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m-i 



JOURNAL OF SHELLFISH RESEARCH 



VOLUME 17, MUMBER 4 



DECEMBER 1998 




The Journal of Shellfish Research (formerly Proceedings of the 

National Shellfisheries Association) is the official publication 

of the National Shellfisheries Association 

Editor 

Dr. Sandra E. Shumway 

Natural Science Division 

Southampton College. Long Island University 

Southampton, NY 11968 



Dr. Standish K. Allen, Jr. (1998) 
School of Marine Science 
Virginia Institute of Marine Science 
Gloucester Point, VA 23062-1 1346 

Dr. Peter Beninger (1999) 

Laboratoire de Biologic Marine 

Faculte des Sciences 

Universite de Nantes 

BP 92208 

44322 Nantes Cedex 3 

France 

Dr. Andrew Boghen (1999) 
Department of Biology 
University of Moncton 
Moncton, New Brunswick 
Canada ElA 3E9 

Dr. Neil Bourne (1999) 
Fisheries and Oceans 
Pacific Biological Station 
Nanaimo, Briti.sh Columbia 
Canada V9R 5K6 

Dr. Andrew Brand (1999) 
University of Liverpool 
Marine Biological Station 
Port Erin. Isle of Man 

Dr. Eugene Burreson (1999) 
Virginia Institute of Marine Science 
Gloucester Point. Virginia 23062 

Dr. Peter Cook (1998) 
Department of Zoology 
University of Cape Town 
Rondebosch 7700 
Cape Town. South Africa 



EDITORIAL BOARD 

Dr. Simon Cragg (1998) 
Institute of Marine Sciences 
LJniversity of Portsmouth 
Ferry Road 
Portsmouth P04 9LY 
United Kingdom 

Dr. Leroy Creswell (1999) 
Harbor Branch Oceanographic 

Institute 
US Highway 1 North 
Fort Pierce. Florida 34946 

Dr. Lou D'Abramo (1998) 
Mississippi State University 
Dept of Wildlife and Fisheries 
Box 9690 
Mississippi State, Mississippi 39762 

Dr. Ralph Elston (1999) 
Battelle Northwest 
Marine Sciences Laboratory 
439 West Sequim Bay Road 
Sequim. Washington 98382 

Dr. Susan Ford (1998) 

Rutgers University 

Haskin Laboratory for Shellfish 

Research 
P.O. Box 687 
Port Norris. New Jersey 08349 

Dr. Raymond Grizzle (1999) 
Randall Environmental Studies Center 
Taylor University 
Upland, Indiana 46989 

Dr. Robert E. Hillman (1998) 

Battelle Ocean Sciences 

New England Marine Research 

Laboratory 
Duxbury. Massachusetts 02332 



Dr. Mark Luckenbach (1999) 
Virginia Institute of Marine Science 
Wachapreague, Virginia 23480 

Dr. Bruce MacDonald (1997) 
Department of Biology 
University of New Brunswick 
P.O. Box 5050 
Saint John. New Brunswick 
Canada E2L 4L5 

Dr. Roger Mann (1998) 

Virginia Institute of Marine Science 

Gloucester Point. Virginia 23062 

Dr. Islay D. Marsden (1998) 
Department of Zoology 
Canterbury University 
Christchurch. New Zealand 

Dr. Kennedy Paynter (1998) 

1200 Zoology 

Psychology Building 

College Park. Maryland 20742-4415 

Dr. Michael A. Rice (1998) 
Dept. of Fisheries. Animal & 

Veterinary Science 
The University of Rhode Island 
Kingston. Rhode Island 02881 

Dr. Tom Soniat (1998) 
Biology Department 
NichoUs State University 
Thibodaux, Louisiana 70310 

Susan Waddy (1998) 
Biological Station 
St. Andrews. New Brunswick 
Canada, EOG 2XO 

Dr. Gary Wikfors (1998) 

NOAA/NMFS 

Rogers Avenue 

Milford, Connecticut 06460 



Journal of Shellfish Research 

Volume 17, Number 4 
ISSN: 00775711 
December 1998 



Joiirmil of Shellfish Research. Vol. 17, No. 4. 9()?-910. 1998. 

PREDATION OF JUVENILE SEA SCALLOPS (PLACOPECTEN MAGELLANICUS) BY CRABS 
{CANCER IRRORATUS AND HYAS SP.) AND STARFISH {ASTERIAS VULGARIS, LEPTASTERIAS 

POLARIS, AND CROSSASTER PAPPOSUS) 



MADELEINE NADEAU AND GEORGES CLICHE 

Ministere de I'Agriciiltiire. des Pechehes et le I'Alimeiilalloii dii Quebec 

Direction de I'liiiuntition et des Technologies 

Cup-aux-Meules. Quebec. Canada GOB I BO ■ -^ .-^y 

ABSTRACT A scallop fishermen's association has seeded sea scallops, Placopecleii iiianellaniciis. off Iles-de-la-Madeleine (Quebec, 
Canada) since 1993. When the .scallops reach the bottom, they fall prey to starfish and crabs. Two size classes are available during the 
iles-de-la-Madeleine seeding period: small scallops from collectors (15 to 25 mm shell height) and large scallops grown-out in pearl 
nets (35 to 45 mm). To evaluate predation on both classes shortly after seeding, experiments were performed under controlled 
laboratory conditions. Testing involved three species of slstrfish. Asterias vulgaris ( 120 to 200 mm diameter), Lepiasterias polaris ( 120 
to 200 mm diameter), and Crossasler papposus ( 1 10 to 130 mm diameter), and two species of crab. Cancer irroratus (85 to 1 20 mm 
carapace width) and Hyas sp. (90 to 1 10 mm carapace length). Scallops from both size classes were presented separately (nonchoice 
treatment) and together (choice treatment) to each predator species. Starfish and Hyas sp. consumed less than one scallop per predator 
per day as compared to C. irroratus. which consumed as many as 12 scallops per predator per day. Starfish and crabs did not show 
a clear prey size preference in both treatments. However, large .scallops tend to be consumed faster by both crab species in choice 
treatments. These results indicate that bottoms w ith high densities of starfish or crab should be avoided in seeding. In addition, bottom 
seeding should be done if possible with scallops greater than the size classes used in this study (over 50 mm), according on these results 
and those of other studies. 

KEY WORDS: Placopeclen nwfiellanicus. scallop, enhancement, predation 



INTRODUCTION 

Sea scallops, Placopecten mageUanicu.s (Gmelin), have been 
seeded commercially off Jles-de-la-Madeleine (Quebec, Canada) 
in the Gulf of St-Lawrence since 1993. The profitability of these 
operations hinges on scallop survival rates 4 years after seeding, 
until scallops reached the commercial shell height of 90 mm. Pre- 
dation is an important factor affecting the survival of juvenile 
scallops between seeding and hai-vest. In Nova Scotia, Hatcher et 
al. ( 1996) showed that 50% of their seeded scallops were killed by 
crabs and starfish within 2 weeks. Experimental summer and win- 
ter seedings performed by Barbeau et al. ( 1996) indicated survival 
rates of 1 and 10%, respectively, after only 8 weeks. Crab (Cancer 
irroratus Say) predation was identified as the prime factor of scal- 
lop mortalities. Cliche et al. (1994) determined during an experi- 
mental seeding performed in Iles-de-la-Madeleine that 1 1 .5% of 
seeded scallops were killed by crabs within 44 days. Haugum et al. 
(1997) lost all of their seeded scallops to crab (Cancer paguni.s) 
predation within 3 weeks during an experimental bottom seeding 
in Norway. 

Many factors may affect the impact of predation after seeding. 
Size of seeded scallops is generally considered important (Elner 
and Jamieson 1979, Morgan et al. 1980, Lake and Jones 1987, 
Minchin 1991. Barbeau and Scheibling l_994a, Barbeau et al. 1994. 
Arsenault and Himmelman 1996). In iles-de-la-Madeleine. two 
size classes are seeded in late fall. Scallops kept in collector bags 
attain 15 to 25 mm shell height after 1 year, and those transferred 
to peari nets, 35 to 45 mm. Production costs (labor and equipment 
costs) of scallops grown-out in pearl nets are five times higher than 
scallops grown directly in collector bags. Thus, it is essential to 
estimate the survival rate of both classes when choosing an optimal 
growing strategy. 

Previous surveys around Iles-de-la-Madeleine showed that star- 
fish are abundant (-0.5 starfish/m") in natural scallop grounds. 
\N\\h Asterias vulgaris (Verrill) and Lepiasterias polaris (Miilleret 



Troschel) predominating and Crossasler papposus (L.) regulariy 
observed. Low densities of two crab species (-0.05 crabs/m"). C. 
irroratus and Hyas sp., are also reported. Predation of juvenile 
scallops by A. vulgaris and C. irroratus has been documented by 
Elner and Jamieson (1979). Jamieson et al. (1982). Lake et al. 
(1987), Barbeau and Scheibling (1994a), Barbeau and Scheibling 
(1994b), Barbeau et al. (1994), and Arsenault and Himmelman 
(1996). However, few studies have examined predation involving 
sea scallops (P. magellanicus) of 15 to 50 mm shell height (Elner 
and Jamieson 1979). Barbeau and Scheibling ( 1994a) and Barbeau 
and Scheibling ( 1994b) and Barbeau et al. ( 1994) used scallops of 
5 to 28 mm shell height, and Jamieson et al. (1982) used scallops 
of 40 to 55 mm and 80 to 110 mm. In addition, very little docu- 
mentation exists on the predatory capacity of L polaris. C. pap- 
posus and Hyas sp. on scallops (Arsenault and Himmelman 1996). 

Scallop vulnerabihty is high the first few days after seeding 
(Cliche et al. 1994, Barbeau et al. 1996). Stress induced during 
handling, exposure to air, and transportation to the seeding site 
may affect scallop vitality (Fleury et al. 1996). After seeded scal- 
lops reach the bottom, the time required to turn up their superior 
valve and find refuge may have an impact on their survival. Ar- 
senault and Himmelman (1996) concluded that refuge use de- 
creased the risk of predation for smaller scallops. Thus, studies 
conducted to increase the seeded scallops survival must target this 
period in particular. 

The objective of this study was to evaluate the impact or pre- 
dation by starfish. A. vulgaris. L polaris and C. papposus and 
crabs. C. irroratus and Hyas sp.. on small scallops from collector 
bags and larger ones from pearl nets under controlled conditions. 
This information is important when planning a seeding bottom 
strategy for a profitable commercial operation. 

MATERIALS AND METHODS 

Experiments were conducted in 2,000-L (I.I in x 2.3 m x 0.7 
m) and 1,500-L (I.I m x 1.6 m x 0.7 m) fiber glass tanks with 



905 



906 



Nadeau and Cliche 



circulating sea water. Temperature was maintained at 1 1.3 ± 3.0°C 
(mean ± SD), oxygen at 8.4 ± 0.7 mg/L, and salinity at 30.3 ± 0.9 
ppt. A 12:12 light:dark regime was simulated by fluorescent lights 
over each tank (-250 lux). Red bulbs were used to allow video 
recording in the dark, as used in Barbeau and Scheibling's ( 1994b) 
experiments. 

The cultivated juvenile scallops were taken from commercial 
operations of the scallop fishermen's association of Iles-de-la- 
Madeleine. Specimens measuring 15 to 25 mm shell height came 
from spat collectors, and those measuring 35 to 45 mm from 
intermediate culture in pearl nets. The shell height was measured 
from the middle of the dorsal hinge to the farthest point of the 
ventral shell edge. A plastic tag (glue-on flexible polyethylene 
shellfish tag. Hallprint Ltd) of 4 x 8 mm was glued with cy- 
anoacrylate glue on the upper valve. C. inonitus and Hyas sp. 
were caught with rock crab or lobster traps. C. inoratits averaged 
109.8 ± 6.6 m (mean ± SD. n = 45. range = 95 to 120 mm) 
carapace width, and Hyas sp. were 96.4 ± 7.7 mm (n = 6, range 
= 85 to 1 10 mm) carapace length. Similar to Barbeau and Scheib- 
ling's ( 1994a) experiments, only male crabs were used to eliminate 
potential sex-related biases (differences in morphology and preda- 
tion behavior). Starfish A. vulgaris of 153.3 ± 15.9 (n = 47, range 
= 120 to 200 mm) diameter. L. polaris of 151.9 ± 21.2 (n = 45, 
range = 120 to 200 mm), and C. papposus of 1 18.5 ± 6.6 mm (n 
= 6, range = 1 10 to 130 mm diameter) were collected by divers 
and scallop drags. The size classes of each predator species were 
selected according the most abundant predator class evaluated at 
the actual seeding sites (Roussy et al. 1994). 

Scallops, crabs, and starfish were maintained in 650-L seawater 
tanks for 2 to 10 weeks before testing began. Each species was 
kept in separate baskets. Scallops were fed on phytoplankton 
(Monochiysis httheri and Thalassiosira pseudonana) at concentra- 



tions of 5x10 cells/mL. Each predator was fed once a week on 
two living mussels (Mytilus edulis) weighing 10 to 20 g. Before 
experiments, predators were starved for 72 hours to standardize 
hunger levels. 

Two series of experiments were performed. The first was con- 
ducted between August and October 1994 in two 2,000-L tanks, 
divided using plexiglass separators to obtain four experimental 
sections of 0.8 m"* (Table 1). Predatory activity of C. irroratiis. A. 
vulgaris, and L. polaris was evaluated and compared. Three preda- 
tors of the same species were placed with 16 scallops of either 15 
to 25 mm or 35 to 45 mm (nonchoice treatment) or eight scallops 
of each class together (choice treatment). Predator density was 
2.4/m~. Scallop density was similar to the commercial seeding 
target density. 10/m". Control treatment involved eight scallops of 
each class without predator. Each treatment was repeated three 
times over a maximum of 5 days. In crab treatments, replicates 
were stopped earlier if all scallops had died. 

A video camera (Panasonic, Lunar Lite) fitted over the tanks 
filmed only one replicate of each experimental treatment because 
of logistical limitations. Starfish behavior was recorded up to 88 
hours and crab behavior, up to 22 hours, because the latter was 
more active. During frame analysis, the time each predator devoted 
to searching for prey and the number of encounters between preda- 
tor and prey were noted. Prey search by starfish was defined as 
displacement on the tank floor toward .scallops with arms tips 
tumed up. Because prey search by crabs was more difficult to 
determine, all ciab movement was considered searching behavior. 
Any contact between predator and prey was considered an encoun- 
ter. The capture occurred when starfish arms attached a scallop 
with their tubefeet or when crabs chelae grabbed a scallop. The 
number of active scallop escapes after encounters was also 
counted. An active escape was noted when scallops jumped or 



TABLE I. 
Laboratory experiments performed from August to October 1994 and .June to November 1995. 









Tank or 




n prey/replicate 


Predi 


ator/Replicate 


Predator 


Experimental 




Section 


II 








Size (mm) 


Species 


Year 


Treatment 


Size (m') 


Replicate 


Small 


Large 


n 


Mean ± SD 


A. vulgaris 


1994 


Nonchoice 


0.8 


3 


16 




3 


148.2 + 12.6 






Nonchoice 


0.8 


3 




16 


3 








Choice 


0.8 


3 


8 


8 


3 






1995 


Nonchoice 


1.8 


2 


24 




T 


160.9 ± 17.5 






Nonchoice 


1.8 


2 




24 


2 








Choice 


1.8 


2 


12 


12 


2 








Choice 


1.2 


4 


8 


8 


2 




L. polaris 


1994 


Nonchoice 


0.8 


3 


16 




3 


146.4 ±2 1.6 






Nonchoice 


0.8 


3 




16 


3 








Choice 


0.8 


3 


8 


8 


3 






1995 


Nonchoice 


1.8 


3 


24 




2 


163.5 ± 15.4 






Nonchoice 


1.8 


3 




24 


2 








Choice 


1.8 


3 


12 


12 


2 




C. papposus 


1995 


Choice 


1.2 


3 


8 


8 


2 


118.5 + 6.6 


C. irroratiis 


1994 


Nonchoice 


0.8 


3 


16 




3 


108.8 ±6.7 






Nonchoice 


0.8 


3 




16 


3 








Choice 


0.8 


3 


8 


8 


3 






■1995 


Nonchoice 


1.8 


2 


24 




2 


112.1 ±6.0 






Nonchoice 


1.8 


2 




24 


-) 








Choice 


1.8 


2 


12 


12 


2 








Choice 


1.2 


3 


8 


8 


2 




Hyas sp. 


igg.s 


Choice 


1 1 


3 


S 


S 


1 


964 ± 7.7 



Predation on Seeded Sea Scallops 



907 



swam away from starfish or crabs. Passive escape, noted when a 
scallop closed its valves without displacement in a predator con- 
tact, was impossible to detect, because the video camera was too 
far from the subject. The number of retractions defined by a retreat 
of a predator after an encounter was noted. 

The second series of expenments was performed between June 
and November 1995. Some of the trials aimed to repeat the 1994 
experiments in two 2,000-L tanks. However, improvements were 
made to the 1994 experimental design. To increase the surface to 
1.8 m", no .separators were used. Predator (C iironirus, A. vul- 
garis, and L polaris) density was reduced to 0.8/m- to simulate 
natural densities more closely. Observations were extended to 1.^ 
days to collect more information on starfish predation. Scallops 
available from commercial operations were larger (15 to 30 mm 
and 35 to 50 mml than in the 1994 tests. Experimental treatments 
consisted of putting two predators of the same species with 24 
scallops from one or both size classes (12 of both classes). A 
control treatment was conducted with 12 scallops of 15 to 30 mm 
and 12 scallops of 35 to 50 mm v\ithout predator. Number of 
replicates ranged from two to three. 

Second, the predatory activities of crab Hyas sp. and starfish C. 
papposus were compared, respectively, with those of C. iironirus 
and A. vuli;aris in two 1.500-L tanks that offered surface of 1.2 m"\ 
Predator density was 1.1 /m" and scallop density. lO/m". For each 
replicate, two predators were placed in a tank with eight .scallops 
of 15 to 30 mm and eight scallops of 35 to 50 mm. Control 
treatment u.sed eight scallops of 15 to 30 mm and eight .scallops of 
35 to 50 mm without predator. Testing ran a maximum of 13 days 
but was stopped earlier if all scallops had died. Each treatment was 
repeated three or four times. 

Sequence of experiments and tank allocation for each replicate 
were random. To simulate seeding conditions, predators were im- 
mersed 24 hours before the scallops. During daily observations, 
dead scallops were removed. The chi-square test was used to com- 
pare daily mortality in both size classes. Contingency tables (treat- 
ment X mortality) were prepared, and cells with insufficient data 
for test validity were grouped. Fisher's exact test was performed if 
the number of data was still lower than five (Sherrer 1984). Pre- 
dation rates (number of scallops consumed per predator per 24 
hours) were evaluated. All statistical analyses were performed us- 
ing SAS (1982), version 6.03 software. Data collected during 
video recording (encounter, capture, escape, and retraction rates) 
were considered more as an indication, given the absence of rep- 
licates. 

RESULT 

Scallop mortality was related to predation, because no mortality 
occurred in control treatments during 1994 and 1995 experiments. 

Starfish 

Video recordings from 1994 showed that starfish spent less 
than lO'vf of their time searching for prey in both treatments, 
remaining immobile on the tank walls the rest of the time. En- 
counter rates (or A. vulgaris and prey were between 0.9 to 4.1 per 
day (Fig. 1). Contacts tended to be higher with large prey in both 
treatments. L polaris encountered 0. 1 to 3.3 prey per day. Contacts 
between L. polaris and small scallops occuired more often. Active 
scallop escapes were higher for treatments involving A. vulgaris 
(0.8 to 4.0 per predator per day), and larger prey tended to escape 
more frequently. Predator retraction was noted with L. polaris (0.1 
to 1.5 per predator per day) and occurred primarily after encoun- 




Treatment 

Figure 1, Behavior of small (S) and large (L) scallops in the presence 
of Asterias vulgaris, Leptaslerias polaris, and Cancer irroratus in choice 
and nonchoice treatments during 1994 experiments. 

ters invoh'ing smaller prey in choice treatment. All of these factors 
resulted m low capture rates during recording periods (0 to 0.06 
per predator per day). 

In nonchoice treatments in 1994. A. vulgaris consumed more 
larger scallops after 4 days 0.22 ±0.13 (mean ± SD) per predator 
per day than smaller ones, 0.03 ± 0.13 (p = .03) (Fig. 2). How- 
ever, when size classes were presented together, mortality rates 
were similar (0.13 ± 0.12 per predator per day) for both (p = 
0.99). In contrast, in 1995, only small scallops were consumed in 
nonchoice treatments after 1 3 days, with a predation rate of 0.37 ± 

A vulgaris - Non-choice treatment 





1994 




f 10- 






o 
|05- 


_ 


1 


1 T 


1 


0.0- 


■ 
• — 


A 







1995 


> 


1.0- 


< 




05- 






00- 


; 





12 3 1 

Choice treatment 



1,5 



^ 1.0- 



g 

TO 

a> 



05- 







1994 


• Small prey 
■ Large prey 


T I- 

• 1 r 1 





1995 




10- 






05- 






00- 




'1 

1 ' 



1 2 3 

Replicate 



1 2 
Replicate 



Figure 2. Mean predation rates (n scallops consumed/predator/24 
hours) (± 95% CI,) o\ Asterias vulgaris on small and large scallops in 
choice and nonchoice treatments per replicate during 1994 and 1995 
experiments. 



908 



Nadeau and Cliche 



0.45. Statistical analysis revealed a difference between mortality 
rates for both size classes (p = .001). In choice treatments, mor- 
tality rates were similar for both classes. 0.04 ± 0.05 per predator 
per day. 

Comparison between C. papposiis and A. vulgaris in 1995 
choice experiments (Fig. 3). showed that A. vulgaris consumed 
more scallops (p = .04) after 13 days. However, both starfish 
demonstrated similar predation rates on 15 to 30 mm and 35 to 50 
mm scallops (p > .05). During this experiment. A. vulgaris con- 
sumed more small scallops. 0.35 ± 0.37 per predator per day. than 
larger ones. 0.1 ± 0. 12 (p = .002). C. papposus induced similar 
mortality rates (p = .06) on both classes, with predation of 0.05 ± 
0.02. 

Experiments performed in 1994 showed that L. polaris con- 
sumed only larger scallops in nonchoice treatments after 4 days, 
with a predation rate of 0.42 ± 0.33 (Fig. 4). Predation on both size 
classes was statistically different (p = .03). In choice treatments, 
predation rates on both classes were similar. 0.10 ±0.1 1 (p> 0.99). 
In 1995. nonchoice treatments showed that predation rates for 
small scallops (0.56 ± 0.34) were higher than those for larger 
scallops (0.08 ±0.13) after 13 days (p = .001). In choice treat- 
ments, smaller scallop mortality was significantly higher (0.24 ± 
0.18 per predator per day) than larger scallop mortality (0.08 ± 
0.04) (p = .004). 

Crabs 

Video analysis showed that C. irroratus spent 70 to 90% of the 
recorded time searching for prey. Encounter rates between preda- 
tor and prey were high ( 3 1 to 66). Some 7 to 1 1 active prey escapes 
occurred per predator per day. Retraction rates were noted 13 to 56 
times per predator per day. Encounter and retraction rates tended 
to be higher with small scallops, but capture rates were higher for 
larger scallops. 

In both experimental years, C. irroratus consumed almost all 
juvenile scallops available in a few days. Consequently, mortality 
rates were similar for both classes (p > .05) at the end of each 
replicate. Statistical comparison was performed 1 day after seeding 
to identify a preference for a particular class. In the 1994 experi- 
ments, predation rates after 24 hours in nonchoice treatments in- 
volving larger scallops were 4.22 ± 0.84 as compared to 2.89 ± 
1.07 for smaller ones (Fig. 5). Predation rates in choice treatments 



L, polans 



Non-choice treatment 



A. vulgaris 



C. papposus 




25- 


« Small prey 


20- 


■ Large prey 


1,5- 




1,0- 




0,5- 




ao- 


I i \ 



2 3 4 

Replicate 



1 2 3 

Replicate 



Figure 3. Mean predation rates (n scallops consumed/predator/24 
hours) (± 95% C.I.) of Asterias vulgaris and Crossaster papposus on 
small and large scallops in choice treatments per replicate during 1995 
experiments. 





£..\j n 


1994 






15- 






03 








to 




1 


1 


c 
g 

00 


10- 




1 


. 


0} 




■ 






0.5- 




^ T 




00- 


♦ 


. h 





1995 






1.5- 






r 


1.0- 




( 




0.5- 


1 1 

T J 
1 ■ 




OO- 


— ■ — 


1 — 1 


_j 



1 2 3 

Choice treatment 






1995 


15- 


• Small prey 
■ Large prey 


10- 


■ 


\ 




05- 
00- 


& i 


1 



12 3 12 3 

Replicate Replicate 

Figure 4. Mean predation rates (n scallops consumed/predator/24 
hours) l± 95% C.I.) of Leptasterias pularis on small and large scallops 
in choice and nonchoice treatments per replicate during 1994 and 1995 
experiments. 

were 2.67 for larger scallops and 1.44 ± 0.77 for smaller ones. 
Statistical analysis showed that larger scallops were consumed 
more often than smaller ones in both treatments (p < .05). In 1995, 
C. irroratus consumed more smaller scallops after 24 hours. 1 1 ± 
0.71 per predator, than larger ones (3.75 ± 1.06) in nonchoice 
treatments (p = .001). However, larger scallops were consumed 
more quickJy in choice treatments, with a predation rate of 5 ± 0.7 1 
as compared to 2.25 ± 2.47 for smaller ones (p = .001). 

Small scallop mortality was statistically lower with Hyas sp. (p 
= .0001 ). but larger scallop consumption was similar for both crab 
species (p > .05). Hyas sp. consumed more larger scallops than 
smaller ones (p = .014) in 13 days (Fig. 6). C. irroratus had 
consumed all of the scallops presented at the end of each replicate. 
However, I day after seeding, C. irroratus consumed all large 
scallops available, four per predator. Large scallops were con- 
sumed more often than smaller ones. 2.67 ± 1 .04 per predator (p = 
.002). 

DISCUSSION 

In this study, starfish consumed less than one scallop per preda- 
tor per day. In neither test year did starfish show a clear size- 
related preference in choice and nonchoice treatments. However, 
Barbeau and Scheibling (1994a) showed that, in aquaria. A. vul- 
garis of 30 to 150 mm diameter consumed more scallops of 5 to 
8.5 mm shell height than those of 10 to 15 mm or 20 to 25 mm in 



Predation on Seeded Sea Scallops 



909 



C irroratus - Non-choice treatment 



12 



C irroratus 



Hyas sp 



S 6 

nj 
■a 

a 
3- 



1994 



"T r- 




12 3 12 

Choice treatment 



12 



0) 

ra 

c 
o 

ra 
■o 

(U 





1994 


9 


• Small prey 




■ Large prey 


6- 




3- 


■ Ji ■ 




• • 


0- 


1 r 1 ' 




.12 3 
Replicate 

Figure 5. Predation rates (n scallops consumed/predator/24 hours) of 
Cancer irroratus after 1 day on small and large scallops in choice and 
nonchoice treatments per replicate during 1994 and 1995 experiments. 

choice and nonchoice treatments. Under field conditions. Barbeau 
et al. (1994) confirmed that A. vulgaris consumed more scallops of 
5 to 9 mm and 10 to 15 mm than 20 to 25 mm. In addition, tests 
performed by Arsenault and Himmelman (1996) showed that vul- 
nerability of the scallop Chlamys iskmdica (between 10 to 60 mm 
shell height) to L. polaris, C. papposus. and A. vulgaris decreased 
with increasing prey size in choice treatments. 

Crossaster papposus had lower predation activity (0.05 prey 
per predator per day) than A. vulgaris (0.2 prey per predator per 
day) on juvenile scallops. However, A. vulgaris and L polaris 
induced comparable mortality rates for juvenile scallops. These 
last predators spent less than 10% of the recorded time searching 
for prey. Active scallop escapes and predator retractions, espe- 
cially involving L. polaris. kept the consumption rate under one 
scallop per predator per day. These findings were confirmed by 
Barbeau and Scheiblmg (1994a). in whose tests A. vulgaris spent 
1 1 to 27% of time searching for prey, resulting in less than one 
scallop of 20 to 25 mm being consumed per starfish per day. 

Differences between experimental years and replicates could be 
related to various factors. Differences in years may be partly as- 
sociated with the experimental design modifications. In 1994, 
predator density was twice as high and tank sections half as large 
as in 1995. Thus, active escapes induced after starfish encounters 
were probably limited by the walls of the smaller tanks used in 
1994. The 1995 experimental design more closely simulated natu- 
ral conditions. Replicates may also have been affected by the 




10 



6- 



2- 



13 days 



Small prey 
Large prey 



i i t 



2 3 
Replicate 



2 3 
Replicate 



Figure 6. Predation rates (n scallops consunied/predator/24 hours) of 
Cancer irroratus after 1 day and mean predation rates (± 95% C.l.) of 
Hyas. sp. after 13 days on small and large scallops in choice treatments 
per replicate during 1995 experiments. 



variability in individual behavior and in physiological needs dur- 
ing the year. 

Crabs were very effective predators, able to ingest as many as 
12 scallops per predator per day. In both experimental years and 
treatments, C. irroratus consumed all scallops available within 24 
to 72 hours after seeding. In nonchoice treatments, this crab spe- 
cies tended to consume all available prey rapidly. In choice treat- 
ments, the mortality of larger scallops after 24 hours was higher 
than that of smaller ones. Although Hyas sp. also selected larger 
specimens in choice treatments, the predation rates were lower 
than those of C. irroratus. In fact, the predation rates of H\as sp. 
(0.5 scallop per predator per day) were closer to the starfish. Bar- 
beau and Scheibling (1994a) showed that C. irroratus consumed 
more scallops of 10 to 15 mm shell height than of 5 to 8.5 mm or 
20 to 25 mm in nonchoice treatments, and more scallops of 20 to 
25 mm in choice treatments. In contrast, their field testing (Bar- 
beau et al. 1994) on scallops of 7 to 28 mm indicated that prey size 
had little effect on crab predation success. 

Video tapes showed that C. irroratus spent 70 to 90% of the 
time searching for prey. This result may be an overestimate, be- 
cause all crab displacements were related to searching behavior. 
Barbeau and Scheibling ( 1994a) estimated crab searching behavior 
at less than 11% of the recorded time. In the present study, C. 
irroratus encountered as many as 3 1 to 66 scallops per day and a 
reaction of retraction followed in 58.4 to 86.6% of the time. In 
Barbeau and Scheibling's (1994a) study, no predator retraction 
was observed, but passive escapes represented 58 ± 41% of total 
escapes. However, passive escape was impossible to detect on 
recording image. Therefore, this escape may have been confused 
with the retraction behavior. Wilkens (1991) postulated that scal- 
lops often respond to crab encounters by closing their valves (pas- 
sive escape). This behavior may result from scallops detecting crab 
movement with the use of eyes on the mantle edge. 

Elner and Jamieson (1979) showed that C. irroratus had a 
specific scallop (P. magellaiiicus) size preference in multiple prey 
choice treatments under controlled conditions. Larger predators 
( 120 to 130 mm carapace width) preferred larger scallops (40 to 50 
mm shell height), and smaller predators (90 to 100 mm) tended to 
prefer smaller scallops (20 to 30 mm). Based on these results, it is 
possible to hypothesize that the intermediate predator size (100 to 



910 



Nadeau and Cliche 



120 mm), used in the present study, would prefer the intermediate 
scallop size (30 to 40 mm). Lake at al. (1987) also showed that 
Cancer pagurus predation was influenced by predator size and 
scallop size. The number of scallop Pecten maximus (30 to 60 mm 
shell height) consumed increased with predator size (60 to 140 mm 
carapace width) and decreased as prey size increased. However, 
their study did not use scallops smaller than 30 mm. In contrast. 
Arsenault and Himmelman (1996) showed that C irromnis and 
Hyas araneus. size 1 12 ± 2 mm (mean ± SD) carapace width and 
97 ± 1 mm carapace length, respectively, preyed more on scallops 
(Chlamys islandica) of 10 to 30 mm shell height than on scallops 
of 30 to 60 mm in multiple prey choice treatments. Furthermore, 
the feeding rate of H. araneus was about twice that of C. irroratus. 

Effective predators (A. vulgaris. L. polaris, C. papposiis. C. 
irroratus, and Hyas sp.) live on the sea bottom, where scallops 
seeding activities occur in the Iles-de-la-Madeleine. In this study, 
predation rates may have been biased by the absence of refuge or 
alternative prey. In addition, interaction between predator species 
was not evaluated. Nevertheless, we can assume that these preda- 
tors will cause high mortality in seeded scallops under natural 
conditions. Based upon our results and those obtained from other 
studies, bottom seeding should be done, if possible, with scallops 
greater than the size classes used in this experiment (over ?0 mm). 
On bottoms with high crab density, both size classes of scallops 
will be vulnerable. Because crabs are highly effective scallop 
predators, these bottoms must be avoided. 

Alternative solutions must be evaluated to minimize the impact 
of predation. Control of seeding density and predator elimination 



(Ventilla 1982) are solutions used in Japan. As observed by Bar- 
beau et al. (1994), predation increases with scallop density and 
crab density. Barbeau and Scheibling (1994b) also demonstrated 
that the seeding season may have an important impact on preda- 
tion, because temperature affects feeding activities of crabs (C 
irroratus) and starfish (A. vulgaris). Seeding at lower temperatures 
was effective in reducing predation, particularly for starfish. Bio- 
chemical analyses of Pecten nia.\inuis performed by Fleury et al. 
( 1996) showed a very low glucide content in the fall. Lower energy 
reserves may affect the survival rates of seeded scallops. In addi- 
tion, stress induced by the seeding operations can reduce their 
vitality. Fleury et al. (1996) found that seeding stress represented 
high energy expenditures for scallops (Pecten maximus). Finally, 
weak animals escape predators less often and less rapidly (Hatcher 
et al. 1996). Seeding strategies will have to deal with all these 
factors. Seeding operations in the Iles-de-la-Madeleine will be 
financially profitable if 20 to 30% of the scallops seeded are 
caught by scallop fisherman. An effort must be made to ensure 
such survival rates. 

ACKNOWLEDGMENTS 

The authors thank the technical staff of the Station Tech- 
nologique Maricole des Iles-de-la-Madeleine of MAPAQ for their 
valuable participation in this project and the local scallop fisher- 
mens' association, who provided the juvenile scallops. A special 
thanks to Michel Giguere of the Department of Fisheries and 
Ocean from Ste-Flavie for assistance. 



REFERENCES 



Arsenault, D.J. & J. G. Himmelman. 1996. Size-related change.s in vul- 
nerahiiity to predators and spatial refuge use by juvenile Iceland scal- 
lops Chlamys islandica. Mar. Ecol. Prog. Ser. 140:115-122. 

Barbeau, M. A. & R. E. Scheibling. 1994a. Behavioral mechanisms of 
prey-size selection by sea stars Asterias vulgaris (Verrill) and crabs 
Cancer irroratus (Say) preying on juvenile sea scallops Placopecten 
magellanicus (Gmelin). / Exp. Mar. Biol. Ecol. 180:103-136. 

Barbeau. M. A. & R. E. Scheibling. 1994b. Temperature effects on preda- 
tion of juvenile sea scallops Placopecten magellanicus (Gmelin) by sea 
stars Asterias vulgaris (Verril) and crabs Cancer irroratus (Say). J. 
Exp. Mar. Biol. Ecol. 182:27-48. 

Barbeau, M. A., B. G. Marcher. R. E. Scheiblmg. A. W. Hennigar. L. H. 
Taylor & A. C. Risk. 1996. Dynamics of juvenile sea scallops Pla- 
copecten magellanicus and their predators in bottom seeding trials in 
Lunenburg Bay, Nova Scotia. Can. J. Fish. Aquat. Sci. 53:2494-2512. 

Barbeau. M. A.. R. E. Scheibling. B. G. Hatcher. L. H. Taylor & A. W. 
Hennigar. 1994. Survival of tethered juvenile .sea scallops Placopecten 
magellanicus in field experiments — effects of predators, .scallop size 
and density, and site and season. Mar. Ecol. Prog. Ser. 115:243-256. 

Cliche. G.. M. Giguere & S. Vigneau. 1994. Dispersal and mortality of sea 
scallops. Placopecten magellanicus (Gmelin 1791) seeded on the sea 
bottom off iles-de-la-Madeleine. J. Shellfish Res. 13:565-570. 

Elner, R. W. & R. N. Hughes. 1978. Energy maximization in the diet of the 
shore crab Carcinus maenas. J. Anim. Ecol. 47:103-1 16, 

Elner. R. W. & G. S. Jamieson. 1979. Predation of sea scallops Pla- 
copecten magellanicus by rock crab Cancer irroratus and American 
lobster Homarus americanus. J. Fish. Res. Board Can. 36:537-543. 

Fleury. P. G.. C. Mingant & A. Castillo. 1996. A preliminary study of the 
behavior and vitality of reseeded juvenile great scallops of three sizes 
in three seasons. Acpiacull. Int. 4:325-337. 

Hatcher, B. G., R. E. Scheibling. M. A. Barbeau, A. W. Hennigar, L. H. 
Taylor & A. J. Windust. 1996. Dispersion and mortality of a population 
of sea scallop Placopecten magellanicus seeded in a tidal channel. Can. 
./. Fi.ih. Aquat. Sci. 53:38-54. 



Haugum. G. A.. O. Strand. A. Svardal & S, Mortensen. 1997. Survival and 
behavior of scallops Pecten maximus L. after transfer to the seabed — 
effect of emersion treatment. Proceedings Eleventh International Pec- 
linid Workshop, La Paz, Baja California Sur, Mexico. 30 pp. 

Jamieson. G. S., H. Stone & M. Etter. 1982. Predation of sea scallop 
Placopecten magellanicus by lobster Homarus americanus and rock 
crab Cancer irroratus in underwater cage enclosures. Can. J. Fish. 
Aquat. Sci. 39:499-505. 

Lake. N. C. H.. M. B, Jones & J. D, Paul, 1987, Crab predation on scallop 
Pecten maximus and its application for scallop cultivation. J. Mar. Biol. 
Ass. U.K. 67:55-64. 

Minchin, D. 1991. Decapod predation and the sowmg of the scallop, 
Pecten maximus (Linnaeus, 1758). pp. 191-197. In: S. E. Shumway 
and P. A. Sandifer (eds.). An International Compendium of Scallop 
Biology and Culture. World Aquaculture Workshops. No. 1. World 
Aquaculture Society, Baton Rouge, Louisiana. 

Morgan. D. E.. J. Goodsell. G. C. Matthiessen, J. Garey & P. Jacobson. 
1980. Release of hatchery-reared bay scallops (Argopecten irridians) 
onto a shallow coastal bottom in Waterford. Connecticut, Proc. World 
MaricuL Sac 11:247-261. 

Roussy, M., G. Cliche & M. Giguere. 1994. Caracterisation et eradication 
des pr6dateurs du petoncle geant (Placopecten magellanicus) aux Iles- 
de-la-Madeleine. MAPA-Pecheries. D.R.S.T Doc. Rech. 94/15. 85 pp. 

SAS Institute. 1982. SAS user's guide: Statistics. SAS Institute Inc., North 
Carolina. 

Sherrer. B. 1984. Biostalistique. Gaetan Morin Edileur. Chicoutimi. Que- 
bec. Canada. 850 pp. 

Ventilla. R. F. 1982. The scallop industry in Japan, pp. 309-382. In: 
J. H. S. Blaxter, F. S. Russell and M. Yonge (eds.). Advances in Marine 
Biology, vol. 20. Academic Press, San Diego, California. 

Wilkens, L. A. 1991. Neurobiology and behavior of the scallop, pp. 429- 
469. In: S. E. Shumway (ed.). Scallops: Biology, Ecology, and Aqua- 
culture. Developments in Aquaculture and Fisheries Science, vol. 21. 
Elsevier Science. New York. 



.louniiil i>l Shrllfish Reseanh. Vol. 17. No. 4, 911-917, 199S. 

GROWTH, PRODUCTION, AND REPRODUCTION IN BAY SCALLOPS ARGOPECTEN 
IRRADIANS CONCENTRICUS (SAY) FROM THE NORTHERN GULF OF MEXICO 



PAUL A. X. BOLOGNA 

University of South Alabama 
Dauphin Island Sea Lab 
Dauphin Island. Alabama 36528 

ABSTRACT The bay scallop. Argopeclen inadians. is a commercially and recreationally important fisheries .species on the Atlantic 
and Gulf Coasts of the United States. Surprisingly, little information exists on northern Gulf of Mexico populations. This research 
assessed growth, production, and reproduction in a population from St. Joseph Bay. Florida. Specifically, scallops exhibited high initial 
growth rates (Gw day''), with rates declining as individual size increased. Additionally, significant interannual variability existed for 
both scallop growth and production. These differences may be attributable, in part, to a reduction in salinity (2l9rr) associated with 
Tropical Storm Alberto, which resulted in significantly lower growth rates and a mass mortality event. Reproductive assessment oi A. 
irradians showed significant peaks in spawning condition, gonad weight, and gonadal-somatic index (GSI) during the winter (De- 
cember. January. February) compared to other seasons. However, the salinity minima in July 1994 ( 1 IS?c) significantly reduced gonad 
weight and GSI from winter 1994/1993. suggesting that a single storm event had a dramatic but short-term reproductive impact on the 
population. Assessment of gonad condition and GSI, coupled with field observations, showed spawning occurred in the spring and fall 
as well, but the presence of small individuals (<4 mm shell height) during July, August, and September suggests that reproduction may 
occur throughout the year in St. Joseph Bay. Florida. 

KEY WORDS: Argopeclen iiradiuiis. gonad weight. GSI. Gulf of Mexico, salinity effects 



INTRODUCTION 

Bay .scallops. Argopeclen irradians (Lamarck 1819) are com- 
mon members of many shallow water benthic communities along 
the Atlantic and Gulf Coasts of North America. Clarke (1965) 
identified three distinct subspecies of A. irradians based on mor- 
phologic characteiistics, and recent morphologic and genetic stud- 
ies support these distinctions (Wilbur and Gaffney 1997). Al- 
though it is recognized that scallops recrtiit to seagrass habitats 
(Gutsell 1930. Thayer and Stuart 1974. Eckman 1987) and that 
they cling to leaves to escape benthic predators (Pohle et al. 1991. 
Ambrose and Irlandi 1992). little information exists on several key 
aspects of their ecology. Several studies have assessed scallop 
growth rates under experimental conditions detennining the im- 
pact of flow (Kirby-Smith 1972, Cahalan et al. 1989. Eckman et 
al. 1989) or food concentration and stocking density (Rheault and 
Rice 1996), but there are few field estimates of adult growth rates 
under natural, noncaged conditions. Eckman (1987) showed 
greater growth rates for juvenile bay scallops in grass beds with 
low shoot densities, and Ambrose and Irlandi (1992) showed that 
juveniles may trade-off decreased growth rates for increased sur- 
vivorship by altering their placement on seagrass leaves. In addi- 
tion, it has been shown that habitat configuration has an impact on 
the growth and survival of juveniles (Irlandi et al. 1995). Although 
these studies adequately assess growth of juveniles, few published 
studies exist on growth rates of adult scallops under natural field 
conditions. 

Another aspect of A. irradians is the relative lack of estimates 
of natural mortality (but see Marshall 1963). Many studies have 
assessed predation mortality (Tettelbach 1986. Pitcher and Butler 
1987. Peterson et al. 1989. Prescott 1990. Pohle et al. 1991. Am- 
brose and Irlandi 1992. Bologna 1998); however, few estimates of 
natural population mortality exist. Exceptions include clear pat- 
terns of high postspawn mortality (Gutsell 1930, Capuzzo and 
Hampson 1984). mortality associated with 2nd-year individuals 
prior to spawning (Bricelj et al. 1987a). and the severe impacts 



of nuisance algal blooms (Suiinnerson and Peterson 1990. Tettel- 
bach and Wenczel 1993). 

One feature of A. irradians ecology that is relatively well 
known for many populations is reproductive effort. Given that 
scallops suffer high moilality after spawning, significant research 
has focused on reproduction, reproductive conditioning, and 
changes in biomass associated w ith gonad development (Bricelj et 
al. 1987h). It has been shown that scallops show distinct peaks in 
spawning, and these peaks differ temporally based on latitude of 
the populations (Sastry 1970, Barber and Blake 1983, Crenshaw et 
al. 1991). Although scallops do show peaks in reproduction, re- 
cruiting individuals (<10 mm shell height) have been reported 
throughout the year, suggesting that trickle spawning may occur in 
some locations (Gutsell 1930, and see Bricelj et al. 1987b). How- 
ever, in general, bay scallops are considered to be a semelparous or 
short-lived iteroparous species, and reproductive effort tends to 
follow this life history trait (Sastry 1970, Sastry 1979, Barber and 
Blake 1983, Bricelj et al. 1987h). 

Despite a wealth of knowledge on populations of A. irradians 
from the Atlantic Coast and southern Florida, little is known about 
northern Gulf of Mexico populations. The goals of this research 
were to assess several life history traits from a bay scallop popu- 
lation from St. Joseph Bay, Florida. U.S.A. Specifically, research 
focused on the following objectives: ( I ) determine the natural 
growth rate of adult scallops: (2) estimate natural population mor- 
tality; (3) estimate production within the population: (4) determine 
reproductive cycles; and (5) assess reproductive effort of scallops. 

METHODS 

Study Site 

This research was conducted in St. Joseph Bay, Florida, which 
lies in the northeastern Gulf of Mexico (29° N, 85.5° W). It is a 
semi-enclosed lagoonal system with little freshwater input. Salin- 
ity and temperature data were collected from 1992 through 1995 in 
St. Joseph Bay (Fig. I) and show that temperature follows a sea- 



911 



912 



Bologna 



sonal trend; whereas, salinity is normally high and ranges from 25 
to 35%c annually. However, large storm events (e.g.. Tropical 
Storm Alberto) can significantly reduce salinity over short periods 
of time (e.g., to 1 \%c). 

Shallow regions of the St. Joseph Bay benthos primarily consist 
of the seagrass Thalassia testiuliniiiii interspersed with Halodide 
wrightii. Syringodium filifonne, and open sediment. T. testiidiiuim 
is the dominant species and covers approximately 2.300-2,400 
hectares (Savastano et al. 1974, Iverson and Bittaker 1986). 

GROWTH, MORTALITY, AND PRODUCTION 

To determine rates of growth and mortality, a series of mark- 
recapture experiments were undertaken. Scallops were marked by 
cleaning and drying the ventral valve and gluing on a numbered 
tag. Scallop shell height and breadth were then measured using 
vernier calipers to the closest 0.05 inm. Six additional scallops 
were marked and held in aquaria for 2 weeks to assess the impacts 
of handling and marking on survival. All six control individuals 
survived the 2 week trial and were released back into the field. 
These individuals were not used to estimate growth, mortality, or 
production. 

In 1993, 79 scallops (31.3 mm to 44.1 mm shell height) were 
marked and released on two dates in June into an expansive 
Thalassia tesnidiniim grass bed. Scallops were marked and re- 
leased on June 1 1 (n = 31 ) and June 14 (n = 48). During 1993, 
scallops were relocated and measured on June 14. 23. July 8. 22, 
August 25, September 19, November 5, and December II. In 
1994, 95 scallops were marked in three cohorts. In April, 40 scal- 
lops (37.75-66.3 mm shell height) were marked and measured in 
the same manner as above. The second cohort of 33 scallops 
(41-51.6 mm shell height) was marked on June 23, and the final 
scallop cohort (n = 22, 41.8-53.8 mm shell height) was marked 
and released on July 14. All cohorts were released into an exten- 
sive T. tesrudiiuiin grass bed mosaic and periodically sampled 
throughout the year. Specifically, scallops were relocated on May 
5, June 6, 23, July 7, 14, 20, August 10, 18, 31. and October 12. 

Estimates of growth and mortality followed the protocol of 
Cowan and Houde (1990). Instantaneous growth rate was calcu- 
lated using the change in biomass over time. Scallop biomass was 
estimated using the following regression equation from Bologna 
(1998), which provides an estimate of tissue dry weight: (ln[tissue 



3 5- 



I 



• Temperature 
' Salinity 




3 
2 5 .■• 



1992 1993 1994 1995 

Figure 1. Weekly temperature (C) and salinity (*?() values collected 
from St. .Joseph Bay, Florida from January IWI through December 
1995. Source: City of Port St. Joe, Florida industrial waste-water treat- 
ment plant, 1996; ^indicates a salinity minima of ll%fi on July 27, 1994. 



dry wt. (g)] = -9.779 -I- 0.7909* ln[.shell height] + 2.2124* In- 
[shell breadth]; n = 161, r" = .92). Growth rate (Gw, growth in 
weight), expres.sed as day"', was computed using the Eq. 1. 



Gir = 



ln(lV,)-ln(W,_|) 



(1) 



where: C\v = instantaneous growth rate (day"'); W = estimated 
scallop dry weight at time t (ln[grams tissue dry weight]); and, t = 
time in days. Instantaneous scallop mortality rate (|x) was esti- 
mated from recaptures using Eq. 2. 



\n{no) - \n(nt) 



(2) 



where: |j. = calculated instantaneous mortality rate (day"'); n = 
is the number of scallops present at a given time t; and t = time. 

Because scallops were motile (>500 m. pers. obs.. this study), 
calculated mortality actually retJects mortality plus emigration 
and, consequently, is an overestimate. To limit the impact of initial 
emigration bias on mortality estimates by second and third cohort 
marked scallops (e.g.. field release dates June 14. 1993. June 23 
and July 14. 1994). mortality on successive dates (June 23, 1993, 
July 7 and 20, 1994) was calculated using the number of scallops 
present from the previous cohort. Subsequent mortality estimates 
were based on any individual present beyond one sampling date. 

Based on estimated instantaneous growth and mortality rates 
calculated from mark-recapture scallops, production (g dry wt 
day"') for time intervals was calculated using the Cowan and 
Houde (1990) equation: 



P = B * Gw 

where: B = average biomass (g) during a time interval; Gw 
instantaneous growth rate; and B was calculated as follows. 



B = : for {Gw > |ji) 



(3) 



(Cm- n.]r 



-J_l^ 



(^l 



■Cw)r 



■ for (|a, > Gil) 



(4) 



where: B^, = estimated initial biomass of all marked individuals 
for a given time interval; Gw = instantaneous growth rate; ij, = 
instantaneous mortality rate; and. t = time. 

Growth, mortality, and production rates were compared be- 
tween 1993 and 1994 using a nonparametric two-tail trend analysis 
(a = 0.05) for rates on similar calendar dates between years. 

REPRODUCTION 

Scallop reproduction was assessed by visual inspection of the 
gonad condition and resultant gonad weight and relative weight 
ratio (GSI). Scallops were collected on 37 dates from St. Joseph 
Bay, Florida from November 1992 to October 1996. Scallops were 
frozen and returned to the laboratory where their shell height and 
breadth were measured to 0.05 mm. Scallops <22 mm were con- 
sidered juveniles, and body tissue was dissected and weighed as a 
whole. Scallops >22 mm shell height had both gonadal and so- 
matic tissue dissected out. 

Reproductive and somatic tissue were then dried at 80°C for 48 
to 72 hours and weighed (g dry weight). A GSI was calculated for 
each scallop using the following equation: GSI = (gonad dry 
weight/total dry weight) * 100. To assess seasonal timing of maxi- 



Growth. Production, and Reproduction of Bay Scallops 



913 



mal GSI and gonad weight, collections were groups as winter 
(samples collected in December. January, and February), spring 
(March. April, and May), summer (June. July. August) and fall 
(September. October. November). Data were analyzed using a one- 
way analysis of variance (ANOVA; a = CO."!). with season as the 
independent variable and gonad weight and GSI as dependent 
variables. Multiple comparisons were made using Scheffe's F-test. 
In addition, scallop gonad weight was analy/.ed against date of 
collection using one-way ANOVA to determine annual variability 
in reproductive effort. 

Visual condition of gonadal material for scallops greater than 
22 mm shell height was assessed for each individual beginning in 
August 1993. Gonad condition was assessed as undeveloped, rip- 
ening, very ripe, or postspawn. Undeveloped gonads appeared gray 
in color, but were robust in nature. Ripening gonads were robust 
and showed visual development of gonadal material (i.e., female 
portions were, to some degree, red-orange in color and/or male 
portions were white or whitening). Very ripe gonads were robust, 
with female portions fully red-orange and male portions entirely 
white. Gonads form postspawn individuals were either gray and 
flaccid or gray with tinges of red-orange/white and flaccid (as 
opposed to robust, which were associated with undeveloped and 
developing gonads). 

Evidence of reproduction was assessed by collection of small 
individuals (<20 mm shell height). This was accomplished by 
visually locating them in the field and returning them to the labo- 
ratory for morphological measurement and biomass determination. 
In addition, in 1994 and 199.'!. artificial seagrass mats (for a full 
description of seagrass mat construction and characteristics, see 
Bologna 1998) were placed in the field during the summer to 
determine the presence and recruitment density of bay scallops. 

RESULTS 

Growth, Mortality, and Production 

Scallop growth, mortality, and production rates are summarized 
in Table 1. In 1993, scallops showed growth rates that were rela- 
tively high, declining slowly from June to September and dramati- 
cally in the fall. However, scallops marked in 1994 showed a rapid 
decline in growth rate from May to June and maintained very low 
growth rates throughout the summer and into the fall. When the 
data were analyzed using trend analysis, calculated compared 
mean scallop growth rate for 1993 was significantly greater than 
growth rates in 1994 (P [K « 017, 0.475] = 0.01 17). 

Scallop mortality rates were initially relatively high for all se- 
ries of mark-recapture scallops (Table 1 ). This may be attributed 
to relatively high emigration (dispersal) rates after initial deploy- 
ment into the field, and these mortality estimates must be treated 
with caution. The calculated mortality rates for scallops on subse- 
quent cohort release dates in 1993 and 1994 did not show this, 
because mortality calculations were based on previous individuals 
in the field and not the potentially high rates of emigration on 
initial placement in the field. In 1993. mortality was high during 
June, July, and August, but relatively reduced in September- 
November, with major losses by the December collection date. No 
mortality patterns were present in 1994 data. However, mortality 
rates were lower in 1994 than 1993 (Table 1 ), albeit not significant 
(P[K < 317. 0.475] = 0.574). However, the relatively high mor- 
tality rate calculated for August 10, 1994 corresponds to the sa- 
linity minima of 1 19ct seen in 1994 from freshwater input associ- 
ated with Tropical Storm Alberto (Fig. 1). 



TABLE L 

Instantaneous growth, mortality, and production rates for 
mark-recaplure .4. irradians from 1993 and 1994. 









Growth 


Mortality 


Production 


Date 


n 


cd 


Day ' 


Day-' 


g Day ' 


6/14/93* 


24 


a 


0.02509 


0.07380 


0.33560 


6/23/93 


54(19) 


b 


0.01865 


0.02696 


0.68474 


7/8/93 


38 


c 


0.02027 


0.02343 


0.49523 


7/22/93 


26 


d 


0.01638 


0.02711 


0.48080 


8/25/93 


15 


e 


0.01054 


0.01618 


0.27.561 


9/19/93 


13 


f 


0.01064 


0.00572 


0.33591 


1 1/5/93 


9 


g 


0.00539 


0.00782 


0.11650 


12/11/93 


2 




0.00224 


0.04178 


0.01852 


1994 












5/5/94 


21 




0.02603 


0.03391 


0.5998 


6/9/94 


14 


a 


0.00544 


0.01158 


0.1722 


6/23/94** 


11 


b 


0.00417 


0.01723 


0.1049 


7/7/94 


19(9) 


c 


0.0043 1 


0.01433 


0.0905 


7/14/94*** 


18 




0.00632 


0.00772 


0.1889 


7/20/94 


25 (17) 


d 


0.00442 


0.00817 


0.1238 


8/10/94 


17 




0.00469 


0.01836 


0.1589 


8/18/94 


16 


e 


0.00650 


0.00758 


0.1743 


8/31/94 


14 


f 


0.00638 


0.01027 


0.1627 


1 (VI 2/94 


5 


g 


0.00343 


0.02451 


0.0584 



Growth and mortality values expressed as day" , production expressed as 
grams day"'; n indicates the number of recaptured individuals u.sed to 
estimate growth, mortality, and production; values in parentheses indicate 
the number of individuals used to estimate mortality when a successive 
cohort of marked individuals was added to the tleld. thus limiting initial 
emigration bias of mortality hy newly placed marked scallops; cd indicates 
calendar dates of comparison for growth, mortality, and production rates 
between 1993 and 1994; * indicates the date when 48 additional individu- 
als were marked and released into the field; ** indicates the addition of 33 
marked scallops; *** indicates the addition of 22 marked scallops. 



Scallop production in 1993 showed an increase during the ini- 
tial part of the study with declining productivity as the year pro- 
gressed (Table 1 ). Because production was based upon estimates 
of mortality that include both mortality and emigration, calculated 
production is a conservative estimate. In 1994, production was 
considerably lower and remained les than one-third that of 1993. 
Results from trend analysis showed a significant reduction in scal- 
lop production from 1993 compared to 1994 (P[K < 017, 0.475] = 
0.01 17). This reduction in production between years was likely the 
result of the significantly lower growth rates in 1994 compared to 
1993 (p < .023); and not to changes in relative mortality (Table I ). 

A scallop mortality event was observed on July 20, 1994 
throughout the southern half of St. Joseph Bay. Although the ab- 
solute salinity minima ( 1 \%c) occurred on July 27 (see Fig. 1), the 
reduction in salinity attributable to Tropical Storm Alberto on July 
3, not to mention the potential for disturbance and burial, may have 
had a severe impact on the survival of bay scallops. The observed 
salinity minima, however, corresponded well to the high estimated 
instantaneous mortality rate calculated for mark-recapture indi- 
viduals on the following collection date (August 10. 1994. Table 
1). On June 24. 1996. another mortality event was observed in St. 
Joseph Bay. During this time period, numerous sitings of red-tide 
algal blooms occurred along the coastal regions of the northeast 
Gulf of Mexico (J. Stout, pers. comm.) and may have been re- 
sponsible for this mortality event. 



914 



Bologna 



REPRODUCTION 

Reproduction assessment by visual inspection of the gonad 
condition showed several interesting features regarding reproduc- 
tive individuals (Table 2). First, scallops as small as 31 mm shell 
height showed very ripe gonads and scallops as small as 34.1 mm 
shell height showed gonad condition indicative of postspawn (May 
1994. Table 2). Second, scallop gonad condition associated with 
individuals from the winter of 1994/1995 showed relatively low 
proportions of individuals in any reproductive condition, as well as 
few individuals (one of 59) showing a postspawn condition (Table 
2). Last, of the 1.106 scallops assessed for reproductive purposes, 
only one individual (October 1995, Table 2) showed nonhermaph- 
roditic sex development with only male gonadal tissue present. 

When scallop reproduction was assessed using GSI. results 
showed scallops had significantly greater GSI values during the 

TABLE 2. 
Visual gonad condition index. 



Date n Juvenile Undeveloped Ripe 



Very 
Ripe 



Postspawn 



8/25/93 

9/11/93 

9/19/93 

10/1/93 

11/5/93 

11/19/93 24 

12/11/93 32 

1/9/94 

2/25/94 

4/15/94 

5/5/94 

6/8/94 

7/20/94 

8/19/94 

8/24/94 

12/1/94 

1/21/95 

2/1/95 

3/10/95 

4/9/95 

5/17/95 

6/20/95 

7/5/95 

8/6/95 

8/30/95 

9/26/95 

10/28/95 

12/5/95 

1/20/96 

2/15/96 

3/13/96 

4/19/96 

5/22/96 

6/24/96 

8/8/96 

10/18/96 



12 

27 
27 
37 
40 



36 
12 
29 
45 
44 
23 
4? 
45 
6 
25 



37 
42 
34 
31 
32 
17 
37 
22 
6 
13 
20 
28 
24 
21 
21 
16 
19 



0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
0.00 
28.00 
17.86 
10.71 
2.70 
0.00 
0.00 
0.00 
0.00 
0.00 
43.24 
0.00 
0.00 
23.08 
0.00 
42.86 
8.33 
0.00 
0.00 
0.00 
0.00 



66.67 

96.30 

37.04 

45.95 

47.50 

16.67 

15.63 

11.11 

4.55 

44.83 

2.22 

95.45 

.S2.17 

86.67 

42.22 

66.67 

44.00 

57.14 

71.43 

72.97 

83.33 

100.00 

100.00 

68.75 

47.06 

45.9.S 

0.00 

0.00 

0.00 

5.00 

10.71 

62.50 

52.38 

100.00 

68.75 

5.26 



33.33 

3.70 

.59.26 

40.54 

0.00 

41.67 

0.00 

11.11 

63.64 

24.14 

60.00 

0.00 

0.00 

8.89 

35.56 

0.00 

20.00 

2 1 .43 

10.71 

13.51 

7.14 

0.00 

0.00 

15.63 

52.94 

10.81 

9.09 

50.00 

7.69 

0.00 

10.71 

12.50 

0.00 

0.00 

31.25 

21.05 



0.00 

0.00 

3.70 

13.51 

52.50 

25.00 

50,00 

63.89 

27.27 

13.79 

24.44* 

0.00 

0.00 

0.00 

2.22 

33.33 

4.00 

3,57 

7.14 

10.81 

9.52 

0.00 

0.00 

15.63 

0.00 

0.00 

90.91V 

33.33 

69.23 

95.00 

,35.71 

16.67 

4.76 

0.00 

0.00 

73.68 



0.00 

0.00 

0.00 

0.00 

0.00 

16.67 

34.38 

13.89 

4.55 

17.24 

13.33* 

4.55 

47.83 

4.44 

20.00 

0.00 

4.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

0.00 

16.67 

0.00 

0.00 

0.00 

0.00 

42.86 

0.00 

0.00 

0.00 



winter as compared to other seasons (F^qgq = 241.2. p < .0001, 
Fig. 2). In addition, scallop gonad weight was significantly greater 
for samples collected during the winter as compared to other sea- 
sons (Figgg = 107.6. p < .0001). However, both GSI and gonad 
weight varied significantly among dates among years (F,^!,.;^ = 
57.5, p < .0001: F^^^s, = 47.7. p < .0001. respectively). Specifi- 
cally, significant reductions in monthly means occurred in both 
GSI and gonad weight during the 1994/1995 winter (Fig. 3), which 
conesponds well to the poor gonad development .seen in the gonad 
condition inde.x above (Table 2). These reductions in GSI and 
gonad weight may have been a result of osmotic stress associated 
with the salinity minima during 1994 (Fig. 1). 

A clear peak in scallop reproduction existed during the winter, 
with mean GSI values exceeding 15'7r dry tissue weight (Fig. 2). 
However, based on gonad condition and GSI. significant repro- 
ductive effort also occurred in the fall and spring (Table 2. Fig. 3). 
On May 17, 1995 a bay scallop mass spawning event was ob- 
served. This event occuired while collecting individuals for repro- 
ductive assessment and natural population surveys. Specifically, 
scallops held in a catch-bag (n ~ 50-75) spontaneously released 
gametes. Consequently, independent of GSI, this observation 
showed that scallops do spawn in late spring. 

Support of year-long reproductive effort and success was as- 
sessed by the presence of small scallops (<20 mm shell height) 
collected in the field. Based on these data, it seems that scallops 
showed some signs of recruitment throughout he year (Table 3). In 
addition, during the summer of 1994. a companion study assessing 
the effects of seagrass habitat architecture on bivalve recruitment 
collected recruiting bay scallops (0.5-2 mm shell height) on novel 
substrata and from Thalassia testudinum (turtle grass). These data 
showed that scallops were recruiting to novel substrata at densities 
of 8.09. 4.33. and 6.07 individuals m"" for the months of July, 
August, and September, respectively. Based on the length of larval 
development (10-14 days, Sastry 1965, Lu and Blake 1997), these 
periods of recruitment correspond to potential spawning events 
during June. July, and August, when minimum values in both 
gonad weight and GSI existed (Fig. 3). 

DISCUSSION 

For species of economic value assessing growth, production, 
and reproduction in populations are essential for both wise man- 



-H 



•a 



E 

o 



c 
o 

O 




Fall 



Values expressed as percentage of sample classified in each category; n 
indicates the number of scallops collected for reproductive assessment; 
* indicates a scallop with 31 mm shell height exhibiting this condition; 
** indicates a .scallop with 34.1 mm shell height exhibiting this condition; 
¥ indicates a scallop with only male gonadal development. 



Winter Spring Summer 

Season 

Figure 2. Comparison of mean scallop CSI for seasons pooled across 
years: n = the number of scallops collected upon which the means are 
based; letters above seasons indicate significant differences among 
means in GSI (a = 0.05). 



Growth. Production, and Reprodiiction of Bay Scallops 



915 



Vi 

O 



30- 


■J 


GSI Index 
Gonad Weighl 




! 


2 5- 








1 " 


2 0- 




(i) Q 






1 5- 
I 0- 
5 - 
- 


V\ 

1 


1 


[ 



3 

a. 

ITO' 



0.4 a. 
0.2 £ 



1/22 
1993 



1/23 
1994 



1/23 
1995 



1/24 
1996 



Figure 3. .Seasonal patterns in scallop (JSI and gonad weight from 
individuals collected between November 1992 through October 1996. 
Peaks in (I.SI and gonad weight occurred during the winter, and mini- 
mum values were recorded during the summer. Significant interan- 
nual variability in gonad weight occurred and was significantly re- 
duced during the winter of 1994/1995. 



agement of healthy populations and conservation and enhancement 
of endangered populations. Results from this research have shown 
that bay scallops from St. Joseph Bay, Florida exhibit high growth 
rates throughout the summer but have dramatic declines in the fall 
(Table 1). This observation of differential growth as body size 
increases has also been shown for larvae and juveniles (Lu and 
Blake 1996) and was similar to results found by Barber and Blake 
(1983) for scallops collected from the eastern Gulf of Mexico. This 
seasonal trend was evident in both 1993 and 1994, but in 1994. 
reduction in salinity by 21%c may have had a significant impact on 
the natural growth rates of scallops. Although other researchers 
have shown that salinity has an impact on the survival of scallops 
(Duggan 1975. Mercaldo and Rhodes 1982). this is the first case 
where coupled measurements of physical parameters can be ap- 
plied directly to differential natural growth rates. 

Similarly, estimates of natural "mortality" (mortality plus emi- 

TABLE 3. 

Presence of recruiting scallops (<20 mm shell height) during 1994 
to 1996. 



Month 



1994 



1995 



1996 



February 




March 


t 


April 




May 




June 




July 


*-H 


August 


*-!- 


September 


*-H 


October 


t 


November 


t 


December 





t 

t 

NA 

NA 



Presence denoted by *; *-(• indicates recruiting individuals (<4 mm shell 
height) collected from suction dredge samples; t indicates samples were 
not collected during this month; NA indicates the termination of scallop 
collection. 



gration) from field studies ranged from 0.005 to 0.025 for non- 
placement periods (Table 1 ) and were similar to those of Allison 
and Brand (1995), who used a similar mark-recapture technique 
on kequipecten opeirulahs. In addition, the rapid dispersal after 
initial deployment has been recorded from other studies (Barbeau 
et al. 1996, Hatcher et al. 1996). However, the dramatic mass 
mortality seen on July 20, 1994 in the field suggests a direct 
correlation between scallop mortality and the decline in salinity 
associated with Tropical Storm Alberto (Fig. 1). The observed 
mass mortality in the field was also seen in the in.stantaneous 
mortality for mark-recapture scallops but correlates better with the 
salinity minima (Fig. 1, Table 1) and provides experimental evi- 
dence to the observed pattern of high mortality. Based on Mer- 
caldo and Rhodes (1982) estimates, the drop in salinity to \\7<c 
during July 1994 could have created conditions such that less than 
20% of the scallop population within St. Joseph Bay, Florida may 
have survived. This corresponds well with this author's inability to 
locate scallops for reproductive assessment during the fall of 1994 
in St. Joseph Bay and reported low abundances by Arnold et al. 
(1998) for scallop surveys for the same time period. 

The impact of reduced salinity on mortality may have had 
dramatic impacts on the population as a whole. Results from re- 
productive assessment of the population showed a significant re- 
duction in GSI and gonad weight during the following winter of 
1994/1995 (Fig. 3). Because reproductive output is directly asso- 
ciated with gonad development (Sastry 1970). the reduction in 
gonad weight during the winter of 1994/1995 and the relative lack 
of gonad development seen in the condition index (Table 2) sug- 
gests a limited reproductive output during this season. Given the 
short life span of A. irradians (Gutsell 1930) and significant in- 
creases in GSI and gonad weight during the winter of I995/I996, 
it seems that no multiple year impacts were evident and corre- 
.sponds to Barber and Blake's ( 1986) suggestion that west Florida 
populations may be stable given their relatively high reproductive 
effort. In fact, both scallop GSI and gonad weights were the great- 
est during the fall of 1995 and winter 1995/1996 suggesting a full 
recovery of the population, despite losses during the previous year. 

By assessing the reproductive potential of the population using 
a gonad condition index, the data suggest that at least some mem- 
bers of the population show reproductive output throughout the 
year (i.e.. very ripe, postspawn. Table 2), and this response has 
been observed for other scallop species as well (O'Connor and 
Heasman 1996). Data clearly show peaks in reproduction for St. 
Joseph Bay scallops occurring in the winter (Figs. 2, 3), later than 
that of other southern Florida populations (Barber and Blake 1983, 
Arnold et al. 1998) and approximately opposite those of Atlantic 
populanons (Gutsell 1930, Sastry 1970, Bricelj et al. 1987b, Peter- 
son et al. 1996). In addition, the observation of the spawning event 
on May 17, 1995 may correlate with increasing water temperatures 
(Fig. 1), which seem to induce the Atlantic population to spawn 
(Sastry 1963, Sastry 1970, Bricelj et al. 1987b) and gives a proxi- 
mal mechanism to explain the multiple spawning seen in St. Jo- 
seph Bay. Florida. This study differs from others that have as- 
sessed reproduction, because presence of juveniles was also as- 
sessed throughout the year. Consequently, results suggest that this 
population exhibits year-long reproduction, as evidenced by juve- 
nile recruits (Table 3). even when mean GSI and gonad weight 
were at their minimum values during the year (Fig. 3). 

The mass mortality observed in the field on June 24, 1996, 
however, has few direct physical correlates (e.g., salinity). Red tide 



916 



Bologna 



was observed in the northeastern Gulf of Mexico during this time 
period (J. Stout, pers. comm.), but no clear documentation of the 
event currently exists. However. Tester and Steidinger ( 1997) have 
shown that red tide {Gymnodinium breve) is a common member of 
the phytoplankton communities in the Gulf of Mexico and is 
present all year. In addition, Tettelbach and Wenczel (1993) and 
Smolowitz and Shumway (1997) have shown dramatic toxic ef- 
fects of nuisance algal blooms on both juvenile and adult survival 
and growth. Therefore, it might be possible that the mass die-off 
observed in the field on June 24 may have been a result of a local 
red tide event. 

In summary, scallops from St. Joseph Bay showed significant 
seasonal and annual variability in growth and reproduction. Spe- 
cifically, the effects of reduced salinity associated with Tropical 
Storm Alberto had significant impacts on the population through 
direct mortality and reduced gonadal tissue development. How- 
ever, these probably only affected a singe cohort of A. irradians. 
because the population seemed to recover during 1995 and 1996, 
and data showed a significant increase in reproductive output. 
Unfortunately, few studies have assessed bay scallop production. 
Perhaps this is because of the relatively short life span of A. irra- 
dians or the lack of mark-recapture estimated field growth and 
mortality rates from natural populations. Results from this research 
indicate significant annual variation, probably mediated by physi- 



cal parameters, and this information may provide a basis for future 
comparisons among bay scallop populations. In addition, the pres- 
ence of recruiting scallops throughout most months of the year and 
the identification of a significant spring spawning event, suggests 
that future assessments of the reproductive output and population 
structure of Gulf of Mexico bay scallops target the entire year and 
not only winter peaks in reproduction. Last, the identification of 
small scallops involved in reproduction (<35 mm shell height) 
suggests that future research should also address the potential these 
individuals have on the reproductive output and success of popu- 
lations. 

ACKNOWLEDGMENTS 

This re.search was funded by grants from the Mississippi- 
Alabama Sea Grant Consortium and a Lerner-Gray Fellowship 
from the American Museum of Natural History. Logistical support 
was provided by the Dauphin Island Sea Lab. I would thank J. 
Harper and G. Eisel for assistance in the laboratory. J. Zande. C. 
Moncreif. S. Lores. S. Sklenar, and Y. Gonzales for assistance in 
the field, and J. Duffy-Anderson and three anonymous reviewers 
for helpful comments on this manuscript. This manuscript is con- 
tribution #305 for the Dauphin Island Sea Lab. The author's 
present address is Rutgers University Marine Field Station, c/o 132 
Great Bay Blvd., Tuckerton, New Jersey 08087-2004. 



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Growth, Production, and Reproduction of Bay Scallops 



917 



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concenlricus Say, reared in the laboratory. Bull. Mar. Sci. 15:417^35. 



Sastry, A.N. 1970. Reproductive physiological variation in latitudinally 
separated populations of the bay scallop, Aequipeclen irradians 
Lamarck. Biol. Bull. 138:56-65. 

Sastry, A.N. 1979. Pelecypoda (excluding Ostreidae). pp. 113-292. In: 
A. C. Giese and J. S. Pearse (eds.). Reproduction of Marine Inverte- 
brates. Academic Press, New York. 

Savastano, K. J., K. H. Pallet & R. L. Iverson. 1984. Estimating vegetation 
coverage in St. Joseph Bay, Florida, with an airborne multispectral 
scanner. Phologram. Eng. Remole Sensing 50:1159-1170. 

Smolowitz, R. & S. Shumway. 1997. Possible cytotoxic effects of the 
dinoflagellate, Gyrodinium aureolum. on juvenile bivalve molluscs. 
Aquacul. Int. 5:291-300. 

Summerson, H. & C. Peterson. 1990. Recruitment failure of the bay scal- 
lop, Argopeclen irradians concenlricus. during the first red tide, Pty- 
chodiscus brevis. outbreak recorded in North Carolina. Esluaries. 13: 
322-331. 

Tester, P. & K, Steidinger. 1997. Gymnodiniuni breve red tide blooms — 
initiation, transport, and consequences of surface circulation. Limnol. 
Oceanogr 45:1039-1051. 

Tettelbach, S. 1986. Dynamics of crustacean predation on the northern bay 
scaWop. Argopeclen irradians irradians. Ph.D. Dissertation, University 
of Connecticut, Storrs, CT. 229 p. 

Tettelbach, S. & P. Wenczel. 1993. Reseeding efforts and the status of bay 
scallop Argopeclen irradians (Lamarck, 1819) populations in New 
York following the occurrence of "brown tide" algal blooms. / Shell- 
fish Res. 12:423-431. 

Thayer, G. W, & H. H, Stuart. 1974. The bay scallop makes its bed of 
seagrass. Mar. Fish. Rev. 35:27-30. 

Wilbur, A. & P. Gaffney. 1997. A genetic basis for geographic variations 
in shell morphology in the bay scallop Argopeclen irradians. Mar. Biol. 
128:97-105. 



J.ninnil of Shellfish Reseanh. Vol. 17, No. 4. 919-923. 1998. 

GONADAL MATURATION, CONDITIONING, AND SPAWNING IN THE LABORATORY AND 
MATURATION CYCLE IN THE WILD OF CERASTODERMA GLA t/Ct/M BRUGUIERE 



P. TROTTA AND C. A. CORDISCO 

IstituUi per la Studio degli Ecosislenii Costieri 
Consiglio Naziomile delle Riceirhe 
1-7 WW Lesina (Fg). Italy 

ABSTRACT Cerastoderma glaiuiim Briigiiiere has a wide geographical distribution in Europe, from the Mediterranean to the North 
Atlantic and the Baltic Sea. This cockle is found mainly in coastal embayments and lagoons on muddy, soft bottom. The different 
reproductive behavior of cockles is related to latitude. In Lesina Lagoon, cockles initiate gametogenesis several times per year; this has 
been confirmed in laboratory tests where cockle.s are provided ample Tahitian Isochrysis aff. gatbana (clone T-ISO). In the laboratory, 
cockle biomass (density 400-800 g/m") releases viable gametes all year in a relatively wide range of temperatures (9-24°C). During 
12 months of repeated spawning, cockles released a mean of 68,100 eggs/cockle, with an average of 1.13 spawning per week. Because 
of its high genetic variability, which accounts for its adaptability and ease of spreading in environments with striking changes of 
physical and chemical aquatic conditions, cockles may have an excellent potential for mariculture application. In addition cockles may 
play an important ecological role in reducing the particulate organic load of eutrophic lagoons and embayments with a wide range type 
of salinity and thermal characteristics. 

KEY WORDS: cockle, bivalve, mollusc, spawning, conditioning 



INTRODUCTION 

Bivalve mollusks derived from fisheries and fanning are a con- 
siderable part ot the total sea food landed (30Vr of the world 
aquaculture, Manzi and Castagna 1989), With the success of 
worldwide mussel cultivation, research efforts are underway to 
match the production of clams, oysters, and scallops. Indeed, dur- 
ing the last two decades, scientists have successfully reared more 
than 20 new bivalve species (Manzi and Castagna 1989). Despite 
such success, the scientific community is being asked to propagate 
potential new species for fanning and/or restocking overexploited 
areas. In addition to the edible aspect of farmed organisms, bivalve 
molluscs are important as filter feeders for the removal of particu- 
late organic matter load of eutrophic lagoons and embayments 
(Glude 1984, Mann and Ryther 1977). One candidate bivalve to 
this purpose is the dioecious cockle Cerastoderma i;laiieiiiu Bru- 
guiere. This cockle commonly occurs from the Baltic and North 
Seas to the Mediterranean Sea (Boyden and Russell 1972. Zaouali 
1975, Labourg and Lasserre 1980, Brock and Christiansen 1989) 
(Fig, 1). C. glaucum preferentially dwells on muddy bottoms of 
lagoons and estuaries. Because of their wide genetic variability 
(Brock and Christiansen 1989), cockles occur in frontier areas 
where the environmental conditions fluctuate at the extremes 
(Rygg 1970, Zaouali 1975, Labourg and Lassen-e 1980), This 
makes C. glaucum an interesting subject for farming and/or reduc- 
ing the environmental impact of organic loading in estuarine sys- 
tems. This study examines the maturation conditioning and spawn- 
ing of cockles in relation to water temperature and diet regimes, 

MATERIALS AND METHODS 

This study examines the annual gametogenic cycle of C. glau- 
cum from Lesina Lagoon (Fig. 2), In addition some C. glaucum 
specimens were tested in the laboratory for maturation condition- 
ing and successive spawning. Laboratory trials were carried out 
either in 40- to 600-L tanks. These vessels were supplied with 
lagoon water after filtering through a l-|ji,ni pore size cartridge. 
The residence time of the water in the 40- and 600-L tanks was 6 
and 48 hours, respectively. For the conditioning and spawning 
tests, field-collected cockles with minimum average size of 4 g 



were stocked in 40-L tanks with biomass density ranging from 400 
and 8(J0 g/m". They were fed with unicellular algae culture of 
Tahitian Isochiysis aff. galhana Green (clone T-ISO), Dunaliella 
tertiolecta (Butcher), and Tetruselmis sueeica Kylin (Butcher), 
grown in the artificial light (Trotta 1981) and fed to cockles over 
24 hours at the rate of 2,5 to 5% (dw/dw) per day. Photoperiod was 
set at 12 light/12 dark. Incoming water salinity ranged from 18 ppt 
to 38 ppt. 

The maturation phase of the gonads were recorded according to 
the description by Boyden (1971) and Wolowicz (1987). 

• .stage I. undifferentiated; beginning of gametogenesis; gonads 
are not developed and sex is not yet distinguishable; 

• stage II. developmental; gonads start to develop in the foot 
tissue and around the digestive gland; sex is distinguishable; 

• stage III. gonads developed; tnade of a compact tissue; white 
cream streamed line in the male, yellow cream granular isles in 
the female: 

• stage IV. reproductive stage; inflated and ripe gonads; isles in 
the females and stream line in the males; flowing; 

• stage V. rest; this is the postreproductive phase or regressive 
phase; the visceral mass becomes flaccid; gonads are failed in. 
often with a small number of remaining gametes, which are 
distinguishable with difficulty because of the presence of some 
corpi lutei. 

Experiment I. Maturation Conditioning of C. Glaucum Fed T-ISO 
and D. Tertiolecta 

Fifty cockles collected at the Fortore River outlet (average size 
4.05 g-l-/-0.96 (2s) — 95*7? confidence intervals — equivalent to 417 
g/m") were assigned to two 40-L tanks. One tank received a culture 
of T-ISO and the other a culture of D. tertiolecta at a daily rate of 
2.5 to 5% (dw/dw). Cockle mean weight in grams was taken at the 
start and at 30 and 60 days into the experiment. Twenty-five speci- 
mens left after the random selection were not fed and were con- 
sidered as blank (control group in a 40-L tank). At regular inter- 
vals, cockles were biopsied to determine gametogenic state. At the 
end of the experiment, 20 cockles per treatment were taken to 
check the gonadal stage of maturation. 



919 



920 



Trotta and Cordisco 




Figure 1. The reported occurrence of C. glaucum in Europe. 

Experiment 2. Maturation Conditioning of C glaucum /prf T-ISO and 
T. suecica 

A second step of the maturation conditioning started with cock- 
les at the stage I (undifferentiated) and gonadal development. This 
experiment was conducted to confirm the previous test. Sixty 
cockles sampled from the Varano Lagoon (average size 4.54 
g+/-2.6 equivalent to 542 g/m") were assigned to two 40-L tanks 
and fed, respectively, T-ISO and T. suecica at a daily rate of 2.5 to 
5% (dw/dw). 

Experiment 3. Spawning of Cockles Fed T-ISO and 50/50 Mixture of 
T-ISO/T. suecica, but Previously Conditioned to Maturation in a 
600-L tank and Fed T-ISO 

After the confirmation of the maturation conditioning from the 
previous experiment, 500 g of cockles, sampled from the Lesina 
Lagoon, corresponding to 782 g/m", was put in a 600-L tank for 
over 2 months and fed T-ISO at the rate of 1% (dw/dw). Cockles 
were assigned to two 40-L tanks (average size 7.68 g +/-3.94 
corresponding to 800 g/m") at 26 specimens per each vessel. Cock- 
les received T-ISO and a 50/50 mixture of T-ISO/r. suecica. re- 
spectively, at a daily rate of 2.5 to 5% (dw/dw). 

After each spontaneous spawn, eggs were collected on a 80-(j.m 
sieve after draw ing the 40-L tank. Eggs were placed in a graduated 
cylindrical vessel with gentle aeration. A 2.5-mL subsample was 
counted for number of eggs with a 2.5-mL inverted microscope 




Figure 2. C. glaucum used in this study were collected from Lesina 
Lagoon, Italy. 



chamber. The counts were validated with the x' test (a = 0.05). 
The laid eggs were assigned to a four classes ranked as follows: 

• first-class interval ( 1 ) up to 1 50.000 eggs/tank; 

• second-class interval (2) from 150,000 to 300,000 eggs/tank; 

• third-class interval (3) from 300,000 to 600,000 eggs/tank; 
and 

• fourth-class interval (4) more than 600,000 eggs/tank. 

C. glaucum sampled in the wild 

C. glaucum were collected periodically in the Lesina Lagoon as 
well as at the border sites from 1995 to 1996. Collecting in these 
latter sites was made necessary when cockles from Lesina Lagoon 
were lacking. 

RESULTS 

Experiment I. Maturation Conditioning of C. glaucum Fed T-ISO and 

D. tertiolecta 

r-test showed that cockles fed T-ISO (t68 10.1 a = 0.05) 
increased in live weight from 4.05 to 5.37 g in 60 days as com- 
pared to a slight increase of those fed D. teniolecta (t68 2.164 a 
= 0.05) from 4.05 to 4.31 g (Table 1). The difference of weight 
gain in cockles fed T-ISO and D. tertiolecta on day 60 was sig- 
nificant (t38 7.515 a = 0.05) (Table 1). 

The specimens fed T-ISO resulted in 100% of cockles with ripe 
gonads (stage III and IV) and a sex ratio of 12F:8M, i,; however, 
only 45% of the cockles fed D. tertiolecta had recognizable go- 
nads. Most of these latter were in the regression stage (V). The sex 
ratio for cockles fed D. tertiolecta was 5F:4M, , ,, w ith 1 1 cockles 
being undifferentiated. For cockles fed T-ISO, the foot appeared 
fleshy, yellow-orange colored, and the digestive gland showed 
large ocher brown acini; whereas, those fed D. tertiolecta had flat 
pale yellow foot and small pale brown acini. The starved cockles 
(control group), at the end of the experiment, showed developed 
gonads (stage II and 111), with a sex ratio of 9F:9M:1U,,,, but 
reduced size of foot and pale colored digestive gland. 
(F = female, M = male. U = undifferentiated) 

Experiment 2. Maturation Conditioning of C. glaucum Fed T-ISO and 
T. suecica 

Field cockles collected for this study were all in the undiffer- 
entiated stage (I). After 2 months on the microalgae diets, cockles 
commenced spawning (Fig. 3), which lasted for 3 months. By 
midMarch, the surviving specimens (23% T-ISO, 33% T. suecica) 

TABLE 1. 

Average live weight of cockles in grams fed with T-ISO and 
D. tertiolecta 



T-ISO 
Start 
.^0 Days 
60 Days 

D. tertiolecta 
Start 
30 Days 
60 Days 



Average W 


eight 


Standard 


in g 




Deviation s 


4.05 




0.48 


4.62 




0.65 


5.37 




0.52 


4.05 




0.48 


4.25 




0.42 


4.31 




0.36 



Maturation Conditioning of Cerastoderma glaucum 



921 




Figure 3. Egg emission from C. glaucum broodstocli fed either T-ISO or T. siiecica and related water temperature. 




^y^^ 



1 . i 



li ii i H ii i i i ii i ii ii ii i iiiiiiiiiiiiii i iiiiii ii ii ii iiiiiiiiiiiiiiiiiiiiii i iiiiiiiiiii i iiii i i ii iiiiiii i ii i iiiiii i ii^ 




— -^ ■* > 



X X 

vO o 




a es « 



n 



""""^M. 



p ?< s - 



Figure 4A. Egg emission from C. glaucum broodstock fed T-ISO and related water temperature. Figure 4B. Egg emission from C. glaucum 
broodstock fed a 50:50 mixture of T-ISO and T. suecica and related water temperature. 



922 



Trotta and Cordisco 



TABLE 2. 

