche in a habitat (infaunal, oligo-
haline) that no other similar animal tolerates;

(3) Provides shell for storm built marsh
beaches;

(4) Provides a hard substrate in bay bottoms
for attachment of epifaunal species; and

(5) Probably has many unknown effects on sed-
imentation and survival of burrowing species of
other groups.

Rangia have commercial applications both
potential and realized. While some of the more
obvious applications, such as mudshell, receive the
most attention, some unstudied aspects may be
more important. Rangia maintains a productive,
stable area, which produces one of the largest
commercial catches of other animals in the world.
Hopefully future utilization of this clam will be
considered over a long term view since the data
gathered in our studies suggests that rangia may be
very susceptible to rapid depletion.

ACKNOWLEDGMENTS

The start of this study was financed with Sea
Grant funds through Louisiana State University.
Much of the field work was conducted by Edward
Morgan, Carolyn Stone, and Claude Boudreaux.
Mr. Tom Huggins anedyzed the sediment samples.
Mr. Donald Gooch aided in design of the project
and many students, especially Joyce Teerling and
Harry Blanchet, contributed free labor. Mr. Jacob
Valentine arranged a trip over the Sabine Wildlife
Refuge.

LITERATURE CITED

Andrews, J. D. and C. Cook. 1951. Range and

habitat of the clam, Polymesoda caroliniana

(Bosc) in Virginia (Family Cycladidae) Ecology.

32: 758-760.
Behre, E. H. 1950. Annotated list of the fauna of

the Grand Isle region, 1928-1946, Occ. Pap.

Mar. Lab. LSU 6: 1-66.
Coleman, J. M. 1966. Recent coastal sedimentation

of the central Louisiana coast. LSU Coastal

Stud. Ser. 17: 1-71.

Fairbanks, L. D. 1963. Biodemographic studies of
the clam Rangia cuneata. Tulane Stud. Zool.
10: 3-47.

Gooch, D. M. 1970. An inventory of Rangia cune-
ata in White and Grand Lakes, Louisiana. Rep.
to La. Shell Producers Assoc. Part II, p. 11-17.

Gunter, G. 1952. Historical changes in the Missis-
sippi River and the adjacent marine environ-
ment. Publ. Inst. Mar. Sci. Univ. Tex. 2 (2):
121-139.

Gunter, G. and W. E. Shell, Jr. 1958. A study of
an estuarine area with water-level control in the
Louisiana marsh. Proc. La. Acad. Sci. 21: 5-34.

Harry, H. W. 1942. List of MoUusca of Grand Isle,
Louisiana, recorded from the Louisiana State
University Marine Laboratory, 1929-1941. Occ.
Pap. Mar. Lab. LSU 1: 1-13.

Hopkins, S. H. 1970. Studies on brackish water
clams of the genus Rangia in Texas. Proc. Natl.
Shellfish Assoc. 60: 5-6. (Abstract).

Hopkins, S. H., J. G. Mackin and R. W. Menzel.
1954. The annual cycle of reproduction,
growth, and fattening in Louisiana oysters.
Conv. Add. Natl. Shellfish Assoc. 1953: 39-50.

Howe, H. v., R. J. Russell and J. H. McGuirt.
1935. Physiography of coastal southwest
Louisiana. La. Geol. Bull. 6: 1-72.

Kellogg, J. L. 1905. Notes on marine food mol-
lusks of Louisiana. Bull. Gulf Biol. Stn. 3:
69-79.

Mclntire, W. G. 1958. Prehistoric Indian settle-
ments of the changing Mississippi River delta.
LSU Coastal Stud. Ser. 1: 1-128.

O'Neil, T. 1949. The muskrat in the Louisiana
coastal marshes. La. Wildl. Fish. Comm., New
Orieans, La. 159 p.

Perret, W. S., et al. 1971. Cooperative Gulf of
Mexico Estuarine Inventory and Study,
Louisiana. Phase I, Area Description. La. Wildl.
Fish. Comm, New Orieans, La. p. 5-27.

Russell, R. J. and H. V. Howe. 1935. Cheniers of
southwestern Louisiana. Geogr. Rev. 25:
449-461.