Number of emission and total eggs per year laid by cockles fed 

either T-ISO and a 50:50 mixture of T-ISO/7". stiecica in 

Experiment 3 





T-ISO 


T-ISO/T. suecica 


Class interval 4 


3 


3 


Class interval 3 


17 


20 


Class interval 2 


25 


20 


Class interval 1 


14 


15 


Total emis./year 


59 


58 


No emis./week 


1.13 


1.12 


No eggs/specimen/year 


681.000 


687.000 



had ripe gonads (stage III and IV), although those fed the latter 
microalga appeared undernourished after the biopsy test. 

Experiment .?. Spawning of Cuckles Fed T-ISO and 50/50 Mixture of 
T-ISO/T. suecica, and Previously Conditioned for 2 Months to 
Maturation in a 600-L tank with a T-ISO Feeding Regime 

The cockles conditioned in a 600-L tank started to spawn im- 
mediately after stocking in the 40-L tanks. The emission of the 
gametes occurred for 12 months, during which the water tempera- 
ture regime ranged from 9 to 24"C (Fig. 4A, B ). The specimens fed 
either T-ISO or the mixture T-ISO/7". suecica. spawned in total 59 
and 58 times in the course of the 12 months, with an average of 
1.13 and 1.12 times per week, respectively. The mean annual 
number of laid eggs was 681,000 and 687.000 per cockle for 
animals fed T-ISO and T-ISO/F. suecica. respectively (Table 2). 

C. glaucum Sampled in the Wild 

From January 1995 to October 1996, cockles were found in all 
stages of maturation, with individuals occurring in the undifferen- 
tiated to ripe stage at each time period (Fig. 5). 

DISCUSSION 

For many bivalves, the period for hatchery operators to obtain, 
condition, and spawn animals is short. This is because of different 



factors, among which the most important are water temperature 
and feeding regime (Helm et al. 1973, Helm 1977, Rossi et al. 
1994, Heasman et al. 1996, Wilson et al. 1996). Manzi and 
Castagna (1989) make exhaustive mention of difficulties met by 
bivalve mollusks hatchery operators when they want to prolong 
(anticipating and retarding) the natural spawning period. Alterna- 
tive methods for extending spawning imply costly procedures for 
heating and cooling the water during the conditioning or importing 
the broodstock from lower or higher latitude sites. Beattie ( 1995) 
gives an economy view of brookslock management and shows that 
lower costs result when the parent bivalve mollusks are kept in 
captivity and conditioned, as compared to the continuous supply of 
wild caught broodstock. Our study shows that it is possible to get 
viable gametes from C. glaucum. and thereafter seeds, all year at 
a relatively wide range of temperatures (Fig. 4A, B). 

As with most bivalve mollusks, the phase of maturation con- 
ditioning with selected algae feeding is obligatory (Millican and 
Helm 1994. Heasman et al. 1996). For C. glaucum with proper 
conditionmg, as shown in experiment 3, it is possible to maintain 
viable broodstock for seed production all year. This study confirms 
that D. tertiolecta is of poor nutritional value for C. glaucum. as it 
is for other bivalve mollusks (Millican and Helm 1994). However. 
T-ISO is shown to be an adequate food source for the maturation 
conditioning phase as well as for prolonged spawning period of C. 
glaucum. 

C. glaucum is widely distributed throughout Europe. Brock and 
Wolowicz (1994) mention that this species enters sexual matura- 
tion phase once a year in the Baltic Sea and twice a year in the 
north Mediterranean Sea. Zaouali ( 1975) states that the cockle has 
developed gonads all year in the lagoons of the coastal African 
Mediterranean Sea, with the exception of 1 month in autumn. 
Likewise, C. glaucum from Lesina Lagoon and border areas had 
sexually developed specimens all year (Fig. 5). Unfortunately, live 
cockles could not be obtained at each sampling interval in this 
study because of the high mortality of C. glaucum after prolonged 
anossic period typical of eutrophic lagoon or the poor transparency 
of the water caused by wind generated resuspension of silt into the 
water column. From these findings, the reproductive potentiality of 
this filter feeder seem to be important in the trophic economy of 
the Lesina Lagoon in all seasons and makes this lagoon more 



specimen npe 
' specimen undifferentiated 




Figure 5. Maturation phase of gonads in C. glaucum sampled in the wild, during 2 years of survey. To facilitate the figure reading, fertile 
specimens from stage II to IV were reported as ripe. 



Maturation Conditioning of Cerastoderma glaucum 



923 



similar lo the southern Mediterranean wetland ratlier than to the 
northern one. 

CONCLUSION 

By conditioning cockles on T-ISO in the laboratory, cockles 
ripen and are able to produce viable seeds all year. In light of the 
fluctuating presence of live cockles and the abundance of empty 
shells in several places of the lagoon, it seems that, despite the high 
nutritional value of the suspended organic matter (organic particles 
and microphytoplankton) and the frequent finding of veliger stages 
in the plankton sampling (Cordisco 1996), the lagoon does not 
properly support the reproductive potentiality of this organism. 
Thus, this bivalve mollusk might play an important ecological role 
in eulrophic lagoons by converting the energy of organic particles 
into food for such cockle predators as sea bream (Barbaro et al. 
1982). This cockle is found in the Lesina Lagoon in places with 



different salinity (10 ppt and ."iO ppt). m the ponds near Margherita 
di Savoia salterns (60 ppt), in the Bay of Cadiz (Spain) (50-60 
ppt). It also inhabits such frontier as the Fortore River outlet, 
which undergoes frequent daily salinity changes (0 ppt to 36 ppt). 
Considering the temperature range tolerated by these cockles for 
thriving and spaw ning. in this study, it is intriguing to consider this 
bivalve mollusk as useful for restocking and production recovery 
of eutrophic lagoons and embayments with considerable loads of 
particulate organic matter. 

ACKNOWLEDGMENTS 

The authors thank Mr. A. D'Amato, Mr. P. Cammarino, and 
Mr. N. Dentale for their continuous help and Miss E. Carlino for 
her fruitful collaboratiim. The work was supported by the 4th 
Triennal Plan "Aquaculture and Fishery" of the Italian Ministry of 
Agriculture. Food Resources, and Forestry. 



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Beattie, H.J. 1995. Serial spawning of the geoduck clam (Panopea 

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cockles Ceraslodernia ediile and C. t^laitciim. J. Mar. Biol. Ass. U.K. 

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Brock, V. & M. Wolowicz. 1994. Comparisons of European populations of 

the Cerasioderma i^laucmn/C. lamarcki complex based on reproductive 

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ticolare riguardo al moUusco bivalve Cerasioderma glaucum. Docu- 

mento consuntivo relativo all' atdvita di formazione svolta. nell'ambito 

del Programma del Fondo Strutturale Europeo (FSE). marzo 1995- 

febbraio 1996. 
Glude, J. B. 1984. The applicability of recent innovation to mollusc culture 

in the western Pacific island. Aquaculture 39:29—13. 
Heasman, M. P., W. A. O'Connor & A. W. J. Frazer. 1996. Temperature 

and nutrition as factors in conditioning broodstock of the commercial 

scallop Pecleu fiimalus Reeve: Aquaculture 143:75-90. 
Helm, M. M„ D. L. Holland & R. R. Stephenson. 1973. The effect of 

supplementary algal feeding of a hatchery breeding stock of Oslreu 

edulis L. on larval vigour. / Mar. Biol. Ass. U.K. 53:673-684. 
Helm, M. M. 1977. Mixed algal feeding of Osirea edulis larvae with Iso- 



chrysis galbanu and Tetraselmis suecica. J. Mar. Biol. Ass. U.K. 57: 
1019-1029. 

Labourg. P. J. & G. Lasserre. 1980. Population dynamic of Cerastoderma 
glaucum in an artiUcial lagoon of the Arcachon region. Mar. Biol. 
60:147-157. 

Mann, R. & J. H. Ryther. 1977. Growth of six species of bivalve molluscs 
in a waste-recycling-aquaculture system. Aquaculture 11:231-245. 

Manzi. J.J. & M. Castagna (eds.). 1989. Clam mariculture in North 
America. Elsevier, New York. 461 pp. 

Millican. P. F. & M. M. Helm. 1994. Effect of nutrition on larvae produc- 
tion in the European flat oyster, Osirea edulis. Aquaculture 123:83-94. 

Rossi, R.. D. Campioni. A. G. Conte, F. Paesanti & E. Turolla. 1994. 
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risultati. Rivista Italiana Acquaculturu 29:53-62. 

Rygg, B. 1970. Studies on Cerasioderma glaucum (Poiret). Sarsia 43:65- 
80. 

Trotta, P. 1981. A simple and inexpensive system for continuous mono- 
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Wilson, J. A., O. R. Chaparro & R. J. Thompson. 1996. The importance of 
broodstock nutrition on the viability of larvae and spat in the Chilean 
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Wolowicz. M. 1987. A comparative study of a reproductive cycle of cock- 
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34:91-105. 

Zaouali, J. 1975. Study of the sexual cycle of Ceraslodenna glaucum in 
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Jdiinml of Shellfish Research. Vol. 17. No. 4. 925-929, 1998. 

THE OCCURRENCE OF JUVENILES OF THE GRAPSID CRAB CHASMAGNATHUS 
GRANULATA IN SIPHON HOLES OF THE STOUT RAZOR CLAM TAGELUS PLEBEWS 

JORGE L. GUTIERREZ AND OSCAR O. IRIBARNE 

Depurtamento de Biologi'a (FCEyN) 
Universidad Nacional de Mar del Pkila 
CC573 Correo Central 
Mar del Plata (7600), Argentina 

ABSTRACT Previous samplings for megalopae and juvenile instars of the southwestern Atlantic burrowing crab Chasmagnathus 
gramdata showed that they occur at high densities in adult crab burrows. Nevertheless, this study documents the occurrence of 
juveniles of this species in siphon holes of the stout razor clam Tagehis plebeius after a settlement event. Carapace width of the crabs 
collected ranged between 1.9 mm and 4.5 mm (2nd to 8th instari. A significantly higher proponion of crabs were found inhabiting 
inhalant instead of exhalant siphon holes. The siphon holes occupied by crabs showed a significantly larger diameter than those not 
occupied by them, and a significant positive correlation was found between siphon hole diameter and crab carapace width. A lateral 
chamber connected to the inhalant siphon gallery was observed in siphon holes inhabited by larger crabs. No correlation was found 
between the density of pairs of siphon holes and the proportion of pairs of siphon holes inhabited by crabs. However, the proportion 
of pairs of siphon holes occupied by crabs was higher in a site with low density of adult burrows in relation to adjacent sites with high 
density of adult burrows. We propose that the presence of clam siphon holes allows crab larvae to colonize poorly structured habitats 
where adult crab burrows are absent or at low densities, making possible the growth of areas inhabited by crabs. 

KEY WORDS: Habitat structure. Chasmagnathus gramilalu. Tagehis plebeius, settlement, siphon holes 



INTRODUCTION 

In shallow maiine environments, high densities of juvenile life 
stages or small-sized species of decapod crustaceans are com- 
monly as.sociated with habitats that show a complex tridimensional 
structure, such as cordgrass marshes (Zimmerman et al. 1983). 
seagrass meadows (Thomas et al. 1990), mangrove roots or cano- 
pies (Wilson 1989), tubiculous polychaete reefs (Gore et al. 1978), 
beds of mollusk shells (Gunderson et al. 1990). woody debris 
(Everett and Ruiz 1993). or cobble (Wahle and Steneck 1991 ). The 
habitat structuring elements serve as obstacles for predator activi- 
ties and also act to minimize the injurious effects of the different 
disturbance sources and environmental extremes (Kneib 1984). 
Thus, mortality risk for these organisms is lower in structurally 
complex sites with respect to adjacent flat areas (e.g.. Heck and 
Thoman 1981, Fernandez et al. 1993a). Although differential mor- 
tality after settlement may account for the higher density of deca- 
pods in structurally complex habitats (Johns and Mann 1987. 
Fernandez et al. 1993a). decapod larvae may actively select com- 
plex habitats as settlement sites (Botero and Atema 1982, Fernan- 
dez et al. 1993a, Fernandez et al, 1994). This preference may also 
be shown by juveniles (Johns and Mann 1987. Fernandez et al. 
1993a). 

The grapsid crab Chasmagnathus granidata Dana is one of the 
most abundant macroinvertebrates in saltmarsh and estuarine en- 
vironments of the southwestern Atlantic (Boschi 1964). Its distri- 
bution ranges from Rio de Janeiro (23°S. Brazil) to the San Mati'as 
Gulf (41°S. Argentina; Boschi 1964). This gregarious species ex- 
cavates and maintains semipermanent open burrows in the inter- 
tidal, from the soft bare sediment flats to areas vegetated by the 
cordgrass Spartina densiflora (Spivak et al. 1994. Iribame et al. 
1997). and behaves as deposit feeders in mud flats and as herbivo- 
rous-detritivorous in 5. densiflora marshes (Iribame et al. 1997). 
Because of their high density (up to 30 adults ■ m""; J. Gutierrez 
pers. obs.) and the effect of their deposit feeding and burrowing 
activities on sediment composition (Botto and Iribame 1997) and 
benthic community structure (Botto and Iribame 1996). this spe- 
cies is likely to play a key role in determining the structure of SW 



Atlantic marshes and estuarine soft-bottom communities. The stout 
razor clam Tagelus plebeius Solander is a deep burrowing bivalve 
species that constnicts permanent burrows and siphon holes (Hol- 
land and Dean 1977). This species is distributed in estuarine en- 
vironments of the western Atlantic coast from North Carolina 
(34°N, USA; Holland and Dean 1977) to the San Mati'as Gulf 
(41°S. Argentina; Iribame and Botto 1998). attaining densities of 
200 ind. ■ m'- (Holland and Dean 1977. Iribame et al. 1998) and 
coexisting with C. gramdata in intertidal mud flat areas in most of 
their distribution range. Although there is no information, this 
coexistence pattern may produce several direct and indirect inter- 
actions. 

Recently settled megalopae and juvenile Chasmagnathus 
granidata are found at high densities in association with adult crab 
burrows, and dense burrow beds seemed to be the major nursery 
habitat for this species (Spivak et al. 1994). However, during ob- 
servations performed after a settlement event, we noticed the pres- 
ence of juvenile C. gramdata dwelling in siphon holes of Tagelus 
plebeius. Given that this type of shelter may be important, the 
purpose of this work is to describe the pattern of siphon hole use 
by juvenile C. granidata. focusing on; (1) size and molting instar 
of the crabs using the holes; (2) type of siphon hole occupied by 
crab (inhalant or exhalant); (3) differences between siphon holes 
occupied and not occupied by them; and (4) the percentage of 
holes occupied and density of crabs dwelling in siphon holes in 
crab beds with different densities of adult burrows. 

MATERIALS AND METHODS 

This study was carried out in the Mar Chiquita coastal lagoon 
(Argentina. 37°32' to 37°45' S. 57° 19' to 57°26' W). a 46 km= 
body of brackish water (Fasano et al. 1982) affected by low am- 
plitude (<l m) tides (Lanfredi et al. 1987) and characterized by 
mud flats and a large sun'ounding cordgrass (Spartina densiflora) 
area (Olivier et al. 1972. Iribame et al. 1997). In soft sediment flat 
areas of the lagoon, the spatial coexistence between Chasmag- 
nathus granidata and Tagelus plebeius is a common phenomenon 
(e.g.. Olivier et al. 1972). Samplings were conducted during the 



925 



926 



Gutierrez and Iribarne 



late summer of 1998 in two areas (located approximately 10 m 
apart) at the same inteitidal level (0.5 m above mean lower low 
water [MLLW]). Both sites are characterized by sandy-silty sedi- 
ments and the presence of sediment depressions (often associated 
with C. gnuuilata burrows) whose sediment shows a higher or- 
ganic matter content with respect to those of the surrounding mud 
flats. However, they differ in the density of C. gramdata burrows 
(high burrow density site [HBD site]: x = 6.98 burrows • m"'^. SD 
= 5.41; low burrow density site [LED site]: x = 0.58 
burrows ■ m"", SD = 1.72) and the percentage of area covered by 
sediment depressions (HBD site: x = 51.29^. SD = 25.4; LBD 
site: x = 3.2%. SD = 3.01; Gutierrez and Iribarne unpubl. data). 
A random sampling using 4 m" square sampling units (SU) was 
conducted both at the HBD (n = 8) and LBD site (n = 10). The 
total number of pairs of clam siphon holes at each SU was counted. 
At the LBD site. 10 pairs of clam siphon holes were randomly 
taken from each SL'. inhalant and exhalant holes were detemiined 
by the presence of pseudofeces. and their diameters were measured 
to the nearest 0.01 mm. Later, the 10 pairs of measured siphon 
holes were carefully excavated. All crabs obtained were carried to 
the laboratory to be determined to the species level, and their 
carapace widths (CW) measured to the nearest 0.01 mm. The 
presence or absence of lateral burrow chambers was recorded. The 
remaining pairs of siphon holes of each SU of the LBD site and all 
the pairs of siphon holes of the HBD site were also excavated but 
only to determine presence or absence of crabs. The null hypoth- 
esis of no difference in the number of pairs of siphon holes be- 
tween the HBD and the LBD site was contrasted with the Mann- 
Whitney test (Conover 1980). A binomial test (Conover 1980) was 
used to test for differences in the proportion of crabs occupying 
siphon holes corresponding to the inhalant or exhalant siphon. A 
Wilcoxon test (Conover 1980) was used to evaluate for differences 
in the diameter between the hole occupied by the crab and the 
complementary of the pair, and a Mann-Whitney test (Conover 
1980) was employed to test for differences in the relation: diameter 
of the major siphon hole/diameter of the minor siphon hole be- 
tween crab inhabited and vacant pairs of siphon holes. A correla- 
tion analysis (Zar 1984) was perfomred to evaluate the existence of 
an association between crab size (CW) and hole diameter. The null 
hypothesis of no difference in the size (CW) between crabs occu- 
pying holes with and without lateral chambers was contrasted us- 
ing Student's /-test (Zar 1984). The possibility of association be- 
tween the number of siphon holes in a SU and the number of crabs 
per pair of siphon holes was evaluated using a correlation analysis 
(Zar 1984). A Mann-Whitney test (Conover 1980) was applied to 
test for differences in the relation: number of siphon holes occu- 
pied by crabs/total number of siphon holes and in the density of 
crabs occupying holes between the HBD and the LBD sites. Sha- 
piro-Wilk tests (Zar 1984) were used to test for normality of the 
data, and Levene's test (Underwood 1997) was applied to test for 
homocedasticity of the data. In those cases where sample sizes 
were unequal, the calculation of the test statistics were carried out 
using the corrections suggested by Conover (1980) and Zar (1984). 

RESULTS 

The density of pairs of siphon holes of Tagelus pleheius dif- 
fered significantly between sites (HBD: x = 43.56 pairs • m~~, SD 
= 5.41; LBD: x = 12.86 pairs ■ m"", SD = 10.06: Mann- 
Whitney test: Z = -6.42. p < .01). Carapace width of the crabs 
collected ransed between 1.9 and 4.5 mm (2nd to 8th instar; ac- 



cording to Rieger and Nakagawa 1995). A significantly higher 
proportion of crabs (86.677f) was encountered dwelling in the 
inhalant siphon holes (Binomial test: r, = 26, f, = 4, n = 30, p 
< .001) than in the exhalant hole. The siphon holes inhabited by 
crabs showed significantly larger diameter than their complement 
(crab: x = 5.81 mm, SD = 1.62; no crab = 4.46 mm. SD = 0.8; 
Wilcoxon test: Z = 4.31, p < .001; Fig. lA), and the ratio between 
the diameters of the major hole and the minor hole was signifi- 
cantly higher in pairs of siphon holes occupied by crabs (crab: x = 
1.31, SD = 0.27; no crab: 1.06, SD = 0.04: Mann-Whitney test: 
Z = -6.85. p < .001; Fig. IB). A significant positive correlation 
was encountered between crab carapace width and diameter of the 
siphon hole occupied by crabs (r^ = 0.80, df = 1, 28, p < .001, 
Fig. 2). 36.67% of the crabs construct lateral chambers associated 
to the siphon hole at depths ranging between 4.5 and 8 cm, and all 
the chambers were found in association with the inhalant tube. The 
crabs found in holes with chamber showed significantly larger 
carapace width with respect to those dwelling in holes without 
(chamber: x = 3.86 mm, SD = 0.51: no chamber: x = 2.76 mm. 
SD = 0.5; r-test: -5.78, df = 28, p < .001; Fig. 3). No significant 
relationship was found between the number of pairs of siphon 
holes in the sampling units and the number of pairs inhabited by 
crabs (r^ = 0.07, df = 1. 19, p > .05). The percentage of pairs of 
siphon holes occupied by crabs was significantly higher at the 
LBD site (LBD: x = 31.24%, SD = 5.38; HBD: x = 8.52%, SD 
= 2.34; Mann-Whitney test; Z = -3.55, p < .001; Fig. 4). How- 
ever, the density of crabs occupying holes did not differ between 



12 



F 


10 


E 








HI 


8 


\- 




LU 




? 




< 


b 


Q 




LU 




— 1 


4 


o 


X 





I ° I , 



a: 2.5 

LU 



Q 2 



1.5 



< 

o 

LU 

_l 

o 

X 

cr 
O 

z 

in 

o 

I 

o 



0.5 





B 






— \ — 




1 A 





CRAB 



NO CRAB 



Figure \. Median quantile box plots showing: (.\) the diameter of the 
siphon holes occupied by juvenile Chasmagnathus granulata and the 
coniplementaries of each pair: and (B) the ratio between the diameter 
of the major and minor hole for pairs of siphon holes with and without 
crab. 



Juvenile Crabs Dwell in Clam Siphon Holes 



927 




2 3 

CARAPACE WIDTH (mm) 

Figure 2. Carapace width of juvenile Chasmagnathiis graniilata 
against diameter of the siphon hole occupied by them. 



LBD 



HBD 



sites (LBD: x = 
crabs ■ m~'^, SD 



3.87 crabs ■ m 
= 1.51; f-test: t 



SD = 0.83; HBD: x = 3.4 
0.79, df = 16, p > .05). 



DISCUSSION 



Larval decapod settlement is primarily an active process in 
which larvae choose settlement sites based on sediment character- 
istics or chemical cues (Castro 1978. Botero and Atema 1982). 
Flume experiments demonstrated that megalopae of Chasmag- 
nalhiis gramdata actively swim in current conditions similar to 
those encountered in the field, and it was also proposed that they 
are able to select settlement sites (Valero 1998). In addition, the 
higher densities of recently settled megalopae and juveniles occur 
in association with adult crab burrows (Spivak at al. 1994), and 
metamorphosis of C. gramdata megalopae occurs earlier in the 
presence of chemical cues of adult conspecifics than with sea 
water alone (Gebauer et al. 1998). All these data suggest that 
megalopae of this species settle in response to adult-released 
chemical cues. In this context, the occurrence of juvenile C. grami- 
lala dwelling in siphon holes of stout razor clams cannot be ex- 
plained by means of selective settlement of megalopae. An alter- 
native hypothesis may be stated on the basis of competition be- 
tween cohorts. Settlement of C. gramdata megalopae occurs in the 
lagoon between December and June, with peaks in intensity (Spi- 
vak 1994). Thus, there is a potential for interaction between co- 
horts. Eariy cohorts of the Dungeness crab Cancer magister re- 
duces the abundance of subsequent cohorts in intertidal shell habi- 




CHAMBER NO CHAMBER 

Figure 3. Median quantile box plots showing carapace width of juve- 
nile Chasmagnatlius gramilata occurring al siphon holes with and 
without lateral chambers. 



Figure 4. Median quantile box plots showing the proportion of pairs of 
siphon holes occupied bv juvenile Chasmagnatlius granulata at the site 
of low (LBD) and high density of adult burrows. 



tats, and smaller juveniles migrate to the open fiats in response to 
high density of conspecifics (Fernandez et al. 1993b). Eariy juve- 
nile instars of C. granulata are subject of cannibalism by larger 
juveniles (Luppi et al. 1995). and it may be possible that a density- 
dependent migration from adult burrows to siphon holes has been 
carried out by crabs settling in late summer. 

Nevertheless, the possibility of direct settlement of C. granu- 
lata megalopae in siphon holes cannot be discarded. Muddy sand 
substrata (such as those occurring at both study sites) also pro- 
motes an eariier metamorphosis of C. granulata megalopae when 
compared with coarser sediment types. Despite the fact that a 
combination of muddy substrata and adult chemical cues deter- 
mines the shortest time to metamorphic molt, muddy sand sub- 
strata alone have an important effect (Gebauer et al. 1998). If we 
assume that muddy sand substrata alone may also induce settle- 
ment of C. granulata megalopae, settlement in sites other than 
adult burrows is plausible. In addition, Gebauer et al. (1998) found 
that artificial substrata of the same grain size do not have the same 
effect on metamorphic molt, suggesting that characteristics other 
than grain size are relevant for the induction of metamorphosis 
(see Pawlik 1992). As previously mentioned, sediment from adult 
burrows and sediment depressions in association with adult bur- 
rows are organically richer than those of the surrounding flat. 
Taking into account that this species behaves as a deposit feeder in 
unvegetated sediment fiats, it is likely that high levels of organic 
matter may function as a cue for the settlement of megalopae of 
this species. Although no data are available about the small-scale 
distribution of organic matter around bivalve siphon holes, in- 
creased levels may occur as a result of the enhancement of local 
particle deposition caused by flow convergence toward the inhal- 
ant siphon (see Ertman and Jumars 1988). Moreover, larvae may 
respond to physical factors (see Pawlik 1992). Negative bottom 
roughness elements (such as burrows and siphon holes) behave as 
hydrodynamically quiet microhabitats (DePatra and Levin 1989). 
Weak current conditions are commonly associated with habitats 
that have a complex tridimensional structure (e.g.. oyster beds, 
Wright et al. 1990). which may provide refuge for juvenile deca- 
pods. Dungeness crab C. magister megalopae are able to select 
positively for weak current conditions (e.g., Fernandez et al. 1994). 
In addition, metamorphosis of the blue crab Callinectes sapidus 
megalopae is accelerated by textural cues caused by the presence 
of eelgrass (Forward et al. 1994). Burrows or siphon holes may 
produce similar effects on C. gramdata megalopae. 



928 



Gutierrez and Iribarne 



Siphon holes occupied by juvenile Chasinagnatluis graimlata 
appeared modified with respect to the complementary of the pair. 
These crabs enlarge the diameter of the siphon holes occupied by 
them and larger crabs also construct lateral chambers. This may 
occur as a result to the provision of space for both the crab and the 
extended siphon. The thalassinidean shrimp Jci.xea noctiirna in- 
habit burrows of the echiurid Maxmiielleria lankesteii. modifying 
them by the excavation of semicircular side branches (Nickell et al. 
1994). In addition, it was proposed that / noctiirna probably ben- 
efit by the irrigation activities of the echiuran. which supply both 
oxygen and food (Nickell et al. 1994). Pinnotherid crabs Pinnixa 
schmiui and Scleroplax granulata behave as symbionts in burrows 
of the suspension feeding burrowing shrimp Upogebia pugettiensis 
of the Northwestern Atlantic (Kozloff 1987). Thus, the higher 
proportion of juvenile C. gniniihita occurring in siphon holes cor- 
responding to the inhalent siphon may be the result of selectively 
favoring a site with a high inflow of food particles. However, we 
do not know to what extent clam attributes are affected (e.g., 
decreased filtration and oxygen consumption rates in mussels with 
pea crabs in the mantle cavity; Biembaum and Shumway 1988), to 
determine if the relationship is commensalistic of parasitic. 



It is also interesting to notice that the percentage of holes oc- 
cupied by juvenile Chasmagnathiis gninutata was higher in the 
site with a low density of adult burrows than in the site with a high 
density of adult burrows. This pattern indicates that where habitat 
structure is poor, small scale sediment features may be important 
as settlement sites and/or refuges for this species. We also believe 
that the strategy of settling into holes of bivalves or other burrow- 
ing species may be a fairly common phenomenon. For example, 
new settlers of the Dungeness crab Cancer inagister have been 
found in holes of burrowing shrimps in the Grays Harbor estuary 
(Washington, USA: O. Iribarne, pers. obs.) but their importance 
has never been quantified. 

ACKNOWLEDGMENTS 

Support for this project was provided by grants from the 
Universidad Nacional de Mar del Plata. CONICET (PIA No; 
6097) and IFS {Sweden, No. A/2.'iOI-l/2). We very much appre- 
ciate the comments and conections made by two anonymous 
reviewers. 



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Juvenile Crabs Dwell in Clam Siphon Holes 



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Joiiniul nf Shellfish Research. Vol. 17. No. 4, 431-4.^3. 1W8. 



POPULATION BIOLOGY OF XIPHOPENAEUS KROYERI (HELLER 1862) (DECAPODA: 
PENAEIDAE) FROM UBATUBA BAY, SAO PAULO BRAZIL 



J. M. NAKAGAKI AND M. L. NEGREIROS-FRANSOZO 

Depaitaiiicnto de Zoologia 

Instilulo dc Biocieiicias e Centro de Aquivulnira 

Universidade Esladiial Paulista (UNESP) 

18618-000 Botiicani 

Sao Paulo. Brasil 

ABSTRACT The population structure and abundance of Xiphopenaeus kroyeri (Heller, 1862) were analyzed during monthly samples 
from October 1992 to September 199.3 in Ubatuba Bay (23°26'S and 45°02'W). Brazil. Sampling was carried out at two parallel 
transects: one of them located in its midregion and the other at the mouth of the bay. After trawling, shrimps were separated from other 
benthic organisms, sexed. and counted. Their total length was also measured, and the degree of gonadal development was assessed. 
Xiphopenaeus kraxeri. the most common penaeid species in the bay. was recorded in all samples, but its abundance decreased from 
November to March. Size ranged from 14.3 to 1 18.3 min in males and from 12.7 to 133.6 mm in females, suggesting a slight sexual 
dimorphism related to body size. Males prevailed during most of the year; whereas, females predominated during summer and 
midwinter. Based on the percentage of mature females during this study, two main reproductive periods were identified, occurring in 
spring and autumn. Despite some breeding activity throughout the year, such a trend indicates that the population follows a tropical/ 
subtropical reproductive pattern. 

KEY WORDS: Population biology, Penaeidac, Xiphopenaeus kroxeri. reproduction, sex-ratio 



INTRODUCTION 

Xiphopenaeu.s kroyeri is the most intensively exploited shritiip 
species in Sao Paulo State. According to Pires (1992), X. kroyeri 
and the swimming crab Portumis spinicarpus (Stimpson 1871 ) are 
the most abundant species of the benthic niegafauna in the conti- 
nental shelf off the study area. This species represents the second 
most important fishery resource along the coa.st of Sao Paulo State 
(Rodrigues et at. 1993), and its trophic relationships may be es- 
sential in maintaining the stability of benthic communities (Pires 
1992), Despite being an extremely abundant species along the 
Brazilian coast, information on the biology, ecology, and behavior 
of X. kroyeri is scarce (Vieira 1947, Mota-Alves and Rodrigues 
1977, Cortes and Ciiales 1990, Cortes 1991, Rodrigues et al. 1993 
and Branco et al. 1994). 

Because of the complexity of their life cycle, studies on penaeid 
shrimp populations (e.g., migration and reproduction) are needed 
to improve fishery management. Boschi (1969) pointed out its 
importance when he studied Artemesia longinaris Bate 1888 in 
Mar del Plata, noting a continuous change of its age composition 
structure. 

The study of penaeid reproductive cycles is also important, and 
usually is achieved by means of recording the degree of gonadal 
development in sampled specimens. In this procedure, a number of 
development levels are established and described, usually ranging 
trom three to five (i.e,, immature, in maturation, almost mature, 
mature, and spawned). Relevant contributions concerning the re- 
productive biology of different penaeid shrimp species are Vieira 
(1947), Olguin-Palacios (1967), Perez-Farfante (1969), Mota- 
Alves and Rodrigues (1977), Motta-Amado (1978), and El Hady et 
al. ( 1990). This study examines the abundance and the population 
biology of X, kroyeri in Ubatuba Bay, Ubatuba, Sao Paulo, Brazil, 
with emphasis on its population structure and reproductive period. 

MATERULS AND METHODS 

Monthly trawlings were carried out from October 1992 to Sep- 
tember 1993 in Ubatuba Bay. Samples were taken along two 



transects: transect A, located at the shallow midregion of the bay, 
and transect B located at the deeper bay mouth (Fig. 1 ). Trawlings 
were accomplished with an otter trawl net ( lO-mm mesh cod end) 
and were conducted at constant speed for 1 h, covering a 7,400 m" 
area. The shallow region of the bay is strongly affected by coastal 
environmental conditions, receiving freshwater drainage from four 
rivers. Otherwise, the deep stratum is to subject a greater oceanic 
influence. 

Some physical factors were monitored at each transect. Bottom 
water temperature was obtained with a Nansen bottle provided 
with a thermometer (±1.0°C). Depth was obtained by means of a 
marked rope attached to the Nansen bottle, and a VanVeen grab 
sampler was used to obtain sediment samples. Sieving analysis 
according to Wentworth grades were carried out for grain size 
classification. Sediments were .sorted using the phi-.scale (<()) in 
1.0-<t) intervals between -1.0 {j> and 4.0 <|)- including the fractions 
under 4.0 i) (Hakanson and Jansson 1983). Sediment organic con- 
tents were obtained using the loss on ignition method (Hakanson 
and Jansson 1983), 

The shrimps were separated, sexed, and measured (total length, 
TL) with a vernier caliper to the nearest 0. 1 mm. A Student's r-test 
was used to detect size differences between sexes. Box plots were 
performed for males and females in each month to analyze the 
population structure through the study period. Monthly .sex ratios 
were also obtained. 

Median size differences between transects were tested in each 
month using a Mann-Whitney U-test and among months in each 
transect using a Kruskal-Wallis test (Si'egel, 1956). 

In each trawl, subsamples from 200 to 400 individuals were 
separated for examination of gonads. In females, four development 
.stages were considered according to Motta-Amado (1978): I- 
immature, Il-developing, Ill-mature, and IV-spent. In males, the 
presence (or absence) of spermatophores in the terminal ampoule 
was recorded. 

Monthly sex ratios and proportions of mature females were 
statistically compared by means of a Goodman's test ( 1964, 1965), 



931 



932 



Nakagaki and Negreiros-Fransozo 




■25' 



26' 



-27 



Figure 1. Map of Ubatuba Bay showing the position of sampling 
transects. 



This analysis is based on the binomial proportion comparison for 
contrasts between and within multinomial populations. These re- 
suhs were analyzed at the 5% significance level. 

To estimate the mean size of first maturation in each sex. the 
Gallon's Ogive, Fr = 1 - t-""^'-'' (Fonteles-Filho 1989). was 
adjusted to fit the total length (TL. independent variable) versus 
relative frequency of mature individuals (Fr. dependent vanable) 
scatterplots. Mature individuals were defined as male with full 
terminal ampoule and females with gonads in stage II. III. or IV. 

RESULTS 

Water temperature averaged 2.3.16 ± 2.97°C (ranging from 20 
to 28°C). with highest values recorded from February to April. 
Mean depth at transect A is 7.6 ± 1.3 m. Along this transect 
sediments are very poorly sorted (ct, = +2.025), mainly consisting 
of fine sand (Mz = 2.73 <j)) with a high percentage of organic 
contents (11.66 ± 2.149?-). Average depth at transect B is 14.6 ± 
0.74 m. Sediments are moderately sorted (<j = +0.525). composed 
by very fine sand (Mz = 3.40 i>) and low organic contents (2.97 
±0.61%). 

Xiphopenaeiis kroyeri was very abundant in the area (5.027 
individuals in transect A and 5.282 in transect B) during all sam- 
pling periods, e.xcept from December to February in transect B 
(Fig. 2). This fact indicates a seasonal abundance variation. 

Females ranaed from 12.7 to 133.6 mm (74.52 ± 17.47) and 



^^1 Transect A 
I I Transect B 




/\pr Ma> Jun 
Months 



Aug Sep 



Figure 2. Monthly abundance of .V. kroyeri in both transects. 



males from 14.3 to 1 18.3 mm (71.82 ± 14.01), indicating a sexual 
dimorphism, in which females attain a larger size (Student's r-test, 
t = 8.825, p< .0001). 

Mann-Whitney comparative analyses of size frequency distri- 
butions in transects A and B revealed significant size differences in 
Oct. 1992, Apr., May, June, and Aug. 1993 (Table 1. Figs. 3 and 
4), showing that the population is not equally distributed in these 
areas. During Oct. 1992, April, and June 1993. larger shrimps were 
sampled in transect A. and during May and Aug. 1993. larger 
specimens were captured in transect B. 

Differences in shrimp median size were repeatedly verified 
among sampled months (Kruskal-Wallis, p < .001) (Table 2, Figs. 
3 and 4). However, they were too complex to reveal recruitment 
pattern. 

Males were generally slightly predominant, but the sex ratio 
varied throughout the year. In November 1992. January and July 
1993 (Fig. 5). an increase (Goodman's test, p < .05) of the relative 
number of females was observed, when sex ratios attained 1 : 1 .78; 
1:1.28, and 1:1.2, re.spectively. 

Xiphopenaeus kroyeri breeds all year, but higher reproductive 
activity was verified in some periods. Based upon the data ob- 
tained from gonadal analysis in females, it can be concluded that 
higher reproductive activity occurred in November 1992, and 
March, August, and September 1993 (Fig. 6). In the case of males, 
higher proportions of individuals with full terminal ampoule were 
recorded in two main periods (November 1992 and May 1993) 
(Fig. 7). Males achieve sexual maturity (68.02 mm) at a smaller 
size than females (83.19 mm) (Fig, 8). 

DISCUSSION 

The abundance of X. kroyeri showed a marked seasonal varia- 
tion. During the summer period (December to March) this species' 
abundance is lower, mainly in the deeper portion of the bay. Sig- 
noret (1974) observed that X. kroyeri follows a similar pattern in 
the Terminos Lagoon (Mexico), with low abundances from sum- 
mer to fall. 

The low abundance of A', kroxeri during summer may be related 
to the intrusion of a cold current. Castro-Filho et al. (1987) indi- 
cated the presence of three oceanic currents in the Ubatuba region, 
the coastal water (CW) (T > 20°C), the South Atlantic central 
water (SACW) (T < 18°C), and the tropical water (TW) (T > 
20°C). Pires (1992), who studied the benthic megafauna commu- 
nities in the continental shelf of Ubatuba region, observed by 
means of a cluster analysis that there is a close association between 
some species abundance and specific environmental conditions. 
This is the case of positive correlation between X. kroyeri and the 
prevalence of CW during winter. 

Despite different sediment features found in each transect, the 
incoming SACW during summer could be the most important 
physical factor influencing the distribution of this species. The 
physical action of the current itself together with low temperature 
conditions would restrain the population distribution of .Y. kroyeri 
within the study area. 

Statistical differences in shrimp median size among sampled 
months do not support a growth model through time, which could 
have explained the growth pattern in this population. The great 
fishery effort in Ubatuba Bay probably affects the species, as 
observed by Somers et al. ( 1987) in P. escidentus Haswell in the 
ToiTes Strait (Australia), who suggested a continuous recruitment 
and/or the existence of a size-dependent source of mortality. In the 
present study, the comparison of shrimp size in transects A and B, 



Population Biology of Xiphopenaeus kroyeri 



933 



TABLE 1. 
Mann-Whitney analysis in .V. kroyeri size comparisons in eacli montli between transects in Ubatuba Bay. 







Transect A 




Transect B 






Number of 




Number of 






Month 


individuals 


Ranked sums 


individuals 


Ranked sums 


U 


October 1992 


552 


735.56 


719 


559.57 


8.47* 


November 1992 


193 


180.08 


163 


176.63 


0.31 


December 1992 


184 


95.00 


7 


122.28 


1.28 


January 1993 


142 


73.79 


4 


63.25 


0.50 


February 1993 


409 


229.59 


52 


242.08 


0.63 


March 1993 


673 


— 





— 


— 


April 1993 


1 169 


1568.07 


1640 


1288.34 


8.996* 


May 1993 


390 


351.24 


370 


411.34 


3.77* 


June 1993 


275 


816.52 


1235 


741.91 


2.56* 


July 1993 


281 


509.77 


695 


479.90 


1.50 


August 1993 


466 


381.11 


401 


495.46 


6.70* 


September 1993 


293 


296.16 


296 


243. S5 


0.16 



' Statistical significant differences at a = 0.01. 



reveals remarkable differences in the population distribution. 
which are likely to reflect recruitment and migration processes. 

The sex ratio variations observed in this study are supported by 
other results obtained for the same species. According to Signoret 
(1974), the sexual distribution through the year in X. kroyeri is not 
homogeneous, with males and females often strongly segregated. 

According to Wenner ( 1972), mentioning the Fisher theory, the 
1:1 proportion is favored by natural selection. Wenner (1972) 




Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep 
1»'2 Months "" 




o Outlier 

Ncm-outUer max 



Ncm-outlier min 
o Outlier 



Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep 
'"2 Months 1'" 

Figure 3. Series of box plot graphics for monthly size of males (upper) 
and females (below) obtained in transect A. 




Oct Nov Ctec Jan Feb Mar Apr May Jun Jul Aug Sep 
1992 ., , 1993 

Months 




Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep 
1992 ....^. 1993 



o Outlier 

Non-outlier max 



Non-outlier min 
o Outlier 



Months 

Figure 4. Series of box plot graphics for monthly size of males (upper) 
and females (below) obtained in transect B. 



TABLE 2. 
Results of Kruskal-Wallis analysis. 



Transect 



H 



df 



Probability 



5027 

5282 



576.25 
640.68 



11 

in 



p<.001 
P<.001 



934 



Nakagaki and Negreiros-Fransozo 




A 


B 


A 


B 




A 


A 


A 


A 


B 


A 


A 


a 


b 


ah 


" ab ab a 


ab 




hr 


ab ^ 



Female 



Oct 



Nov Dec 
1992 



Jul Aug 
1993 



Sep 



Feb Mar Apr May Jun 

Months 

Figure 5. Monthly sex ratio for V. kroyeri. Capital letters inside the 
bars indicate comparisons w ithin month, and lower case letters outside 
the bars indicate comparisons among months. The same letters indi- 
cate no statistical differences. 

pointed out that sex-dependent mortality, activity, migration, habi- 
tat utilization and al.so the effect of restricted food resources are 
important factors explaining departures from the Mendelian pro- 
portion. Wenner also stated that the 1:1 ratio is an exception rather 
than the rule in crustacean populations. He concluded that sex ratio 
can be a function of size for a given species. 

The temporal sex ratio variation can be related to a seasonal 
reproductive pattern in ,V. kroyeri. The proportion of males was 
higher during most of the year, but females were more abundant in 
November (spring), when it was recorded a 1:1.78 sex ratio. This 
peak coincides with major reproductive activity. Contrarily, Cortes 
(1991) observed that during spawning, males outnumbered fe- 
males in a Colombian Caribbean population. This fact supports the 
hypothesis of sex-dependent pattern of migration, because Cortes 
collected the shrimps near the coast at depths ranging from 1 .5 to 3 m. 



e e 




Nov Dec 

mi 



Jul 
893 



Aug St'p 



Mar Apr May 

Months 

Figure 6. Bar graph showing gonadal maturity in females. Capital 
letters inside the bars indicate comparisons within month, and the 
lower case letters outside the bars indicate comparisons among 
months. The same letters indicate no statistical differences. Data of 
transects A and B are grouped. Females in gonad stage I were con- 
sidered immature, and females in stages II, III, and IV were consid- 
ered mature. 




Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep 

Months 
Figure 7. Terminal ampoule status in males. 



Analyzing both transects, we can assume that X. kroyeri ex- 
hibits a tropical/subtropical reproductive pattern (Dall et al. 1990), 
in which there is a main reproductive period in the spring and a 
secondary one in the fall. The present results are similar to those 
observed by Mota-Alves and Rodrigues (1977). Motta-Amado 
( 1978), and Cortes (1991). It can be assumed that the presence of 
juvenile individuals from February to May is attributable to 
spawning events during spring. 

The onset of sexual maturity can vary between populations. 
The size estimates in this study (68.02 mm in males and 83.19 mm 
in females) are larger than those observed by Rodrigues et al. 
( 1993) (62 mm for males and 71 mm for females) in other locali- 
ties within Sao Paulo State. The determination of this parameter 
can be important for assessment of the reproductive stock in natu- 
ral populations (Fonteles-Filho 1989) and for guidance of future 
governmental fishery control. Establishing a minimum catch size 
and defining the period of lower relative abundance of juvenile 
shrimps will help establish rational management of this species' 
exploitation. 

ACKNOWLEDGMENTS 

CAPES (Coordenadoria de Aperfeigoamento Superior) pro- 
vided financial support as a fellowship to the first author. We are 









O r^9872 






'^f 




A./ 


/ 




A / 


7 • Female 
/ * Male 




A, / 


/ 




/ */ 

»=i^i — 1 r 


^ r-=09823 
1 1 ' 1 ■■ 1 ' 1 



Total Lengh(mm) 

Figure 8. .V. kroyeri. Frequency of morphologically mature males and 
females as a function of total length. 



Population Biology of Xiphopenaeus kroyeri 



935 



also grateful to Carlos Roberto Padovani tor his statistical assis- 
tance and to Adilson Fransozo and Augusto A. V. Flores for manu- 



script suggestions. Thanks are extended to other NEBECC mem- 
bers for helping during fieldwork. 



REFERENCES 



Boschi, E. E. 1969. Estudio biologico pesquero del camaron Arlemesui 
longinaris Bate de Mar del Plata. Bol. Inst. Biol. Mar. 18:1^7. 

Branco, J. O., M. J. Lunardon-Branco & A. de Finis. 1994. Crescimcnto de 
Xiphopenaeus kroyeri (Heller. 1862) (Crustacea: Natantia: Penaeidae) 
da regiao de Matinhos. Parana, Brasil. An/. Biol. Tecnol. 37:1-8. 

Castro-Filho, B. M., L. B. de Miranda & S. Y. Miyao. 1987. Condi96es 
hidrograficas na plataforma conlinental ao largo de Ubaluba: variances 
sazonais e em media escala. Bolm. Inst. Oceanogr. 35:135-151. 

Cortes. M. L. 1991. Aspectos reproductivos del camaron Xiphopenaeus 
kroyeri (Heller) en Costa Verde, Cienaga (Caribe Colombiano). Cal- 
dasia 16:513-518. 

Cortes, M. L. & M. M. Criales. 1990. Analisis del contenido estomacal del 
camaron titi Xiphopeiweus kroyeri (Heller) (Crustacea: Natantia: Pe- 
naeidae). Na. Inst. Invest. Mar Punta Betin 19-20:23-33. 

Dall, W., B. J. Hill, PC. Rothlisberg & D. J. Staples. 1990. The biology of 
the Penaeidae. V.27. //;.• J. H. S. Blaxter and A.J. Southward (eds). 
Advance. Mar. Biol. Academic Press, London. 489 p. 

El Hady. H. A., F. A. Abdcl-Ra/ek & A. Ezzai. 1990. Reproduction of 
Penaeus semisulcatus De Haan en Damman water (Arabia Gulf), King- 
dom of Saudi Arabia. Arch. Hydrobiol. 118:241-251. 

Fonteles-Filho, A. A, 1989. Recurso pesqueiro: biologia e dinamica popu- 
lacional. Imprensa Oficial do Ceara, Fortaleza, Brazil. 296 pp. 

Goodman, L. A. 1964. Simultaneous confidence intervals for contrasts 
among multinomial populations. Ann. Math. Statist. 35:716-725. 

Goodman, L. A. 1965. On simultaneous confidence intervals for multino- 
mials proportions. Technometrics 7:247-254. 

Hakanson, L. & M. Jansson. 1983. Principles of lake sedimentology. 
Springer-Verlag, Berlin. Heidelberg. 316 pp. 

Mota-Alves, M. I. & M. M. Rodrigues. 1977. Aspectos da reprodu^ao do 
camarao sete-barbas, Xiphopenaeus kroyeri (Heller) (Decapoda, 
Macrura) na costa do Estado de Ceara. Arq. Cien. Mar. 17:29-35. 



Motta-Amado, M. A. P. 1978. Estudo biologico do Xiphopenaeus kroyeri 
(Heller, 1862), camarao "sete-barbas" (Crustacea, penaeidae) de Mat- 
inhos, Parana. Master's Thesis. Federal University of Parana, Curitiba, 
Brazil. 94 pp. 

Olguin-Palacios. M. 1967. Estudio de la biologi' del camaron cafe Penaeus 
califoniiensis Holmes. FAO Fish. Repl. 57:331-356. 

Perez-Farfante, I. 1969. Western Atlantic shrimps of the genus Penaeus. 
Fish. Bull. 67:461-590. 

Pires, A. M. S. 1992. Structure and dynamics of benthic megafauna on the 
conlinental shelf offshore of Ubatuba, southeastern Brazil. Mar Ecol. 
Prog. Ser 86:63-76. 

Rodrigues, E. S.. J. B. Pita, R. Gra^a-Lopes, J. A. P. Coelho & A. Puzzi. 
1993. Aspectos biologicos e pesqueiros do camarao sete-barbas (Xi- 
phopenaeus kroyeri) capturado pela pesca artesanal no litoral do estado 
de Sao Paulo. B. Inst. Pesca 19:67-81. 

Siegel. S. 1956. Nonparametric statistics for the behavioral sciences. 
McGraw-Hill. New York. 312 pp. 

Signoret, M. 1974. Abundancia, tamaiio. y distribucion de camarones 
{Crustacea. Penaeidae) de la Laguna de Terminos, Campeche y su 
relacion con algunos factores hidrologicos. Ann. Inst. Biol. Univ. Nal. 
Anion. Mexico 45. Ser. Zoologia 0:119-140. 

Somers. L F.. Poiner. I. R. & Harris, A. N. 1987. A study of the species 
composition and distribution of commercial penaeid prawns of Torres 
Strait. Ausl. J. Mar. Freshwater Res. 38:47-61. 

Vieira, B. B. 1947. Observa^oes sobre a malurafao de Xiphopenaeus kroy- 
eri no litoral de Sao Paulo. Bol. Mus. Nac. 72:1-22. 

Wenner, A. M. 1972. Sex ratio as a function of size in marine Crustacea. 
Am. Nat. 106:321-350. 



Jdiirmil III Slu'llfish Research. Vol. 17. No. 4. 937-944, 1998. 

PRODUCTION AND RESOURCE ALLOCATION IN THE PERIWINKLE, LITTORINA LITTOREA 
(LINNAEUS 1758), ON PENDLETON ISLAND, NEW BRUNSWICK 



M. E. CHASE AND M. L. H. THOMAS 

Biology Department and Centre for Coastal Studies and Aquaculture 

University of New Brunswick 

Saint John, New Brunswick, E2L 4L5 

Canada 

ABSTRACT An intertidal population of Litlonna linnifa L. from a rocky shore on Pendleton Island. New Brunswick, Canada was 
analyzed to determine its demographic structure and the energy allocated to gamete production, somatic growth, and the synthesis of 
organic shell matrix. Total production, calculated as the sum of somatic growth (Pg), shell growth (Ps). and the production of gametes 
(Pr), was 151 kJ/m'^/y'. The percentage of production allocated to Pg. Ps, and Pr was 60.0, 5.9, and 36.1%. respectively. Younger 
cohorts (0 and 1) were responsible for the bulk of shell (41.5 and 20.5%, respectively) and somatic production (58.5 and 79.5%, 
respectively), but for 0% of the reproductive output. As compared to other populations of L lillorea. the proportion of total production 
allocated to gamete production by the population on Pendleton Island was higher. 

KEY WORDS: Liuorina linorea. production, resource allocation, reproduction 



INTRODUCTION 

Estimates of production are useful in assessing contributions of 
marine species to energy flow through the ecosystetn (Rodhouse 
1979, Griffiths 1981a, Griffiths 1981b, Vahl 1981 ) in addition to 
determining the suitability of different habitats to producers 
(Bayne and Worrall 1980). Among mollusks, bivalves have re- 
ceived the most attention (Cerastodenna edule, Ivell 1981, Pla- 
copecten magellanicus. MacDonald and Thompson 1986, Mytilits 
edidis. Bayne and Worrall 1980, Thompson 1979, and Gardner and 
Thomas 1987a) largely because of their commercial and ecological 
importance. 

The common periwinkle, Littorina littorea (L.), is one of the 
most widely studied marine gastropods. Extensive literature exists 
regarding its biology (Gegenbaur 1852, Caullery and Pelsener 
1910, Linke 19.33, Fretter and Graham 1962), breeding cycle (Tat- 
ter.sall 1920, Elmhirst 1923, Moore 1937. Williams 1964, Fish 
1972. Chase and Thomas 1995a) and growth (Hayes 1927. Moore 
1937, Ekaratne and Crisp 1982. Gardner and Thomas 1987b). 
However, there is a paucity of data on its production and allocation 
of resources. Grahame (1973) examined the importance of lepro- 
duction as a pathway of energy flow when he calculated both the 
production of .somatic tissue and gamete production of L. littorea 
in North Wales, Hughes and Roberts (1980) compared the repro- 
ductive effort of L. littorea to that of L. neritoides, L. nigrolineata 
and L ritdis in North Wales. In Canada, the only study that has 
calculated secondary production of L littorea was that of Gardner 
and Thomas (1987b) on a population at Welch's Cove, Bay of 
Fundy. In their study, however. Gardner and Thomas (1987b) 
considered only the partitioning of energy between somatic and 
shell growth; reproductive output was not considered. No study to 
date has looked at all components of production (somatic growth. 
shell growth, and the production of gametes) and the allocation of 
resources to those components. L littorea is the most dominant 
mollusk within the midlittoral zone of rocky shores on Pendleton 
Island and throughout most of the Bay of Fundy (Gardner and 
Thomas 1987b). Knowledge of the production and allocation of 
resources in populations of L littorea will provide information 
critical to the understanding its life history, and importance to the 
rocky shore community. The objective of this paper is twofold. 
The first is to calculate secondary production as the sum of all 



components (somatic, shell, and gamete) to detemune the propor- 
tion of total production allocated to gamete production. The second 
is to deterinine the age-specific pattern of energy partitioning be- 
tween growth (somatic and shell) and reproduction. 



MATERIALS AND METHODS 



Study Site 



Pendleton Island is located in New Brunswick, Canada at 
45°02'N and 67''56'W within the Deer Island Archipelago (Fig, 1), 
The climate and oceanographic setting have been summarized by 
Thomas et al. (1990). The collection sites were on an approxi- 
mately 100 m long section of shore in Pendleton Passage, a shel- 
tered channel with high tidal currents ranging up to 0.47 and 0.60 
m/s' for the ebb and flood of spring tides, respectively (Thomas 
et al. 1990). The upper shore is predominately sedimentary, with 
scattered rocks and outcrops, and L. littorea are abundant through- 
out this area. 

Demographics 

Monthly samples of L littorea were collected between May 
and September, 1988 and May and October, 1989. Samples com- 
prised approximately 1,000 individuals, collected randomly from 
all tidal levels along three transects. The spire height was measured 
using computer-assisted vernier calipers. Gardner (1986) has 
shown that age of Z.. littorea cannot be ascertained accurately from 
growth ring analysis. Therefore, length frequency histograms were 
plotted, and the cohort placement of Cassie (1954) was used to 
analyze the demographic structure of the population. Although it is 
accepted that cohort identification by such a means may be sub- 
jective, we feel that regular sampling of the population and the 
incorporation of a settlement study (Chase and Thomas 1995a) 
more than compensated for the subjectivity of this method. 

Production 

Secondary production was calculated using the following rela- 
tionship, 

P = Pg 4- Pr -I- Ps ( I ) 

where; P = total production; Pg = energy incorporated into so- 



937 



938 



Chase and Thomas 




Figure 1. Location of Pendleton Island in tlie Deer Island Archipelago, New Brunswick, Canada. 



matic growth; Ps = energy incorporated into the shell matrix; and 
Pr = energy expended on gamete production. 

Secondary production for the somatic and shell parts for the 
1988 and 1989 study periods was estimated using the increment 
summation method outlined by Rigler and Downing (1984). This 
method calculates production of cohorts as the sum of the change 
in biomass over specified time intervals. 

The density of the population was estimated from a population 
census along each of three transects running perpendicular to the 
shore line. The number of L. liaorea in every 5th m" were counted, 
and a random sample of 25 were measured for spire height. Bio- 
mass was determined monthly for each size class or cohort (from 
demographic analysis) m grams of ash-free dry weight (AFDW). 
This was done by interpolating the mean spire height of each 
cohort onto the graph of the log regression of the AFDW (somatic 
tissue and shell) on the log spire height of 50 individuals in the 
population at each sampling time. All regression equations were 
highly significant (R" > 0.8). This estimate of weight was then 
multiplied by the cohort density to give an estimate of biomass in 
gAFDW/m~. Biomass determination formed the basis of produc- 
tion calculations. Biomass estimates were converted to energy 
units using the following conversion of Grahame (1973) of 24.7 
kJ/mg' AFDW. 

The reproductive output was calculated directly for females 
using the direct method of Crisp ( 1984). L. liuorea females, of a 
range of spire heights, were housed individually in plastic jars, and 
the spawn from each was collected (Chase and Thomas 1995b). 
Four samples of approximately 2,000 eggs were incinerated in a 
muftle furnace at 435°C for 3 h for ash-free determinations. Be- 
cause there was little variation in the calculated weights, the mean 
of the samples were used in all calculations. 



The reproductive output of males could not be calculated di- 
rectly as in the females. Indirect analysis of fecundity was based on 
the mean gonad weight before and after spawning was known to 
have occurred (weight loss following spawning). For comparison 
purposes, the female reproductive output was also calculated using 
the indirect method (Crisp 1984). From May to November, 1989 
samples of approximately 230 L. linorea were collected from 
Pendleton Island and taken to the laboratory, where they were 
maintained without food in running aerated seawater for 48 hours 
to allow the gut to clear (Grahame 1973). Histological analysis of 
the gonads of L. littorea in Pendleton Island revealed that the 
breeding season was between May and October, and spawning 
generally occurred between June and September (Chase and 
Thomas 1995a). Samples of approximately 100 each of males and 
females were killed by boiling for 1 minute. The gonadal region 
(including the penis and prostrate in the males and the shell gland 
of the females) was dis.sected out and weighed before and after 
drying for 48 h at 60"C. In L. littorea. the gonadal tissue is found 
closely associated with the digestive system and could not be 
separated for individual measurement. It was assumed that by 
emptying the gut and consistently taking the same tissue for each 
sample, any difference in weight would be attributed to changes in 
the gonad. However, estimates of reproductive output may be 
more variable as a result of this approach. Samples were inciner- 
ated in a muffle furnace at 435°C for 3 h for ash-free determina- 
tions. Covariance analysis of gonad weight versus spire height was 
used to test for significant changes in gonad weight between sam- 
pling dates. This may indicate spawning. Further evidence of 
spawning was obtained through histological examination of the 
gonadal tissue (Chase and Thomas 1995a). The differences in 
gonad weight between these data was used in the calculation of 



Production of Littorina 



939 



gamete production. Bioniass estimates for gonadal tissue were 
converted to energy units using the conversion of Grahame { 1973) 
of 26 kJ/mg' AFDW. 

RESULTS 

Demography 1988 

Figure 2(a-c) are histograms of the population of L liltorea on 
Pendleton Island at peak reproductive times (i.e.. settlement); May 
(Fig. 2a), July (Fig. 2b). and August (Fig. 2c) 1988. Summary of 
mean spire height (mm ± SD) and density (number of individuals/ 
m") of each cohort are shown in Table 1. Cassie (1954) analysis 
revealed that the population was composed of four cohorts from 3 
years in 1988. In May, the population was dominated by mature 
individuals (>13 mm), which represented l\9'c of the population. 
The percentage of mature individuals in the population declined 
throughout the season to 46 and i29c of the population in July and 
August, respectively, as a result of settlement of new cohorts and 
subsequent reduction of older individuals from the population. 



Settlement on Pendleton Island seems to in\ olve the recruitment of 
two distinct cohorts, cohorts 0+ in July (Fig. 2b) and 0++ in 
August (Fig. 2c). Two cohorts per year remained discemable until 
the end of the first year, after which only one cohort per year could 
be identified. This is because older individuals have a very slow 
growth rate as opposed to the young, faster growing cohorts (Wil- 
liams 1964), resulting in a merging of the younger cohorts with the 
older. In 1988, the newly settled cohorts (0+ and 0++) comprised 
22.87f of the population at the end of the breeding season. 

Demography 1989 

Figure 2(d-f) are histograms of the population at peak repro- 
ductive times in May (Fig. 2d), Augu.st (Fig. 2e), and October (Fig. 
2f) 1989. Summary of mean spire height (mm ± SD) and density 
(number of individuals/m") of each cohort are in Table 1. Cassie 
( 1954) analysis revealed that the population was composed of four 
cohorts from 3 years [ 1 a and 1 b, ( 0++ and 0-H of the previous year), 
2, and 3]. A cohort four was detected in May and August, but 
comprised only 5 and 0.3'7r of the population in each month. In 



1988 



1989 



(A) .MAY 4 




14 21 28 

LENGTH (mm) 




14 21 28 35 





(D) MAY 23 


lb 

1 






1 


200 - 
150 ■ 


la 


2 


3 


Q 


100 ' 






^^L 


u 


50 ' 


lj 


1 


Mr 


A 






7 




14 


21 








LENGTH (mm) 






(E) ALGUSl 


22 


2 



1> 1 

7 14 21 2« 35 



LENGTH(mm) 



(C) AUGUST 25 






14 !1 U 35 



LENGTH (mml 



(F) OCTOBER 30 




14 21 2J* 35 



LENGTH(mm) 

Figure 2. Length-frequency histograms of the Littorina littorea population at Pendleton Island at sampling intervals in 1988 (2 a-c) and 1989 (2 
d-f ). .\rrows indicate the mean spire height (mm) of the component cohorts in the population. 1988: (A) May 4, (B) July 6, and 1989: (C) August 
25, (D) May 23, (E) August 22, (F) October 30. 



940 



Chase and Thomas 



TABLE 1. 

Summary of the mean spire height (mm ± SDl and density (number of individuals/m") of the component cohorts in the Littoriiia littorea 

population at Pendleton Island at sampMng intervals in 1988 and 1989. 



1988 




May 






July 






August 






Mean Spire 






Mean Spire 






Mean Spire 






Height (mm) 


Density 




Height (mml 


Density 




Height (mm) 


Density 


Cohort 


(±SD) 


(#/m-| 


Cohort 


(±SD) 


(#/m-) 


Cohort 


(±SD) 


(#/m') 














0++ 


2.5 ±0.9 


20 








0+ 


3.7 ±0.6 


3 


0+ 


8.5 ±1.6 


4 


IB 


44 ± 1.0 


2 


IB 


10.0 + 2.4 


1 


IB 


12.6± 1.7 


2 


lA 


10.6 ±2.3 


I 


lA 


14.6 ±1.8 


4 


lA 


17.2 ± 1.3 


4 


1 


17.2 ±2.6 


7 


2 


19.4 ± 0.9 


6 


2 


22.5 ± 2.2 


18 


3 


24.2 ± 2.2 


18 


3 


26.4 ± 2.0 
1989 


19 


3 


26.0 ±0.8 


4 




May 






.August 






October 






Mean Spire 






Mean Spire 






Mean Spire 






Height (niml 


Density 




Height (mm) 


Density 




Height (mm) 


Density 


Cohort 


(±SI)) 


(#/ni-) 


Cohort 


(±SD) 


(#/m-) 


Cohort 


(±SD) 


(#/m-) 














0++ 


2.5 ± 0.9 


73 








0+ 


4.9 ± 1.9 


12 


0+ 


7.3 ± 1.4 


1 


IB 


4,1 ± 1.2 


2 


IB 


10.7 ±1.5 


2 


IB 


12.8 ±2.6 


2 


lA 


10.8 ±2.2 


1 


lA 


17.1 ± 1.5 


8 


lA 


20.0 ±2.0 


3 


2 


14.3 ±2.1 


2 


2 


22.7 ± 1.6 


12 


2 


23.8 ±1.0 


3 


3 


24.1 ±2.5 


27 


3 


26.4 ± 1.2 


5 


3 


26.5 ± 0.5 


4 


4 


27,8 ± 1.5 


5 















May. the population was dominated by mature individuals (>13 
mm), which represented 60% of the population. The percentage of 
mature individuals in the population declined throughout the sea- 
.son to 54 and 459c of the population in August and October, 
respectively, as a result of the settlement of new cohorts and the 
subsequent reduction in the older individuals (cohort 3: 34% in 
May to 2% in October) and the loss of cohort 4. Settlement of 
cohorts occurred in August (Fig. 2e) and October (Fig. 2f), 1989. 
Experimental data have shown that the differential timing may be 
the result of local temperature variations (Chase and Thomas 
1995b). In 1989. the newly settled cohorts (0+ and 0++) comprised 
31% of the population at the end of the breeding season. 

Somatic and Shell Production 

Table 2 contains the values calculated for production of so- 
matic tissue (Pg) and shell (Ps). in kJ/m", for the 1988 and 1989 
population of L littorea on Pendleton Island. Somatic production 
(Pg) for the 1988 season was 70.265 kj/nr. Shell production (Ps) 
for the same time period was 16,859 kJ/m". An analysis of the 
production per cohort revealed the largest contribution was from 
the larger, hence older, cohorts 2 and 3. Production of these two 
cohoits lepresented 91.7% of Pg and 88.6% of Ps. 

Somatic production (Pg) for the 1989 season was 87.764 kJ/nr. 
Shell production (Ps) for the same time period was 8.974 kJ/m". 
Analysis revealed the majority of Pg was from cohorts 2 and 3 
(74.5%); whereas, the majority of Ps was from cohorts I and 2 
(88.8%). The decrease in Pg of cohort 4 and the negative Ps for 
cohorts 3 and 4 were probably a reflection of the decrease in 
density of each cohort (cohort 3: 1988. 18 individuals/m" to 4 



individuals/m- in 1989: cohort 4; 5 individuals/m' in 1988 to 
individuals/m" in 1989). 

A two-way analysis of variance (ANOVA) on the mean so- 
matic production (Pg) of each cohort, for the population in 1988 
and 1989 revealed no significant year effect (df = 1,3; F = 0.966; 
p > .5); however, there were significant differences among cohorts 
(df = 1.3; F = 25.95; p > .02). Production of cohorts and I was 
higher in 1989. A two-way analysis of variance on the mean shell 
production (Ps) of each cohort, for the population in 1988 and 
1989 revealed no significant effect of year (df = 1.3; F = 0.546; 
p > .5) or cohort (df = 1.3; F = 1.18; p > .5). 

Production of Gametes (Pr) 

Female Reproductive Output 

The mean (±SE) AFDW of four batches of approximately 
2.000 eggs was 1.15 x 10"* ± 0.2 x 10^'' g AFDW per egg. Table 
2 shows the production of gametes for females (Prf) using both 
direct and indirect methods of determination. Estimated Pr in 1989 
using the direct method was 46,160 kJ/m". Pr was high at the 
onset of maturity (>I3 mm, cohort 1) at 13.661 kJ/m". and in 
cohort 3 (22.038 kJ/m") but decreased in the oldest cohort. This 
reduction in Pr is probably a reflection of the decrease in density 
of this cohort in the population. The estimated Prf in 1989 using 
the indirect calculation was 33.020 kj/m". Production estimates 
using this method increased with the age of each cohort 3 (20.865 
kJ/m") and then decreased in cohort 4 (11.496 kJ/nr). A two- 
way ANOVA revealed that there was no significant difference 
effect of method (df = 1.4; F = 0.72; p > .5) or cohort (df = 1,4; 



Production of Littorina 



941 



TABLE 2. 

Efjtimated production (kj/m') of shell (Ps. 1988 and 1989), somatic tissue (Pg, 1988 and 1989), and the production of gametes (Pr, 1989) of 

Littorina liltorea on Pendleton Island, 



Production (KJ/m'/v') 






1988 








1989 














Prf 

(Indirect) 


Prf 

(Direct) 


Prm 
(Indirect) 


Pr(m + f) 
(Total) 




Cohort 


Pg 


Ps 


Pg 


Ps 


Pt 





0.565 


0.304 


1.741 


1.235 














2.976 


1 


5.268 


1.621 


15.637 


4.039 


0.002 


13.661 


0.003 


0.005 


19.681 


2 


31.469 


7.870 


26.981 


5.716 


0.657 


4.084 


0.438 


1.095 


33.792 


3 


3:.%3 


7.064 


38.290 


-0.962 


20.865 


22.038 


15.740 


36.606 


73.933 


4 


— 


— 


5.115 


-1.054 


1 1 .496 


6.378 


5.410 


16.906 


20.967 


Total 


70.265 


16.859 


87.764 


8.974 


33.020 


46.160 


21.591 


54.114 


151.349 




(80.6%) 


(19.4%) 


(60.0%) 


(5.9%) 








(36.1%) 





Percentage total production allocated to each component in parentheses (/). 

Production gametes (Pr) presented for feinales (Prf I and males (Prm) separately and together [Pr(m + t") 

Prf calculated using both indirect and direct methods (Crisp 1984). 



F = 5.83; p > .1 ). However, the indirect metho(j seems to tinder- 
estimate the Pr of the younger cohorts. Indirect estimates seem to 
be inappiopriate for young cohorts, and fecundity estimates ba.sed 
on such will underestimate Pr. especially for the tnales. 

Male Reproductive Output 

The total annual production of male gametes (Prm) was calcu- 
lated to be 21.591 kJ/nr (Table 2). Production increased to a 
maximum at cohort 3 ( 15.740 kJ/m-. 72.9% of the total Pr). A 
two-way ANOVA on Pr of male and females using the indirect 
calculated values revealed no significant effect of se.x (df = 1.4: 
F = 2.81; p > .2); however, cohort was significant (df = 1.4: F = 
27.68; p > .01). Pr for the older females (cohorts 2 and 3) was 
larger than that of the males. The total estimate of Pr for the L 
litlorea population (males and females) on Pendleton Island based 
on the indirect calculations of male and female reproductive output 
was 54.114 kJ/nr. 

Total Production 

The total production for the breeding season in 1989. calculated 
as the sutn of Ps, Pg. and Pr, was 151,349 kJ/nr (36.1% Pr. 60.0% 
Pg, and 5.9% Ps). Total production could not be calculated for the 
1988 season, because no data were available on production allo- 
cated to reproduction. Figure 3 shows the percentages of produc- 
tion allocated to each of Ps. Pg. and Pr in each cohort. In the newly 
settled cohort (0) all of the production was allocated to either Ps 
(41.5%) or Pg (58.5%). The percentage of production allocated to 
Ps and Pg in each cohort decreased once maturity was reached 
(cohort 1 ). The percentage of the production allocated to Pr in- 
creased with age to age 4. with Pr comprising 76.8% of the pro- 
duction of that cohort. 



DISCUSSION 



Demography 



The structure of the intertidal population of L. litlorea on 
Pendleton Island changed seasonally as a result of recruitment and 
growth (Fig. 2). At the beginning of each of the 1988 and 1989 
seasons, the population of L. Uttorea was dominated by mature 
individuals (>13 mm). However, at the end of the season, the 



Z 

o 



u so 



a. 




AGE (years) 

Figure 3. Percentage of total production in each age class that is al- 
located to shell growth (Ps). somatic tissue growth (Pg), and produc- 
tion of gametes (Pr) in 1989. 



percentages of the population composed of immature and mature 
individuals were almost equal (% immature:% mature; 48:52 in 
1988 and 45:55 in 1989). Recruitment of L. Uttorea was tempo- 
rally variable but consisted of two pulses per year, in July and 
August 1988 and in August and October 1989. The newly settled 
cohorts comprised 23 and 3 1 % of the population at the end of each 
season in 1988 and 1989. Recruitment was much higher on Pendle- 
ton Island than recorded in other studies of L. Uttorea (8.4% Gard- 
ner and Thomas 1987b at Welch's Cove. Bay of Fundy; 1.6% 
Lambert and Farley 1968 at Ketch Harbour, Nova Scotia; and 
0.4-4.4% Smith and Newell 1955 at Whitsable. England). Larger 
recRiitment densities may be the result of the existence of two 
pulses of recruitment per year. In addition. L. Uttorea on Pendleton 
Island were observed to recruit at high shore levels only (80% tidal 
level) (Chase and Thomas 1995a). Low recruitment densities in the 
studies of Gardner and Thomas {1987b) and Lambert and Farley 
(1968) were attributed to recruitment from subtidal regions that 
was not accounted for in fall estimates of recruitment density. 



942 



Chase and Thomas 



Production 

Secondary production estimated for L litiorea on Pendleton 
Island in 1988 was measured as the production of somatic tissue 
and shell. Reproductive output was not included. In 1989. gamete 
production comprised 36.1% of the total production; thus, any 
calculations fori., littorea in 1988 were underestimated. Compari- 
son of the somatic and shell production estimates in 1988 and 1989 
revealed that there was no difference between years, despite a 
difference in the duration of the sampling season. In 1988, pro- 
duction was measured from early May to late August as compared 
to early May to late October in 1989. a difference of approximately 
8 weeks. A breakdown of production in 1989 into two time peri- 
ods: May to late August and late August to late October; however, 
revealed that the majority of shell (64.7%) and all of the somatic 
production ( 100%) occurred in the May to late August time period. 

Comparisons of total secondary production values of L littorea 
are difficult, because no studies have examined all components of 
total production (somatic tissue, shell growth, and reproductive 
output). There are. however, .studies where somatic tissue and/or 
gamete production have been measured, so some comparisons are 
possible. Gardner and Thomas (1987b) measured somatic tissue 
production for a population of L. littorea at Welch's Cove in the 
Bay of Fundy. Their calculated value was approximately 3X larger 
than our figures; although the populations at each location were 
very different, which may prevent comparisons between the stud- 
ies. The study of Gardner and Thomas ( 1987b) was restricted to a 
small portion of the intertidal zone and had much higher densities 
than our study (657 to 934 individuals/ni" versus 28 to 86 indi- 
viduals/m"). In contrast, our study examined the entire intertidal 
zone. 

Grahame (1973) calculated somatic production and gamete 
production of a population of L. littorea at Anglesey, Wales. Total 
production of these two measures was 852.51 kJ/ni" [somatic pro- 
duction = 629.510 kJ/m" ( 138 kcal/nr) and gamete production = 
223 kJ/m" (46.7 kcal/m~)]. The estimate of gamete production in 
this study was only 54 kJ/m". only 24% of that reported by Gra- 
hame (1973). The population in that study, however, contained 
many larger individuals (spire height > 25 mm). On Pendleton 
Island in 1989, the percentage of the population comprising indi- 
viduals with a spire height > 25 mm was less than 3%. Because 
fecundity increases with spire height in L. littorea (Grahame 1973, 
Hughes and Roberts 1980, Chase and Thomas 1995b), it would be 
expected that we would observe a larger Pr for a population com- 
posed of larger, more fecund individuals. 

No estimate of the production of the shell matrix for L. littorea 
was found in the literature. Values from other studies indicate that 
shell production in bivalves is usually <5'7r of total production. For 
L. littorea, the organic component of the shell represented 6% of 
the total production in 1989. As such, it seems that, for L. littorea. 
the majority of the secondary production, was attributed to the 
production of somatic tissue and gametes. 

Considering such a large amount of energy is required for the 
production of gametes, it would be expected that there would be a 
close coupling between the reproductive cycle and energy avail- 
able for growth. Our data are in keeping with the general obser- 
vation that animals devote a greater share of the production to 
reproduction as they age (Fig. 3). However, the proportion of total 
production allocated to reproduction in the L. littorea population 
on Pendleton Island was higher as compared to other studies. 

Hughes and Roberts (1980) examined the proportion of total 



production allocated to reproduction (Pr/Pr -i- Pg) for different age 
classes of four Littorinid species, including L. littorea. They ob- 
served, for all species, that the proportion of total production al- 
located to reproduction increased linearly during a phase of rapid 
growth, but began to level off abruptly toward its asymptotic value 
of 80 to 100% (Hughes and Roberts 1980). For L. littorea. spe- 
cifically, the proportion allocated to reproduction increased from 
to 100% in 9 years, with the asymptote occurring at age 5 (Hughes 
and Roberts 1980). In this study, only four cohorts were discem- 
able. either because older individuals were not present in the popu- 
lation, or they were so few in numbers so as not detectable using 
this method of cohort determination. Over the 4 years, however, 
the proportion of total production allocated to reproduction in- 
creased linearly once maturity was reached (cohort 1 ). For an age 
4 individual, the proportion of total production allocated to repro- 
duction (Pr) in L. littorea was approximately 60%' in the study of 
Hughes and Roberts ( 1980). In this study. Pr in an age 4 individual 
was 76.87r. 

Alternative estimates of the proportional allocation to repro- 
duction include the ratio of gamete to somatic production (Pr/Pg). 
In this study. Pr/Pg was 61.7%. This estimate is high as compared 
to studies on other gastropods, including L. littorea. where Pr/Pg 
was calculated; 34% (L. littorea. Grahame 1973). 30 to 40% (La- 
cuna vincta. Grahame 1982), 25% (Lacuna pallidula. Grahame 
1982). and 11.7% (Fissurella barbadensis. Hughes 1971). 

Developing hypotheses to explain the higher allocation to re- 
production (Pr) observed in this study is partly hampered by only 
1 year of measurement. In many studies, the larger Pr may reflect 
older populations, because Pr generally increases with the age of 
an indi\idual (Hughes and Roberts 1980. this study). The popula- 
tion monitored in this study was made up of young individuals 
(only four cohorts detected in each year). A high allocation to 
reproduction in such young animals might be expected if this 
population is subject to high mortality and lowered life expectan- 
cy. This reduced life expectancy may be the result of environmen- 
tal conditions. The population of L. littorea on Pendleton Island is 
situated in a passageway where there is a very strong current. 
Average surface currents 3 hours before and after low tide gener- 
ally exceed 1.3 m/s'. bottom currents have been measured at 0.47 
to 0.60 m/s' (Thomas et al. 1990). Many studies examining the 
effect of wave exposure on the population energetics of gastropods 
have found greater mortality and a larger amount of energy de- 
voted to reproduction (e.g.. Hughes and Roberts 1980; Hart and 
Begon 1982; Etter 1989). In addition to mortality caused by en- 
vironmental conditions, it was found that at least 25% of the larger 
animals examined had parasitic infestations of the digenetic treraa- 
tode larvae. Cryptocotyle lingua (Crepling) (Chase and Thomas 
1995b). Similar infestations have resulted in the destruction of the 
digestive gland, castration, and change in the migration pattern 
(Fretter and Graham 1962; Lambert and Farley 1968). Such infes- 
tations may explain the absence of larger/older individuals on this 
shore. 

SUMMARY 

The results of this study revealed that estimates of population 
production of L. littorea were much lower than values reported in 
the literature. Rates of secondary production are known to vary 
widely in nature and are affected by a variety of biotic and abiotic 
characteristics of the environment (Plante and Downing 1989). 
Such variation in production may be the result of independent or 



Production of Littorina 



943 



combined effects of annual differences in such environmental fac- 
tors as water temperature and/or food supply, variation in density, 
age structure, and allocation to growth and reproduction. Density 
and age structure have been proposed as factors causmg the lower 
somatic and gamete production values observed in this study. 
However, despite lower estimates of production, the proportion of 
total production allocated to reproduction (Pr) measured as both 
the proportion of total production (Pr/Pg + Pr + Ps) and somatic 
production (Pr/Pg) was higher in this study by approximately 20% 
as compared to estimates for L Httorea in the literature. 

The Bay of Fundy exhibits a great diversity of marine .species, 
a combination of the influx of larvae, propagules. and adults from 
the Labrador Current and the Gulf of Marine waters, and the high 
productivity as a result of the vigorous tidal mixing (Thomas et al. 
1990). Prior research on the population of L. liitorea in Pendleton 
Passage revealed high growth rates, large recruitment densities, 
and high reproductive outputs (Chase and Thomas 1995a. b). How- 
ever, analysis of the demography, production, and resource allo- 
cation of the population of L. Httorea in Pendleton Passage seems 



to be more characteristic of an environment with very harsh con- 
ditions; that is, fewer older individuals, faster turnover rates, and 
lower total production, with a greater proportion of total produc- 
tion allocated to reproduction. It is likely that the high tidal cur- 
rents are a major influence on the population of L. Httorea in 
Pendleton Passage. However, additional information will have to 
be gathered before we can speculate on the effects of high tidal 
energy on this population of L. Httorea. 

ACKNOWLEDGMENTS 

We are grateful to G. Bacon, C. Hatfield, and R. Bosien for 
assistance in the field, to W. Morris for computer and drafting 
assistance. Special thanks to M. G. Topping and B. A. MacDonald 
for reading drafts and offering constructive criticism. This research 
was funded through a Natural Science and Engineering Research 
Council of Canada (NSERC) grant to M. L. H. Thomas and an 
NSERC postgraduate scholarship to M. E. Chase. Current address 
for MEC: Department of Zoology. Miami University. Oxford. 
Ohio 45056, USA. 



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Cassie, R. M. 1954. Some uses of probability paper in the analysis of size 

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Chase. M. E. & M. L. H. Thomas. 1995a. Evidence for double recruilmenl 

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Httorea L. J. E.xp. Mar Biol. Ecol. 186:277-287. 
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Holme and A. D. Maclntyre AD (eds.). Methods for the Study of 

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Ekaratne. S. U. K. & D.J. Crisp. 1982. Tidal microgrowth bands in an 
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Elmhirst. R. 1923. Notes on the breeding and growth of marine animals in 
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Fish. F. D. 1972. The breeding cycle and growth of open coast and estua- 
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Fretter. V. & A. Graham. 1962. British prosobranch molluscs: their func- 
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Gardner. J. P. A. 1986. Demography, Growth, and Production of Mytilus 
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Jaiinuil ofShetlthh Research. Vol. 17, No. 4. 945-953, 1998. 

IDENTIFICATION OF STOCKS OF THE EXPLOITED LIMPETS PATELLA ASPERA AND P. 
CANDEI AT MADEIRA ARCHIPELAGO BY ALLOZYME ELECTROPHORESIS 



LAURA I. WEBER,' JOHN P. THORPE,' RICARDO S. SANTOS,^ 
AND STEPHEN J. HAWKINS' 

Port Erin Marine Lxiboratory 
University of Liverpool 
Port Erin, Isle of Man 
'Departamento Oceanografm e Pescas 
Universidade dos A(^ores 
Horta. Fcdal. A(;ores 

ABSTRACT Allozyme electrophoresis was used to investigate stock integrity of Patella aspeni ( = P. iily.ssiponeiisis) and Patella 
camlet {Molliisca. Patellogastropoda) from the Madeira Archipelago. Samples from the north of the island of Madeira (Pono Moniz) 
and the north of Deserla Grande were taken for both species, and a sample of P. aspera was also taken from the south of the island 
of Madeira (Canine). Twenty-one putative loci were resolved and analyzed for both species. Significant differences in allele frequencies 
were found between locations in each species, suggesting the presence of different genetic stocks. P. candei was found to be slightly 
structured with a small standardized variance in allele frequencies between samples from Porto Moniz and Deserta Grande (Fsy = 
0.036); whereas, P. aspera was highly structured, with an Fsj value over all subpopulations of 0.157. Our results indicate more genetic 
interchange between the populations from the north of Madeira and Desena Grande than between north and south Madeira. Our 
findings are consistent with surface water-mass circulation patterns from northwest to southeast along the Madeira Archipelago, 
determined mainly by the Canary Current. Species with low dispersal and a high degree of spatial genetic structuring among 
subpopulations are more susceptible to collap.se caused by overfishing than those wnh high dispersal capabilities; therefore, the 
individual management and control of these biological units is recommended. 

KEY WORDS: Mollusca, patellogastropoda. Patella, population genetics, stocks, allozymes 



INTRODUCTION 

The stock concept was first desciibed by Larkiti ( 1972) as: "a 
population of organism.s shainng a common gene pool, that is 
di.screte to warrant consideration as a self-perpetuating sy.stem that 
can be managed." This concept, primarily applied to fish species, 
has been widely used with different connotations by the fisheries 
community, politicians, and biologists. Stock, as a tnanagement 
unit, may also be defined as the extent of a population or mi.xed 
populations over which a fishing activity occurs. Therefore, it may 
comprise more than one biological stock or subpopulation (Hedge- 
cock 1986). 

In the 1970s, allozyme electrophoresis emerged as a powerful 
tool to recognize biological stocks by means of genetic informa- 
tion (Gall 1986, Hedgecock 1986, Utter 1986, Utter 1991, Shep- 
herd and Brown 1992. Avi.se 1994). Once the genetic structure of 
an exploited species is understood, the fishery can be regulated so 
that harvesting of each subpopulation can be managed and con- 
trolled individually (Allendorf et al. 1988). However, despite its 
importance, the "stock-composition" strategy has been practiced 
rarely in fisheries management (Pella and Milner 1988). 

P. aspera Roding, 1798 (= P. tilyssipimensis Gmelin, 1791) 
and P. candei d'Orbigny, 1840 are two palellogastropod limpets 
distributed throughout the Macaronesian Archipelagos, where they 
represent an important food resource. In recent years, they have 
been intensively exploited for subsistence and for commercial pur- 
poses. In the Azores, both species have been declining for many 
years (Martins et al. 1987. Menezes 1991. Corte-Real et al. 1996). 

Recent works on the population genetics of these limpets, using 
allozyme electrophoresis (Corte-Real 1992, Corte-Real et al. 1996. 
Weber et al. in preparation), have shown high macroscale struc- 
turing of their populations. The aim of the present work is to study 
the structuring of these species within the Madeira Archipelago, to 



find out if there are different biological stocks. This work forms 
part of a fisheries management strategy by the regional govern- 
ment. 



MATERIALS AND METHODS 



Sampling Sites 



Samples of Patella aspera and Patella candei were taken from 
the Madeira Archipelago (Fig. I ). Samples of both species were 
taken in July 1994 from Porto Moniz (north Madeira) and from the 
north of Deserta Grande in March 1995. An additional sample of 
Patella aspera was taken from Canifo (south Madeira) in July 
1994. Locations in Madeira were chcsen from those points where 
human activity is concentrated. Sample sizes (n) are specified in 
Tables 2 and 3. All samples were transported live to the Port Erin 
Marine Laboratory, where they were frozen at -78°C until re- 
quired for electrophoresis. 

Electrophoresis 

Homogenates were prepared by macerating foot muscle in 
buffer Tris-HCl. pH 8.0, and then centrifuged at 10,000 rpm for 5 
min. Supematants were used afterward for electrophoresis. Stan- 
dard horizontal 12.5% starch gel (Sigma-Aldrich Co, Ltd,) elec- 
trophoresis was carried out using the following buffer systems: (I) 
Tris-citrate, pH 8.0 (Siciliano and Shaw, 1976) and fll) Tris- 
citrate-EDTA. pH 7.0 (Ayala et al. 1972); (III) Discontinuous 
Tris-citrate-borate, pH 8.2-8.7 (Poulik 1957); (IV) Tri.s-citrate- 
borate-LiOH, pH 8.26-8.31 (Redfield and Salini 1980); (V) Tris- 
borate EDTA, pH 9.0 (Ayala et al. 1974). Specific staining pro- 
cedures for the enzyines analyzed (Table I) followed the tech- 
niques of Brewer (1970), Shaw and Prasad (1970), Harris and 
Hopkinson (1976) and Murphy et al. (1990). 



945 



946 



Weber et al. 



MADEIRA ISLANDS ^ 

PORTO SANTO 



33"N 

Porto Moniz 



MADEIRA 





Figure 1. (*) Sampling sites for P. caitdei and P. aspera (see text for 
details). 



Allele frequencies, mean expected heterozygosity, Nei's ( 1978) 
genetic identities, and x' tests for homogeneity in allele frequen- 
cies were calculated using the statistical package BIOSYS-1 
(Swofford and Selander 1981). For those individual tests of het- 
erogeneity that showed p < .05, contingency tables were checked 
using G (log-likelihood ratio)-test with pooling when more than 
two alleles, and expected frequencies less than five were observed. 
Yate's correction was also applied for those 2x2 tables, p-values 
after G-test were reported in Tables 2 and 3 only when they were 



notably different from those obtained by the common x"- Unbiased 
inbreeding coefficients, F,s and F,y (Weir and Cockerham 1984) 
and the genetic variance between populations (F^-,). with their 
respective 95% confidence intervals, estimated from a bootstrap 
procedure of 15,000 resamplings, were obtained by the FSTAT 
Program, version 1.2 (Goudet 1995). Numbers of migrants be- 
tween populations per generation (n^^) were calculated by using 
Slatkin"s ( 1993) formula: n^.,„ = (I/F^t-I )/4. Loci were numbered 
according to increasing anodal mobility. 

RESULTS 

Twenty one loci were resolved for both species. Est-D was only 
resolved for P. candei and LAh-l only for P. aspera. Allele fre- 
quencies, variability measures, F-statistics. and results of the x" 
test for the homogeneity in allele frequencies are summarized in 
Tables 2 and 3 for P. candei and P. aspera, respectively. 

Variability 

The locus Gludh was fixed for the same allele in all populations 
of both species, and Mdh-l was fixed in all the populations of P. 
aspera. High levels of polymorphism (76-85.7%) and heterozy- 
gosity were registered for both species. P. aspera populations 
showed higher values of heterozygosity (all over 21%) than P. 
candei {around 10 to 12%). Mean number of alleles per locus was 
also higher in P. aspera (4.4) than P. candei (2.7). 

Differentiation among Subpopiilations 

Both species showed significant differentiation in allele fre- 
quencies between locations (see x~ tests in Tables 2 and 3). 



P. candei Subpopulations 



candei samples 



An FsT value of 



The genetic identity (Nei. 1978) between P. 
from Porto Moniz and Deserta Grande was 0.995 
0.036 was found for the analyzed P. candei samples, indicating the 
subdivision of the total population. The bootstrap 95% confidence 
interval showed that the F^y value was close to but different from 



TABLE 1. 
Name, E.C. number, abbreviation, buffer system and number of loci for each enzyme analyzed 



Enzvme Name 



E.C. 




Number 


Buffer 


Number* 


Abbreviation 


of loci 


System 


2.6.1.1 


AAT 


2 


ni 


3.1.1.- 


EST 


2 


rv 


3.1.- 


EST-D 




m 


4.1.2.13 


FBALD 




V 


1.2.1.12 


GAPDH 




V 


1.4.1.- 


GLUDH 




V 


5.3.1.9 


GPI 




I 


1.1.1.42 


IDHP 


2 


I 


1.1.1.27 


LDH 


2 


n 


1.1.1.37 


MDH 


2 


IV 


1.1.1.40 


MEP 


2 


I 


5.3.1.8 


MPl 


1 


IV 


5.4.2.2 


PGM 


1 


u 


1.1.1.44 


PGDH 


1 


I 


24.2.1 


PNP 


1 


I 


1.15.1.1 


SOD 


2 


m 



Aspartate aminotransferase 
Esterase 
Esterase-D 

Fructose-biphosphate aldolase 
Glyceraldehyde-3-phosphate dehydrogenase 
Glutamate dehydrogenase 
Glucose-6-phosphate isomerase 
Isocitrale dehydrogenase (NADP+) 
L-Lactate dehydrogenase 
Malate dehydrogenase 
Malic Enzyme (NADP-h) 
Mannose-6-phosphate isomerase 
Phosphoglucomutase 
Phosphogluconate dehydrogenase 
Purine-nucleoside phosphorilase 
Superoxide dismutase 



■ 1UBNC(1984); Shaklee et al. (1990). 



Patella spp., genetic stocks at Madeira 



947 



TABLE 2. 

Patella candei: allele frequencies, F-statistics per locus, and over-all loci (with 95% confidence intervals 
Bootstrap procedure) and contingency x'' test for the homogeneity in the allele frequencies for each 



in parentheses obtained by 
locus and over-all loci. 



Allele frequencies 



F statistics per locus 



Homogeneity Test 



Locus 



Allele 



DES (n = 50) 



MNZ (n = 50) 



F,,s 



Aat-1 
Aat-2 
Est-1 

Est-2 

Est-D 

Fbald 

Gapdh 

Gpi 

Idhp-1 

hihp-2 

Uh-2 

Mdh-1 
Mdh-2 

Mep-I 
Mep-2 



A 

B 

All 

A 

B 

All 

A 

B 

C 

All 

A 

B 

All 

A 

B 

All 

A 

B 

All 

A 

B 

All 

A 

B 

C 

D 

All 

A 

B 

C 

All 

A 

B 

C 

D 

All 

A 

B 

C 

All 

A 

B 

All 

A 

B 

C 

All 

A 

B 

All 

A 

B 

C 

D 

E 

All 



0.1 ?0 
0.850 

1.000 
0.000 

0.110 
0.770 
0.120 

1.000 
0.000 

0.080 
0.920 

0.960 
0.040 

0.000 
1.000 

0.020 
0.930 
0.040 
0.010 

0.010 
0.990 
0.000 

0.030 
0.670 
0.290 
0.010 

0.060 
0.010 
0.930 

0.000 
1.000 

0.170 
0.820 
0.010 

0.010 
0.990 

0.010 
0.970 
0.010 
0.010 
0.000 



0.020 
0.980 

0.950 
0.050 

0.110 
0.820 
0.070 

0.980 
0.020 

0.060 
0.940 

1.000 
0.000 

0.010 
0.990 

0.010 
0.980 
0.000 
0.010 

0.010 
0.980 
0.010 

0.030 
0.450 
0.500 
0.020 

0.050 
0.020 
0.930 

0.010 
0.990 

0.000 
1.000 
0.000 

0.010 
0.990 

0.010 
0.920 
0.040 
0.000 
0.030 



-0.010 0.085 



-0.043 



0.171 



-0.010 



-0.032 



0.167 



0.152 0.174 



0.000 0.000 



0.094 



-0.000 0.041 



-0.005 



0.000 0.010 



0.026 



-0.000 0.031 



0.000 



-0.034 -0.019 0.014 



-0.003 -0.010 -0.007 



-0.081 0.149 0.074 



0.241 0.232 -0.013 



0.000 0.000 0.000 



-0.196 -0.000 0.164 



-0.000 -0.010 -0.010 



10.865 



5.128 



1.473 



2.020 



2.083 



4.082 



1.005 



4.464 



1 .005 



10.237 



0.424 



1.005 



19.780 



0.000 



0.00191 1 
0.00098* 



0.05353t 
0.02354 



0.47878 



0.15522 



0.14892 

0.11434t 
0.04335 



0.31610 



0.21550 



0.60499 



0.01755t 
0.01666 



0.80887 



0.31610 



0.00005* 



0.99835 



-0.039 -0.027 



0.011 



5.932 0.20426 
continued on next page 



948 



Weber et al. 



TABLE 2. 
continued 



Allele frequencies 



F statistics per locus 



Locus 


Allele 


DES (n = 50) 


MNZ (n = 50) 


Mpi 


A 


0.020 


0.010 




B 


0.980 


0.970 




C 


0.000 


0.020 




All 






Pgdb 


A 


0.000 


0.020 




B 


0.010 


0.030 




C 


0.990 


0.950 




All 






Pf;m 


A 


0.000 


0.020 




B 


0.210 


0.120 




C 


0.790 


0.860 




All 






Pnp 


A 


0.230 


0.170 




B 


0.770 


0.830 




All 






Sod-2 


A 


0.010 


0.020 




B 


0.990 


0.980 




All 






Overall 


Ho-' 


0.117 


0.105 


loci 


He'' 


0.126 


0.109 




^0 9y 


76.2 


85.7 



F,s 



F., 



Homogeneity Test 



0.192 



-0.024 



0.109 



0.130 



-0.007 



0.187 



-0.014 



0.120 



0.130 



-0.014 



-0.006 



0.011 



0.012 



-0.000 



-0.007 



0.071 0.105 

rO.006-0.113) (0.057-0.132) 



.338 0.31061 



3.082 0.21412 



4.752 0.09294 



1.125 0.28885 



0.338 0.56075 



0.036 81.240 0.00001* 

(0.006-0.065) 



■* Mean observed heterozygosity (direct-count). 

''Mean unbiased heterozygosity based on Hardy-Weinberg expectation (Nei 1978). 

■■ Percentage of polymorphic loci: a locus is considered polymorphic if the frequency of the most common allele does not exceed 0.99. 

* Significant at a = 0.05 after a Bonferroni procedure for multiple tests was applied (a' = a/1 + k-i: for k = number of tests, and i = rank of the values 

of p when ordered from the smallest to the largest; when pi s a', then the corresponding test indicates significance at the "table-wide" a level (Rice. 

1989). 

t P after G test and Yates correction. 

Key: F,s Mean heterozygote defficiency within populations; Fn- Heterozygote deficiency in the total population; Fj^, Degree of differentiation between 

subpopulations: MNZ: Pto. Moniz; DES: Deserta Grande; (n) Sample size. 



zero (Table 2 1. The results of the x~ test corroborated the presence 
of different subpopulations although with only slight differences. 
The significant differentiation found in P. candei between 
Porto Moniz and Deserta Grande was largely a result of the varia- 
tion in allele frequencies of two loci: Aat-1 and Mdh-2 (Table 2). 
These showed a geographic pattern in allele frequencies, with a 
tendency toward the fixation of the most common allele at Deserta 
Grande Island (see Fig. 2). with a resulting loss of genetic vari- 
ability at Deseita Grande (see tiieasures of genetic variability in 
Table 2). The number of migrants estimated between these sub- 
populations was 6.7 individuals per generation. F,s values were 
high for various loci indicating a tendency of heterozygote deficit 
(see Table 2). 

P. aspera Subpopulations 

The genetic identities and distances between samples of P. 
aspera are shown in Table 4. The identity values ranged from 
0.930 to 0.959 (see also UPGMA dendrogram in Fig. 4). The total 
Fs-p estimated over-all samples was 0.136 (0.026-0.307). and 
0.126 (0.013-0.262) between the most clo,sely related locations 
(Canigo and Deserta Grande), both values being different from 
zero. The estimated numbers of migrants obtained from the ¥^y 
values are 1.7 individuals per generation between Caniijo and De- 



serta Grande, 1.1 between Porto Moniz and Canit^o, and 1.5 be- 
tween Porto Moniz and Deserta Grande. 

A X" te^t applied to over-all samples was highly significant, 
showing that seven of the 21 loci analyzed were responsible for the 
genetic differentiation among populations (see Table 3). Even be- 
tween the most closely related pair of samples (Canigo and Deserta 
Grande), there was highly significant differentiation in allele fre- 
quencies (X" = 256.50; p < lO"*^). which was mainly attributable 
to the contributions of 4 loci: Est-1 (p < lO""^), Aat-1 (p < 10^"'); 
Est-I (p < 10"-*), and Idhp-2 (p < lO""*). 

It was possible to detect geographic patterns in allele frequen- 
cies (Fig. 3) as in P. candei. Figure 3 shows the si.x loci that 
displayed the most significant differentiation between subpopula- 
tions. We could distinguish three different patterns. The first, de- 
tected only at the Me-2 locus, showed that the less common allele 
of Porto Moniz. "B." became the most common one in Canigo 
and Deserta Grande subpopulations. As a second geographic pat- 
tern, it was possible to detect a dine in three loci. Aat-1. Est-I. and 
Spd-2. At \he Aat-1 locus, the allele "C" of Porto Moniz increased 
its frequency from 23 to 36% in Canijo and to 75% in Deserta 
Grande; at the Est-1 locus, the most common allele "B" in Porto 
Moniz, also increased its frequency from 62 to 85% in Cani90 and 
to 87% in Desena Grande; and at the Sod-2 locus, the most com- 



Patella spp., genetic stocks at Madeira 

TABLE 3. 



949 



Patella aspera: allele frequencies, F-statistics per locus, and over-all loci (Hith 95% confidence intervals. bet\»een brackets, obtained by 
bootstrap procedure) and contingency X' <est for the homogeneity in the allele frequencies for each locus and over-all loci. 



Allele frequencies 



F statistics per locus 



Homogeneity Test 



Locus 



Aaf-I 



Aat-2 



Est- 1 

Est-2 

FhaUl 

Giipcth 
Gpi 



hlhp-l 



lilhp-2 



Uh-1 



Ldh-2 



Allele 



A 

B 

C 

D 

All 

A 

B 

C 

D 

E 

All 

A 

B 

C 

D 

All 

A 

B 

C 

D 

All 

A 

B 

C 

D 

All 

A 

B 

All 

A 

B 

C 

D 

E 

F 

G 

All 

A 

B 

C 

D 

All 

A 

B 

C 

D 

E 

F 

All 

A 

B 

C 

D 

E 

All 

A 

B 

C 

D 

E 

All 



MNZ(n = KM)) 



0.025 
0.005 
0.230 
0.740 

0.005 
0.080 
0.790 
0.115 
0.010 

0.115 
0.625 
0.115 
0.145 

0.045 
0.790 
0.155 
0.010 

0.000 
0.250 
0.745 
0.005 

0.250 
0.750 

0.005 
0.005 
0.895 
0.025 
0.000 
0.065 
0.005 

0.010 
0.980 
0.005 
0.005 

0.010 
0.010 
0.010 
0.000 
0.670 
0.300 

0.010 
0.955 
0.025 
0.005 
0.005 

0.045 
0.260 
0.595 
0.070 
0.030 



CNC (n = 49) 


DES (n = 50) 


0.000 


0.040 


0.000 


0.000 


0.357 


0.750 


0.643 


0.210 


0.000 


0.000 


0.031 


0.090 


0.867 


0.780 


0.102 


0.130 


0.000 


0.000 


0.010 


0.130 


0.847 


0.870 


0.133 


0.000 


0.010 


0.000 


0.000 


0.030 


0.306 


0.940 


0.694 


0.030 


0.000 


0.000 


0.082 


0.000 


0.214 


0.230 


0.704 


0.770 


0.000 


0.000 


0.265 


0.240 


0.735 


0.760 


O.OIO 


0.020 


0.000 


0.000 


0.929 


0.910 


0.000 


0.010 


0,000 


0.020 


0.061 


0.040 


0.000 


0.000 


0.000 


0.010 


1.000 


0.990 


0.000 


0.000 


0.000 


0.000 


0.000 


0.000 


0.010 


0.040 


0.020 


0.010 


0.000 


0.010 


0.969 


0.760 


0.000 


0.180 


0.000 


0.000 


0.980 


1.000 


0.10 


0.000 


0.010 


0.000 


0.000 


0.000 


0.061 


0.050 


0.276 


0.220 


0.643 


0.730 


0.010 


0.000 


0.010 


0.000 



Fis Fr 



FsT 



0.159 0.382 0.265 



83.914 0.00000* 



0.077 0.077 0.000 6.924 0.54490 



0.262 0.316 0.073 55.329 0.00000* 



0.437 0.652 0.381 139.439 0.00000* 



0.137 0.136 -0.001 



0.30174t 
26.155 0.00021 



0.126 0.118 -0.008 0.172 0.17660 



0.015 0.013 -0.002 13.218 0.35337 



-0.006 -0.007 -0.002 2.988 0.81041 



0.197 



0.306 



0.136 



47.490 0.00000* 



-0.019 



-0.014 



0.006 



0.282 



0.285 



0.004 



7.110 0.52481 



0.07090t 
17.992 0.02128 
continued on next page 



950 



Weber et al. 



TABLE 3. 
continued 



Allele frequencies 



F statistics per locus 



Homogeneity Test 



Locus 


Allele 


MNZ (n = 100) 


CNC (n = 49) 


DES (n = 50) 


Mdh-2 


A 


0.005 


0.000 


0.010 




B 


0.965 


0.969 


0.970 




C 


0.030 


0.031 


0.020 




All 








Mep-1 


A 


0.010 


0.082 


0.030 




B 


0.990 


0.918 


0.970 




All 








Mep-2 


A 


0.000 


0.010 


0.080 




B 


0.075 


0.929 


0.810 




C 


0.895 


0.061 


0.110 




D 


0.025 


0.000 


0.000 




E 


0.005 


0.000 


0.000 




All 








Mpi 


A 


0.000 


0.102 


0.030 




B 


1.000 


0.898 


0.970 




All 








Pgdh 


A 


0.005 


0.000 


0.020 




B 


0.040 


0.031 


0.100 




C 


0.955 


0.969 


0.880 




All 








Pgm 


A 


0.015 


0.000 


0.000 




B 


0.185 


0.133 


0.210 




C 


0.020 


0.000 


O.OIO 




D 


0.250 


0.306 


0.280 




E 


0.515 


0.531 


0.500 




F 


0.015 


0.031 


0.000 




All 








Pup 


A 


0.005 


0.000 


0.000 




B 


0.000 


0.000 


0.020 




C 


0.005 


0.000 


0.070 




D 


0.980 


0.990 


0.900 




E 


0.010 


0.010 


0.000 




F 


0.000 


0.000 


0.010 




All 








Sod-2 


A 


0.195 


0.255 


0.120 




B 


0.090 


0.000 


0.000 




C 


0.660 


0.745 


0.790 




D 


0.010 


0.000 


0.050 




E 


0.025 


0.000 


0.040 




F 


0.020 


0.000 


0.000 




All 








Over-all 


Ho-' 


0.228 


0.176 


0.181 


loci 


He" 


0.254 


0.217 


0.224 




P0.99' 


85.7 


85.7 


85.7 



F,s 



0.316 



0.109 



0.048 



0.250 



0.111 



0.070 



-0.051 



0.055 



0.155 



0.310 



0.139 



0.702 



0.307 



0.1; 



0.066 



-0.008 



0.074 



0.287 



-0.009 



0.034 



1.270 0.86643 

0.00799t 
10.711 0.00472 



0.688 



0.075 



0.020 



293.090 0.00000* 



21.705 0.00002* 



9.249 0.05518 



-0.004 



10.859 0.36858 



0.042 



0.00277t 
28.109 0.00173* 



0.020 



0.156 



0.01 130t 
40.258 0,00002 



815.981 0.00000* 



(0.100-O.214) (0.154-0.423) (0.026-0.307) 



"'Mean observed heterozygosity (direct-count). 

"Mean unbia.sed heterozygosity based on Hardy-Weinberg expectation (Nei 1978). 

' Percentage of polymorphic loci: a locus is considered polymorphic if the frequency of the most common allele does not exceed 0.99. 

* Significant at a = 0.05 after a Bonferroni procedure for multiple tests was applied (see Table 2). 

t P after G test and Yate's correction. 

Key: F,s Mean heterozygote defficiency within populations; V^f heterozygole defficiency in the total population; Fgj degree of differentiation between 

subpopulations; MNZ: Pto. Moniz; CN(^: Cani^o; DES: Deserta Grande; (n) sample size. 



mon allele "C" in Porto Moniz also increased its frequency in the 
same geographic direction, from 65 to 74% and to 79%. Finally, 
the third pattern was mainly characterised by Est-2 and Idhp-2 loci, 
where the frequency patterns are more similar between Porto 



Moniz and Deserta Grande than to the Canifo subpopulation. This 
last pattern results in the greater genetic identity found between 
Porto Moniz and Deserta Grande samples than between the former 
and the Canigo subpopulation (see Table 4). F,s values were even 



Patella spp., genetic stocks at Madeira 



931 



Aat-1 



Mdh-2 




Porto Moniz 



\ \ 




Deserta Grande 



■ Other Alleles 

Figure 2. P. candei; geographic pattern (if allele frequencies along 
Madeira archipelago at the Aat-I and Mdh-2 loci. 



higher than in P. candei. indicating a higher tendency toward 
heterozygote deficit with a mean over loci of 0.155 (see Table 3). 

DISCUSSION 

The mean observed heterozygosity obtained over all subpopu- 
lations of P. candei (0. 1 11 ) is very close to the mean obtained over 
subpopulations of the same species (0.117) by Corte-Real et al. 
( 1996). The mean value of heterozygosity over the populations of 
P. aspera at the Madeira Archipelago (0.195) is higher than the 
value (0.1.37) obtained for the Macaronesian Islands by Corte-Real 
(1992). Differences found between these values may be attribut- 
able to different number of loci analyzed and the different number 
of populations. 

F,s values were high in both species, indicating an heterozygote 
deficit. E.\cess of homozygotes is not uncommon in mollusks. 
being already found in P. candei and P. caendea by Corte-Real et 
al. (1996). It has been explained by the presence of null alleles, 
inbreeding, negative heterosis, aneuploidy, and population mixing 
(see further references in Corte-Real et al. 19961. However, we do 
not have enough evidence to establish which is the case for ex- 
plaining P. candei and P. aspera heterozygote deficit. 

Our results showed that both species are structured in the Ma- 
deiran Archipelago. Although P. candei was slightly structured, 
maintaining le\ els of gene flow of about 6.7 migrants per genera- 
tion between Porto Moniz and the north of Deserta Grande. P. 
aspera was highly structured, with a gene flow of only 1 .5 indi- 
vidual per generation between the same localities, 1.1 between the 



north (Porto Moniz) and south (Cani(;o) of Madeira, and 1.7 be- 
tween Caniij'o and the north of Deserta Grande. 

The lenght of the pelagic phase during larval development of P. 
aspera and P. candei is not known. Nevertheless, a larval dispersal 
phase of about 4 days after fertilization has been described for P. 
vulgaia and P. caendea (Dodd 1956). From the 4th day after 
fertilization the veliger starts to become more sedentary until be- 
coming completely benthic after metamorphosis, around the 9th 
day after fertilization (Dodd 1956). A dispersal time of 4 to 9 days 
suggests restricted dispersal capabilities. 

Patella viilgata showed levels of structuring (F^y = 0.027) at 
a small and large geographic scale in the northeast of England and 
in south Wales (Hurst and Skibinski 1995) similar to those of P. 
candei in the Madeira Archipelago. Similar lengths of planktonic 
larval life have been described for the highly structured popula- 
tions of Tridacna gigas. suggesting that those lengths of larval 
dispersal may not be sufficient to allow dispersal over large 
stretches of the ocean (Benzie and Williams 1995). 

When the dispersal capability of a species is considerably less 
than its geographic range, genetic differences between subpopula- 
tions should increase with the distance separating them (Slatkin 
1987. Hellberg 1994). The pelagic larvae of P. aspera and P. 
candei would have to travel at least 1 74 nautical miles from Porto 
Moniz to reach the north of Deserta Grande, approximately 177 
nautical miles to reach Canifo, but to cross from Canigo to the 
north of Deserta Grande would have to travel only 16 nautical 
miles. The surface water circulation along the Madeira Archi- 
pelago is mainly caused by the Canary Current, which flows north- 
west to southeast, at around 0.72 km/h (Lalli and Parsons 1995). 
Pelagic larvae carried by this current would have to travel at least 
18 days from the northwest of Madeira Island (Porto Moniz) to the 
southeast in direction to Deserta Grande to reach the north of this 
island. This period of time for limpet larvae to be in the plankton 
seems too long to allow them to travel from one island to the other 
before metamorphosis occurs. Nevertheless, more rapid transport 
could occur during storms, and, perhaps larvae can extend their 
pelagic phase for a time when no suitable substrate for settling is 
available. Smith (1935) obtained metamorphosis in few individu- 
als of P. viilgaia, around 2 to 3 weeks after fertilization. Certain 
gastropods, as well as the larvae of many other invertebrates, can 
delay settlement if a habitat suitable for postlarval survival is not 
available ("delay period," Scheltema 1978). 

Our data and the surface water circulation pattern suggest that 
the gene flow between Porto Moniz and Canigo should be mainly 



Me-2 Aat-1 Est-1 Sod-2 Est-2 ldhp-2 



PortoMoniz 




Deserta Grande ^ 



^C^CD© 



■ other Alleles 
Figure 3. P. aspera: geographic patterns of allele frequencies along Madeira Archipelago al the Me-2. Aal-I. Est-l. Sod-2. Est-2. and Idhp-2 loci. 



952 



Weber et al. 



• Porto Moniz 
. CanJqo 

■Deserta Grande 



94 



—I 
1 00 



92 94 96 98 

Genetic Identity (Nei,1978) 

Figure 4. P. aspera; UPGMA dendrogram by using Nei's genetic iden- 
tities between subpopulations. 



from the former to the latter around the northern end of Madeira, 
presumably occurring by genetic interchange of neighboring sub- 
populations following an isolation by distance model (Slatkin 
1993). Deserta Grande should receive migrants through the Canary 
Current, with major contributions from the south of the Island. 
Gene flows between Porto Moniz and the north of Deserta Grande 
may be greater than between Porto Moniz and Caniijo. possibly as 
a results of a more direct contribution of the water-mass move- 
ments in the direction of Deserta Grande around the north of 
Madeira (Fig. ."i). An upwelling of trigged water, a consequence of 
wind stress during summer, could partially prevent the dispersion 
of larvae through the southern side of Madeira. 

Within islands, the structure of subpopulations should follow 
an isolation by distance model (see Slatkin. 1993); whereas, over 
a larger scale, between islands, it should follow a stepping-stone 
model (see Kimura and Weiss 1964). Further analysis using popu- 
lations from the other archipelagos (Azores and Canaries) is 
needed to test this hypothesis. 

The difference found on the degree of structuring between P. 
candei and P. aspera may be explained by one of the following 
hypotheses. First. P. aspera is older than P. candei; therefore, it 
has had more time to diverge intraspecifically and to increase their 
variability by having more time to incorporate new alleles. Con- 
sequently, allowing it even more chances of divergence between 
localities by either genetic drift or selection. Second. P. aspera 
with a more restrictive habitat (low intertidal to upper subtidal 
range) has been subject to stronger divergent selection at different 
localities than P. candei. with a broader habitat (from upper inter- 
tidal to upper subtidal range). Third, different life histories, could 
also partly explain this pattern, such as differences in the length of 
larval life, behavior, and reproductive patterns. 

The present work showed us that different biological stocks of 
the limpets P. candei and P. aspera are present in Madeira Archi- 
pelago. These stocks showed higher variability (for mean number 
of alleles per locus) than those from the other Macaronesian ar- 
chipelagos (Weber et al. in prep.). This fact is a further reason to 
encourage the conservation of Madeiran stocks, because they may 



T.\BI.E 4. 

Unbiased genetic identity (above the diagonal) and distance (below 
the diagonal) between P. aspera subpopulations. 



Subpopulations 



Pto. Moniz 



Cani^o 



Deserta Grande 



Pto. Moniz 
Canii^o 
Deserta Grande 



0.073 
0.061 



0.930 



0.042 



0.941 
0.959 




«v^Deserta 
\ Grande 



Figure 5. P. aspera; probable gene flow patterns at .Madeira .Archi- 
pelago (arrows). Values correspond to number of migrants between 
localities; ( ) summer island mass effect. 



constitute sources of variability for other Macaronesian popula- 
tions. 

The difference found in the degree of variability and structuring 
of populations between P. candei and P. aspera adds to the list of 
biological differences that characterize these species. P. candei is 
mainly midtidal: whereas, P. aspera is mainly subtidal. Whereas in 
the Azores. P. aspera has a discrete reproductive period, beginning 
in late summer from August to April. P. candei shows spawning 
for most of the year with only a very short summer resting period 
(Martins et al. 1987, Menezes, 1991 ). P. aspera is a partial protan- 
drous hermaphrodite (Thompson 1979. Guerra and Gaudencio 
1986. Corte-Real 1992). with males first appearing in the 2nd year, 
and females appearing during the 3rd year, increasing their pro- 
portion to the subsequent year classes. Males are predominant at 
13 to 20 mm length size classes and females at 18 to 55 mm length 
size classes. P. candei has no sex change, and individuals reach 
their maturity between 16 to 20 mm length. 

P. aspera subpopulations. with their reproductive mechanism 
and their restricted gene interchange between them, need special 
care in their regulation. A " "minimum size class" strategy be in- 
adequate for P. aspera. Nevertheless, such strategies as that 
adopted by the regional government from Azores might be ad- 
equate for these cases. They imposed laws of rotational openings 
and closing of grounds. For example, the collection of limpets in 
the eastern and central group of Azores islands was forbidden 
during 1989 and 1990. allowing only noncommercial harvest dur- 
ing 1990 in the western groups of islands (see Menezes 1991 and 
Corte-Real 1992). 

ACKNOWLEDGMENTS 

We thank Peter Wirtz, Thomas Dillinger, and Norberto Serpa 
who provided valuable assistance, the Madeira Regional Govern- 
ment for providing samples, and A. O. Rogers for commenting on 
the manuscript. S. J. H. was supported by grants from JNICT and 
the British Council. Present address for L. I. W.: Laboratorio de 
Bioqui'mica Marinha, Depto. Quimica, Fundagao. Universidade de 
Rio Grande, C.P. 474. 96201-900. Rio Grande-RS. Brazil. Present 
address for S. J. H.: Division of Biodiversity & Ecology. School of 
Biological Sciences. University of Southampton. S016 7PX, 
Southampton, UK. 



Patella spp., genetic stocks at Madeira 



953 



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Join mil of Shellfish Research. Vol. 17. No. 4. 9?5-9hy, 1998. 

MESOSCALE DISTRIBUTION PATTERNS OF QUEEN CONCH (STROMBUS GIGAS LINNE) IN 
EXUMA SOUND, BAHAMAS: LINKS IN RECRUITMENT FROM LARVAE TO 

FISHERY YIELDS 



A. W. STONER,' N. MEHTA,^ M. RAY-CULP^ 

Northeast Fisheries Science Center 
National Marine Fisheries Sen'ice 
Highlands. New Jersey 07732 
'Caribbean Marine Research Center 
Vero Beach. Florida 32963 

ABSTRACT Populations of benthic species that produce pelagic larvae are sustained through a complex interaction of factors, 
including larval supply, variable transpon mechanisms, and a host of postsettlement processes that affect differential recruitment and 
abundance. We report distributional data for the larvae, juveniles, adults, and a time-averaged index of fishery yield (shell middens) 
of the economically important marine gastropod Sirombus gigus (queen conch) in the Exuma Sound, Bahamas. All life history stages 
and the fishery yields were heterogeneously distributed around this semienclosed system, with higher densities of benthic stages in the 
northern part of the sound than in the south and east. Distribution of shell middens closely reflected abundance patterns of shallow- 
water juvenile aggregations and abundance of adults in depth-stratified suireys; therefore, midden distribution provided a good 
indicator of long-term productivity around the periphery of the sound. Although patterns of fishery productivity around the system were 
closely related to both juvenile and adult distributions, and density of newly-hatched larvae refiected the distribution of adults and shell 
middens, as would be expected, benthic stages and the fishery yields were completely decoupled from the abundance of settlement- 
stage larvae. When transplants of newly settled conch were made to four seagrass sites in the eastern Exuma Sound with characteristics 
typical of conch nurseries, low growth rates resulted in all but one location. All of these results suggest that conch abundance and 
distribution in Exuma Sound is determined in the benthos, either during settlement or in the first year of postsettlement life. Therefore, 
although larva! supply has been shown to influence benthic recruitment on a small scale (i.e., size and location of juvenile aggrega- 
tions), and juvenile populations will always depend upon a reliable source of competent larvae, high quality habitat plays an equally 
important role in the recruitment of this important fishery resource. 

KEY WORDS: Fishery, habitat, larval supply, mesoscale, oceanography, postsettlement. presettlement, recruitment 



INTRODUCTION 

Many marine animals have complex life cycles in which they 
release large quantities of planktonic propagules that are trans- 
ported by currents to habitats distant from where they were 
spawned (Doherty and Williams 1988, Roughgarden et al. 1988). 
Although some marine fishes and invertebrates reduce presettle- 
ment losses of larvae by releasing eggs or larvae into transport 
pathways that favor delivery away from predators and on to ap- 
propriate juvenile habitats (Johnson and Hester 1989, Hensley et 
al. 1994, Morgan and Cristy 1995), the vast majority of these 
propagules are advected away from suitable settlement habitat, or 
die during the planktonic or early postsettlement periods. Although 
all populations that produce pelagic larvae are sustained, to some 
degree, by the transport of larvae from upstream sources, predation 
rates on settling and newly settled invertebrates can be very high 
(Woodin 1976, Osman and Whitlatch 1995, Gosselin and Qian 
1997, Stoner et al. 1998), and a host of postsettlement processes 
can also influence spatial distribution (Hunt and Scheibling 1997). 
Consequently, the relative importance of pre- and postsettletiient 
processes on distribution has been a subject of much recent re- 
search related to invertebrate recruitment (e.g., Olafsson et al. 
1994, Eggleston and Armstrong 1995, Wahle and Incze 1997). 
One way to examine the significance of pre- and postsettlement 
processes is to test geographic coherence of abundance patterns in 
all of the life stages, as recommended by Hunt and Schieblmg 
(1997). 

The large gastropod mollusk Srroinhus ^igas Linne (queen 
conch) is a convenient model for examining relationships between 
life history stages for several reasons. First, larvae of 5. gigus are 



readily identifiable at all stages and are relatively large, hatching at 
about 0.3 mm shell diameter and settling to the benthos at over 1 .0 
mm in shell length (Davis et al. 1993). Second, juveniles occur in 
large aggregations, primarily in shallow coastal habitats making 
them relatively easy to survey. Third, the adults are slow moving, 
large (to 30 cm), and easily surveyed to their typical depth limit of 
-30 m depth. Juvenile and adult conch normally inhabit clear 
oligotrophic waters, which also facilitates survey work. Fourth, 
fishers ordinarily land the heavy shells of queen conch on beaches 
near the collection sites where they extract the edible meat. Be- 
cause the shells persist on the beaches for at least several hundred 
years, the shell middens provide an index of historical di.stribution 
patterns. 

In 1992, the multidisciplinary program FORECAST (Fisheries 
Oceanography and Recruitment in the Caribbean and Subtropics) 
was developed at the Caribbean Marine Research Center. The goal 
of this 5-year program was to provide an understanding of recruit- 
ment sufficient to explain me.soscale distribution patterns and in- 
terannual variation in economically significant species in Exuma 
Sound. We have collected distributional data on conch larvae 
(Stoner and Ray 1996, Stoner et al. 1996a, Stoner and Davis 
1997a), juveniles (Stoner et al. 1994, Stoner et al. 1995, Stoner et 
al. 1996b), adults (Stoner and Schwarte 1994, Stoner and Ray 
1996), and shell middens (Stoner 1998) in the Bahamas from as 
long ago as 1989. In this study, we focus on findings from the 
FORECAST program related to mesoscale spatial variation in 
populations of queen conch. We expand our previous analyses of 
the fishery record and benthic populations to include the entire 
Exuma Sound system, report new data on synoptic surveys for 



955 



956 



Stoner et al. 



queen conch larvae, and investigate patterns of distribution among 
the interconnected populations of queen conch in this system. We 
hypothesized that larval production and transport are the dominant 
factors controlling spatial variation in the distribution of conch 
larvae, juveniles, and adults in Exuma Sound, and that, as a result, 
long-term fisheries for conch reflect the general larval supply pat- 
tern. We further hypothesized that mesoscale patterns of abun- 
dance and distribution may be set during the juvenile stage and 
mediated by density-dependent postsettlement processes. 

Study Site and Background Information 

The Exuma Sound is a deep, semienclosed basin located in the 
central Bahamas that extends for 250 km along an axis, oriented 
southeast to northwest (Fig. 1). It is bordered to the south, west, 
and north by the Exuma Cays and the shallow Great Bahama Bank, 
most of which is sand and seagrass habitat less than 4 m deep. 
Eleuthera and Cat Island bound the eastern edge of the sound. The 
only deep-water O200 m) connection to the Atlantic Ocean is at 
the southeast end of the sound, between Cat Island and Long Island 
through a pass that is 30-km wide. The pass between Little San 
Salvador Island and Eleuthera, 16 km wide, is characterized by a 
sill with depths to approximately 35 m. West of Cat Island a broad 
island shelf (to IO-kni wide) borders the Exuma Sound. This shelf 
grades slowly from the intertidal through shallow sand and sea- 
grass habitats to sand and coral near the shelf edge in 15 to 30-m 
depth. The island shelf along the west coast of Eleuthera and the 
Exuma Cays is narrow (<l-km wide), grading rapidly from the 
island shores to 30-ni depth. The shelf edge begins at 30 to 35-m 
depth throughout the sound. Steep slopes descend to depths >2,000 
m in the southern part of the basin and to near 1.000 m in the 
northern part. Because the shelf edge provides a curvilinear bound- 
ary between shallow-water conch habitats and the deep Exuma 



Sound, distance along the shelf edge was used to standardize abun- 
dance patterns for juveniles, adults, and shell middens (see below). 

The total human population around the periphery of Exuma 
Sound is < 1 0,000 people, centered primarily in George Town on 
Great Exuma. As a result, there are few sources of pollution, 
fishing pressure on queen conch is relatively low, and the ecology 
of the system is relatively unspoiled. The semienclosed nature of 
the sound and the presence of suitable conch habitat make this 
system a natural laboratory for the study of fishery recruitment 
processes and for analysis of distribution of conch from larva to 
adult. 

Along the Exuma Cays, juvenile conch live primarily in sea- 
grass meadows on the shallow bank, and adult conch live primarily 
offshore in the deeper waters of the sound to 30 m (Stoner and 
Schwarte 1994, Stoner and Ray 1996). Adult conch lay eggs from 
April through October (Stoner et al. 1992). The prevailing current 
on the shelf near the Exuma Cays runs alongshore from the south- 
east to northwest (Colin 1995) and plays an important role in 
transporting conch larvae to the northwest. Larvae are advected 
through the numerous tidal passes between the cays and onto the 
bank (Stoner and Davis 1997a), where competent larvae settle 
selectively and metamorphose in nursery grounds that have been 
well studied (Davis and Stoner 1994). Juveniles live in aggrega- 
tions at densities of 0.1 to 2 individuals/m" (Stoner and Ray 1993, 
Stoner et al. 1996a). As they mature into adults, juveniles migrate 
back through the tidal passes and out to the deepwater reproductive 
areas (Stoner and Ray 1996). Most conch fishers free -dive for their 
catch from small boats that have limited range. After removing the 
meat, they discard the shells along the shores of Exuma Sound, 
thereby creating ever-growing piles near the site of capture. These 
piles of discarded shells, hereafter referred to as middens, provide 
a time-averaged record of large-scale conch distribution, and a 
history of the fishery for at least 500 years (Stoner 1998). 



SAIL 
ROCKS 




STOCKING 
ISLAND 

GREAT EXUMA 
76 






SAN 


.o*- 




SALVADOR 


■v » 




ISLAND 






CONCEPTION 




.'"' 


■■, ISLAND 




't. 


.,200m 

, joo?;- ( 






(e^ ( 


t 


LONG 
, ISLAND 


N 

10k 



Figure 1. Map of the Exuma Sound system in the central Bahamas. The periphery of the sound was divided into U sectors (boxed numbers). 
The values below the sector numbers indicate the volume of queen conch shell middens expressed in m' per km of shelf edge (dashed line). 
Asterisks indicate the five stations at which newly settled conch were transplanted, four at Cat Island (CT-1 to CI-4) and one station at Shark 
Rock (SR) near Lee Stocking Island. The letter "P" near the island of Eleuthera indicates Powell Point, referred to in the text. 



Distribution of Queen Conch 



957 



METHODS 

Spatial relationships between the abundance of queen conch 
larvae, juveniles, adults, and discarded shells in middens were 
examined in and around the Exuma Sound. Surveys for the various 
life stages spanned several years, and some components of the 
results, as noted, have been published in previous studies. The 
methods and results sections will describe the results for different 
conch stages in reverse ontogenetic order, beginning with mid- 
dens, because it is this time-integrated spatial record that reflects 
the long-term fishery that we wish to explain. Furthermore, we 
were able to quantify shell middens around the entire rim of the 
Exuma Sound, thereby surveying the entire system. Quantifying 
all three living conch .stages was much more labor intensive, and 
only regional surveys could be accomplished. A similar survey 
strategy was used by Lipcius et al. ( 1997) in an analogous study of 
spiny lobster (Panulirus argus) populations in Exuma Sound. 

The purpose of this investigation was not to follow a single 
cohort of queen conch from larvae to adult stage or to the fishery 
in the Exuma Sound. Rather, our intent was to examine the long- 
term record of fishery yields over a relatively large scale (i.e.. 
Exuma Sound) and to interpret it in terms of cunent abundance 
patterns observed for early life stages and adults. 

Long-Term Record of the Conch Fishery 

Shell midden data used in this study were modified from Stoner 
(1948). where the survey methods were described in detail. 
BrieHy, all of the islands facing the Exuma Sound were searched 
for shell remains in a clockwise direction from the northern tip of 
Great Exuma to the southern end of Cat Island between 1989 and 
1994 (Fig. I). The shoreline of Great Exuma was not surveyed, 
because this island has the largest human population on the pe- 
riphery of the Sound, and middens have been removed or disturbed 
by development. Small settlements occur around the rest of the 
sound, but most of the extensive shoreline is undisturbed. 

For this study, the shelf around the sound periphery was di- 
vided into sectors that were -20-km long (Fig. I ). Not included in 
ihe division of the periphery were deep-water passes between Cat 
Island and Long Island, the open-water pass between Little San 
Salvador and Eleuthera, and the bank periphery where there were 
no islands for landing conch (i.e., in the extreme northern sound 
and between Long Island and Great Exuma). Distances were mea- 
sured at the edge of the shelf and varied somewhat to separate 
nurseries for queen conch that are as.sociated directly with the tidal 
flow fields between islands at the edge of the bank (Jones 1996, 
Stoner et al. 1996b). 

Shell middens ranged in size from a few scattered shells to 
accumulations that were 3 to 4-m high. Estimates of the total 
volume of individual accumulations were made by measuring their 
basic dimensions as described by Stoner (1998). Notes were also 
made on the apparent age of the shell middens. For example, some 
were composed primarily of very old and eroded shells, and the top 
layers of others were covered with recently landed shells from 
which the bright shell nacre had not yet faded. The middens were 
mapped, volumes were summed for each sector, and the volume of 
shells per kilometer of shelf periphery was used as a standard 
index of historic fishery yield from individual bank sectors. 

Adult Surveys 

Labor-intensive diving surveys for adults were concentrated in 
four sectors of the sound chosen on the basis of general geographic 



positions and known productivity patterns in queen conch revealed 
in the midden survey. They included: ( I ) the conch-poor area at the 
southern end of Cat Island (Fig. I. sector 1 1 ); (2) the well-studied 
area near Lee Stocking Island in the southern Exuma Cays (sector 
I), where conch productivity is moderate; (3) an area inside the 
Exuma Cays Land and Sea Park near Waderick Wells (sector 4). 
where Stoner and Ray (1996) found very high densities of adult 
conch; and (4) an area between the Schooner Cays and the south- 
east tip of Eleuthera (sector 7), where the highest concentrations of 
shell middens were located (Stoner 1998). 

Stoner (1998) found a positive correlation between middens 
and juvenile conch abundance, and we hypothesized a similar 
relationship between middens and adult conch abundance. If such 
a relationship exists, midden volume could be used as an indicator 
of living adult conch distribution. Extensive adult surveys were 
made during the summer 1991 near Lee Stocking Island (Stoner 
and Schwarte 1994), and near Waderick Wells (Stoner and Ray 
1996), Cat Island, and Eleuthera in 1994. Although surveys for 
conch were made during two different years, the conch populations 
in the Exuma Sound seem to be relatively stable over the long 
term. Annual surveys for adult conch conducted at selected sites 
off Lee Stocking Island between 1988 and 1994 (Stoner and Sandt 
1992, unpubl. data) revealed that maximum variation from the 
mean population size and density was just 19%, and the population 
was only 4% above average in 1991. This stability is probably a 
function of low fishing pressure, particularly in depths below the 
reach of the average free-diving fishers (>I0 m). and a queen 
conch life span of at least 12 years (Coulston et al. 1987). 

Depth-stratified surveys for adult conch were conducted in 
each of the four sectors described above. Seven depth intervals 
were examined: to 2.5 m (where present), 2.5 to 5 m. 5 to 10 m, 
10 to 15 m, 15 to 20 m, 20 to 25 m, and 25 to 30 m. The deepest 
interval was not surveyed at either Eleuthera or Cat Island because 
of a very steep grade in depths >25 m that did not seem to support 
adult conch. The intervals were surveyed along nine offshore 
transect lines pei-pendicular to Lee Stocking Island, six lines per- 
pendicular to Waderick Wells, four lines perpendicular to Eleuth- 
era, and three lines pei-pendicular to Cat Island. Because of ex- 
tremely low conch densities in the shallow waters (<I0 m) near 
Cat Island, standard swimming transects (described below) were 
supplemented by extensive observations made by towing one or 
two divers behind the boat. Densities of approximately zero at 
most depth intervals (see Results) reflected Ihe results from this 
more extensive survey method. Total numbers of transects and 
dives made in each of the four sectors were dependent upon ship 
time available and logistics. Land-based operations at Lee Stock- 
ing Island and Waderick Wells permitted the most intensive sur- 
veys. 

In each depth interval, two divers swam for 8 to 30 minutes, 
depending upon depth, holding a taut line (8 m) between them and 
counting the number of adult conch that lay beneath the line 
(Stoner and Schwarte 1994. Stoner and Ray 1996). One diver 
carried a calibrated low-velocity flow meter to calculate the dis- 
tance traveled. To compensate for the potential influence of current 
on distance measured, the divers swam into any discernible current 
and back, covering two parallel, nonoverlapping paths that nor- 
mally ran parallel to the isobaths. Mean swim distance was 360 m 
(SD = 106 m), for a typical sampling area of nearly 3 ha. Conch 
densities were standardized to numbers per hectare. Mean adult 
conch density was calculated from the replicates at each depth 



958 



Stoner et al. 



interval at each sector and then used to generate a final mean 
representative of that sector for all depths. 

Juvenile Surveys 

Surveys for juvenile queen conch were conducted in the four 
sectors of Exuma Sound described above for adults and in sector 
5. Juvenile conch in the Exuma Sound region occur in high density 
(0.1 to 2.0 conch m"") aggregations and are found almost exclu- 
sively in shallow (<5 m-deep) bank habitats (Stoner and Ray 1993, 
Stoner et al. 1996b). Consequently, aggregations are usually easy 
to locate in the clear water, but estimations for juvenile abundance 
require survey techniques different from those used for adults. A 
detailed description of juvenile mapping technique can be found in 
Stoner and Ray (1993). In brief, divers were towed systematically 
over the bank with sufficient intensity to locate aggregations larger 
than -1.0 ha in surface area. Once located, the boundaries within 
which density was greater than -0.1 conch m"" were determined 
and marked with buoys. Buoy positions were then determined 
using hand-held GPS (Global Positioning System). Aggregations 
were plotted on a small-scale chart, and their surface areas were 
determined with a calibrated digitizing board. We made exhaustive 
surveys for juveniles along known lengths of the shelf edge within 
each of the five sectors, and surface areas of aggregations were 
standardized per unit of distance along the shelf (i.e., ha of juvenile 
aggregation/km of shelf). 

In the Exuma Cays, juvenile aggregations were located in shal- 
low seagrass meadows to the west of the islands and were directly 
associated with flood tidal pathways. They were relatively rare in 
the high energy, windward (east) side of the islands (Stoner et al. 
1996b). On the eastern side of the sound, the western shores of the 
islands are protected from the prevailing tradewind and wave en- 
ergy, and juvenile aggregations again occur in shallow seagrass 
meadows immediately to the west of the islands. Searches for 
juveniles near Eleuthera were concentrated on the shallow bank 
areas surrounding the Schooner Cays, and on the open, seagrass- 
covered shelf adjacent to Powell Point. Because aggregations in 
this area were very large, they were relatively easy to locate and 
map. Virtually all of the southern bight of Cat Island and 3 km of 
the southern shore between the shoreline and 5-ni depth was 
searched by towing divers behind small boats in transects sepa- 
rated by no more than 0.75 km. Many days of towing near Cat 
Island over the entire length of sector 1 1 (20 km) produced only 
scattered juvenile conch and no aggregations. All of the surveys 
were conducted between 1991 and 1993, depending upon the 



availability of ship time and other logistics. The general strategy 
was to search a section at least lO-km long in each of the five 
selected geographic .sectors (see Table 1). 

Veliger Suneys 

Plankton surveys for conch larvae were conducted between 
1993 and 1995. in an attempt to explain the large-scale distribution 
of queen conch around the periphery of the Sound. The first survey 
in 1993 comprised simple transects across the Sound in the south- 
west to northeast direction. In 1994 and 1995. the surveys were 
made in conjunction with physical oceanographic studies and were 
expanded for a more synoptic view of the Sound. 

Size-specific larval density data are useful tools for interpreting 
larval production and understanding transport processes. Early- 
stage, newly hatched queen conch veligers provide an indication of 
local larval production, and late-stage veligers (2 to 3 weeks old), 
which may have originated from a distant reproductive population, 
yield information on the number of conch available to settle into 
the benthos (Davis et al. 1993; Stoner et al. 1996a). 

Queen conch larvae are relatively easy to sample, because they 
are photopositive (Barile et al. 1994) and most abundant near the 
sea surface (<5-m depth) when conditions are relatively smooth, as 
is typical during summer in the Bahamas (Stoner and Davis 
1997b). All of the plankton samples collected during the surveys 
described below were made by towing nets in a stepwise oblique 
fashion from a depth of 5 m to the surface at -1 m/sec during 
daylight hours (except for a subset of 15 stations sampled during 
the night in 1994, see below). During the first 2 years (1993 and 
1994) collections were made with standard conical nets (diam. = 
50 cm. mesh size = 0.202 mm) that collect all queen conch larvae 
including the smallest (-0.3 mm shell length) newly hatched stage. 
These nets were towed for an average time of 36 minutes, with 12 
minutes each at 5 m. at 2.5 m below the surface, and just below the 
surface. The volume of water sampled, typically 250 to 300 m , 
was calculated from a calibrated General Oceanics flowmeter sus- 
pended in the mouth of the net. Two tows were made at each 
station. 

In 1 995, the primary objective was to sample higher volumes of 
water for late-stage larvae; therefore, net diameter and mesh size 
were increased to 75 cm and 0.333 mm, respectively. The tow 
strategy was similar to that used in 1993 and 1994. but total tow 
time was increased to 45 minutes. Tow volumes with the larger 
nets were typically 1000 to 1200 m\ All plankton samples were 
preserved in a buffered 5% formalin-seawater mixture. 



TABLE 1. 

Surface area and concentration of shallow-water juvenile queen conch aggregations in five sectors around the periphery of the 

Exuma Sound. 









Total 


Kilometers 


Aggregation 




General 


Survey 


Aggregation 


of Shelf 


Concentration 


Sector No. 


Location 


Date 


Area (ha) 


Surveyed 


(ha/km) 


1 


Lee Stocking 
Island 


7/93 


129 


11 


1 1.7 


4 


Waderick Wells 


2/91 


431 


17 


25.4 


5 


Norman's Cay 


9/91 


269 


13 


20.7 


7 


Schooner Cays 


8/93 


650 


10 


65.0 


n 


Cat Island 


7/93 





20 






Juveniles were *1 year old and were aggregated at densities of 0.1 and 1.0 conch/m". See Figure I for location of each sector. 



Distribution of Queen Conch 



959 



In 1993, five cruises were made during the peak reproductive 
season, 12 June to 23 August, on hoard R/V Shadow. During eacli 
cruise, a total of 13 stations was sampled along two transects that 
ran east to west, with one transect across the northern end of 
Exuma Sound from Waderick Wells to Schooner Cays and the 
other across the southern sound from Lee Stocking Island to Cat 
Island (see Results). The four stations at the end of the two transect 
lines were located in the four sectors that were surveyed for both 
juvenile and adult conch (sectors 1,4,7,11). The temporal patterns 
observed during the 1993 plankton surveys (see Results) facilitated 
the planning of subsequent cruises so that specific larval stages 
could be targeted, early-stage larvae in the month of June, and 
late-stage larvae in late August. 

In June 1994, two cruises were conducted to provide a synoptic 
view of veliger density in the Exuma Sound early in the spawning 
season. On the first cruise (5 to 13 June). 32 stations were sampled 
from R/V Sea Diver along six transects that ran east to west across 
the sound (see Results). One of the physical oceanographic objec- 
tives to be accomplished during this multidisciplinary cruise was 
the analysis of upper water column circulation. Therefore, 15 .sta- 
tions had to be sampled at night. Although this was not an optimal 
sampling strategy, Stoner and Davis (1997b) have shown that 
conch larvae occur in the upper 5 m of the water column during 
both day and night, under calm conditions. When they occur below 
5 m depth, they do so more in response to high wave action and the 
associated turbulence than to light conditions. On the second cruise 
(22 to 24 June), 12 additional stations (total of 44) were sampled 
from RA' Shadow at the 20-m isobath along the length of the 
Exuma Cays (see Results). All samples during this second cruise 
were collected during daylight hours. 

In 1995, two cruises (25 to 31 August and 15 to 17 September) 
were conducted to obtain a synoptic view of the distribution of 
late-stage larvae in Exuma Sound during peak settlement period. A 
total of 41 stations were sampled from R/V Cyclone (see Results). 
The station plan was similar to that employed in 1994, and all 
collections were made during daytime hours. As mentioned earlier, 
these collections were made with larger nets and mesh size, to 
sample late-stage larvae better. Sampling was suspended during a 
high wind period in early September. 

Plankton samples were sorted in their entirety for strombid 
veligers with the aid of a dissecting microscope. All strombids 
were identified to species (see Davis et al. 1993, for descriptions), 
counted, and measured for maximum shell length (SL), but only 
queen conch veligers (S. gigas) are discussed here. The veligers 
were divided into three general age classes on the basis of size: 
early-stage (<500 |xm SL), midstage (500 to 900 |jLm SL), and 
late-stage larvae (>900 |xm SL), which were at or near metamor- 
phic competence. Abundance was calculated as numbers of ve- 
ligers per unit \olume of water sampled ( veligers/100 m") for each 
age class. Data are reported as the mean of two tows for each 
station for individual cruises and as mean of means for 1 993 when 
cruises were pooled. 

Relationships Among Different Ontogenetic Stages 

The abundance and distributional data for middens, adults, ju- 
veniles, and larval conch were collected to examine the relation- 
ships among distinct ontogenetic stages in different geographic 
sectors around Exuma Sound. Tests of correlation were performed 
between conch midden volume and adult conch density, and be- 
tween midden volume and juvenile conch density. We hypoth- 



esized that, if a positive relationship exists between the fishery 
yield and benthic stages, then midden volume would retlect living 
adult conch populations and could be used as an index of living 
conch abundance around Exuma Sound. We also tested the rela- 
tionship between midden volume and density of early-stage conch 
veligers along the shelf periphery to determine if newly hatched 
larvae reflected large-scale distribution of the reproductive stock. 
To explore the potential importance of larval supply to distribution 
of benthic populations around the sound, we also tested for cor- 
relations between midden volume and density of late-stage larvae 
and between late-stage larvae and juvenile conch. Individual sec- 
tors were often represented by more than one plankton sampling 
station, providing increased confidence in the values used in the 
regressions. This varied with year and sampling strategy; however, 
all plankton stations inside a sector boundary and within 5 km of 
the shelf edge were included in a mean value. 

Transplant Experiment 

We observed very low densities of both juvenile and adult 
conch in the shallow shelf environment at the southern end of Cat 
Island (see Results). Because late-stage larvae were relatively 
ubiquitous throughout the sound, it is unlikely that such low den- 
sity is explained by a low supply of settlement stage larvae to that 
location. Therefore, we hypothesized that the habitat in this area 
was unsuitable for juvenile growth and survival. To test this habi- 
tat-limitation hypothesis, we conducted a transplant experiment 
during the summer of 1995 to measure postlarval growth. If the 
Cat Island habitat was suitable for newly settled conch growth, 
then postlarvae transplanted there should grow at rates similar to 
those transplanted in a conch nursery area near Lee Stocking Is- 
land called Shark Rock, where a well-studied juvenile aggregation 
has persisted for over 10 years (Stoner and Waite 1990, Stoner and 
Ray 1993, Stoner et al. 1994). Although growth rates in enclosures 
give no indication of predation-induced mortality, they do provide 
a good index of habitat suitability in terms of food quality and 
availability (Stoner and Sandt 1991). 

Three queen conch egg masses were collected from a repro- 
ductive site on the shelf off Lee Stocking Island on 18 June 1995. 
The eggs hatched 5 days later, and the larvae were cultured ac- 
cording to well-established procedures (Davis 1994). Briefly, lar- 
vae were held in 20-L plastic buckets filled with seawater collected 
daily from the bank west of Lee Stocking Island. Natural foods in 
the seawater were supplemented with cultured Tahitian Isochrysis 
spp. Metamorphosis was induced on 21 July, at -1 mm shell length 
(SL). and postlarvae were raised in plastic trays with aerated sea- 
water on a diet of seagrass detritus {Thalassia testudinuin) col- 
lected from the field. 

Five enclosures were deployed at one Shark Rock station in an 
area of uniform habitat characteri.stics at a depth of 4.1 m MLW 
(Fig. 1 ). It was our intent to deploy the Cat Island enclosures in 
similar habitat, and. after extensive surveying, four stations were 
selected along the southwest shore at 3.2-5.3 m depth (Fig. 1). 
Sediment and seagrass (Thalassia teslmliiuim) detritus samples 
were collected, and living seagrass shoot density was counted near 
each enclosure to characterize the station (Table 2). 

Enclosures were pvc cylinders (diameter = 16 cm, height = 
25 cm) with abundant large holes (diameter = 5.5 cm) cut from 
each to allow for water circulation (after Ray and Stoner 1995). 
Each cylinder was lined with a polyester mesh ( 1 mm) sleeve, 
pushed into the substrata, secured to reinforcement bars driven into 



960 



Stoner et al. 



TABLE 2. 

Mean growth rate and survival of postlarval queen conch transplanted at four stations near Cat Island (CI) and one station near Lee 

Stocking Island at Shark Rock (SR). 





Growth Rate 


Survival 




Seagrass Density 


Seagra.ss Detritus 


Station 


(mm/da.v) 


(%) 


Sediment Type 


(Shoots/m-| 


(g) 


CM 


0.12 + 0.01- 


90 ±9 


Medium sand 


570 ± 182 


1.35±0.17 


CI-2 


0.10 ±0.03-' 


100 ±0 


Coarse sand 


950 ±178 


0.71+0.18 


CI-3 


0.16±0.01'' 


90± 11 


Coarse sand 


750 ± 257 


0.68 ± 0.28 


CI-4 


0.30 ± o.o:"- 


99 + 2 


Medium sand 


680 ± 144 


0.19 + 0.13 


SR 


0.28 + 0.02" 


92 ± 12 


Medium sand 


710± 167 


4.5 1 ± 0.94 



Values are mean + SD; n = 5 at each station except CI-2, where n = 4 for conch growth rale. Growth rale data were homogeneous (Cochran's test, 
p > .05, and differences in the means were determined by one-way ANOVA (F|4 |^, = 1 14; p < .001 ) followed by Tukey HSD multiple comparison test. 
Means that are not significantly different (p > .05) are designated by similar lower case letters. Habitat characteristics including sediment type, seagrass 
(Thalassis testudinwn) shoot density, and seagrass detrital biomass (dry weight) (n = 5) are also given for each station. See Figure 1 for location of each 
station. 



the sediment, and covered with a mesh top. Large predators were 
removed prior to introduction of postlarvae. 

Prior to transplanting, subsamples of the cultured postlarvae. 
which were relatively uniform in size, were measured for shell 
length with dial calipers. Po.stlarvae were introduced into enclo- 
sures (see below) at Shark Rock on 20 August 1995 at 6.0 mm SL 
(SD = 0.4, n = 40) and at Cat Island on 24 August at 6. 1 mm SL 
(SD = 0.4. n = 80). Each enclosure contained 18 animals. 

Postlarvae were recovered from Shark Rock on 9 September, 
after 20 days in the field, and from Cat Island on 16 September, 
after 23 days. They were measured for shell length immediately 
after recovery. Mean daily growth rates were calculated from the 
living individuals in each cage using the initial shell length from 
the appropriate subsample. To test for differences among stations, 
one-way analysis of variance (ANOVA) was performed followed 
by Tukey HSD tiiultiple comparison test (Day and Quinn 1989). 

RESULTS 

Long-Term Record of the Conch Fishery 

Surveys of shell middens revealed the highly variable nature of 
the conch fishery yield around Exutna Sound (Fig. 1). Highest 
shell concentrations ( 198 m' shells/km) occuiTed in the northeast 
region (sector 7), located between two regions with low concen- 
trations (-0.2 m'/km). A high concentration (133 m"" shells/km) 
was also observed in sector 4 in the north central Exuma Cays near 
Waderick Wells. All four sectors in the eastern sound, from 
Eleuthera to Cat Island, had very low concentrations of conch 
shells (^2 m^ shells/km), corroborating the low productivities of 
conch reported by fishers interviewed at Cat Island. Shells were 
abundant all along the Exuma Cays island chain on the western 
boundary of the sound, except at the extreme north. 

Most of the shell middens in the Exuma Sound contained both 
very old and recently collected shells. Important exceptions to this 
were enormous accumulations of recently collected conch in the 
vicinity of Powell Point on Eleuthera. These shells had bright color 
indicating capture over the last few years, and most had the thin 
shell lips indicative of relatively young adults. The largest indi- 
vidual accumulations (>1000 m') occurred in sector 4 near Wad- 
erick Wells, but it was apparent that most of these shells were 
collected much earlier than those on Eleuthera. This finding was 



not unexpected, because sector 4 lies within the Exuma Cays Land 
and Sea Park, where all fishing has been prohibited since 1985. 

Adult Siineys 

In general, densities of adult conch were highest at Waderick 
Wells and Schooner Cays, intermediate near Lee Stocking Island, 
and very low (except in the 15 to 20-m depth interval) at Cat Island 
(Fig. 2). Highest density of adults occurred at 10 to 15 m near 
Waderick Wells (270 conch/ha), at 15 to 20 m near Lee Stocking 
Island (88 conch/ha), and at 15 to 20 m off Cat Island (84 conch/ 
ha). Distribution at Schooner Cays was bimodal. with density 
maxima in shallow water (2.5 to 5 m-228 conch/ha) and in rela- 
tively deep water (20 to 25 m-93 conch/ha). Most of the conch in 
the 2.5 to 5-m interval at Eleuthera were very young adults (with 
thin shell lips), with a high density of large, late-stage juveniles 
mixed in as noted below. The adults at most other locations and 
depths were older. 

The benthic habitat within the to 2. 5-m depth inter\al cotn- 
prised very little surface area in each of the four regions surveyed. 
Adult conch in this nanow band were rare, and, therefore, consid- 
ered to be negligible. At all four sites, the two depth intervals 
between 20 and 30 m represented relatively small proportions of 
the total habitat occupied by adults; therefore, densities of conch in 
the depth intervals with largest surface area (2.5 to 20 m) were 
used to test for correlations between adults and other ontogenetic 
stages (see below). 

Juvenile Sun'eys 

Surveys for juvenile aggregations were conducted in five sec- 
tors along the periphery of the Exuma Sound (Table 1 ), The 
lengths of shelf edge, corresponding to the shallow-water areas 
surveyed, ranged from 10 km near the Schooner Cays, where 
conch juveniles were abundant, to 20 km near Cat Island. Scattered 
juveniles were observed in the bight of Cat Island and along the 
westernmost third of the south shore, but no aggregations were 
found during our extensive systematic surveys conducted in 1993 
or during numerous visits to the area between 1993 and 1995. 
Largest aggregations of juvenile conch occuired in the vicinity of 
the Schooner Cays just north of Powell Point on the island of 
Eleuthera and on the shelf immediately west of the Point (Fig, I), 
where young adults were also abundant. In August 1993, a single 
aggregation in the seagrass bed extending south from the Schooner 



400 



Distribution of Queen Conch 

400 
Waderick 
Wells 

Sector 4 

300 



200 



961 




100 - 




Schooner 
Cays 
Sector 7 



u 

c 
o 
o 



300 



200 



100 



Lee 

Stocking 
Island 
Sector 1 




400 



300 



200 



100 



c\i 



o 


in 


o 


in 


O 






(M 


<M 


CQ 


in 


o 


in 


O 


in 








C\J 


CM 



in 


o 


m o in o 
-- c\j CM rj 


c\j 


in 


o un o in 

T- T- CM CM 

Cat Island 
Sector 1 1 



J 



o in o in o 

-^ T- CVJ CM CO 

in o in o in 

■.-•.- CM CM 



Depth (m) 

Figure 2. Density of adult queen conch at six depth intervals in the Exuma Sound. Surveys were conducted at four locations, shown here by 
sector. Values are mean ± SE. Data for Waderick Wells and Lee Stocking Island are modified from Stoner and Schwarte ( 1994) and Stoner and 
Rav (1996). 



Cays comprised 610 ha of juvenile conch in densities of at least 0.5 
conch/m". Another large aggregation (431 ha) occurred near Wa- 
derick Wells. Intermediate concentrations of juvenile aggregations 
occurred near Norman's Cay (sector 5) in the northern Exunia 
Cays and near Lee Stocking Island (sector 1) at the southern end 
of the island chain. Most of the individual aggregations in the 
Exuma Cays covered between 10 and 100 ha and all were asso- 
ciated with the tidal flow fields immediately west of the inlets and 
islands in shallow seagrass beds. Repeated observations revealed 
that the locations of these aggregations were persistent over time. 

Veliger Surveys 

In 1993, early-stage larvae were most abundant near Waderick 
Wells (159 to 197 veligers/100 m'), followed by Schooner Cays 
(29 to 45 veligers/100 m'), Lee Stocking Island (7 to 13 veligers/ 
100 m'), and Cat Island (0 to 5 veligers/100 m') (Fig. 3A). Early- 
stage larvae were nearly absent offshore in the open waters of 



Exuma Sound. Midstage larvae were concentrated offshore in the 
northern sound with a mean density of 24 to 41 veligers/100 m' 
(Fig. 3B). The mean density of late-stage larvae varied between 
to 8 veligers/100 m"* at all stations except one offshore in the 
northern Sound, where mean density was 30 veligers/100 m' (Fig. 
3C). Mid- and late-stage larvae were rarely found along the pe- 
riphery of the sound during the five surveys conducted in 1993. 
As in 1993, early-stage larvae were abundant all along the 
northern Exuma Cays in 1994, with highest mean densities (208 to 
929 veligers/100 m') near Waderick Wells (Fig. 4A). Stations 
north of Waderick Wells, near Sail Rocks (see Fig. 1), had mean 
densities of 65 to 70 veligers/100 m^'. The southern part of the 
sound and the entire eastern periphery yielded low densities of 
early-stage veligers. For example. Lee Stocking Island, Great 
Exuma, and Little San Salvador all had intermediate densities of 
early stages (25 to 59 veligers/100 m'): whereas, Eleuthera and Cat 
Island had very low densities (0 to 2 veligers/100 m'). As in 1993, 



962 



Stoner et al. 



ELEUTHEB. Early-stagB 
(< 500 urn) 




TOHOUe 
OF THE 
OCEAN 



ELEUTHEn. Mid-Stage 

(500 - 900 nm) 





*;■■. 





WAOEHICK 
WELLS 


■g 
H, 



ELEUTHERA Latc-stage 
(> 900 jam) 



TONOUE 
OF THE 
OCEAN 



«. 



Figure 3. Density of (A) early-stage, (B) midstage, and (C) late-stage queen conch veligers collected during five cruises in 1993 (12 June to 23 
August) at 13 stations. Plankton tows were made at each station with 202-|iim mesh nets. Values represent the mean of means for each station. 



very few early stages were collected at the offshore, open-water 
stations. 

Although mid- and late-stage larvae were widespread through- 
out Exunia Sound in 1994, they were usually collected in relatively 
low densities (Figs. 4B.C). Moderate densities of midstage larvae 
were found along the shelf edge of the northwest sound near Sail 
Rocks (45 veligers/100 m'), near Waderick Wells (24 veligers/100 
m^), and at one station in the center of the Sound (12 veligers/100 
m^) (Fig. 4B). The rest of the sound, including its periphery, 
yielded a mean density of <10 midstage veligers/100 m\ Highest 
densities of late-stage larvae were found near Sail Rocks (52 ve- 
ligers/100 m"^). the pass between Eleuthera and Little San Salvador 
(34 veliger.s/100 m"*), the outer shelf edge of Cat Island (56 ve- 
ligers/100 ni"*), and one station in the center of the sound (10 
veligers/100 m') (Fig. 4C). The rest of the sound .stations yielded 
to 6 veligers/100 m'^. Thus, despite high concentrations of early 
stage larvae near the large reproductive populations in the north- 
central Exuma Cays, settlement-stage queen conch larvae were 
found throughout the sound in relatively low densities. 

Veliger distribution was explored along the Exuma Cays in two 
subsequent cruises, in July and August 1994. and the spatial pat- 
terns were remarkably similar to those reported above. For ex- 
ample, highest densities of early-stage larvae were always most 



abundant from the middle Exuma Cays to the north, and late-stage 
larvae were always highest in the extreme northern Exumas. 

More intensive surveys for late-stage larvae from late August to 
mid-September 1995 revealed that these settlement-ready stages 
were ubiquitous throughout the Exuma Sound, except in the ex- 
treme southern sound, in the opening between Cat Island and Long 
Island, and at numerous stations on the shelf along the Exuma 
Cays (Fig. 5). Highest densities (10 to 28 veligers/100 m') oc- 
curred in the extreme northern sound, near Waderick Wells, and at 
a station south of Little San Salvador. The rest of the Exuma Sound 
had late-stage densities of 1 to 10 veligers/100 m\ with the ex- 
ception of one station in the central basin (12 veligers/100 m" ). 

Relationships Among Different Ontogenetic Stages 

When the abundance of shell middens in a sector was compared 
with the inean density of adult conch on the adjacent shelf <20 
m in depth (Sectors 1. 4. 7. and 1 1). there was a highly signifi- 
cant correlation (r = 0.973, p = .03) (Fig. 6A). Midden 
abundance was also closely correlated with the abundance of 
juveniles in adjacent waters (Sectors 1. 4, 5. 7, and II) (r = 
0.915. p = .03) (Fig. 6B). The correlations between shell midden 
volumes and living populations of both juvenile and adult conch 



Distribution of Queen Conch 



963 



TONGUE 
OF THE 
OCEAN 



ELEUTHERA Eafly-stage 
(< 500 )im] 




TONGUE 
OF THE 
OCEAN 



ELEUTHERA Mld-stagc 

(500-900 Jim) 




...jog,. 



WAOERICK N ^^ 
WELLS 



TONOUE 
OF THE 
OCEAN 



jELEUTHEBA Lats-Stage 
(> 900 urn) 




. 





» 


1-10 


© 


10-50 


o 


SO- 100 


o 


1O0-2O0 


G 


j >2O0 



Figure 4. Density of (A) early-stage, (B) midstage, and (C) late-stage queen conch veligers collected during two cruises in 1994 (5 to 13 June, and 
22 to 24 June) at 44 stations. Plankton tows were made at each station with 202-nm mesh nets. Values represent the mean for each station. 



over the mesoscale validates the use of middens as a proxy indi- 
cator of living conch abundance around the perimeter of Exuma 
Sound. 

Abundance of early-stage, newly hatched larvae at any one 
location should reflect the size and/or density of the reproductive 
population in the general vicinity. The most synoptic data for early 
stages were collected in 1994 (Fig. 4). and there were 10 sectors 
for which we had both veliger and midden data. Very high con- 
centrations of larvae were collected in the north-central Exuma 
Cays and near Lee Stocking Island in the southern Exumas. Un- 
expectedly, the correlation between early-stage larval densities and 
midden abundance was low and not significant (r = 0.413. p = 
.24). The poor correlation was a function of one extreme outlier 
representing sector 7, near the Schooner Cays, where very large 
populations of adult conch and large middens were found, but few 
early-stage veligers. As mentioned above, most of the adult conch 
at this site were very young adults, which may not have been in 
reproductive state in the summer of 1994. Also, unlike other in- 
shore shelf stations around the sound, the stations that we sampled 
for veligers near the Schooner Cays were swept by very strong 
tidal cunents; therefore, it is possible that sampling at this site 
during the flood tide resulted in low larval densities. The flood tide 
would carry locally spawned larvae onto the adjacent bank and 
away from the sampling stations. When sector 7 was removed 



from the analysis, there was a highly significant positive conela- 
tion between the abundance of early larval stages and middens (r 
= 0.964, p < .001 ). as was predicted. Best distribution of residuals 
occurred with a natural log transformation of the data (r = 0.746, 
p = .02) shown in Figure 7. 

We also hypothesized that the juvenile abundance pattern 
(Table 1 ) would reflect densities of late-stage larvae (i.e.. those 
that are at or near metamorphic competence and ready to settle). 
However, using the juvenile abundance data available for five 
sectors (Table 1 ), the correlations were low and not significant (p 
> .35) in all 3 years in which larval data were collected (r = 0.431 
in 1993, r = 0.512 in 1994. r = 0.220 in 1995) (Fig. 8). In 1994, 
high densities of late-stage larvae were found at the south end of 
Cat Island (sector 11) (Fig. 4). where juvenile populations were 
typically very small. In the same year, sectors with large juvenile 
populations (e.g.. sectors 4 and 5) had low densities of late-stage 
larvae. In 1995. late-stage larvae were relatively high in sectors 4 
and 5. but also common in sector 1 1 near Cat Island (Fig. 5). 
Larval supply was not a good predictor of juvenile concentration. 

To complete the analysis of the relationship between middens 
and mesoscale distribution of conch around the Exuma Sound, we 
examined midden volume as a function of late-stage, competent 
larvae. The correlations were negative and not significant in 1994 
(r = 0.420, p = .23. n = 10) and in 1995 (r = 0.312, p = .35, 



964 



Stoner et al. 










.% 


<H ® 




'^0 

WADERICK Vj' 




WELLS ;® 




V.X 
-24- 








TONGUE 




OF THE 




OCEAN ; 






CAT 
rSLANO 




<Vf., 



GREAT 
EXUMA 




,00 f,- 



200 



N 



Figure 5. Density of late stage queen loncli veligers collected during two cruises in 1995 (25 to 31 August and 15 to 17 September) at 41 stations. 
Plankton tows were made at each station with 333-Mm mesh nets. Values represent the mean for each station. 



n = 1 1) (Fig. 9). As with juvenile distribution, larval supply was 
not a good predictor for the distribution of fishery yields. 

Transplant Experiment 

Most (90 to 100%) of the conch transplanted at Cat Island and 
in the Shark Rock conch nursery were recovered from their en- 
closures alive, except for one cage at station CI-2. where all conch 
were lost for unknown reasons (Table 2). Growth rates were sig- 
nificantly higher (0.3 mm/day) at both CI-4 and Shark Rock than 
at the three other Cat Island stations (0.1 to 0.2 mm/day) (Table 2). 
Growth was independent of seagrass shoot density and seagrass 
detritus. 

DISCUSSION 

It is widely recognized that fishing is better at some locations 
than others and that this variation occurs over both small and large 
scales. Variation in the abundance of exploited benthic animals 
that have pelagic larvae can be explained by differences in: ( 1 ) 
larval supply: (2) larval settlement: (3) the amount and quality of 
habitat for juveniles: (4) survival to the size at which the animals 
enter the fishery; and (5) fishing mortality. It is clear that long-term 
landings of queen conch are not homogeneously distributed around 
Exuma Sound (Stoner 1998. this study). The puipose of this in- 
vestigation and the following discussion is to exainine the meso- 
scale patterns of abundance and distribution of all queen conch 
stages and to determine the point in the life history at which the 
observed patterns of fishery landings are set. 

Radiocarbon dates for shells in middens along the periphery of 
the Exunia Sound show that these middens provide a historical 
record of the queen conch fishery spanning several hundred years 
(Stoner 1998). Although the pattern of conch exploitation is inde- 
pendent of the distribution of human settlements around the sound, 
there were high conelations between the volume of shell middens 
and the abundance of both living adults (r = 0.97) and juveniles 
(r = 0.92). Thus, midden volumes provide a long-term record of 
fishing productivity around Exuma Sound as well as an indirect 
index of living conch distribution. The close conelations between 



cumulative landings and local conch populations suggest that the 
mechanism of distribution occurs somewhere in the life history of 
conch prior to the age- 1 and age-2 year classes that were quantified 
in the juvenile surveys. 

Spatial variation in the adult conch populations was reflected in 



E 



0) 

E 

3 

o 

> 
c 
o 
•o 
■a 



200 r 

150 ■ 

100 ■ 

50 



y = 1.7305x- 38.028 
R = 0.973 
P = 0.03 



• 4 



50 



100 



150 



Adult density (no./ha) 



200 



150 



100 ■ 



y = 3.1467x- 0.039 
R = 0.915 
P = 0.03 

• 4 



• 7 




Concentration of juveniles (ha/km) 

Figure 6. Conch midden volume plotted as a linear function of (A) 
adult conch density at 2.5 to 20-m depth and (B) concentration of 
juvenile conch. Pearson correlation coefficients (R) and p-values are 
given for each regression equation. The number above each point 
represents the sector at which surveys were conducted in Exuma 
Sound. 



Distribution of Queen Conch 



965 



a 






y = 0.845 x + 1.35 




n 

E 

o 






R = 0.746 








P = 0.021 




? 6 


- 






• 


■■*. 










d 










c^ 








-^ 










^/^ 


>« 








^ 


«rf 






j< 




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0) 






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^,.^ • 




0) 


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w 






^^ 




o 






^^ 




> 






j,^ 




u 




• 


^^ 




O) 




V- 


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ra 




^ — 






*-• 










w 2 


- 


y^ 






>. 






• 




k. 










w 










LU 

n 


1 


< 


1 1 1 


' 







1 



Midden volume (m /km) 

Figure 7. Relationship between mean density of early-stage (<5()(l jim) 
conch veligers (natural log transformed) and conch midden volume, 
surveyed at nine sectors in Exuma Sound in 1994. The Pearson cor- 
relation coefficient (Rl and p-value are given for the regression equa- 
tion. 

the production of early-stage larvae around the periphery of 
Exuma Sound. These newly hatched larvae were most abundant in 
the nearshore areas where adults live and in regions known for 
high adult concentrations, such as near the Schooner Cays and in 
the Exuma Cays Land and Sea Park near Waderick Wells. The 
positive correlation between adults (i.e., spawner abundance) and 
early-stage larvae was predictable and not surprising. 

Ultimately, however, it is settlement-stage larvae that supply 
and sustain benthic populations, and examples of correlations be- 
tween larval supply and settlement and/or recruitment to various 
benthic stages are known for a variety of marine invertebrates 
(Caffey 1985. Keough. 1988, Bertness et al. 1992) and fishes 
(Milicich et al. 1992, Doherty and Fowler 1994). Relationships 
between late-stage larval concentration and juvenile population 
size have been explored for queen conch in several different lo- 
cations and on different scales. Stoner and Davis (1997a) have 
shown that aggregations of juvenile conch were directly associated 
with local concentrations of late-stage larvae within a tidal current 
flow field (<10-km long) on the Great Bahama Bank near Lee 
Stocking Island. Significant positive correlations have also been 
found between densities of late-stage larvae and juvenile popula- 
tion size on a 10 to 5()-km scale across multiple nursery grounds in 
the Exuma Cays and in the Florida Keys, although the pattern was 
not coherent across the two locations (-500 km) (Stoner et al. 
1996a). 

Because of the semienclosed circulation pattern in the Exuma 
Sound (Colin 1995, Lipcius et al. 1997). it is probable that conch 
larvae produced in the sound could be retained in the system for 
the duration of their developmental period. However, the potential 



tn 

0) 

'E 

> 
p 

»^ 
o 

c 
,o 

+3 
CC 

k. 

c 
<u 
o 
c 
o 
o 



70 
60 

50 h 
40 
30 
20 
10 




- 


7 
• 


y = 7.1153x + 13.265 


1993 


. 




R = 0.431 




- 




P = 0.57 








4 








• 




.• 1 








11 

• 1 






, 



^ 01 23456789 10 

E 
.be 

■J3 



70 


- 7 

• 


y = 


-1.0294X + 31.115 


1994 


60 


r 




R = 0.512 




50 


h 




P = 0.38 




40 


. 








30 


> 4~~~--~ 








20 ^ 5 


^^~~~~~~~ 


--.^.^^^ 




10 



.• 1 


1 


^^^^ 


--.-.1^1 
—J • — 1 



10 



15 



20 



25 



30 



• 7 




y = -1.7072x + 29.717 
R = 0.220 


1995 


■ 




P = 0.72 




■ 





4 
—_____• 5 




_• 1 

1 


11 


_] 1 1 1 1 1- 


< ' 



70 
60 
50 
40 
30 
20 
10 



0123456789 10 

Late-Stage veliger density (no./100 m^) 

Figure 8. Linear relationship between the concentration of .ju>enile 
conch and mean density of late-stage (>900 (ini) conch veligers from 

1993 to 1995. Pearson correlation coefficients IR) and p-values are 
given for each regression equation. The number above each point 
represents the sector al which the survey was conducted in Exuma 
Sound. Note extended x-axis for 1994 data. 

for dispersion within the sound over the 2 to 4 week precompetent 
period (Davis 1998) is very large. Although late-stage conch larvae 
were consistently abundant in the northern Exuma Sound during 

1994 and 1995. they were relatively ubiquitous throughout the 
sound. Correlations between the concentrations of settlement-stage 
larvae and either juvenile or midden distributions were never sig- 
nificant in any of the 3 survey years despite consistent spatial 
patterns of larval density. These results suggest that the regional 
pattern of distribution in benthic life stages within Exuma Sound is 
set by settlement processes and/or early postsettlement processes 
during the first year of life, and not by differences in larval supply. 
Lipcius et al. ( 1997) arrived at similar conclusions about the large- 



966 



Stoner et al. 



250 
200 
150 
100 



y = -2.5411x + 62.841 
R = 0.420 
P = 0.23 



1994 




o 

> 

C 250 

a> 
•a 

■a 200 



150 



100 

50 





:-3.6896x-f 56.161 
R = 0.312 
P = 0.35 



25 30 



1995 



10 



12 



14 



16 



18 



Late-Stage veliger density (noVlOO m ) 



Figure 9. Linear relationship between queen conch midden volume 
and mean density of late-stage (>900 pm) conch veligers in 1994 and 
1995. Pearson correlation coefficients (Rl and p-values are given for 
each regression equation. Symbols represent sectors surveyed in 
Exuma Sound for the 2 years. 



scale distribution of spiny lobster (Painilinis argiis) populations in 
Exuma Sound. Settlement stage lobster were abundant at Cat Is- 
land, yet benthic populations were small. The decoupling between 
larval supply and juvenile and adult stages was attributed to habitat 
limitation for early juvenile lobster at Cat Island. 

Miron et al. (1995) pointed out the inherent methodological 
difficulties in correlating larval supply and larval settlement or 
recruitment. They noted that competent larvae must be quantified 
and that they must be sampled properly (i.e., with proper respect to 
location in the water column and settlement substratum). Although 
settlement-stage queen conch larvae have never been collected in 
high densities, compared with densities of other mollusks in tem- 
perate waters, they are relatively easy to sample, because they 
occupy near-surface waters in most circumstances (Barile et al. 
1994, Stoner and Davis 1997a, Noyes 1996). Furthermore, it is 
relatively easy to identify competent forms on the basis of size, 
pigmentation, and other features (Davis 1998). Consequently, we 
believe that we have sampled the correct larval stages using an 
appropriate technique. 

It can also be argued that larval supply is best measured as a 
rate of delivery of competent larvae to a potential settlement site 
(Olmi et al. 1990, Yund et al. 1991 ). Measuring this is particularly 
difficult for queen conch on the Great Bahama Bank because of 
strong tidal currents (see Stoner and Davis 1997b). However, the 
difficulty is lower in the Exuma Sound, and we have high confi- 
dence in the regional patterns of larval abundance reported for two 
reasons. First, currents in the sound are much weaker (<20 cm/sec; 
Colin 1995), than those on the Bank (often >100 cm/sec; N. P. 
Smith, unpubl. data), so the issue of larval flux is less complicated 
in the .sound than on the nursery grounds of the Bank. Second, and 



more importantly, multiple visits to selected stations throughout 
the sound between 1993 and 1994 revealed that the regional pat- 
terns in veliger distribution were consistent over time. For ex- 
ample, five cruises over 13 stations in 1993 showed that early- and 
midstage larvae were always abundant in the northern sound, and 
always highest on the shelf adjacent to Waderick Wells. Late-stage 
larvae were always most abundant in the sound offshore from 
Waderick Wells. Three cruises along the island shelf east of the 
Exuma Cays in 1994 (Stoner and Mehta. unpubl. data) confirmed 
the pattern of maximum abundance of early- and midstage larvae 
in the vicinity of Waderick Wells and to the north, and late-stages 
were most abundant near Sail Rocks in every case. Larvae of all 
stages were always rare in the extreme south section of the sound. 
Therefore, because of the consistency of larval distribution, both 
within and between years, we believe that the regional patterns of 
larval abundance reported in this study are representative for the 
sound. 

Given that the abundance of late-stage larvae did not explain 
mesoscale variation in the abundance of juveniles, adults, or fish- 
ery yields of queen conch in the Exuma Sound, we conclude that 
the regional distribution of benthic stages is regulated by settle- 
ment and/or postsettlement processes associated with some ele- 
ment of the habitat. Similar conclusions have been drawn for a 
large number of other marine invertebrates (Keough and Downes 
1982, Luckenbach 1984, Connell 1985, McGuinness and Davis 
1989, Osman et al. 1992. Olafsson et al. 1994. Eggleston and 
Armstrong 1995, Hunt and Scheibling 1997, Lipcius et al. 1997). 