Shlemon, R. J. 1971. Hydrologic and geologic
studies of coastal Louisiana. LSU Center for
Wetland Resources. Rep. to Corps of Engineers.,
55 p.

Thompson, W. C. 1955. Sandless coastal terrain of
the Atchafalaya Bay Area, Louisiana. In Finding
Ancient Shorelines. Soc. Econ. Paleon. Min.
Spec. Publ. 3: 52-77.

Van Lopik, J. R. 1955. Recent geologic and geo-
morphic history of central coastal Louisiana.
LSU Coastal Stud. Inst. Tech. Rep. 7: 1-89.

Proceedings of the National Shetlfisheries Association
Volume 63 - June 1973

PATTERN OF DISTRIBUTION OF THE SURF CLAM

(SPISILA SOLIDISSIMA) IN THE POINT JUDITH,

RHODE ISLAND HARBOR OF REFUGE'

John M. Flowers

MARINE EXPERIMENT STATION

UNIVERSITY OF RHODE ISLAND

KINGSTON, RHODE ISLAND

ABSTRACT

The object of this study was to determine the nature of the distribution of
patches of the surf clam, Spisula solidissima solidissima. Two areas were selected in-
side the breakwater at Point Judith, Rhode Island. A statistical method designed for
plant populations was utilized and a method of solving for the unknown parameters
was developed. The collection of data was carried out by scuba divers. From the
analysis and observations it was concluded that the pattern of patches of Spisula
were density dependent, with high density areas tending toward complete aggregation
while medium and low density areas consisted of randomly distributed discrete
patches.

INTRODUCTION

Surveys of commercially important shellfish are
made frequently to determine numerical abundance
for management purposes. Most survey methods in-
volve collecting individuals within a specified
quadrat cast in a statistically valid manner. From
the number collected, inferences are made as to
the distribution of individuals and their numerical
abundance. All of these sampling methods are car-
ried out from the surface using mechanical
sampling devices. Hard clams exhibit various de-
grees of contagion. That is, they are not dis-
tributed randomly on the bottom but are in
patches. Previous work by Saila, Flowers and
Campbell (1966) indicated that the quahog,
Mercenaria mercenaria is contagiously distributed
and further work by Saila and Gaucher (1965)
confirmed this contagious nature for other marine
pelecypods. Little has been done to determine the
nature of the distribution of these patches of hard
clams. Surface sampling techniques are inadequate
for this determination in that they do not yield

' This work is a result of research sponsored by
NOAA, Office of Sea Grant, Department of
Commerce, under Grant #2-35190.

the required precision. In recent years underwater
photography has been used in studying benthic
organisms. This method is effective on an organism
which is readily identifiable from a photograph.
For the hard clam this method was found to be
ineffective in that the only visible indication is the
presence of a siphon hole. Identification of the
clam is difficult even with a practiced eye. Vari-
able environmental conditions confound the prob-
lem by controlling the clams' condition. Under the
proper conditions the clams pumped and the
siphon holes were visible. At other times the clams
were not pumping and the siphon holes were not
visible.

As a first attempt at evaluating the nature of
the patch and gap patterns of pelecypod moUusks,
the surf clam, Spisula solidissima solidissima was
selected. Spisula was chosen for its large physical
size and ease in identification. The sampling was
conducted by scuba divers using hand rakes in a
depth of water varying from 18-25 ft. Identifi-
cation of the clam in hand is an important factor
in this type of sampling.

Two areas inside the breakwater at Point
Judith, Rhode Island were selected. This location
was selected for several reasons: a) virtually no
commercial fishing of the surf clam has been done

107

108

J.M. FLOWERS

in the areas selected for the past 8-10 years; b)
the density, as well as the physical size of the surf
clams in the areas chosen differed; and c) minimal
distrubance of the natural distribution of the surf
clam is afforded by the wave force reduction of
the Point Judith breakwater.