Many invertebrates settle and metamorphose in the presence of 
certain chemical agents in or on the substratum (Morse and Morse 
1984, Hadfield and Scheuer 1985, Burke 1986, Butman and 
Grassle 1992, Pawlik 1992), and Mianmanus (1988) has shown 
that phycobiliproteins associated with red algae are active agents 
in conch settlement and metamorphosis. We know from extensive 
dredge sampling for newly settled queen conch (both live and 
recently killed) in a tidal flow field near Lee Stocking Island that 
settlement is not random and that it occurs in specific locations 
(Stoner et al. 1998). This confirms earlier laboratory experiments 
showing that competent queen conch larvae settle in response to 
specific biological cues found within nursery grounds (Davis and 
Stoner 1994). Queen conch larvae are. in fact, capable of testing 
the substratum, returning to the water column multiple times, and 
delaying metamorphosis for long periods of time (for at least 60 
days after competence is achieved) (Noyes 1996). Experimental 
laboratory work shows that the larvae settle and metamorphose 
only in habitats where sub,sequent growth rates are high (Stoner et 
al. 1996c). Consequently, it is possible that variation in the abun- 
dance of queen conch populations on the Great Bahama Bank 
surrounding the Exuma Sound is related to either the quality or 
quantity of habitat with appropriate settlement cues and high 
growth potential for postlarvae. 

Habitat-limitation is the most plausible explanation for the low 
abundance of juvenile and adult conch west of Cat Island, because 
competent larvae were present in substantial numbers. Frequency 
of settlement was not tested, because the only way to ascertain this 
is by dredging, which is extremely labor intensive. However, trans- 
plant experiments provide important insights into the nutritional 
quality of potential nursery sites. Two lines of reasoning suggest 
that habitat at Cat Island is limiting for conch. First, the type of 
habitat that typically supports juvenile conch on the Great Bahama 
Bank (moderate density seagrass with accumulations of decom- 
posing detritus and red and green algae) has been studied exten- 



Distribution of Queen Conch 



967 



sively (see Stoner 1997). and was uncommon on the bunk west of 
Cat Island. Seagrass was found in relatively small patches ( 1 to 10 
ha), and much of this was exposed to higher physical energy than 
is typical for conch nurseries. Second, only one of the four sites 
assumed to be suitable for juvenile conch provided for growth 
rates similar to those in a known nursery near Lee Stocking Island. 
Therefore, it is likely that the small queen conch population and 
the poor fishery for conch near Cat Island is habitat-hmited. 

Differential mortality of young conch could also explain re- 
gional variation in recruitment to the age- 1 year class. There are a 
host of predators on juvenile conch (Randall 1964), including a 
large variety of recently discovered micropredators such as xanthid 
crabs and certain polychaetes that feed on newly settled conch 
(Ray-Culp et al. 1997). It is now recognized that mortality rates in 
newly settled invertebrates can be very high (Osman and Whitlatch 
1995. Gosselin and Qian 1997), and queen conch are no exception 
(Ray et al. 1994. Stoner and Glazer 1998). Although we did not 
test for regional variation in mortality of juvenile conch, this is a 
possible explanation for the population patterns observed. 

Conclusions and Fishery Management Implications 

Genetic analysis of queen conch collected from 22 populations 
throughout the greater Caribbean region, including the Bahamas 
and south Florida indicate a high rate of gene flow among the 
populations (Mitton et al. 1989, Campton et al. 1992), and certain 
populations may depend entirely upon upstream reproductive 
sources (Stoner et al. 1997c). It is clear, therefore, that sound 
fisheries management will demand good knowledge of larval drift 
and associated metapopulation dynamics (Berg and Olsen 1989. 
Appeldoom 1994, Stoner 1997). However, the direct correlation 
between the quantity of larvae supplied to the nurseries and the 



subsequent abundance of juvenile queen conch in the benthic 
population that occurs at a local .scale (Stoner et al. 1996a) seems 
to break down at the large scale. In Exuma Sound ( 180-km long) 
the abundance of early-stage larvae was positively correlated with 
regional abundance of adults. However, the distribution of juve- 
niles, adults, and fishery yields was independent of the abundance 
of competent larvae, and processes of settlement and postsettle- 
ment seem to regulate benthic population size. We have shown in 
the past that conch nursery grounds have unique physical and 
biological features that enhance larval settlement, provide high 
nutritional qualities, and promote high survivorship (Stoner et al. 
1995, Stoner 1997). It is now clear that high abundance of com- 
petent lar\ae does not guarantee high queen conch production, and 
that fisheries management for the species must consider both 
qualitative and quantitative elements of habitat for young conch. 
Because vast shallow-water areas within the biogeographic range 
of queen conch are. in fact, not suitable for production of the 
species, both local- and large-scale mechanisms of population dy- 
namics and habitat use need to be understood, and the ecological 
integrity of key nursery habitats needs to be preserved. 

ACKNOWLEDGMENTS 

This research was supported by grants from the National Un- 
dersea Research Program of NOAA (U.S. Department of Com- 
merce) to the Caribbean Marine Research Center. Many people 
participated in the field work and plankton sorting for this long- 
term study including P. Bergman. L. Cowell, M. Davis, G. Dennis, 
L. Hambrick, R, Jones, C. Kelso, S. O'Connell. H. Proft, V. Sandt, 
J. Waite, and E. Wishinski. Crews from the research vessels Un- 
dersea Hunter. Chiillenger, Shadow. Sea Diver, and Cyclone pro- 
vided ship support, and B. Bower- Dennis drafted the maps. 



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Distribution of Queen Conch 



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Joiinud of Shellfisli Research. VoL 17, No. 4. 971-477. 199X. 

ALLOZYME AND MORPHOLOGICAL EVIDENCE SUPPORTING THE SEPARATION OF 
BABYLONIA FORMOSAE FORMOSAE FROM B. FORMOSAE HABEI AT SPECIFIC LEVEL 

(PROSOBRANCHIA: BUCCINIDAE) 



LI-LIAN LIU AND VUH-WEN CHIU 

Institule of Murine Biology 
National Sun Yat-Sen Universit}- 
Kaohsiung, Taiwan 804, Republic of China 

ABSTRACT Bubylonui Jormoiue is a common specie;, on the west coast of Taiwan. From its shell color pattern and shape, two 
subspecies have been classified: B. fonnome formosae (Sowerby 1856) and B. formosae habei (Altena and Gittenberger 1981). 
Recently, both subspecies were collected from the same area, which was not in accordance with previous recorded harvests. Therefore, 
the present study was undertaken to evaluate the validity of the subspecies. B. areolata occurring in the same area was also examined 
for comparati\'e purpose. Samples of B. formosae fiirmostu: B. formosae habei. and B. areolalu were collected between September 
1990 and March 1991. Si.\ shell characters (shell length, shell width, spire length, apertural length, fasciole ridge width, and shoulder 
width), radulae. and allozymes were analyzed. The shoulder width of shell could separate species with B. formosae habei > B. areohila 
> B. formosae formosae. Fixed allelic differences were observed at loci of ark. gol-\. mpi. and pi^m between B. formosae and B. 
areolala, and at loci of ark. got-\. and mpi between B. formosae formosae and B. formosae habei. Nei's genetic distances (D) were 
0.25 for B. formosae formosae vs. B. formosae habei. 0.35 for B. formosae formosae vs. B. areolala. and 0.37 for B. formosae habei 
vs. B. areolata. The mean heterozygosity among populations were low in B. areolata (Ho = to 0.06). B. formosae formosae (Ho 
= 0.07 - 0.08). and B. formosae habei (Ho = 0.06 to 0.07). All the above results indicated that the two subspecies deserve to be 
recognized as full species: B. formosae and B. habei. 

KEY WORDS: Babylonia, ivory snail, allozyme. shell, radula 



INTRODUCTION 

Babylonia formosae (Sowerby 1866) belongs to the family of 
Buccinidae. Its distribution was limited to the west coast of Taiwan 
(Altena and Gittenberger 1981). Within this small region, two 
subspecies have been classified: B. formosae formosae (Sowerby 
1866) and B. formosae habei (Altena and Gittenberger 1981 ) (see 
Table 1). According to Altena and Gittenberger ( 1981 ). B. formo- 
sae habei was found only in the northeast coast of Taiwan. How- 
ever, our investigation in late 1990 revealed that B. formosae for- 
mosae and B. formosae habei both were commonly caught on the 
southwest coast of Taiwan. Moreover, Lan (1990) mentioned that 
B. formosae habei sold in Taiwan may be imported from China. 
Ke and Li (1991) and (Ke and Li 1992) also reported that B. 
formosae habei is a commercially important shellfish on the south- 
east coast of China. Meanwhile, studies of reproduction found that 
B. formosae habei spawns between June to September (Ke and Li 

TABLE 1, 

Diagnostic Subspecies Characters of Babylonia formosae Based on 
Altena and Gittenberger (1981). 



Diagnostic 
Characteristics 



B. formosae formosae B. formosae habei 



Last body whorl 
Shoulder on the last 

body whorl 
Suture on the last body 

whorl 
Spots on the last body 

whorl 
Color pattern of the spots 
Distribution 



Evenly rounded 
Narrower 



Less evenly rounded 
Narrow 



Narrow 



Distinct 



Bright 



Narrower 



Less distinct 



Dull 



Northwest to southwest Northeast coast of 



coast of Taiwan 



Taiwan 



1991) (Ke and Li 1992): whereas, B. formosae formosae spawns 
between October to January (Chiu and Liu 1994). Although the 
two subspecies have been studied since 1991 by Ke and Li ( 1991 ) 
(Ke and Li 1992) and Chiu & Liu (1994), it is still difficult to 
evaluate the systematic status of the two subspecies at this mo- 
ment. Hence, the present paper studied the systematic status of the 
two subspecies using morphological, radula, and allozyme char- 
acters. B. areolata (Link 1807) was also studied for comparative 
purpose. B. areolata is the most common Babylonia species in 
Taiwan. Its distribution is from Ceylon and the Nicobar Islands 
through the Gulf of Siam, along the Vietnamese and Chinese 
coasts to Taiwan (Altena and Gittenberger 1981 ). 

Both B. formosae and B. areolata live in sandy or muddy 
subtidal areas and can be caught by either bottom trawling or in 
baited baskets at depths of 15 to 50 m (Lai 1987). 

MATERIALS AND METHODS 

Samples of S. areolata. B. formosae formosae. and B. fonnosae 
habei were collected from coastal waters of Taiwan (Fig. 1) be- 
tween September 1990 and March 1991. Collected snails were 
stored at -70°C for later use. Voucher specimens were deposited 
in the Molluscan Collection. University of Colorado Museum 
(UCM), with the catalog numbers UCM 37665 for B. areolata. 
37666 for B. formosae formosae, and 37667 for B. formosae habei. 

Species were identified preliminarily using the color-pattern cri- 
teria (Altena and Gittenberger 1981). Species of B, areolata has 
three rows of large reddish brown squarish spots on the last body 
whorl (Fig. 2a,b). B. formosae has four rows of violet-brown spots 
on the last body whorl. The spots clearly contrast with the light 
background, and the color is brighter in B. formosae formosae 
(Fig. 2c,d) than in B. formosae habei (Fig. 2e,f). 

Shell characters (shell length, shell width, spire length, aper- 
tural length, fasciole ridge width, and shoulder width) (Fig. 3) and 
total wet weight of individual snails were determined. Duncan's 



971 



972 



Liu and Chiu 



CHINA 




118 



119" 



120 



121 



2S2 



24 



2# 



22 W 



122"E 



Figure I. Sampling localities of Babylonia. KH: Kaohsiung: TK: Tungkang; PH: Penghu; CC: Chungchou; Pt: Pyngtan. 




multiple comparison tests were used for data analysis (SAS Insti- 
tute. Inc. 1985). 

For radula examination, six buccal masses of each species were 
put in a lOVr KOH solution overnight to resolve muscle and con- 
nective tissue around the radula. Each radula was then rinsed in 
distilled water and ultrasonically cleaned for 30 seconds. The 




|lill|lll!l!lllllli;|ii!'|iiil!li|i|illlji;'ipli!'2!l!. :..'."': 

'' ^' ^' ' ''' ^' ^' ^ "" -^ Figure 3. Measurements of the shell of Baftv/oma. SH: shell height; 

Figure 2. SheWs o( Babylonia areolata {a, b), B. formosae foniiosae ic, SW: shell width: SPL: spire length: AL: apertural length; SHW: 

d), and B. formosae liabei (e, f). shoulder width: FW: fasciole ridge width. 



Separation of B. Formosae Formosae from B. Formosae Habei 



973 



TABLE 2. 
Measurements of Shell Characteristics of Babylonia Species (Mean ± SD). 



Species/ 




Shell 


Shell 


Spire 


Apcrtural 


Fasciole 


Shoulder 


Total 


Sample 




Length 


Width 


Length 


Length 


Ridge 


Width 


Weight 


Location 


n 


(mnil 


(mm) 


(mm) 


(mm) 


Width (mm) 


(mm) 


(g) 



Bahxlonict ureoUtttt 
















KH 30 


54.7 ± 3.3 B 


33.0 ± 1.4 B 


29.0 ± 2.2 B 


32.6+ 1.2 B 


4.7 ±0.4 A 


3.0 ± 0.5 C 


29.2 ± 3.7 B 


TK 28 


55.3 ± 16.2 3 


32.2 ± 8.6 B 


29.7 ± 9.6 B 


30.6 ± 8. 1 C 


4.4 ± 1.2 B 


3.3 ± 1.1 C 


31.7 ± 19.3 B 


PH 30 


42.9 ± 8.5 C 


26.8 ± 4.9 C 


21.9 ± 5.3 D 


26.5 ± 4.6 D 


3.8 ± 0.8 C 


3.0 ± 0.6 C 


17.7 ± 9.6 C 


FT 28 


65.2 ± 7.7 A 


38.2 ± 3.6 A 


35.3 ± 4.8 A 


37.1 ±3.4 A 


5.0 ± 0.7 A 


3.7 ± 0.6 B 


46.3 ± 11.9 A 


B. Itinnositf formosae 
















KH 55 


45.0 ± 6.0 C 


26,1 ±2.9 CD 


24.9 + 3.7 C 


24.7 ± 2.9 E 


3.6 ± 0.5 CD 


2.3 ± 0.5 D 


15.9 ±5,5 CD 


TK 50 


41.6 ±6.2 CD 


24.6 ± 3.3 D 


22.3 ± 4.0 D 


23.2 ± 3.0 EF 


3.3 + 0.6 E 


2.0 ± (15 E 


13.0 ±5.1 DE 


B. formostif habei 
















CC 66 


43.0 ± 5.3 C 


25.5 + 3.2 CD 


22.2 ± 3.2 D 


24.5 ± 3.0 E 


3.9 + 0.6 C 


4.3 ± 0.8 A 


14.4 ± 5.7 CDE 


PT 30 


38.7 ±4. ID 


22.3 ± 2.5 E 


20.0 ± 2.4 D 


22.1 ± 2.3 F 


3.4 ± 0.6 DE 


4.1 ±0.5 A 


10.2 ± 3.5 E 


„. .-„.^„i., .,:^., T^ '^ 

















n: sample size. Duncan's test for significant variation is indicated by capital letters (p < .05) 



cleaned radulae were dried and mounted on SEM stubs. These 
specimen were then gold-coated and examined v. ith a Hitachi 450 
scanning electron microscope at 15 KV. 

For allozyme studies, foot tissue (0.2 to 0.5 g) was taken and 
homogenized in a Tekmar tissuniizer with an equal volume of 10 
mM Tris-HCl buffer (pH 7.0) containing 1% Triton X-100. Ho- 
mogenates were centrifuged at 5.000 g for 10 min. and the super- 
nates were stored at -70 "C. Horizontal starch-gel electrophoresis 
with buffer systems Tris-citrate pH 8.0. Tris-citrate pH 6.3/6.7. 
Tris-maleate-EDTA pH 7.4. and lithium hydroxide pH 8.1/8.3 was 
used. Enzyme-staining methods followed Richardson et al. (1986) 
and Murphy et al. (1990). 

Multiple loci encoding the same enzyme (isozymes) were des- 
ignated by consecutive numbers, with I denoting the slowest mi- 
grating isozyme. Twelve enzyme loci were scored: arginine kinase 
(ark, EC 2.7.3.3); esterase (est-\.2. EC 3.1.1.1); glutamate- 
oxaloacetate transaminase (g«r-l,2, EC 2.6.1.1); isocitrate dehy- 



drogenase (idh. EC 1.1.1.42); maiate dehydrogenase [mdh. EC 
1. 1. 1. 37); mannose-6-phosphate isomerase (mpi, EC 5.3.1.8); oc- 
topine dehydrogenase {opdh. EC 1.5.1.11); 6-phosphogluconate 
dehydrogenase [b-pgdh. EC 1.1.1.44); phosphoglucomutase (pgm. 
EC 2.7.5.1); and sorbitol dehydrogenase (,sy//p. 1.1.1.14). Alleles at 
each locus were scored by designating the most common allele of 
B. areohita as 100. All other alleles were numbered according to 
their relative anodal distance from the reference allele. Chi-square 
goodness-of-fit tests were computed to determine if there were 
significant deviations from Hardy-Weinberg equilibrium between 
observed and expected heterozygote genotype frequencies at each 
locus (Nei 1978). The mean observed and expected heterozygosity 
in each population was also calculated (Nei 1978). Nei's genetic 
distance coefficients (D) were calculated and clustered by the un- 
weighted pair-group method with arithtnetic means (UPGMA) al- 
gorithm (Sneath and Sokal 1973). These analyses were performed 
with BIOSYS-I (Swofford and Selander 1989). 




Figure 4. Micrographs of the radula of Babylonia: (a) male B. areolala; (b) female B. areolala; (c) female B. formosae formosae: and (d) female 
B. formosae habei (scale bar = 500 pm). 



974 



Liu and Chiu 



TABLE 3. 
Allele Frequencies of Babylonia Species. 



Species/ 










B. fonnosae 


B. 


fonnosae 


Allele 




B. 


areolata 




formosai 






habei 


Population 


KH 


TK 


PH 


PT 


KH 


TK 


CC 


PT 


ARK 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


189 




















0.118 


0.100 


156 




















0.875 


0.900 


100 


0.917 


1 


0.950 


1 








0.007 





89 


0.083 





0.050 

















56 














0.982 


0.900 








40 














0.018 


0.100 








Ho 


0.100 





0.100 





0.036 


0.040 


0.221 


0.200 


He 


0.155 

* 





0.097 





0.036 


0.182 

** 


0.222 


0.183 


EST-1 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


100 


1 


1 


1 


1 


1 


0.990 


1 


1 


94 

















0.010 








Ho 

















0.020 








He 

















0.020 








EST- 2 


















(n) 


29 


28 


30 


27 


55 


48 


67 


30 


111 








0.017 








0.031 


0.015 





100 


0.983 


0.982 


0.766 


0.981 


0.991 


0.948 


0.948 


1 


89 


0.017 


0.018 


0.217 


0.019 


0.009 


0.021 


0.037 





Ho 


0.003 


0.004 


0.433 


0.037 


0.018 


0.063 


0.075 





He 


0.003 


0.004 


0.371 


0.037 


0.018 


O.IOI 

** 


0.101 

** 





GOT-1 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


176 














0.091 


0.070 








151 














0.609 


0.530 








128 














0.027 


0.030 


0.956 


0.917 


112 














0.218 


0.360 


0.044 


0.083 


100 


0.983 


1 


0.950 


0.963 


0.055 


0.010 








94 


0.017 





0.050 


0.037 














Ho 


0.003 





0.100 





0.418 


0.400 


0.088 


0.167 


He 


0.003 





0.097 


0.073 

** 


0.575 

** 


0.589 


0.085 


0.155 


GOT-2 


















(n) 


30 


28 


30 


27 


55 


50 


66 


30 


160 




















0.030 


0.017 


100 


1 


1 


1 


0.944 


1 


1 


0.955 


0.983 


40 











0.056 








0.015 





Ho 











0.111 








0.091 


0.033 


He 











0.107 








0.088 


0.033 


IDH 


















(111 


30 


28 


30 


27 


55 


50 


68 


30 


100 


1 


1 


1 


1 


1 


1 


1 


1 


MDH 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


115 














0.082 


0.060 








100 


1 


1 


1 


1 


0.918 


0.940 


1 


1 


Ho 














0.091 


0.040 








He 











. 


0.152 

** 


0.114 








MPI 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


100 


1 


1 


1 


1 















continued on next page 



Separation of B. Formosae Formosae from B. Formosae Habei 



975 



TABLE 3. 
continued 



Species/ 










B. formosae 


B. 


formosae 


Allele 






B. areolata 




formosae 






hahei 


93 














1 


1 








76 




















1 


1 


OPDH 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


100 


1 


1 


1 


1 


1 


1 


1 


1 


6-PGDH 


















(n) 


29 


28 


30 


27 


55 


50 


68 


30 


ISO 


0.034 

















0.015 





100 


0.966 


1 


1 


1 


1 


1 


0.978 


1 


67 




















0.007 





Ho 


0.007 

















0.044 





He 


0.007 

















0.044 





PGM 


















(n) 


30 


28 


30 


27 


55 


50 


68 


30 


327 














0.091 


0.240 


0.176 


0.116 


303 








0.017 


0.019 


0.809 


0.690 


0.706 


0.800 


252 














0.091 


0.060 


0.118 


0.067 


170 


0.033 





0.033 


0.019 





0.010 





0.017 


100 


0.950 


1 


0.950 


0.962 


0.009 











79 


0.017 























Ho 


0.100 





0.100 


0.074 


0.291 


0.400 


0.206 


0.333 


He 


0.098 





0.098 


0.073 


0.332 


0.467 

** 


0.460 

** 


0.347 


SDH 


















(nl 


30 


28 


30 


27 


55 


50 


68 


30 


100 


1 


1 


1 


1 


1 


1 


1 


1 



(n; sample size. 

Ho: observed heterozygosity. 

He: expected heterozygosity. 



Significant deviation from Hardy- Weinberg proportion at p = .05 and p = .01. respectively). 



RESULTS 

Quantitative measurements of shell characters are indicated in 
Table 2. Shell lengths of the examined species were from 39 to 65 
mm. Shoulder width was the onlv character shown significant 



variation among species: B. formosae habei > B. areolata > B. 
formosae fonnosae. respectively. 

No significant difference in the radulae was found among the 
Babylonia species and between radulae of male and female B. 
areolata (Fig. 4). The radulae belong to the rachiglossan type. The 



TABLE 4. 
Summary of Genetic Variation in Babylonia Species. 



Species 
and 




Mean Number 
Allelles per 


Percentage of 
Polymorphic 


Mean Heterozygosity 








Population 


Number of Loci 


Locus ± SE 




Loci* 


Observed Mean ± 


SE 


Expected Mean ± SE 


B. areolata 
















KH 


12 


1 .5 ± 0.2 




16.7 


0.028 ±0.012 




0.032 ±0.015 


TK 


12 


1.1 ±0.1 




0.0 


0.003 ± 0.003 




0.003 ± 0.003 


PH 


12 


1.5 ±0.2 




33.3 


0.061 ±0.036 




0.055 ± 0.03 1 


PT 


12 


1.4 ±0.2 




8.3 


0.019 ±0.01 1 




0.024 ±0.011 


B. fonnosae formosae 
















KH 


12 


1.8 + 0.4 




25.0 


0.071 ±0.040 




0.093 ± 0.052 


TK 


12 


2.0 ± 0.4 




41.7 


0.080 ± 0.044 




0.123 ±0.058 


B. formosae hahei 
















CC 


12 


1.9 ±0.3 




25.0 


0.060 ± 0.023 




0.083 ± 0.039 


PT 


i: 


1.6 ±0.3 




25.0 


0.067 ± 0.036 




0.068 ± 0.039 



* a locus is considered polymorphic if the frequency of the most common allele does not exceed 0.95. 



976 



Liu and Chiu 





B. areolata \ 




B. formosae formosae 




B. formosae habei . 









{\K (Tungkang) 
- PT (Pyngtan) 
KH (Kaohsiung) 
PH (Penghu) 

KH (Kaohsiung) 
TK (Tungkang) 

CC (Chungchou) 
PT (Pyngtan) 



0.4 0,3 0.2 0.1 

Nei's genetic distance 

Figure 5. The UPGMA cluster analysis of Nei's (1978) unbiased ge- 
netic distance (D) among Babylonia species. 

central tooth has five cusps. The lateral teeth have two curved 
cusps: the inner cusp short; and the outer cusp is long. 

Among the 12 loci examined, using 0.95 as the criterion for 
polymorphism. si.\ were polymorphic: ark, est-2, got-1.2. mdh. and 
pgm. Detailed allelic frequencies of Babylonia species are shown 
in Table 3. Fixed allelic differences were observed at ark, goi-\. 
nipi. and pgm between B. formosae and B. areolata and at ark, 
got- 1 . and mpi between B. formosae formosae and B. formosae 
habei. Heterozygote deficiencies among populations and species 
were found in all the polymorphic loci. Mean heterozygosities 
among populations of B. formosae formosae and B. formosae ha- 
bei varied from 0.060 to 0.080. which were higher than the popu- 
lations of B. areolata (0.003 to 0.061 ) (Table 4). 

Nei's genetic distance (D) between B. formosae formosae and 
B. formosae habei was 0.25. Comparison with other marine inver- 
tebrate genetic distance values, the difference between B. formosae 
formosae and B. formosae habei could be interpreted at a specific 
rather than a subspecific level. In addition. B. areolata was sepa- 
rated from B. fonnosae formosae and B. formosae habei at the 
distances of 0.35 and 0.37. An UPGMA cluster phenogram is 
shown in Fig. 5. Only minor differentiation existed among the 
populations in each of the three species (D < 0.0002 to 0.0030). 

DISCUSSION 

Our results indicated the shoulder width differed among spe- 
cies: B. formosae habei > B. areolata > B. formosae formosae. 
respectively. The fixed allelic difference between B. formosae for- 
mosae and B. formosae habei was observed in three of the 12 
examined loci. The Nei's genetic distance between B. formosae 
formosae and B. formosae habei was 0.25. These allozyme differ- 
ences are well above the specific level (Thorpe 1983; Richardson 
et al. 1986). Therefore, they should be recognized as two full 
species: B. formosae and B. habei. 

By using the allozyme electrophoretic technique, several gas- 
tropod species previously considered to be polytypic or subspecies 
are actually separate species: Oncomelania hupensis hupensis and 
O. hupensis quadrasi (with Nei's genetic distance [D] = 0.62) 
(Woodruff et al. 1988). Stramonita haemastoma canalictdata. and 
S. haemastoma floridana (with D = 0.28) (Liu et al. 1991). Nii- 



cella emarginata complex (with D = 0.16) (Palmer et al. 1990), 
Crepidula conve.xa complex (with D = 0.76) and C. plana com- 
plex (with D = 0.39) (Hoagland 1984). Although no simple re- 
lationship exists between genetic distance and taxonomic level, 
Thorpe (1983) found the Nei's genetic distances range from 0.19 
to 2.59 for >95% of the congeneric invertebrates. Richardson et al. 
( 1986) also suggested that fixed allelic difference can be diagnos- 
tic in separating species if the loci with fixed allelic differences are 
>209'f of the examined loci. In the present study, Nei's genetic 
distance (D = 0.25) and the level of fixed allelic differences 
(257^) all indicated that the difference between B. formosae for- 
mosae and B. formosae habei is on the specific level. 

According to the records of Altena and Gittenberger ( 1981 ), S. 
formosae habei are distributed on the northeast coast of Taiwan; 
however, we were unable to locate them. From a reliable record 
indicating the natural distribution of B. fonnosae formosae is on 
the southwest coast of Taiwan and that of B. fonnosae habei is on 
the southeast coa.st of China (Ke and Li 1991) (Ke and Li 1992). 
The occurrence of B. formosae habei in southern Taiwan was 
observed in 1987; since then it has been a very coinmon shellfish 
in fish markets. It has become very rare after 1996. with one or two 
individuals mixed in hundreds of B. areolata. Because of this 
unusual change in abundance, we suspected that B. formosae habei 
might have been introduced from China through fisheries opera- 
tions and is not a resident species in southern Taiwan. It is also 
speculated that range expansion could be caused by a temporal 
change in hydrographic condition. 

Introduction of an exotic species either by an accident or com- 
mercial purpose is quite common in Taiwan. For example, Perna 
viridis was believed to be imported by ship industry (Lai 1987). A 
South America freshwater apple snail, Ampullarius insularus was 
imported from Argentina for commercial use in 1980 (Chang 
1985). Both species are now widespread in Taiwan. In addition, 
marine bivalve Mytilopsis sallei. freshwater snail Pila leopordvil- 
lensis. and land snails Achatina fnlica and Bradybaena similaris 
are also introduced species in Taiwan. The presence of B. formo- 
sae habei in Taiwan might be another case of an importation from 
southeast coast of China. 

The reproductive season of B. fonnosae formosae is known to 
be from October to January, with average egg diameter 0.55 mm 
(Chiu and Liu 1994); whereas, B. formosae habei spawns from 
June to September with average egg diameter 0.26 mm (Ke and Li 
1991) (Ke and Li 1992). The colors of the female sperm-ingesting 
gland are also different, being dark brown and brown, in B. for- 
mosae formosae and B. fonnosae habei. respectively (personal 
observation). Although the breeding compatibility of these two 
subspecies is unknown, the differences in allozyme patterns indi- 
cated they are different and should be elevated to full species level; 
that is, B. formosae and B. habei. 

ACKNOWLEDGMENTS 

We thank Dr. H. Y. Chen for help with collecting ivory snails. 
We also thank Dr. C. C. Lu and S. K. Wu for their constructive 
comments on this manuscript. 



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Journal of Slwllfish Research. Vol, 17. No. 4, 474-98^, 1998. 

CHARACTERIZATION OF THE DIGESTIVE TRACT OF GREENLIP ABALONE, HALIOTIS 
LAEVIGATA DONOVAN. I. MORPHOLOGY AND HISTOLOGY 



JAMES O. HARRIS, CHRISTOPHER M. BURKE, AND 
GREG B. MAGUIRE 

School of AijiuiciiltiiiT 

University of Tasmania 

Launceston. Tasmania. Australia. 7250 

ABSTRACT Australasian abalone such as the greenlip abalone, Haliulis laevigata, prefer a diet ol red algae (Rhodophyta); whereas, 
abalone from elsewhere more commonly prefer brown algae (Phaeophyta). Because of this feeding preference, the structure of the 
digestive tract of A/, laevigata was investigated using histological and scanning electron microscopy (SEM) techniques. The digestive 
tract of both starved and fed adult H. laevigata revealed the presence of ciliated, mucus, and secretory cells throughout the digestive 
tract. The esophagus contained secretory, ciliated, and large mucous cells, with fragmentation spherules also present. The crop extended 
from the esophagus to the stomach. It was surrounded by thin muscularis and consisted mainly of secretory cells, although some 
phagocytes were present. The stomach possessed mainly secretory cells, although some ciliated cells, mucous cells, and phagocytes 
were present. The style sac differed from the stomach, having more ciliated cells. In mtestinal regions I to III, the epithelium was shorter 
than in previous regions. Few cilia were present on the ridges, although many were observed in the gutters. Intestinal regions IV to 
V contained more mucous cells than intestine III. and more bactena were observed associated with the fecal string than in other regions. 
The low incidence of bacterial association with the gut epithelium was attributed to the occurrence and number of mucous cells, 
common throughout the digestive tract. Spherical bodies present in the lumen are believed to be fragmentation spherules involved in 
waste removal and enzyme release. Starved abalone contained fewer mucous cells in the esophagus, had less pronounced staining 
reactions in the stomach, contained large amounts of granular inclusions in the style sac. and had fewer phagocytes in the intestines. 

KEY WORDS: Abalone. Haliotis laevigata, histology, digestive tract, starvation 



INTRODUCTION 

Abalone are herbivorous archaeogastropods whose diet con- 
sists mainly of macroalgae, although diatoms and some detritus, 
including sand, are also ingested (Campbell 1965, Garland et al. 
1985). Australasian abalone prefer red algae (Rhodophyta) and 
will consume brown algae (Phaeophyta) only when preferred spe- 
cies are less common (Poore 1972, .Shepherd 1973, Shepherd and 
Steinberg 1992). Brown algae are digested much more slowly 
(Foale and Day 1992). Abalone from elsewhere more commonly 
prefer brown algae (Shepherd and Steinberg 1992), suggesting 
possible differences in digestive strategy. Several enzymatic stud- 
ies of abalone from the northern hemisphere are available, but few 
data are available for Australasian species (Clark and Jowett 
1978). Feeding preference differences for Australasian species 
have been attributed to algal toughness (McShane et al. 1994) or 
phenolic content (Shepherd and Steinberg 1992), although Shep- 
herd and Steinberg ( 1992) considered that feeding preferences are 
primarily attributable to the selective nature of environments on 
available algae. 

In such primitive gastropods as abalone, digestion begins with 
extracellular digestion followed by phagocytosis, or cellular up- 
take of particles, in the digestive gland and ingestion by mobile 
amoebocytes in some cases (Owen 1966). Intracellular digestion 
occurs within the duct cells of the digestive gland (Fretter and 
Graham 1962). Secretory cells occur throughout the digestive tract 
of Haliotis spp. They have been documented in the buccal cavity, 
crop, stomach, cecum, digestive gland, style sac, and intestine by 
Crofts (1929). Other locations for secretory cells include the buc- 
cal pouches (Fretter and Graham 1962) and esophagus (Beve- 
lander 1988). 

A wide variety of digestive enzymes have been identified from 
the gut of abalone with the carbohydrates fucoidan, carboxymeth- 
ylcellulose (CMC), and algin being among the most common sub- 



strates used for detecting enzyme activity (Duffas and Duffas 
1968, Elyakova et al. 1981, Yamaguchi et al. 1989, Boyen et al, 
1990). Protease, alginase, and amylase activity also appear in the 
crop fluid o'i Haliotis rufescens Swainson (McLean 1970). 

The nature of the digestive tract of Haliotis spp. was examined 
by Crofts (1929) (drawings for Haliotis tiiberculata Linnaeus). 
Campbell (1965) (drawings for Haliotis cracheroclii Leach), and 
Bevelander ( 1988) (a photographic study for H. tiiberculata). Ex- 
amination of the greenlip abalone digestive tract, by histology and 
scanning electron microscopy (SEM), strengthens our knowledge 
of normal abalone gut structure, particularly for species that prefer 
red algae. It also complements current gut physiology research 
(Hanis et al. 1998a). In addition, it facilitates the detection of 
dietary or toxicant-induced alterations in structure (Harris et al. 
1998b). 

Contributions to host animals from digestive tract bacteria can 
come from either resident or transient populations. Alimentary 
tracts offer many habitats conducive to microbial activities, such as 
fermentation of complex organic molecules, the products of which 
can be used by the host. Intestinal surfaces are often colonized by 
bacteria, which then make up the autochthonous flora of the host 
(Savage 198.^), and which can contribute to the nutrition of the 
host (McBee 1971). Transient bacteria are ingested with, or as, 
food and encounter both physical or biological events during pas- 
sage that protect the resident populations from displacement (Or- 
pin and Anderson 1988). Scanning electron microscopy has been 
used on the oysters Crassostrea virginica Gmelin and Crassostrea 
gigas Thunberg to examine bacterial associations (Tall and Nau- 
man 1981. Garland et al. 1982a) and has revealed physical attach- 
ment by resident microbes to internal digestive surfaces of other 
invertebrates (Harris 1993. Jolly et al. 1993). 

In this study, the gut structure of greenlip abalone is charac- 
terized using histological and/or SEM techniques for fed and 
starved individuals. Because the digestive gland is the most com- 



979 



980 



Harris et al. 



prehensively studied organ involved in digestion in abalone 
(Campbell 1965. McLean 1970. Bevelander 1988) and is known to 
be free of bacteria (Erasmus et al. 1997). it was not considered in 
this study. Our emphasis was on epithelial function, because this 
can influence bacterial associations with the gut wall. It comple- 
ments a study of the gut microenvironment in this species (Harris 
et al. submitted). 

MATERIALS AND METHODS 

Adult specimens of H. laevigata were maintained in a recircu- 
lating system consisting of 6 x 20 L plastic containers and a 
biofilter for up to 3 weeks prior to histological sampling. Greenlip 
abalone were collected from several locations in northern Tasma- 
nia, 40° to 4I°50'S, 146°50' to I48°50'E, (Petal Point, Foster 
Islands, Port Sorell, and Flinders Island) and consisted of adults of 
135 to 185-mm length. Macroalgae were collected in southern 
Tasmania, 42°50' to 43°50'S, 147°50' to 148°E (Blackman Bay 
and Port Arthur). Macroalgae of the genera Polysiphonia sp.. Ulva 
sp., and other epiphytic algae associated with the macroalgae Am- 
phibohis spp. were collected by divers and used as food. Epiphytic 
algae of seagrasses are known to comprise up to 859f of the green- 
lip abalone diet in wild conditions (Shepherd 1973). The algae 
were added to the maintenance tank and left for 10 to 14 days. The 
abalone fed rapidly when Polysiplumia sp. were added, although 
they were "messy"" feeders. To remove algal debris, tanks were 
siphoned every second day. A diatom film, which was grazed by 
the abalone, developed within the tank during the study period. 

Abalone were removed from the maintenance tank by either a 
commercial abalone iron, a warm water siphon or a flat plastic 
spatula with grease. Several abalone were dissected for prelimi- 
nary investigation of the digestive tract. For histological examina- 
tion, separate groups of fed and starved animals were used. No 
macroalgae were added for 10 days to tanks containing starved 
abalone, allowing sufficient time for physiological changes to oc- 
cur within the abalone (Carefoot et al. 1993). 

A scalpel was used to cut the foot as close as possible to the 
shell without disrupting the mantle and visceral mass. The mantle 
surrounding the mantle cavity was removed to facilitate access to 
the digestive tract, taking care not to puncture the rectum (intestine 
V). Two parallel incisions were made in the integument covering 
the digestive tract (Fig. 1 ), joined by an additional two longitudinal 
cuts in the integument to complete a rectangular section. This layer 
was peeled away from the digestive tract with scalpel, tweezers, 
and a blunt probe. The intestine (sections 11, III. IV, and V) were 
teased with a blunt probe from the connective tissue and mem- 
branes, and excised with scissors and scalpel. Because the oe- 
sophagus lies under the intestines, it was carefully separated by 
teasing with a blunt probe. The digestive gland and gonad were 
removed by scraping them from the surface of the crop and stom- 
ach. 

Short lengths of gut (about 10 mm) were removed from the 
esophagus, the crop, the stomach wall, the style sac, and intestinal 
regions III and IV and placed into either phosphate-buffered for- 
malin, Zenker's fluid, or Bouin"s fluid during daylight hours. 
Samples were fixed for 24 hours at room temperature ( 15 to 18°C) 
then dehydrated through a graded ethanol series to xylene in a 
Tissue-Tek II tissue processor. Dehydrated tissue samples were 
embedded in paraffin resin on a Shandon Histocentre 2 and sec- 
tioned on a Microm HM 340 microtome at 4 p,m. Sections were 
oven dried overnight at 37"C. Routine Harris" Haematoxylin and 




Figure L Schematic diagram of greenlip abalone showing locations 
for incisions into integument (a.b — lateral cut site; c,d — longitudinal 
cut site). 



Eosin (H & E) staining in a Shandon Linistain GLX automatic 
tissue stainer was carried out on all tissues processed. 

Five abalone. all of which had been maintained in recirculating 
aquaria and fed with red, filamentous algae Polysiphonia sp. were 
used for SEM. Abalone were removed from the shell, and the 
digestive tract was exposed. To preserve the contents, whole gut 
sections were removed aseptically. Where necessary, regions of 
the gut were teased from the integument with a blunt probe and/or 
forceps. Samples were taken from the esophagus, crop, stomach, 
style sac, and intestines III and IV. Samples were immediately 
transferred into 2.5% glutaraldehyde fixative in 0.2 m cacodylate 
buffer, pH 7.2 containing the major salts present in sea water: 1.6% 
NaCl, 0.6% MgCU, and CaCU (Garland et al. 1982b). Samples 
were fixed for 24 h at 4°C then rinsed through three O.IM caco- 
dylate buffers of decreasing osmolality (Lewis et al. 1985). At this 
stage, the samples were trimmed and cut open to expose internal 
surfaces. The samples were then dehydrated through a graded 
ethanol series (Hodson and Burke 1994). Absolute ethanol and 
acetone were prepared by storing the commercial grade chemicals 
over anhydrous copper sulphate (in dialysis tubing). Dehydrated 
samples were immediately transferred to acetone prior to being 
critical point dried in liquid CO,. Samples were dried using a 
Balzers CPD 020 Critical Point Dryer (CPD) apparatus. The 
samples were mounted onto aluminium SEM stubs with carbon 
paint and/or double-sided tape. Samples were then kept in a plastic 
desiccator and stored over CaCL at a vacuum pressure of 25 to 30 
psi, to prevent rehydration of the dried samples (Garland et al. 
1982b), and sputter coated with gold (Balzers coater) within 24 h. 
Gut sections were viewed with a Phillips 505 SEM at operating 



Gastrointestinal Structure of Greenlip Abalone 



981 



TABLE 1. 

Cellular types distributed nithin the digestive tract of greenlip 
abalone. H. laevigata. 



Gut Region 


External Color 


Cell Types Observed 


Esophagus 


Pale 


Mucus, ciliated, and secretory 


Crop 


Blue-gray 


Secretory, phagocytes 


Stomach 


Green-gray 


Secretory, ciliated, mucus, 

phagocytes, amoebocytes, muscularis 


Stvie sac 


Green-gray 


Secretory, ciliated 


Intestine 11 


Light brown 




Intestine III 


Gray-black 


Ciliated, phagocytes, secretory 


Intestine TV 


Brown 


Mucus, secretory, ciliated 


Intestine V 


White 





voltages between 15 to 20 kV, using llford FP4 120 lllm for 
micrographs. 

RESULTS 

The epithelium of the greenlip abalone digestive tract varied in 
shape, cellular types, composition, and staining reaction (Table 1 ). 
The efficacy of fixative type had a marked influence on cellular 
appearance between and within different regions of the gut. Star- 
vation caused very minor effects to the epithelia of the esophagus, 
style sac, and intestines, with little effects noted elsewhere (Table 
2), SEM of digestive tract epithelium revealed few bacterial cells, 
with most in the intestines. What is apparent from this investiga- 
tion is the widespread occurrence throughout the digestive tract of 
spherical bodies, 5. to 8-|xm in diameter, corresponding to the 
fragmentation spherules described by Morton (1953). believed to 
be involved in waste removal and/or delivery of enzymes to the 
lumen. Although their size and spherical nature suggested the pos- 
sibility of these spherules being bacterial, visible evidence of 
spherules being shed into lumen from the crop (see Fig. 7) and 
style sac (see Fig. 13) indicates otherwise. A general plan of the 
digestive tract of H. laevigata was developed from the morpho- 
logical observations (Fig. 2). 

Esophagus 

The esophagus of the greenlip abalone is oriented posterior to 
the cephalic region. The entrances to the esophageal pouches lie 
immediately posterior to the salivary glands. The right esophageal 
pouch is twisted over the esophagus, following the rest of the 
digestive tract posteriorly, to the region where intestine 111 begins. 

TABLE 2. 

Changes in the digesti>e epithelium of starved greenlip abalone, H. 
laevigata, as indicated by histology. 



Tissue 



Starved 



Esophagus 
Crop 
Stomach 
Style sac 
Intestine II 
Intestine III 
Intestine IV 
Intestine V 



Fewer mucous cells on ridges 

No difference 

Less pronounced staining 

Large amounts of granular inclusions 

Fewer phagocytes 

No difference 

More intense staining 

No difference 




Figure 2. General layout of greenlip abalone digestive tract, showing 
(left) digestive tract in situ, and (right) schematic diagram of the di- 
gestive tract, in dorsal viev* (a = cephalic region; b = right buccal 
pouch: c = esophagus; d = crop; e = stomach; f = stomach cecum; g = 
style sac; b = intestine I; i = intestine II; j = intestine III; k = intestine 
I\ : I = intestine V). The esophagus and crop are equivalent to the 
postesophageal regions I and II as described in Campbell (1965). 

The left esophageal pouch is smaller and is located underneath the 
right esophageal pouch. The midesophagus extends to the posterior 
end of the esophageal pouches, where it then becomes paler and 
continues to the crop. This paler section corresponds to the post- 
esophagus region 1 of H. cracherodii (Campbell 1965). but is 
referred to as the esophagus in this study. No muscularis was 
observed in this region. The oesophagus contains mucous cells, 
secretory cells, and ciliated cells (Fig. 3). Large mucous cells occur 
on the ridges of the esophagus, among secretory cells containing 
granules. Granular inclusions occurred in the distal cell tips of both 
fed and starved animals. Ciliated cells occurred on both the ridges 
and gutters. Comparison with starved abalone revealed tnany more 
mucous cells on the ridges of the esophageal cells of fed animals. 
Within the esophagus, fragmentation spherules were evident 
among mucus within the lumen (Fig. 4). Neither the gut wall nor 
ingested food showed bacterial association. Food particles were 
associated with the mucus (Fig. 5). 




Figure 3. Esophageal epithelium from fed abalone in histological sec- 
tion; tissue was fixed in Zenker's fluid; magnification 400 x (a = granu- 
lar inclusions; b = ciliated epithelium; c = mucous cell). 



982 



Harris et al. 





"\. 



Figure 4. Epithelial surface of esophagus using SEM; Bar = 10 uni (a 
= fragmentation spherules: b = mucous; c = cilia I. 

Crop 

The posterior oesophagus I expands into the crop, which is 
distinguished by its deep blue-gray color. The crop extends to the 
most posterior point of the right foot muscle, then narrows and 
twists 180° into the stomach. Two large folds extend from the crop 
into the stomach, forming a valve that controls entry of material 
into the stomach. Thin muscularis surrounded the crop. The epi- 
thelium of the crop contained mainly columnar secretory cells, 
although phagocytes were also present (Fig. 6). Observed within 
the crop were cells in various stages of constriction and fragmen- 
tation, demonstrating the stages involved in release of fragmenta- 
tion spherules (Fig. 7). including bulging through the mucus. The 
cells" surface also had a striated border. Granular inclusions were 
prevalent toward the distal cell tips in both fed and starved ani- 
mals. Nuclei were mainly located in the basal half of the cells. 
Heavily ciliated regions contained both debris and fragmentation 
spherules (Fig. 8). In the crop, greater cell definition was found 
with Zenker's fluid than other fixatives. An isolated helical bac- 
terial cell was observed in this region. 

Stomach 

The stomach of H. laevigata follows the general pattern for 
prosobranch gastropods as described by Morton (1953). A gastric 



Figure 6. Crop epithelium of slar\ed ahaione in histological section; 
tissue Has tl.ved in Zenker's fluid: niagniflcalion 4(10 x (a = nucleus of 
mucus cell: b = non-nucleated fragmentation spherules; c = muscula- 
ris). 

.shield is present on the anterior, right side wall. A furrowed ciliary 
sorting area takes up the floor of the stomach. The stomach cham- 
ber narrows anteriorly into the heavily ciliated style sac. in which 
is located the protostyle. The style sac continues to narrow into the 
intestine. The ducts known to lead to the digestive gland in other 
species of Haliotis were difficult to locate in H. laevigata, al- 
though these ducts are presumably present. Muscularis was also 
found below the epithelial cells. Cells coinprising the stomach 
epithelium included ciliated cells, mucous cells, secretory cells, 
and phagocytes (Fig. 9). Secretory cells constituted the majority of 
the epithelium, with mucous cells occurring consistently. Phago- 
cytes, although infrequently observed, were present both in basal 
and distal regions of the cells. Nuclei were located within the basal 
third of the cells. Amebocytes occurred in the stomach epithelia 
and were distinguishable from phagocytes by their irregular mor- 
phology. Some cilia were also visible under the spherules (Fig. 
10). Bulging of the secretory cells through the epithelium and 
cavities in the mucus occurred in the style sac (Fig. 1 1). similar to 
the observations of the crop. Secretory cells involved in fragmen- 
tation appear uniform in view from the epithelial surface, but side 
views of fragmented cells indicate more variety in shape. The 
club-shaped tips of the cells were observed protruding from the 




Figure 5. Esophageal epithelium, showing mucopolysaccharide matrix 
and algal fragments using SEM. Bar = 50 \im (a = algal fragments in 
mucus). 



Figure 7. Epithelium of crop wall, showing secretory cells in various 
stages of constriction and fragmentation using SEM; Bar = 10 ]xm (a 
= constricting cell; b = fragmented cell; c = cilia). 



Gastrointestinal Structure of Greenlip Abalone 



983 




Figure S. Crop epithelium, sliowinj; a renioii where cilia are located. 
Fragmentation spherules are collected together in a furrow of the 
epithelium using SEM. Bar = 10 pm (a = fragmentation spherule; b = 
algal debris; c = cilia). 

mucus. The style sac has an evenly ciliated, striated epithelium 
(Fig. 12). although epithelia toward the stomach showed fewer 
cilia and more secretory cells. Bouin's fluid produced more defi- 
nition in cellular structure than other fixatives, although formalin- 
fixed tissue enabled phagocytes to be distinguished more easily. 
Interestingly, formalin-fixed stomach tissue retained a large layer 
of mucus that also showed separation from the epithelial surface. 
In star\ed animals, tissue anterior to the stomach showed large 
amounts of granular inclusions within the distal third of the cells 
and in the striated. borders. Nuclei of starved tissue cells also 
showed a less pronounced staining reaction (Figs. 13). 

Intestines I to III 

Intestine I continued from the narrowed style sac, crossed the 
oesophagus dorsally, into the first of the vertically oriented 180° 
intestinal twists. The second 180° twist and a change in exterior 
color to light brown/beige marked the beginning of intestine II, 
which extended to about the midpoint of the foot. Here the intes- 
tine abruptly changed exterior color to almost black, indicating the 
start of intestine III. No muscularis was observed in this region. 



Figure 10. Epithelial surface of the left stomach wall using SEM. Note 
the occurrence of secretory cells involved in fragmentation as the dom- 
inant cell types. Bar = 10 pm (a = fragmentation spherules; b = cilia). 

The epithelium was most similar to that of the crop. Cells within 
intestine III were more cuboidal than in intestines I and II. Few 
ciliated cells were present on the ridges (Fig. 14), though more 
occurred in the gutters, and were observed supporting fragmenta- 
tion spherules within the intestine III. Phagocytes were widespread 
among the cells, although mainly confined to the distal cell tips. 
Formalin-fixed tissues revealed more mucous cells in ridge regions 
than other fixatives, as well as phagocytes located in the distal 
parts of the cells. Few phagocytes were evident within the starved 
tissue samples (Fig. 15), although several phagocytes occurred in 
the fed tissue samples. SEM revealed large areas of the epithelium 
covered by fragmentation spherules, and a layer of mucus. Where 
this layer of mucus was removed from the intestinal surface, the 
fragmentation spherules were apparent, indicating mostly secre- 
tory cells as the dominant cell type (Fig. 16). This removal of 
mucus may have been an artifact of the glutaraldehyde fixation 
process, but it shows the arrangement of the cells beneath the 
mucous coat. Epithelial cells can be seen in cross section where 
tissue was trimmed after fixation (Fig. 17). These cells were over- 
laid with mucus, some debris, and fragmentation spherules. On 
epithelial surfaces, more fragmentation spherules were found, 
along with rod-shaped bacteria (Fig. 18). 




Figure 9. Stomach epithelium iif led al)al<inc in histological section; 
tissue was fixed in Bouin's fluid; magnification 400 x (a = fragmenta- 
tion spherule; b = club-shaped tip of secretory cell bulging towards 
lumen; c = phagocyte; d = nucleus of secretory cell). 



Figure 11. Epithelium of posterior style sac. showing mucous- 
associated secretory cells using SEM. Bar = 10 pm (a = secretory cells; 
b = mucus). 



984 



Harris et al. 




Figure 12. Style sac epithelium ol ted ahalone in lii.sloiojjicai section; 
tissue was fixed in Bouin's fluid; magnification 400 x (a = ciliated 
cells). 



Figure 14. Intestine III epithelium from fed abalone in histological 
section; tissue was fixed in Bouin's fluid; magnification 400 x (a = 
nucleus of ciliated cell; b = ciliated cell; c = cilia; d = mucous cell). 



Intestines IV to V 



DISCUSSION 



The intestine continues anteriorly to the most anterior point of 
the foot, where intestine IV begins. The exterior of intestine IV 
was mostly light brown in color. The intestine turns 180° and 
follows the previous regions of the gut. dorsal to intestine III and 
the esophagus. This continues to close to the intestinal bends (in- 
testines I and II). Intestine V is defined by another 180° turn, 
passing through the ventricle to end within the mantle cavity as the 
white-colored rectum. Intestine IV contained more mucous cells 
than samples from intestine III (Fig. 19). Secretory cells and cili- 
ated cells were also widespread, although ciliated cells were more 
common within some gutters. Cells within the gutters were also 
more cuboidal than those occurring in ridges (Fig. 20). Bouin- 
fixed tissues had more defined nuclei than either Zenker's or for- 
malin-fixed tissues. In formalin-fixed samples, starved abalone 
tissues stained more intensely than fed abalone tissues. Granular 
inclusions appeared inore prevalent within starved tissue samples. 
Some bacterial cells were evident, associated with the mucus of 
this region. These were mostly rod-shaped cells, and were few in 
number. These cells all appeared associated or entangled with the 
preserved mucus of the fecal string. 



Gut Structure 

Cellular type and location within the digestive tract of H. lae- 
vigata illustrate digestive processes occurring in discrete regions. 
The presence of secretory, inucous. and ciliated cells in the epi- 
thelium of different regions enabled functions for each organ to be 
postulated. Distinguishing between secretory cell types requires 
further histological investigation of the cells and the roles they 
play in digestion. Each fixative considered here proved effective at 
highlighting certain structural details in particular areas. No gen- 
eral pattern emerged as to which fixative was suited to each se- 
lected area, although Bouin's fluid seemed best at fixing tissues of 
the stomach and style sac. Formalin-fixed tissues showed a more 
intense staining reaction, sometimes making subtle features, such 
as cytoplasmic granules, difficult to see. 

The frequency and large size of mucous cells found within the 
esophagus indicate that this region is responsible for substantial 
mucus production. Mucous secretions aid in entangling food par- 
ticles, thus enhancing movement of food through this region and 
into the crop. Food movement through the esophagus is also fa- 




Figure 13. Style sac epithelium of star^ed abalone in histological sec- 
tion; tissue was fixed in phosphate-buffered formalin; magnification 
400 X (a = phagocytes; b = granular inclusions). 



Figure 15. Intestine 111 epithelium of starved animals in histological 
section; tissue was fixed in phosphate-bulTered formalin; magnifica- 
tion 400 X (a = phagocytes; b = mucous cell; c = ciliated cell). 



Gastrointestinal Structure of Greenup Abalone 



985 







.^jt^S^t^vasr^ 



Figure 16. \intral surface of intistinv 111 using SEM. Bar = 100 pm 
la = mucus; b = epithelial surface with large numbers of secretory 
cells). 



cililated by widespread cilia. The similar incidence of mucous cells 
in the intestinal region relates to the need for abalone to have 
cohesive fecal pellets, because feces are discharged directly into 
the mantle cavity, where disintegration of feces would be disad- 
vantageous. Mucous secretion thus serves two purposes: it helps to 
lubricate passage of the fecal string, and it coals the fecal pellets in 
mucus that becomes more viscid with increasing pH typical of 
seawater (Morton 1968). 

Large numbers of secretory and mucous cells were present 
within the crop, but very few ciliated cells were observed. The 
physical nature of the esophageal folds within the crop prevent 
uncontrolled food movement from the crop into the stomach. Be- 
cause the crop receives the contents of the esophagus, including 
food, mucus, and secretory cell products, and contains few cilia, it 
seems likely that its function differs from the oesophagus. The 
internal pressure of the crop causes rapid leakage of gut contents 
if any part of its surface is punctured. Therefore, food is likely to 
be retained within the crop for some time, which, together with the 
expanded walls of the crop, suggests that it is a storage organ, 
although there may be some food digestion before the food-mucus 
mixture enters the stomach. Analysis of crop contents has revealed 
algal degradation within the crop (Shepherd 1973. Foale and Day 



Figure 18. Epithelial surface of intestine III using .SEM: note the oc- 
currence of fragmentation spherules, debris, and some rod-shaped 
bacteria. Bar = 50 pm (a = fragmentation spherules; b = fecal debris; 
c = rod-shaped bacterial cells). 

1992). implying some enzyme production to degrade algae beyond 
the particle size rasped into the digestive tract by the radula. Amy- 
lases have been recorded from the esophageal epithelium of H. 
riifi'sceiis (McLean 1970). 

The large number of secretory cells observed in the crop and 
stomach suggest that these regions are important in digestion by H. 
laevigata. Crofts (1929) observed secretory cells in the crop, stom- 
ach, and digestive gland of H. tubercidata. Fragmentation spher- 
ules were widespread in the esophagus, crop, stomach, style sac, 
and intestine of all animals examined. The.se cells are not believed 
to be bacterial, because "pinching-off of cells within the crop 
and style sac was observed in this study and by Campbell (1965) 
in H. cracherodii. These cells bulge into the lumen, becoming 
rounded and club-shaped and surrounded by mucous-bound food 
(Morton 1953). The tips of the cells, or whole cells, are constricted 
into the lumen. In this study, spherules were observed still intact in 
the intestine, supporting one view held by Owen (1966) that these 
spherules aid in removing indigestible wastes. The presence of 
spherules in the esophagus is unlikely to be associated with waste 
removal, so it is tnore likely that they carry digestive enzymes into 
the lumen to facilitate extracellular digestion. Breakdown of these 
spherules would, thus, be a means of introducing enzymes to the 









Figure 17. Intestine III, showing both a cross section of the epithelial 
cells and the surface mucus coating using SEM. Bar = 50 pm (a = 
fragmentation spherules; b = mucus; c = epithelial cells). 



Figure 19. Intestine 1\ epithelium of fed abalone In histological sec- 
tion; tissue was fi.xed in Zenker's fluid; magnification 400 x (a = basal 
lamina; b = phagocyte: c = ciliated cells: d = mucous cell). 



986 



Harris et al. 




Figure 20. Intestine I\ epitiieliuni ot led abalone in tiistolojjical sec- 
tion; tissue was fixed in Bouin's fluid: magnification 400 x (a = ciliated 
cells; b = nucleus of ciliated cell). 

large substrate volume of the food mass. Further definition of the 
role of spherules is required. 

The widespread occurrence of cilia within the style sac and the 
presence of a protostyle within this region are typical features of 
gastropods (Morton 1953). The protostyle is known to receive 
indigestible wastes from the stomach sorting area and, because of 
the rotating action of cilia, bind these wastes onto the protostyle 
surface (Morton 1953). The extensive ciliation in the style sac 
suggests that H. laevigata has a stomach stnjcture typical of other 
primitive archaeogastropods. such as Palella spp. (Fretter and Gra- 
ham 1962). The high incidence of secretory cells would contribute 
to the digestive processes involved with the action of the proto- 
style, because chemical breakdown would greatly enhance the me- 
chanical action of the protostyle on the gastric shield. 

The presence of phagocytes near the distal epithelial cell tips in 
the stomach suggests a role in either waste rejection or food uptake 
(Owen 1966). When comparing intestine III to the esophagus, 
fewer cilia are visible. In view of this, food passage through in- 
testine III would be much slower, allowing ample time for phago- 
cytes to interact with the lumen contents. It would follow that this 
region has a role in food absorption and waste rejection. Because 
fewer phagocytes were recorded in the stomach and intestine of 
starved abalone. this could be seen as a response to decreased food 
availability. Intestinal region IV has distribution of cilia similar to 
intestine III. Thus, slow passage time through the intestines pro- 
vides both ample time and surface area for either absorption or 
phagocytosis. The presence of granules in epithelial cells alludes to 
digestive activity, instead of simply the consolidation of the fecal 
string, as suggested by Campbell ( 1965). 

Starvation of the abalone produced changes in some of the 
digestive tissues. Compared to fed animals, periods of starvation 
are known to decrease the rate of digestion of algae by H. rubra 
after feeding recommences (Foale and Day 1992). Both blood 
glucose levels and stored glycogen content were depleted within 6 
days of starvation (Carefoot et al. 1993). Similar responses in 
cellular structure have been elicited by other causes, such as poor 
water quality (Harris et al. 1998b). Therefore, the structural 
changes could have several possible explanations. 

Microbial Aspects 

Overall, few bacteria were observed within the digestive tract 
of W. laevigata. Areas likely to be harboring microorganisms, such 



as folds and crevices (McBee 1971). showed little evidence of 
colonization. In this study, most bacteria were evident in the in- 
testine of H. laevigata. The isolated spirilloid bacterium observed 
within the crop and the several rod-shaped bacteria observed 
within the intestine all seemed to be associated more with mucus 
than directly with epithelial surfaces. Interestingly, only in the 
intestine were any bacteria observed that could be regarded as 
attached. These were located on the epithelial surface with frag- 
mentation spherules and debris. Because this was only observed in 
one abalone. it suggests that bacterial attachment to intestinal sur- 
face may occur, but not to any great extent. 

Mucus was observed closely associated with the epithelial sur- 
faces, including ciliated surfaces. Detachment of some mucus in 
the intestine showed the surface of secretory cells with and without 
mucus; in neither case were bacterial associations evident. The 
presence of mucus after glutaraldehyde fixation is in contrast to 
Gariand et al. (1979). who found tissue surfaces relatively free of 
mucus when fixed in this way. The secretion of copious quantities 
of mucus is believed to remove microorganisms from epithelial 
surfaces (Gariand et al. 1979). Prosobranchs such as abalone se- 
crete large amounts of mucus throughout the digestive tract, 
mainly from the esophageal glands and style sac (Crofts 1929. 
Fretter and Graham 1976). Mucus within C. gigas had an effect on 
microbial attachment, because of its viscosity, by entangling mi- 
crobes and preventing their attachment (Gariand et al. 1982a). The 
few bacterial cells observed among mucus in the intestines and 
esophagus of H. laevigata are unlikely to be attached populations, 
because microbes on the surface of a mucous layer are more likely 
to be originate from either food or feces (Harris 1993). 

The few food particles observed in the esophagus of H. laevi- 
gata also revealed no bacteria, and only an isolated bacterial cell 
in one esophagus sample. The absence of bacteria in this region 
requires explanation, because abalone are known to consume mi- 
croorganisms with ingested algae (Gariand et al. 1985). The visible 
absence of bacteria may be attributable to the rapid passage time of 
food into the crop, because ciliary currents move food quickly 
through the esophagus of W. tuberculata (Crofts 1929). Movement 
of uigested. radio-labeled food into the blood of H. rufescens oc- 
cun-ed within 15 minutes of feeding (McLean 1970). suggesting 
that movement of food and absorption through this region can be 
rapid. Because abalone feed soon after dusk (Shepherd 1973). food 
may be in the guts for up to 12 hours before collection, allowing 
adequate time for algae to fragment to an unrecognizable state 
(Foale and Day 1992). This may have influenced the results: how- 
ever, the presence of isolated food particles within the oesophagus 
suggests other reasons for the absence of visible bacteria. 

The lack of bacteria observed in this study within the gut of H. 
laevigata could he attributed, in part, to the physiological aspects 
of digestion associated with the three main cell types. Within the 
digestive tract. H. laevigata has a large surface area taken up by 
secretory, ciliated, or mucous cells. Mucous and secretory cells, 
through secretion, provide a surface area that would be unsuitable 
for microbial association (Harris 1993). Surfaces covered by cilia 
are unsuitable for microbial attachiuent. because it is through the 
movement of cilia that food is directed through all gut regions 
except the crop and stomach (Crofts 1929. Campbell 1965). The 
major means of food passage through the digestive tract of oysters 
is entanglement in mucus, directed by ciliary movement (Garland 
et al. 1982a). These authors found few bacteria within the digestive 
tract of the oyster C. gigas and attributed this to the extensive 
ciliation within the oyster. 



Gastrointestinal Structure of Greenlip Abalone 



987 



The greenlip abalone seems to have a transitory relationship 
with its ingested bacterial populations, similar to C. gigas (Garland 
et al. 1982a). The nature of the gut of H. laevigata, with few 
visible bacterial associations, suggests that for possible bacterial 
contribution, bacteria within the food may be more important at 
breaking down carbohydrates than surface-associated bacteria. 
Both these possibilities have been suggested for other aquatic in- 
vertebrates (Harris 1993). Because bacterial populations within the 
lumen may be either transient populations or colonizers, signifi- 
cant contributions from these populations would require prolonged 
retention of food within the latter regions of the gut. Because feces 
are produced for several days postfeeding (Wee et al. 1992). this 
situation is entirely possible. 

CONCLUSION 

The gut structure of H. laevigata does not differ greatly from 
that of other haliotids that chiefly consume brown macroalgae. It 
has a complex gut with different regions being characterized by 
specific cellular composition indicati\e of region-specific gut 



functions. SEM analysis suggests that relatively little of the gut 
epithelial surface is suitable for colonization by bacteria. The con- 
tribution of transient bacteria in the lumen of the gut could not be 
assessed in this descriptive study, but the contributions of bacteria 
to particular aspects of digestion are addressed in a complementary 
physiological study (Harris et al. 1998a). 

ACKNOWLEDGMENTS 

This work was supported by the School of Aquaculture. Uni- 
versity of Tasmania at Launceston. The authors thank Mr. James 
Mason of Fumeaux Aquaculture for providing the abalone. The 
authors also thank Mr. Mark Heather. Mr. Brad Adams, and Mr. 
Nick Savva for providing much of the algae fed to the abalone. and 
to Dr. Stephen Hodson and Mr. Wiecslaw Jablonski (Central Sci- 
ence Laboratories. Hobart) for assistance with SEM techniques. 
We also thank Dr. Judith Handlinger for critical assessment of this 
manuscript. Present address of G. B. M.: Fisheries Western Aus- 
tralia, Research Division. P.O. Box 20. North Beach. WA. 6020. 
Australia. 



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Erasmus. J. H.. P. A. Cook & V. E. Coyne. 1997. The role of bactena in the 
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Fretter. V & A. Graham. 1962. British prosobranch molluscs. Their func- 
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Garland. C. D.. A. Lee & M. R. Dickson. 1979. The preservation of surface 
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Garland. C. D.. G. V. Nash & T. A. McMeekin. 1982a. Absence of surface 
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Garland. C. D.. G. V. Nash & T. A. McMeekin. 1982b. The preservation of 
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digestibility studies with abalone. 1. Preliminary studies on the feeding 
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Jimnuil of Shellfish Research. Vol. 17. No. 4, 989-944. 1998. 



CHARACTERIZATION OF THE DIGESTIVE TRACT OF GREENLIP ABALONE, HALIOTIS 
LAEVIGATA DONOVAN. II. MICROENVIRONMENT AND BACTERIAL FLORA 



JAMES O. HARRIS, CHRISTOPHER M. BURKE, AND 
GREG B. MAGUIRE 

School of Aqiiacninire 

University of Tasmania 

Lawweston, Tasmania. Australia. 7250 

ABSTRACT Microelectrodes were used to measure pH and dissolved oxygen within the gut environmenl of adult greenlip abalone 
(145 to 160 mm), Haliotis laevigata Donovan. Oxygen levels were found to be below the limit of detection for the oxygen micro- 
electrode (0.38 mg DO.L"'), suggesting either mieroaerophilic or anaerobic conditions. The pH profile of the gut revealed a decrease 
from the external environment (pH = 8.20) to pH 5.31 within the crop, increasing through the intestine to 6.64 in the rectum. 
Enrichment cultures of bacteria from within the abalone gut revealed mostly isolates from the family Enterobacteriaceae. These isolates 
occurred throughout all regions of the abalone gut. and almost all showed hydrolytic ability for one or more carbohydrates. Cxtophaga 
spp. isolates appeared from esophageal and intestinal enrichments of the digestive tract and were all capable of both carboxymeth- 
ylcellulose and agar hydrolysis. A decrease in diversity of bacterial tvpes m the stomach, crop, and style sac corresponded with reduced 
pH. 

KEY WORDS: Abalone. Halunis laevigahi. ditiestive tract 



INTRODUCTION 

The gastrointestinal tract is a microenvironmeiu that has been 
e.xaniined in marine invertebrates in terms of pH (Mathers 1974) 
and dissolved oxygen or redox potential (Plante and Juniars 1992). 
As well as having a significant effect on enzyme activity, high or 
low pH can favor maintenance of symbiotic microbes (Plante and 
Jumars 1992). The selective nature of the niicroenvironment di- 
rectly influences microbial composition and activity. In turn, it is 
possible for the microorganisms, through their metabolism, to 
modify the microenvironment. 

The most commonly reported association between microbes 
and in\ertebrates involves the ingestion of bacteria (e.g.. Garland 
et al. 1985, Vitalis et al. 1988). Bacterial associations with diges- 
tive tracts of marine animals have revealed a restricted range of 
microorganisms, suggesting the existence of strong selective pres- 
sures within the gut that result in characteristic gul microflora 
(Unkles 1977. Sochard et al. 1979, Tall and Nauman 1981 ). Stud- 
ies on the bacterial flora of digestive tracts require an understand- 
ing of the microenvironment in order to mimic these conditions in 
culture. Microelectrodes are ideally suited for studying. (;; siiii. 
some aspects of the physiology of undisturbed microbial commu- 
nities, such as microbial respiration (Revsbech and Jurgensen 1986). 

Nearly all terrestrial herbivorous animals have one or more 
parts of the digestive tract expanded into an organ that accommo- 
dates a microbial population valuable in the digestion of food, for 
which the host animals do not necessarily produce the correct 
complement of enzymes (McBee 1971). The activity of such bac- 
teria often benefits the host through cellulose breakdown, nitrogen 
fixation, increased host resistance to toxins, or preconditioning of 
food (Harris 1993). In some herbivorous animals, there exists a 
specific fermentation organ, in which food (cellulose) is subjected 
to highly reduced conditions arising as a result of microbial me- 
tabolism. Abalone are aquatic herbivores. If an analogy can be 
drawn between herbivorous animals that ferment and abalone in 
terms of microbial cellulase activity, then the conditions prevalent 
in these fermentation chambers should also be repeated. These 
conditions would include a highly reduced environment in which 
oxygen has been removed. The most effective means of detecting 



these conditions is with microelectrodes because of their small 
size, accuracy, and sensitivity. Oxygen depletion as seen within the 
digestive tract of vertebrates does occur in some aquatic deposit 
feeders (Plante and Jumars 1992). 

Less is understood about the relationships that microbes have 
with invertebrate hosts than with vertebrates, with the exception of 
the cellulose-degrading bacteria found within Teredinidae (Bi- 
valvia) (Morton 1978). Associations between microbes and aquatic 
invertebrates have been reviewed by Harris ( 1993). If not digested, 
microbes can travel the length of the gut and pass out unaffected. 
or may proliferate in a favorable region of the gut. Attachment 
often leads to the development of residential populations. The 
importance of the role these microbes play is unclear. Vitalis et al. 
( 1988) found that bacteria that degraded algae contributed signifi- 
cantly to the nutrition of the sea-hare, Aplysia spp. However, Galli 
and Giese (1959) described several isolates from within the gut of 
the herbivorous aquatic snail, Tegida fimelirulis. few of which 
could degrade either agar, alginic acid, or carrageenan. 

Algal carbohydrates (Table 1 ) have been used as substrates to 
examine the role of microbes in aquatic herbivore digestive physi- 
ology. Alginate lyase, amylase, cellulase, agarase, laminarinase, 
carrageenanase, and b- 1 ,4-glucanase activities have all been evalu- 
ated (Galli and Giese 1959, Vitalis et al. 1988, Harris 1993, 
Sawabe et al. 1995, Erasmus et al. 1997). Bivalves that exhibit 
cellulase activity have been shown to possess a cellulolytic micro- 
flora (Crosby and Reid 1971 ). Other bacterial strains isolated from 
aquatic invertebrate guts have shown agarase. protease, lipase, 
laminarinase, amylase, alginase, and chitinase activities (Harris 
1993). 

Most commonly, facultatively aerobic bacteria have been iden- 
tified from the digestive tracts of invertebrates (Galli and Giese 
1959. Prim and Lawrence 1975. Musgrove 1988). However, few 
attempts to isolate strict anaerobes have been made (Harris 1993, 
Sawabe et al. 1995). In vertebrates, facultative aerobes are present, 
but in lower numbers than other types. The activities of these 
facultative aerobes quickly deplete the oxygen within the digestive 
tract, thus providing favourable conditions for growth of obligate 
anaerobes. 



989 



990 



Harris et al. 



TABLE 1. 

Common polysaccharides found in some marine algae (Kreger 1962, 
McCandless 1981, Craigie 1990). 



Polysaccharides 



Algal Division 



Storage 



Structural 



Rhodophyceae 
Chlorophyceae 
Phaeophyceae 



Floridean starch Cellulose, agar, carrageenan, mannans 

Starch Cellulose, xylans 

Laminarin Cellulose, alginic acid, fucoidan 



Preparation of Abalone for Experiments 

Abalone were removed from the maimenaiice tank w ith either 
a commercial abalone iron, a warm water siphon, or a flat plastic 
spatula with grease. Abalone were anesthetized in 1 mL/L of ethyl 
p-aminobenzioc acid (benzocaine) solution for 15 minutes to pre- 
vent any movement. The stock benzocaine solution was made up 
from 100 g ethyl p-aminobenzoic acid dissolved in 1 L of 95% 
alcohol (Hahn 1989). 

Microprohe Analysis of the Abalone Digestive Tract Microenrironment 



In aquatic invertebrate guts, bacteria can occur within the 
esophagus, the stomach, intestine, midgut, style sac, cecum, and 
hindgut (Harris 1993). In bivalves, the hindgut is the most heavily 
colonized region (Harris 1993). Accumulation of bacteria in the 
hindgut of bivalves occurs because of the extended passage time of 
food (Prieur et al. 1990). Bactena have doubling times that range 
from 15 minutes up to several days, so passage times of up to 3 
days through bivalve guts are sufficient for adapted bacteria to 
survive and grow (Prieur et al. 1990). Observations of blacklip 
abalone. H. rubra, indicate that feces are produced up to 7 days 
after feeding (Wee et al. 1 992 1. suggesting ample time for bacterial 
colonization. Similarly, the relatively long intestine in abalone. 
with numerous folds and grooves (Campbell 1965). provides 
ample surface for bacterial colonization (Harris 1993). However. 
Harris et al. (submitted) found relatively few bacteria within the 
gut of H. laevigata. 

The abalone digestive tract contains several functional regions 
through which there is a continuous, one-way flow of ingested 
material (Harris et al. submitted). Characterization of the micro- 
environment of these areas would enhance understanding of the 
microbial and physiological processes occurring within the diges- 
tive system of abalone. The nature of the microbes from the green- 
lip abalone, H. laevigata, also requires characterization to deter- 
mine their potential importance to the digestive physiology of the 
host. The purpose of this study is to determine the physical con- 
ditions within the gut of the greenlip abalone and to examine the 
ability of microbes isolated from the gut to digest algal carbohy- 
drates. This inforination complements another study on the gut 
structure of this species (Hanis et al.. submitted). 

MATERIALS AND METHODS 

Maintenance System 

Adult greenlip abalone (135 to I85-mm length) were collected 
from several locations in northern Tasmania, 40° to 41°50'S. 
146°50' to 148°50'E (Petal Point. Foster Islands, Port Sorell, and 
Flinders Island) and maintained in recirculating systems. Macroal- 
gae were collected in southern Tasmania, 42°50' to 43°50'S. 
147°50' to 148°E (Blackman Bay and Port Arthur). Macroalgae of 
the genera Polysiphnnia sp., Ulva sp.. and other epiphytic algae 
associated with the macroalgae Amphiholu.s spp. collected by 
divers were used as food for the abalone (Harris et al. 1998). The 
algae were added to the maintenance tank and left for 10 to 14 
days. To remove algal debris, tanks were siphoned every second 
day. A diatom film, which was grazed by the abalone. developed 
within the tank during the study period. 



Seven adult abalone. 145 to 160-mm. were used for pH analy- 
sis. Two of these were also used for determining dissolved oxygen 
levels in the gut. Once anesthetized, the abalone were removed 
from their shells, and the integument was removed to expose the 
gut (see Harris et al. 1998). All measurements were recorded after 
the electrodes were fully inserted into the lumen and the electrode 
response stabilized, which took less than 4 seconds. 

The pH microelectrode was a MI-413 microcombination pH 
electrode in an 18-gauge needle (Microelectrodes Inc., New 
Hampshire. USA). It was connected to a Hanna Instruments (HI 
9017) microprocessor pH meter, accurate to ±0.0001 mA. The pH 
electrode was calibrated with buffered solutions of pH 7.00 and pH 
4.00, at 20°C. pH measurements were performed at 18 to 20°C and 
35.5 ppt. 

The dissolved oxygen probe was a Clark-type oxygen micro- 
electrode (OME) with guard cathode (Diamond General Corp., MI, 
USA). It was used with a Keithley 485 autoranging picoammeter. 
The dissolved oxygen electrode was prepared for calibration by 
immersion in air-saturated water for 30 min. The current output 
was measured for 1 hour after saturating aquarium water of 35.5 
ppt and held at 16.1 ± 0.05°C with N,, air or O,. The electrode had 
good linearity in its response and a low stirring effect of 1.2%, 
which was considered negligible (Revsbech and Jorgensen 1986). 
Before testing each animal, water samples were taken for Winkler 
analysis to check electrode calibration. Electrodes were held in 
position using a micromanipulator (Maerzhauser, Germany), at- 
tached to a heavy stand (Fig. 1 ). Any lateral stress on an OME may 
cause it to crack, so the abalone were held in position by pinning 
them to a soft polystyrene base. The base was fixed to a wire rack 
and positioned within a small (30 x 30 x 20 cm) aquarium con- 




Figure 1. Experimental setup (a = 15 kg stand base: b = 
abalone: d = seawater aquarium; e = dissolved oxygen 
micromanipulator). 



airline; 
OME; 



Digestive Physiology of the Greenlip Abalone 



991 



taining seawater. The gut wall was punctured with the electrode, 
which was then inserted into the lumen. Positioning the OME in 
the lumen was achieved once the gut wall slid up the probe. 

OME's give an output in pA which is directly related to the 
partial pressure of oxygen. Because temperature and salinity were 
constant, the dissolved oxygen concentration was calculated as: 

I sample 

DO sample = — - x DO standard 

I standard 

(I sample = current output in pA; I standard = current output 
from aquarium water; DO standard = DO of aquarium water as 
measured by Winkler titration; DO sample = DO of sample in 
mg/L.) 

The limit for detection by the OME was \5 pA. or 0..^8 mg 
DO.L"'. The oxygen saturation can be calculated by dividing the 
calculated concentration in mg/L by the saturated concentration of 
oxygen at IbA C and 35.3 ppt. which is 7.93 mg/L. 

Bacterial Flora of the Abalone Digestive Tract 

To obtain bacterial samples from the abalone, sections of the 
digestive tract (postesophagus region 1. crop, stomach, style sac. 
and intestines) were excised with scissors, scalpel, and tweezers 
(Harris et al. 1998) and placed into each of three enrichment broths 
(CarboxyMethylCellulose (CMC), starch, or agar). Smaller sec- 
tions, such as the esophagus, were sampled as whole gut sections: 
whereas, larger areas, such as the stomach, required the removal of 
one wall. Before use, these broths were boiled for up to 30 minutes 
to remove dissolved oxygen, then placed in an ice-bath to cool 
rapidly. Enrichments were performed in anaerobic and microaero- 
philic conditions according to methods modified from Lewis et al. 
(1992). Carbohydrate enrichment broths were incubated at 2rC 
and examined on the third day. 

Loops of enrichment broth were transferred to solid media 
containing either starch, agar, or CMC. CMC and starch plates 
were made using modified seawater, agar, and vitamins (SWAV) 
medium with agar at 10 g/L and CMC or starch at 10 g/L. pH 
concentrations of enrichments broths and solid media were ad- 
justed to 6.5 for esophagus and intestines II and IV, and 5.5 for 
crop, stomach, and style sac samples. Plates were incubated in 
anaerobic and microaerophilic conditions at 2rC and examined 
after 4 days. Visible colonies were subcultured onto the same 
media to purify the isolates. Bacteriological peptone and yeast 
extract were omitted from the (SWAV) medium of Lewis et al. 
(1992) to enable single carbon sources (starch, agar, or CMC) to be 
added. 

CMC was dissolved in distilled water before adding to seawa- 
ter. CMC degradation was detected visually by observing clear- 
ance zones around colonies. Localized clearance of the medium 
was taken as hydrolysis. Hydrolysis on agar plates was seen by a 
lowering of colonies into the agar. Combined ingredients for solid 
media were autoclaved at I2rC and 15 psi for 15 minutes. 

Tests performed on pure isolates were: gram reaction and cel- 
lular morphology, colonial morphology, OF test, Craigie tube mo- 
tility test, catalase, oxidase, and susceptibility to the antibiotic 
0/129 (150 (jLg) (Oxoid). Organisms found to be fermentative were 
tested against this antibiotic for presumptive Vibrio spp. identifi- 
cation. All three media types were tested for their effect on the 
catalase and oxidase reactions and were found to have no effect. 



Statistical Analysis 

Data were subjected to single, fixed factor analysis of variance 
(ANOVAl after meeting assumptions of normality using the Sha- 
piro-Wilk test (Zar 1996) and homogeneity of variance using 
Cochran's test (Underwood 1981). Multiple comparison of means 
(Tukey-Kramer HSD) was performed on data that showed a sig- 
nificant ANOVA result (Sokal and Rohlf 1995). All analyses were 
conducted using JMP 3.0 software (SAS Institute). 

RESULTS 

MicroemironmenI of the Abalone Digestive Tract 

Readings below the limit of detection for the OME (0.38 mg 
DO.L"') indicated that conditions were, at least, microaerophilic. 
This was consistently found throughout the length of the digestive 
tract, with little variation evident. All the dissolved oxygen con- 
centrations were calculated to be =£5.7% oxygen saturation at 
16.1°C and 35.5 ppt. The crop was significantly (p < .05) more 
acidic than the esophagus and the intestines (Table 2). 

Bacterial Flora of the Abalone Digestive Tract 

The attempt at isolating anaerobic bacteria produced few or- 
ganisms. The microaerophilically incubated plates showed growth 
after 4 days incubation and were subcultured after 7 days. Ap- 
proximately three bacterial types were evident on each plate for a 
total of 51 isolates. 

Subculturing revealed both pigmented and nonpigmented colo- 
nies, mostly as Gram-negative rods. Strains showed both negative 
and positive catalase reactions, and all strains examined were oxi- 
dase negative. Physiological types included mostly fermentative 
reactions (36 isolates), although no reaction (13 isolates), and oxi- 
dative reactions (two isolates) were also observed. Only two iso- 
lates were obligate microaerophiles, both being members of the 
family Enterobacteriaceae; the remainder were facultati\'ely aero- 
bic. Most fermentative strains were resistant to 0/129, although six 
isolates showed inhibition zones ranging up to 20 mm. 

Most of the microbial isolates showing positive hydrolytic ac- 
tivity occurred within the oesophagus (II isolates) and intestines 
( 17 isolates). Fewer isolates were recovered from the crop, stom- 
ach, and style sac. Hydrolytic activity varied among the isolates 
(Table 3), being prevalent among those isolates identified as Cy- 
tophaga spp. and Enterobacteriaceae. Degradative ability on agar 
and CMC was common among the isolates, although no isolates 
were observed that could degrade both starch and agar. 



TABLE 2. 
pH profile of the digestive tract of greenlip abalone. H. laevigata. 



Sampling Site 



Mean ± SE 



Esophagus 
Crop 
Stomach 
Style sac 
Intestine II 
Intestine III 
Intestine IV 
Intestine V 



6.20 ±0.16'' 
5.28 ± 0.08'' 
5.53 ± 0.1 0"" 
5.49 ±0.12"" 
5.80 ±0.12" 
6.34 ± 0.04° 
6.65 ± 0.06" 
6.64 ± 0.04" 



Means sharing a common superscript are not significantly different (p > .05). 



992 



Harris et al. 



TABLE 3. 

Genera, location, and hydrolytic activity ol' bacterial isolates from 
the digestive tract of H. laevigata. 









Polymer Degrading 






No. 




Activity" 




Site 


Bacterial Groups 


Isolates 


CMC 


Agar 


Starch 


Esophagus 


Enterobacteriaceae 


6 


4 


3 


■) 




Cytophaga 


4 


4 


4 






Altcnmionas 


1 


1 


1 




Crop 


Enterobacteriaceae 


6 


4 


3 






Aerococcus 


2 


■y 






Stomach 


Enterobacteriaceae 
Neisseriaceae 


6 
1 


3 

1 


I 


3 

1 


Style sac 


Enterobacteriaceae 
Neisseriaceae 
Alleroiuonas 


6 
1 

1 


4 


I 


2 


Intestine I] 


Enterobacteriaceae 
Alferomonas 
Listeria 


6 

1 
I 


5 


1 

1 


3 
1 


Intestine IV 


Enterobacteriaceae 


4 


-) 


1 






Cytopluiga 


3 


3 


3 






Acinetobacter 


I 


1 




1 




Aerococcus 


I 








Total 




51 


34 


19 


13 



■' Number of isolates showing positive hydrolytic activity. 

DISCUSSION 

Within the abalone gut. dissolved oxygen levels were below the 
detection level of this OME and the gut should, therefore, be 
regarded as anoxic, or at least niicroaerophilic. Low dissolved 
oxygen levels, similar to those found within the abalone digestive 
tract, have been reported in other invertebrates. Plante and Jumars 
(1992) found that even within the digestive tracts of deposit- 
feeders known to have consumed oxygenated sediment, oxygen 
levels were similar to animals known to have consumed anoxic 
sediments. From this. Plante and Jumars ( 1992) proposed that the 
low dissolved oxygen levels were attributable to biological or 
chemical processes in the foregut that quickly consumed added 
oxygen, with the gut contents effectively acting as an oxygen sink. 

The low oxygen tension and weakly acidic conditions within 
the digestive tract of H. laevigata provide a selective environment. 
Prieur et al. ( 1990) reviewed the microbiology of bivalve digestive 
tracts and noted a higher proportion of fermentative bacteria than 
in the surrounding seawater. Most of the bacteria isolated from the 
guts of aquatic invertebrates have been facultative aerobes, al- 
though obligate aerobes and anaerobes have been reported (Harris 
1993). The metabolism of facultative aerobes quickly depletes the 
available oxygen, thereby creating conditions favorable for anaero- 
bic fermentation. However, anoxia is an insufficient variable with 
which to define microbial activity, and fermentation in particular, 
in an environment such as the abalone gut. because an anoxic 
environment tnay still have oxidizing conditions. Combined Eh 
and dissolved oxygen measurements provide a better understand- 
ing of the inicrobial environment (Plante and Jumars 1992). 

The pH profile along the greenlip abalone digestive tract is 
similar to other gastropod and bivalve mollusks. The lowest values 
recorded are in the stomach of Patella sp. (.'i.S.'i) (Hyinan 1967), 
Crepidula sp. (6.U0) (Hyman 1967). Buccinum sp. (5.6) (Hytnan 



1967). Ostrea ediilis sp. (6.02) (Mathers 1974). and in the style sac 
oi Mya sp. (4.4) (Owen 1966). although few authors have reported 
pH levels in the crop of mollusks. The lower pH in the crop and 
stomach reduces the viscosity of mucus, allowing the gut contents 
to mix readily. Raising pH increases the viscosity of the mucus in 
the intestine, helping to consolidate the loosely bound mucus string 
into cohesive pellets (Morton 1968). Crop contents in H. cra- 
cherodii are considerably less viscid than in other regions of the 
gut (Campbell 1965). The only direct measurement of pH within 
the digestive tract of abalone was described by Gomez-Pinchetti 
and Garcia-Reina ( 1993). They measured the pH of digestive gland 
homogenates from Haliati.s coccinea ca)niriensis. and recorded 
values between 5.5-6.0. This suggests that the crop is the most 
acidic region in haliotids in general and specifically in H. laevi- 
gata. This organ is believed to act as a food storage and digestion 
organ, because both recognizable food pieces up to 3-cm long and 
unrecognizable food have been found (Campbell 1965). Esopha- 
geal valves restrict the tnovement of larger food particles from the 
crop into either the stomach or the stomach cecum (Crofts 1929). 

Natural seawater has pH values varying between 7.5 to 8.5 
(Austin 1988). The decrea.se in pH within the abalone gut portrays 
an environment that differs from relatively stable, alkaline seawa- 
ter. The acidic abalone gut provides an environment that would 
select against organisms unable to tolerate acid pH. The genus 
Vibrio, for example, is tolerant of mildly alkaline conditions and is 
generally grown on media of pH 8.6 (Baumann and Schubert 
1984); whereas, other marine bacteria such as Alcaligenes are 
commonly isolated in neutral pH (Kersters and De Ley 1984). 

The enzyme activity peaks found in other abalone also illustrate 
the pH changes found throughout the gut of H. laevigata. Diges- 
tive activity in the esophagus of H. rufescens is highest at pH 
levels between that of seawater and 6.8 (McLean 1970). Peak 
alginase activity in H. ntfescens and H. corrugata occurs from pH 
7.4 to 7.6 (Nakada and Sweeney 1967). However, the lower pH 
levels found in the crop of H. laevigata are still within the range 
of pH that enables efficient amylase and protease activity in H. 
rufescens (McLean 1970). even though different enzymes with 
different pH optima are likely to be present in H. laevigata. 

The stomach functions to collect food and secretions from the 
salivary glands, cecum, and the digestive glands (Crofts 1929), so 
the pH within the stomach should also be a mixture of these 
influences and the secretions of the stomach. Observations from H. 
laevigata indicate that the stomach has similar pH to that of the 
crop and style sac. In the digestive diverticula of Haliotiis sp. 
( =Haliotis). the maximum activity of enzymes such as fucoidan- 
ase occurs at pH = 5.4 (Thanassi and Nakada 1967). a value of pH 
similar to that found in the crop, stomach, and style sac of H. 
laevigata. 

In some invertebrates, pH and redox conditions are sometimes 
at unusual levels that favor association between the host and spe- 
cific microbial communities (Hatris 1993). The selective nature of 
these changes in pH imparted on the microbial communities will 
favor those microbes best adapted to the conditions. The different 
pH readings and the microaerophilic environment found in the 
abalone digestive tract, therefore, provide several niches for mi- 
crobes to exploit and grow. 

■ Most of the bacteria found within the abalotie gut were able to 
degrade starch. CMC. or agar. The ability of several different 
isolates to degrade both agar and CMC indicates that these bacteria 
were capable of growth on two of the more common substrates 
available in this environment. Althouiih the isolation media were 



Digestive Physiology of the Greenlip Abalone 



993 



as close as possible to the conditions within the gut environment. 
the use of selective media can sometimes fail to detect some bac- 
teria capable of hydrolytic activity (Harris 1993). Therefore, it is 
likely that there are some bacterial types present that were not 
isolated through the enrichment process. The larger diversity of 
bacteria isolated from the esophagus of the abalone. with a de- 
crease in species in the crop and stomach, is directly related to the 
selective conditions of the gut environment. Dissolved oxygen and 
pH are two variables likely to influence microbial growth strongly 
in the abalone. 

Bacterial genera found within the digestive tracts of bivalve 
molluscs include: Achromobacier, Flavohacterium/Cytophaga. 
Pseudomonas, Vibrio. Corynebacterium, Arthiobacter. Escheri- 
chia. Neisseria, Streptococcus. Micrococcus. Moraxetla. Acineto- 
bacter. and Aeromonas spp. (Prieur et al. 1990). Juvenile blacklip 
abalone. Haliotis rubra, have been shown to consume bacteria 
with coralline algae (Garland et al. 1985). These bacteria were 
predominantly Moraxella. although Pseudomonas, Vibrio. Altero- 
monas. and smaller numbers of Flavobacterimn/Cytophaga and 
Aeromonas spp. were also present. Bacterial isolates obtained from 
the South African abalone. H. midae. showed an ability to use a 
range of complex polysaccharides (Erasmus et al. 1997). In terms 
of hydrolytic capabilities, the types of bacteria found within the 
abalone gut are similar to those found in the sea hare (Gastropoda). 
Aplysia Juliana. (Vitalis et al. 1988). However, not all the bacteria 
ingested by abalone may be able to exploit the gut environment. 
From our study, it seems that the marine bacteria capable of 
growth at reduced pH are different in composition to those isolated 
by other authors at higher pH (Sawabe et al. 1995, Erasmus et al. 
1997). Consequently, the reports of other authors may have re- 
vealed bacterial populations that are present, but not necessarily 
capable of contributing to the digestive ability of the host in a 
typical gut pH regime. It may be that the bacteria reported in this 
study differ from those reported elsewhere by being capable of 
digesting algae within the gut environment, from the wider variety 
of bacteria ingested by the abalone. 

Vibrio spp. have been recorded as predominant microorganisms 
in several marine invertebrate digestive tracts (Unkles 1977, So- 
chard et al. 1979. Hartis 1993). including abalone (Erasmus et al. 
1997, Sawabe et al. 1995). It may seem surprising that so few 
isolates of Vibrio spp. were obtained from the greenlip abalone. 
However. Vibrio spp. are usually isolated on alkaline media (Bau- 
mann and Schubert 1984), suggesting growth is reduced or pre- 
vented in acidic conditions. The microbial isolates from the most 
acidic region, the crop, were almost entirely from the family En- 
terobacteriaceae, suggesting that these bacteria are well adapted to 
the acidic gut environment. The Enterobacteriaceae are rarely re- 
corded from the marine environment or from the guts of inverte- 
brates (Harris 1993). The occurtence of the Enterobacteriaceae in 
H. laevigata may represent a normal bacterial flora that specifi- 
cally developed within the gut and adapted to the reduced pH and 
microaerophilic environment. Their presence throughout the di- 
gestive tract suggests that these bacteria may be indigenous. 

Wild greenlip abalone are obligate drift algae consumers, able 
to consume many different types of algae (Shepherd 1973). Inges- 
tion of diatoms, detritus, bacteria, and sand also occurs as a result 
of the mode of feeding (Campbell 1965). The diet fed to the 
abalone during this study was limited in diversity as compared to 
that of abalone in the wild. The restrictions this would place on 
microbial growth may be subtle, although some decrease in normal 
microbial species diversity could be expected. By restricting avail- 



able food types, this may also reduce bacterial diversity. Digestive 
tract analysis of bacterial biota in other animals maintained in 
laboratory systems supports this theory, because the selective pres- 
sures imposed by the artificial environment influence the normal 
bacterial flora occurring in the gut of aquatic invertebrates (So- 
chard et al. 1979). 

Seaweeds are known to have epiphytic colonies of diatoms, 
yeasts, and bacteria (Austin 1988), some of which have known 
algal cell-degrading abilities, such as Cytophaga spp. Mechanical 
breakdown of algae by the radula would release cellular contents 
previously unavailable to epiphytic or free-living bacteria, and this 
would be expected to slunulate microbial growth. However, few 
bacteria were seen to be associated with the gut surface of the 
greenlip abalone. This may be caused by the action of cilia and the 
presence of mucous and secretory cells (Harris et al. 1998). and the 
results from this study that suggest that variation in pH from the 
external environment may also be a factor. We obtained 44 isolates 
of bacteria from the gut of H. laevigata. These bacteria were 
capable of degrading algal polysaccharides at levels of pH and 
dissolved oxygen similar to gut values. Therefore, bacteria may 
contribute to H. laevigata nutrition. Because the bacteria do not 
seem to be strongly associated with the gut epithelium, then bac- 
terial digestive activity is likely to be restricted to the lumen (Har- 
ris et al. 1998). Some output of feces still occurs several days after 
feeding has ceased (Wee et al. 1992). allowing the possibility for 
sustained bacterial activity within the intestines. The less intimate 
association of bacteria with bivalves as compared to terrestrial 
animals (Kueh and Chan 1985) also seems apparent in the greenlip 
abalone. Kueh and Chan (1985) suggested that, for oysters, the gut 
flora are mainly derived from the external environment and a more 
indigenous population of bacteria dominate the lower digestive 
tract because of selective pressures and multiplication. This situ- 
ation seems analogous to that of the greenlip abalone. 

CONCLUSION 

The presence of bacteria within the digestive tract of the green- 
lip abalone, and their ability to break down algal carbohydrates at 
pH levels found within the gut, suggests that bacteria are capable 
of contributing to the nutrition of their host, although the amount 
remains in question. The lack of physical association of these 
bacteria with gut epithelium suggests a different digestive strategy 
to terrestrial herbivores. If bacteria contribute significantly to host 
nutrition, they are more likely to contribute through activity within 
the gut lumen. The selective pressures of the gut environment give 
rise to bacterial populations that are different in composition to 
those reported from the external marine environment. 

ACKNOWLEDGMENTS 

This work was supported by the School of Aquaculture. Uni- 
versity of Tasmania at Launceston. The authors to thank Mr. James 
Mason of Funieaux Aquaculture for providing the abalone. Also, 
the authors thank Mr. Mark Heather, Mr. Brad Adams, and Mr. 
Nick Savva of Tasmanian Tiger Abalone for providing much of 
the algae fed to the abalone. We also thank Dr. Judith Handlinger 
for critical assessment of this manuscript. Present address for 
G.B.M.; Fisheries Western Australia. Research Division, P.O. Box 
20, North Beach, WA. 6020. Australia. 



994 



Harris et al. 



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Journal of Shcllfnli Rcn'orch. Veil. 17, Nii. 4. 445-1002. IWS. 

BACTERIAL COLONIZATION OF A FORMULATED ABALONE DIET DURING 

EXTENDED IMMERSION 

ANDREW BISSETT.' CHRIS BURKE,' " * 
GRAEME A. DUNSTAN,' ' GREG B. MAGUIRE' ^ 

'School of Aqiiacitlture 

University of Tasnuiiiia 

Launcestoii, Tasmania. 7250. Australia 
'The Cooperative Research Centre for Aqitacidtitre 

P.O. Box 123 Broadway 
Sydney. 2007. Australia 
^CSIRO Division of Marine Research 

Hohart. Tasmania. 7001. Australia 

ABSTRACT The characteristics of the microbiota of a formulated abalone (Haliotis laevigata) diet were studied by scanning electron 
microscopy (SEM) and standard bacterial culture and taxonomic techniques. Microbes colonizing the diet (ABCHOW) were enumer- 
ated by SEM and partly identified after immersion of the diet in seawater for 0, 2, and 4 days with and without abalone. The fatty acid 
composition of the diet was also analyzed, after similar treatments, for biomass estimates and bacterial biomarker identification. 
Bacterial numbers on unimmersed diet and diet immersed in sterile seawater for 2 and 4 days were negligible. Bacteria proliferated 
after 2 days immersion in seawater with abalone ( 1.2 x 10' cells/mnr ) and without abalone (5.7 x lO'' cells/mnr) (p < .05). Numbers 
continued to rise between 2 and 4 days for diet immersed without abalone (6.5 x 10"* cells/mm"). However, a decrease in bacterial 
numbers was observed between 2 and 4 days immersion in seawater with abalone (7.7 x lO"* cells/mm" after 4 days), and this was 
accompanied by an increase in ciliate numbers (from to 10" ciliates/mm"). Ten distinct taxonomic groups of bacteria were identified 
from the diet after immersion; Cylophaga spp. was the most abundant group. Chemotaxonomic analysis, including fatty acid profiling, 
failed to provide microbial biomass estimates or bacterial biomarkers. The majority of the microbes were found to have the capacity 
to degrade a protein and a lipid source within the diet, but not two carbohydrate sources, including the binder. Bacteria were found to 
affect the physical form of the diet, but it is unlikely that they affected its macronutritional value to any great extent. 



INTRODUCTION 

Worldwicje decline in abalone fisheries has accelerated the de- 
velopment of abalone mariculture (Coote et al. 1996). One of the 
major constraints to the industry is the provision of an economi- 
cally viable, nutritionally suitable, formulated diet (Fleming et al. 
1996). Given the high cost and logistical difficulties of supplying 
natural seaweeds to abalone (Coote et al. 1996), the industry pref- 
erence is for a cost-effective formulated diet. The development of 
such a diet depends not only on an understanding of the nutritional 
requirements of the abalone and their digestive processes, but also 
on the diet's performance in the culture system. 

Most formulated diets for fish are consumed rapidly, but, large 
industries now exist for slow-feeding invertebrates such as marine 
shrimp and marine gastropods. Abalone graze on food slowly, so 
it is not uncommon in cominercial situations for diet to be im- 
mersed for up to 4 days before it is consumed. Given the rapid 
leaching of water-soluble nutrients from formulated diets (Goldb- 
latt et al. 1979) and the ample time for microbial colonization, 
costly feeding strategies, based on frequent input, may be adopted. 
However, growth trials have suggested the opposite may actually 
be the case: faster growth rates were observed with less frequent 
feeding and cleaning intervals and therefore extended immersion 
(Maguire et al. 1996). 

When considering the effects of microbes on the nutritive value 
of formulated diets, several possibilities exist. They may have 
detrimental effects: by consuming the nutrients meant for the aba- 
lone, or decreasing the stability of the diet by facilitating its physi- 
cal breakdown and exacerbating water quality problems (Shigueno 



*Present address: Fisheries Western Australia, Research Division, PO Box 
20, North Beach, WA 6020. Australia. 



1975, Moriarty 1986). The diet may also act as a reservoir for 
pathogenic microbes (Moriarty 1986, Muir and Sutton 1994). Con- 
versely, bacteria may be beneficial by forming extracellular par- 
ticulate matter after uptake of dissolved nutrients (Pearl 1978). 
which may then become available to the abalone. Dietary constitu- 
ents may be broken down and consumed by bacteria, which may 
in turn be consumed by the abalone. Both Garland et al. ( 1985) and 
Harris (1993) state that digestive enzymes may be supplied to 
abalone by bacteria. Finally, bacteria may produce certain feed 
attractants or micronutrients that make the diet more palatable or 
nutritionally adequate (Sakata 1987, McShane et al. 1994). 

The aim of this study was to quantify the microbial population 
colonizing a formulated abalone diet after periods of extended 
immersion and to isolate and partially identify members of this 
microbial population in order to examine their exoenzyme activity 
in relation to diet constituents. This information was sought using 
three distinct methods: scanning electron microscopy (SEM) enu- 
meration, traditional biochemical methods, and chemotaxonomic 
techniques. Results from this study could allow more informed 
decisions regarding feed strategies and diet formulation. 

MATERIALS AND METHODS 



Diet 



The formulated diet was a proprietary formulation (ABCHOW) 
produced with a pasta maker and subsequently dried as a biscuit 
(14 X 9 X 1 mm). ABCHOW was supplied by the South Australian 
Research and Development Institute (SARDI), Adelaide, Australia 
and was derived from diet 9 as used by Fleming et al. ( 1996). Diet 
was stored in a domestic freezer and added to treatment tanks 
manually. 



995 



996 



BiSSETT ET AL. 



Abalone Culture Systems 

Treatment tanks comprised 70-L. round, aerated, center- 
draining, tlow-through, fiberglass aquaria supplied with approxi- 
mately 1.5 L/min of sand-filtered (40 to 50 (xm) sea water. Each 
treatment utilized three replicate tanks. Tanks were continously 
shaded in a black plastic (100% shade) enclosure to prevent ex- 
cessive benthic diatom colonization. The entire system was housed 
within a translucent fiberglass building. Light intensity was < 0.03 
microEinsteins/m". Treatment tanks using abalone contained a bio- 
mass of approximately 100 x 1 g abalone/tank. Trials were con- 
ducted at ambient conditions (typically about 14°C, pH 8.2 and 
salinity 34 to 35 ppt) at a commercial abalone farm. Marine Shell- 
fish Hatcheries P/L, Bicheno, Tasmania, Australia. Tanks were 
cleaned by rapid draining before the sample diet was added. 

Treatments 

Seven treatments were employed in this study: 

1 . Two days immersion in the presence of abalone 

2. Two days immersion, abalone absent 

3. Four days immersion in the presence of abalone 

4. Four days immersion, abalone absent 

5. Zero days immersion 

6. Two days immersion in sterile seawater 

7. Four days immersion in sterile seawater 

For treatments 1 to 4. samples were collected for both SEM 
enumeration and bacterial isolation directly from the aquaria. For 
treatments 6 and 7. 200 mL of coarse-filtered, aged seawater was 
added to 500 mL glass beakers and autocla\ed for 15 min at 
121°C, 15 psi. Diet was added aseptically, and the beakers were 
then incubated under similar conditions to those experienced by 
other treatments, except they were static. 

Bacterial Isolation and Identification 

For isolation of bacteria, five pieces of the feed were collected 
from each replicate tank or beaker. Samples were collected using 
methylated-spirit disinfected filter forceps and placed in 10 mL of 
sterile saline. They were then hand homogenized and a further 
four, tenfold serial dilutions performed. Samples from the five 
dilutions were subsequently inoculated onto general carbon source 
plates and restricted carbon source plates and incubated at room 
temperature for 1 to 14 days. Restricted carbon source plates were 
used in the study to isolate bacteria utilizing nutrients specific to 
the ABCHOW diet. The restricted carbon source plates comprised 
20 g agar, 10 g carbon source, and 500 mL each of distilled and 
filtered seawater. The nutrient source for each plate consisted of 
one of the principal ingredients of the ABCHOW diet: two carbo- 
hydrates (semolina), including the binder (sodium alginate), one 
high protein meal (casein), and one lipid (fish oil) source. Follow- 
ing incubation, plates were inspected daily for growth. Discrete 
colonies were removed and transferred to separate abalone-feed- 
nutrient-agar plates (identical to those above, substituting homog- 
enized ABCHOW diet for the individual nutrient sources) for pu- 
rification. Subculturing continued until pure cultures were ob- 
tained, and these were maintained on OrdaPs medium (Atlas 
1993). 

Identification tests performed on the pure cultures were: Gram 
reaction, cellular and colonial morphology on Ordal's medium 
after 5 days incubation; glucose utilization (OF) test for metabolic 
type using a modification of the method of BariDW and Feltham 
(1993), whereby 500 niL of filtered seawater was substituted for 



the equivalent amount of distilled water; Craigie tube motility test 
(similarly modified from Barrow and Feltham 1993); oxidase and 
catalase (Barrow and Feltham 1993); anaerobic growth (Oxoid, 
Anaerogen system) on Ordal's medium; sensitivity to the antibi- 
otic 0/I29 (Oxoid). and growth on the restricted carbon sources 
specific to the ABCHOW diet. 

SEM Enumeration 

For each replicate tank or beaker, three whole pieces of feed 
were used. These were fixed in 2.5% glutaraldehyde in 0.2M cac- 
odylate buffer (pH 7.4) containing the major marine salts (Garland 
et al. 1982) immediately upon removal from experimental tanks. 
Samples were thus fixed for 2 h at room temperature, rinsed for 20 
min in 0.1 m cacodylate buffer (Hodson and Burke 1994) and 
dehydrated through a graded ethanol (EtOH) series (Hodson and 
Burke 1994). The dehydration series was suspended at 70% EtOH 
until critical point drying was possible. 

Dehydrated samples were transferred to acetone and critical 
point dried using liquid CO,. Samples were dried using a Baltec 
CPD 030 or a Polaron E3000 CPD. Following drying, samples 
were mounted onto aluminum SEM stubs with conductive carbon 
paint as the adhesive. Samples were then sputter coated with gold 
(Balzers type coater) twice, to improve sample stability (Rosowski 
et al. 1981 ). as soon as practicable after drying. Samples were then 
stored in a vacuum desiccator (25-30 psi) over CaCI, (Garland et 
al. 1982). Samples were viewed and photographed under a Philips 
505 SEM at a voltage of 15 kV. 

Random fields on the sample surface were chosen at low mag- 
nification (Lewis et al. 1985). then examined at a magnification of 
2500 X. giving a viewing field of 1322 \x.m' (although it should be 
noted that the viewing field was not flat). The surface was focused 
and aligned at 90° to the viewing plane, and all organisms within 
the viewing field (including those that intersected the top and left 
sides) were counted. For each replicate, at least 10 full fields were 
counted. Representative micrographs were taken of each replicate. 

Data were transformed with a square root transformation 
(Sokal and Rohlf 1987) prior to statistical analysis, with a one-way 
analysis of variance ( ANOVA), to meet assumptions of normality 
and homogeneity of variance. For all tests, a significance level of 
p < .05 was adopted. Data for each immersion period were ana- 
lyzed separately, and environment (abalone present or absent or 
sterile seawater) was considered as a fixed factor. Pairs of means 
were compared using Fishers LSD (Sokal and Rohlf 1987). 

Fatty Acid Analysis 

The method of Bligh and Dyer ( 1959), as modified by Dunstan 
et al. (1995). was used for extraction. Fatty acid methyl ester 
(FAME) samples were analyzed with a Hewlett-Packard 5890 gas 
chromatograph (GC) that was equipped with a flame ionisation 
detector (FID). FAME samples were injected using an air-cooled 
on-column injection into a polar BPX-70 fused silica column (50 
m X 0.32 mm ID). High-purity H, was the carrier gas. The GC 
oven temperature was initially held at 45°C for 2 min after injec- 
tion and then increased at 30°C/min to I20°C and at 3°C/min to 
240°C, and was then held constant for 10 min. The retention index 
on both polar and nonpolar columns was used to identify fatty 
acids. Fatty acid identifications were verified with a Hewlett- 
Packard 5970B GC/MS system. 



Bacterial Colonization of Immersed ABCHOW 



997 




Figure I. A split view of the surface of diet immersed for 4 days 
without abalone. The left of the micrograph shows the interface be- 
tween areas of confluent growth and very little growth (arrows indi- 
cate boundary). The right demonstrates the density of cells within the 
area of growth. (Bar = 0.5 mm and refers to the left of the micrograph). 



RESULTS 



Enumeration 



At low magnification, tlie surface of eacli sample was observed 
to be typically undulating with irregular depressions. The inegu- 
larity of the diet's topography was accentuated with immersion 
tiine. At high magnification (2,500 X) it was possible to distin- 
guish individual bacterial cells, despite the surface corrugations 
and often dense mucilage. Cells colonized different areas of the 
diet to differing degrees; lipid globules (identified visually under 
EM) were less densely colonized than the rest of the diet matrix 
(Fig. 6). Areas of confluent growth, adjacent to areas of no growth, 
were also evident (Fig. 1 ). These areas of confluent growth seemed 



S3 ^ 

« s 




Immersion T 
(D avs) 



m e 



Figure 2. Mean bacterial numbers on ,\BCH()\V diet at (I, 2, and 4 
days immersion with abalone, without abalone, and in sterile seawater. 
Means for the same immersion time that share a common superscript 
are not significantly different (p > .05). Vertical lines represent stan- 
dard errors of the means (n = 3). 



Figure i. \ typical random counting area from unimmersed diet 
(2500 X, Bar = 10 fiml. No bacterial cells were present. 

to be the result of bacteria spreading from initial sites that were 
favorable for growth. SEM examination showed negligible bacte- 
ria colonizing treatments 5 to 7, but a substantial proliferation in 
bacterial numbers for treatments 1 to 4 (Figs. 2^). 

One-way ANOVAs, based on all treatments except 5. con- 
firmed a significant treatment effect (p < .001) on bacterial abun- 
dance for 2- and 4-day data. Comparisons of means for the same 
immersion time showed that bacterial numbers for all treatment 
levels (n = 3) (abalone absent = 5.7 x 10"* cells/mm", abalone 
present = 1.2 x 10' cells/mm", sterile = 1.5 x 10^ cells/mnr) 
were significantly different after 2 days immersion (p < .05). There 
was no significant difference between the bacterial densities for 
nonsterile treatments after 4 days, with (mean = 7.7 x 10"* cells/ 
mm-) or without (mean = 6.5 x lO"* cells/mm^) abalone. but mean 
counts from diet immersed in sterile seawater (mean = 7.0 x lO' 
cells/mm") were significantly different (p < .05) to other treat- 
ments after this immersion time. 

SEM also revealed the presence of ciliates (Fig. 5) on the diet 
subjected to treatment 3. The average number of ciliates present on 
treatment 4 diet was 0.8 ciliates/counling field. The ciliates were 
approximately 30 x 20 |jLm in size, were all of the same morpho- 
type and were observed on all replicate samples from treatment 3, 
but no other samples. 




Figure 4. A typical random counting area from diet immersed for 4 
days, without abalone. (2500 X, Bar = 10 ^m) showing massive bacte- 
rial colonization. 



998 



BiSSETT ET AL. 




Figure 5. A micrograph of an unidentifled ciliate (193000 X, bar = 10 
fim), typically observed on treatment 4. 



Identification 

A large number of bacteria (108) were isolated directly from 
agars containing ingredients of the ABCHOW diet. No bacteria 
were isolated from diet immersed in sterile seawater, because no 
colonies had formed on plates from these treatments after 14 days. 
Bacteria were identified and placed into one of 10 taxonomic 
groups (Table 1 1. Most groups were observed in all treatments; 
bacteria falling into the group Cytophaga being the most numer- 
ous. 

The identification scheme used was adopted from Cropp and 
Garland ( 1988). All isolates were Gram-negative rods, catalase {+) 
and grew aerobically on Ordal's medium. Isolates were initially 
separated on their ability to produce acid from glucose (OF test) 
and on their ability to grow anaerobically on Ordal's medium. 
Three distinct groups resulted (Table 2). The first group were able 
to produce acid from glucose aerobically, but were incapable of 
anaerobic growth on Ordal's medium. The second group did not 
produce acid from glucose aerobically or anaerobically. hence they 
were oxidative, but some were capable of anaerobic growth on 
Ordal's medium. The final group were fermentative, producing 
acid from glucose both aerobically and anaerobically and were 
capable of anaerobic growth on Ordal's medium. 

All bacteria isolated demonstrated the ability to degrade a wide 
range of carbon sources (Table 3). Results were similar for both 

TABLE 1. 

Bacterial types isolated and identified from treatments 1 to 5, 
showing the total numbers of isolates in each taxonomic group. 



Bacterial Type 



Number of 
Isolates 



Treatments 



Cytophaga 27 

Mesophilohacter or Monuella 19 

Vibrionaceae 17 

Alcaligeiies or P.seiidomoiias 12 

Enterobacteriaceae 13 

Acinetobacter 8 

Moraxella or Paracoccus 5 

Aeromunas 4 

Flavobacterium or Phenylobaclenitm 2 

AUeronumas 1 

Total 108 



1 to 5 
1 to 5 
1 to 5 

1 to 5 

2 to 5 

2 to 5 
1,2,3,5 

1,2,3 

3 &4 
4 



Figure 6. Lipid globules in the diet from 2 days immersion, with aba- 
lone (2500 X, Bar = 10 fim), showing typical sparse colonization of the 
lipids. 

carbohydrates. All isolates were able to use protein meal as a 
nutrient source. At least one isolate from each treatment was able 
to degrade either the carbohydrate or the lipid sources. At least one 
isolate from each taxonomic group m treatment 1 was able to 
utilize all nutrient sources. However, members of the families 
Vibrionaceae and Enterobacteriaceae demonstrated poor utiliza- 
tion of the binder. No Pseiidomonas or Acinetobacter spp. was 
observed to utilize the carbohydrate source. All other groups de- 
tennined to have oxidative metabolism were generally poor users 
of carbohydrate energy sources. Lipase activity was expressed by 
a large proportion of isolates of most taxonomic groups. 

Fatty Acid Analysis 

Analysis of the ABCHOW diet's fatty acid content after 0. 2. 
and 4 days immersion (with and without abalone) showed an in- 
crease in total fatty acids from 3.26 g/100 g dry wt at day 
immersion, to 4.55 g/lOOg dry wt at 4 days immersion without 
abalone (Table 4). Analysis of straight and branched-chain fatty 
acids in the ABCHOW diet revealed no change in relative com- 
position after extended immersion (Table 5. 6). 



DISCUSSION 



Enumeration 



Microorganisms are known to colonize many natural and arti- 
ficial marine surfaces, hence it is not surprising that this study 

TABLE 2. 

Metabolic types of bacteria isolated from treatments 1 to 5 as 
determined by glucose utilization (OF test! and anaerobic growth 





Oxidative 


Oxidative 




Total 




Obligate 


Facultativelv 




Number 


Treatment 


.Aerobe 


■Anaerobic 


Fermentative 


of Isolates 



I 

1 

3 

4 

5 

Total 



13(9) 


10 


23 


13(2) 


7 


20 


16(2) 


6 


22 


20(3) 


4 


25 


13(2) 


5 


18 


75(18) 


32 


108 



Figures in parentheses indicate numbers of organisms capable of anaerobic 
growth. 



Bacterial Colonization of Immersed ABCHOW 



999 



TABLE 3. 

Bacterial types isolated frum treatments 1 to 5, showing total number of Isolates in each group and the number of isolates capable of 

degrading specific nutrient sources found in the ABCHOW diet. 







Number 


Binder 






Lipid 






of 


(Sodium 


Carbohydrate 


Protein 


(Fish 


Treatment 


Bacterial Group 


Isolates 


Alginate) 


iSemolinal 


(Casein) 


Oil) 




Aeromonas spp. 


2 


1 


1 


-) 


2 




Akaligenes or Pseiidomoiun spp. 


3 


2 


1 


3 


3 




Cytophaga spp. 


4 


2 


1 


4 


4 




Mesophilohacler spp. 


4 


4 


2 


4 


4 




Moraxella or Paracoccus sp. 


1 


1 


1 


1 


1 




Pseudomonas sp. 


1 


1 


1 


1 


1 




Vibrio spp. 


4 


4 


2 


4 


4 




Vibrionaceae 


4 





1 


4 


4 


2 


Acinetobacter sp. 


1 


1 





1 


1 


2 


Aeromonas spp. 


1 





1 


1 


1 


2 


Akaligenes or Pseudomonas sp. 


1 








1 


1 


2 


Cytophaga spp. 


7 


1 


4 


7 


7 


2 


Enterobacteriaceae 


4 





1 


4 


4 


2 


Mesophilobacter or Moraxella spp. 


4 


3 


2 


4 


4 


2 


Moraxella or Paracoccus sp. 


1 





1 


1 


1 


2 


Vibrionaceae 


3 





3 


3 


3 


3 


Acinetobacter sp. 


3 


3 





3 





3 


Aeromonas spp. 


1 





1 


1 


1 


3 


Cytophaga spp. 


9 


2 


3 


9 


9 


3 


Enterobacteriaceae 


T 








1 


2 


3 


Flavobaclerium or Phenylobacterium sp. 


1 


1 





1 


1 


3 


Mesophilobacter or Moraxella spp. 


1 


1 





1 


1 


3 


Moraxella or Paracoccus sp. 


1 








1 





3 


Pseudomonas sp. 


1 


1 




1 


1 


3 


Vibrionaceae 


3 







3 


3 


4 


Acinetobacter sp. 


2 







2 


2 


4 


Alcaligenes or Pseudomonas spp. 


4 








3 


3 


4 


Alteromonas sp. 


1 







1 


1 


4 


Cytophaga spp. 


4 


1 




4 


4 


4 


Enterobacteriaceae 


4 





2 


3 


2 


4 


Flavobacterium or Phenylobacterium sp. 


1 


1 




1 


I 


4 


Flavobacterium sp. 


1 


1 




1 


1 


4 


Mesophilobacter or Moraxella spp. 


7 


1 




6 


6 


4 


Pseudomonas sp. 


1 







1 


1 


4 


Vibrionaceae 


1 







I 


1 


5 


Acinetobacter sp. 


2 








2 


1 


5 


Alcaligenes or Pseudomonas spp. 


2 


1 





2 


2 


5 


Cytophaga spp. 


6 


3 





6 


6 


5 


Enterobacteriaceae 


3 








3 


3 


5 


Mesophilobacter or Moraxella spp. 


2 


1 


1 


2 


2 


5 


Vibrio sp. 


1 


1 




I 


1 



demonstrates microbial colonization of the surface of the AB- 
CHOW diet after 2 to 4 days immersion. Microorganisms were 
clearly visible on the surface of the diet, under SEM examination, 
despite the fragility of the diet. The diet's postimmersion fragility 
was also expected, especially given dry matter loss of 2'&'7c after 48 
h immersion (Maguire 1996). This fragility and dry matter loss are 
noteworthy for two reasons, the first being the relationship be- 
tween dry matter loss and available surface area. Dry matter loss 
does not necessarily imply a decline in the surface area. The loss 
of food particles and the expansion of food particle size following 
hydration may actually increase the surface area available for mi- 
crobial colonization. This point should be considered when inter- 



preting bacterial density figures. Second, the fragility of the diet 
postimmersion presented a problem in handling for SEM exami- 
nation. It may be of benefit to stop the dehydration process at 70% 
EtOH and store the samples until ready for SEM examination. 
Samples that were viewed immediately after the dehydration and 
critical point drying processes were completed, appeared more 
stable under SEM. 

Bacterial numbers observed ranged from lO" to 10'' cells/mm". 
It is not surprising to find low bacterial numbers on samples from 
treatments 5 to 7. The diet contains very little moisture (approxi- 
mately 6%) (Maguire unpublished data) and was stored in a freezer 
before application to the culture environment. Such conditions will 



1000 



BiSSETT ET AL. 



TABLE 4. 

Total fatty acids (g/100 g \vt dry \vt) for the ABCHOW diet after 
various periods of immersion, with and without abalone (n = 3). 





Immersion 


Abalone 


Total Fativ 


Treatment 


Time (d) 


Present ( 


+)/Absent (-) 


Acids 


5 







- 


3.26 


1 


2 




+ 


3.86 


2 


2 




- 


3.98 


3 


4 




+ 


4.5.'; 


4 


4 




- 


4.44 



cause many bacteria to become donnant. if not render them unvi- 
able. The small increase in bacterial numbers over 4 days immer- 
sion in sterile seawater demonstrates the very low initial numbers 
present. 

The bacterial densities observed from treatments 1 to 4 are 
similar to those reported in the literature. Lewis et al. (1985) re- 
ported bacterial numbers in the order lO"* cells/mm' on crustose 
coralline algae, a preferred natural settlement substrate of juvenile 
abalone. The microbial community existing on natural seaweeds is 
well established and stable, although seasonal fluctuations do oc- 
cur (Lewis et al. 1985). Bacteria examined in this study have had 
only 2 to 4 days to colonize the substrate and proliferate. The fact 
that the microbial biomass is higher on the formulated diet after 4 
days than on the abalone's natural diet indicates the suitability of 
the diet as a substrate for bacteria. 

The difference between bacterial numbers observed in treat- 
ments I and 2 (p < .05) (Fig. 2) is a result of the presence of 
abalone. Bacteria are known to be associated with abalone, both 
externally and internally (Prieur et al. 1990. Harris 1993). The 
association of bacteria with abalone places more bacteria in con- 
tact with the diet than if the abalone were absent. Abalone may 
also transfer bacteria between diet pieces. Free-floating bacteria 
are reliant upon chance contact with the diet before they can locate 
a suitable substrate (via chemotaxis). Although movement of mo- 
tile bacteria is relatively fast (Schlegel 1993). their movement is 

TABLE 5. 

Distribution and total branched-chain fatty acids from the 

ABCHOW diet after various periods of immersion, with and 

without abalone. 









Immersion Time (dl 








2 


2 


4 


4 


Branched-Chain 




With 


Without 


With 


Without 


Fatty Acids 





Abalone 


Abalone 


Abalone 


Abalone 


il4:0 


0.0 


0.0 


0.0 


0.0 


0.0 


il5:0 


0.2 


0.2 


0.2 


0.2 


0.2 


a 15:0 


0.1 


0.1 


0.1 


0.1 


0.1 


il6:0 


0.1 


0.1 


0.1 


0.1 


0.1 


il7:0 


0.4 


0.4 


0.4 


0.4 


0.4 


al7:0 


0.0 


0.0 


0.0 


0.0 


0.0 


il7:l 


0.2 


0.2 


0.1 


0.1 


0.2 


il8:0 


0.5 


0.5 


0.5 


0.5 


0.5 


il8:l 


0.1 


0.1 


0.1 


0.1 


0.1 


brl9:l 


0.8 


0.7 


0.7 


0.7 


0.8 


Total 


2.4 


2.3 


2.2 


2.2 


2.4 



Figures represent branched-chain fatty acids as a percentage proportion of 
total fatty acids (n = 3). 



effectively confined to very small areas and does not play any 
major role in the distribution of bacteria over large areas. How- 
ever, the water column itself is also an important microbial source, 
as is indicated by the difference (p < .05) in bacterial numbers 
between treatments 4 and 7. This suggests that bacteria that come 
into contact with the diet are able to move, via chemotaxis. to a 
suitable substrate and proliferate. 

Bacterial numbers were not significantly different between 
treatinents 3 and 4 (p > .05 ). This apparent loss of treatment effect 
was brought about by an increase in bacterial numbers in treatment 
4 and a decrease in treatment 3 (Fig. 2). It is unlikely that the 
nutrient content of the diet would be exhausted in such a small 
sampling time, so it was expected that bacterial numbers would 
continue to increase over this period. A factor that may have 
contributed to the decline in bacterial numbers is the presence of 
protozoan ciliates (6.2 x lO'/mm") in treatment 3. Ciliates are 
known to graze heavily on bacteria; indeed, it has been shown that 
ciliates can clear approximately 10^ bacteria/ciliate/h (Iriberri et al. 
1994. Solic and Krustulovic 1994). This being the case, the ciliates 
observed on treatment 3 could be capable of consuming 10^ cells/ 
mm-/h. This figure equates to the whole standing crop of bacteria 
and thus, may explain how bacterial numbers declined in treatment 
3. The abalone themselves may also have been ingesting bacteria 
from the diet surface or disturbing surface films. 

Identification 

One of the aims of this study was to categorize, to a degree that 
allowed an assessment of metabolic activity and capacity, the bac- 
terial microflora colonizing the ABCHOW diet. The majority of 
the bacteria found on the diet demonstrated an ability to degrade a 
range of nutrient sources presented to them. 

All isolates demonstrated protease activity (Table 3). Although 
proteins are among the most expensive of the diet's components, 
their degradation may not be deleterious. Fleming et al. (1996) 
suggested that protein partially digested by bacteria may be more 
efficiently digested by abalone. It should be noted, however, that 
faster protein decomposition generally leads to water quality prob- 
lems. 

The binder was poorly utilized by many of the bacterial isolates 
(Table 3). This is surprising given that many of the binders used in 
formulated diets, for example alginate and cellulose, are readily 
available in the marine environment. If binder is not readily ac- 
cessible to the bacteria, it may be able to perform its task of 
holding water-soluble nutrients longer. 

Lipase activity was demonstrated by most of the bacterial iso- 
lates (Table 3). It is interesting to note, however, that SEM ex- 
amination of the diet revealed that lipids were not very heavily 
colonized in relation to other areas (Fig. 6). This indicates that 
although most of the isolates were able to utilize lipids, they may 
not have been the preferred nutrient source. The lack of bacterial 
colonization of lipids seen under SEM examination may be attrib- 
uted to the hydrophobic/hydrophilic interactions that must be over- 
come by the bacteria at the lipid/water interface. 

Because many of the oxidative organisms were unable to pro- 
duce acid from glucose, it is reasonable to suggest that they are 
likely to be poor users of complex carbohydrates, which are ini- 
tially hydrolyzed to glucose. No isolates identified as Pseudomo- 
nas or Acinetobacter were able to use the carbohydrate source 
supplied. Abalone consume a natural diet that is high (40-50%) in 
carbohydrates and possess many enzymes capable of carbohydrate 
hydrolysis (Fleming et al. 1996). As a result, many formulated 



Bacterial Colonization of Immersed ABCHOW 



1001 



TABLE 6. 

Straight-chain fatty acids from the ABCHOW diet after various immersidn times with and without abalone. 

Immersion Time (d) 



Straight-Chain 




2 


Fatty Acids 





With Abalone 


Saturated 






12:0 


0.1 


0.1 


14:0 


4.1 


4.2 


15:0 


0.4 


0.4 


16:0 


17.4 


17.9 


17:0 


0.2 


0.2 


18:0 


3.2 


3.2 


Total 


25.4 


26.0 


Monoenoic 






16:l(n-9) 


0.2 


0.1 


16:l(n-7) 


4.4 


4.5 


18:l(n-9) 


10.6 


10.6 


18:l(n-7) 


1.8 


1.8 


18:l(n-5) 


0.2 


0.2 


Total 


16.2 


17.2 



Without Abalone 



With Abalone 



Without Abalone 



0.1 
4.1 
0.4 

IS.O 
0.2 
3.3 

26.1 

0.1 
4.5 

10.9 
2.0 
0.2 

17.7 



0.1 
4.2 
0.4 

18.1 
0.2 
3.3 

26.3 

0.2 
4.6 

11.0 
2.0 
0.2 

17.8 



0.1 
4.2 
0.4 

18.2 
0.3 
3.3 

26.5 

0.2 
4.8 

11.0 
2.1 
0.2 

18.3 



Figures represent straight-chain fatty acids as a percentage proportion of total fatty acids (n = 3). 



abalone diets compiise up to 60% carbohydrate. The bacteria as- 
sociated with surface of abalone would also be expected to per- 
form well on carbohydrates, but this does not seem to be the ca,se. 
It is unlikely, then, that the predominantly oxidative isolates in this 
studs would have a great effect on carbohydrate availability dunng 
4 days immersion. 

Fatty Acid Analysis 

The quantitative increase in total fatty acids is thought to have 
resulted from a decrease through leaching in amounts of other food 
components. Many lipids are not water soluble, so a rise in lipid 
content and total fatty acids (g/lOO g dry wl). as water-soluble 
nutrients are lost, is expected. 

Many Gram-negative bacteria, including: Pseudomonas, Al- 
teromonas, Moraxella, Cytophaga, and Flavobactehwn (Kaneda 
1991), all of which have been isolated from the ABCHOW diet, 
contain greater than 20'7f branched-chain fatty acids. It has been 
noted, however, that branched-chain fatty acids are more common 
in Gram-positive bacteria; whereas, straight-chain fatty acids are 
more common in Gram-negative bacteria (Kaneda 1991 ). Bacterial 
growth on the diet should, then, be indicated by an increa.se in 



these fatty acids. No such change was demonstrated in the present 
study. 

SEM analysis has demonstrated clearly that bacterial coloniza- 
tion of the diet occurs after immersion. Chemotaxonomic tech- 
niques have previously been utilized for ecological studies inves- 
tigating relatively low-nutrient environments, so very small shifts 
in the fatty acid spectrum have been observable. However, the 
chemotaxonomic method chosen failed to demonstrate an obvious 
increase in microbial biomass. The most likely explanation for this 
is that it is inadequate for use on samples of high original lipid 
content. Table 5 indicates that the ABCHOW diet was relatively 
high in initial fatty acid levels. This high lipid content masks the 
presence of bacterial lipids, which make up only a very small 
percentage of total lipids. 

ACKNOWLEDGMENTS 

Thanks are due to the University of Tasmania and the Coop- 
erative Research Centre for Aquaculture for funding and facilities. 
Marine Shellfish Hatcheries Ltd., for use of their research facility, 
Stephen Hodson for SEM advice and comments, and Stephen Hin- 
drum and Deon Johns for technical assistance. 



LITERATURE CITED 



Atlas, R. M. 1993. Handbook of microbiological media. CRC Press. Lon- 
don, pp. 222. 

Barrow, G. I. & R. K. A. Feltham. 1993. Cowan and Steele's manual for 
the identification of medical bacteria. Cambridge University Press, 
Cambndge, UK. pp. 189-238. 

Bligh, E. G. & W. J. Dyer. 1959. A rapid method of total lipid extraction 
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Coote, T. A., P. W. Hone, R. Kenyon & G. B. Maguire. 1996. The effect 
of combinations of dietary calcium and phosphorus on the growth of 
juvenile Haliolis laevigata. Aquaculture 145:267-279. 

Cropp. C. M. & C. D. Garland. 1988. A scheme for the identification of 
marine bacteria. Ausl. Microhini 9:27-34. 

Dunstan, G. A.. H. J. Bailie. S. M. Barrett & J. K. Volkman. 1995. Effect 
of diet on the lipid composition of wild and cultured abalone. Aqua- 
culture 140:115-127. 



Fleming, A. E., R. J. Van Bameveld & P. W. Hone. 1996. The develop- 
ment of formulated diets for abalone: a review and future directions. 
Aquaculture 140:5-53. 

Garland, C. D., G. V. Nash & T. A. McMeekin. 1982. The preservation of 
mucus and surface associated microorganisms using acrolein vapor 
fixation. J. Microscopy 128:307-312. 

Garland, C. D., S. L. Cooke, J. F. Grant & T. A. McMeekin. 1985. Inges- 
tion of the bacteria on and the cuticle of crustose (nonarticulated) 
coralline algae by post larval and juvenile abalone (Haliotis ruber 
Leach) from Tasmanian waters. / E.xper. Mar. Biol. Ecol. 91:137-149. 

Goldblatt. M. J.. D. E. Conklin & W. D. Brown. 1979. Nutrient leaching 
from pelleted rations. Proceedings of the World Symposium on Finfish 
Nutrition and Fishfeed Technology. Hamburg. Germany. 1978. vol. 11. 
pp. 117-129. 



1002 



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Harris. J. 1993. The presence, nature, and role of gut microflora in aquatic 
invertebrates: a synthesis. Microh. Ecol. 25:195-231. 

Hodson. S. L. & C. Burke. 1994. Microfouling of salmon cage netting: a 
preliminary investigation. Biofoiilmg 8:3-105. 

Iriberri. J., B. Ayo, E. Santamaria. 1. Barcina, & J. Egea. 1994. Influence 
of bacterial density and water temperature on the grazing activity of 
two freshwater ciliates. Freshw. Biol. 33:223-231. 

Kaneda. T. 1991. Iso- and anteiso-fatty acids in bacteria: biosynthesis, 
function, and taxonomic significance. Microbiolog. Rev. 55:288-302. 

Lewis, T. E., C. D. Garland & T. A. McMeekin. 1985. The bacterial biota 
on crustose (nonarticulated) coralline algae from Tasmanian waters. 
Microh. Ecol. 11:221-230. 

Maguire, G. B. 1996. Effects of extended feed immersion, feeding fre- 
quency, and feed rate on growth of juvenile green-lip abalone Haliotis 
laevigata and on composition and microbial colonisation of a formu- 
lated diet. pp. 89-91. In: P. Hone and A. Fleming (eds.). Proceedings 
First and Second Annual Abalone Aquaculture Workshops, South Aus- 
tralian Research and Development Institute. Adelaide. SA. 

Maguire, G. B., S. Hindrum, D. R., Johns. G. A. Dunstan & M. Cropp. 
1996. Effects of tank drainage frequency on growth of juvenile greenlip 
abalone Haliotis laevigata, pp. 74-83. In: P. Hone (ed.). Proceedings of 
the Third FRDC/CRC Abalone Aquaculture Workshop. August 1996. 
Port Lincoln. South Australia. South Australian Research and Devel- 
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McShane, P. E., H. K. Gorfine & I. A. Knuckey. 1994. Factors influencing 
food selection in the abalone Haliotis nihra (Mollusca: Gastropoda). J. 
Exper. Mar. Biol. Ecol. 176:15-26. 



Moriarty, D. J. W. 1986. Bacterial productivity in ponds used for culture of 
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Interactions between bivalve molluscs and bacteria in the marine en- 
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proving the image of complex and delicate biological surfaces. 5a;ii- 
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Schlegel, H. G. 1993. General microbiology, 7th ed. Cambridge University 
Press, Cambridge UK. pp. 606. 

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Technical Promotion, Tokyo. 153 pp. 

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Jounud of SlwUfisb Research. Vol. 17. No. 4. KJO.VIOOS. 1998. 



ABUNDANCE, RECRUITMENT, AND MORTALITY OF PACIFIC LITTLENECK CLAMS 
PROTOTHACA STAMINEA AT CHUGACHIK ISLAND, ALASKA 



WILLIAM R. BECHTOL AND RICHARD L. GUSTAFSON 

Alaska Department of Fish and Game 
Division of Commercial Fisheries 
Homer. Alaska 99603 

ABSTRACT From 1992 to 1996, the Alaska Department ot Fish and Game annually surveyed the Pacific littleneck clam Pruloiluica 
shiminea (Conrad, 1857) population at Chugachik Island, Alaska. Estimates based on randomly placed survey quadrats indicated the 
population declined from 7,2 million clams in 1992 to 3.3 million clams in 1995 and increased to 5.5 million clams in 1996. Survey 
hiomass estimates similarly declined from 136,887 kg in 1992 to 65.852 kg in 1995, and then increased to 1 15,495 kg in 1996. Annual 
harvest hiomass. mean weight-at-age, and survey abundance and age composition data from 1992 to 1996 were also used as inputs in 
an age-structured model of the Chugachik clam population. The age model used independent selectivity curves to estimate age-specific 
recruitment to the fishery and the survey. Age of 505^ selectivity to the fishery ranged from 7 to 11 years, depending upon model 
emphasis of survey age composition data. This agreed well with size-at-age data, indicating few clams recruited to legal harvest size 
of 38 mm prior to age 5 and only 50% had recruited by age 7. Length-based estimates of annual recruitment to legal size ranged from 
6 to \2% of the population, averaging 10%. Greater age model emphasis on survey age data generally increased both estimated survival 
and the age when 50% of a cohort recruited into the fishery, and decreased the age when 50% of a cohort recruited into the survey. 
Age-model estimates of population abundance also varied with weighting applied to survey age composition data. Population abun- 
dance trends from the model agreed well with survey trends from 1992 to 1995. although the model usually exceeded survey estimates. 
Model estimates were 15 to 25% less than survey estimates for the 1996 population, probably because of a lag in model response to 
abundance trend changes. 

KEY WORDS: Littleneck clams, Protothacu suimmeu, age-model. Alaska 



INTRODUCTION 

Hardshell clams have long been an important component of the 
recreational and commercial fisheries in Cook Inlet. Alaska. The 
commercial fishery dates to the 1950s, when butter clams Saxido- 
mus giganleiis (Deshayes, 1839) were sold in canned and fresh 
markets. Sales of canned clams contaminated with paralytic shell- 
fish poisoning (PSP) from southeast Alaska subsequently led to a 
market collapse in the late 1950s. The commercial hardshell clam 
fishery in Cook Inlet re-emerged, targeting Pacific littleneck clams 
Protothaca staminea (Conrad, 1857) in 1986 after the Alaska De- 
partment of Environmental Conservation (DEC) certified the 
Chugachik Island beach in Kachemak Bay, Alaska, for commer- 
cial harvesting. Commercial harvests during 1986 to 1991 aver- 
aged 7.107 kg of clams annually from Chugachik Island (Fig. I). 
In addition, recreational diggers accounted for 77% of all hardshell 
clam harvests in southern Cook Inlet from 1986 to 1996. although 
beach-specific harvest information is unavailable (Scott Meyer, 
Alaska Department of Fish and Game. Homer. Alaska, unpub- 
lished data). 

The Alaska Department of Fish and Game (ADF&G) initiated 
annual surveys on Chugachik Island in 1992. because this area had 
the longest history of DEC certification for commercial clamming 
in Kachemak Bay. Average harvests increased to 14.876 kg annu- 
ally during 1992 to 1994 (Gustafson 1995). The commercial fish- 
ery was closed in 1995 and 1996 after ADF&G surveys indicated 
3 consecutive years of abundance declines. Management strategies 
for the commercial hardshell clam fishery in Kachemak Bay now 
include: minimum legal size of 38 mm ( 1.5 inches); 1 April annual 
registration deadline; fishing district segregation into two groups 
that open on alternate years; quarterly harvest allocations with 
maximum quotas for specific beaches; closures in areas of high 
recreational use; and closures during 1 November to 15 March 



when ambient air temperatures are below freezing and during 
weekends from 15 May to 15 September. 

To evaluate commercial fishery impacts on Pacific littleneck 
clams at Chugachik Island, we used survey data in a length-based 
inodel to estimate age-specific recruitment to legal size. Our field 
surveys were designed to estimate legal clam abundance in the 
study area. Most clam studies wash the removed substrate through 
screens (Paul and Feder 1973). However, because our budget and 
survey time was limited, we did not wash the removed substrate 
through screens. As a result, as the survey design developed, we 
were able to sample more quadrats during a site visit, but with the 
recognition that our survey was biased toward larger clams be- 
cause of a lack of substrate screening. To back-calculate the sub- 
legal component of the population and estimate the true clam 
population abundance, recruitment, and mortality, we developed 
an age-structured model that accommodated survey bias through 
selectivity functions. 



MATERIALS AND METHODS 



Survey Data 



Abundance, biomass. and age composition of the hardshell 
clam population at Chugachik Island in Kachemak Bay. Alaska 
were estimated from surveys conducted in May during 1992 to 
1996 (Gustafson 1995). The littleneck clam bed. defined to include 
habitat located between the -1.5 m mean low water level and the 
blue mussel Mytihis ediiliis (Linnaeus. 1 758) bed. was estimated to 
encompass 61.254 m''. Substrate in the clam bed was a mi.xture of 
1 to 8-cm coarse rock and muddy sand (Gustafson 1996). During 
tides exposing the beach to the -1.5 m mean low water level, 
substrate was removed by hand digging with a rake to a depth of 
30 cm within randomly placed quadrats measuring 0.5 m x 0.5 m. 
The number of quadrats sampled ranged annually from 12 in 1992 



1003 



1004 



Bechtol and Gustafson 



ISO 



1211 







1 — ^Hmtst 






^ SurHEJ BOITBSS 


■u 'Z 




-.- Effort ((Sggers) 





0. n in .n I ifl .n .f 



. , ..20 



Year 
Figure 1. Estimates of survey biomass and commercial fishery harvest 
and effort for Pacific littleneck clams at Chugachik Island, Alaska. 
1986 to 1996. 



to 33 in 1995 (Table 1). During substrate removal and replace- 
ment, all observed clams were retained. Clams were transported to 
the laboratory' to obtain age, weight, and length data; weighl-at-age 
was estimated from samples collected in 1997 only. Clams were 
aged by counting concentric growth rings on the external surface 
of the clamshell. The use of these rings to indicate annual growth 
in littleneck clams has been validated through mark-recapture and 
size distribution studies (Houghton 1973, Paul and Feder 1973). 
Although cautioning against the use of growth lines as annuli for 
littleneck clams in a clean-sand habitat. Peterson and Ambrose 
(1985) found that specimens deposited a single growth line over 12 
months in a muddy-sand environment, as is found at Chugachik 
Island. All age and size-at-age data used in the model were derived 
from survey samples. 

A simple random sample design was used to estimate litdeneck 
clam abundance at Chugachik Island. Abundance was calculated 
by multiplying the mean density of littleneck clams in sample 
quadrats (0.25 m") by the total area where littleneck clams were 
found. Standard variance estimates for simple random sampling 
were used to calculate the variance (Cochran 1977). The sampling 
fraction of quadrats was less than Kf at Chugachik Island. Finite 
population corrections were not included in the variance estimate 
of abundance, because finite population coiTCctions can generally 
be ignored if the sampling fraction does not exceed 5% (Cochran 
1977). 

We estimated annual fishery recruitment using length-at-age 
survey data and assuming knife-edged recruitment at the minimum 
legal size for the fishery. For the length-based model we calculated 




Mainland 



25 5 Km 



Figure 2. Study area showing sequential quadrat selection during the 
1996 survey as an example of the simple random survey design at 
Chugachik Island. Alaska. 

recruitment as differences in mean percentage legal clams by age 
class. For age class <(. the mean proportion of legal clams p„ among 
survey years was calculated from the following: 



(1) 



where l„ ,, is the number of legal clams age a clams in year y. and 
n,, , is the abundance of age a clams in year y. Mean annual rate of 
recruitment to age class a was estimated as the difference between 
the proportions of legal clams in ages a and a - I using the fol- 
lowins: 



= /'„ -/'„-! ■ 



(2) 



TABLE 1. 
Estimates of annual sur\ey abundance and length-based recruitment for Pacific littleneck clams at Chugachik Island, 1992 to 1996. 





Sample 

Quadrats 

n 


Sample 


Densities 




Annual Abundance 


Annual Recruitment 


Year 


Mean (Clams/m") 


SD 


(Clams) 


95% CI. 


(Clams) 


Percentage 


1992 


12 


117.7 




62.98 


7.207.5(12 


±2.923.9.^0 


858,548 


11.9% 


1993 


16 


89.8 




52.68 


-5.497.507 


±1.767.208 


463.089 


8.4% 


1994 


33 


79.3 




74.35 


4.888.737 


±1.791.592 


278.764 


5.7% 


1995 


35 


53.3 




38.24 


3.262.213 


±1,021.592 


369.098 


11.3% 


1996 


33 


88.2 




67.62 


5.405.201 


±1.886.510 


628.649 


1 1 .6% 


Mean 


26 


85.7 






5.252.232 




519.6.W 


9.9% 



LiTTLENECK ClAMS AT CHUGACHIK ISLAND 



1005 



The total year y recruitment R^ was the cumulative products of age 
class abundance and age-specific recruitment rates as in the fol- 
lowina: 



^('\, X "<,.,)■ 



(3) 



Age-Structured Model 



Our primary objectives in using an age-structured model were 
to estimate natural mortality, fishery selectivity, survey selectivity, 
and annual population abundance for 1992 to 1996. Information 
supplied to the age model included survey data and commercial 
harvest data. Commercial harvest weights were obtained from 
ADF&G fish tickets during 1992 to 1994 when the littleneck clam 
fishery occurred. Age-structured models that incorporate hetero- 
geneous data have been reviewed by Hilboni and Walters ( 1992). 
Megrey (1989), and Quinn and Szarzi (1993). The Chugachik 
model incorporated auxiliary information, similar to age-based 
models developed by Deriso et al. ( 1985). In our conceptual model 
of the annual cycle of events affecting Pacific littleneck clams at 
Chugachik Island (Fig. 3). age increments occur at the end of 
winter to coincide with the approximate time of annulus formation. 
The population is then subjected to age-specific mortality through 
commercial fishing, followed by natural mortality prior to again 
incrementing to the next year class. 

The Chugachik Island resource also incurs unquantified. rec- 
reational and subsistence harvests. It is likely that strong year 
classes, once recruited to legal size, are subjected to greater non- 
commercial mortality than weak year classes. Although noncom- 
mercial mortality abundance varies annually, annual mortality 
rates may be moderately stable, because the abundance removals 
from strong year classes are greater than abundance removals from 
weak year classes. Thus, the proportion of clams dying from non- 
commercial sources is assumed to be stable among years and is 
treated as natural mortality. Natural mortality is described by a 
single exponential decay function for all years and cohorts. Our 
age-structured model used a reduction equation to describe annual 



survival. The number of age-rt clams in a cohort in the spring of 
year y after winter annulus formation was the following: 



^„.i.,., = 5(N.,,, - C„,,) 



(4) 



where S is the annual survival rate, a model-estimated parameter, 
and C„ ,, is commercial fishery harvest. The population model as- 
sumes that clams from age 2 to 14 are present in the estimated 
population. Although age classes outside this range were observed 
in all Chugachik surveys, clams younger than age 2 have not 
consistently appeared in surveys, and cohorts older than age 14 are 
a minor component of the population. Age 2 recruitment in 1996 
was calculated as the median of age 2 clam abundance estimates 
for the 1992 to 1995 population years. 

Through independent logistic functions describing fishery and 
survey selectivity, the model accommodated differences among 
age compositions of the underlying population, the field surveys, 
and the commercial fisheries. Relationships between clam age and 
fishery and survey selectivity were as.sumed to be constant among 
years. Annual age compositions in the commercial fishery were 
estimated by the age-structured model, because commercial har- 
vests were not sampled. Composition of the annual commercial 
harvest/, , was estimated from an age-specific selectivity function 
.V, , and model-estimated cohort abundance usina the followina: 



L.y = — • and 



X [^<A.v] 



l-f-e' 



,pUv-al 



(5) 



where a was the age of 50% selectivity, and 3 was a steepness 
parameter. Given the fishery selectivity function and mean weight- 
at-age. we calculated the number of clams that produced the ob- 
served harvest biomass for each calendar year. 

Survey selectivity was similarly described by the following 
loeistic function. 



Annual 
Survey 




/ 


\ 






Suney 
.Abundance 



Age 2 
Recruitment 



\ 



/ 



I Expected 
_ Sur\ey 
I .Abundance 



Sur\ey 
Selectivity 



/ 



\ 



\ 



Survey Age 
Composition 



Biological Loop 
Measured 
Model Calculated 
Model Selected 

Model Tuning 



• Summer 
I Population 



Fishery 
Selectivitv 



Winter 
Survival 



I Expected Sur\ey ' 
I Composition ■ 



I Harvest 
. .\bundance I 



/ 



HanesI 

Biomass 
1992-1996 



\ 



W'eight- 
at-.Age 



Figure 3. Components of the age-structured model used to evaluate the Pacific littleneck clam population at Chugachik Island. Alaska. 



1006 



Bechtol and Gustafson 



Pa = 



1 + e* 



(6) 



where t was the age of 50% selectivity by the survey gear, and <j) 
was a steepness parameter. 

Measurement errors in each of the data sources are assumed to 
be independent. We also assume the model is correctly specified 
with respect to the amount and type of available data so that 
parameter estimates are not correlated and differences between 
model estimates and observed values are caused by measurement 
error, not errors in correctly specifying mathematical forms of the 
underlying processes. This age-.structured model was applied to a 
variety of survey and fishery size and abundance data measured in 
different units and of varying utility in identifying true parameter 
values. Nonlinear least-squares techniques were used to minimize 
sums of squares constructed with heterogeneous auxiliary data 
from the Chugachik Island population. Unlike least-squares linear 
regression, there is no rigid statistical theory underlying the pa- 
rameter estimation procedure. The rationale is that the best esti- 
mates of model parameters should provide a reasonable fit to all 
available data. In some cases, data are arc sine-transformed to 
achieve symmetric and approximately normal error distributions, 
although robustness of parameter estimates to departures from nor- 
mality is unknown (Funk 1994). However, various weighting sce- 
narios were applied to error terms from data sources to examine 
the utility of these data in the model. 

One measure of age-structured model fit was obtained by com- 
paring annual age compositions observed by the surveys to those 
estimated by the model. The sum of squares 5S2j„„,p^_„^,„ mea- 
sured the goodness of fit of the age composition of the survey and 
was computed as follows: 



55e„„-,,.v_<,,..= SE (sin"' VpI.- 



(7) 



where p^ ^ was the model estimate and p^ ^ the survey observation 
of the proportion in year v comprised by age a. To stabilize the 
variance, the age compositions were transformed by taking the arc 
sine of the square root of the proportions. The fishery age com- 
position was fit across ages 2 to 14 and years 1992 through 1996. 



The age model also minimized the sums of squares between 
model-estimated abundances and survey-estimated population 
abundances. The sum of squares was calculated by the following: 



id - 2j 



= 1W2 



„,) - in(iV,,.,„„^^,)]- 



where A', „„.,,,,> vvas the survey estimate and A', ,„,„;_., the model 
estimate of abundance in year y. We used the natural log of clams 
numbers because a log-normal error structure is commonly asso- 
ciated with abundance data (Funk 1994). 

Model sensitivity was examined through several scenarios that 
varied the emphasis, or weighting, on available data sources. In 
some cases, model runs were rejected, because they yielded unre- 
alistic results, such as the age of 50% selectivity being greater than 
15 or a population abundance estimate that was negative. 

RESULTS AND DISCUSSION 

Siiney and Length-Based Estimates 

Survey estimates showed population abundance declined from 
7.2 million clams in 1992 to 3.3 million clams in 1995 before 
staging a moderate increase to 5.4 million clams in 1996 (Table 1, 
Fig. 4). Survey biomass similarly declined from 136.887 kg in 
1992 to 65,852 kg in 1995, and then increased to 1 15,495 kg in 
1996. 

Based on size-at-age of littleneck clams among all survey 
years, the length-based model indicated that few clams recruited to 
legal size prior to age 5 (Fig. 5). Age-specific clam recruitments 
were 0.4% for age 5, 5.8% for age 6. 44.3% for age 7, 39.1% for 
age 8. 9.2% for age 9, and 1.1% for age 10. Cumulative increases 
in the legal component of the surveyed population indicated age 7 
to be the age of 50% recruitment to the commercial fisher)'. Ap- 
plication of age-specific mean recruitment to estimated annual age 
composition produced annual recruitment rates ranging from 5.7% 
to 11.9% and averaging 9.9% during 1992 to 1996 survey time 
series (Table 1 ). Annual recruitment as a percentage of the total 
abundance declined during 1992 to 1994. the years of the com- 




Figure 4. Comparison of Pacific littleneck clam abundance estimates from field surveys (thick line) and from an age-structured model with 
different weighting of the survey age composition data, Chugachik Island, .\laska, 1986 to 1996. 



LiTTLENECK CLAMS AT ChUGACHIK ISLAND 



1007 



100%- 






80%. 


/ / / '^ 


Selected 








1 r 


^- 0.1 
• ••- 2 




Percent 


l/h ' 


-o- 10 
—4- 50 






/ /' / ' 


- «- 100 




20% ■ 


/ » b ' j 






...» _yC^ O 1 






( 


) 2 4 6 8 10 12 14 16 




Clam Age (>ears) 



Figure 5. Fishery selectivity for Pacific littleneck clams calculated from a length-based model (thick line) and from an age-structured model 
applying different weights to survey age composition data. 



mercial fishery, and increased in 1995 and 1996 when the fishery 
was closed. 



Age-Structured Model Estimates 

An age-structured model was previously used to estimate sus- 
tained recreational fishery yield for Pacific razor clams Siliqiia 
panda (Dixon. 1788) in Cook Inlet (Quinn and Szarzi 1993). This 
model relied heavily on fecundity data and spawner-recruit rela- 
tionships. In contrast, our approach for Chugachik clams was more 
generic in dealing with known commercial harvests but unknown 
recreational removals to evaluate the underlying population abun- 
dance. Althouch model estimates of the Chugachik Island clam 



population varied with weighting applied to survey age composi- 
tion data, results agreed well with the 1992 to 1995 population 
decrease observed in surveys (Fig. 4). For most weighting options, 
model estimates of the population slightly exceeded survey esti- 
mates. This supports the assumption of systematic survey selec- 
tivity. Some studies both within and outside of the Cook Inlet area 
have attempted to reduce selectivity by passing the removed sub- 
strate through mesh screens to reduce the clam nondetection 
(Gustafson 1996). However, using screens also decreases the num- 
ber of sample quadrats that can be dug. Through the logistic func- 
tion, our model accommodates systematic survey selectivity that 
results from a greater sample rate but with slightly less scrutiny of 
quadrats. The primary exception to model estimates slightly ex- 
ceeding survey estimates was 1996. when all model runs suggested 



12 T 



lO-: 



2-. 



t1.0 




— •— Surv e> Selectivity 
— »— Fishery Selectivity 
— »- Annual Survival 



■+- 



-+- 



10 20 30 

Survey Age Composition Weighting 



40 



■ 0.2 



0.0 



50 



Figure 6. The effect of increased weighting of survey age composition data on the estimated annual survival and the ages of 50% selectivity in 
the commercial fishery and in abundance surveys for Pacific littleneck clams at Chugachik Island, Alaska. 



1008 



Bechtol and Gustafson 



the true population was 15 to ISVc. or up to 1.0 million clams, less 
than the survey estimate. Because the model tracks abundance 
throughout the life of a cohort from the age of recruitment to the 
survey or fishery, a long time series is typically required to follow 
changes in population trends reliably. Thus, the model may lag 
behind trend changes detected by the survey. The lack of model 
response to the 1996 increase could reflect either a response lag or 
errors in survey estimation. Interestingly, preliminary results from 
the 1997 survey showed a slight increase over the 1996 survey 
estimate. 

The age model estimated that about 50% of a cohort recruited 
into the fishery at ages ranging from 7 to 1 1 years, depending upon 
weighting applied to survey age composition data (Fig. 5). In 
general, increased weighting resulted in increases to estimated 
survival and age of 50*^ selectivity by the fishery, and decreased 
the age of 50'7f selectivity by the survey (Fig. 6). Annual survival 
generally ranged from 50 to 609c. The age of 509^ selectivity in the 
fishery exceeded that in the survey by I to 6 years, a result con- 
sistent with field observations. 

In summary, the age-structured model estimates agreed mod- 
erately well with length-based model estimates. The age model 



was found to be sensitive to starting parameters and some anoma- 
lies observed in model run results were probably attributable to 
inappropriate initial parameters. Fit of an age-structured model to 
available data may improve if: ( I ) age composition estimates of 
the commercial harvests were based on commercial fishery 
samples rather than model estimates; and (2) noncommercial har- 
vests could be separated from natural mortality. 

ACKNOWLEDGMENTS 

The comments of Steve Fried, Robert Wilbur, Tim Baker, Pat- 
rick Sullivan, and several anonymous reviewers helped clarify this 
manuscript. Individuals contributing to field data collection in- 
cluded Tom Sigurdsson. Greg Demers, Trish McNeill, Ted Otis, 
Daisy Morton, Henry Yuen, Phil Cowan, Mike Parish, Mike Hol- 
man, Tracy Gotthardt. Nicky Szarzi, and Scott Meyer. The ASA 
mode! from which this clam model was derived was originally 
developed by Fritz Funk as a spreadsheet application for Pacific 
herring. This manuscript is contribution PP-165 of the Alaska 
Department of Fish and Game. Commercial Fisheries Division, 
Juneau, Alaska. 



LITERATURE CITED 



Cochran. W. G. l')77. Sampling techniques. 3rd ed. John Wiley & Sons, 
New York. 

Deriso. R. B., T. J. Quinn, II & P. R. Neal. 1985. Catch-age analysis with 
auxiliary information. Can. J. Fish. Aqual. Sci. 42:8215-824. 

Funk, F. 1994. Forecast of the Pacific herring biomass in Prince William 
Sound, Alaska, 1993. Alaska Department of Fish and Game, Commer- 
cial Fisheries Management and Development Division. Regional Infor- 
mation Rep. 5J94-04. Juneau. Alaska. 

Gustafson. R. 1995. Kachemak Bay littleneck clam assessinents, 1990- 
1994. Alaska Department of Fish and Game, Commercial Fisheries 
Management and Development Division, Regional Information Report 
2A95-19, Anchorage. Alaska. 

Gustafson. R. 1996. Kachemak Bay littleneck clam assessments. 1995. 
Alaska Department of Fish and Game. Commercial Fisheries Manage- 
ment and Development Division, Regional Information Rep. 2A96-12. 
Anchorage. Alaska. 

Hilborn. R. & C. J. Walters. 1992. Quantitative fisheries stock assessment: 
choice, dynamics, and uncertainty. Chapman & Hall. New York. 



Houghton. J. P. 1973. The intertidal ecology of Kiket Island. Washington, 
with emphasis on age and growth of Pioiorluica staininen and Saxido- 
niis giganteus (Lamellibranchia: Veneridae). Ph.D. thesis. University 
of Washington, Seattle, WA. 

Megrey. B. A. 1989. Review and comparison of age-structured stock as- 
sessment models from theoretical and applied points of view. Am. Fish. 
Soc. Syinp. 6:8^8. 

Paul. A. J. & H. M. Feder. 1973. Growth, recruitment, and distribution of 
the littleneck clam. Protothaca shiinineu. in Galena Bay. Pnnce Wil- 
liam Sound. Alaska. Fish. Bull 7\:6b5-6n. 

Peterson, C. H. & W. G. Ambrose, Jr. 1985. Potential habitat dependence 
in deposition rate of presumptive annual lines in shells of the bivalve 
Protothaca staminae. Lethaia 18:257-260. 

Quinn, T. J., n & N. J. Szarzi. 1993. Determination of sustained yield in 
Alaska's recreational fisheries, pp. 61-84. In: Proceedings of the In- 
ternational Symposium on Management Strategies for Exploited Fish 
Populations. Alaska Sea Grant Rep. 93-02. University of Alaska, Fair- 
banks. 



Journal of Shellfish Research. Vol. 17. Nii. 4, 11)04-11)1.^, 1998. 

REPRODUCTIVE CYCLE OF THE GIANT REEF CLAM PERIGLYPTA MULTICOSTATA 
(SOWERBY, 1835) (PELECYPODA: VENERIDAE) AT ISLA ESPIRITU SANTO, BAJA 

CALIFORNIA SUR, MEXICO 



FEDERICO GARCIA-DOMINGUEZ, 
BERTHA PATRICIA CEBALLOS-VAZQUEZ, 
MARCIAL VILLALEJO-FUERTE. AND 
MARCIAL ARELLANO-MARTINEZ 

Ccnlro IiUcrdiscipUnario de Ciencias Marinas 
liisliliilo Polileciiico Nacional 
La Paz. BCS 23000. Mexico 

ABSTRACT The reproductive cycle of Perii;hpta muliieostata (S.). was studied at Isla Espiritu Santo, Gulf of California. Mexico, 
from October 1992 to December 1993. The reproductive activity was present throughout the study period, except in February 1993, 
nevertheless a distinct seasonality was ob.served with three distinct peaks of spawning activity. A clear relationship between spawning 
and temperature or photosynthetic pigments concentration was not observed. Spawning occurs all year, but at a lower rate in the months 
with the lowest water temperatures. 

KEY WORDS: Reproductive cycle, gametogenesis, bivalves, Veneridae. Perighpta 



INTRODUCTION 

The giant reef clam, Periglypta multicostata (Sowerby, 1835), 
is the heaviest if not the largest of the Panamic members of the 
family Veneridae. They inhabit the sand ainong rocks at extreme 
low tide from Gulf of California to Peru (Keen 1971 ). This species 
is a dominant cotnponent of the zone of the coral Pocillopora 
elegans in the rocky substrata communities of Isla Espiritu Santo 
and coexists with three bivalve species. Veiuricokiria isocardia. 
Megapitaria uurantiaca. and Chione tumens. 

In Baja California Sur, wild stocks of giant reef clams remain 
practically untouched and are considered as a potential fisheries 
resource; however, aquaculture is not recommended (Baqueiro 
1989). No research has been done on the biology or life history of 
this species. Clams are gathered by free diving and are dug out 
with hands, knifes, or forks. 

The synchronization of reproductive activity in local popula- 
tions is very important for successful fertilization. Reproduction 
seems to be cyclic, with events coordinated on an annual cycle 
(Eversole 1989). Environmental factors may intluetice the timing 
of reproduction in clams. The most commonly cited are food avail- 
ability and temperature (Bayne and Newell 1983. MacDonald and 
Thompson 1985. Jaramillo et al, 1993). The water temperature and 
its variation with latitude is used by many authors to attempt to 
explain reproductive timing in bivalves (Newell et al. 1982. 
Lozada and Bustos 1984. Manzi et al, 1985, Maiachowski 1988, 
Hesselman et al. 1989. Garci'a-Domi'nguez et al, 1993), The food 
availability is used to attempt to explain spawning timing in bi- 
valves in the sense of that if the spawning coincides with the 
highest food availability, this enables the larvae to exploit the 
phytoplankton bloom (Jaramillo et al, 1993, Villalejo-Fuerte et al, 
1996a), 

The lack of biological information for proper management has 
led to overexploitation and misuse of stocks. It is essential to know 
the life cycle of the target species, and documentation of the re- 
productive cycle in a fishery is one necessary step in determining 
when recruitment might occur. This study describes the reproduc- 
tive cycle and the spawning season of P. multicostata in relation to 
the temperature and food availability. 



MATERIAL AND METHODS 

Monthly, 20 to 25 specimens of a wild and unexploited popu- 
lation of P. tnidticoslaia were collected from October 1992 to 
December 1993 at Isla Espiritu Santo, Bahia de La Paz. Gulf of 
California. Mexico ( 1 10°24'27"W. 24''28'54"N) by a scuba diver 
at 3- to 6-m depth, A total of 310 organisms were captured. When 
the biological samples were collected, water temperature was re- 
corded. The photosynthetic pigment concentration (mg chloro- 
phyll/m') in Bahi'a de La Paz. Gulf of California was obtained 
from satellite-derived information (Trant et al. 1993). this was 
considered to be an estimation of the food availability for the 
clams. 

Mantle, adductor muscles, gills, labial palps, and siphons were 
removed, keeping only the visceral inass (gonad, liver, and gas- 
trointestinal tract) and the foot. These tissues were fixed in buff- 
ered lO^f formalin, A slice of tissue of each clam was obtained 
from the dorsal area of the visceral mass and embedded in paraffin. 
Sections 7- to 9-[j,m thick were stained with hematoxylin and eosin 
(Luna 1968). This method was adopted after verifying, in 25 speci- 
mens, that gonadal maturity was uniform in different parts of 
gonad. 

The reproductive process (either spemiatogenesis or oogenesis) 
of P. multicostata was categorized in five stages based solely on 
morphological observations, characterized by the structure of the 
gonad, presence, absence, and quantity and development of ga- 
metes (Table 1. Figs. 1. 2. 3). In all the stages of gonadal devel- 
opment, phagocytes were present in varying proportions. 

Individuals were sexed by microscopic examination of histo- 
logical slides. Sex ratios were analyzed with chi-square to test the 
significance of the deviation from the expected sex ratio of 1 : 1 , for 
the total sample. The indifferent stage clams \vere not considered. 

Mean oocyte diameters, and their standard deviation, of six 
females selected randomly per month were determined from his- 
tological sections using an eyepiece graticule calibrated with a 
stage micrometer. At least 100 oocytes sectioned through the 
nucleus (i.e,. near the maximum diameter) per individual were 
measured along the longest axis. Individuals with few measurable 
oocytes and extensive phagocytosis ( "spent" specimens) were not 



1009 



1010 



Garci'a-Dominguez et al. 



TABLE 1. 
Developmental Stages of P. multicostata Gonads. 



Maturity Stage 



Female 



Male 



Indifferent Characterized by presence of acinis with total absence 

tissue is abundant. 
Developing Oocytes inside of follicles are conspicuous, 

young oocytes with pear shape growing 

attached to folicular walls. The area of 

connective tissue decreasing. 
Ripe Free large oocytes present in the lumen with 

maximum size, few oocytes with pear shape 

attached to folicular walls. Connective tissue 

absent. 
Partially spawned Follicles containing some oocytes and large 

spaces, while others were empty. Some 

connective tissue visible. 
Spent Few residual oocytes, being phagocytized by 

amebocvtes. No evidence of active oosenesis. 



of gametes. It is not possible to distinguish the sex. The connective 

A variable quantity of germinal cells and 

spermatozoa were present inside the follicles. The 
area of connective tissue decreasing. 

Follicles filled with spermatozoa. Other 

spermatogenic cells restricted to a thick layer on the 
folicular walls. Connective tissue absent. 

Follicles partially empty. A marked decrease in the 

number of spermatozoa filling the lumen. Some 

connective tissue visible. 
Follicles collapsed, amebocytes phagocytizing 

residual spermatozoa. No evidence of active 

spermatogenesis. 



considered, using the criteria of Gratit and Tyler ( 1983a) and Grant 
and Tyler (1983b). 

RESULTS 

Spawning activity was present throughout the study period, 
except in February 1993 (Fig. 4). Nevertheless, there seems to be 
a distinct seasonality in the reproductive cycle, because P. multi- 
costata has fluctuations in its reproductive intensity, showing three 
distinct peaks of spawning activity during the study period: Octo- 
ber to December 1992, July to September 1993, and November to 
December 1993. 

Giant reef clams in the indifferent stage were observed every 
month except October 1992. Developing clams were present over 
the year except in February, September, November, and December 
1993. The highest frequency of ripe organisms was present in 
October 1992. March, June to September, and November 1993. 
Partially spawned individuals were observed all year, except in 





Vl'^'K 



Figure 1. Indifferent stage; scale bar = 5(1 nm. 



February. The highest frequency of partially spawned individuals 
was observed from June to September. The spent stage was re- 
corded throughout the year, except in April. 

Of 310 ciatns examined. 149 (48.1%) were females and 83 
(26,8%) males. The remaining 78 (25,1%) were undifferentiated. 
The sex ratio of the sexed sample (1,8 F: 1 m, n = 232) differs 
significantly (p «,05) from the expected ratio of 1:1. 

During the year, the mean oocyte diameter was > 47 |jim, 
except in October, when it was 41 \x.m (Fig. 5). In February, all the 
clams were spent and indifferent, so there were no oocytes. The 
pattern observed in oocyte diameters was consistent with the his- 
tological observations, which suggests spawning throughout the 
year, with the exception of February 1993. The standard deviations 
were wide in all months, indicating the presence of both small and 
large oocytes characteristics of ripe and spawning stages. 

The water tetnperature during the study period varied from 
22°C to 31°C. The highest values were in October 1992 and Au- 
gust 1993. and the lowest values were in February and March 1993 
(Fig. 5). 

Photosynthelic pigment concentration (mg chlorophyll/m'') in 
Bahi'a de La Paz was greater in the colder months than in the 
wanner ones. The maxiiTiuin value was in January (4.87 mg chlo- 
rophyll/m''). and the minimum was in Septetnber (1.36 mg chlo- 
rophyll/in'') (Fig. 5). 

DISCUSSION 

The annual reproductive cycle of P. midticoslata at Isla Espi'ritu 
Santo showed seasonality, with a protracted period of reproduction 
indicated by the consistent presence of spawning activity through- 
out the study period, with the exception of February 1993. Other 
species of bivalves, abundant in this locality, such as Megapitaria 
iiiirantiaca (Garci'a-Dominguez et al. 1994) or Pinctada mazat- 
Uiiiica (Garcia-Dominguez et al. 1996), have no .sea.sonal repro- 
dtictive cycles, and their spawning activity is continuous. In other 
bivalves, such as Meicenaria spp.. the spawning is essentially 
continuous in lower latitudes, but there are still cycles (Hesselman 
et al. 1989). 

Among other venerid clams from other localities along the 
Mexican Pacific coast, several other species lack distinct seasonal 



Reprodlictive Cycle of Periglypta multicostata 



1011 



<£f^' 






^^ 



,-^^**^<* 




r 








Figure 2. Photomicrographs of gonadal stages of the fetnale giant reef clam, P. multicostata. (A) developing stage, (B) ripe stage, (C) partially 
spawned stage, and (D) spent stage; scale bar = 50 pm. 













Figure 3. Photomicrographs of gonadal stages of the male giant reef clam, P. multicostata. (Ai developing stage, (B) ripe stage, (C) partially 
spawned stage, and (D) spent stage; scale bar = 50 pm. 



1012 



Garci'a-Dominguez et al. 



100 




N D J F 


M 


A 


M 


J J 


1992 








1993 


□ INDIFFERENT 




DEVELOPING 



RIPE 



I PART SPAWN □ SPENT 



Figure 4. Reproductive cycle of P. multicostata at Isla Espi'ritu Santo. 
Relative frequency of gonadal stages between October 1992 and De- 
cember 1993. Observations of males and females are combined. 

reproductive cycles; Megapitaria aurantiaca. M. sqiialida. 
Dosinia ponderosa (Baqueiro and Stuardo 1977). Chione unda- 
tella (Baqueiro and Masso 1988). and M. sqiialida (Villalejo- 
Fuerte et al. 1996b). Other bivalves, such as Meixenaria merce- 
naria. displayed a synchronized polymodal breeding pattern, al- 
though not every year. In some years, it is polymodal and in others, 
it is bimodal with continuous spawning (Hetfeman et al. 19891. 

The sex ratio of the sample differs significantly from the ex- 
pected ratio of 1:1, with females being dominant, which suggests 
females outnumbered males in the population. The same was ob- 
served for the pearl oyster P. mazatkinka in the same locality 
(Garcia-Dominguez et al. 1996). this condition may be related to 
the fact that P. mazatlanica is a protandric hermaphrodite (Sevilla 
1969, Saucedo and Monteforte 1994. Garcia-Dominguez et al. 
1996). However, in the case of P. multicostata. evidence of he- 
maphroditism was not observed. Also, the fact that within the 
population as a whole, the majority are females is considered 
typical of freshwater and brackish water bivalves (Morton 1985). 

Oocyte diameters reflect the gametogenic cycle, thus minimum 
diameters coincide with the developing stage, and maximum di- 
ameters coincide with the mature and partially spawned stages. 
This pattern is similar for other species such as Argopecten ciirii- 
laris. Glycynieris gigantea, and Laevicardium elatum (Villalejo- 
Fuerte and Ochoa-Baez 1993. Villalejo-Fuerte et al. 1995. Villa- 
lejo-Fuerte et al. 1996a). 

MacDonald and Thompson (1985) suggested that bivalve ga- 
mete production is strongly influenced by such environmental fac- 
tors as temperature and food availability set in a seasonal context. 
The reproductive cycle of Periglypta multicostata was not clearly 
related to the water temperature. The same has been observed for 
such other venerid clams as Megapitaria aurantiaca. M. squalida. 
and D. ponderosa from Bahi'a Zihuatanejo (Baqueiro and Stuardo 
1977), M. aurantiaca from Isla Espi'ritu Santo (Garcia-Dominguez 
et al. 1994). and in other bivalves such as Pinctada mazatlanica 
(Garci'a-Dominguez et al. 1996) from Isla Espiritu Santo. The re- 
lation between the temperature and spawning of other bivalves of 
the Mexican Pacific coast has been well documented in several 
species; Modiolus capax (Garza-Aguirre and BUckle-Rami'rez 
1989), Chione californiensis (Garcia-Dominguez et al. 1993). Gly- 
cymeris gigantea (Villalejo-Fuerte et al. 1995), and Laevicardium 



elatum (Villalejo-Fuerte et al. 1996a). Accordingly with Sastry 
(1970). although temperature affects reproduction, other environ- 
mental factors seem to interact with it in determining the pattern of 
annual gonad activity in a given geographical area. It is likely the 
variation in annual reproductive activity of a species will be the 
phenotypic response of a single genotype. 