METHODS
Statistical Methods

Before sampling was begun the degree of ag-
gregation or patchiness of the Spisula was empiric-
ally determined. Several small areas (20 x 20 ft)
were completely searched for Spisula. Each clam
was extricated from the bottom and placed by its
respective hole. By rising 6-10 ft off the bottom,
patch shapes and dimensions were clearly visible.
The shapes of the patches varied. Some formed
clusters where each individual was separated from
another by a nearest neighbor distance. Others
were in single file forming irregular curved pat-
terns. Two instances where a patch consisted of a
circle with the individual clams making up the
perimeter of the circle were observed. Although
the area within the circles was much greater than
the nearest neighbor distances, this area could not
be regarded as belonging to a gap. Maximum dis-
tances across patches were measured and visual
notes on patch distribution were taken. From this
empirical data a rough estimate of the mean patch
radius r was established.

The method of sampling was one adapted from
Pielou (1964) who used paired circular quadrats
for estimating the patch and gap patterns of vege-
tatively reproducing plants. Pielou's basic methods
and assumptions have been included as an explana-
tion of the technique. On the basis of the
empirical data gathered the patches and gaps were
defined as follows: any point on the bottom at a
distance greater than r from the nearest clam is to
be regarded as belonging to a gap; and any point
whose distance from the nearest clam is not great-
er than r is in a patch.

Although the density of clams in the two areas
sampled varied, the mean patch sizes in each area
was approximately the same. Hence, r for both
areas was set at 20 in. since the mean diameter of
patches was approximately 40 in.

If paired circular quadrats, each of radius, r are
set dowTi in an area, where d > 2r is the distance
between centers we have a possibility of four
events, HH, HM, MH and MM. H denotes a hit or
the presence of a clam within a circle and M
denotes a miss or the absence of a clam within a
circle. The events HM and MH are to denote the
order of occurrence which will be dropped. When

a quadrat pair is set down in an area it may be
thought of as constituting two observations of a
two state discrete Markov Process. The states are a
hit (state 1) and a miss (state 2). If the circles are
tangent (d = 2r) a single transition is assumed to
have occurred. When d = 4r two transitions have
occurred and so on.

The matrix of transition probabilities may be
wrritten as:

Second Quadrat

First
Quadrat

P. 2

= 1 and p^

(1)

1. Here

where p -^ p . ^

*^l 1 '^l 2 "^2 1 "^2 2

p is the probability that the second quadrat
scores a hit given that the first one did, and the
other three probabilities are similarly defined. To
determine the four possible events HH, HM, MH,
MM, it is necessary to know the probability a,
that the first quadrat scores a hit, and the proba-
bility (b = 1-a) that it scores a miss. The vector
(a, b) is the limiting probability vector of the
Markov chain with transition matrix P. Thus a and
b may be expressed in terms of the transition
probabilities Pjj by solving the matrix equation
(a,b) P = (a,b). Whence

a = Pj,/(P,2 + Pjj) and b = P^^lip^^ + P2i)(2)

The probability that both quadrats score hits is then

Pr(HH), =ap,, =p,j p^,/(p,^ +p^^) (3)

where the suffix 1 in the first member denotes the

single transition pair (d = 2r) is being used. Also

Pr(MM), =bp^^=p,^p^^/(p,^+p^,) (4)

and

Pr(HM)j = Pr(HM)j + pr(MH)j = ap^ ,

Pr(HM), =2p.,p^,/(p.^.p^,)

The absence of an arrow from the first member of the

above equation signifies that the order in which the

two quadrats are observed is disregarded.

When a pair of quadrats of length d = 2nr is used
n transitions are assumed to have occurred between
each quadrat of the pair. The n step transition prob-
abilities are given by the elements of P". Putting 1 -

P. 2

+ bp

I 2 ^2 1

(5)
(6)

p = k, we have

(n)

P2,

^k\

I P,2('-k")

Pi.

Pi 2

P. 2

P2,

P21

d-k")

Pl^^k^p^

(n)
P2.

(n)
P22
(7)

PATCH AND GAP PATTERN OF MOLLUSKS

109

Then , , n v

Pr(HH) =ap (") = P2i(P2i ^ '^ P12' (8)

(p +p n

^*^12 ^21'

Pr(MM)„ = bp^ ^ ("> = P,,(P,,+kyj (9)

(P,2^P2,)'
Pr(HM)„ - ap, , (n) + bp^ , '"^ = 2p^ ^p, , (1-k") (10)

(P + P )"
Once the two parameters, p^ ^ and p, ^ , are known it
is possible to calculate the probabilities of the events,
HH, HM and MM for any quadrat pair whose length
is an integral multiple of the shortest pair.