Food availability has been related to the timing of reproduction 
in some bivalves (Sastry 1979, Bayne and Newell 1983, Mac- 
Donald and Thompson 1985, Jaramillo et al. 1993). For example, 
in Chlamys amandi. the spawning time seemed to be related to 
food availability (Jaramillo et al. 1993); whereas. Hinnites gigan- 
teus showed no correlation between food availability and spawn- 
ing (Malachowski 1988). The reproductive cycle of P. multicos- 
tata at Isla Espi'ritu Santo did not exhibit a clear relation with food 
availability, because the spawning extends all year, independent of 
food availability, expressed as photosynthetic pigment concentra- 
tion. In Pinctada mazatlanica from the same location, maximum 
spawning did not coincide with maximum food availability (Gar- 
cia-Dominguez et al. 1996). 

Although, spawning of P. multicostata did not seem to be re- 
lated to temperature or food availability, its spawning may be 
triggered by other factors, such as day length, a particular lunar 



E 
^ 

tu 
\~ 
tu 

< 

Q 

tu 

I- 
> 

o 
o 
o 



80 



70 . 
60 . 
50 . 
40 
30 
20 





■ ' ■ ■ 



o 

tu 
en 

< 
tr 

LU 26 

Q. 

LU 24 

I- 

22 



^t5 




■ ■ ■ ■ 



J ' 




O N D J 
1992 



M A M J J 
1993 



O N D 



Figure 5. Mean oocyte diameters of P. multicostata (bars = standard 
deviation), water temperature, and photosynthetic pigment concentra- 
tion from Isla Espiritu Santo, BCS, Mexico. 



Reproductive Cycle of Periglypta multicostata 



1013 



phase, salinity fluctuations, tidal cycle, or a combination of se\ eral 
of these. Unfortunately, in this study did not consider these other 
environmental factors. 

ACKNOWLEDGMENTS 

Our gratitude to the Direccion de Estudios de Posgrado e In- 
vestigacion del Instituto Politecnico Nacional (IPN), who gave us 



the funds for this work, to Ciro Arista. Arturo Tripp Q.. and Jose 
Luis Castro O. for their help in collecting samples, and Dr. Ellis 
Glazier for his editorial help on the English manuscript. We ac- 
knowledge the fellowships of Comision de Operacion y Fomento 
de actividades Academicas del IPN to F. Garcia-Dominguez and 
M. Villalejo-Fuerte. F. Garci'a-Domi'nguez is a Ph.D. student from 
PICP of the Universidad de Colima. Mexico. 



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perature and photoperiod. in Bahi'a Concepcion. B.C.S.. Mexico. Cien- 
cias Marinas 19:181-202. 

Villalejo-Puerte. M.. p. Garcia-Dominguez & R. I. Ochoa-Baez. 1995. Re- 
productive cycle of Glycxmeris gigantea (Reeve. 1843) (Bi\alvia: Gly- 
cymendidae) in Bahia Concepcion. Baja California Sur. Mexico. Ve- 
/iger 38:126-132. 

Villalejo-Fuerte. M., B. P. Ceballos-Vazquez & F. Garcia-Dominguez. 
1996a. Reproductive cycle of Laevicardium elatmn (Sowerby. 1833) 
(Bivalvia: Cardiidae) in Bahi'a Concepcion, Baja California Sur, 
Mexico. ,/. Shellfish Res. 15:741-745. 

Villalejo-Fuerte. M., G. Garci'a-Melgar, R. I. Ochoa & A. Garci'a-Gasca. 
1996b. Ciclo reproductivo de Megapitaria squalida (Sowerby, 1835) 
(Bivalvia: Veneridae) en Bahia Concepcion, Baja California Sur, 
Mexico. Boletin Cientifico (Santa Fe de Bogota) 4:29-39. 



Journal of Shellfish Research. Vol. 17, No. 4. 1015-1036. 1998. 



THE EVOLUTION OF A MUNICIPAL QUAHOG (HARDCLAM), MERCENARIA MERCENARIA 
MANAGEMENT PROGRAM, A 20- YEAR HISTORY: 1975-1995 



SANDRA L. MACFARLANE 

Town of Orleans ConseiTcition Department 
Orleans. MA 02653 

ABSTRACT Local municipal control of shellfisheries has been in existence in Massachusetts since 1942. The shellfish management 
programs of Orleans, MA. a town located at the elbow of Cape Cod. have evolved from transplants of native stock to use of 
hatchery-raised seed. For the period 1975 to 1995, the town utilized several forms of nursery culture in three separate estuaries 
including bottom culture, raft culture, a municipal hatchery, a land-based upweller system, tidal upweller. and floating trays. Financial 
constraints, as well as political and social perceptions determined the extent of the program at any given time. Management decisions 
were based primarily on survival of the seed rather than such factors as fast growth. The most successful method was a land-based 
upweller system with which we raised 1 million seed per year at 95% survival that were transplanted throughout the town. Survival 
in the field was directly related to water temperature at time of planting, which was most successful when water temperature was about 
45=F(7=Cl. 



KEY WORDS: Quahog. hard clam seed. Mercenaria mer 



xenana, si 



hellfish management, nursery culture, aquaculture 



INTRODUCTION 

Efforts to observe growth of northern quahogs. also known as 
hard clams (Mercenaria mercenaria Linne) or to increase the natu- 
ral production have been attempted since the early part of this 
century. Belding (19121 described both bottom culture and off- 
bottom methods used in four separate locations in Massachusetts. 
Haskin ( 1952). Carriker ( 1959), and Caniker ( 1961 ) added data to 
our understanding of environmental aspects of water and sediment 
that increase quahog growth, survival, and abundance. 

Larval culture began with Wells, who cultured five molluscan 
species through metamorphosis and patented his methods in 1933 
(Manzi and Castagna 1989). Loosanoff and his colleagues at the 
Bureau of Commercial Fisheries Laboratory in Milford. Connecti- 
cut (now National Marine Fisheries Service Laboratory) are cred- 
ited with numerous developments in rearing of bivalve moUusks 
from spawning through the juvenile stage (Loosanoff and Davis 
1950), (Loosanoff and Davis 1951). and (Loosanoff and Davis 
1963). Once larvae and juveniles were readily available, knowl- 
edge of quahog culture expanded enormously (Judson et al. 1977. 
Manzi and Castagna 1989. Rice, 1992). 

As a direct result of the culture efforts, entrepreneurs developed 
commercial hatcheries and field grow-out businesses. Towns in 
Massachusetts that manage their own shellfish resources pur- 
chased seed from hatcheries. Municipal shellfish programs, such 
as those on Cape Cod and Martha's Vineyard. Massachusetts, 
utilized this source of hatchery seed to develop their propagation 
schemes. 

George Souza. Shellfish Constable of Falmouth. MA was the 
first shellfish officer to take advantage of these seed. Working 
with staff biologists from the Massachusetts Division of Marine 
Fisheries, in 1972, he developed an off-bottom culture system for 
raising small seed to be planted in the wild. By 1977. eight Cape 
Cod towns: Bourne. Barnstable. Chatham. Dennis. Eastham. Or- 
leans. Wellfleet. and Yarmouth and the Martha's Vineyard Shell- 
fish Group followed his example. Information was shared among 
the towns through workshops sponsored by the Division of Ma- 
rine Fisheries. The focus of this paper is to describe the munici- 
pal propagation efforts that took place in Orleans from 1975 to 
1997. 



Municipal Management 

Massachusetts is one of the few states where municipal man- 
agement of shellfish resources is the norm. Towns are encouraged 
to promote and protect those resources under broad guidelines set 
by the Commonwealth. Towns appoint a shellfish constable and 
staff, promulgate and enforce local regulations, issue harvest li- 
censes, determine harvesting techniques, determine areas suitable 
for harvest, conduct experiments in propagation, and issue licenses 
for private aquaculture. The state sets size limits, determines the 
species to be regulated, administers all aspects of contaminated 
areas, sets maxitnum fees for shellfish leases, and surveys potential 
lease sites for suitability and compliance with state law. 

Belding (1912) examined many aspects of quahog life history 
and concluded that, without culturing this animal, the fishery 
would collapse. 

Over half a century later, in 1975. the town of Orleans pur- 
chased 10.000 seed quahogs from Coastal Zone Resources, a 
hatchery in North Carolina to begin propagation experiments. Our 
primary objectives were to determine: 

1. if hatchery-raised seed would survive transplant into a pro- 
tected environment; 

2. if seed would survive and grow under varying environmen- 
tal conditions; 

3. limiting factors of seed growth, including density; 

4. if seed quahogs would survive in areas devoid of native 
stock: and 

5. predators and estimate loss. 

Initial field trials were somewhat successful, and we continued 
and expanded the program throughout the next 14 years. During 
this period, we developed a number of secondary objectives. These 
were: 

6. to monitor transplants and determine survival; 

7. to find economical methods for growing seed; 

8. to produce as many quahogs as funds would allow; and 

9. to increase quahog stocks in areas of Pleasant Bay that had 
been marginally productive for 20 or more years. 

The program is presented chronologically. This emphasizes the 
transition as we built on success, learned from mistakes, and ex- 
perimented with alternatives. Each phase of the program was gov- 



1015 



1016 



Macfarlane 



emed by logic, financial constraints, and management principles. 
Much of the discussion is based on the observations of the author, 
only some of which were quantified. The program is divided into 
eight separate sections as follows: 

1. bottom culture; 

2. raft (off-bottom) culture; 

3. hatchery; 

4. upweller; 

5. transplants to the natural environment; 

6. changes in direction; 

7. private aquaculture; and 

8. budget constraints. 

Methods for evaluation varied according to the specific propa- 
gation culture used at the time. Volumetric counts were made for 
each shipment of seed delivered. Square foot samples were dug 
with bottom culture, and survival and growth were estimated; 
samples of seed (appro.ximately 100 randomly gathered) from the 
rafts were measured for growth, and the entire harvest was volu- 
metrically counted to arrive at survival percentages; and volumet- 
ric counts were made from the upweller technique. Lack of tech- 
nical staff and a desire to transplant as soon as possible from 
harvest limited our ability for more detailed statistical analysis. 
Seed were measured in metric units; construction details are dealt 
with in standard units. 

Study Area 

Orleans is located at the "elbow" of Cape Cod, MA (Figs. 1 
and 2). It has three separate embayments within its boundaries; 
Cape Cod Bay, Nauset/Town Cove, and Pleasant Bay. Although 
different from one another, all support, or historically have sup- 
ported, natural populations of quahogs. 

The Orleans municipal jurisdiction in Cape Cod Bay extends 
six miles (9.7 km) from shore and includes approximately \.5 
miles (2.4 km) of intertidal sand flats that are nonproductive for 



quahogs because of harsh environmental conditions, including 
heavy ice buildup and shifting sands. The depth offshore ranges 
frotn to 25-1- feet (7.5 ml. Abundant beds of quahogs have his- 
torically been found in much of the deeper waters of the bay and 
are still harvested commercially by a few medium-sized (35 ft, 
10.5 m) draggers. 

The Nauset estuary (2.333 acre) is a very productive area (Ro- 
man et al. 1989), shared by the towns of Orleans and Eastham and 
protected from the Atlantic Ocean by a migrating barrier beach. 
Approximately 1,150 acres are in Orleans, but Orleans and East- 
ham share a reciprocal fishing agreement. Quahogs are found 
along the edge of the Town Cove, in eelgrass (Zoslera marina), 
sand/silt/mud combinations, and along the steep gradient that leads 
to deeper water (6-18 ft; 2-6 m), as well as many parts of Nauset 
Harbor. They are harvested by hand scratchers or bull rakes. 

Pleasant Bay. a larger estuary, is shared with Chatham. Har- 
wich, and Brewster (no reciprocal fishing agreements); 3.500 acres 
are within the boundary of Orleans. A migrating barrier beach 
extending approximately 12 miles (19 km) to Chatham Inlet pro- 
tects the bay from the Atlantic Ocean. Of the three estuaries. 
Pleasant Bay has had the least stable quahog population (based on 
landings). This may result from the barrier beach dynamics. A 
large set of seed (less than 2" legal size. [50 mm] in longest 
diameter) was discovered in Pleasant Bay in the late 1950s. Gates 
(1964) conducted a survey of Big Bay. making 33 samples in 27 
acres, and found up to 1 80/0.3 m" with an average density of about 
50/0.3 m". He estimated a standing crop of 60.7 million animals 
worth $1,026 million at that time, providing a steady supply of 
shellfish and employment. They were well known in the market- 
place for their long shelf life and could be easily identified as 
originating from Pleasant Bay. because their growth rings were 
almost indistinguishable from one another. The population lasted 
until the early 1970s, at which time, quahogs became rare in the 
bay. with no recurring set. 




Figure I. Locus map of area, 
Cod. 



Cape Cod is the easternmost land mass of Massachusetts. The Town of Orleans is situated at the "elbow" of Cape 



A Municipal Quahog (Mercenaria mercenaia) Management Program 



1017 




Figure 2. Map of Orleans, Cape Cod Bay, Nauset/Town Cove and Pleasant Bay. 



BOTTOM CULTURE 

We used bottom culture of seed for 3 years at a total o'i 20 
locations (Fig. 3). Three different enclosure designs were deployed 
in all three estuaries. 



1975 



We chose 10 separate locations, with varying sediment, current, 
and other environmental conditions, to plant the 10,000 seed qua- 
hogs, approximately 8-mm in size. Frames, 3' x 6' were con- 
structed of 1" X 2" '"strapping" on end to which a 3/16" mesh 
netting was stapled. Each intertidal area was raked to loosen the 
substrate and remove any visible predators, and the seed was 
broadcast within the raked area (Fig. 4). The frame was placed 
over the seed with the edges buried and was secured by stakes at 
the corners and attached to the frames. 

Results 

Table 1 provides details of the experimental planting for 1975. 
We considered survival above 90% to be excellent, 75 to 907r very 



good, 50 to 75% good, 25 to 50% poor, and less than 25% very 
poor. 

With the exception of the frame at Namequoit Point, which was 
lost in a storm, survival in the summer was excellent. Quahogs 
grew from 8 mm in July to 1 1 to 18 mm in October, depending, in 
part, on location. The seed at Snow Shore exhibited the least 
growth. Native stock in the general vicinity also had slow growth 
(and unusually thick shells, often associated with slow growth). 

Survival after the winter was excellent in two locations, very 
good at two, and disappointing in five locations where the survival 
was poor or very poor, despite an unusually mild winter. Most of 
the stock at Snow Shore and Meetinghouse River and half from 
Asa's Landing and the Yacht Club had died. The frame at Skaket 
had disappeared. We observed no correlation between sediment 
type or specific estuary and loss. In the spring at Asa's Landing, 
we observed live animals interspersed with empty shells in the top 
25 mm of substrate. Below them, at around 33 mm, patches of 
black empty shells were found in black sulfurous smelling sand, 
adjacent to live seed in nonblack sediment. This initial observation 
was seen throughout the years in relation to the phenomenon 
known as "winter kill." 



1018 



Macfarlane 



ORLEANS SEED BOTTOM CULTURE 
QUAHAUGS 




Figure 3. Bottom culture experiments were conducted in 20 separate locations in all three estuaries over a 3-year period. 



1976 

In 1 976, we expanded the program and purchased 280,000 seed 
from two sources: 150,000 from Coastal Zone Resources and 
130,000 from ARC (Aquacultural Resources Corp.). a local hatch- 
ery. Nearly 150.000 were used for intertidal bottom culture. A 
portion. 30,000, were large enough to plant directly into the natural 
environment, and the remaining 100.000 were grown in floating 
rafts (see next section). 

The bottom culture enclosures were larger. 9' x 6' constructed 
of 1" X 6" wooden boards. Each box was divided longitudinally for 
strength. We added netting to the bottom to enhance recovery, 
because a few quahogs had been found outside the frames in 1975. 
We also built smaller boxes, 4' x 3', installed at seven additional 
sites with 5.000 ARC seed at each site. 

Two boxes were installed side by side at three locations: Doane 
Way, Yacht Club, and Lonnie's Pond. One box was planted with 
ARC stock and the other with CZR stock in equal numbers to see 
if there was a variation in growth or survival depending on the 
source. 

Results 

Table 2 provides details of the plantings for 1976. 

1. The ARC stock was smaller at the beginning of the season 



(6-8 mm) and delivered 2 weeks later than the CZR stock 
(8-10 mm): 25,0(W seed from .ARC was delivered in mid- 
July (8-10 mm). 

2. At Meetinghouse River and Quanset Pond, seed grew faster 
than those at other sites. 6 to 8 mm when planted to 19 mm 
in October. 

3. In the side-by-side experiment with equal densities, the CZR 
stock, beginning at 8 to 10 mm grew to an average of 15 mm 
(average 6 mm of growth), and the ARC stock beginning at 
6 to 8 mm grew to an average of 1 1.5 mm (average 4.5 mm 
of growth). The boxes at Lonnie's Pond could not be ad- 
equately evaluated because of the time difference of plant- 
ing. 

4. The average rate of growth at the other sites, 6.3 mm. did 
not seem to be dependent on the source of seed. 

5. In the smaller boxes that survived, the lower density seemed 
to have a positive affect on growth. 

6. The type of substrate did not seem to affect growth. 

7. Where the boxes were tight enough to exclude predators, 
summer survival was excellent. Exceptions at Tonset, Pleas- 
ant Bay, and Barley Neck where boxes were lost, the loss 
was caused by boats, human interference, and storms. 

8. Winter survival at Quanset Pond and Mill Pond was >80%, 



A Municipal Quahog {Mercenaria mercenma) Management Program 



1019 




Figure 4. Ten intertidal locations chosen in 1975. were raked to loosen 
the suhstrate and remove any visible predators. About 1,()(H) seed qua- 
hojjs {Mercenaria mercenaria) were broadcast within the raked area 
and a frame, 3' x 6' to which a 3/16" mesh netting! was stapled, was 
placed over the seed and staked in place. Edges were then backHUed. 

and >50'7f survived in Meetingliouse River and Jack Knife 

Cove boxes, but survival was very poor at the remainder of 

sites. 

The winter of 1976 to 1977 was exceptionally severe. Some 

bodies of water were frozen over for 14 consecutive weeks, and 

most of the boxes were severely damaged or destroyed. As an 

experiment, we had retained some covers and removed others 

before winter's onset. The boxes without covers sustained less 

winter damage than those with covers. A covered box at Doane 

Way was lifted out of the bottom leaving only the bottom netting. 

and a covered box at Meetinghouse River was physically moved 

3.3 m. The covered box at Quanset Pond sustained no damage, but 

it had been installed on a fairly steep slope, which may have saved 

it from ice damage. 

1977 

Because of the damage to intertidal boxes from the severe 
previous winter, we modified the box design again for subtidal 
culture. Boxes were identical except that bottom netting was de- 
leted and covers that could be handled under water were con- 
structed. The covers were made of two layers of 1" x 3" strapping 
laid flat with netting securely sandwiched between. These were 
attached to the boxes by rubber hinges and held down by clasps. 
Boxes were trenched at ground surface but rocks and clay deposits 
sometimes prevented complete trenching. In these cases, boxes 
were above ground and had to be filled around the base. 

Results 

Table 3 provides details of the plantings for 1977. Quanset 
Pond. Meetinghouse River, and Jack Knife Cove showed contin- 
ued excellent summer survival and good growth. Except for the 
one at Meetinghouse River, they also showed very good winter 
survival. Two anomalies occurred this year. First, large 14 to 16- 



mm CZR stock was purchased and planted in May, hut m three 
locations. Mill Pond. Little Mill Pond and Hopkins Island Channel. 
it only grew to 17 or 18 mm by November. Second, at Lonnie's 
Pond, stock that was 8 to 12 mm in May only grew to 13 mm. We 
do not know the reasons for such limited growth except that in 
Little Mill Pond and Lonnie's, the shells began to erode by the end 
of the summer. 

The hinged cover design did not exclude predators, and above- 
ground boxes led to poor water exchange within tlie boxes, serious 
predation. and over-all poor results occurred. 

Discussion 

Seed quahogs were planted at 20 .separate locations over a 
3-year period with three different enclosure designs. Two sources 
of seed were used. CZR from North Carolina and ARC from 
■Massachusetts. Size at planting ranged from 6 to 16 mm. Density 
ranged from 55 to 230/0.3 m~, with one box at 456/0.3 m". Sub- 
strate ranged from coarse sand and cobbles to clay/mud, with the 
majority being a mixture of sand and silt. Most locations had either 
natural quahogs or soft-shell clams in the general vicinity, but a 
few locations had neither. Except for storm damage and human 
interference, most animals survived well over the course of the 
summer. Survival after the winter varied from a high of 50 to 90% 
in some areas to to \0% in others. Some of this was caused by 
the severity of the winter. 

Summer survival of s;50%, was generally attributable to pre- 
dation. Inadequate installation of the boxes resulted in washouts 
around the edges leaving holes under the boxes. Covers that were 
not tight allowed invertebrates to lodge between the box and cover. 
In both cases, predators entered and consumed the quahogs. We 
suspect that predators also may have entered the boxes as larvae 
and grew within the enclosure based on the time of year of de- 
ployment. 

The bottom culture project as described employed many vari- 
ables, and there were no "controls" as part of the program. This 
is because the primary objective was to increase quahog stock 
throughout the town. 

Winter conditions proved to be the greatest obstacle to inter- 
tidal quahog culture that relies on structures and netting. Removing 
top netting just prior to a freeze lessened the physical damage but 
did not assure seed survival. Seed from North Carolina survived 
equally as well as local stock. Cost of materials was about $ 1 .000 
for the 3 years of bottom culture and cost of seed was about 
$4,000. 

RAFT CULTURE 

Raft culture methods were used in 1976 to 1987. Although the 
methods were essentially the same for the entire period, the 
amount planted, source of supply, and amount of growth varied 
during this time span. 

Prior to 1975, George Souza, Falmouth Shellfish Constable and 
Arnold Carr, Massachusetts Division of Marine Fisheries, experi- 
mented with floating trays to grow quahog seed. They found that 
trays without sediment did not work well. Souza designed suc- 
cessful floating sand box rafts in Falmouth, In 1976. we decided to 
build rafts, but Souza's design was unwieldy for Orleans' depart- 
mental equipment to handle. We modified his design to our needs 
(Fig. 5). 

The inside of the 9' x 6' wooden frame was covered with 1" 
vinyl-coated galvanized wire, 1/8" plastic netting, and window 



1020 



Macfarlane 



TABLE 1. 
Ten sites in tliree estuaries with varying environmental conditions chosen for initial intertidal bottom culture nursery plantings in 1975. 













1 


1975 Bottom Culture 


















Date 


Density 




Natural 


Survival 


Size (MM) 


Survival 


Location 


Estuary 


Source 


Size 


Planted 


Ft 2 


Substrate 


Clams/Q 


Summer 


Oct. 


Spring "76 


Skaket 


CCB 


CZR* 


8-10 mm 


Julv 75 


55 


Sand 


None 


90% 


15 


LOST 


Doane Way 


N 


CZR 


8-10 mm 


July 75 


55 


Coarse sand/silt 


SS/Q 


95% 


17 


80% 


Robert's Cove 


N 


CZR 


8-10 mm 


July 75 


55 


Silt/niud/sand 


SS 


95% 


13.5 


90% 


Snow Shore 


N 


CZR 


8-10 mm 


Julv 75 


35 


Coarse sand 


QNear 


75% 


11 


5% 


Asa's Landing 


N 


CZR 


8-10 mm 


Julv 75 


55 


Sand 


SS 


95% 


18 


55% 


Yacht Club 


N 


CZR 


8-10 mm 


July 75 


55 


Coarse sand 


SS/Q 


95% 


18 


50% 


Meetinghouse River 


PB 


CZR 


8-10 mm 


July 75 


55 


Sand/silt 


None 


95% 


18 


10% 


Namequoit Point 


PB 


CZR 


8-10 mm 


July 75 


55 


Coarse sand/grave 


None 


0% 


N/A 


0% 


Quanset Pond 


PB 


CZR 


8-10 mm 


July 75 


55 


Coarse sand 


QNear 


95% 


17 


90% 


Pochet Landing 


PB 


CZR 


8-10 mm 


July 75 


55 


Med. fme sand 


SSNear 


95% 


16 


80% 



CCB = Cape Cod Bay; N 
Zone Resources. 



Nauset; PPB = Pleasant Bay; SS = soft shelled clams {A/v<i arenuriu): Q = quahogs (Mercenaria mercenarily. *Coastal 



screen material. Each raft was filled with at least 4" ( 100 mm) sand 
from an inland site. Styrofoam blocks were attached to two long 
sides. Two short sides were open to allow for a tlow-through of 
water. Bright orange floatation blocks (9' x 1' x 1') were painted 
with green nonantifouling paint so the rafts would blend with the 
landscape. Each raft was moored by 5/8" galvanized chain to a 50 
lb (23 kg) mushroom mooring on the short end to allow the raft to 
swing in the tide. Threaded rods, used to assemble the structure, 
held galvanized chicken wire to prevent bird predation. 

The rafts were planted with 25,000 seed at a density of about 
400/0.3 m". Two salt ponds were selected for deployment: Mill 
Pond (Nauset) and Lonnie's Pond (Pleasant Bay) (Fig. 3). Mill 
Pond was chosen, because it has native populations of shellfish, is 
protected from motor boat activity by a rock dam, and is protected 
from most winds e.xcept NE. A second raft was deployed in Little 
Mill Pond, a small shallow. <3' (<l m) pond adjacent to Mill Pond 



that is not productive for shellfish, but is secluded and protected 
from strong winds. Lonnie's Pond was chosen because of limited 
boating activity, protection from wind, and natural production of 
soft-shell clams. The water in Mill Pond was consistently 4°F 
(2.2 C), and the salinity was about A%c higher than Lonnie's Pond. 

1976 

The rafts were planted in May with 100.000 seed and remained 
in the water until October. They were emptied using a Venturi-type 
hydraulic pump with a 1/2" mesh hardware cloth basket attached. 
The mesh allowed the sand to wash through but retained the qua- 
hogs. Harvest mortality was negligible, except in cases where sand 
had not been carefully screened and contained large stones. Seed 
was counted volumetrically, and the quahogs were then trans- 
planted to the natural environment (see separate section on trans- 
plants). 



TABLE 2. 
Twelve sites were chosen for intertidal bottom culture plantings in 1976. 















1976 Bottom Culture 






























Size 












Date 




Density 




Natural SS 


Survival 


(MM) 


Survival 


Location 


Estuary 


Source 


Size 


Planted 


Amount 


Ft 2 


Substrate 


Clams/Q 


Summer 


Oct. 


Spring '76 


Doane Way 


N 


CZR 


8-10 mm 


}> June 


12.500 


230 


Sand/silt 


SS/Q 


90% 


15 


0% 


Doane Way 


N 


ARC 


6-8 mm 


17 June 


12.500 


230 


Sand/silt 


SS/Q 


85% 


11.5 


10% 


Yacht Club 


N 


CZR 


8-10 mm 


3 June 


12.500 


230 


Coarse sand 


Q 


98% 


15 


10% 


Yacht Club 


N 


ARC 


6-8 mm 


17 June 


12.500 


230 


Coarse sand 


Q 


98% 


11.5 


10% 


Town Cove 


N 


ARC 


6-8 mm 


17 June 


12.500 


230 


Coarse Sand 


SS 


98% 


13 


0% 


Meetinghouse River 


PB 


ARC 


6-8 mm 


17 June 


12.500 


230 


Sand/silt 


None 


98% 


19 


50% 


Quanset Pond 


PB 


ARC 


6-8 mm 


17 June 


12,500 


2.W 


Coarse sand 


QNear 


98% 


19 


90% 


Nau.set Bar 


N 


ARC 


6-8 mm 


17 June 


5,000 


90 


Sand 


SS 


90% 


13 


0% 


Mill Pond 


N 


ARC 


6-8 mm 


17 June 


5,000 


90 


Sand/silt 


SS 


98% 


15 


85% 


Tonset 


N 


ARC 


6-8 mm 


17 June 


5.000 


90 


Coarse sand 


Q 


0% 





0% 


P. Bay 


PB 


ARC 


6-8 mm 


17 June 


5.000 


90 


Coarse sand 


None 


0% 





0% 


Jack Knife Cove 


PB 


ARC 


6-8 mm 


17 June 


5.000 


. 90 


Sand/silt 


None 


98% 


15 


50% 


Barley Neck 


PB 


ARC 


6-8 mm 


17 June 


5.000 


90 


Coarse sand 


SS 


0% 





0% 


Lonnie's Pond 


PB 


ARC 


6-8 mm 


17 June 


5.000 


90 


Silt/sand 


None 


98% 


17 


0% 


Lonnie's Pond 


PB 


CZR 


8-10 mm 


15 July 


25.000 


465 


Silt/sand/clay 


None 


98% 


17 


0% 



N = Nauset; PB = Pleasant Bay; SS = soft shelled clams (Mya arenaria): Q = quahogs (Mercenaria mercenaria); CZR = Coastal Zone Resources 
and ARC = Aquacultural Resources Corp. as sources of seed. 



A Municipal Quahog (Mercenaria mercenaia) Management Program 



1021 



TABLE 3. 
Eight sites were selected for subtidal bottom culture plantings for 1977. 















1977 Bottom C 


jlture 
































Size 














Amount 






Substrate 


Natural 


Survival 


(mm) 


Survival 


Location 


Estuary 


Source 


Size 


Date 


Planted 


Boxes 


Density 


Ft 2 


SS Clams/Q 


Summer 


Oct. 


Spring '76 


Jack Knife Co\e 


PB 


CZR 


X- 1 2 mm 


IS Mav 


27.000 


3 


165 


Sand/sill 


None 


90-95<>r 


20 


75'7r 


Meetinghouse River 


PB 


CZR 


8-12 mm 


18Mav 


18,000 


2 


165 


Sill/sand 


None 


90-95 Vr 


19 


0% 


Lonnie's Pond 


PB 


CZR 


S-12 mm 


18 May 


18.000 


1 


165 


Sand/clay 


SS 


80% 


13 


20% 


Quanset Pond 


PB 


CZR 


8-12 mm 


IS May 


18.000 


2 


165 


Silt/sand 


QNear 


90-95% 


18 


75% 


Mill Pond 


N 


CZR 


14-16 m 


26 May 


9.500 


1 


90 


Coarse sand/ 
Rocks 


SS 


15-20% 


17 


5% 


L. Mill Pond 


N 


CZR 


14-16 m 


26 May 


9.500 


1 


90 


Silt/sand 


SSNear 


50% 


17 


10% 


Hopkins Is. Chan. 


N 


CZR 


14-16 m 


26 May 


4.700 


1 


90 


Sand/rocks 


Q/SS Near 


40% 


18 


5% 


Asa's Landing 


N 


CZR 


14-16 m 


26 May 


19,000 


2 


175 


Sill/sand 


SS 


50% 


21 


10% 



N = Nauset: PB = Pleasant Bay: SS = soft shelled clams (A/mi ureiuiriu I; Q = quahogs (Mercenaria mercenaria). All stock originated from Coastal Zone Resources (CZR). 



Results 

Table 4 summarizes the raft culture program in Orleans from 
1976 10 1980. 

1976 Results 

There was negligible summer mortality, no evidence of pretfa- 
tion, an(j excellent growth. Fouling was a problem, especially in 
Lonnie's Pond, where seaweeds attached to the wood or appeared 
as clumps within the rafts. Seaweed became dense enough to cause 
smothering if the rafts were improperly maintained. Barnacles, 
tunicates, and bryozoans were the principle animal fouling com- 
munities, but we have no evidence that they adversely affected the 
seed growth or survival. The rafts in Mill Pond had less seaweed 
or organism fouling, but blue mussels {Mniliis edulis Linne) set 
prolifically on the undersides of the rafts. This added substantial 
weight. 





Birds, principally gulls and ducks, sat on the floats and left their 
droppings, but did not predate them. The mesh holding the sand in 
place was too fine to allow for organic waste removal. Black sand 
built up by the end of the season but had no apparent negative 
effect on the animals. In Lonnie's. animals that were 8 to 10 mm 
when planted in May were 25 to 30 mm by October. In early 
■September, many of the animals were on top of the sand because 
of crowding, but growth and/or survival did not seem to be af- 
fected. We harvested these rafts by the end of September to pre- 
vent potential crowding-related problems. The seed planted on the 
Mill Pond rafts exhibited a slower growth of 20 to 23 mm. This 
was despite their having been planted a week earlier than the seed 
in Lonnie's and harvested almost a month later. 

At the end of the sea.son. those quahogs that were less than 15 
mm were put back on the raft to hold over for the winter. As 
previously noted, the winter of 1976 to 1977 was unusually severe. 
The rafts were frozen in ice for 12 to 14 consecutive weeks. We 
estimated 20% mortality when we could get out to the rafts and an 
additional 20% when the water temperature increased to about 
50°F. Black sand was found throughout the raft (winter kill). 

1977 

In addition to rafts in Lonnie's Pond and Mill Pond, a raft 
placed in Quanset Pond (PB). was painted with antifouling paint 
and planted with 20,000 seed, as an additional experiment. The 
paint did not seem to affect growth or survival. 

1977 Results 

See Table 4 for details on raft culture for 1978. A strong NE 
storm damaged the rafts in the Mill Pond and 10 to 20% of the seed 
washed out of the rafts. 

1978 

We ordered 400,000 seed quahogs but only received 17,000. 
Coastal Zone Resources went out of business, and ARC could not 
supply our needs. A third hatchery. Shellfish Inc. (SI) in Sayville. 
NY shipped 17,000 in August. They did not grow large enough to 
be transplanted and were overwintered on the rafts. Approximately 
60% survived. 



Figure 5. Rafts were deployed in Mill Pond (Nauset) and Lonnie's 
Pond (Pleasant BaM. The rafts were planted with 25,000 seed at a 
density of about 400/0.3ni-. 



1979 



We ordered 300,000 and received 275,000 (see Table 4), 



1022 



Macfarlane 



TABLE 4. 
Summary of raft culture in Orleans from 1976 through 1980 at two sites; rafts were not deployed in 1978 because of unavailability of seed. 













1976 Raft Culture 






















Date 


Density 


Survival 


Size 


Survival 


Location 


Estuary 


Source 


Amount 


Rafts 


Size 


Planted 


Ft2 


Summer 


Oct. 


Spring 


Mill Pond 


N 


CZR 


25.000 


1 


8-10 mm 


20 May 


400 


95% 


25-30 


65% 


Little Mill Pond 


N 


CZR 


25.000 


1 


8-10 mm 


20 May 


400 


95% 


25-30 


65% 


Lonnie's Pond 


PB 


CZR 


50.000 


T 


8-10 mm 


26 May 


400 


95% 


26-30 


65% 












1977 Raft Culture 










Mill Pond 


N 


CZR 


60.000 


") 


8-12 mm 


1 8 May 


475 


80% 


25 


N/A 


Mill Pond 


N 


CZR 


20.000 


1 


8-12 mm 


26Mav 


315 


80% 






Lonnie's Pond 


PB 


CZR 


30,000 


1 


8-12 mm 


18 May 


475 


95% 


30 


N/A 


Lonnie's Pond 


PB 


CZR 


50.000 


T 


8-16 mm 


26 May 


400 


95% 


30 


N/A 


Lonnie's Pond 


PB 


ARC 


35,000 


1 


5-9 mm 


21 July 


180 


95% 


12-18 mm 


N/A 


Quanset Pond 


PB 


ARC 


20.000 


1 


5-9 mm 


21 July 




95% 


10-16 mm 


N/A 












1979 Raft Culture 










Mill Pond 


N 


ARC 


100.000 


3 


5-6 mm 


31 Mav 


617 


95% 


15-25 mm 


N/A 


Lonnie's Pond 


PB 


ARC 


100.000 


3 


5-6 mm 


7 June 


617 


95 


25 mm 


N/A 


Lonnie's Pond 


PB 


SI 


75.000 


1 


2 mm 


28 June 


1400 


40% 


6-15 mm 


N/A 












1980 Raft Culture 










Lonnie's Pond 


PB 


BSP 


100.000 


3 


8-19 mm 


1 Apr 


617 


95% 


25-30 mm 


N/A 


Lonnie's Pond 


PB 


ARC 


100.000 


4 


5-6 mm 


30 Mav 


463 


95% 


18-30 mm 


N/A 


Lonnie's Pond 


PB 


ARC 


100.000 


4 


5-6 mm 


2 July 


463 


95% 


18-30 mm 


N/A 



N = Nauset; PB = Pleasant Bay; CZR 
= Bristol Shellfish Products. 



Coastal Zone Resources; ARC = Aquacultural Resources Corporation; SI = Shellfish Incorporated; BSP 



1979 Results 

The small size of the SI seed may have resulted in low survival 
(40%) in the raft by the end of the season. We observed animals 
being washed out with boat wakes and during high winds and 
believe the balance of seed was washed out. The remaining stock 
grew to 6 to 15 mm. The ARC stock growth was comparable to 
previous years, and survival was 95%. 



1980 



We bought 100.000 seed from a fourth hatchery. Bristol Shell- 
fish Products (BSP) in Maine, which we planted very early, April 
1. We purchased 200,000 from ARC and planted half on May 30 
and half on July 2. All were planted in Lonnie's Pond rafts. 

1980 Results 

Fouling was extremely heavy. See Table 4 for growth and 
survival results. 

Discussion 

From 1976 to 1980. we used two ponds, four suppliers of seed. 
with size at planting ranging from 2 to 19 mm for the rafting 
program. Seed was planted as early as April 1 and as late as July 
21, with optimal planting in May and June, when weather had 
stabilized. Predation was negligible. Summer survival was 95% or 
more, regardless of the density, except in three instances, where 
the shellfish were lost because of washout in stonns. Seed, less 
than 5 mm at planting time, were too small for this system, because 
they were easily washed out by waves, including boat wakes. 

The major advantages of the raft system was increased growth 
rates and ability to concentrate density of plantings. Most seed 



reached the state-recommended plantable size of 25 mm by the 
fall, and thus overwintering problems associated with gear in the 
water could be avoided. 

We continued to use the rafts through 1987. Lonnie's Pond had 
proved to be superior for growth and survival. Although we were 
concerned about putting all our seed in one location, we used only 
Lonnie's from 1980 to 1987. Growth was still good, and mortality 
was negligible. Fouling in 1980 was severe, and we had noticed 
that the water quality in Lonnie's was beginning to show signs of 
decline. Average salinity dropped by l%f, water color was often 
tinged red (monospecific blooms of dinoflagellates), and the 
amount of floating seaweeds, both green filamentous (Enteromor- 
pha sp.) and red (Calithainnion) increased dramatically. Taken 
together, these are signs of eutrophication. As the 1980s pro- 
gressed, fouling increased. The Pleasant Bay circulation was af- 
fected by a southward migration of the protective barrier beach. 
This diminished the exchange of oceanic water that reduced the 
salinity. In 1987. a breach occurred in the barrier beach at 
Chatham. MA. This drastically changed the hydrodynamics of the 
bay. including a 1-ft (0.3 m) change in tidal amplitude. By that 
time, we had changed our nursery culture methods (see section on 
Hatchery /Lab below). 

Mill Pond continued to have good water quality, but because of 
potential storm damage and slower growth, we were reluctant to 
use Mill Pond extensively. 

The primary problem with the rafts was their size and amount 
of clean sand required. Lonnie's was small, and we had maximized 
the amount of area we felt should be devoted to this project. The 
pond was also used by recreational boaters, and the number of 
moorings in the pond was increasing each year. The rafts had been 
damaged through boating activity, and we feared vandalism or 
accidents if the number of rafts was increased. 

During the course of the program, the seed cost increased sub- 



A Municipal Quahog {Merci^nar/a mercenaia) Management Program 



1023 



stantially. and the availahilil\ iii spring lluLtuatcd greatly. The 
only way we could obtain the number of seed required was to 
purchase smaller animals; however, with seed less than ? mm. 
losses in the raft system were severe. The Mill Pond was reason- 
able for growth and survival but was not secure from wind dam- 
age, and removing the annual mussel set was labor intensive. 

The rafting method of nursery growout was more expensive 
and labor intensive than bottom culture, but, in Orleans, consistent 
high growth and survival were achieved in both Lonnie's Pond and 
Mill Pond. The rafts cost about $250.00 each ( 1978) for materials, 
and they had a life span of at least 5 years. We preferred a density 
of about 450/0.3 m~. but we planted as many as 30,000/raft, 555/ 
0.3 nr, without impeding growth or survival. The highest density 
bottom culture was close to 200/0.3 m". We did not determine the 
maximum density with these systems. 

Clearly, growth rate was greater on the rafts than in the bottom 
boxes. The boxes were intertidal and, thus, exposed for .several 
hours each tide, and the seed were unable to feed; whereas, on the 
rafts, the animals were submerged. The sand in the rafts seemed to 
have an affect on growth, because seed observed on rafts with sand 
washed out and density of animals sparse, the seed seemed to 
siphon less vigorously, and growth was less than seed observed in 
the sand. 

After the winter of 1976 to 1977. we determined that seed 
should be planted in the fall, and we began to determine factors for 
best transplant survival. We realized, after several years, thai if we 
planted the largest seed in September, it would survive fairly well, 
and if we planted the smallest seed in November, it also would 
survive well, but if we planted small seed in September, it would 
perish. Thus, we revised our thinking about the minimum size seed 
that could be successfully transplanted in the fall. (See section on 
transplants). 

We could not increase the number of rafts in Lonnie's Pond 
because of other public uses. This limited the amount of seed that 
we could raise to 300,000. After 1980, we raised approximately 
250.000 to 300,000 per year in rafts. The rafting program was 
halted in 1987. 

HATCHERY/LAB 

Site Selection 

In 1979, the Shellfish Department office, a 16 ft x 24 ft (4.8 m 
X 7.2 m) building had to be relocated. The rapidly rising cost of 
seed in the late 1970s, unreliable seed supply from commercial 
hatcheries, and a requirement by Massachusetts Division of Ma- 
rine Fisheries that seed could only be imported from hatcheries 
north of, but including. Long Island, prompted us to consider 
raising our own seed. Matthiessen and Toner (1966) developed 
hatchery methods for Martha's Vineyard, and we thought these 
would work in Orieans. Suitable waterfront land was limited to 
two parcels, neither of which was ideal or optimal. A small area on 
the shore of Lonnie's Pond was particularly appealing, but the site 
was located in a residential district. There was opposition from the 
neighbors who feared such potential negative aspects as unsightly 
gear, traffic congestion, and unknown problems. 

The second site was on the shore of Town Cove. This included 
a public boat launching ramp in a business-zoned area. We moved 
the building to the Town Cove site (Fig. 6). In 1981, a bulkhead 
was constructed in front of the building, and a ramp and floats 
were added for public access to the water. This improvement in- 
creased the public usage of the landing. Site selection is critical to 




Figure 6. A 16' x 24' (5.1 x 8 ml building was moved to the shores of 
Town Cove where a seawater system was installed. Within 2 years 
after moving the building, the town constructed a bulkhead that sub- 
stantially enhanced the area for recreational boating. This led to in- 
compatibility with the shelirish lab. 

hatchery success, and our experience with site-related problems 
are illustrated. 

Construction of a Seawater System 

The budget of $7,000 to equip the building for the project 
required installation of a single intake system using 1.5" pipe and 
a I hp swimming pool pump. Tanks were constructed of wood and 
fiberglassed. The tanks were 8 ft long and 4 ft wide, divided in the 
middle lengthwise. Valves and drains were installed in each com- 
partment to allow for flexibility. Three tanks or six compartments 
were constructed so stock could be held and manipulated for vari- 
ous purposes. 

When the bulkhead was being planned, it was recommended to 
install a dual intake system with pumps located at the bulkhead 
rather than in the building (90 ft [27 rn] landward), but we were not 
pemiitted to do so. Instead, a single 2" (2.5 cm) pipe set within a 
6" (1.2 cm) PVC pipe was buried under the parking area to guard 
against potential damage from automobile traffic and a larger 
pump (2 hp) was purchased. Although the larger pipe and pump 
were improvements, the system was difficult to maintain because 
of its inaccessibility. In addition, because we were pulling water 
rather than pushing it, air in the vacuum side of the pump was a 
constant problem. 

HATCHERY METHODS 

Algae Production 

Glass bottles ( 12 narrow mouth 5 gallon |22 L] carboys and a 
number of 1 gallon |4.4 L]) were donated. Starter cultures of 
Monochrisis. Isochrisis, 3-H (Thalasiosira). and Dunaliella were 
obtained from the NOAA National Marine Fisheries Service labo- 
ratory in Milford Connecticut. Bigelow Laboratory for Ocean Sci- 
ences, and the Woods Hole Oceanographic Institute. 

The cultures were grown in an illuminated, temperature con- 
trolled room (6' x6' [I.8mx I.8m]), with an air supply following 
the procedures detailed in Guillard (1974). Culture densities were 
estitnated and recorded daily using standard methods. The general 
layout, equipment, and operation followed the design of algae 
rooms in similar sized hatcheries in Maine, (Smith, Heinig, pers. 



1024 



Macfarlane 



comm.) We tried outdoor algae production, but the lack of a secure 
area resulted in accidents that were too numerous to overcome. 

Quahog Culture 

A hatchery in New England can operate either using natural 
ripened stock and commence spawning in June or condition the 
animals by slowly raising water temperature to commence spawn- 
ing in January or February. Cost of winter spawning is high, be- 
cause heated water is necessary to condition the quahogs. We 
chose using the natural spawning time of summer. 

Quahogs used for spawning, came from 20 ± feet (6 ml depth 
in Cape Cod Bay, native stock from the Town Cove, and trans- 
planted hatchery stock from Pleasant Bay. Each aniinal was 
marked according to its origin. 

Loosanoff and Davis (1950), (Loosanoff and Davis 1951), 
Loosanoff and Davis (1963), Loosanoff et al. (1953). and 
(Loosanoff and Davis 1955) developed hatchery procedures for 
bivalve mollusks. Other researchers, such as Castagna and Kraeu- 
ter (1977) and (Castagna and Kraeuter 1981) have refined the 
process to suit individual needs. We generally followed prescribed 
procedures for hatchery methods, substituting low-cost containers 
(see Table 5) whenever possible. 

Castagna and Kraeuter (1981) used mass spawning methods, 
and researchers at the Milford Laboratory used nidividual spawn- 
ing animals. Because we needed several spawns so that high larval 
or set mortalities would not preclude loss of the entire year's 
production, we chose the individual spawning method. 

To induce spawning, two or three quahogs (from 37 to 100 
mm) were placed in a Pyrex loaf pan with 1 micron filtered sea- 
water (Fig. 7). Food was added when the quahogs began pumping. 
Hot tap water was added to the tank to raise the water temperature 
to a minimum of 27°C. If spawning did not occur, the tank water 
was cooled with ice and the process was repeated. Sperm suspen- 
sion, either fresh or frozen, was added occasionally as rec- 



ommended by Loosanoff and Davis (1963) and Castagna and 
Kraeuter (1981). 

When spawning occurred, males and females were separated. 
Males were removed from the loaf pans after a few minutes, the 
shells were dried and marked as male. When the females were 
spent, they were inarked con'espondingly. 

The eggs were sieved through a 50 micron sieve and placed in 
30-gallon (32-liter) plastic trash cans as larval containers (Fig. 8). 
We followed the advice of other researchers regarding the viability 
of eggs as related to size (Castagna, Kraeuter, Gibbons, Rhodes, 
Goldberg. Chapman. Karney and others, pers. comms). We tried to 
ensure that several males were used and also several females to 
diversify the genetic pool. 

Fertilized eggs were sieved after 48 hours. Low-cost sieves (see 
Table 5) were constructed from 5-gallon (22-L) plastic buckets 
with tight fitting lids donated by the food service industry. We 
removed the center of the lid leaving only the rim. We placed 
Nytex netting tightly over the bucket top, replaced the lid rim, and 
sealed the inside edge with aquarium sealant. We cut off the bot- 
tom of the bucket so that when the bucket was inverted, the netting 
was on the bottom (Fig. 9). 

Larvae were sieved every other day and were fed a diet of 
Monochhsis. Isochrisis. and Dunaliela. depending on the size and 
food availability. We did not try to increase production through the 
use of antibiotics (Hidu and Tubiash 1963. Castagna and Kraeuter 
1981 ). because we did not know if there would be ramifications for 
future generations using antibiotics. 

Our postset system is described in Table 5. setting system ( 1 ). 
The cascading trays (Fig. 9) were based on a design by Lind (Town 
of Eastham Dept. Natural Res. pers. comm.) in Eastham. This 
system was easier to handle than raceways described by Castagna 
and Kraeuter ( 1981 ) and Rhodes et al. (pers. comm.) However, our 
second type of systein (Table 5) worked even better. While the 
veliger larvae were in the trash cans but ready to metamorphose, 
we added stacks of the trays (three trays per trash can) 




Figure 7. Spanning in Pyrex loaf pans filled with seawater. Marks Figure 8. Eggs were placed in 30 gallon (32 liter) plastic trash cans at 
indicate estuary of origin and after spawning, individuals were also a density of 10 to 20 eggs/ml,. Larvae were siphoned to sieves; the 
marked with gender symbols. "dregs" remained on the bottom preventing batch contamination. 



A Municipal Quahog (Mercenaria mercenaia) Management Program 



1025 




Figure 9. Inexpensive sieves were made from 5 gallon (19 liter) plastic 
buckets with tight fitting lids. Connector rings, (12" high) from the 
buckets ensured that no larvae were lost in the sieving process. Cas- 
cading glass "shelving" trays, (12" x 10" x 2") with attached mesh 
inside plastic "kittv litter trays" (15" x 12" x }"), constructed for 
postset juveniles. 



lined with appropriate sized mesh (Fig. 10). As metamorphosis 
occurred, the animals set on the mesh as well as on the bottom. 
Animals that set on the bottom were moved to the trays and kept 
in the trash cans until they were large enough to go into upwellers. 
We treated these postset animals as larvae until transferral. 

UPWELLER TECHNOLOGY 

Bayes. (1981) described a method for using "forced plankton- 
rich seawater up through a partially fluidized bed of filter feeding 
molluscs," which became known as the "upweller technique." 
The upweller technique had the potential of increasing the capacity 
at our site, and we developed a design based on standard fish lotes 
(80 cm X 45 cm X 29 cm) placed in the tanks. 

Figure 1 1 shows our upweller system. Each tote had two cham- 
bers (often referred to as "silos") made from the same type of 
5-gallon plastic buckets as our sieves for a total of 36 silos. The 
bottom 3 inches of each bucket was cut off and the solid bottom 
was removed from that piece, resulting in a ring. Holes drilled in 
the ring (resembling a work ring) allowed water to flow up through 
the chambers placed on the ring. Mesh size was increased as the 
seed grew. 

The system allowed us to eliminate the hatchery and purchase 
small, low-cost seed (1-2 mm) from commercial hatcheries. We 
purchased a million seed that were equally distributed among the 
silos. Silos were added as the seed grew, ending with a density of 
about 25,()00/silo. 

The chambers were rinsed with freshwater daily to remove 
waste products, and every other day, the chambers were rinsed, 
seed were removed, and the silo was washed with soap and water. 
Depending upon the biofouling severity, the chambers were 
cleaned with bleach and rinsed thoroughly. The same procedure 



was used for the totes. The intake pipe was back flushed weekly 
with freshwater. 

Food for the seed in the upwellers was primarily natural plank- 
ton; however, we supplemented this food with approximately I 
liter/tote of cultured algae per day. The water was shut off during 
feeding (just after cleaning in an attempt to maximize the benefit), 
and once the seed cleared the algae from the water, the How was 
resumed. 

On July 31, 1985. our technician noticed an oil slick next to the 
dock, within 100 ft (33 m) of our intake pipe. The slick was traced 
to a trash compactor behind a large supermarket and a trail of oil 
from the compactor lead to a lagoon with a direct discharge to 
Town Cove. Within 10 days. 90% of the seed quahogs were dead. 

The small seed (2-6 mm) did not have enough tissue remaining 
to perform a bioassay (Tripp, pers. comm.). After a literature 
search and assistance from Dr. Judith (Capuzzo) MacDowall. we 
assembled enough information for the town to sue the supermar- 
ket. We claimed loss of the seed cost, the wholesale market value 
of the seed at maturity at 60% survival, and the cost of the equip- 
ment. 

In 1987, we were awarded an out-of-court settlement of 
586,000 for the oil spill. A portion of the funds were used to install 
a full dual-intake system with new, larger (5 hp) pumps. We re- 
designed our tanks to a total of 48 individual chambers in which 
1.0 million seed were grown, allowing us to decrease the density 
to 20.000/silo. 

Discussion: Hatchery and Upwellers 

We spent 4 years learning and perfecting methodologies for 
both algae and bivalve culture with our limited system. By 1984, 
we had over 150.000 seed that reached 3.0 mm by late August; 
however, when hatchery production did not approach expectations 
of millions of seed, we were required to abandon the building as a 
hatchery. 

Seed in all postset systems during late July and early August 
seemed to be stressed. Survival during this stage was difficult, 
especially with the constant threat and reality of gas bubble disease 
resulting from the intake pipe design. Water temperature was high; 
80 to 82°F (27-28°C) was not uncommon: oxygen was low; plank- 
ton in the seawater was predominantly dinoflagellates (a poor 
food); and any mesh smaller than window screen trapped air under 
the netting. Work by Anderson ( 1978) and Anderson ( 1979) on the 
local red tide organism Gonyaulax tamarensis (now Alexandrium 
tainarensis) suggested that M. mercenaria do not actively feed 
when A. tamarensis is in the water column. We suspect that other 
dinoflagellates may have the same effect. We aerated the tanks and 
added our own food as a supplement. Adding the food after clean- 
ing seemed to be effective. 

When we changed to upwellers, we were able to culture the 1 
to 2 mm seed up to 12 to 18 mm by the fall. In 1986, 1987, 1988. 
and 1989, we raised a million seed per year, over 95% of which 
were transplanted to the natural environment. 

Although the site was not conducive for a shellfish nursery, it 
had a major advantage over the Lonnie's Pond site — visibility. 
With a town landing, a marina, a large sporting-goods store, and a 
restaurant/inn all adjacent to the lab, it was located in a very public 
area. The lab hosted between 250 to 400 visits per year between 
June 15 and October 15, allowing us the opportunity to provide 
education to tourists and residents alike regarding the operation 
and general marine related topics. Table 6 summarizes hatchery/ 
onshore nursery problems encountered and benefits to the town. 



1026 



Macfarlane 



TABLE 5. 

Summary of onshore hatchery/nursery methods including larval and postset containers, sieves and upwellcrs; sieves and upwellers were 

same basic design (see text). 



System 


Material 


Shape 


Size 


Mesh 


Density 


Number 


Larval containers 


Plastic 


Round 


30 Gal. (132L) 


N/A 


0.5-1.0 MiUion 


S 


Sieves 


Plastic 


Round 


5 Gal. (22 Ll 


Variable 


N/A 




Postset ( 1 ) 


Glass with mesh attached 


Square 


\' X \' (300 mm x 300 mm) 


225 m 


75-100.000/Tray 


16 




Plastic 


Rectangular 


r X 1.5' (30 cm X 45 cm) 






16 


Postset (2) 


Plastic setting trays 


Round with 
4 compartments 


3' (0.9 m) Diameter 


Variable 


0.5 Million 


3/Trashcan 


Upwellers 


Plastic 


Round 


5 Gal. (22 L) 


Variable 


125.000-20,000 
size dependent 


12-48 



TRANSPLANTS TO THE NATURAL ENVIRONMENT 

Stock raised in intertidal bottom culture boxes remained in the 

general vicinity. Some were transplanted to reduce the density and 
some were planted in more subtidal locations. Stock grown on the 
rafts and in upwellers at the lab. was transplanted to many loca- 
tions within the Town (Fig. 12). 

The transplants were the final program stage. We needed to 
determine if planted, unprotected seed would survive in the wild, 
because the program goal was to augment native stock. We could 
not afford to protect the seed for a second year while tnaintaining 
seed production. Because this was a municipal program, survival 
was of paramount importance: whereas, both rapid growth rate and 
survival are important foci for pri\'ate enterprise. Survival experi- 
ments by Flagg (1981). Castagna and Kraeuter (1977). (Kraeuter 
1 98 1). Menzel ( 1 976 ). Carriker (1 959), (Carriker 1 96 1), and many 
others suggested that survival of seed mollusks was related to 
density and size. 

Seed from raft culture attained a transplant size of 20 to 30 mm. 
Seed planted in the bottom boxes and those grown in the lab were 



much smaller, averaging 12 to 18 mm at transplant time. Massa- 
chusetts Division of Marine Fisheries biologists Carr and Hickey. 
(pars, comm.) and McKenzie ( 1977) suggested that 25 mm was the 
minimum size needed for a high percentage of survival in the field. 
Because our seed did not always reach 20 tnm. we needed to learn 
if a smaller size would survive in the field and the requiretiients for 
survival of small seed. 

To distinguish propagation program stock from wild stock, we 
purchased M. inercenaria (notala) whenever possible. This stock 
had been bred with a getietic tracer of brown zigzag marks or 
stripes on the shell that remained throughout the animal's life. 
Approximately 50 to 60% of our quahogs exhibited the markings. 

Method of Transplant 

We broadcast quahogs over a wide geographic area. The bot- 
totii culture experiments and the literature suggested that dense 
plantings were highly preyed upon; whereas, predation might be 




Figure 10. Mesh liners within ccjniparlnienls of plastic tiered setting 
trays placed in the trash cans as an alternative setting and postset 
system. 



tiyure IL Upweller system of common plastic fish totes with bucket 
"silos." Constructed in the same manner as the sieves, each silo was 
placed on a bucket ring that had holes drilled (resembling a wok ring) 
to allow water to flow through the ring and up through the silo. Water 
pumped into the fish totes, flowed through the silos, and was dis- 
charged. 



A Municipal Quahog (Mercenaria mercenaia) Management Program 



1027 



TABI,E 6. 
Summary of hatchery/onshore nursery problems and benefits in Orleans. 



Problem 



Funding 



Site Selection 



Intake 



Food Supply 



Spawning 



Poslsel 



Insufficient 

Inconsistent 

Jury-rig 

Inadequate equipment 



Construction of 

hulkhead: 
Intake damage 
Commercial area 

Manna 

Highly used town 
landing 

Commercial 
businesses 
Oil spill 

Loss of 1985 season 

Lack of e\idence 



Single pipe 



Inadequate 



Inaccessibility No outdoor production 

Pulled vs. pushed water Lack of security 

Pump size 

Impractical to clean 

Gas bubble disease 
Pumps too small 



Natural spawning time 



lndi\idual spawning 



Env. conditions 
July/Aug. 

High water temp. 
Low DO 
Poor natural food 
Uneven food 
distribution 



Air bubbles under mesh 
Insufficient water flow 
Inexplicable mortalities 



\'islbililv 



Upwellers 



:5(l— too Msiiors per 

year 
Public education 

regarding shellfish 

life history and 

ecology 
Focal point and pride 

of town 



Program expansion to 
one million seed/year 
with high survival 

No winter operation 



Insufficient funding and site selection were primary factors in degree of operational success but public nature of site raised public perception of program through interaction 
with cullurists. 



less at lower density. Because survival was the most important 
result, spreading seed over wide areas seemed to be the best so- 
lution. Monitoring the results of such transplants was difficult. To 
facilitate tracking survival, seed were often planted near a large 
rock or piling, or within certain ranges including a set distance 
from the shoreline and in water shallow enough for future moni- 
toring. For several years, we transplanted .seed by walking along 
the shoreline with buckets broadcasting it freely. Once we knew 
that even the 1 2 to 20 mm seed could yield adequate survival, we 
broadcast larger numbers from a boat. 

From the bottom culture, we learned that certain areas seemed 
to be more conducive to optimal growth or survival. We needed to 
find additional areas where quahog transplants would survive and. 
we hoped, produce future "beds"" of quahogs. We noted that 
shoreline with red sand and visible fresh water rivulets from the 
land were unsuitable for transplants. The combination of red sand, 
an indicator of excessive iron, and the freshets, an indicator of 
groundwater inputs, yielded unacceptable quahog survival. Areas 
of high concentrations of macrophytes in the summer {Ulva lac- 
tiica. Gnicilaria. Enlennnorpha. Calitluiiiiiiii>ii and Afiardhiella) 
species were also poor choices for transplants, because they smoth- 
ered the seed. Sediment in these areas often developed to a heavy 
organic anaerobic mud as part of the eutrophication process. There 
were other areas where an inhospitable environment was more 
subtle and difficult to define, but when the result was poor, we 
abandoned those areas. 

Seed quahogs were planted in September, October, and No- 
vember. When the seed were close to 23 mm (from the rafting 
techniques!, they could be planted in September without much 
problem. They dug in within 2 hours and often within 30 minutes 



and did not seem to be bothered by the major predators. Smaller 
seed, planted in September, were heavily preyed upon. The pri- 
mary predators were baitfish (primarily Euspira). which ate the 
foot as the quahog was digging in. and crabs of various species. 

Laboratory experiments revealed that quahogs stop siphoning 
at 38°F (3.5°C). We experimented with planting times and found 
that if the water temperature had dropped to about 45°F (7.5°C), 
the seed had enough inobility to dig in, but the predators were 
fairly inactive. We often watched quahogs after planting to see 
what happened or came back in a few hours to dig them up again 
and check for evidence of predation. We felt confident, after many 
trials, that this method worked well for us. Thus, holding the seed 
until November became routine practice. 

Planting was done along stretches of shoreline. Because inter- 
tidally transplanted quahogs may not survive well in winter (bot- 
tom experiments), neariy all the seed were planted below the low- 
tide mark. In most of the areas planted, there was a narrow band of 
fringe salt marsh (Spartina aherniflora). and the seed were usually 
planted about 20 to 30 ft (7-10 m) from the marsh edge. The 
subtidal zone that begins 50 to 80 ft (16-24 m) from the marsh 
edge is either eelgrass or silty organic mud substrate and corre- 
sponds with a depth of 4 to 8 ft ( 1-3 m). Quahogs survive in these 
areas, but they grow more slowly and are inaccessible to harvest by 
the general public. Therefore, planting was done in the narrow 
band of 2 to 5 ft ( 1-2 m) depth, about 20-40 ft (7-12 m) from the 
edge of the marsh. 

Site Selection 

Through a process of elimination, we found numerous suitable 
areas for transplant (Fig. 13), some of which were used repeatedly 



1028 



Macfarlane 



ORLEANS 

SEHD TRANSPLANTS 



QUAHAUGS J 976 -1989 1 Jj '^- 

4 




Figure 12. Seed, transplanted in all three estuaries, was the final phase of the program. 



in the transplant procedure. Many of these areas had been produc- 
tive, and we theorized that if they were productive at some point, 
gregarious setting (Hidu 1969) would restore natural production, 
given an infusion of seed. The Town also planted many bushels of 
adult spawner quahogs in the same areas to increase the chances of 
larvae settling in the general vicinity. Amounts planted in all three 
estuaries are as follows: 

1 . Cape Cod Bay was unsuitable for transplants primarily be- 
cause ice build-up scoured the flats when the ice moved. 
Although the seed may have survived, it disappeared from 
the transplant location, and a transplant could not be justi- 
fied. 

2. The Nauset system received a moderate amount of seed. The 
estuary is naturally productive, especially in the Town Cove 
and Mill Pond, both of which are heavily used by harvesters. 
Seed planted there was intended to augment the natural 
production. The Nauset system has several areas reserved 
for family permit holders, where commercial fishing was 
prohibited. We felt that if those areas. Yacht Club, Asa's 
Landing, and Mill Pond were seeded, it would show recre- 



ational users that the program was working. Also, recre- 
ational users would tend to stay in the seeded areas and 
would not be competing with commercial fishermen in other 
areas open to all harvesters. By 1981, natural stock in the 
Town Cove areas traditionally fished by commercial fish- 
ermen was dwindling, and we began planting seed there as 
well. Therefore, we planted a total of 2. 1 million seed in the 
Nauset system. 
3. Pleasant Bay. had exhibited poor natural production for 
many years. The majority of the seed from our program was 
transplanted to the bay. Upper regions of Pleasant Bay. from 
Meetinghouse Pond to Namequoit Point, received the most: 
2.8 million. We had some success in Little Bay outside Pau 
Wah Pond (355.000). but the seed were preyed upon by 
small knobbed whelks {Biisycon carica). The remainder 
were planted in other parts of Pleasant Bay including the 
perimeter of Big Bay. 
Quahogs were harvested in shallow water using "scratchers" 
(short-handled rakes) and in deeper water using "bullrakes" 
(long-handled rakes with baskets) (See Schwind 1977. for ex- 



A Municipal Quahog (Mercenaria mercenaia) Management Program 



1029 



amples of harvesting gear). The deeper waters, ( 12-20 ft, 4-7 ni) 
of Big Bay, had provided employment to about 40 bullrakers for 
some 20 years. Monitoring quahogs planted in the deep water 
would have been difficult, so we limited planting in Big Bay. 

The hydrodynamics of Pleasant Bay changed abruptly and 
drastically in January of 1987, when a breach occurred ni Chatham 
Harbor, resulting in higher tides and greater tidal exchange. Old 
maps of the area indicated the new inlet location after the breach 
approximated that of the 1950s, when the large set occuned in the 
bay. 

MONITORING 

Monitoring was performed as a qualitative check on survival 
and growth, not as a quantitative measure. Survival was estimated 
based on repeated sampling of the transplant areas. Broadcasting 
seed quahogs over large expanses of shoreline to prevent predation 
and enhance survival precluded accurate tracking of the large num- 
ber of seed. However, we observed that in areas where small 
plantings were done near rocks, pilings, or within known ranges, if 
the seed survived the initial transplant and the first winter, they 
were likely to survive to legal size. Our best estimate, is that %) to 
757r survived the transplants, especially if we waited until No- 
vember to plant. 

The author repeatedly sampled most transplant sites, using a 
basket scratcher with 1/4" (6 mm) hardware cloth lining designed 
to catch shell fragments and small seed. At each location, approxi- 
mately 200 ft (60 m) of shoreline was sampled by pulling the 
scratcher through about 6 ft (2 m) of sediment per sample. Seed, 
adult quahogs, and evidence of problems were recorded. The pro- 
cess was repeated several times to cover the distance from the 
marsh edge that had been planted. When we used M. menenaha 
notata. identification of planted seed was easy. Hatchery stock 
without the genetic markings was different in appearance (wider, 
thinner shells, less highly raised ridges), from the natural stock and 
could be easily identified. 

Discussion 

Planting seed by walking the shoreline was fairly accurate, but 
the optimum strip that met the criteria was more difficult to define 
when seed were planted by boat at high tide, and some seed were 
inevitably planted outside the optimum area. We felt that these 
would survive but not be readily harvested and might therefore, 
provide parent stock for repopulation of the area. 

We heavily planted the Town Cove, (Nauset e.stuary) from 
Hopkins Island to the head of the cove at the Yacht Club and Mill 
Pond. We had hydrographic information that suggested turnover 
rate for the Town Cove to be 2 days (Teal 1983, Aubrey et al. 
1997). We planted the area from Meetinghouse Pond to the end of 
Barley Neck and Lonnie's Pond. We surmised from the hydro- 
graphic data that all these areas might retain larvae, because they 
are seniienclosed ponds with narrow openings that lead to the 
mouths of the estuaries. We felt that if those areas were heavily 
planted, and if the conditions were conducive for setting, natural 
productivity could be augmented. We planted the perimeter of Big 
Bay from Quanset to the Town Line because of the large set in the 
1960s, theorizing that it is more likely now for Big Bay to be 
productive again, especially if there are larvae in the water to assist 
in establishing a new set. The change in inlet location could pro- 
vide the conditions conductive for a new set. 

Fishers routinely found planted stock but kept that information 



to themselves. The areas of Pleasant Bay that were heavily planted 
are not easily accessible except by boat and are visually inacces- 
sible to observe fishing activity. Therefore, we did not know who 
was fishing where until after the fact. 

Virtually all littleneck (2" legal stock) quahogs harvested from 
Little Pleasant Bay resulted from this program. Many of them 
showed M. Mercenaria notata stock markings; however, trans- 
plant success was difficult to document, because the transplant 
method and the time lag between transplant and attaining legal 
harvesting size was long (4-7 years). Moreover, the harvest sta- 
tistics did not provide a means of measurement because of: 

1 . lack of differentiation between cultured and wild stock; and 

2. lack of cooperation from fishers to divulge whether they had 
harvested cultured stock, and if so. where they had found 
them. 

The program has been criticized both by fishers and the scien- 
tific community for lack of definitive information regarding the 
survivability of seed transplanted to the natural environment. Field 
trials of small seed (10-18 mm), approximately 500 per trial, 
planted at various locations at varying densities ( 10 to 25/0.3m". 
without any form of protection, were planted in September. Octo- 
ber, and November. Those planted in September were heavily 
preyed upon, and except for extremely low-density plantings (ssl 
per square foot) regardless of size, approximately 90% were dead 
within 1 week. Those planted in October had about 75% mortality, 
but it took about 2 weeks for the mortalities in high density plant- 
ings. Those planted at lower densities exhibited similar mortalities, 
but it took about 1 month. The larger ones (15-18 mm) showed 25 
to 50% mortality. Those planted in November exhibited 10 to 25% 
mortality within the first 2 weeks and nothing thereafter, regardless 
of the density or size, as long as the initial transplant was done 
when the water temperature had dropped to below 50°F. We ob- 
served that seed moved from the place of initial transplant, which 
further complicated recovery and analysis. Often, "lost" animals 
were later found nearby. Most designated as "lost" were predated, 
because .shell fragments were often found in the planting locations. 
Had we used enclosures to track mortality in a more statistically 
accurate manner, we felt we would have been creating an artificial 
situation that would not represent what would happen once the 
animals were freely broadcast. 

The trial plantings suggested that time of planting was as im- 
portant as density and size at planting. A high density in a rela- 
tively small area seemed to draw predators to the area, but broad- 
casting seed in a wider area at very low densities (often «5 per 
square ft) increased survivability. Further increases in survival 
were achieved by planting all seed when the water temperature 
was between 45 to 50°F (7.5-IO°C). Based on the trials, the trans- 
plants from the upwellers, where the seed was between 12 to 20 
mm, were done in November, and transplants of the larger seed 
from the rafts were done in October. 

A total of 6,297,000 seed were broadcast in Orleans, which is 
10,495 bushels of quahogs at a legal size (2" at 600/bushel), if all 
survived. We estimate we added between 5,200 to 7,900 bushels of 
quahogs to the fishery at 50 to 75% survival, with an estimated 
wholesale value of $800,000 to $1,170,000 (average price from 
1980-1990). The seed cost the town $135,000 (exclusive of bi- 
ologist's salary and amortization of equipment. Table 8). 

From 1994 to 1996. we estimate 40 to 60% of the quahogs 
harvested from the Nauset system resulted from seed planted in the 
late 1980s. Those harvested from the Yacht Club, the east shore of 
Town Cove, and Nauset Harbor were especially noticeable. In the 



1030 



Macfarlane 



winters of 1992 and 1993, buUrakers who had moved to Orleans 
from Long Island, NY, were fishing in Town Cove. Local fisher- 
men joined them and worked in the Town Cove for three succes- 
sive winters. Up to 20 boats worked each day. Both the tlshemien 
and Shellfish Department recognized the notata quahogs and knew 
that they were not native stock. Slowly and individually, the local 
fishermen began to acknowledge the success of the propagation 
program. 

CHANGES IN THE PROPAGATION PROGRAM 

In most wild fisheries, the commercial vision and focus is on 
short-term gains rather than long-term benefits. This was a key 
point of Hardin (1968). This was true for our program, where it 
was perceived as a failure by the local fishermen. They had an 
overwhelming desire to see immediate tangible results, such as the 
1" seed from the rafts, but it took 4 to 7 years for the seed to attain 
legal size in many locations. This was too long to wait for visible 
results. 

In 1989, the state withdrew financial assistance to municipali- 
ties for propagation. This had funded a major portion of the pro- 
gram in Orleans. Aside from the biologist's salary, the major pro- 
gram expense was seed cost and the seasonal technician's salary. 
Operating the lab by volunteers was suggested, but was not ac- 
cepted because of perceived liability problems. 

The town had purchased two open space parcels on Pleasant 
Bay that would be suitable as shellfish propagation facilities. After 
the oil spill settlement, plans were drawn to construct a new fa- 
cility at Kent's Point that would include a public education com- 
ponent. The land was purchased for "conservation, open space, 
and passive recreation" with a caveat in the deed that a shellfish 
facility could be the only additional building built on the property. 
A benefactor offered to construct the building and create an en- 
dowment. A second benefactor was ready to lend support. The 
Selectmen would not entertain either offer, stating that, at some 
point, the town would have to pay something. A management plan 
for the property included a shellfish grow-out facility, but the idea 
was not pursued becau,se of fears that the remote area, accessed by 
a private residential road, would be "overburdened" if the shell- 
fish facility were there. The Conservation Commission, who ad- 
minister and oversee the property use, opposed a shellfish facility. 
They added it to the management plan only because the deed 
allows such activity, and it was voted favorably by the citizens at 
a town meeting. 

The decrease in funding, the oil spill of 1985, a smaller incident 
in 1987 that did not cause mortalities but indicated the undepend- 
able nature of the site, the change in Shellfish Department Man- 
ager to one who had no experience with shellfish propagation, the 
fishers' perception that the program was a failure, the success of 
private shellfish farmers in Wellfieet (a neighboring town) using 
bottom culture (see below), and a disturbing trend of degraded 
water quality (Macfarlane 1996), all converged in 1989. The po- 
sition of shellfish biologist was changed to shellfish biologist/ 
conservation administrator. The duties changed from shellfish 
propagation and management to administering the state and local 
Wetlands Protection regulations and environmental planning. No 
further responsibility for work with shellfish propagation was in- 
cluded. The laboratory was closed and razed in 1993. 

Shellfish Propagation after 1990 

The municipal propagation program (1990-1994) consisted of 
floating trays deployed at Asa's Landing (Nauset) and subtidal 



bottom boxes at six other areas. Trays received 10 to 20.000 (2.5 
mm) quahogs per tray at $10.00/1,000, and bottom boxes were 
stocked with 10 to 15,000 quahogs (2.5 mm) per box. 

The floating trays were used, becau.se they were easier to 
handle than the old rafts, and another town had indicated success- 
ful results with the floating trays. They were 3' x 4', constructed 
of wood and netting with solid covers to prevent bird damage. The 
covers reduce water flow, but the seed are not predated and do not 
wash out. Seed ordered for May delivery was usually received in 
late June or July. The seed were placed in the floating trays the first 
summer. They transfer the 10 to 12 mm seed to subtidal bottom 
boxes in late summer or early fall. They are retained in the bottom 
boxes for a second season and winter until they are about 18 mm, 
when they are finally transplanted to the natural environment. 
Those transplants take place in the spring. 

The bottom boxes were constructed of 2" x 6" stock, 5' x 10' 
with 1" galvanized coated wire bottom mesh covered with land- 
scape cloth. They are filled with sand, planted with seed, and 
covered with 1/4" plastic mesh covers constructed with laths (1" x 
2") attached to the sides. 

The stock bought in 1994 remained in bottom boxes for 3 years 
before transplant. Covers remained on the boxes during the winter, 
and there was little damage, because there was little ice cover in 
those years. In 1997, the Shellfish Department deployed 10 float- 
ing trays in Lonnie's Pond and 25 bottom boxes. Approximately 
50% of the 1991 stock was lost in a hurricane. There is no record 
of whether the loss was actual mortality or displacement of the 
seed. 

In 1996 to 1997. the Department purchased "field planting"- 
size quahogs. about 12 to 20 mm at $25.00/1.000. They changed 
their strategy for nursery culture and now buy small seed 1 year 
and large field plant seed the next to stagger the nursery require- 
ments. Lack of funding for personnel to care for smaller seed has 
resulted in losses caused by heavy fouling, predation if holes de- 
velop in protective netting, and other problems. From 1990 to 
1997. 5.4 million quahogs were grown in nursery culture. They 
estimate survival from purchase to transplant in the field to be 
about 50 to 60% or 2.7 million seed. There are no estimates of 
survival once the seed are transplanted. Larger seed can be planted 
without the nursery phase and with a propagation budget of 
$9,000, but options are limited. Bay scallop (Argopecten irradians 
irradians) seed (865,000 since 1992) are also purchased with the 
funds. 

Commercial fishers have recently assisted the Shellfish Depart- 
ment in harvesting and transplanting seed, making it a community 
project rather than a departmental one. 

Orleans constructed a tidal upweller in 1990 based on plans by 
Mook (1988). Although a smaller version worked well in the 
neighboring town of Eastham, it did not work well in Orleans in 
the initial year, and the idea was never modified. It is difficult to 
find an area with enough tidal current necessary to operate the 
upweller successfully in Orleans that does not have substantial 
boating activity. Without that method. Orleans must rely on bot- 
tom or off-bottom culture or on a shore-based nursery for propa- 
gation methods. 

PRIVATE AQUACULTURE IN ORLEANS 

The first private aquaculture authority was specifically granted 
to Eastham, Orleans, and Wellfieet ( 1904) to allow "bedding" of 
quahogs until the market was more favorable (Belding 1912). In 



A Municipal Quahog {Mekce\akia mercenaia) Management Program 



1031 



1909. the legislature granted licensing authority tor planting and 
cultivating quahogs to all the towns in Massachusetts. For many 
years, the only propagation in most towns was public and consisted 
of transplanting adult "spawning stock"" to stimulate a set of qua- 
hogs. as similarly described by Kassner and Malouf ( 1982). 

In the late 1980s, private aquaculture developed on the Cape. 
The success of municipal efforts described for Orleans prompted 
private shellfish farmers in Welltleet to adopt low-cost intertidal 
bottom culture. Their success increased requests for leases or shell- 
fish grants in many towns. Modified predation enclosures were 
improved (iron rebar rather than wood) for predator e.\clusion. 

In Orleans, the town adopted a shellfish management plan 
(Macfarlane 1986) that outlined a system for private aquaculture to 
be initiated in Orleans. The plan included several recommenda- 
tions. 

1 . Three areas to be considered for grants: 

A. From Old Field Point to Pochet Inlet (Pleasant Bay); 

B. Pleasant Bay west of Nauset barrier beach; 

C. Cape Cod Bay flats; 

2. Grants to be 1/2 acre for the first year, with an option for 
renewal and expansion; 

3. upper limit of 5 acres/grant; 

4. upper limit of 3 grants/area, with buffer zones between 
grants; 

5. grants to be self-policing; 

6. all grants to be surveyed by a registered land surveyor and 
survey to be recorded and on file at the Town Hall; and 

7. subleasing of grants to be prohibited. 

After 2 years of debate, the plan was implemented. The issue of 
designating public bottom for private enterprise was the primary 
focus of the debate. It was a common public perception that grants 
in existence in the 1960 to 1970s had been mismanaged. The 
pervasive feeling voiced strongly at public meetings was that pub- 
lic waters should not be leased to private entrepreneurs, except for 
small isolated cases. At the time, Orleans had eight grants ranging 
in size from 0.06 acre to 1.0 acre (most were 0.5 acre). A single 
10-acre grant lease had not been renewed because of nonuse. Lease 
applications meeting state criteria were usually approved by the 
town, because there was no plan to determine where additional 
grants could or should be sited. 

The Shellfish Department determined that two areas in Pleasant 
Bay could be set aside for shellfish grants. The primary area (from 
Old Field Point to Pochet Inlet north of Sampson Island) has a 
maximum depth of 2 ft (0.6 m) at low tide, was not under riparian 
rights of upland property owners, was not heavily used for boating, 
was "out of the way"" for most bay users, and importantly for 
Massachusetts laws, had been unproductive of shellfish resources 
for at least the last 40 years. 

Shortly after adopting the Management Plan, the Selectmen 
changed the regulations to two acres per grant (maximum), and the 
total grant area in the primary zone was expanded to 30 acres. By 
1990. there were three new grants, all in the primary area in Pleas- 
ant Bay; two grantholders relinquished their acreage by 1993. New 
grants were not surveyed, because grantholders argued that the 
requirement was prohibitively expensive. Buffer zones were es- 
tablished haphazardly, if at all. As a result of this activity, the 
subject of shellfish grants has remained contentious over the past 
decade. 

A portion of the grant debate has centered on determining grant 
use. By 1994, there were 15 new grants. The Shellfish Advisory- 
Committee tried to ascertain how people were using the grants. 



The following year, six new individuals requested 1/2 acre 
grants. The Shellfish Department recommended against the pro- 
posed leases, because if the existing 15 grantholders were allowed 
to expand to the legal 2 acre maximum, there would not be enough 
room to accommodate 2 1 grants. The Selectmen were pressured 
into allowing the additional licenses and amended the grant regu- 
lations. The primary reason for the increased activity and political 
pressure was the availability of Federal funds to aid displaced 
fishers in the Northeast. The six were granted licenses, but only 
one received Federal funding. 

With a waiting list for btnh grant expansions and for new 
grants, it was determined that current license holders must prove 
that they are actively engaged in aquaculture either by seed re- 
ceipts or by compliance with a "'density requirement" enacted to 
ensure that the grant is being "worked."" Those who opposed 
adding new grants suggested that if the grants were not "worked,"" 
the bottom should be relinquished. After considerable debate, 
regulations were adopted by the Selectmen to include a density 
requirement (minimum 50 quahogs/square foot). To maintain an 
existing grant for the 5-year lease, 75% of the area had to be 
planted with the minimum density. Furthermore, no expansion 
would be considered unless 75% of the existing grant area were 
planted with the minimum density. Compliance was required by 
December 31. 1996. 

Before the deadline, aquaculturists requested a review of the 
regulations and proposed an alternative. New regulations were 
enacted in May. 1997. including a 3-year planting schedule, by 
which 50% of the grant must have a density of 30 quahogs per 
square foot or an expenditure of $2,500 per 1/2 acre expended 
annually (on shellfish only), with proof given through seed re- 
ceipts. A hardship clause was also included. If grants were relin- 
quished, expansion preference for the area would be given to origi- 
nal grantholders. Only after expansion would a potential aquacul- 
turist on the waiting list be given an opportunity. Sale and/or 
transfer of licenses was prohibited. 

As of 1997. there were 26 grants in Orleans. Those who re- 
ceived 1/2 acre from changes in regulations, insist that the town 
find suitable acreage for expansion. To date, the town is waiting 
for a regional resource management plan to be completed and 
adopted. The proposed "density requirement"" has sparked con- 
tinual controversy and has yet to be resolved. 

Resource-Based Management Plans 

In 1995. Orieans entered into an agreement with the neighbor- 
ing towns of Brewster. Harwich, and Chatham to develop a Re- 
source Management Plan for Pleasant Bay. a state-designated Area 
of Critical Environmental Concern (ACEC). The plan was divided 
into five issues: structures (docks, piers, and erosion control struc- 
tures), shellfish/aquaculture, biodiversity, boating and navigation, 
and public access. Five working groups met during the winter of 
1997 to discuss key issues, and each group provided recommen- 
dations and/or comments. No consensus was reached on aquacul- 
ture. 

Orleans is the only town of the four that allows private aqua- 
culture. Working groups independently suggested formation of a 
scientific panel/oversight group that would initiate requests for 
specific studies and review documentation as it became available. 
One key data gap is an ecological assessment of the bay: the latest 
information is from the 1960s (Fisk et al. 1968), (U.S. Army Corps 
of Engineers 1968). 



1032 



Macfarlane 



Not all the dynamic fisheries in the Bay are regulated. For the 
past 2 years ( 1995 to 1 997), razor clams (Ensis directus) have been 
harvested in quantity, and one fisher has harvested horseshoe crabs 
iLinniliis pitlyphemiis) for about 20 years. The crabs are trucked to 
Falmouth, bled for their medically important lysate, and returned 
to the Bay. The fisherman has harvested crabs throughout the 
shallow bay waters (Harrington, pers. comm.). These are the same 
areas requested for grant expansion in both the primary and sec- 
ondary areas allowed for grants in Pleasant Bay. Neither of the 
above fisheries is regulated, but state law. significantly modified in 
1995. requires that a grant cannot be issued for an area that would 
affect the town's natural resources. Before the newest changes, 
grants could not affect a town's shellfish resources, narrowly de- 
fined as clams (Mya arenaria Linne), quahogs (Mercenaria iner- 
cenaria). scallops {Argopecten irradians irradians). mussels 
{Mytilus ediilis). and oysters t.Crasostrea virginica Gmelin). In 
addition, horseshoe crabs have been listed as shelltlsh predators. 
Although this would seem to preclude all leasing, the regulations 
have not had that effect, but there has been controversy over what 
would constitute significant adverse effect to the town's natural 
resources. 

Quahog Diseases 

In 1995, a phenomenon was described in Duxbury and Prov- 
incetown. MA where cultured quahog stock was dying just before 
reaching market size. In their paper Smolowitz et al. (1997) de- 
scribed the parasitic infestation QPX (Quahog Parasite Unknown) 
as the primary cause for the mortalities. 

Smolowitz et al. (1996) described the history, symptoms, and 
possible protective measures in an advisory bulletin. The organism 
was first identified in a dense natural set of wild hard clams in New 
Brunswick, Canada in 1959. and in 1989. at a nursery on Prince 
Edward Island, NB. Unpublished observations of Ford et al. (1976) 
and Smolowitz ( 1 992) found QPX-like organisms associated with 
dead and dying hard clams in natural populations from Barnegat 
Bay. NJ and Mitchell Ri\er. Chatham. MA. The Provincetown 
infestation occurred to hard clams being cultured and the QPX-like 
organism was the only microorganism found in the tissues. 

The life history of the QPX organism is unknown. The organ- 
ism may be present in the water column or sediment of growing 
areas. It is unlikely that the infestation originates from hatcheries 
(Ford et al. 1997). Crowding can be a form of stress that may 
compromise animals, and optimum planting density for each area 
is unknown. 

The ramifications of this disease are germane to the shellfish 
propagation efforts of both the town and the private cultunsts. At 
this point in the investigation, density may play a role, but its 
effects are unknown. 

If density proves to be a factor, this poses somewhat of a 
dilemma for Orleans. The town wants proof that leased bottom is 
worked and have required a "density" regulation. But high den- 
sity may encourage proliferation of the QPX. Grants squeezed into 
a relatively small area and located on "barren" ground as defined 
by the state may add stress to the animals and may increase the 
probability of infection. With 75% of the grant area required to be 
worked, crop rotation is limited. 

BUDGET/FINANCIAL CONSTRAINTS 

In 1974. the state provided financial assistance for shellfish 
propagation and established a reimbursement program. A percent- 



age of a town's shellfish department operational budget was re- 
imbursed for the shellfish projects. 

Funding was limited and a continual hindrance throughout. The 
increased lab operations costs from 1985 to 1989 were attributable 
to continuing the technician's salary through October. An operat- 
ing budget of $12,000 produced wild fishery quahogs worth 
$150,000 wholesale. 

The total propagation budget for 1997 was S9.000. With higher 
seed costs ($10.00/1,0(30 for 2.4 mm quahogsl. including scallops, 
the town has a limited propagation effort. The Shellfish/ 
Aquaculture workgroup for the Pleasant Bay plan placed a high 
priority on increased funding for shellfish propagation. This pri- 
oritization was based on shellfish catch statistics since 1970 (Table 
7). Although propagation costs have been modest, the value of 
shellfish to the town is considerable. 

The funding was the rationale for Orleans to hire a shellfish 
biologist and start the seed program. The funding decreased over 
time but lasted until 1989 (Table 8). It ended because of fiscal 
constraints at the state level and perceived misuse of funds by 
many towns from the intended purpose. 

Compilation of these data was stimulated because of the need 
to assist Orleans Shellfish Department and others seeking to justify 
funding shellfish propagation. The economic returns to the town 
are significant, but tangible benefits also accrue because of the 
need to maintain water quality in shellfish areas. In addition, local 
propagation efforts can serve as a catalyst to reconnect town resi- 
dents with their harvesting of natural resources nearby. 

CONCLUSIONS 

Orleans demonstrated that municipal propagation of hatchery- 
reared nursery cultured quahogs (Mercenaria mercenaria) is a 
feasible and economically viable way to augment natural produc- 
tion. Orleans was the first town in Massachusetts to specifically 
hire a biologist for shellfish propagation and management. Over a 
15-year period, from 1975 to 1990, we developed and utilized 
techniques that including bottom, rafts, a hatchery, and upweller 
culture with a maximum annual budget of slightly over $12,000 
(exclusive of the biologist's salary). The town successfully trans- 
planted over 6.."? million seed from 1975 to 1989. 

All methods showed promise, but the upweller technique 
proved to be the most successfijl in terms of survival and the 
number of animals that could be cultured. gi\'en the program con- 
straints. All methods are labor mtensive, demand close attention, 
and all are dependent on natural conditions. The latter are erratic, 
and disaster can strike with little or no notice. 

Our original intent was to discover if hatchery-raised quahogs 
could be utilized for propagation efforts. We showed that regard- 
less of seed supply, hatchery-raised seed survived and grew. 

The discovery that temperature plays a critical role in surviv- 
ability of transplanted stock was crucial to our success. The ob- 
servations, in the lab and in the field, on activities of the seed and 
other biota resulted in determining 45°F (7°C) as the optimal tem- 
perature for transplant. Transplants of different seed sizes led us to 
conclude that we could successfully transplant seed as small as 12 
to 15 mm, with no additional protection, as long as we waited until 
November to plant. 

. We experimented with three different types of bottom nursery 
enclosure designs at 20 separate locations over a 3-year period. 
Enclosure design is critical to the success of bottom culture. Some 
areas were clearly better than others, but reasons for success were 
poorly understood. 



A Municipal Quahog (Mercenaria mercenaia) Management Program 



1033 



TABLE 7. 

Total hardest (commercial and recreational) of lour shellllsh species: clams = soft-shelled clams {Mya arenaria); quahogs = hard-shell clams 
(Mercenaria mercenaria): scallops = ba) scallops {Argopeclen irradians irradians). and mussels = blue mussels {Mytilus edulis): value = 

wholesale value at the time of harvest. 



Shellfish Harvested in Bushels 






Clams 






Quahogs 






Scallops 




Mussels 




Value 


Total 


Year 


Com 


Rec 


Total 


Com 


Rec 


Total 


Com 


Rec 


Total 


Com 


Rec 


Total 


Com 


Rec 


Value 


1970 


730 


930 


1 .660 


17,085 


140 


17.225 


1,255 


485 


1 ,740 


6,100 


125 


6,225 


$195,069 


$15,861 


$210,930 


1971 


464 


1.134 


1.598 


14,186 


250 


14.43f) 


1 ,540 


286 


1.826 


9,150 


125 


9,275 


$308,937 


$7,822 


$316,759 


1972 


637 


524 


1,161 


5,840 


151 


5.99] 


3.567 


700 


4,267 





35 


,15 


$167,328 


$23,102 


$190,430 


1973 





195 


195 


1 7,395 


95 


17.490 


425 


5 


430 











$219,525 


$5,025 


$224,550 


1974 


5.699 


2.170 


7,869 


12,460 


1,143 


13.605 


2,050 


210 


2.260 


1,200 


10 


1,210 


$263,910 


$67,815 


$331,725 


1975 


1.760 


360 


2,120 


12.577 


855 


1 3,432 


915 


110 


1,025 


1,800 


50 


1.850 


$364,615 


$70,505 


$435,120 


1976 


2.603 


893 


3,496 


10.945 


716 


11,661 


31,490 


757 


32,247 


5,030 


450 


5.480 


$760,521 


$75,536 


$836,057 


1977 


3.098 


1.017 


4,115 


9.912 


1 .007 


10,919 


8,372 


122 


8,494 


12,632 


163 


12.795 


$570,887 


$58,518 


$629,405 


1978 


2.404 


981 


3.385 


5,633 


764 


6,397 


160 


38 


198 


3,090 


231 


3.321 


$324,094 


$57,826 


$381,920 


1979 


6,118 


1.344 


7.462 


5,014 


1.339 


6,353 


240 


60 


300 


2,440 


406 


2.846 


$508,196 


$89,958 


$598,154 


1980 


6,499 


1.336 


7.835 


5.612 


971 


6,583 


15,2.^0 


612 


15,842 


6,150 


550 


6.700 


$1,243,005 


$121,256 


$1,364,261 


1981 


5,285 


718 


6.003 


3.665 


738 


4,403 


3,455 


65 


3,520 


10,100 


800 


10.900 


$600,912 


$71,058 


$671,970 


1982 


2.7SI 


522 


3.303 


3,264 


727 


3,991 


4,958 


760 


5,718 


19,400 


540 


19,940 


$606,748 


$87,424 


$694,172 


1983 


3,684 


206 


3.890 


3,746 


592 


4,338 


40,000 


4,000 


44,000 


10,200 


340 


10,540 


$1,908,264 


$181,984 


$2,090,248 


1984 


2,599 


193 


2.792 


2,458 


342 


2,800 


821 


70 


891 


1,145 


104 


1,249 


$278,402 


$24,246 


$302,648 


1985 


1,006 


111 


1.117 


2,924 


489 


3,413 


6,000 


3,545 


9,545 


8,105 


115 


8,220 


$131,761 


$487,150 


$618,911 


1986 


648 


79 


727 


2,181 


280 


2,461 


235 


31 


266 


2,390 


215 


2,605 


$176,896 


$20,597 


$197,493 


1987 


880 


88 


968 


2,023 


428 


2,451 











605 


103 


708 


$173,181 


$25,033 


$198,214 


1988 


615 


50 


665 


2,310 


382 


2,692 


2,5.10 


101 


2,631 


500 


100 


600 


$291,980 


$26,043 


$318,023 


1989 


545 


50 


595 


3,800 


350 


4,L50 


990 


50 


1,040 


3,510 


20 


3,530 


$326,170 


$20,310 


$346,480 


1990 


803 


75 


878 


3,889 


375 


4,264 


860 


50 


910 


3,261 


20 


3,281 


$463,073 


$26,610 


$489,683 


1991 


1,826 


75 


1,901 


5.796 


400 


6,196 


1,888 


475 


2,363 


4,740 


50 


4,790 


$507,320 


$51,500 


$558,820 


1992 


2.325 


100- 


2,425 


3.258 


250 


3,508 


2,450 


250 


2,700 


4,375 


50 


4,425 


$597,430 


$34,150 


$631,580 


1993 


1.187 


100 


1,287 


3.006 


300 


3,306 


355 


25 


380 


2,866 


25 


2,891 


$346,788 


$27,550 


$374,338 


1994 


1.867 


175 


2,042 


3,588 


450 


4,038 


701 


5 


706 


1 ,635 


15 


1,650 


$436,206 


$41,550 


$477,756 


1995 


9.233 


500 


9,733 


4,072 


450 


4,522 


298 


4 


302 


1,659 


10 


1,669 


$1,038,210 


$67,404 


$1,105,614 


1996 


5.0S0 


1 .5.50 


6,630 


1 3,567 


625 


14,192 


1,286 


110 


1,396 


1,112 


10 


1,122 


$1,441,932 


$171,140 


$1,613,072 



Where mortality was attributable to predation, numerous preda- 
tors were found, including crabs (many species), drills (Urosalpin.x 
cinerea and), moon snails (Liinatia lieros). and several species of 
fish, including Euspira. 

Floating sand box rafts worked very well, but the deployment 
and hauling were unwieldy. Growth and survival were far superior 
to the bottom boxes, and the results were reproducible and de- 
pendable year after year without change in design or implemen- 
tation. Our problems with the rafts centered on the necessity to 
plant smaller animals each year (because of rising costs of seed), 
and our reluctance to field plant anything less than 20 mm in the 
fall (before we had discovered temperature as the crucial factor) 
for fear of poor survival. Using the rafts earlier in the year, when 
the weather had not stabilized, increased losses. Additional raft 
problems came from decreased flushing and increased nutrient 
enrichment in the deployment area. Although growth was still 
good, fouling of the rafts increased maintenance. In addition, to 
increase seed output to a million per year or more could not be met 
with the rafts, because they were too big, and we were unwilling 
to add additional rafts to the pond. 

Our efforts to operate a hatchery were not highly successful. 
The refusal to install a dual-intake flowing water system and the 
purchase of pumps that were too small were critical mistakes. The 
decision to construct a bulkhead and parking area creating a de- 
sirable public access point created havoc in our system. Mass 



spawning may have been preferable. We felt we would have more 
control over individual spawnings, but this was difficult to achieve 
with our system. The real obstacle to hatchery success was cul- 
turing the postset animals. 

Upweller methodology allowed us to use our flow-through sys- 
tem, and it allowed us to raise a million seed per year, at 95% 
survival, within the building. Expansion within a secured enclo- 
sure outside could have doubled our output, but lack of additional 
personnel and budget constraints prevented this. Volunteer labor, 
(presently the backbone of many community efforts) was not al- 
lowed for fear of liability. It is the author's opinion, judging by the 
number and frequency of comments heard, that volunteers would 
have been willing to work. The location of the facility fueled the 
perception that the entire project was foolhardy. The loss of a 
year's work in 1985 because of an oil spill pointed to the fragility 
of our site. Measures were taken to reduce the threat of human 
interference, but the area was still highly used and highly visible. 
We had prepared plans to move the facility to a more hospitable 
site, but that option was not supported. 

A major problem in raising quahogs in our locale is that they 
take at least 4 years to mature. Politically, 4 years is longer than 
policy makers can wait to see tangible results. Every year added to 
our knowledge of how to increase survival in our area, but any- 
thing that went wrong meant a "wasted"" year. Damaging winters, 
oil spills, poor summer weather, human error, and human inter- 



1034 



Macfarlane 



TABLE 8. 
Orleans quahog propagation budget including costs of program and income from permit fees. 







Cost 










Income 










1 


Permits 










Lab 
(Includes 


Rafts/ 






Com 




Rec 


Total 
















Year 


Seed 


Labor 1 


Boxes 


Total 


No. 


Fees 


No. 


Fees 


Income 


1975 


$80 




$100 


$180 


91 


$910 


979 


$4,291 


$5,201 


1976 


$3,637 




$1,250 


$4,887 


140 


$1,400 


924 


$3,084 


$4,484 


1977 


$6,994 




$1,250 


$8,244 


114 


$1,140 


979 


$4,178 


$5,318 


1978 


$1,040 




$250 


$1,290 


107 


$1,070 


1121 


$4,684 


$5,754 


1979 


$4,500 


$7,060 


$1,000 


$12,560 


142 


$1,420 


1.285 


$6,180 


$7,600 


1980 


$3,500 


$8,912 


$0 


$12,412 


248 


$2,480 


1.533 


$8,068 


$10,548 


1981 


$4,500 


$8,000 


$0 


$12,500 


167 


$6,782 


1.581 


$9,658 


$16,440 


1982 


$2,100 


$6,400 


$0 


$8,500 


180 


$9,000 


1,928 


$8,475 


$17,475 


1983 


$3,500 


$5,000 


$0 


$8,500 


336 


$16,800 


1,428 


$12,725 


$29,525 


1984 


$2,000 


$5,000 


$0 


$7,000 


354 


$17,700 


1.409 


$8,355 


$26,055 


1985 


$6,000 


$5,500 


$0 


$11,500 


225 


$12,200 


1,253 


$9,223 


$21,423 


1986 


$6,200 


$5,700 


$0 


$11,900 


167 


$11,250 


1.192 


$7,194 


$18,444 


1987 


$6,200 


$5,700 




$11,900 


167 


$8,350 


1,059 


$6,900 


$15,250 


1988 


$6,200 


$5,900 




$12,100 


285 


$8,350 


1.113 


$6,380 


$14,730 


1989 


$6,200 


$6,000 




$12,200 


195 


$14,250 


1.248 


$6,155 


$20,405 


TotuI 


$62,65 1 


$69,172 


$3,850 


$135,673 




$113,102 




$105,5.50 


$218,652 



vention all took their tolls on the product. These factors made 
budgetary justification difficult; neither finance committee mem- 
bers or Selectmen were willing to take positive results on faith. 
There was also a feeling that the town should not be conducting 
research, because leaders felt that the state should be doing the 
work. Because shellfish management is a town responsibility, the 
Shellfish Department felt justified and mandated by law to manage 
shellfish to the best of our ability, including experimentation. 

Several decisions were made that may have contributed to the 
program's decline. One was to plant many "family permits only" 
areas in certain high visibility locations. Although the program 
gained support and admiration from many recreational shellfish 
permit holders, it did not gain credibility with the commercial 
fishers, who were not allowed to fish in those areas. 

The second decision was to plant areas that were sparsely popu- 
lated with quahogs but not to advertise (hat they had been planted 
in those areas. General planting locations were given to anyone 
who asked, but we were not specific. Because the seed was broad- 
cast at low densities along long stretches of shoreline, and it took 
4 to 7 years to reach harvestability. a lack of confidence developed 
among the commercial fishers that anything was being done. 

The third was to plant all the seed without enlisting the services 
of any other user groups. Without that interaction, there was only 
criticism and lack of support from all directions. 

Although we stand by our original intent to try to augment the 
natural production of Pleasant Bay, which was sparsely populated 
with quahogs, the amount of seed produced for such an extensive 
area proved to be inadequate to make a difference visually or 
statistically. We believed that when the transplanted seed matured. 
it would reproduce, and when of legal size, it would be harvested. 
We had hoped that enough quahogs would be harvested from our 
program to prove its worth to the town. However, our intent was 
not a "put and take" program, where every quahog that was 
planted was harvested several years later. Parts of the bay now 
producing quahogs were not producing any when we started the 
program. A total of about 9.0 million seed added to the waters 



from 1975 to 1995 were more tangible than relying on spawner 
transplants. 

The Shellfish Departmenl was. is. and will be short staffed. 
With the change in position of the biologist to conservation ad- 
ministrator, the Shellfish Department lost an employee whose re- 
sponsibility was strictly shellfish propagation and management. 
The Shellfish Constable is (and was) also the Harbormaster and, 
therefore, has a dual role in a town with three separate estuaries, a 
migrating barrier beach with one of the three most dangerous inlets 
on the East Coast, and a harbor that boasts being the largest charter 
fishing fleet in New England. The Department Manager from 1974 
to 1983 called himself Shellfish Constable first and Harbormaster 
second. The next three succeeding Department Managers switched 
the order of their title. Although possibly insignificant, it subtly 
shows which duty is first priority. 

Shellfish records kept after 1990 were inconsistent, and propa- 
gation program records were very difficult to obtain. The depart- 
ment duties overwhelm the staff, and propagation program record 
keeping is not high on the priority list. No one person has respon- 
sibility for records or for maintaining nursery culture systems, and 
inaintenance is "as needed" or when time permits. Research for 
this document has heightened the necessity of good records for 
accountability and historical perspective. Records also project a 
positive professional perspective to the public when requesting 
funding. 

We remain optimistic about the future of shellfish in Orleans. 
Our experiments of the mid-1970s using hatchery-raised seed have 
become routine grow-out procedures for both municipal programs 
and private aquaculture. Both Chatham and Harwich, towns that 
border Orleans on Pleasant Bay. have municipal programs. East- 
ham has continued its efforts. Orieans has continued to use nursery 
grow-out techniques to supply seed for the public fishery. 

This paper documents work involved in nursery culture of qua- 
hogs and the management options used by the town. Economics, 
politics, and social pressures played enormous roles in the opera- 
tion and evolution of the program. Shellfish were, and still are. an 



A Municipal Quahog {Mercenaria mercenma) Management Program 



1035 



imponaiit facet of the town character, and ahhouyh interest in 
shellfish seems to be waning with the gentrification of the town, 
surveys continue to show that whether people actually go shell- 
fishing, the fact that shellfish are there to take as a continuing 
natural resource is important, and citizens are willing to pay for the 
privilege. 

In retrospect, Belding (1912) was right: "growth in any par- 
ticular locality can be determined only by experiment" " and with- 
out culturing. the fishery will collapse. What Belding could not 
have foreseen was the enormous social change and attendant eco- 
nomic and political forces that become part of the "experiment." 

Dedication 

This manuscript is dedicated to Gardner Munsey, Orleans 
Shellfish Constable from 1974 until his retirement in 198.3. With- 
out his vision for Orleans as the shellfish Mecca of Massachusetts, 
his inquisitiveness. and his never-ending support for all aspects of 
a program to attain that goal, none of the preceding would have 
been possible. 

ACKNOWLEDGMENTS 

Many individuals from myriad institutions, research centers, 
and private hatcheries assisted us in developing our program and 
we are grateful to all, especially those in the following list and 
those who may have been inadvertently omitted. Dick Krause 



(Aquacultural Research Corp.); Robert Guillard, (Bigelow Labo- 
ratory for Ocean Sciences); Jeff Kassner (Brookhaven NY Depart- 
ment of Environmental Protection); Kass Abreu, (Chatham, MA 
Shellfish Warden); Henry Lind (Eastham, MA Natural Resources 
Department), Scott Colby, (Edgartown, MA Shellfish Depart- 
ment), George Souza (Falmouth, MA Shellfish Department), Chris 
Heinig and Brian Tarbox (Intertide) Dana Wallace and Walter 
Foster, (Maine Department of Marine Resources), Jim Chalfant 
(Maritec, Inc.), Rick Karney (Martha's Vineyard Shellfish Group), 
Anthony Calabreeze, Ed Rhodes. Jim Widman, Ron Goldberg, 
Warren Landers, Steve Tettleback. Jim Hanks. Walter Blogoslaw- 
ski. Gary Wikfors and Ravena Ukeles (NOAA Milford CT Labo- 
ratory), Bob Malouf, Paul Flagg, Scott Sidell, Monica Bricelj 
(SUNY Stony Brook). Jim and Cindy Smith; Sam Chapman, Herb 
Hidu, Bemie McAlice, and Dave Dean (University of Maine, Dar- 
ling Marine Center), Mike Castagna, John Kraeuter, and Mary 
Gibbons (Virginia Institute of Marine Science), Scott Gallagher, 
Roger Mann, John Ryther, Judy (Capuzzo) McDowell, (Woods 
Hole Oceanographic Institute), and especially to my husband, 
Bruce Macfarlane, for his patience, technical acumen, power of 
observation, and genuine interest in all the permutations of this 
program. 

Author's present address; P.O. Box 1 164, Orleans. MA 02653. 
E-mail: sandymac@capecod.net 



LITERATURE CITED 



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Phycol. 14:224-234. 

Anderson, D. M. 1979. Toxic dinonagellate blooms in the Cape Cod region 
of Massachusetts, pp. 145-150. /;;.■ D. L. Taylor and H. Seliger (eds.). 
Toxic Dinotlagellate Blooms, Developments in Marine Biology, vol. I. 
El.sevier/Nonh Holland. New York. 

Aubrey. D.. G. Voulgaris. W. D. Spencer & S. O'Malley. 1997. Tidal 
residence times within the Nauset Marsh system. Rep. suhmilted to the 
Town of Orleans. Woods Hole Oceanographic Institution. Department 
of Geology and Geophysics. Woods Hole. MA. 

Bayes. J. C. 1 98 1. Forced upwelling nurseries for oysters and clams using 
impounded water systems, pp. 73-83. In: C. Claus. N. DePauw and E. 
Jaspers (eds.). Nursery Culturing of Bivalve Molluscs. Spec. Publ. 7. 
European Mariculture Society. Bredene. Belgium. 

Belding. D. L. 1912. A report upon the quahog and oyster fisheries of 
Massachusetts, including the life history, growth, and cultivation of the 
quahog. Department of Conservation. Commonwealth of Massachu- 
setts. Wright and Potter Printing Co.. Boston. MA. 1 134 pp. 

Carriker. M. R. 1959. The role of physical and biological factors in the 
culture of Crassostrea and Mercenaria in a salt water pond. Ecol. 
Mono. 29:219-266. 

Carriker. M. R. 1961. Interrelation of functional niorpholog\. beha\ior. 
and autoecology in early stages of the bivalve Mercenaria mercenaria. 
J. Eltsha Mitchell Sci. Sac. 77:168-241. 

Castagna. M. & J. N. Kraeuter. 1977. Mercenaria culture using stone ag- 
gregate for predator protection. Proc. Natl. Shellfish. A.ssoc. 67:1-6. 

Castagna. M. & J. N. Kraeuter. 1981. Manual for growing the hard clam. 
Mercenaria. Special Rept. 249 in Ocean Engineering and Applied Ma- 
rine Science. Virginia Institute of Marine Science. 110 pp. 

Fisk. J. D.. C. E. Watson & P. G. Coates. 1967. A study of the marine 
resources of Pleasant Bay. Commonwealth of Massachusetts Division 
of Marine Fisheries, Monograph Series 5. 56 pp. 

Flagg. P. J. 1981. Effects of environmental factors and methods of protec- 
tion on the growth and survival of hatchery-produced seed clams Mer- 
cenaria mercenaria Linne (1758). Master's Thesis. Marine Environ- 
mental Sciences Program. State University of New York at Stony 
Brook, December. 1981. 109 pp. 



Ford. S. E.. R. Smolowitz. L. M. Ragone Caivo, R. D. Barber, & J. N. 
Kraueter. 1997. Evidence that QPX (quahog parasite unknown) is not 
present in hatchery-produced hard clam seed. J. Shellfish. Res. 16:5 19- 
521. 

Gates, D. A. 1964. A preliminary report on the feasibility of oyster propa- 
gation in Paw Wah Pond and a Pleasant Bay survey indicating the 
extent and condition of the quahog IVeniis mercenaria) resources. 
Rept. to the Orieans Board of Selectmen. October 15. 1964. 

Guillard. R. L. 1975. Culture of phytoplankton for feeding marine inver- 
tebrates. In: W. L. Smith and M. H. Chanley (eds.). Culture of Inver- 
tebrate Animals. Plenum. New York. 

Hardin, G. 1968. Tragedy of the Commons. Science 162:1243-1248. 

Haskin, H. H. 1949. Growth studies on the Quahog, Venus mercenaria. 
Proc. Natl. Shellfish. Assoc. 39:67-75. 

Haskin, H. H. 1952. Further growth studies on the quahog. Venns merce- 
naria. Proc. Natl. Shellfish. Assoc. 42:181-187. 

Hidu. H. 1969. Gregarious setting in the American oyster Crassostrea 
virginica Gmelin. Chesapeake Sci. 10:85-92. 

Hidu. H. & H. S. Tubiash. 1963. A bacterial basis for the growth of anti- 
biotic-treated bivalve larvae. Proc. Natl. Shellfish. Assoc. 54:25-39. 

Judson, W. I.. R. Macpherson. P. Stewart & W. N. Carver. 1977. Culture 
of the quahog from hatchery-spawned seed stock. Prince Edward Island 
Department of Fisheries. Charlottetown. Price Edward Island. Tech. 
Rept. Series 185. 

Kassner. J. & R. E. Malouf. 1982. An evaluation of "spawner transplants" 
as a management tool in Long Island's hard-clam fishery. J. Shellfish 
Res. 2:165-172. 

Loosanoff & H. C. Davis. 1950. Conditioning V. Mercenaria for spawning 
in the winter and breeding its larvae in the laboratory. Biol. Bull. 
98:60-65. 

Loosanoff. V. L. & H. C. Davis. 1951. Delaying spawning of lamelli- 
branchs by low temperature. J. Mar. Res. 10:197-202. 

Loosanoff. V. L. & H. C. Davis. 1963. Rearing of bivalve molluscs. Adv. 
Mar Biol. 1:1-136. 

Macfarlane, S. L. 1986. A comprehensive shellfish management plan for 
the Town of Orleans. Rept. to the Orleans Board of Selectmen and 
Commonwealth of Massachusetts Division of Marine Fisheries. Or- 
leans, MA. 40 pp. 



1036 



Macfarlane 



Macfarlane, S. L. 1989. Shellfish as the impetus tor embayment manage- 
ment. Estuaries I9:pp. 311-319. 

Manzi, J. J. & M. Castagna (eds.). 1989. Clam culture in North America, 
introduction. Developments in aquaculture and fisheries science. Num- 
ber 19. Elsevier Science. New York. pp. 1-22. 

Manzi. J. J.. N. H. Hadley. C. Battey. R. Haggerty, R. Hamilton & M. 
Carter. 1984. Culture of the northern hard clam Mercenaiiu merceimria 
in a commercial scale upflow nursery system. J. Shellfish. Res. 4:1 19- 
124, 

Matthiessen. G. C. & R. C. Toner. 1966. Possible methods of improving 
the shellfish industry of Martha's Vineyard. Duke's County. Massa- 
chusetts. A publication of the Marine Research Foundation, Inc, 

McKenzie. C, L. 1977. Predation on hard clam Mercenariu mcrcenaria 
populations. Trans. Am. Fish. Soc. 106:530-537. 

Menzel, R, W. 1976, Growth and mortality of northern hard clams in 
Florida waters, Assoc. S.E. Biol. Bull. 7:34—35, 

Mook, W, 1988, Guide to construction of a tidal upweller, Mook Sea 
Farms, Bristol, Maine. 

Rice, 1992, The northern quahog: the biology oi Merceimria inercenaria. 
Rhode Island Sea Grant Publ, RIU-B-92-001 (P1276), 

Roman, C, T,. K, 'W, Able, K, L, Heck. Jr,. J, 'W, Portnoy, M, P, Fahay. 



D, G. Aubrey & M, A, Lazzari, 1989, An ecological analysis of Nauset 
Marsh. Cape Cod National Seashore, National Park Service Coopera- 
tive Research Unit. 'Wellfleet. MA, 181 pp. 

Ropes. J, 'W,, D, S, Jones. S, A, Murawski, F, M. Serchuk & A, Jearid, Jr, 
1984, Documentation of annual growth lines in ocean quahogs. /4rcr/aj 
islaiulica Linne, Fish. Bull. U.S. 82:1-19, 

Schwind, P, 1977, Practical shellfish farming. International Marine Pub- 
lishing Co,, Camden, ME. 1 12 pp, 

Smolowitz. R,. S, Ford, J, Kraeuter, W. Canzonier, R, Rheault & D, Leav- 
itt, 1996, QPX Advisory Bulletin, Ocean State Aquaculture Associa- 
tion. Special Publ,. May. 1994, 4 pp. 

Smolowitz. R,, D, Leavitt & F, Perkins, 1998, Observations of a protistan 
similar to QPX in Mercenaria mercenaria (hard clams) from the coast 
of Massachusetts, / Invert. Pathol. 71:9-25, 

Teal, J, 1983, The coastal impact of ground water discharge: an assessment 
of anthropogenic nitrogen loading in Town Cove, Orleans. M.'k, Woods 
Hole Oceanographic Institution Proposal 2778, 

LIS, Army Corps of Engineers, 1968, Pleasant Bay, Chatham, Orleans. 
Harwich. Massachusetts. Survey Report, Department of the Army. 
New England Division. Corps of Engineers. Waltham, MA, 61 pp, plus 
appendices. 



Jininml of Slwllthh Research. Vol. 17. No. 4. 1037-1042. 1998. 

REPRODUCTIVE CYCLE OF SPONDYLUS LEUCACANTHUS BRODERIP, 1833 (BIVALVIA: 
SPONDYLIDAE) AT ISLA DANZANTE, GULF OF CALIFORNIA 



MARCIAL VILLALEJO-FUERTE AND 
FEDERICO GARCIA-DOMINGUEZ 

Ceuiro Intevdisciplinario de Ciencias Marinas 
Institiito PoUtecnico National A. P. 592 
La Paz. BCS 23000. Mexico 

ABSTRACT The reproductive cycle ot Spoiiilylii}, leiicciciuithuy at Isia Danzaiite. Cult ot Calitornm. was studied from January 1994 
to January 1996. Microscopic analysis established that the female specimens had male gametes in the gonads during the starting of 
ganietogenesis (April 1994. March 1995). The individuals were fully ripe from June, coincidmg with the highest values of gonad index. 
In both the years investigated, spawning occurred during August to October (23 to 26°C). when the gonad index was falling. Spawning 
occurred at the mean shell height of 75 mm. 

KEY WORDS: Reproductive cycle, bivalves. Spoiul\U(.s leucacanihus. histology. Gulf of California 



INTRODUCTION 

Sponilylus leucacanthiis Broderip. 1833 is commonly named 
"Viejita" or "Concha China."" h is distributed from the Gulf of 
California to Ecuador and can reach a length of 150 mm (Keen 
1971). Its main habitat is the bottom at about 40-m depth, although 
organisms can be gathered from shallow water up to 3-m depth. As 
with the majority of the bivalves, this is a filter-feeding species, 
feeding mainly on detritus and phytoplankton (Villalejo-Fuerte 
and Muneton-Gomez 1995). 

Anatomical and morphological descriptions of Spondyhis spp. 
have been published by Yonge ( 1973), Dakin ( l92Sa) and Dakin 
(1928b). A small number of studies on other topics have been 
made by Mata et al. ( 1990), Parth ( 1990). and Okutani ( 1991 ). A 
recent review of the taxonomy, habitat, and distribution of Genus 
Spoiulyliis of the Panamic Province was made by Skoglund and 
Mulliner (1996). 

Despite the wide distribution of this species around the islands 
of the Gulf of California, there are no published records on its 
biology. This study describes the reproductive cycle and the 
spawning season of 5. leucacanthiis from subjective analysis of 
histologic gonad sections, measurements of oocyte sizes, and cal- 
culated gonad index from weight measurements. 

MATERIALS AND METHODS 

Monthly, from January 1994 to January 1996 ut Isla Danzante. 
Gulf of California (25 ■48'54". 1 1 P15'45"l (Fig. 1 ). 30 to 60 speci- 
mens oi Spondyhis leucacanthiis were collected by trawling with a 
net at 40-m depth. The bottom water temperature was recorded at 
the time of sampling using a Van Dom bottle and a protected 
thermometer with range of-10 to 1 10°C. Shell heights and the wet 
weights of the gonad and total soft body were recorded for each 
clam after fixation in a neutral I07f formalin solution prepared 
with seawater. 

The sex ratio of the population in the study period was obtained 
from histological analysis of all the clams collected. The females 
and the males were separated, and the percentage of each sex in the 
study period was determined. The significance was tested with a 
chi-square analysis. The null hypothesis of 1:1 sex ratio was es- 
tablished, and the observed value was compared with the theoret- 
ical value of x" = 3.84, a = 0.05 (Sokal and Rolf 1979). 

The gonad index was calculated using the criteria of Sastry 



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L 
F 

O 




Yj' 


'- ^ A 












26° 49 


V 


> 


F 

C 
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F 
O 
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25° 45 




1 


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Sampling /Vrcj 



Figure 1. Location of the sampling station near Isla Danzante, Gulf of 
California. 



(1970), which uses the weight of gonad and soft body, and is 
expressed as percentage. For histological studies, a piece of the 
gonad (one cubic centimeter) was dehydrated in alcohol and em- 
bedded in paraffin. Sections (7 \xm) were placed on slides and 
stained with hematoxylin-eosin (Humason 1979). 

The gonad developmental stages of the individuals sampled 
were defined from the histological preparations using categories 
comparable to those species that have previously been studied in 
the Gulf of California, such as Megapitaria aurantiaca (Garci'a- 
Domi'nguez et al. 1994), Argopecten cinuhiris (Villalejo-Fuerte 



1037 



1038 



ViLLALFJO-FUERTE AND GaRCI'a-DOMi'nGUEZ 



and Ochoa-Baez 1993), Glycyineris gigautea (Villalejo-Fuerte et 
al. 1995), and Laevicardium elatum (Villalejo-Fuerte et al. 1996a). 
The classification of gonad condition consisted of five main 
stages: indifferent, developing, ripe, spawning, and spent. The 
stages are described below. 

Indifferent Stage 

This stage is characterized by a total absence of gametes, there- 
fore, it is not possible to distinguish between the sexes. Empty 
follicles are seen, and the connective tissue occupies almost all the 
space (Fig. 2A). 

Developing Stage 

Female 

The germinal cells can be observed, and developing oocytes are 
attached to the follicle walls (Fig. 2B). 



Male 



Varying quantities of spermalogenic cells and spermatocytes 
were present (Fig. 3A). Interfollicular connective tissue begins 
decreasing. 

Ripe Stage 

Female 

All follicles are filled with ripe oocytes of polygonal shape. 
Some oocytes remained attached to the follicle walls (Fig. 2C). 



Male 

Follicles filled with spermatocytes, spermatid, and spemiatozoa 
are aiTanged in characteristic bands (Fig. 3B). Interfollicular con- 
nective tissue is absent. 

Spawning Stage 

Female 

The centers of follicles are partially empty, and there are large 
spaces between the free oocytes that were present. Developing 
oocytes are present at the follicle walls (Fig. 2D). 

Male 

Follicles are partially empty, with large spaces inside the fol- 
licles and a marked decrease in the quantity of spermatozoa. The 
spermatocytes remain at the walls of the follicles (Fig. 3C). Inter- 
follicular connective tissue' is scarce. 

Spent Stage 

Female 

At this stage, the follicles are empty, but some unspawned 
oocytes were observed within them (Fig. 2E). 

Male 

In some follicles, only a few residual spermatozoa were present 
(Fig. 3D). In both sexes, the gametes were being phagocytosed by 
amebocytes. There is abundant interfollicular connective tissue. 

To analyze the sex of this species, females with spermatozoa 




'^'^-Sk 



Figure 2. Photomicrographs of gonadal stages of female S. leiicacanlliiis; a) Indifferent; b) de\eloplng; c) ripe: d) spawning; e) spent; f) 
hermaphrodite; scale = 50 fim. 



Repro[3uctive Cycle of Spondylus leucacanthus 



1039 




Figure 3. Photomicrographs of gonadal stages of male S. leucacanthus; a) developing; b) ripe: cl partially spawned; e) spent; scale = 50 (im. 



occupying the center of follicles were classified as hermaphrodites 
(Fig 2F). Size at maturity was estimated by plotting the relative 
cumulative frequency of spawning organisms against shell height 
measurements and tracing a line from 507c cumulative frequency 
of spawning to the shell height measurements. 

To obtain the mean size of the oocytes at each saiiipluig date, 
the diameters of at least 100 oocytes, from six randomly selected 
females, were measured using an eyepiece graticule calibrated 
with a stage micrometer. The measurements were made along the 
longest axis of the oocyte sectioned through the nucleus. Individu- 
als with few measurable oocytes and extensive phagocytosis were 
not used, following the criteria of Grant and Tyler (1983a) and 
Grant and Tyler (1983b). 

RESULTS 

The range in shell height of captured male organisms was from 
30 to 120 mm. with the mode at 83 mm. Females had shell heights 
between 55 and 120 mm. with the mode at 85 mm. The females 
with spermatozoa in the center of follicles were considered her- 
maphrodites and had shell heights from 55 to 105 mm. with the 
mode at 75 mm (Fig. 4). The sexual ratio of 1.128 organisms was 
213 (19%) females, 516 (45.79f) males, and 43 (3.89f) hermaph- 
rodites. The remaining (356) were undifferentiated. The sex ratio 
of the total sample (X~ = 125. n = 729) differed significantly (p 
< = .05) from the expected ratio of 1:1. 

The reproductive cycle of S. leucacanthus was remarkably 
similar over the 2-year study (Fig. 5). From November to March 
each year the population was inactive, as determined by the pres- 
ence of spent or indifferent stages. The developing stage started in 
March and April, and the population was fully ripe from June to 
August, when spawning began. In November, all the individuals 



were spent. Figure 6 shows that the size at spawning in the popu- 
lation of S. leucacanthus is 75 mm (shell height); however, indi- 
vidual organisms may start spawning at 40 mm shell height. 

Figure 7 shows the monthly mean diameter of intraovarian 
oocyte measurements of 5. leucacanthus from April 1994 to Oc- 
tober 1995. The mean diameter of mature oocytes is 56.4 fjim (SD 
= 10 p.m). Monthly measurements from 1994 show that the de- 
velopment of oocytes in the follicles have two periods of growth 
corresponding to gonad classification (Fig. 7 1. The first period of 
growth was from March to June and coiTesponds with the devel- 




2^7=?=- MALE 
' ^// ^ FEMALE 



HERMAPH. 
25 45 65 85 105 125 
35 55 75 95 115 135 
SHELL HEIGHT 
Figure 4. Frequency distribution for 5 mm size classes (shell height) of 
male, female, and hermaphroditic organisms of S. leucacanthus. 



1040 



ViLLALEJO-FUERTE AND GaRCIA-DOMINGUEZ 



u 

2 

U 
D 
O 
W 

OS 




J/94 M M J S N J/95 M M J S N J/96 

FAJAODFAJAOD 

MONTHS 



ffffl INDIFFERENT ^^ DEVELOPING | | RIPE 
H^ SPAWNING ; '--I SPENT 



Figure 5. Reproductive cycle of S. leucacanthus from January 1994 to 
January 1996. 




A/94M J J A S OM/95A M J J A 
MONTHS 



S 



Figure 7. The mean (± SDl diameter of oocytes from S. leucucanthiis 
from April 1994 to October 1995. 



oping stage. Tlie second slower growth was from June to Septem- 
ber corresponding to maturity of the oocytes. However, in 1995. 
the oocytes seem to have reached the maximum size in June. 

During the study period, the bottom water temperature at Isia 
Danzante varied from 15 to 26°C. The highest value measured was 
in November 1994 (25°C), and in September 1995 (26°C). The 
lowest temperature measured was from January to May in both 
years investigated (Fig. 8). 

The gonad index has a cyclic oscillation (Fig. 9). A single 
period of maximum value occurred during July in both years in- 
vestigated, coinciding with the occurrence of highest frequences of 
maturity (Fig. 5). The index declined from August to October 
during spawning. The lowest values measured were from Novem- 
ber to February, followed by a period of recuperation from April 
to June (Fig. 9). 

DISCUSSION 

The characteristics of gametogenesis and the description of 
gonadic development stages in S. leucacanthus were similar to 
those described for the pearl oysters Pinctada mazatlanica (Gar- 



cia-Domi'nguez et al. 1996). Pinctada alhina (Tranter 1958a). P. 
margaritifcra (Tranter 1958b), P. fucata (Tranter 1959). and P. 
maxinui (Rose et al. 1990). hi S. leucacainhus. the gonads increase 
in weight as the ova and spermatozoa grow during the developing 
and the ripening stages. The gametes of both sexes were spawned 
about the same time. 

In this species, all females collected during April 1994 and 
March 1995 were in the development stage and had spermatozoa 
in the lumen of follicles. Similar gonadic conditions are recorded 
for P. cdbina (Tranter 1958a), P. margaritifera (Tranter 1958c), P. 
maxima (Rose et al. 1990), and P. mazatlanica (Garcia- 
Domfnguez et al. 1996). In addition, the predominance of small 
(<45 mm shell height) male clams and the lack of female or her- 
maphroditic clams <45 mm shell height suggests that this species 
may be a protandric hermaphrodite. 

There is one major spawning period annually during August to 
October, so recruitment occurs once each year. The size at spawn- 
ing in the population is defined as the smallest height at which 
50'7f of females and males sampled are spawning (Somerton 
1980). In this study, some individuals exhibit signs of spawning at 



< 

in 

b 
O 

u 

IZ 

w 

D 

o 




s 

U 

20 30 40 50 60 70 80 90 100110120 
SHELL HEIGHT (mm) 

Figure 6. The spawning size of 5tl% of the population of .S. leucacan- 
thus from Isia Danzante. 




J/94 M M J S NJ/95M M J S NJ/96 
FAJAODFAJAOD 

Figure 8. Variation in bottom temperature at Isia Danzante, Gulf of 
California, and frequency of spawning of Spoudylus leucacanthus from 
January 1994 to January 1996. 



Reproductivk Cycle of Spondylus leucacanthus 



1041 




J/94M M J S NJ/95M M J S NJ/96 
FAJAODFAJAOD 

Figure 9. The mean gonad index value ± SD in S. leucacanthus from 
January 1994 to January 1996. 



40 mm shell height; however, the main spawnini; in population 
occurs at 75 mm shell height. 

The mean diameter of fully ripe oocytes of S. leucacaiilluis is 
similar to oocytes of Modiolus capax (Ochoa-Baez 1985). Ar- 
gopecten circidaris (Villalejo-Fuerte and Ochoa-Baez 1993), P. 
mazallanica (Garci'a-Dominguez et al. 1996), Laevicardiuin ela- 
tum (Villalejo-Fuerte et al. 1996a), and Megapitaria squcdida (Vil- 
lalejo-Fuerte et al. 1996) found in the Gulf of California. When we 
compare oocyte diameters with gonadal stages in S. leiicaciiiitluis, 
it is clear that tlie minimum diameters coincide with the develop- 
ing stage and maximum diameters coincide with the ripe and 
spawning stages. The oocyte diameters are reflective of the game- 
logenic cycle, similar to Mercenaria spp. (Hesselman et al. 1989), 
PUicopecten magellankus (Dibacco et al. 1995). Glycymeris gi- 



ganteci (Villalejo-Fuerte et al. 1993). and L. elciliiin (Villalejo- 
Fuerte et al. 1996a). 

Temperature is an important environmental factor in the regu- 
lation of bivalve reproduction (Sastry 1979). The reproductive 
cycle of 5. leucacuntluis al Isla Danzante shows a clear seasonality 
related to the bottom water temperature. The inactive period is 
from November to March with water temperatures decreasing 
from 25 to 18°C. Gametogenesis occurs at the lowest temperature 
(18°C), with ripening coinciding with the increasing water tem- 
peratures ( 18 to 23°C) and spawning with the highest values (23 to 
26°C). A similar relationship between the temperature and gonadal 
activity has been observed in such other bivalve species as Perna 
picia (Shafee 1989), A. circidaris (Villalejo-Fuerte and Ochoa- 
Baez 1993). G. gigantea (Villalejo-Fuerte et al. 1995). and L. 
clatiun (Villalejo-Fuerte et al. 1996a). 

In S. leiicacunduis. the mean values of the gonad index are 
representative of the gonad development and can be associated 
with the reproductive condition. The lowest values of gonad index 
occur during the inactive period (November to March), when the 
gametes are absent. The increase in number and volume of ga- 
metes during the development and ripening stages yields the high- 
est values of the gonad index during June to August when the 
individuals start spawning. A similar relation between this index 
and the reproductive condition has been observed for other bi- 
valves (Sastry 1979. Dibacco et al. 1995, Villalejo-Fuerte and 
Ceballos-Vazquez 1996). 

ACKNOWLEDGMENTS 

We are grateful to Direccion de Estudios de Posgrado e Inves- 
tigacion del Instituto Politecnico Nacional (IPN) for funding this 
work, and to Comision de Operacion y Fomento de Actividades 
Academicas del IPN for the grants to M. Villalejo-Fuerte and F. 
Garcia-Dominguez. Thanks to Dr. Ellis Glazier. CIBNOR for his 
help in editing the English language text. Marcial Villalejo Fuerte 
participated in this research in meinory of Ramon Villalejo Fuerte. 



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kyo, Japan. Nautilus 105:165 pp. 

Parth. M. 1990. Spondylus pratii. spec, nov., a new species from Somalia 
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Rose. R. A.. R. E. Dybdalh & S. Harders. 1990. Reproductive cycle of the 
Western Australian silverlip pearl oyster Pinctada alhina (Jameson) 
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Sastry. A.N. 1979. Pelecypoda (excluding Ostreidae). pp. 131-192. In: 
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Skoglund, C. & D. K. Mulliner. 1996. The genus Spondylus (Bivalvia: 
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Somerton. D. A. 1980. A computer technique for estimating the size of 
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Tranter, D.J. 1958b. Reproduction in Australian pearl oysters (Lamelli- 
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.Iininuil ,>l Shcllthh Research. Vol. 17, No. 4, I(I4.V|(144. 1998. 

EFFECTS OF BAITWORM DIGGING ON THE SOFT-SHELLED CLAM, MYA ARENARIA, IN 
MAINE: SHELL DAMAGE AND EXPOSURE ON THE SEDIMENT SURFACE 



WILLIAM G. AMBROSE. JR., MAX DAWSON, CHRIS GAILEY. 
PETRA LEDKOVSKY. SHAWN O'LEARY, BENJAMIN TASSINARI. 
HEIDI VOGEL, AND CLIFF WILSON 

Depuitmcnt of Biology 
Bates College 
Lewiston, Maine 04240 

ABSTRACT Experiment> conducted during the fall of 1997 on an intertidal flat in Maine determined the extent of shell damage and 
exposure of Mya arenana on the sediment surface resulting from commercial bloodworm (Glycera dihranchiata) digging. We 
conservatively estimate that worm diggers dig up and expose on the sediment surface approximately 6% of the greater than 2 mm 
fraction of the clam population each time they turn over the sediment. Twenty percent of the clams had at least one valve damaged. 
Fifteen percent of intact clams exposed were found with their siphon up (normal living position). 41% with their siphon down, and 44% 
were horizontal on the sediment surface. Large clams (5.7 cm average shell length) placed on the sediment surface in the siphon up 
position reburied faster and to greater depths than tho.se in horizontal or inverted positions. Small clams (2.7 cin shell length) buried 
faster than large clams, and those placed horizontally or with their siphons up reburied faster than clams placed w ith their siphons down. 
We detected no difference in reburial patterns between large clams exposed on undug and recently dug sediment. Our recovery of large 
clams after 10 days, however, was much greater (91.8%) from undug sediment than dug sediment (59.4%) and we found twice as many 
clam shells exhibiting evidence of predation in the dug than the undug area. Only about 50% of the small clams were recovered live. 
Shell damage of recovered dead clams indicated that predators consumed some missing clams. Our results suggest that baitworm 
digging negatively affects the survival of Myci areimria by directly damaging shells and by exposing clams to increased risk of 
predation. 

KEY WORDS: Mya arenaria. Glyceia clihniihiiuiki. baitworm digging, shell dainage, predation 



INTRODUCTION 

Intertidal sand and mud flats are harvested for infaunal clams 
and worms by commercial and recreational diggers world wide 
(Jackson and James 1979. Blake 1979. Cryer et al. 1987. van den 
Heiligenberg 1987, Brown 1993. Jamieson 1993, Olive 1993, Wal- 
lace 1997. Hylleberg et al. 1986). Harvesting usually involves 
manually turning over the sediment with a hoe or shovel (see van 
den Heiligenberg 1987, Hall et al. 1990 for discussions of me- 
chanical harvesting). Moderate, animal-tnediated sediment distur- 
bance can have large effects on .soft-sediment communities (Po.sey 
1987, Peterson 1991. Wilson. 1991. Flach 1992. Ambrose 1993. 
Hall 1994, Commito et al. 1995a). so the effects of the massive 
sediment disruption associated with digging for clams and worms 
has long been of concern (Jackson and James 1979, Cryer et al. 
1987. van den Heiligenberg 1987. Brown and Wilson 1997). Stud- 
ies examining the impact of digging on intertidal soft-sediment 
systems all show a dramatic impact on the distribution and abun- 
dance of many infaunal taxa. with rates of recovery dependent on 
species' recolonization abilities (Jackson and James 1979, 
McLusky et al. 1983. Cryer et al. 1987, van den Heiligenberg 
1987. Brown and Wilson 1997). 

In Maine, three commercially important species co-occur on 
intertidal mud and sand flats: Mya arenana L. (soft-shelled clam), 
the polychaete worms Glycera dihranchiata Ehlers (bloodworm), 
and Nereis virens Sars (sandworm). Worms are used as bait in 
recreational fisheries (Brown 1993). In Maine in 1997, soft-shelled 
clams ranked eighth in landed value among marine species, and 
both worms together ranked fourteenth (National Marine Fisheries 
Service Commercial Landings). All three species are harvested 
manually. Clatii and worm diggers often find themselves digging 
next to each other, and conflicts between the two fisheries date 



back to the 1950s (Maine Legislative Research Committee 1957. 
Wallace per. com.). 

Worm harvesting may affect clams in several ways: direct shell 
damage, displacement below their natural burial depth in the sedi- 
ment, or exposure on the sediment surface. Harvesting soft-shelled 
clams with a hoe typically results in the breakage and death of 14 
to 20% of the clams (Dow et al. 1954, Glude 1954. Medcof and 
MacPhail 1964. Robinson and Rowell 1990). Clam hoes in Maine 
typically have 4 to 5. 15-cm long, tines (Wallace 1997). Worm 
diggers also use hoes to turn sediment over, but worm hoes usually 
have more tines (5 to 7 for bloodworm hoes, 5 to 6 for clamworm 
hoes), and the tines are longer (21 to 22 cm for bloodwonn hoes. 
34 to 39 cm for sandworrn hoes) (Creaser et al. 1983) as compared 
to clam hoes. There has been no assessment of damage to clams by 
worm digging. 

Turning over the sediment can bury clams deeper than they 
normally live. When suffocation and exposure are included as 
sources of mortality, studies indicate a wide range of mortality 
rates ranging from 2 to 50% (Medcof and MacPhail 1964. Rob- 
inson and Rowell 1990). Clam burial experiments clearly show 
that a clam's survival declines with increased depth below its 
natural depth in sediment (Glude 1954. Emerson et al. 1990). 

Exposure of clams on the sediment surface increases their 
chances of freezing or becoming desiccated and greatly increases 
their susceptibility to avian predators during low tide and to de- 
mersal fish and crustacean predators during high tide (Medcof and 
MacPhail 1964). When the anterior edge of Myt;'.s shell is not in 
contact with the sediment surface, clams cannot rebury efficiently 
(Emerson et al. 1990). Consequently, a clam's orientation on or 
near the sediment surface is likely to affect reburial rate pro- 
foundly. Reburial rate is also dependent on sediment type (Emer- 
son et al. 1990). but none of the studies examining reburial rate 
were conducted in the field with natural sediment conditions (Bap- 



1043 



1044 



Ambrose et al. 



tist 1955. Pfitzenmeyer and Droebeck 1967. Emerson et al. 1990). 
Furthermore, sediment compaction on digging tailing is very dif- 
ferent than on undug sediment (Ambrose pers. obs.) and might 
effect rates of reburial. 

Only one study (Beal 1996) has addressed the effects of womi 
digging on Mya populations. Beal ( 1996) examined the effects of 
bloodworm and clam digging on the survival and growth of cul- 
tured and wild Mvrt juveniles (average shell length 12.5 mm). He 
found that predation effects during the summer masked any effects 
of digging on the survival of juveniles. Beal's experiment did not 
address the effects of digging on larger individuals (greater than 35 
mm shell length) that have reached a size/depth refuge from most 
predators (Commito 1982). 

The purpose of our study was to determine the extent of shell 
damage and exposure of Myii aieiuiria on the sediment surface 
resulting from commercial bloodworm digging. Factors likely to 
contribute to mortality that we examined include: shell damage, 
exposure and orientation on the sediment surface, reburial rates, 
and depth of reburial. Experiments were conducted on an intertidal 
mud flat in Maine on undug and recently dug sediment, providing 
insight into the potential importance of predation on the survival of 
clams disturbed by digging. 

MATERIALS AND METHODS 

Study Site 

Observations on the impact of bloodwomi digging on Mya 
arenaria and reburial experiments were conducted on an intertidal 
mudflat at the head of Maquoit Bay, Maine (43°55'N Lat., 
70°00'W Long.). A tidal range of 3 to 4 meters exposes an inter- 
tidal area of about 81 hectares at mean low water (Heinig and 
Campbell 1992). Our work was conducted in the middle intertidal. 
where the sediment is coarse silt, with an average grain size of 
about 30 p.m (Ambrose unpublished data). During our work from 
September to November 1997. the sediment (1-cm depth) main- 
tained a temperature of 16^C. while air temperature durmg daytime 
low tides ranged from 14 to 18°C. Salinity of the incoming water 
was typically 34 ppt. Maquoit Bay has been highly productive for 
shellfish (Heinig and Campbell 1992). and both soft-shelled clams 
and bloodworms are harvested from the tlat. During recent years 
( 1994 to 1998). worm diggers have outnumbered clam diggers on 
the tlat, and as many as 40 worm diggers have been observed at 
one time (Ambrose pers. obs.). 

Siin'ey of Undug and Recently Dug Areas 

The number, orientation, and shell damage of Mya exposed by 
bloodworm digging was asses.sed by surveying newly dug areas. 
Surveys were conducted immediately following commercial dig- 
ging. Bloodworm diggers turn over sediment methodically, leav- 
ing long rows of sediment completely disturbed (see Brown and 
Wilson 1997 for photograph). The length and average width (at 
least three measurements) of dug areas were measured to the near- 
est 5 cm between 19 September and 29 October 1997. We exam- 
ined 13 recently dug areas for exposed Myu. Two people visually 
searched and lightly probed the sediment surface of each dug area 
to locate exposed clams. The length ( measured to the nearest 0. 1 
cm with calipers), shell damage (one or two valves damaged), and 
siphon position (up. down, horizontal) was noted for all exposed 
clams. We used chi-square analysis to compare observed clam 
orientations to a distribution of equal numbers of clams in each 



orientation that would be expected if orientation of exposed clams 
was random. The number of clams exposed in each dug area was 
converted to a number per I m" and averaged over all areas sur- 
veyed to determine the average density of clams exposed by worm 
digging. 

The density and size frequency of clams in areas that showed 
no evidence of recent commercial clam or bloodworm digging 
were determined based on 25. 0.06-m" cores inserted to a depth of 
about 15 cm (the depth of a hard clay layer below which on this 
flat clams do not burrow, and worm diggers do not dig). Undug 
areas were at the same tidal height and often adjacent to dug areas. 
The contents of each core were sieved in the field through a 2-mm 
mesh, and clams retained on the sieve were measured. The size 
frequencies of exposed clams collected from dug sediment and 
buried clams from undug areas were compared using chi-square 
analysis to determine if all size classes of clams are equally likely 
to be exposed by digging. 

Reburial Experiments 

Reburial experiments were designed to determine the influence 
of siphon orientation on the rate clams exposed by digging disap- 
peared from the sediment surface and the depth of reburial of these 
clams. Experiments were conducted separately for large commer- 
cial-sized (mean shell length = 5.7 cm, SE = 0.4) and small 
(mean shell length = 2.7 cm, SE = 0.5) clams. 

Large clams, which had been harvested the previous day, were 
purchased from a retail dealer. Clams were measured with calipers 
and those with cracked shells or that did not respond to touch by 
closing their valves were discarded. Two 4x4 matrices of 1 ni" 
plots were established on 25 September 1997, one on recently dug 
sediment and one on sediment showing no evidence of recent 
digging. Matrices were about 10 m apart and at the same tidal 
height. The three treatments were: (1) siphon horizontal, (n = 5 
replicate plots); (2) siphon vertical and up in the normal living 
position (n = 6); and (3) siphon vertical and down (n = 5). 
Siphon-up and siphon-down clams were pushed one-half shell 
length into the sediment. Clams placed horizontally were pressed 
slightly into the sediment. The top 2 to 3 cm of the sediment was 
very soft, so there was minimal sediment compaction. Treatment 
replicates were randomly assigned to plots. In each plot. 10 clams 
were spread evenly on the sediment surface. Changes in siphon 
position, number of clams still visible on the sediment surface, and 
the condition of clams (shell damage) were recorded after 24 
hours, 3 days, and 10 days. After 10 days, each plot was excavated 
in 3-cm depth intervals and the sediment in each interval was 
sieved through a 2-mm mesh. Experimental clams could very eas- 
ily be distinguished from natural clams, because experimental 
clams had a much lighter shell color as compared to the black 
shells of natural clams that had resided longer in anoxic sediment. 
This difference in shell color was obvious even after 10 days. 

The number of exposed clams was compared for each time 
period separately using a one-way analysis of variance (ANOVA). 
An F-max test (Sokal and Rohlf 1981 ) revealed no significant (p > 
.05) differences among variances. When an ANOVA was signifi- 
cant (p < .05). differences among treatment means were compared 
using a Tukey multiple comparison test. These analyses were con- 
ducted separately for the dug and undug site, because lack of 
replication of dug and undug sites as a variable precluded a two- 
way ANOVA with replication. We used a 3 x 3, depth x siphon 
orientation, contingency table to examine the relationship between 



Effect of Baitworm Digging on Soft-Shelled Clam 



1045 



y 15 





10 15 20 25 30 35 40 45 50 >50 
Size (mm) 

Figure 1. Size frequency distribution of Mya arenaria recovered from 
the sediment surface of recently dug areas (n = 173 individuals! and of 
all individuals sampled with a 0.(l6-m- core (n = 25l from undug areas 
(n = 29 individuals) of Maquoil Bay, intertidal Hat. There was no 
significant difference between the two distributions (x" = 6.64, p > .21. 

siphon orientation and final burial depth. Numbers of clams re- 
covered were compared among treatments u.sing a one-way 
ANOVA. 

Small clams were dug from the flat and held overnight in 20^C. 
aerated water. Each clam was marked with nail polish to distin- 
guish experimental clams from natural ones. The same experimen- 
tal design and statistical analyses were used for small clams as for 
large clams, except that a limited number of small clams only 
allowed us to establish the matrix on recently dug sediment. The 
experiment began on 14 October 1997, and sampling, using the 
same methods used for large clams, occurred 1 2 h. 24 h. and 3 days 
later. 

RESULTS 

Survey of Undug and Recently Dug Areas 

A total of 178 m" of freshly dug sediment was surveyed in 13 
recently dug areas. Dug areas ranged in length from 10 to 25 ni 
(mean = 1 1.9 m, SE = 1.4) and had an average width of 1.2 ± 
0.15 m (SE), the average arc length of a diggers arm. All diggers 
were professional bloodworm diggers and were turning over the 
top 15 cm of sediment. 

We found an average of 1.12 clams ±0.19 (SE)/m" exposed on 
freshly dug sediment. These clams ranged in length from 1.1 cm to 
6.0 cm. and their size frequency distribution was not significantly 
different from clams sampled by core from nearby undug areas (x" 
= 6.64. p > .20; Fig. I). In total. 199 clams were collected, and 
22.6% of these had at least one valve damaged. This damage was 
clearly caused by digging, because the breaks were fresh, and in 
many cases, valves had a hole, probably made from a bloodworm 
hoe tine. Of the remaining whole clams. 43.7% were found in the 
horizontal position. 41.1% had their siphon down, and 15.2% were 
in the normal, siphon up, anterior down, position. This distribution 
of orientations is significantly different from random (x" = 29.9, 
p< .001). The density of Mya in undug areas was 1.16 individuals 
±0.24/0.06 m~ or 19.3/m-. Few of these clams were of commercial 
size 05.1 cm. Fia. 1). 



Large Clam Experiment 

A significant difference was found among siphon orientations 
in the mean number of large clams per plot remaining exposed for 
each time period for both dug and undug locations (Fig. 2). With 
the exception of the undug location at 10 days, differences in the 
mean number of clams exposed among siphon orientations were 
the same at each sampling and location. Clams beginning the 
experiment in the normal siphon up position disappeared from the 
sediment surface faster than clams with their siphons in either the 
horizontal or down positions. There was no significant difference 
among these last two orientations. At 10 days in the undug sedi- 
ment, there was no significant difference in the number of exposed 
clams between siphon up and horizontal orientations, both of 
which were different from clams beginning with their siphons 
down. After 10 days. 19% of the clams remained exposed on the 
dug site and 24% on the undug site. 

More of the clams experimentally exposed were recovered live 
from undug sediment (n = 147. 91.8%) than dug sediment (n = 
95, 59.4% ). Most of the unrecovered clams could not be accounted 
for. but six dead clams with cracked shells were recovered from 
the undug site (leaving only seven unaccounted clams) as com- 
pared to 13 with cracked shells from the dug site (52 unaccounted 
clams). No significant difference was found in the mean number of 



24 Hours 



I I Siphon Up 
[ , , , I Horizontal 
^^ Siphon Down 




D- 

D. 

T3 

0} 
03 
O 
Q. 
X 

tu 

a3 



c 



72 Hours 



1 



A A 





10 Days 






*** 




*** 




B 


A 

B 




A A 


^ n^ 


^^i_ 


' 


— a: ^^^H 



Undug 



Dug 



Figure 2, Mean number (-f 1 SE) per 1 m" experimental plot of large 
Mya arenaria remaining exposed on the sediment surface of recently 
dug and undug sediment after 24 hours, 72 hours, and 10 days. Each 
plot began with 10 clams. Mean number of clams exposed per plot was 
compared among siphon orientation (up, horizontal, down) for undug 
and dug sediment and each time period separately using one-way 
ANOV.A (** = p < .01, *** = p < ,001), Bars with the same letter over 
them indicate that the means they represent are not significantly dif- 
ferent from each other (Tukey multiple comparison test, p > .05). 



1046 



Ambrose et al. 



clams per plot recovered from the three treatments from the dug 
area (up = 6.5. SE = 1.5. down = 5.6. SE = 1.1, horizontal = 



5.6, SE = 0.5; F, , 



0.21. p > .81). There was. however, a 



significant difference in the mean number of clams per plot recov- 
ered from the undug area (up = 1 1.3, SE = 0.7. down = 9.6, SE 
= 1.4, horizontal = 6.2, SE = 0.6: F, ,, = 8.01, p < .005). 
Significantly (p < .05) fewer individuals were recovered from the 
horizontal treatment than the siphon-up treatment, but there were 
no other significant differences among treatment means. 

For large clams, there was a significant relationship between 
siphon orientation and the depth in the sediment where clams were 
recovered after 10 days for both undug (x" = .^2.49. p < .001 ) and 
dug (x" = 15.47, p < .004) sites (Fig. 3). A greater number of 
clams beginning with their siphon in the up position reached a 
sediment depth of 6 to 9 cm than clams placed on the sediment 
surface with their siphon down or horizontal. This pattern was the 
same for dug and undug sediment. 

Small Clam Experiment 

All small clams had disappeared from the sediment surface by 
3 days. Siphon orientation had a significant effect on the mean 
number of individuals remaining exposed on the surface after 12 
and 24 hours (Fig. 4). For both observation periods, no significant 
difference in the number of exposed clams between siphon-up and 
siphon-horizontal treatments was found, but significantly fewer 
clams in these two treatments were exposed than clams placed on 
the sediment surface with their siphons down. 

Approximately half (53.7%) of the 160 small clams were re- 
covered live after 10 days. During the experiment. 2.5% of the 
clams were recovered dead on the sediment surface. Some of these 
dead clams had cracked shells, and others had no shell damage and 
articulated valves. One marked, cracked shell was found 5 m from 



Large Clams-Undug 



Siphon Up 
K 1 Horizontal 
^H Siphon Down 




- 3 cm >3 - 6 cm >6 - 9 cm 

Large Clams-Dug 




3 cm 



>3 - 6 cm 
Depth Range 



>6 - 9 cm 



Figure 3. Mean number {+ I SEl per I m' experimental plot of large 
Mya arenaria witli different initial siphon orientations (up, horizontal, 
down) recovered from three different depth ranges (0 to 3 cm, >3 to 6 
cm, >6 to 9 cm) of recently dug and undug sediments. Each plot began 
with 10 clams. 




"D 


W 

o 

Q-o 

X 

LU 

8 



Siphon Up Horizontal Siphon Down 



24 Hours 




Siphon Up Horizontal Siphon Down 

Siphon Orientation 

Figure 4. Mean number 1+ 1 SE) per I nr experimental plot of small 
Mya arenaria remaining exposed on the sediment surface of recently 
dug sediment after 12 and 24 hours. Each plot began with 10 clams. 
Mean number of clams exposed per plot was compared among siphon 
orientation (up, horizontal, down) for each time period separately us- 
ing one-way .\NOV.V (** = p < .01, *** = p < .001 ). Bars with the same 
letter over them indicate that the means they represent are not sig- 
nitlcanlly different from each other (Tukey multiple comparison test, 
p > .05). 



the experimental matrix. No significant difference in the mean 
number of clams recovered from the three treatments was found, 
even though about half as many clams with their siphons oriented 
down were recovered or compared to those with their siphons up 
(up = 5.7. SE = 1.0, down = 2.8, SE = 0.4, horizontal 5.2, SE 
= 1.2; F, ,3 = 2.41, 0.1 < p < .2). The depth in the sediment at 
which small clams were recovered was only marginally dependent 
on their initial siphon orientation (Fig. 5; x" = 8.07, 0.05 < p < 
.09). Most individuals were found shallower than 6 cm deep. 

DISCUSSION 

Our results offer the first experimental evidence that worm 
digging negatively affects Mya arenaria survival by both damag- 
ing shells and exposing clams on the sediment surface. Our esti- 
mate of shell damage, 22.6%, is similar to the incidence of shell 
damage reported from clam digging (Dow et al. 1954, Medcof and 
MacPhail 1964, Robinson and Rowell 1990). Clams with shell 
damage have less than a 1% chance of survival (Glude 1954), so 
the shell damage we observed can be assumed fatal. Our estimate 
of the nuinbers of clams exposed by digging (1.12/m") is almost 
certainly low. Our survey was probably not 100% efficient, par- 
ticularly for small individuals. In addition, gulls occasionally re- 
moved clams before we began surveying. 

• Clams deposited on the sediment surface by digging can suffer 
two fates before they rebury: they may die from physical stress 
(desiccation, freezing, starvation) or predators may consume them. 
Other researchers (Medcof and MacPahil 1964. Emerson et al. 
1990) have remarked that exposure during low tide is often not 



Effect of Baitworm Digging on Soft-Shelled Clam 



1047 



tutaL The survival of some large clams on the sediment surface for 
10 days (Fig. 2) supports this hypothesis. Death from exposure is 
certainly more of a problem during the summer and winter when 
air temperatures can be substantially higher (temperatures higher 
than 32°C are common) and lower (temperatures below 0°C are 
common) than those recorded during our fall experiment. Never- 
theless, in general, clams on the sediment surface are probably at 
much greater risk of mortality from predation than from exposure. 

The cracked shells we recovered from experimental plots sug- 
gest predation by crustaceans and we also observed gulls preying 
upon clams. Crabs, particularly the green crab (Carciniis maenas 
L.) are major predators on Mya in the Northeast United States 
(Lindsay and Savage 1978. Hidu and Newell 1989. Wallace 1997). 
Gulls are prominent predators on Maine intertidal flats year round 
(Ambrose 1986) and have previously been observed to prey on 
Mya (Medcof 1949, Medcof and MacPhail 1964). The nemertean 
Cerebratidus huleiis (Verrill) is also present at the experimental 
site and can be a voracious predator on Mya (Rowell and Woo 
1990, Rowell 1992). It cannot be assumed, however, that all miss- 
ing clams were preyed upon, because Mya. particularly small in- 
dividuals, can move short distances along the sediment surface 
(Baptist 1995). Waves and currents also transport clams (Emmer- 
son and Grant 1991. Commito et al. 1995b). Nevertheless, unless 
we assume very different rates of migration or passive transport 
from dug and undug areas, the large difference in recovery of large 
clams between dug (59.5%) and undug (91.8%) areas, although not 
tested statistically, is probably largely a con.sequence of different 
levels of predation. Gulls often followed diggers and flocked to 
dug areas after diggers left and twice as many cracked shells were 
recovered from the dug than the undug area. Subtidally, predators 
are attracted to recently trawled areas (Kaiser and Spencer 1994. 
Kaiser and Spencer 1996, Kaiser and Ramsay 1997, Ramsey et al. 
1996. Ramsey et al. 19981. Our results suggest that predators may 
be attracted to intertidal areas disturbed by baitwonn diggers. The 
lower recovery of small clams (approximately 50%) as compared 
to large clams is likely a consequence of both greater rates of 
predation on these sized clams than on large ones and migration or 
passive transport out of experimental plots. Loss of clams to pre- 
dation would certainly have been much greater than we observed 
had we conducted our experiments in the summer when intertidal 
densities of green crabs are greater than in the fall (Ambrose pers. 
obs.). In Nova Scotia and New Brunswick, Canada, Mya mortality 
is higher in the summer (August) than winter (February) (Robin- 
son and Rowell 1990). 

A clam's risk of predation by epibenthic predators is propor- 
tional to the time it is exposed on the sediment surface and the 
depth to which it reburies (Zwarts and Wanink 1989, Emerson et 
al. 1990, Zaklan and Ydenberg 1997). Reburial rate is related to 
siphon orientation (Figs. 2 and 4), and baitworm digging deposits 
clams on the sediment surface with different orientations. It does 
not even deposit them equally by siphon orientation, leaving only 
15% in a normal siphon-up position. Large clams disappeared 
from the sediment surface faster when they were placed in an 
upright position than when in horizontal or down positions (Fig. 
2). There was no significant difference among treatments in re- 
covery of clams from the dug area, so this difference is attributable 
to differences in reburial rate and not to differences in migration or 
predation among treatments. There was, however, a significant 
difference in recovery among treatments in the undug area, with an 
average of more than 10 clams per plot recovered from the upright 



treatment and significantly fewer recovered from the horizontal 
treatment. Clams placed horizontally were possibly most prone to 
passive transport by waves and currents because their shells were 
not anchored in the sediment as well as clams in other orientations. 
Even with this bias, clams still disappeared faster from the upright 
treatment than from the horizontal treatment (Fig. 2). Large clams 
w ith horizontal and inverted orientations have a hard time reaching 
the sediment with their foot, which is necessary for reburial (True- 
man et al. 1966, Emerson et al. 1990). There was no difference in 
recovery of small clams ainong treatments and no difference be- 
tween upright and horizontal treatments in the rate at which clams 
disappeared from the sediment surface (Fig. 4). Smaller clams in a 
horizontal position find it easier to reach the sediment surface with 
their foot than large clams in the same position (Ambrose pers. 
obs.). Inverted small clams still had a very difficult time reburying. 

Large clams placed on the sediment surface with their siphon 
horizontal or down did not burrow as deeply as those beginning 
with their siphons up (Fig. ?i). Deep-dwelling clams are at a lower 
risk of being preyed upon by epibenthic predators than are shal- 
lower-dwelling individuals (Zwarts and Wanink 1989), so these 
differences in reburial depths could have consequences for sur- 
vival. We sampled burial depth after only 10 days, however, and 
it is possible that clams beginning with their siphons horizontal or 
down might burrow deeper after a longer time. But even after 6 
weeks in muddy sediment, exposed Mya reach shallower depths 
than natural clams (Emerson et al. 1990). Small clams, possibly 
because they reburrow faster than large clams, did not show a 
strong relationship between reburial depth and initial siphon ori- 
entation (Fig. 5). 

Our study may have underestimated reburial rate and, conse- 
quently, overestimated mortality if experimental clams were 
stressed by our manipulations. Two factors might have stressed 
clams and reduced their reburial rate: ( I ) clams purchased or drug 
by ourselves were held 1 day before being placed in the field; and 
(2) large clams had light shells and, therefore, came from an oxic 
sediment but were placed in anoxic sediment. The effects of these 
factors on reburial rate should be considered in future studies. 

Most studies of the impact of clam digging on soft-shelled 
clams have not related harvest effects on clams to the clam popu- 



Small Clams 



o 



0) 
Q. 



> 



0) 

E 




Siphon Up 
I7~~i Horizontal 
^^ Siphon Down 



0-3cm 



>6 - 9 cm 



>3 - 6 cm 
Depth Range 

Figure 5. Mean number (+ 1 SE) per 1 m' experimental plot of small 
Mya arenaria with different initial siphon orientations (up, horizontal, 
down) recovered from three different depth ranges (0 to 3 cm, >i to 6 
cm, >6 to 9 cm) of recently dug sediment. Each plot began with 10 
clams. 



1048 



Ambrose et al. 



lation (but see Robinson and Rowell 1990). Using our estimates of 
clam density in areas not recently dug and the density of exposed 
clams, we estimate that at least 5.8% of the greater than 2 mm 
fraction of the clam population is exposed each time sediment is 
dug for worms. The proportion of clams affected by worm digging 
may vary depending on clam size and density and sediment char- 
acteristics. Large clams live deeper in the sediment than small 
clams (Emerson et al. 1990. Zaklan and Ydenberg 1997. Ambrose 
unpub. data), and some may live deep enough to avoid bloodworm 
hoes. At Maquoit Bay, no clams burrowed deeper than the 15 cm 
necessary to avoid bloodworm hoes, and all sizes were exposed 
with equal frequency (Fig. 1 1. If shell breakage as a result of worm 
digging increases with clam density as it does for clam digging 
(Dow et al. 1954), the percentage of clams affected by worm 
digging may be greater in areas with higher clam densities than the 
relatively low density at Maquoit Bay. Finally, our estimate is for 
only one turning of the sediment. Worm diggers turn Maquoit Bay 
over at least three times a year (Ambrose pers. obs.), and some 
flats are reported to be turned over more (Maine Department of 
Marine Resources public hearing, April 30, 1998. Wiscasset 
Maine). 

Only one other study has assessed the effects of worm digging 
on Mya arenaria. In a study also done at Maquoit Bay, Beal ( 1 996 ) 
found that in the absence of predators, moderate bloodworm dig- 
ging enhanced juvenile ( 12.5 mm shell length) survival. Effects of 



digging on all sizes of clams, however, need to be determined to 
assess the impact of baitworm harvesting accurately on Mya popu- 
lations. Effects of worm digging on larger clams are likely influ- 
enced by sediment type, clam density, predator type and abun- 
dance, season of digging, worm hoe design, and individual digging 
styles and experience. Clearly, more study is needed before we can 
fully evaluate the effects of baitworm digging on Mya populations. 
Nevertheless, our results indicate that baitworm digging negatively 
effects the survival of Mya larger than about 2 cm (the smallest 
experimental clam) and should be considered in their management 
when both species are present in commercial densities. Further- 
more, some of our results can be applied to Mya harvesting that 
leaves behind sublegal-sized clams exposed on the sediment sur- 
face and to other harvesting practices that deposit infaunal bivalves 
on the sediment surface. 

ACKNOWLEDGMENTS 

Our research was supported by the Department of Biology, 
Bates College. We appreciate insights into worm and clam digging 
from Ron Aho, Ted Creaser, Dave Mercier, and Dana Wallace. We 
thank B. Brown, J. Commito, T. Creaser, S. Fegley, P. Renaud, 
Les Watling, W. H. Wilson, and an anonymous reviewer for com- 
ments on an earlier draft of this manuscript. We also thank the 
Maine Department of Marine Resources for issuing us a collecting 
pemiit for this work. 



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.loiinuil ,>) Slu'llfish Research. Vol, 17. No, 4. I()?1-I()S6. 1998. 

TRANSMISSION ROUTES AND TREATMENT OF BROWN RING DISEASE AFFECTING 

MANILA CLAMS (TAPES PHILIPPINARUM) 



EDUARDO MARTINEZ-MANZANARES,' DOLORES CASTRO,* 
J. IGNACIO NAVAS,- M. LOURDES LOPEZ-CORTES,' AND 
JUAN J. BORREGO' 

^Department of Microbiology 

Faculty of Sciences 

Universit}- of Malaga. Campus Universitario Teatinos 

2907 1 -Malaga. Spain 

-C.I.C.E.M. -'Agua del Pino" 

Junta de Andalucia 

Huelva. Spain 



ABSTRACT The potential tran.smission route.s of Vibrio nipetis. the causative agent of the hrown ring disease affecting manila clams. 
Tapes philippiiiarum. have been studied under laboratory conditions. The results obtained indicate thai the most probable transmission 
route is by means of direct contact with infected clams. The main factors affecting the incidence of brown ring disease in cultured 
manila clams were evaluated in normal aquaculture conditions. No significant differences were obtained in regard to clam seed density, 
but the type of substrate in clam beds significantly affected the disease. Chemotherapeutic treatment of infected clams is proposed, on 
the basis on the antimicrobial susceptibility of V. tapetis. antibiotic solubility in seawater, differential clam toxicity and cost, as being 
the most adequate treatment the contact of the clams for 1 to 2 h previously to their seeding with nitrofurantoin ( 10 mg/L seawater for 
3 days), nuniequine (1.5 mg/L seawater for 3 days), or o.xolinic acid (1 mg/L seawater, only one bath treatment). 

KEY WORDS: Brown ring disease. Vibrio lapelis. manila clams. Tapes philippiiiarum, disease transmission, treatment, epizootic 



INTRODUCTION 

A disease affecting cultured manila clams (Tapes philippi- 
nanim) associated with mortalities (about 709f ) was first reported 
in summer 1987 in Brouenou (Finisterre, France) (Flassch 1987). 
Moribund clams appeared on the clam bed surface before their 
death, and more than 80% of the affected clams presented a char- 
acteristic organic brown deposit on the inner surface of the shell 
margins, generally located between the pallial line and the edge of 
the shell, which frequently extended toward the pallial cavity. 
Because of these typical signs of the disease, it is called brown ring 
disease (BRD) (Paillard et al. 1989). 

BRD appeared in Spain in the summer of 1989 associated with 
massive mortalities of cultured manila clams of several commer- 
cial production areas in southwestern Spain (province of Cadiz). 
Later, several outbreaks of the disease were recorded in Cantabria 
(northern Spain), Galicia (northwestern Spain), and Andalusia 
(provinces of Cadiz and Huelva. southwestern Spain) (Castro et al. 
1992, Castro et al. 1993). For this reason, the disease constitutes a 
major obstacle to expansion of shellfish aquaculture on the Atlan- 
tic coasts of France and Spain. Several sanitary measures based on 
epizootic data have been proposed to decrease this disease, such as 
the control of BRD incidence in seed lots, reduction of the seeding 
density, and adaptation of the seeding to optimal season. The use 
of chemotherapeutics in intertidal shellfish culture zones is im- 
practical because of its high cost and low efficacy. For this, pre- 
vention criteria are the only mechanistns to control diseases in the 
extensive cultures of shellfish (Alderman 1992), Similar prophy- 
lactic measures have proved to be effective in the control of other 
epizootic shellfish diseases, such as bonamiasis and marteliasis in 
Ostrea edidis (Grizel et al. 1986. van Bannina 1988). Perkinsus 



spp. infection, and the MSX disease caused by Haplosporidium 
nelsoni in the case of Crassostrea virginica (Andrews and Ray 
1988, Ford and Haskin 1988). 

The incidence of BRD was previously studied by Castro 
(1994). who analyzed several manila clam populations seeded on 
different areas of Cadiz Bay. This study established a characteristic 
temporal pattern for BRD. The incidence of the disease increased 
in the first months of culture, achieving a peak in summer or 
aututnn (between 10 and 209^ of affected specimens, depending 
upon the population and zone). In the following months, the inci- 
dence of BRD maintained or decreased, but in the second summer 
of culture, substantial increase in BRD occurred again. The sea- 
sonal nature of BRD may be explained by the negative effect that 
high temperatures produced on the physiology of manila clams 
(reduction of the growth and increase in the metabolic rate), which 
provokes a decrease in the defense response of the clams (Fisher et 
al. 1987, Newell and Barber 1988). In addition, the maximal in- 
cidence of the BRD symptoms is coincident with the reproductive 
period of this clam species on the south Atlantic coast (Morel 
1988. Devauchelle 1990. Sarasquete et al. 1990), which con-e- 
sponds to a drop in the clam condition index. 

The causative agent of the BRD has been described as a new 
bacterial species, designated Vibrio tapetis (Borrego et al. 1996). 
Although several studies have been carried out to determine the 
antigenic, genetic, and molecular relationships between V. tapetis 
and other pathogenic Vibrio species (Castro et al. 1995. Castro et 
al, 1996. Castro et al. 1997a), little information is available re- 
garding transmission mechanisms and factors that affect the inci- 
dence and prevalence of BRD in clam culture. For these reasons, 
we studied transmission routes of V. tapetis as well as factors 



1051 



1052 



Martinez-Manzanares et al. 



TABLE 1. 
Conditions in Vibrio tapetis transmission experiments. 



Tanl< No. 



v. lapetis Dose 



Clam Separation 



2.5 X 10'-' 
2.5 X 10' 
2.5 X 10' 
2.5 X 10' 
Control 
Control 



Yes 
No 
Yes 
No 
Yes 
No 



° Colony-forming units (CFU) per clam. 

affecting prevalence of the disease. In addition, several antimicro- 
bial treatments were tested, and their efficacy was evaluated. 

MATERIALS AND METHODS 

Factors Affecting Disease Incidence 

Seed Density 

The influence of seed density on the prevalence of BRD signs 
was studied in an immersed cylindrical containers with forced 
upwelling of the Research Center "El Torufio" (Cadiz, Spain). 
Manila clam seed (about 10,000 specimens) of 0.2 to 0.5 g were 
tested previously to detect \'. tapetis. using the technique described 
by Castro et al. ( 1995). Healthy seed were placed in four contain- 
ers at two different densities: ( 1 ) high (600 seeds/nr). and (2) low 
(200 seeds/m"). The clam seeds were then allowed to grow for 3 
months, and sampling extended over the 4th, 5th. and 6th months. 
About 100 specimens per month per container were analyzed for 
the following parameters: wet weight, anteroposterior length, BRD 
signs, shell deformity, and mortality. BRD signs were recorded by 
examination of the inner surface of the clam shells under a ste- 
reomicroscope. 

Substrate Types 

Two populations of clams from the same stock of seed (wet 
weight 0.3-0.5 g) without BRD symptoms, were used to study the 
influence of the substrate type on disease prevalence. Population A 
(about 800,000 specimens) was seeded on transformed substrate 
consisting of sand and fine gravel at a density 425 clams per m^ 
and with a coefficient of tide (semidiurnal type) of 0.4 to 0.7. 
Population B (about I million specimens) was seeded on natural 



mud substrate at a seed density of 400 clams per m" and with the 
same coefficient of tide. Seven samples (about 100 clam speci- 
mens) of each population were collected during a 13-month period 
and analyzed for the same parameters as those for the seed density 
experiments. 

BRD Transmission Routes 

To establish the potential pathogen transmission routes. (1) 
direct contact (by contact with clams or sediment) or (2) water 
route, 600 specimens of T. phiUppinanim (5-7 g wet weight) were 
experimentally inoculated with V. tapetis strain CECT 4600^. fol- 
lowing the methodology described by Castro et al. (1997b). Two 
doses of V. tapetis were used to inoculate the clams: 2.5 x 10' 
colony forming units (CFU)/clam and 2.5 x 10' CFU/clam. 

The transmission experiments were carried out in the research 
center "Agua del Pino" (Huelva. Spain), using methacrylate tanks 
with a bed of about 10 cm of autoclaved marine sediment and 
recirculating water system (36% salinity and 24°C). For each 
pathogen dose, two kinds of tanks were used, one with two com- 
partments separated by a methacrylate sheet from the bottom of the 
tank to 2 cm below water level and sealed with silicone, and the 
other without separation (Table I ). In first kind of tank (nos. 1 and 
3), 100 labeled injected clams and 100 uninoculated clams were 
placed in each compartment. In tanks without separation (nos. 2 
and 4) the same number (n = 100) of labeled injected and non- 
injected clams were held together. Tanks 5 and 6 contained 100 
clams injected with sterile artificial seawater and 100 clams that 
were not injected. Clams were fed twice a week with 50 mg/tank 
of lyophilized Tetrasebnis spp. After 30 days, the clams were 
examined for survival and incidence of BRD signs. 

Antimicrobial Susceptibility of V. tapetis 

The antimicrobial susceptibility of I', tapetis was evaluated 
using the disk diffusion technique described by Barry and Thorns- 
berry (1991) using Mueller-Hinton agar no. 2 (BioMerieux) 
supplemented with NaCI to achieve a final concentration of 2% 
(w/v). The following antimicrobial agents and concentrations (sup- 
plied by BioMerieux) were used: ampicillin (Ap. 10 |j.g), amyka- 
cin (An, 30 jig), streptomycin (Sm, 10 |jig), kanamycin (Km. 30 
Jig), neomycin (Nm. 30 |xg). gentamycin (Gm. 10 (jLg), tobramycin 
(Tm, 10 ixg), oxolinic acid (OA, 2 |jLg), tetracycline (Tc, 30 |j.g), 
oxytetracycline (OT. 30 |xg). erythromycin (E. 15 p.g), chloram- 
phenicol (Cm, 30 jjLg), nitrofurantoin (Fm, 300 lU), and fiume- 
quine (UB. 30 p.g). Twenty-two strains of V. tapetis isolated from 



TABLE 2. 
Growth and pathological parameters of Tapes philippinarum seeds cultured at different densities. 







Low Density (200 Clams/m") 






High Density 


(600 Clams/m-) 




Parameters 


Dec. 


Jan. 


Feb. 


Mar. 


Dec. 


Jan. 


Feb. 


Mar. 


Wet weight (g) 


0.54 


0.58 


(1,54 


0,67 


0..54 


0,60 


0.61 


0.50 


Length (mm) 


















Mean 


13.81 


14.28 


14,60 


14,70 


13,18 


14.40 


14.50 


14.50 


SD 


1.92 


1.86 


1.60 


1.96 


2,21 


1.89 


1.26 


1.38 


BRD signs (%) 


4.23 


4.34 


3.52 


2.57 


2.54 


4.50 


ND 


5.72 


Shell defomiity (%) 


7.62 


2.60 


10.13 


4.13 


13.55 


4.51 


13.52 


11.25 


Mortality rate C*) 


7.62 


ND 


1.98 


4.50 


1.27 


1.52 


3.88 


5.71 



ND: No data. 

SD: Standard deviation. 



Transmission and Treatment of Brown Ring Disease 



1053 



different zones in northwestern France (Borrego et al. 1996; were 
used in these experiments. 

Serial dilution of the antimicrobial agents on agar petri dishes 
(Sahm and Washington 1991 ) was performed to determine experi- 
mentally the minimal inhibitory concentration (MIC) of the anti- 
microbials assayed. Serial dilutions of the antimicrobial drugs 
were carried out on Mueller-Hinton agar no. 2 supplemented with 
2% NaCI. 

To study the potential application of the antimicrobial agents 
effective against V. tapetis as a treatment tool, the following fac- 
tors were taken into consideration: antibiotic solubility and stabil- 
ity in seawater, and cost. For these assays, the antimicrobial con- 
centrations recommended for aquaculture practices in European 
Union (Schnick et al. 1997) were used. The to,\icity to clams was 
tested recording the mortality rate of the clams (0.3-0.5 g wet 
weight) at the end of the treatment period (maximum 3 days). The 
efficacy of antimicrobial treatments was evaluated on injected 
clams ( 10'' CFU of V. tapetis strain CECT 4600"^ per clam) treated 
and nontreated with the antimicrobial tested. After treatment. 
clams (100 per experimental group) were placed in aquaria with 40 
L of 5-|jLm filtered seawater and maintained at 19 ± 1°C with 
aeration. BRD signs were recorded after a 1 -month period, as 
above mentioned. 