Estimation of f>^^ and p^ ^

Using the method of maximum likelihood, Pielou
developed a set of equations for the estimation of
p and p . To solve these equations for P, 2 and
p a computer program was written in Fortran IV
using Newton's method of solving systems of
non-linear equations (McCalla, 1967).
Let

9 'ogL (11)

(Pl2-Pl2,0'*f'2l(Pl2,O'P2.,o) (P2.-P21,o)

+ . . .

glPjjiPjj) SvPj 2 ^o'Pj 1 ,0 ^ 1 2^Pl 2,0'P2 1 ,0'

(p -p ) + e' (P ,P ) (P -P ) + .

^'^l 2 *^1 2 ,0 ' ^21 '*^1 2 ,0 '^2 1 ,0 ' ^*^2 1 ^2 1 ,0 '

(17)

(18)

f(P,2.P2,)
g(g, 2'P2 1^

9P.2
: 9 log L

then

9P.

*^l 2

g' =9g(P,2,P2,) = 9^ logL

2 1 — :;^ "^ 2~

dp 9 P

^^2 1 '^2 1

(12)

(13)

(14)

and
f',.=g',

9^ logL
9 P Pp

^12 *^2 1

9^f(P.2-P,.)^9^g(P..-P.,)- (15)

9P2l9P.2 3P.2^P2.

as first approximations of p^ ^ and p^ j let
Pi2'o=.!^ andP2,'o=^!^ (16)

(2n +n J

' 1 2 '

(2n3+n^)

These are the estimates obtained by equating to
expectation the observed frequencies using the
shortest quadrat pair (d = 2r).

Expanding f and g in a Taylor series about

<P.2,0'P2.,0»^^^'^^^
f(P,2.P2,) = f(Pl2,0'P21,o)^f'.2<P,2,0'P21,o)

We want to find (Pi2'P2i) ^"*='^ ^^^^ ^(Pi2'P2i^ "
g^Pi 2 ,P2 1 * = 0. Using only linear terms of the above
series expansion and setting them to zero will give us
an approximate solution.

LetAp^, = (p^^-p,2,o)andAp2,=(Ap2,-P2,,o)

(19)

and

0=f<P.2,0'P2,,o)^f'.2(P.2,O'P2.,o)^P>2^
f'2,(P.2,0'P2>,o)^P2, (20)

0 = g(P.2,O'P21,o'^g'l2(Pl2,O'P2.,o)^P.2 ^

g' (p ,p )Ap (21)

^21^^12,O'*^2 1,0' *^2 1 '

The solution to this system of linear equations, using
Cramer's rule is found to be:

Ap ^ =_g 2 1 ^^21

12^21 ' 2 1 " 1 2

.2 ±^I,ll^i-^(P.2,0'P2.,0)
t.-g-. -I., g..

Ap^,= ^g'l2 -gf'l2 (P2..o.P2i,o)

f g -f g

12^21 2 1^12

If P = P . + ^P, , and P, , , =
P + Ap

1^2 1,0 *^2 1

(22)
(23)

(24)

then (Pj ^ J ,P2 J J ) is an approximation of (p^ ^ p^ ^ ).
Using (p ,p ) as the new approximation the
prodedure' was repeated. Four iterations were neces-
sary to obtain six place accuracy.

To obtain the variances of p ^ and ^ ^ letting p^ ^
= P P

'^l 2

Var($,^) =

9^ log L
9 p

'^l 2

(25)

and similarly for Var(p2 , ) (Kendall and Stewart,
1967).

Pielou's assumptions in developing this two state
discrete Markov Process were:

1) The probability of a hit or miss with one
quadrat depends only on the result with the other
quadrat.

110

J.M. FLOWERS

FIG. 1. Harbor of Refuge, Point Judith, Rhode

Island.

Area 1, 18' depth at mean low water

d = 2r area sampled with circles tangent

d = 4r area sampled with circles separated by one

diameter

Area 2, 24' depth at mean low water

d = 2r area sampled with circles tangent

d^ = 4r area sampled with circles separated by one

diameter

2) These probabilities are constant throughout the
area sampled.

If the observed frequencies of hits and misses show
these assumptions to be justified, the pattern may be
regarded as being random.
Sampling Methods

In the first area sampled the depth at mean low
water was approximately 18 ft (Fig. 1) and the bot-
tom was composed of fine sand. Spisula size in this
area ranged from 5.5 - 6.8 in., this measurement being
the maximum diameter across the shell. Nearest
neighbor distances within a patch varied from 10-14
in. Using the radius r established from empirical ob-
servations as a basis, circular quadrats were con-
structed of heavy gauge iron wire each having radius
r. One pair of quadrats was attached tangent to each
other and a second pair separated by one diameter.
Grid patterns 150 x 100 ft were constructed on the
bottom using two foot sections of iron reinforcing
rod and heavy fishing twine. The grid interval was set
at 10 ft, the maximum distance across the largest
quadrat pair. A total of 300 samples were taken, 150

samples were independent of each other. Each grid
was sampled by dropping a quadrat pair at random
within the 10 x 10 ft area and digging within each
circle with hand rakes to determine the presence (H =
Hit) or absence (M = Miss) of a clam.

Complete rather than random sampling was used
to minimize the basic problems encountered by the
scuba divers during the sampling. Visibility was never
more than 20 ft and a great deal of the time was less
than 10 ft. The poor visibility and dulled mental state
of the divers made the problem of random sampling
extremely time consuming. Complete sampling
actually consumed less time than random sampling
would have.

The second area chosen was in a depth of 24 ft at
mean low water. The bottom was made up of a
silt-sand mixture. Spisula in this area varied from
4.5-6 in. in largest diameter. Nearest neighbor dis-
tances varied from 4-10 in. Grid patterns were set up
and the sampling was carried out in the same manner
as in the first area.

RESULTS AND CONCLUSIONS

The following estimates for p and p and their
variances were computed for each area.

Estimates for the first area were calculated such
that

Pj 2 =0.498579 var(P| J = 0.0014182

■p =0.382237

var (p^ I ) = 0.0009293

which gives a matrix of transition probabilities

Second Quadrat
H M

First
Quadrat

0.50142 0.498579

0.382237 0.617763

Estimates for the second area were calculated such
that

2 1

= 0.324245
= 0.652231

var (p, J = 0.0006641

var (Pjj)

0.0021517

giving us a matrix of transition probabilities

Second Quadrat

P =

Using the maximum likelihood estimate

First

0.675755

0.324245

Quadrat

0.652231

0.347769

with d.

2r and 150 with d, = 4r. The individual

^ 1

2 (Fhh + (./.) Fh^)

(26)

PATCH AND GAP PATTERN OF MOLLUSKS

111

TABLE 1. Fit of the theoretical distribution to obser-
vations.

AREA 1
Length of Quadrat Frequency

Pair d = 2ir Event Observed Expected

assumed to be random.

Denoting the martix of transition rates by R where

32.639

64.909

52.452

28.771

72.645

48.584

x^ = 2.07297 < F(x^ .70,2) = 2.41

AREA 2

Length of Quadrat

Pair dj= 2ir

Event

Observed

Expected

67.705

64.973

17.322

66.941

66.502

16.502

x^ = 8.1747 < f(x^

99,2)

= 9.21

where Fuu and Fu«« are the observed frequencies of
the events HH and HM and N is the size of the total
sample, the proportion of the total area a, occupied
by patches may be calculated.

For the first area sampled, again using the com-
puter program developed for this problem

a = .43500
and for the second area

a = .66833
A (Pearson's X^ ) goodness of fit test (Fisz, 1963) was
used to test the validity of assuming randomness for
the patches of surf clams in each area (Table 1).

A good fit of the observed to expected frequencies
was obtained for Area 1. For the second area the
hypothesis of randomness is acceptable at the ijS = .01
level.

Pielou (1964) described a method for determining
the mean patch and gap size for a pattern which is

R =

we have
whence

and
X = "P2.

-K

R = InP

•P,2

P + P

*^1 2 ^2 1

P + P
^12 ^^2 1

^*^1 1 ^2 1 '

(27)

(28)

.ln(P,,-p,,)
(29)

(30)

The length of the intervals along a random line
transect that lie in patches or gaps are exponentially
distributed with mean 1/X or 1/X where the unit is
the length of the shortesl quadr^ pair used in es-
timating the transition probabilities of P.

For the first area a patch size 1/X = 33.222 in.and
a gap size 1/X = 43.334 in. were computed. Computa-
tions for Area 2 yielded a patch size of 1/X = 32.126
in. which is very close to patch size for Area 1 and a
gap size 1/X = 15.971 in. Noticing that the com-
puted mean gap size for the second area is not much
larger than the nearest neighbor distance between in-
dividual clams indicating a tendancy toward complete
aggregation.

From the assumptions and methods used in this
experiment it may be inferred that the distribution of
patches of the surf clam, S. solidissima solidissima, is
density dependent. That is, areas of high density tend
toward complete aggregation and areas of medium to
low density are composed of discrete randomly distri-
buted patches. The observed differences in the size
range of the clams in each area apparently had little
effect on patch size. This implies that the second area
having smaller clams and less distance between near-
est neighbors would have more clams per patch. The
determination of this implication would involve
within patch distributions.

The techniques developed in this experiment
coupled with a dredging technique to assess density in
an area could be used effectively in making popula-
tion assessments. It could also be used in determining
the effectiveness of dredges and dredging techniques
by sampling before and after an area has been
dredged.

112

J.M. FLOWERS

LITERATURE CITED

Fisz, M. 1963. Probability Theory and Mathematical
Statistics. 3rd ed. John Wiley and Sons, N. Y.
Chapter 12.

Kendall, M. G. and A. Stuart. 1967. Advanced
Theory of Statistics. Vol. 2, statistical Inference
and Statistical Relationship. 2nd ed. Hafner Pub-
lishing Co., N. Y. Chapter 18.

McCalla, T. R. 1967, Introduction to Numerical
Methods and FORTRAN Programming. John

Wiley and Sons, N. Y.

Pielou, E. C. 1967. The spatial pattern of two phase
patchworks of vegetation. Biometrics 20: 156-157.

Saila, S. B. and T. A. Gaucher. Estimation of the
sampling distribution and numerical abundance of
some mollusks in a Rhode Island salt pond. Proc.
Natl. Shellfish Assoc. 56: 73-80.

Saila, S. B., J. M. Flowers and R. Campbell. 1966.
Application of sequential sampling to marine re-
source surveys. Ocean Sci. Ocean Eng. 2: 782-802.

ASSOCIATION AFFAIRS

ANNUAL CONVENTION

The 64th annual meeting of the National Shell-
fisheries Association and the Shellfish Institute of
North America was held jointly 25-29 June 1972
at the Williamsburg Logde, Williamsburg, Virginia.

Officers and Executive Committee members
elected for 1972-1973 were:

President R. Winston Menzel

President-Elect Ronald Westley

Vice-President Dexter Haven

Secretary-Treasurer Michael Castagna

Member-at-large Herbert Hidu

Editors of the Proceedings .... William N. Shaw

Sara V. Otto

Mr. Darryl J. Christensen, National Marine
Fisheries Service, Oxford, Maryland 21654, is
custodian of back issues of the Proceedings, and
John Ropes is archivist.

A change in dues from six to eight dollars per
year was passed effective January 1973.

An amendment to the constitution was passed
to include a President-Elect instead of two
Vice-Presidents. It was moved that Vol. 63 be
dedicated to Dr. Imai (deceased) in recognition of
his contribution to shellfish biology. A resolution
was passed in recognition of Mrs. Haynie's work as
Secretary-Treasurer of the organization.

Twenty-nine new members were accepted mak-
ing a total of 349 general members, 7 honorary
members and 3 life members as of May 1st.

The Pacific Coast Section of NSA and the
Pacific Coast Oyster Growers Association met
August 18-19 at the Evergreen Inn, Olympia,
Washington. New officers are:

Chairman , Herb Tegelberg

Vice-Chairman Robert Herrmann

Secretary-Treasurer Gerald Lukas

Section dues were reduced from $2.00 to $1.00
per year.

113

MBI. WHDI I IBRARY

bJH lABX

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