RESULTS 

Factors Affecting Incidence of the Disease 

Results obtained for growth (wet weight and anteroposterior 
length) and pathological (BRD signs, shell deformity, and mortal- 
ity rate) parameters in both clam populations tested (low and high 
seed density) were examined using a one-way analysis of variance 
(ANOVA) (Table 2). Only in the last sampling (March), were 
significant differences between the populations (low and high den- 
sity) obtained for clam length (p = .0079). BRD signs (p = 
.0468). and shell deformity (p = .0158). 

The growth rate of clams in different substrate types was ana- 
lyzed by wet weight, length, and productivity per ni" of substrate, 
which was estimated from the values of density and mean weight 
of clams. Weight and length of the cultured clams in both sub- 
strates was similar throughout the experiment (Table 3). with no 
significant differences (p > .01) between clam populations. How- 
ever, a significantly lower biomass in natural substrate was re- 
corded (p = .0145). largely caused by the higher clam mortality 
rate (Fig. I ). 

Mortality rate was clearly different between clam populations 
(Fig. 2). In the natural substrate, the mortality increased quickly in 





7n-i 






F* 






60- 


i^, 












n 


51.1 1 














j: 


411 - 






i^ 




,^ 


iU - 






s 








s 


'() - 






c 




3 


10 - 


O 






Figure 1. Cumulative mortality of clams cultured in two substrate 
types (T.S.: transformed substrate: N.S: natural substrate). 



the first months of culture, achieving a cumulative mortality rate of 
48.8% after 6 months (January), with a peak of mortality of 27.1% 
in November. Monthly mortality percentages in subsequent sam- 
plings were lower than 13%. with a minimum of 2.6% (Fig. 2-A). 
At the end of the experiment, cumulative mortalities on natural 
substrate were 62%. In contrast, low monthly mortality rates were 
recorded in transformed substrates during the first months of cul- 
ture (lower than 5%). Higher mortality peaks appeared in spring 
and summer months 8-13 with mortalities lower than 20% (Fig. 
2-B). The cumulative percentage in the transformed substrate was 
41.2%, which was significantly lower (p < .01 ) than that obtained 
for natural substrate (62%). 

The percentage of BRD signs during the sampling period var- 
ied between and 15% (mean value 6.4%) in transformed sub- 
strate (Fig. 2-B). with increases in the percentages in both sum- 
mers. A siinilar rate was observed in the natural substrate, varying 
between 9 and 45%. with a mean value of 19.2% (Fig. 2-A). A 
chi-square test showed that there was a significant difference (p < 
.05) between substrates for BRD signs. Shell deformity was simi- 
lar in both substrates (Fig. 2), with high occurrence at the end of 
the sampling (52% in transformed substrate and 74% in natural 
substrate). 

BRD Transmission Experiments 

The incidence of the BRD was dependent on the V. tapetis 
concentration and on separation of tanks (Table 4). No significant 
differences (p > .05) were obtained between the mortality rate of 
the uninoculated and inoculated clams, although for the experi- 
mentally infected clams, the pathogen doses as well as the sepa- 
ration in the tanks significantly affected mortality rate (p = .013). 



TABLE 3. 
Growth of Tapes philippinarum in natural and transformed substrates. 





Biomass 


(Kg/m-| 




Length (mm) 




Wet Weight (g) 


Months Sampling 


Natural 


Transformed 


Natural 


Transformed 


Natura 


Transformed 


July 


0.60 




0.75 


18.6 


19.6 


1.7 


1.8 


November 


0.60 




0.80 


19.6 


20.1 


2.0 


2.2 


January 


0.65 




0.91) 


20.2 


21.8 


2.3 


2.4 


March 


0.70 




0.90 


22.2 


22.3 


2.5 


2.6 


May 


0.80 




1. 00 


24.0 


23.7 


4.0 


3.2 


July 


0.80 




2.20 


25.0 


27.0 


4.4 


4.6 


August 


0.90 




2.30 


27.0 


29.0 


5.7 


5.9 



1054 



Martinez-Manzanares et al. 



Natural substrate 



m 


Mortaliiv 


-^ 


Brown Ring 


-KD- 


Shell deformity 




TABLE 5. 

Characteristics of the selected antimicrobials effective against 
Vibrio tapetis. 



Antimicrobials 



MIC (|jg/mL) 
Serial Dilution 



Ampicillin 


2.0 


Chloramphenicol 


4.0 


Erythromycin 


4.0 


Flumequine 


2.0 


Nitrofurantoin 


2.0 


Oxolinic acid 


2.0 


Oxytetracycline 


2,0 



Seawater 
Solubility 



Cost 



L 
H 
H 
L 
L 
L 
H 



+: Positive; -: negative; H: high; L: low. 



Transformed substrate 



n 


Mortalm 


-*- 


Brown Ring 


-■o- 


Shtll defomm\ 




Figure 2. Temporal evolution of the pathological parameters studied 
in clams seeded in two substrate types (natural and transformed). 



Susceptibility to Antimicrobial Agents 

The susceptibility of V. tapetis to chemotherapeutic agents was 
established by determining the MICs of the most effective agents 
against the pathogen. All strains of V. tapetis tested (n = 22) 
showed resistance to streptomycin, amykacin. kanamycin, genta- 
mycin, and neomycin. They were sensitive to seven other antimi- 
crobial agents, with MIC ranging from 2 to 4 (xg/mL (Table 5). 
Those antimicrobials belonging to the same antibiotic group 
showed similar MIC for V. tapetis. 

To study the potential application of antimicrobials as a treat- 
ment tool, the following factors were taken into consideration: 
antiinicrobial MIC for V. tapetis. antibiotic solubility and stability 
in seawater. and cost (Table 5 ). On the basis of these factors, three 



antimicrobials were selected and tested to evaluate their efficacy to 
prevent the development of BRD. The results showed that the bath 
treatment was effective to reduce the BRD signs under 7.3 vs. 38% 
for injected clams without antibiotic treatment, in the following 
conditions: 10 mg/L for nitrofurantoin (bath during 1-2 h for 3 
days). 1.5 mg/L for flumequine (bath during 1-2 h for 3 days), and 
I mg/L for oxolinic acid (only one bath during 2 h). The mortality 
recorded at the end of the experiment ranged between 2.0 and 
8.9% in antibiotic-treated clams, and 8.2% in the nontreated group. 

DISCUSSION 

In the present study, two substrate types were evaluated in 
relation to the occurrence of BRD. a natural mud substrate and a 
transfonned (sand and fine gravel) substrate. The results (Fig. 2) 
demonstrated a higher BRD occurrence and lower clam survival in 
natural substrate as compared to transformed substrate. These find- 
ings confirm the results obtained by Muiioz et al. (1993) and 
Castro (1994). The physical characteristics of the seeded sediment, 
as well as the biogeochemical cycle of the organic matter in the 
substrate, significantly intluence the physiology and susceptibility 
of cultured clams to pathogenic agents. Barillari et al. (1990), in a 
study carried out in nine sedimentary beds of the Venezia Lagoon, 
deinonstrated that the mortality of T. philippiiianon was higher in 
natural substrates of fine particle size than in substrates with 
greater size particles. In the latter, better circulation of the water 
and oxygen is produced, avoiding the anoxic process and the mac- 
roalgal sedimentation, two parameters that exerted a negative ef- 
fect on bivalve mollusk growth. Moreover, in fine mud sediments, 
there is a higher concentration of organic matter (Dahlback and 
Gunnarsson 1981). which increases the oxygen consumption and 



TABLE 4. 
BRD prevalence and mortality rate of clams in transmission experiments. 



Inoculated Clams 



Noninoculated Clams 



Tank No./Clam Separation/Pathogen Doses" 



BRD Signs ( % ) 


Mortality ( % ) 


BRD 


Signs ( % ) 


Mortality ( % ) 


13 


10 




8 


16 


17 


28 




8 


18 


24 


22 




14 


16 


36 


28 




38 


8 





2 







6 


2 


10 







9 



l/Yes/2.5 X 10' 
2/N0/2.5 X 10' 
3/Yes/2.5 x 10' 
4/N0/2.5 X 10' 
5/Yes/Control 
6/No/Control 



' Expressed as colony-forming units (CFU) per clam. 



Transmission and Treatment of Brown Ring Disease 



1055 



the subsequent growth of anaerobic bacteria with H,S procluctit)n 
(Fenchel and Riedl 1970). The negative effect exerted by the 
muddy substrata on the clam physiology may also be explained in 
terms of the energetic costs that require the constant release by the 
clam of inorganic particulate matter from the sediment. This 
inechanism involves a weakening of the shellfish that prevents 
them from responding adequately to external stress. 

Epizootic studies on shellfish are based on the population re- 
sponse to a pathogenic agent, but resistance capability to the dis- 
ease is an individual characteristic. In addition, several environ- 
mental factors act as stressing agents, negatively affecting the 
physiology and defense mechanisms of the host (Sindermann 
198.^). Therefore, in deficient culture conditions, such as mud 
substrates, the percentage of highly susceptible clams will be large, 
and the disease will progress quickly without typical signs until a 
large proportion of the clam population develops the disease. In 
contrast, in populations with better physiological conditions, mor- 
tality will be associated with advanced steps of the disease and. 
therefore, there will be a delay between the first signs of the 
disease and the clam mortality. 

Flassch ( 1989) demonstrated the existence of a close and direct 
relationship between the seed density and the incidence of the 
BRD. In the present study, the results obtained (Table 2) do not 
establish a clear relationship between the seed density and the 
pathologic parameters tested, except for the last sampling (month 
of March). Similar results were found by Mufioz et al. (1993) using 
clam densities of 200 to 600 units/m" for transformed substrate, 
and 30 to 200 units/m" for natural substrate. In contrast to the 
results reported by Flassch (1989) in France, the clam seed density 
does not seem to play an important role in the prevalence of BRD 
on the Spanish south Atlantic coasts. 

The results of the transmission experiments (Table 4) suggest 
that V. tapetis infects manila clams under the laboratory conditions 
assayed. It seems that direct contact in the sedimentary bed be- 
tween inoculated and healthy clams enhances the transmission of 
the infective agent and the development of the disease. Similar 
results were obtained by Raghukumar and Lande (1988), who 
experimentally infected rock oyster Crassostrea cucuUala with the 
fungus Ostrocoblabe implexa. This pathogenic microorganism is 
the causative agent of the "shell disease," and provokes a bio- 
degradation of calcareous substrates of the oyster shells, symptoms 
similar to those caused by V. tapetis both in T. philippiiuiniin and 
T. decussatus (Novoa et al. 1998). 

The classical practical procedures for controlling the bacteria in 
juvenile shellfish, on which bacterial populations have established 
themselves, are based on treatment with dilute solutions of sodium 
hypochlorite ( 10 ppm) followed by a thorough seawater rinse (El- 
ston et al. 1982). However, the capacity of seed clams to tolerate 



such treatment requires that the valves are intact enough to seal 
well on closure, and sometimes the above-mentioned treatment 
causes a higher mortality rate of clams than the pathogenic agent. 
For this reason, in the present study and according to Sindermann 
and Lightner ( 1988), we have evaluated several chemotherapeutic 
agents on the basis of their efficiency to inhibit V. tapetis. their 
solubility and stability in seawater, their toxicity and accumulation 
in clams, and applicability and cost. 

Few studies have been focused on the antibiotic treatment of 
shellfish affected by an infectious disease because of the potential 
risk posed by the fact that drugs may enter the environment as a 
result of drug released from the treated-animal feces or dissolved 
in seawater (Ervik et al. 1994). In the present study, 14 chemo- 
therapeutic agents were assayed, and three of them were selected 
for specific treatment of affected manila clams with BRD. On the 
basis of the effectiveness of the bath treatment to clam seeds to 
prevent the development of BRD, the three chemotherapeutic 
agents selected were nitrofurantoin ( 10 mg/L seawater for 3 days), 
tlumequine ( 1 .5 mg/L seawater. I h, for 3 days), and oxolinic acid 
(only one bath treatment of 1 mg/L seawater for 2 h). None of 
antibiotic treatments negatively affect the seed clams (mortality 
rate lower than 10% and similar to control population) and were 
very effective to control V. tapetis. 

The use of antimicrobial drugs as a method to eliminate patho- 
genic bacteria from shellfish systems has been reported previously 
(Le Pennec and Prieur 1977, Kraeuter and Castagna 1984). More 
recently. Brown and Tettelbach (1988) used streptomycin sulfate 
(30 mg/L) and neomycin sulfate ( 100 mg/L) treatments against a 
vibriosis affecting larvae of Mercenariainercenaria and Crassos- 
trea virginica. However, there is a danger of resistance associated 
with the use of these antibiotics, both in naturally occurring Vibrio 
anguillaruin (Aoki et al. 1974) as well as other vibrios (Koditschek 
and Guyre 1974), because R plasmids, which confer resistance to 
a wide spectrum of antibacterials, has been reported for these 
Vibrio species (Jeffries 1982). 

Although the antimicrobial treatments tested in this study have 
proved to be adequate to prevent BRD. large-scale experiments 
would be conducted to confirm their applicability in aquaculture 
facilities. In addition, the treatments proposed must be applied in 
a controlled system, such as shellfish nurseries, which would avoid 
the potential risk of the antibiotic dissemination into natural envi- 
ronment. 

ACKNOWLEDGMENTS 

This paper has been supported by a grant from DGICYT (Ref 
PB93-0467). We thank M. J. Navarrete for her help in the English 
revision of the manuscript. 



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Joiinuil of Shellfish Research. Vol. 17. No. 4. 1057-1064, 1998. 



GENETIC STUDIES OF THE VENERID CLAM GENUS KATELYSIA 



SHIRLENA W. L. SOH,' - GREG B. MAGUIRE,' AND 
ROBERT D. WARD' 

'CSIRO Division of Marine Research 

Huharl. Tasmania 7001, Australia 
'Department of Applied Biomedical Science 

University of Tasmania 

Lcmnceston, Tasmania, Australia 
^Department of Aquaculture 

University of Tasmania 

iMunceston, Tasmania, Australia 

ABSTRACT Two samples of Katelysia scalarina. one of A', rhyuphora. and one of K. peronii were compared for 16 allozyme loci. 
Diagnostic loci were identified, one of which (SOD*) provided unambiguous discrimination of all three clam species. Katelysia 
rlnnphora and K. peronii were found to be the most similar pair (D = 0.34). Vanability levels were high, with average observed 
heterozygosities per locus ranging from 0.257 to 0.314, and percentage polymorphism from 62.5 to 81.3. Six samples of K. scalarina 
(three from Tasmania and one each from Victoria, South Australia, and Western Australia), examined for six variable loci, revealed 
three distinct groups: the three Tasmanian samples; the Victorian and South Australian samples; and the Western Australian sample. 
Differentiation between the Tasmanian and mainland subpopulations was striking, especially for the PGM* locus. Coastal gene flow 
mediated by stepping-stone migration between adjacent subpopulations may account for the similarities between subpopulations on the 
same land mass; whereas, the 10 to 12-day larval period is likely to hmder gene flow across the Bass Strait between Tasmania and the 
mainland. It is suggested that any future Tasmanian hatchery production should use Tasmanian rather than mainland broodstock, and 
vice versa, to prevent the introduction to native populations of possibly ill-adapted genotypes. 

KEY WORDS: Katelysia. clam, allozyme. diagnostic loci, stock-structure, gene flow 



INTRODUCTION 

The worldwide aquaculture production of clams, cockles, and 
ark shells is on a par with that of oysters: about 1.23 million tons 
in 1994, as compared with 1.1 million tons for oysters (FAO 
1996). Wild fisheries production is also substantial — in 1992 only 
about 40% of mollusk production came from aquaculture (FAO 
1995). In Australia, mainly wild clam populations are harvested, 
but the sustainability of this practice is uncertain and so aquacul- 
ture production is being considered. 

Clams of the genus Katelysia are among those most heavily 
exploited (both as food and fish bait) in the temperate waters of 
southern Australia. They are some of the most abundant shallow 
water bivalves in these waters, frequently constituting major or 
dominant faunal components of soft-bottomed intertidal and shal- 
low subtidal zones of sheltered bays and estuaries (Bellchambers 
1998). The three species, K. scalarina. K. rhytiphora. and K. pero- 
nii. are frequently sympatric and can be difficult to distinguish 
morphologically (Roberts 1984, Lamprell and Whitehead 1992). 
The first two species have very similar distributions, ranging from 
Western Australia to New South Wales and including Tasmania; 
whereas, the less abundant K. peronii is absent from New South 
Wales (Nielsen 1963, Lamprell and Whitehead 1992). 

In Tasmania, the main clams harvested are Katelysia (princi- 
pally K. scalarina) and Ruditapes largillierti (thought to have been 
introduced from New Zealand). Both inhabit similar habitats: 
Katelysia the intertidal and shallow subtidal zone of sheltered bays 
and estuaries, Ruditapes the subtidal zone. The total Tasmanian 
clam catch rose from 15.56 tons in 1990 and 14.00 t in 1991 to 
51.44 t in 1992 and 60.97 t in 1993 (Anon. 1994). From 1994 to 
1996, between 23 and 45 t per year of K. sccdarina alone were 
harvested (S. Riley pers. comm.). Most are exported live to fish 
markets in Melbourne and Sydney. 



With the increased utilization of the Katelysia resource, a 
hatchery-based industry to supplement or replace the wild fisheries 
is being considered. Experimental hatchery production in New 
South Wales of K. rhytiphora to metamorphosis has been reported 
(Nell et al. 1994). and similar work with K. scalarina is under way 
in Tasmania (Kent et al. submitted). In Asia, "almost all of their 
cockle and clam landings involve some aspect of culture in the 
grow-out process" (Manzi 1991). 

Although there has been considerable ecological research into 
these species (Nielsen 1963, Coleman 1982, Roberts 1981, Roberts 
1984. Bellchambers and Richardson 1995). there have been very 
few biochemical genetic studies. Nielsen (1963) carried out some 
preliminary chromatography research but was unable to differen- 
tiate Katelysia scalarina from K. rhytiphora. Roberts (1984) ex- 
amined protein-stained isoelectric focusing gels, and found some 
apparent species differences but did not assess within-species 
variation. 

This paper investigates the allozyme genetics of Katelysia. The 
first part examines the genetic relationships of the three taxa, prin- 
cipally to find diagnostic allozyme markers to assist in species 
identification. The second part examines the genetic population 
structure of K. scalarina in Tasmania and the mainland of Aus- 
tralia, principally to assist fisheries management and any future 
aquaculture operations. A knowledge of the genetic diversity of 
wild stocks can help minimize any deleterious effects of introduc- 
ing hatchery-bred animals or translocating stock (Allendorf and 
Waples 1996. Rhymer and Simberloff 1996). 

MATERIALS AND METHODS 

Samples of Katelysia .sccdarina (Fig. 1 ) were collected from six 
sites: three in Tasmania and one each in Victoria. South Australia, 
and Western Australia (Fig. 2. Table I ). Samples oi K. peronii and 



1057 



1058 



SOH ET AL. 




Figure 1. Vencrid clams included in this study. From left to right: Katelysia rhytiphora {^■in mm shell length, from Portland Harbour, Victoria: 
K. scalarina (41.2 mm, from Macrae, Port Phillip, Victoria: A', peroiiii (43.7 mm, from St. Kilda, Victoria). Specimens from the Western 
Australian Museum collection of venerids. 



K. rhytiphora (Fig. 1) were collecte(J from the saine site in South 
Australia (Table I ). The clams were transported live to the labo- 
ratory in cool-boxes with ice-packs. On arrival, adductor muscles 
and digestive gland tissues were dissected and stored at -80°C in 
2 mL microcentrifuge tubes. The siphon tissue of small individuals 
was pooled with their adductor muscle tissue. 

Morphological Identification 

The following descriptions are from Lamprell and Whitehead 
( 1992). The genus Katelysia is characterized by subovate to elon- 
gate shells with angular posterior: concentric ridges predominating 
over radial ribs, radial sculpture being weak or absent; pallial sinus 
medium sized to short, horizontal or ascending; anterior laterals 
absent. K. scalarina: shell length to 40 mm; subovate with angular 
posterior; close-set. sharp concentric ridges; color white or cream 
with brown or gray; interior white, often purple posteriorly. K. 
rhytiphora: shell length to 60 mm; ovate to elongate; concentric 
ridges overlaid by fine radial lirae; color white or light brown, 
sometimes with dark brown zig-zag pattern; interior white, yellow 
centrally, purple at muscle scars. K. peronii: shell length to 38 mm; 
ovate and similar to scalarina. but finer concentric ridges and 
posterior less angular; color cream or fawn, sometimes with darker 
zig-zag patterns; lunule and escutcheon dark; interior purple, 
sometimes yellow or orange centrally. It should be noted that size 
varies from site to site: our photographed specimens (Fig. 1) of 
Katelysia scalarina and K. peronnii are a little larger than the 
maximum sizes given by Lamprell and Whitehead (1992). 

Genetic Analysis 

Before electrophoresis, w hole tissues were homogenized manu- 
ally with 2 to 4 drops of distilled water and centrifuged for 5 min 
at 10.000 rpm: the supernatants were used for electrophoresis 
(Table 2). Either starch or cellulose acetate gels were used (Table 
2). Starch gels used 9% Connaught starch with a discontinuous 
histidine-citrate buffer system run at 100 V for 4.5 h (gel buffer 
O.Od^M histidine HCI pH 7.0: electrode buffer 0.4 IM trisodium 
citrate pH 7.0); the cellulose acetate gels were Helena Titan III 
plates run at 150 V for I h (gel and electrode buffer 0.075 M Tris 
and 0.025 m citric acid, pH 7.0). Staining techniques followed 
Richardson et al. (1986) and Hebert and Beaton (1989). Hetero- 
zygote banding patterns were consistent with known quaternary 
structures (Ward et al. 1992). 

Sixteen loci were examined in the between-species compari- 
sons (Table 3), and six (see Table 6) were examined in the Kately- 
sia scalarina stock-structure analysis. Five of the latter loci were 
chosen for their variability and their reliability (DIA*, ESTD*, 
GPI*, MDH-1*. and PGM*), the sixth locus (MDH-2*) was 



scored on the same gels as MDH-I*. The MDH patterns immedi- 
ately enabled any nonA'. scalarina specimens to be eliminated 
from the stock-structure analysis. 

Where there were multiple loci, the locus encoding the fastest 
migrating allozyme was designated "I." In the between-species 
comparisons, alleles were lettered from "a" for the fastest migrat- 
ing allozyme. In the Katelysia scalarina stock-structure analysis, 
alleles were numbered according to the anodal mobility of their 
product relative to that of the most common allele observed in the 
Cockle Creek (TAS2) sample, which was designated "'lOO"'. (Al- 
lele identities between the two studies, where known, are identified 
in Table 6. Sample sizes were substantially larger for the stock 
structure analysis; see Table 5.) 

Statistical Analysis 

For the between-species comparisons, mean sample sizes, num- 
bers of alleles, percent polymorphism (a locus was considered 
polymorphic if the most common allele had a frequency less than 
0.95) and heterozygosities (both observed and unbiased Hardy- 
Weinberg expected values) were estimated by the BIOSYS-1 
package (Swofford and Selander 1981). 

For the A', scalarina stock-structure analysis, individual 
sample, and locus tests for goodness-of-fit to Hardy-Weinberg 
expectations used Fisher's exact test after all but the most common 
allele had been pooled at each locus. Two genetic diversity pa- 
rameters were estimated: F,s (the correlation between two uniting 
alleles relative to the subpopulation and defined as the ratio of the 
difference between the expected and observed heterozygosities to 
the expected heterozygosity) and F^y (the correlation between two 
gametes drawn at random from each subpopulation. defined as the 
ratio of the difference between the expected total heterozygosity 



30-S - 


WA 


SA 


V 


K.^TAS3 


40"S - 






/ 

VIC 


W^TAS2 
TASl 




5(X1 km 


lO'S 












1 


1 


1 


1 



120*E 



130"E 



140"E 



150"E 



Figure 2. Southern Australia, showing approximate locations of 
sample sites. 



Genetic Studies of K.\TELysiA 



1059 



TABLE 1. 
Collection details for Kalelysia species; n = number of clams collected. 



Site 


State 


Abbreviation 


Location 


Dale Collected 


n 


K. scaUiriiui 












Cockle Creek 


Tasmania 


TASl 


43°33'S. 146°52'E 


Aug. 95 


120 


Ansons Bay 


Tasmania 


TAS2 


41°03'S. 148°2rE 


Sept. 95 


120 


Smithton 


Tasmania 


TAS3 


40°50'S. 145°00'E 


Sept. 95 


120 


Queenscliff 


Victoria 


VIC 


38°16"S, I44°39-S 


Nov. 95 


81 


Ceduna 


South Australia 


SA 


32°07'S. 133°46'E 


Dec. 95 


58 


Albany 


Western Australia 


WA 


SS-OrS. 117°58-E 


Dec. 95 


110 


K. rlixtiphora 












Ceduna 


South Australia 


SA:RHY 


32°07-S. 133°46-E 


Dec. 95 


16 


K. peroiiii 












Ceduna 


South Australia 


SA:PER 


32°07-S. 133°46-E 


Dec. 95 


16 



and the average expected subpopulation heterozygosity to the e.x- 
pected total heterozygosity). F,,; essentially estimates deviations 
from Hardy-Weinberg expectations, and F^t estimates the extent 
of genetic differentiation of subpopulations. Values of Fisher's 
exact test. F,s. and F^y were estimated by BIOSYS-1 (Swofford 
and Selander 1981 ). Allele frequency homogeneity across samples 
was tested by the randomised Monte Carlo chi-square procedure of 
Roff and Bentzen (1989). which obviates the need to pool rare 
alleles. For each test, 1 ,000 randomizations of the data were car- 
ried out. 

For both sets of comparisons, BIOSYS-I was used to calculate 
unbiased genetic distances between samples (Nei 1978) and to 
cluster the resulting distance matrices with the UPGMA (un- 
weighted pair-group method with averaging) algorithm. Sample 
sizes were smaller for the between-species comparisons, because 
estimates of genetic distance are far more dependent on numbers 
of loci than numbers of individuals (Nei 1978, Gorman and Renzi 
1979). 

When multiple tests of a single hypothesis were carried out, the 
standard Bonferroni procedure was applied. The p value for a 
specific test had to be less than, or equal to, 0.05/n (where n is the 
number of tests) to be deemed statistically significant (Miller 
1981). 

RESULTS 

Comparison Between Species 

Sixteen loci were examined in four samples, one each of 
Katelysia rhytiphora and K. peronii and two of K. scalahna (Table 
3). Levels of variation in each sample were high, despite quite 
small sample sizes, with an average of 2.5 to 2.8 alleles per locus 
and between 62.3 and 81.257f polymorphism. Observed and ex- 
pected heterozygosities per locus ranged from 0.257 to 0.314 and 
from 0.283 to 0.377, respectively (Table 4). Sample sizes were too 
small to warrant Hardy-Weinberg tests. 

All species pairs showed diagnostic loci (Ayala and Powell 
1972), although only SOD*, which was nearly monomorphic and 
different in each species, allowed all three species to be discrimi- 
nated unambiguously. Other diagnostic loci were: K. scalarina — 
K. rhytiphora. AK*, APK*, MDH-2*, PGDH*. K. scalarina~K. 
peronii. ADA*, AK*, MDH-2*, PGM*: K. rhytiphora— K. pero- 
nii. APK*. Here, a diagnostic locus is defined as one that will 
enable individuals to be identified with a s99% chance of correct 



identification, given that the two taxa are represented in an un- 
known sample in equal frequencies. 

Katelysia rhytiphora and K. peronii were more closely related 
to one another (D = 0.337) than either was to K. .scalarina (mean 
D = 1.047, range 0.859-1.238) (Table 5, Fig. 3). The within- 
species genetic distance of K. scalarina (D = 0.068) was much 
less than any of the between-species genetic distances (mean D = 
0.905, range 0.337-1.238) (Table 5, Fig. 3). 

Katelysia scalarina Population Structure 

Three samples of Katelysia scalarina from Tasmania and three 
from the mainland of Australia were examined to assess popula- 
tion structure (Table 6), both on medium scales (comparisons of 
Tasmanian samples) and broad scales (comparisons among main- 
land samples, and of mainland with Tasmanian samples). 

Thirty-six tests of Hardy-Weinberg equilibrium (6 loci x 6 

TABLE 2. 
Enzymes used in this study. 





EC 


Locus 






Enzyme 


Number 


Abbreviation 


Tissue 


Gel 


Adenosine deaminase 


3.5.4.4 


ADA* 


m 


s 


Adenylate kinase 


2.7.4.3 


AK* 


m 


s 


Arginine phosphokinase 


2.7.3.3 


APK* 


m 


ca 


Aspartate aminotransferase 


2.6.1.1 


AAT* 


m 


s 


Diaphorase 


1.8.1.4 


DIA* 


d 


s 


Esterase-D (UV. umb. acetate I 


3.1.-.- 


ESTD* 


m 


s 


Fumarate hvdratase 


4.2.1.2 


FH* 


d 


s 


GIucose-6-phosphate isomerase 


5.3.1.9 


GPI* 


m 


s 


Isocitrate dehydrogenase 


1.1.1.42 


IDHP-1* 


d 


ca 






IDHP-2* 


d 


ca 


Malate dehydrogenase 


1.1.1.37 


MDH-1* 


m 


s 






MDH-2* 


m 


s 


Peptidase (val-leu) 


3.4.-.- 


PEP* 


m 


ca 


Phosphogluconate 










dehydrogenase 


1.1.1.44 


PGDH* 


m 


ca 


Phosphoglucomutase 


5.4.2.2 


PGM* 


d 


s 


Superoxide disinutase 


1.15.1.1 


SOD* 


d/m 


s 



Tissue: d = digestive gland (visceral mass), 
ca = cellulose acetate, s = starch (see text), 
same enzyme are designated by consecutive 
the fastest migrating system. 



m = adductor muscle. Gel: 
Multiple loci encoding for the 
numbers, with "I" denoting 



1060 



SOH ET AL. 



TABLE 3. 

Allele frequencies at 16 loci in Katelysia rhytiphora, K. peronii. and 
two populations of A', scalarina; n = number of individuals. 



TABLE 3. 
continued 







A', rhytiphora 


A', peronii 


A', scalarina 








Locus 


Allele 


SA:RHV 


SA:PER 


SA 


TAS2 


ADA* 


a 


0.031 


0.219 








b 


0.219 


0.531 








c 


0.063 


0.250 


0.031 






d 


0.656 




0.031 


0.333 




e 


0.031 




0.938 


0.667 




n 


16 


16 


16 


12 


AK* 


a 


0.958 


1.000 








b 


0.042 










c 






0.969 


1.000 




d 






0.031 






n 


12 


15 


16 


10 


APK* 


a 


0.036 










b 


0.179 










c 


0.786 


0.042 








d 




0.125 


0.906 


1.000 




e 




0.833 


0.094 






n 


14 


12 


16 


12 


AAT* 


a 






0.031 






b 


0.893 


0.367 


0.906 


1.000 




c 


0.107 


0.633 


0.063 






n 


14 


15 


16 


12 


DIA* 


a 


0.156 


0.219 


0.094 


0.042 




b 


0.594 


0.250 


0.531 


0.333 




c 


0.219 


0.188 


0.281 


0.500 




d 




0.094 








e 


0.031 


0.250 


0.094 


0.125 




n 


16 


16 


16 


12 


ESTD* 


a 




0.031 


0.031 


0.167 




b 


0.969 


0.844 


0.969 


0.708 




c 


0.031 


0.063 




0.125 




d 




0.063 








n 


16 


16 


16 


12 


FH* 


a 


0.844 


0.938 


0.594 


0.583 




b 


0.156 


0.063 


0.406 


0.417 




n 


16 


16 


16 


12 


GPI* 


a 




0.469 








b 




0.094 








c 


0.625 


0.438 


0.188 


0.208 




d 


0.125 




0.344 


0.333 




e 


0.094 




0.063 


0.333 




f 


0.094 




0.406 


0.042 




g 


0.063 






0.083 




n 


16 


16 


16 


12 


IDHP-1* 


a 






0.063 






b 




0.083 


0.250 


0.042 




c 


1.000 


0.667 


0.656 


0.875 




d 




0.250 


0.031 


0.083 




n 


14 


12 


16 


12 


IDPH-2* 


a 


0.036 




0.063 






b 


0.929 


0.531 


0.125 






c 


0.036 


0.031 


0.813 


1.000 




d 




0.438 








n 


14 


16 


16 


12 


MDH-1* 


a 




0.031 


0.031 


0.U45 




b 


1.000 


0.969 


0.031 


0.227 




c 






0.938 


0.727 




n 


16 


16 


16 


11 


MDH-2* 


a 


0.094 










b 


0.906 


1.000 


0.063 


0.042 



Locus 



Allele 



A. rhytiphora 
SA:RHV 



A. peronii 
SA:PER 



A. scalarina 



SA 



TAS2 



PEP* 



PGDH* 



PGM* 



SOD* 



16 



0.179 
0.786 
0.036 

14 

0.107 
0.250 
0.643 



14 

0.031 
0.219 
0.563 
0.188 



16 



1.000 



15 



16 



0.031 
0.438 
0.469 
0.063 
16 

0.292 
0.667 
0.042 



0.438 
0.531 
0.031 
16 

0.03 1 
0.969 



16 



0.93S 
16 

0.219 
0.281 
0.438 
0.063 

16 



0.083 
0.917 



12 

0.156 
0.813 
0.031 



0.958 
12 

0.208 
0.417 
0.292 
0.083 



12 



0.042 
0.792 
0.125 
0.042 
12 

0.417 
0.458 
0.125 



16 



0.969 
0.031 
16 



1 ,000 



subpopulations) were performed (data not shown). Two had P 
values less than .05 (GPI* at TAS3, p = .030. and DIA* at SA. p 
= .013). but neither was significant after Bonferroni corrections 
for 36 tests. Six tests (one per locus) of whether the average value 
of F|s was significantly different from zero were performed (Table 
7); one was significant after Bonferroni corrections. This was for 
DIA*. and at 0.177 (p = .003) indicated an over-all homozygote 
excess. 

All loci showed significant heterogeneity in allele frequencies 
(p < .005) across the six subpopulations (Table 7). For five loci — 
DIA*, MDH-1 *, MDH-2*, GPI*, and ESTD*— the extent of dif- 
ferentiation, although significant, was low. with F^^ values of 
about 0.05 or less. For one locus. PGM*, the extent of differen- 



tiation was much more extensive (F<. 



0.298). At this locus. 



nearly 30% of the allele frequency variation could be attributed to 
differentiation among subpopulations. and there was very little 
overlap in the allele frequencies of the Tasmanian and mainland 
subpopulations. The two common alleles in Tasmania (PGM* 120 
and PGM* 100) were uncommon on the mainland, and the two 
common mainland alleles (PGM*85 and PGM*75) were uncom- 
mon in Tasmania (Table 6). 

Comparing the three Tasmanian subpopulations with one an- 
other found only DIA* to show evidence of spatial differentiation 
(p = .038), but this result became nonsignificant after Bonferroni 
corrections for six tests. Only one of the 1 8 pairwise subpopulation 
tests gave a p value less than .05 (DIA*, TAS2-TAS3, p = .047), 



Genetic Studies of Katelysia 



1061 



TABLE 4. 

Summan of genetic variability values at 16 loci in Katelysia rhytiphora. K. peronii, and two subpopulations of A', scalarina. 



Locus 



A', rhytiphora 
SA:RHV 



A', peronii 
SA:PER 



A. scatarina 



SA 



TAS2 



Mean sample size per locus 
Mean number of alleles per locus 
Polymorphism <* (0.95) 
Observed heterozygosity per locus 
Expected heterozygosity per locus" 



14.9 + 0.3 

2.7 ±0.3 

68.75 

0.291 ±0.059 

0.283 ± 0.058 



15.1 ±0.4 

2.8 ± 0.3 

75.00 

0.314 ±0.063 

0.377 ±0.065 



15.8 + 0.3 

2.8 ±0.2 

81.25 

0.276 ± 0.059 

0.299 ± 0.059 



11.3 + 0.7 

2.5 ±0.3 

62.50 

0.257 ± 0.070 

0.333 ±0.071 



' Unbiased estimate (Nei 1977). 



a result clearly nonsignificant after Bonferroni corrections. Thus, 
no significant heterogeneity was observed among the Tasmanian 
subpopulations. 

The three mainland populations showed more differentiation. 
Three loci — DIA*. MDH-2*. and PGM* — showed evidence of 
spatial differences in allele frequencies, and all remained signifi- 
cant after Bonferroni corrections (p = .001, .006, and .002, re- 
spectively). The three mainland populations were compared pair- 
wise for these three loci. This showed that the Western Australia 
subpopulalion was the most distinct; all comparisons with p < .05 
included WA (DIA*. WA-VIC, p = .027. WA-SA. p = .014; 
MDH-2*. WA-VIC. p = .003: PGM*, WA-VIC, p = .010. WA- 
SA. p = .001). In none of these pairwise tests was the VIC-SA 
comparison significant. 

Clustering the pairwise subpopulalion genetic distances over all 
six loci (Fig. 4) confirmed these general findings. The three Tas- 
manian subpopulations clustered together (mean D = 0.001) and 
away from the mainland subpopulations (mean D of Tasmanian vs. 
mainland comparisons = 0.146. n = 9). Of the mainland sites, the 
SA and VIC subpopulations were genetically very similar (D = 
<0.00l). with the WA subpopulation more distinct (mean D = 
0.003. n = 2l. 

DISCUSSION 

All three species of Katelysia are genetically highly variable. 
Sixteen loci were examined. The average observed heterozygosi- 
ties per locus ranged from 0.257 to 0.314. and percent polymor- 
phism from 62.5 to 81.3 (Table 4). The average heterozygosity for 
mollusks, assessed from 105 species and an average of nearly 22 
loci per species, is about 0.145 (Ward et al. 1992). 

The Nei genetic distance estimates between the different spe- 
cies pairs ranged from 0.337 to 1.238. with the corresponding 
identity values ranging from 0.714 to 0.290 (Table 5). These val- 

TABLE 5. 

Unbiased genetic distance (Dl (above diagonal) and identity (I) 

values (below diagonal) (Nei, 1978) between Katelysia rhytiphora, K. 

peronii, and two subpopulations of A. scalarina. 



A. rhytiphora 
SA:RHV 



A. peronii 
SA:PER 



A. scalarina 



SA 



TAS2 



SA:RHY 
SA:PER 
SA 

TAS2 



0.714 
0.424 
0.399 



0.337 

0.309 

0.290 



0.859 
1.173 



0.934 



0.919 
1.238 
0.068 



ues fall within the typical invertebrate between-species distance 
range of about 0.20 to 1.60, corresponding to an identity range of 
about 0.80 to 0.20 (Thorpe 1983). Katelysia rhytiphora and K. 
peronii are the most closely related species-pair, a conclusion 
reached earlier by Roberts ( 1984) from comparing protein-stained 
isoelectric focusing gels of the three species. These two species, 
with D = 0.337 and I = 0.714, are unusually closely related, 
although some bivalve (oyster) species show similarly limited di- 
vergence (e.g., three Sacco,strea species, D = 0.171 to 0.454, 
Buroker et al. 1979). Congeneric clam species typically have D 
values around 1.0 or greater (e.g., three Ruditapes species, D = 
1.050 to 1.840. Borsa and Thiriot-Quievreux 1990; two Chamelea 
species. D = 1.138. Backeljau et al. 1994), similar to the values of 
K. rhytiphora and K. peronii to K. scalarina (c. 0.90 and 1.20, 
respectively). 

Diagnostic allozyme loci were identified to enable unambigu- 
ous species identification — knowledge that proved useful for di.s- 
tinguishing several K. rhytiphora in the putative K. scalarina 
samples. Although sample sizes in the between-species study were 
quite small, the overlaps in allele frequencies at the diagnostic loci 
are very small, and the loci are likely to remain diagnostic, at least 
at the 99% level, in larger samples. 

The data were examined for the possible occurrence of be- 
tween-species hybridization. This seemed possible, because the 
samples of Katelysia rhytiphora, K. peronii, and one of the K. 
scalarina samples were sympatric. and the spawning periods, at 
least of K. rhytiphora and K. scalarina, can overlap (Nielsen 1963, 
Roberts 1984). However, in the between-species study, no het- 
erozygotes of the apposite hybrid Fl genotypes were observed for 
the diagnostic SOD* locus, although sample sizes were small, and 
rare hybrids would not have been detected. 

In the Katelysia scalarina stock-structure analysis, which used 

- K. scalarina TAS2 



K. scalarina SA 



K. rhytiphora 



K. peronii 



1.0 



— r— 

0.8 



0.2 



— I 

0.0 



06 0.4 

Genetic distance 
Figure 3. UPGM.A cluster analysis of Nei's (1978) unbiased genetic 
distance (D) among three species of Katelysia, based on 16 allozyme 
loci. 



1062 



SOH ET AL. 



TABLE 6. 
Allele frequencies at six loci in six populations of A', scalarina. 



Locus 


Allele 


TASl 


TAS2 


TAS3 


VIC 


SA 


WA 


DIA* 


125 






0.004 


0.025 








120(a) 


0.103 


0.092 


0.081 


0.075 


0.107 


0.05 1 




110(b) 


0.310 


0.298 


0.275 


0.342 


0.286 


0.280 




100(c) 


0.413 


0.447 


0.449 


0.308 


0.429 


0.364 




90(e) 


0.136 


0.158 


0.140 


0.217 


0.161 


0.178 




75 


0.022 


0.004 


0.05 1 


0.033 


0.018 


0.121 




60 


0.016 










0.005 




n 


92 


114 


118 


60 


56 


107 


ESTD* 


150 






0.008 


0.019 


0.009 


0.005 




120(a) 


0.183 


0.157 


0.171 


0.179 


0.155 


0.141 




100(b) 


0.731 


0.712 


0.729 


0.790 


0.836 


0.836 




65 (c) 


0.075 


0.127 


0.083 


0.012 




0.018 




50 


0.01 1 


0.004 


0.008 










n 


93 


118 


120 


81 


58 


110 


GPI* 


145 


0.082 


0.065 


0.060 


0.050 


0.018 


0.047 




125(c) 


0.212 


0.178 


0.282 


0.094 


0.140 


0.142 




100(d) 


0.321 


0.300 


0.312 


0.331 


0.333 


0.392 




80(e) 


0.228 


0.252 


0.192 


0.206 


0.167 


0. 1 5 1 




55(0 


0.114 


0.152 


0.124 


0.294 


0.316 


0.255 




35(g) 


0.043 


0.048 


0.030 


0.025 


0.026 


0.014 




n 


92 


115 


117 


80 


57 


106 


MDH-1* 


165 


0.063 


0.064 


0.042 


0.012 


0.035 


0.037 




135(b) 


0.195 


0.182 


0.229 


0.056 


0.053 


0.023 




100(c) 


0,742 


0.754 


0.729 


0.926 


0.912 


0.936 




65 








0.006 




0.005 




n 


95 


118 


120 


81 


57 


109 


MDH-2* 


140 












0.005 




120(b) 




0.008 


0.004 


0.044 


0.009 






100 (c) 


0.995 


0.992 


0.992 


0.956 


0.991 


0.991 




70 


0.005 




0.004 






0.005 




n 


93 


118 


120 


80 


57 


107 


PGM* 


125 


0.011 


0.008 


0.004 










120(a) 


0.317 


0.314 


0.383 


0.006 


0.035 






100(b) 


0.548 


0.610 


0.533 


0.086 


0.096 


0.046 




85 (c) 


0.091 


0.059 


0.075 


0.790 


0.728 


0.713 




75(d) 


0.032 


0.004 


0.004 


0.111 


0.140 


0.222 




55 




0.004 




0.006 




0.019 




n 


93 


lis 


120 


81 


57 


108 



n = number of individuals. 

Allele homologies with Table 3, where known, are in parentheses. 

much larger sample sizes, there was evidence of possible Fl hy- 
brids between K. scalarina and K. rhytiphora. Two of the three 
Tasmanian animals that were MDH-2*b/c heterozygotes were also 
MDH-1 *b/c heterozygotes (one in TAS2 and one in TAS3) — an 
unlikely chance occurrence given the low frequency of MDH-1 *b/ 
c heterozygotes. In Tasmania, the expected frequency of such a 
double heterozygote in K. scalarina would be about O.l'/c. only 
about one-third the observed frequency of 0.7%. Possibly one or 
both these animals were Fl hybrids between K. rhytiphora and K. 
scalarina. despite the failure of laboratory experiments to produce 
such hybrids (Nielsen 1963). No such evidence for hybridization 
was recorded for the mainland subpopulations — none of the six 
MDH-2*b/c heterozygotes observed in the VIC sample nor the 
single SA MDH-2*b/c heterozygote was an MDH-1 *b/c hetero- 
zygote. 

The stock-structure analysis of Katelysia scalarina revealed 
three distinct genetic groups: the three Tasmanian samples; the 
Victorian and South Australian samples; and the Western Austra- 



lian sample. The lack of significant differentiation, at least at the 
scale of resolution afforded by this study, between the three well- 
separated Tasmanian subpopulations (and between the Victorian 
and South Australian subpopulations) suggests significant levels of 
coastal gene flow. On the other hand, the sizeable differences 
between the Tasmanian and mainland subpopulations. especially at 
the PGM* locus, suggest minimal gene flow across Bass Strait. 
The differences at the PGM* locus seemed so large that initially 
the hypothesis of two unrecognized and allopatric sibling species 
was entertained, but the Nei genetic distance between the SA and 
TAS2 subpopulations, 0.068 over the 16 loci, suggests a level of 
differentiation more typical of within- than between-species dif- 
ferentiation (Thorpe 1983). Similarly, the limited divergence evi- 
dent in the Western Australia sample does not support the exis- 
tence of a western Australian subspecies, Katelysia scalarina 
polita. as proposed by Nielsen (1963). 

Coastal gene tlow mediated by stepping-stone migration be- 
tween adjacent subpopulations probably accounts for the similari- 



Genetic Studies of Katelysia 



TABLE 7. 



Genetic diversity statistics for six luci and six pupulutions of 
Katelysia scalarina. 



Locus 


F,s 


P 


Fs. 


P 


DIA* 


0.177 


0.003 


0.008 


<.001 


MDH-I* 


0.030 


0.575 


0.053 


<.001 


MDH-2* 


-0.027 


0.090 


0.015 


.005 


GPI* 


0.009 


0.692 


0.017 


<.001 


ESTD* 


-0.030 


0.195 


0.013 


<.001 


PGM* 


0,043 


0.217 


0.298 


<.0(11 



F^r data analyzed by Mests of Individual suhpopulations against expected 

value of 0. 

FjT data analyzed by Monte-Carlo chi-sijiiare tests of allele frequency 

homogeneity. 



1063 
TAS 1 

TAS2 
>- TAS 3 



WA 



SA 



L VIC 



ties between subpopulations on the same land mass; whereas, the 
short larval life of the species (only c. 10 to 12 days; Katelysia 
scalarina, Kent et al. submitted; K. rhytiphora. Nell et al. 1994) is 
likely to restrict gene flow severely across the Bass Strait between 
Tasmania and the tnainland. Gene flow between the WA and SA 
subpopulations might be hindered by the intervening presence of 
long sections of exposed coastline dominated by cliffs and without 
sheltered bays; these may have fragmented possible clam habitats 
between these two sampled localities. 

The pattern of population differentiation in Kiitelysia scalarina 
is broadly similar to that seen in other tnarine clams. For example, 
populations of giant clams (Tridacna i;ii;as) on the same reef, even 
on extensive reefs such as the Great Barrier Reef, show little 
differentiation at seven polymorphic loci (mean D c. 0.001, F^^ c. 
0-0.01); whereas, regionally separated populations are far more 
divergent (mean D c. 0.069. Fg-p c. 0.15, Benzie and Williams 
1995). The commercial hard clam or quahog of the North Ameri- 
can Atlantic coast, Mercenaria mercenaria. shows little differen- 
tiation froin Florida to Massachusetts, but in the north of its range 
(New Brunswick and Nova Scotia), where populations are dis- 
junct, differentiation is more marked. Comparison of Massachu- 
setts. Maine. Prince Edward Island, and New Brunswick popula- 
tions gave an F^^ estimate for seven polymorphic loci oi 0.050 
(Dillon and Manzi 1992), only a little less than the mean of 0.067 
observed over the six polymorphic loci in our study. 

What are the implications of these results for the clam industry? 
The lack of significant differentiation among the Tasmanian sub- 
populations suggests that these can be managed as a single stock, 
although the standard caveats must be made that it takes only a 
little gene flow to render subpopulations effectively panmictic, and 
that larger sample sizes and more loci might reveal heterogeneity. 
A second point is that the very low levels of gene flow between 
Tasmanian and mainland subpopulations, indicated by their sub- 



I 
0.150 



0.145 



I 
0.140 



0.010 



0.005 



1 

0.000 



Genetic distance 

Figure 4. IIPGM.'V cluster analysis of Nei's (1978) unbiased genetic 
distance (D) among six populations of Katelysia scalarina, based on six 
allozyme loci. 

stantial allozyme differences, would facilitate regional co- 
adaptation of genes and gene complexes. If this scenario is correct, 
then any Tasmanian hatchery production would be advised to use 
Tasmanian rather than mainland animals as broodslock. and vice 
versa. This would help to prevent the introduction of possibly 
ill-adapted genotypes into the wild via farm escapes. Similarly, 
any stock enhancement programs that may be embarked on in the 
future would be wise to use local broodstock to maintain biodi- 
versity levels. 

ACKNOWLEDGMENTS 

This work is part of FRDC project 93/232. We would like to 
thank the following for helping with sample collections: Lynda 
Bellchambers, Elizabeth Cox. Robert Green. Greg Kent. Sean Ri- 
ley (Tasmania); Craig Lowe (South Australia); Jonathon Bitton 
(Western Australia). Sue Boyd, and Louise English (Victoria). 
Shirley Slack-Sinith and Clay Bryce (Western Australia Museum) 
kindly assisted with venerid photography. Thanks also are due to 
Liz Turner (Tasmanian Museuin and Art Gallery) for assistance in 
species identification and to Alastair Richardson (University of 
Tasmania). Nick Elliott, Peter Rothlisberg, Vivienne Mawson 
(CSIRO) and two anonymous reviewers for comments on an ear- 
lier version of the manuscript. Present address for G. B. M.: Fish- 
eries Western Australia. Research Division. P.O. Box 20. North 
Beach, WA6020, Australia. 



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Anon. 1994. Policy Document and Fishery Development Plan for the Tas- 
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Ayala. P. J. & J. R. Powell. 1972. Allozymes as diagnostic characters of 



sibling species o{ Drosoi'liila. Pruc. Nail. Acad. Sci. USA 69:1094- 
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Backeljau. T.. P. Bouchet. S. Gofas & L. de Bruyn. 1994. Genetic varia- 
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Bellchambers. L. M. 1998. Ecology and ecophysiology of Katelysia sca- 
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Beilchambers. L. M. & A. M. M. Richardson. 1995. The effect of substrate 
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Borsa. P. & C. Thiriot-Quievreux. 1990. Karyological and allozymic char- 
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Dillon, R. T., Jr. & J. J. Manzi. 1992. Population genetics of the hard clam, 
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Hebert. P. D. N. & M. J. Beaton. 1989. Methodologies for allozyme analy- 
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Kent, G. N., G. B. Maguire. M. John, M. Cropp & K. Frankish. 1998. 
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Nell, J. A., W. A. O'Connor, M. P. Heasman & L. J. Goard. 1994. Hatch- 
ery production for the venerid clam Katelysia rhytiphora (Lamy) and 
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156. 

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Richardson. B. J.. P. R. Baverstock & M. Adams. 1986. Allozyme elec- 
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Journal of ShellfLsb Research. Vol. 17, No. 4. 1063-1070. 1998. 

BROODSTOCK CONDITIONING, SPAWNING INDUCTION, AND LARVAL REARING OF THE 
STEPPED VENERID, KATELYSIA SCALARINA (LAMARK 1818) 



GREG N. KENT,' GREG B. MAGUIRE,' MARTIN JOHN,^ 
MILES CROPP,' AND KEN FRANKISH'"* 

School of Aquaciihiire 
University of Tasmania 
Launceston, Tasmania, Australia. 7250 
-Shellfish Culture Pty. Ltd. 
Bicheno, Tasmania, Australia, 7215 
Marine Shellfish Hatcheries 
Bicheno, Tasmania, Australia, 7215 
Fremantle Maritime College 
Fremantle. Westenj Australia, Australia, 6160 



ABSTRACT In a series of trials, broodstock venerid clams Kalelysia scalarina were conditioned, or obtained directly from fishing 
grounds, and stimulated to spawn using thermal cycling. Broodstock. initially with only moderately developed gonad, were sufficiently 
conditioned for mass spawnings after 8 weeks at I3"C in an indoor recirculating seawater system. Ripe wild broodstock maintained 
at 20'C could still be spawned after 6 weeks in a similar system. Mean fecundity from mass spawning trials ranged between 0.7 and 
2.4 X 10'' eggs/female, and fecundity estimates for strip spawned individuals varied from 0.3 to 2.2 x 10'' eggs/female. Neither 
intramuscular serotonin injections (6 x 10"" to 1 x 10"'' mol. total dose), nor strip spawning were reliable methods for producing 
significant numbers of healthy larvae. Eggs (69 ± 2 (im) developed to D veliger larvae (110 ± 1.3 jjim) within 48 h at 20°C. 
Metamorphosis to spat (210.9 + 2.1 p.ni) was observed from day 20 following treatment with 10"^ m norepinephrine for 60 min on 
day 19. Larval rearing was not always successful because of bacterial and viral problems and the extended settlement period for this 
species. 

KEY WORDS: Clam, venerid. Karelxsia scalarina. broodstock conditioning, spawning, larval rearing 



INTRODUCTION 

The stepped venerid. Kalelysia scalarina (Lamark 1818) is a 
relatively small clam of 35 to 50 mm shell length (Lush 1992). K. 
scalarina is a shallow (2—1 cm) burrower (Nielsen 1963. Bell- 
chambers and Richardson 1995) and is found throughout both 
sheltered and moderately exposed intertidal waters of Tasmania 
and southern mainland Australia (Gabriel and Macpherson 1962). 
K. scalarina may be distinguished from another closely related 
species K. rhytiphora (Lamy 1937) by the absence of radiating 
striae and more oblique shape (Gabriel and Macpherson 1962). In 
addition, K. .scalarina is often distributed higher up the intertidal 
zone than A', rhytiphora (Roberts 1984). 

In Tasmania. K. scalarina is the major species in a small clam 
fishery and is exported to mainland niche markets and occasionally 
overseas (Treadwell et al. 1992. Zacharin 1993). In recent years, 
concerns over a decrease in abundance of this species in some 
areas have necessitated a reduction in catch quotas and given im- 
petus to research into the feasibility of producing hatchery-reared 
stock for culture or fisheries enhancement. 

Currently, there is no commercial aquaculture production of 
Kalelysia spp. within Australia, although research into hatchery 
production and growout of A', rhytiphora has been undertaken 
(Nell et al. 1994, Paterson and Nell 1997). Although oyster hatch- 
ery techniques have much in common with clam culture (Maguire 
1992). and in the United States have required little modification for 
clam production (Castagna and Manzi 1989), further investigation 
into hatchery techniques for temperate clams, including these en- 
demic species, is still required (Maguire 1992, Nell et al. 1995). 
Potential bottlenecks exist with supply of broodstock, spawning. 



larval rearing, and metamorphosis, and this study addresses as- 
pects of these topics for K. scalarina. 

MATERIALS AND METHODS 

All clams collected from natural habitats were transported to 
Tasmanian culture facilities in plastic containers without water or 
sediment. All seawater used was drawn from exposed sections of 
coastline largely unaffected by freshwater runoff. However the 
quality of seawater for the University of Tasmania's (UTAS) bi- 
valve hatchery at Launceston is periodically affected by seaweed 
debris. 

Broodstock Conditioning (Broodstock Trial I ) 

Two separate groups of 200 broodstock clams were collected 
from Ansons Bay in August 1994 (Broodstock Trial 1 ) and trans- 
ported to Marine Shellfish Hatcheries. Bicheno (Fig. 1 ). Gonad 
condition was visually assessed as intermediate (++) (Garland et 
al. 1993). Clams were scrubbed and rinsed with fresh water and 
placed in an indoor conditioning system consisting of a 1,100 L 
holding through, recirculating pump, and header tank. Clams were 
held in plastic mesh baskets lined with 400 |xni screen and filled 
with 2 to 4 mm quartz gravel. Water temperature was maintained 
at 13°C. and clams were fed a mixture of Pavlova lulheri (Droop) 
and Tahitian Isochrvsis sp., supplemented with Chaeloceros miiel- 
leri (Lemmermann) at approximately 8 to 12 x 10'° cells/day. 
After approximately 8 weeks conditioning, clams were assessed as 
ripe (-H-I-) (Garland et al. 1993), and 50 animals from each group 
were successfully spawned by cycling water temperatures between 
13"C and 20°C. 



1065 



1066 



Kent et al. 




Figure 1. Collection and larval rearing sites within Tasmania, Aus- 
tralia for the clam Kalelysia scalarina. 



Broodstock Maintenance (Broodstock Trial 2) 

In December 1996 approximately 200 clams were collected 
from Ansons Bay. Twenty clams were fixed in 10% phosphate 
buffered formalin for later histology, and the remainder were re- 
productively conditioned in an indoor recirculating seawater sys- 
tem. The system at UTAS Launceston consisted of a 300-L res- 
ervoir and 4 X 50 L plastic bins (45 animals per bin) each with a 
floor area of 0.19 m". Beach sand (6-cm deep) was placed on the 
floor of two bins, so that clams could burrow, while clams in the 
other two bins were maintained without substrate. Water tempera- 
ture was maintained at 20°C ± 0.5°C, and salinity remained con- 
stant at 34.5 ± 0.2 ppt. Clams were fed a daily ration of P. lutheii. 
Tahitian Isochrysis sp., and Tetraselmis siiecica (Butcher) so that 
all feed was cleared within 24 h (Toba et al. 1992). 

In January 1997 (6 weeks later), another sample of 20 clams 
was collected from the same site at Ansons Bay and preserved. A 
sample of 10 clams from each bin was also taken and preserved as 
above for later histology. A further 72 clams from the substrate 
bins were held out of water overnight and placed in 1 jjim (nomi- 
nal) filtered seawater (FSW) at I9°C the following morning, 45 
minutes after which spawning commenced. Animals were allowed 
to spawn out in the tray yielding a total of 24 x 10" eggs, with a 



fertilization rate of 99%. These subsequently developed into ac- 
tive, healthy D larvae within 48 h. 

Standard 4-|jLm paraffin sections were prepared from a 3-mm 
slice of the preserved samples, using the anterior edge of the foot 
as a reference (Howard and Smith 1983). These were stained using 
Mayer's hematoxylin and eosin Y. Gonads were staged for game- 
togenic development, using a modified staging system based on 
Dinamani (1974). 

Spawning Induction 

In summer 1995, clams from three sites (Georges Bay, Ansons 
Bay, and Swanwick) were collected for spawning trials 1 to 3 at 
UTAS, Launceston. Gonad condition by visual observation was 
assessed as moderate (-i-i-) (Garland et al. 1993). Clams failing to 
spawn after temperature cycling (20°C-26°C, at 30 min intervals), 
were injected via the anterior adductor muscle with various con- 
centrations and volumes of serotonin (5-HT) (Table 1 ) (Gibbons 
and Castagna 1984, Gibbons and Castagna 1985, Heasman et al. 
1994, O'Connor and Heasman 1995) and placed in 1 |j.m FSW at 
25=C. 

In spawning trial 4, strip spawnings were also attempted with 
moderate-to-ripe stock {++!+++) (Garland et al. 1993) that failed 
to spawn with temperature fluctuations. Ten females and six males 
were strip spawned by lacerating the gonad with sterile scalpel 
blades and flushing eggs and sperm into separate 1-L beakers with 
1-fjim FSW. Eggs were collected and screened through a 63-|j.m 
screen and resuspended in 1 L fresh FSW. Eggs were then treated 
with 0.25 mL of 2 M NH4OH (0.5 x 10"' M final concentration) 
for 45 minutes to aid breakdown of vesicle after Wada ( 1953) and 
Minaur (1969). Following this, eggs were resuspended in fresh 
FSW, and a dilute sperm solution was added. Fertilized eggs were 
placed in a 300-L lar\'al rearing tank (LRT) at 20°C with low 
aeration. 

Larval Rearing 

Larval trials 1 to 4 (Table 2) were conducted in summer 1993 
to 1994 at Shellfish Culture Pty. Ltd., a commercial bivalve hatch- 
ery at Bicheno. Broodstock were collected from various estuaries 
(Fig. I), including Great Musselroe Bay (north coast). Swanwick 
on the east coast, and Cockle Creek in the south of the state. 

Water used in spawning and larval rearing was l-|jLm FSW 
(spawning and day 0) and lO-jxm FSW thereafter. Disodium eth- 
ylenediaminetetra-acetic acid (EDTA) was added to day water at 
1 mg/L as per Utting and Helm (1985). With the exception of 



TABLE \. 
Numbers of male and female Katelysia scalarina induced to spawn by injection of serotonin at different volumes and concentrations. 



Spawning 
Trial 



Number of 
Broodstock" 



Collection 
Site 



Injection 
Volume (jiL) 



Concentration'' 
(mm I 



Number Spawned 



Male 



Female 



10 


Georges Bay 


100 


10 


Georges Bay 


30 


10 


Ansons Bay 


30 


10 


Swanwick 


30 


40 


Ansons Bay 


30 


39 


Ansons Bay 


40 



10 
10 
10 
10 

2 
15 



lid not be visually differentiated prior to spawning; hence, number of broodstock 



■■ Sexes could not oe visually aillerentiaiea prior to spa 
'' Range of total amount of serotonin injected was (6 x 



S(male + female). 



10"" to 1 



' mole). 



Conditioning. Spawning, and Rearing A', scmarina 



1067 



TABLE 2. 
Mean fecundity and numbers <if' male and female Katelysia scalarina induced to spawn by thermal fluctuations. 



Larval 
Trial 


Number of 
Broodstock 




U V 


Number Spawned 






Stimulus ( C) 


Light 


Male 


Female 


Eggs (xlO") 


(Eggs X lOVCIam) 


1 


No data'' 


20 


No 


No data" 


No data'' 


4.5 


No data"' 


2 


186 


24-27 


Yes 


50 


62 


150 


2.4 


3 


98 


20-26 


Yes 


40 


29 


20 


0.7 


4 


230 


20-26 


Yes 


22 


26 


26 


1.0 



■■ Fertilized eggs collected following inadvertent overnight spawning of captive broodstock. 



trial 1. ripe broodstock were held out of water for 12 h (Castagna 
and Manzi 1989) before being placed in a dark flat fiberglass 
through (100 x 600 x 900 mnt) filled with recirculating filtered 
.seawater. Clams were encouraged to spawn by variously elevating 
water temperature by 3 to 6°C, as indicated in Table 2 (Loosanoff 
and Davis 1963). A UV light was included in the recirculating 
system to help maintain low bacterial levels and stimulate spawn- 
ing (Bourne et al. 1989). Numbers of broodstock and spawning 
details are outlined in Table 2. 

Larvae for trial 1 were obtained after broodstock spawned over- 
night in their holding tank. Eggs were collected the following 
morning and were rinsed through a 53-(j,m screen and collected 
on 25-|xm mesh. Larval rearing then proceeded as for other trials. 
Larvae were cultured in 3,000-L and 15,000-L tlat-bottom 
fiberglass tanks. For each trial, eggs were stocked at 7 to 10 egg.s/ 
mL. and larval densities were between 3 to 4 larvae/mL. Water 
temperatures were rnaintained at 24°C ± 0.5°C. Tanks were 
cleaned and water was changed every 48 h after larvae had been 
retained on appropriate-sized nylon mesh screens. Larvae were fed 
a 1:1 (by cell number) mixture of P. lutheri andTahition Isochrysis 
sp. at approximately 10.000 cells/larvae/day (Toba et al. 1992). 

For larval trial 5 in February 1996. ripe broodstock from An- 
sons Bay were encouraged to spawn at UTAS. Launceston via 
thermal fluctuations. Males and females were left to spawn to- 
gether in the spawning tray. Fertilized eggs were screened through 
a 75-|jt,m screen to remove feces and other debris and retained on 
25-|ji,m screen. Eggs were resuspended in fresh FSW and stocked 
in 300-L tanks at 25 eggs/mL. Equal quantities (by cell number) of 
P. hitheri, Tahitian Isochrysis. and Chuetoceros calcitnms 
(Paulsen) were fed at approximately 5.000 cells/larvae/day. Water 
temperatures were maintained at 20.5°C ± 0.2°C. Tanks were 
cleaned and. water was changed every 48 h with l-|j.m UV ster- 
ilized FSW. On day 8. larvae were transferred to a 1,000-L fiber- 
glass flat-bottom tank, and stocking densities were reduced to 1.2 
larvae/mL. At day 18. 20.000 larvae were placed in a 200-mm 
diameter plastic downweller pot (with 132-p.rn mesh) and treated 
with lO"'* M solution of the catecholamine norepinephrine for 60 
min (Coon et al. 1986) to promote metamorphosis. Water was 
circulated within a 40-L bin by means of an air lift pump. Spat and 
juvenile clams were maintained for a further 48 days in the above 
system, with cleaning, feeding, and water changed every 48 hours. 



tological examination of clams collected from the wild for brood- 
stock trial 2 in December 1996. revealed gonads to be ripe, with 
large numbers of mature ova or sperm in the gonad follicles. 
Subsequent retention of these animals for 6 weeks in a condition- 
ing system did little to improve the extent of gonad develop- 
ment. However, examination of gonad tissue from wild stock in 
January 1997. revealed that at least a partial spawning had oc- 
curred, which was not mirrored by animals held in the broodstock 
system (Fig. 2). 

In spawning trials 1 to 3. intramuscular injection of 5-HT en- 
couraged some moderately conditioned males to spawn, releasing 
sperm with normal activity. However, in all cases, females failed 
to spawn (Table 1), despite variations in concentration and volurne 
of 5-HT administered. Controls that were not injected did not 
spawn. 

Stripped females (35—45 mm shell length) in spawning trial 4 
yielded a total of 8 x 10'' eggs, with a mean diameter of 70 (j.m ± 



100 



C 60 



•S 50- 



O 20 




Dec '96 



I 
Jan. '97 (+ 6 weeks) 



RESULTS 

K. scalarina can be reproductively conditioned in a relatively 
simple recirculating seawater system, and ripe broodstock can be 
maintained for more than a month for subsequent spawnings. His- out substrate: n = 10. 



B Stage 4: Ripe gonad with mature gainetes 

Stage 5: Post spawned gonad, ruptured folbcles. phagocytes present 

Figure 2. Gonad development in wild and captive Katehsia scalarina 
held for 6 weeks in a recirculating conditioning system, with and with- 



1068 



Kent et al. 



2.5 |jLm. Fecundity of individuals ranged from 0.3 x lO*" to 2.2 x 
10^. Observed eggs were pear-shaped, with a prominent germinal 
vesicle. Survival of larvae from eggs treated with NH4OH, was 
very low. falling to less than \% by day 2 postfertilization. More- 
over, many of the larvae that remained alive were abnormal and 
e.\hibited extensive fouling of the velum. 

In larval rearing trials 2 to 5, ripe K. scalarina spawned readily 
in response to themial stimulations of 3 to 6°C above ambient. 
Mean egg diameter was 69 |xm ± 2 |jim, and average fecun- 
dity ranged between 0.7 x 10" and 2.4 x 10'' eggs/female 
(Table 2). Controls, not subjected to thermal stimulation, did not 
spawn. 

Larval rearing was not always successful (Fig. 3). However, 
in all trials, development of fertilized eggs to trochophore stage 
occurred within 24 h and to D veligers, with a mean shell 
length of 110 |j,m ± 1.3 \i.m, by 48 h. Survival of larvae to 
day 5 was between 30 to 50%. but in all cases, was less 
than 1% by settlement. Growth of K. scalarina was rapid at 
24°C, with larvae reaching a mean shell length of 200 (xm by day 
8 (Fig. 3a). Pediveligers of approximately 200 |xm shell length, 
were first observed between day 9 and day 15, depending upon 
rearing temperatures, which were 24°C and 20.5°C. respectively 
(Fig. 3b, 4), and metamorphosis to spat was first observed by day 
20 at 20.5°C (Fig. 4). Following metamorphosis, growth was ex- 
ponential until day 53, after which it slowed abruptly, possibly in 
response to reduced water flows caused by fouling of the down- 
weller screen. 



00 

c 




Days from spawning 

Figure 4. Growth (expressed as shell length) of Katelysia scalarina 
reared at UTAS. Launceston (trial 5) during February to April 1996: 
points are mean ± SE, (n = 5-20). 



DISCUSSION 



Broodstock 



Broodstock conditioning systems are advantageous for matur- 
ing bivalves both within (Castagna and Kraeuter 1981, Castagna 



a. Trial 1 



b. Trial 2 





Age (days) 



10 12 14 16 18 20 
Age (days) 



c. Trial 3 



d. Trial 4 




2 4 6 8 10 12 14 16 18 20 
Age (days) 




2 4 6 



10 12 14 16 18 20 



Age (days) 



Figure 3. Results of four larval rearing trials of Katelysia scalarina, conducted at Bicheno during summer 1993. Points are mean ± SD, n = 20-38: 
a. and b. indicate rapid development (trials 1 and 2): c. and d. indicate characteristic larval crashes around days 6 to 8 (trials 3^). 



Conditioning. Spawning, and Rearing K. scaiarina 



1069 



and Manzi 1989. Toba et al. 1992). and outside ot the normal 
spawning season (Numaguchi 1997). K. scalurina broodstock held 
in clean sediment, could be conditioned at a relatively low tem- 
perature ( \y'C) (broodstock trial I ). Also, mature clams collected 
from the wild could be maintained in ripe condition within a 
recirculating system, despite indication of spawning of stock in 
the fishery during the experimental period (broodstock trial 2). 
(Fig. 2). 

Spawning 

Fecundity of K. scalarina was comparable to that for K. 
rhytiphora (Nell et al. 1994) and the Manila clam Ruditapes phil- 
ippinarum Adams and Reeve (Utting et al. 1996, Laing and Lopez- 
Alvarado 1994). but less than that of the hard clam Meneimrici 
mercenaria (Castagna and Kraeuter 1981). 

During spawning trials, both isolation of spawning male and 
female stock and mass spawnings in the tray were undertaken. We 
detected no deleterious effects from the latter, although more than 
50 sperm per egg were observed on some occasions. Indeed, larvae 
displaying the greatest degree of activity were produced using this 
method. Nevertheless. K. scalarina has not always responded well 
to spawning induction protocols. On occasions, spawning has oc- 
cuiTed up to 2 days after thermal stimulation, and neither serotonin 
injections nor strip spawnings have provided appropriate results. 
These difficulties may have arisen largely from variability in the 
quality of broodstock obtained directly from the wild. Spawning in 
twii additional trials in the summer of 1996 to 1997 was relatively 
predictable (within 60 min of initiation of thermal stimulation) 
when broodstock in excellent condition were available. 

Gibbons and Castagna (1985) found that injections of between 
80 to 800 [jLUiol of 5-HT into the adductor muscle of gravid M. 
mercenaria encouraged spawning; however, males were more re- 
sponsive than females, and both concentration and absolute dose 
significantly affected spawning success. Numaguchi (1997) in- 
duced spawning in the common oriental clam Merelri.x liisoria 
with a 125-fjLmol intragonadal injection of 5-HT. and other work- 
ers have induced spawning in bivalves with 10"' M external ap- 
plications (Ram et al. 1992). During our trials, the application of 
various concentrations of 5-HT failed to induce spawning in fe- 
male broodstock. although .some males did spawn. This may have 
been as a result of inadequate broodstock condition, although Ram 
and Nichols ( 1993) reported that injection of serotonin into zebra 
mussels induced ripe males to spawn but not females. 

Strip spawnings using moderate-to-ripe broodstock were not 
successful, and larval mortality was very high (99'/r). In addition, 
many of the remaining larvae were deformed. In the rearing of 
Pinctada maxima, using stripped eggs treated with ammonium 
hydroxide, Minaur (1969) also reported up to 95% mortality of 
larvae to pediveliger stage, with some larvae showing deformed 
valves or other gross abnormalities. At this stage, it is unclear 
whether the deficiencies of this technique are primarily attributable 
to the method itself, such as possible toxic side effects of the 
NHjOH used to promote breakdown of the germinal vesicle, or 
inadequately matured gametes. Stripped gametes from both this 
study and Minaur (1969) were primarily oocyte ?< as defined by 
Dinamani (1974). These oocytes are smaller and more irregular in 
shape as compared to free oocytes at final maturity (Dinamani 
1974). and. as such, may have contributed to poor larval survival. 

Lanal Rearing 

Our results suggest that K. scalarina cannot be reared reliably 
in hatcheries by simply adopting existing oyster hatchery tech- 



niques. However, at 20"C, in trial 5, larval growth was comparable 
to K. rhytiphora (Nell et al. 1994) and similar to that for the Manila 
clam R. philippinarum (Utting and Spencer 1991). 

In a number of the early trials mn at 24°C, mass mortality 
occurred between days 8 and 9 (Fig. 3c,d). Larval crashes (be- 
tween days 5 and 8) of commercial cultures of K. scalarina run at 
16 to 18°C have also been reported (R. Pugh, pars. comm.). In both 
instances a herpes-like, viral pathogen has been found in larvae 
from some of the cultures investigated (J. Handlinger pers. 
comm.). Other failed cultures have been associated with bacterial 
infestations, a common problem with rearing of bivalve larvae 
(Gibbons and Blogoslawski 1989). Optimal temperatures for each 
successive stage in the early life history of K. scalarina must be 
identified. This should facilitate improved reliability of hatchery 
rearing as has been the case for such other bivalves as the com- 
mercial scallop Pectin fumatus (Reeve) (Heasman et al. 1996). 

Results from larval trial 2 (Fig. 3b), suggest that further inves- 
tigation of setting behavior should also be undertaken. Unlike 
Crassostrea gif;as larvae, which may settle over a 24 to 48 h period 
(Beiras and Widdows 1995). our observations of A', scalarina 
seem similar to data for Manila clams R. philippinarum. which 
may indicate an extended settlement period (Utting and Spencer 
1991 ). Transferring K. scalarina pediveligers to downweller oyster 
set systems containing a layer of ground scallop shell cultch for 
extended periods (as adopted in trial 2) proved to be unsatisfactory 
for this species, because larvae were eventually challenged by 
ciliates. Similarly, larvae from broodstock trial 1 developed well 
but did not metamorphose. 

Although norepinephrine hastened permanent settlement (trial 
5, Fig. 4), larval survival was low. Many larvae were invaded by 
a marine ciliate (resembling Uronenui nigricans, see Munday et al. 
1997) probably in response to increased bacterial numbers (Plun- 
ket and Hidu 1978). Because the effect of epinephrine or norepi- 
nephrine can vary with different .species of oyster (Coon et al. 
1986). specific work to determine appropriate concentration and 
duration for K. .Kalarina should be undertaken. In addition, larvae 
must be competent to metamoiphose before inducement with epi- 
nephrine (Coon et al. 1986). With low larval numbers, this proved 
difficult to achieve for all larvae, because animals need to be 
graded into tight size groups before induction. 

CONCLUSION 

Based on some of the criteria outlined in Kraeuter and Castagna 
(1989). Katelysia scalarina have the potential of being an eco- 
nomically viable culture species. However, although broodstock 
can be adequately conditioned and subsequently spawned, larval 
rearing, and sur\ival through metamorphosis, are still unreliable. 
The latter has primarily been because of the extended settlement 
period observed for this species, and the former because of the 
presence of a herpes-like viral pathogen. Further investigation of 
alternate types and dosing regimes for catecholamines as a means 
of enhancing settlement response is needed. Additional research is 
also required into the prevention and epidemiology of the herpes- 
like virus encountered during the course of this study. It is also 
recommended that the optimal rearing temperatures for the early 
life stages of this clam be identified. 

ACKNOWLEDGMENTS 

This work was funded by the Fisheries Research Development 
Corporation. The authors also thank Ms Elizabeth Cox and Mr. Ian 
Duthie for their assistance with spawning and larval rearing trials. 



1070 



Kent et al. 



Mr. Derek Cropp for help with broodstock conditioning, and Mr. 
Dale Ridges and Ms Lynda Bellchambers who assisted in collec- 
tion of broodstock. We are also indebted to Dr. Mike Heasman for 



his valuable editorial comment^ 
manuscript. 



during the preparation of this 



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Joimuil of Shellfish Research. Vol, 17. No. 4, 1071-1080. 1998. 

ESTIMATING TIME TO CRITICAL LEVELS OF PERKINSUS MARINVS IN EASTERN 

OYSTERS, CRASSOSTREA VIRGINICA 

THOMAS M. SONIAT' AND ENRIQUE V. KORTRIGHT' 

'Depariiucii! <if Biological Sciences 
'Department of Computer Science 
Niclu>lls State Universit}' 
Tliihddaiix, Louisiana 70310 

ABSTRACT A Visual-Basic program was developed as pari of an Excel spreadsheet to estimate the time to a critical level (tc,,,) of 
Perkinsus marimis. in eastern oysters Crassostrea virginica. The estimate is accomplished by assummg that a weighted incidence (WI) 
of disease of 1.5 is critical, converting measured WI values and the critical WI to parasite number, calculating a rate of change (r) of 
the parasite population using measured values of water temperature (T) and salinity (S). and solving for t^,.,, by simulation. The model 
produces estimates of t(-,„ and r using a long-term data set of T. S, and WI from the Terrebonne estuary of Louisiana. The model does 
not predict future values of WI since it cannot predict future trends in T and S; however, regularly determining T and S. considering 
their interaction in a model, measuring WI at reasonable intervals, and iteratively estimating l,^,„. should be useful to oyster manage- 
ment. Estimates of t^n, would support decisions concerning transplanting infected oysters to lower salinity areas, harvesting heavily 
infected populations early, and diverting freshwater into high-salinity estuaries. 

KEY WORDS: Oyster, Crassostrea virginica. Perkmsus marimis. epizootiology, rnanagement, mathematical model, estuary 



INTRODUCTION 

Throughout its range, from Mexico (Burreson et al. 1994) to 
Maine (Ford 1996), Perkinsus marimis ( = Dermocystidiwn mari- 
mim. Mackin et al. 1950) is an important cause of mortality (Ray 
and Mackin 1955, Andrews 1965) in eastern oysters, Crassostrea 
virginica. The success of oyster farming, therefore, often depends 
on the ability to manage oyster populations in estuaries where the 
parasite is widespread and/or abundant (Andrews and Ray 1988, 
Krantz and Jordan 1996). Management techniques of relevance to 
this study include transplanting infected oysters to lower salinity 
areas, early harvest of heavily infected populations, and freshwater 
diversion into high-salinity estuaries (Andrews and Ray 1988). 
These practices would clearly benefit by an estimate of the time to 
critical levels of infection. This estimate might be achieved by 
tneasuring water temperature and salinity, detertnining level of 
infection, calculating a rate of change of the parasite population, 
establishing a critical level of parasitism, and solving for the time 
to reach that critical level. The critical level of parasitism is set at 
a weighted incidence (WI) of 1.5, since a population of oysters 
with a WI of 2.0 contains an intense epidemic (Mackin 1962). 

The purposes of this paper are to ( 1 ) present a long-term data 
set of temperature, salinity, and level of infection: (2) apply equa- 
tions from an existing model (Hofmann et al. 1995, Powell et al. 
1996) to a personal computer/spreadsheet format; (3) calculate 
time to critical level of infection using the data presented; and (4) 
evaluate the utility of the approach. 

MATERIALS AND METHODS 

Environmental parameters (temperature and salinity) and oys- 
ters were sampled from February 1992 to November 1997 at a site 
in the Terrebonne Basin of south central Louisiana. The reef was 
chosen to provide an accessible, unharvested. unplanted, subtidal, 
and persistent population of oysters. The site is at the junction of 
Bay Tambour and Bayou Petit Caillou. a local source of fresh 
water (Global Positioning System coordinates; 29°iril.5"N, 
90°39'56.0"W). Water depth varied from 0.3 to 0.6 m. Environ- 
mental parameters were measured weekly, whereas oysters were 



sampled monthly. (Samples were not taken during the weeks of 
Hurricanes Andrew and Danny.) Water temperature (T) was mea- 
sured to the nearest O.l'C with a inercury thermometer, whereas 
salinity (S) was determined (nearest 0.5 ppt) using a refractometer 
(Behems 1965). 

Oysters were tonged at midmonth. Ten commercial-sized (75- 
124 mm) oysters were selected; a small piece of mantle tissue 
(about 4 mm") was removed from each oyster and assayed for P. 
marinus after incubation in fluid thioglycollate for 1 week (Ray 
1966). The intensity of infection was scored using Mackin's 
(1962) O-to-5 scale as modified by Craig et al. (1989). Weighted 
incidence (WI) was calculated as the sum of the disease code 
numbers divided by the number of oysters in the sample, and 
percent infection (PI) of the sample was recorded. 

A specific rate of parasite division (rj) was related to T and S 
according to the formula of Hofmann et al. (1995): 



rJT] = rJTo] e' 



0.0693(T(t)-To) 



(1) 



where e = 2.718. To = 20°C. rj[T] = the specific rate of cell 
division (day"'). rj[Tf|] is the rate at 20°C and the exponent cor- 
responds to a Qio of 2.0. (At Tq = 20°C and S = 20 ppt, rJXo] 
= 0.555 day"'; that is, the population doubles in size in about 2 
days.) At 10 ppt or less, the rate of cell division is decreased and 
the equation takes the form; 



rJT.S] = rJT„.S„l (S/10) e' 



0.0693(T(t)-To) 



(2) 



idi'"^i - 'd 

where Sq = 20 ppt and rj|T„.S„] = rJT„] = 0.555 day"' (Hof- 
mann et al. 1995). When parasite density is high, the specific rate 
of cell division decreases according to the equation; 

rd(p),,. = 3 r,(T,S) (C,AVj)- (3) 

where rj(T,S) is determined from equation (2), j is oyster size, and 
k is infection class such that j,k is the infection level at a given 
size; (3 is a parasite scaling factor equal to 2.454 x 10* g ash-free 
dry weight (AFDW) cell"', and 7 is a unit-less parasite density 
scaling factor equal to -1.5 according to Hofmann et al. (1995). 
The parasite number (C,,) was calculated from a measured WI 
value (X) according to the conversion of Choi et al. ( 1989): 



1071 



1072 



SONIAT AND KORTRIGHT 



Ck = 1409.9 V W, (10" 



^). 



(4) 



The oyster weight (Wj) is 3.0 g AFDW. appropriate for oysters 
from 75 to 124 mm in length, and v is 5.0, which converts oyster 
AFDW to wet weight (Hofmann et al. 1995). 

A specific rate of parasite mortality (r^J was likewise calcu- 
lated according to Hofmann et al. (1995) as: 



r„(T,S) = r,„„ e 



-6(S(l)/Soe) -6(To-10/So) (S(t)-So) 



(5) 



where 5 = 0.081530°C"'. Density effects on mortality rate follow 
the same relationship as was used for the density effect (equation 
3) on parasite division rate: 

UpV. = P r,„(T,S) (C,AV,)\ (6) 

The specific rate of change (r) was thus calculated as the appro- 
priate rj (equation 2 or 3) minus the appropriate r,„ (equation 5 
or 6). 

A time to a critical level of parasitism is calculated by assuming 
that at a WI of about 1.5 an oyster population is experiencing 
mortalities. Mackin (1962) states: "a population of live oysters 
with a weighted incidence of 2.0 contains an intense epidemic, and 
more than half the population may be in advanced stages of the 
disease, with all of the individuals infected." A WI of 1.5 corre- 
sponds to a C|^ of 194.500 hypnospores per oyster of 3.0 g AFDW 
(equation 4). 

The measured WI is converted to C^^ according to equation 4. 
r is calculated as explained above using measured values for T and 
S. and the time to reach a critical level of parasitism (tcnt) is found 
by simulation. A Visual-Basic program, used to calculate r and as 
part of an Excel spreadsheet module, is shown in Table 1. 

RESULTS 

Table 2 provides weekly measured environmental data (T and 
S). midmonthly measured levels of infection (WI and PI), and 
conversions and calculations generated by the model (C^, t^n,, r^. 
r„, and r). Water temperature varied from 6.5°C (12/19/96) to 
3I.9°C (7/30/93), whereas S ranged from 1.0 ppt (12/23/93 and 
12/30/93) to 25.0 ppt (5/18/95). (Mid-month T and S are plotted in 
Fig. la and lb, respectively.) Weighted incidence was highest 
(2.66) on 11/14/96 and lowest (0.00) on 3/19/92 (Fig. Ic): PI 
ranged from to 100%. Values of C^, calculated from measured 
WI values, ranged from 21,148 (even when WI was 0.00) to 
1,085,308 (at WI = 2.66). (That C, = 21,148 when WI = 0.00 
is implied in equation 4 and is due to false negatives of the thiogly- 
collate method.) Greatest values of parasite division rate (r^) were 
typically found in the late summer and early fall (as high as 0.582 
on 8/17/95). Likewise, greatest values of parasite mortality rate 
(r„,) were commonly found during the winter and early spring (as 
high as 0.553 on 4/7/94 and 12/19/96). The specific rate of change 
(r), the difference between tj and r^, is thus generally greatest in 
the late summer and early fall (Fig. Id). The t^r,, is. of course, 
lowest when r is highest (Fig. le). 

The behavior of the model was observed (Fig. 2) by calculating 
ten, at various combinations of T (5, 10, 15, 20, 25, 30, and 35°C) 
and S (5, 10, 15, 20, 25, 30 and 35 ppt) using initial C,, values of 
10.000 (Fig. 2a), 50,000 (Fig. 2b), 100,000 (Fig. 2c), and 150,000 
hypnospores (Fig. 2d). (To make the graphs easier to read, values 
of t^rii ^60 were assigned a value of 60 days; this is also a 
practical upper limit, since with monthly sampling, values s60 
days should not be of critical concern.) As T, S, and initial C^ 



increase, t^n, progressively decreases. For example, consider con- 
ditions representing a cool, wet winter (1 0°C, 10 ppt) versus a hot. 
dry summer (30^C, 25 ppt) at different initial values of C,^ (Fig. 2). 
With an initial C^, of 10,000 hynospores (Fig. 2a) at 10"C and 10 
ppt, ter„. is 3=60 days: at 30°C and 25 ppt, tcn, is <10 days. With 
an initial C^ of 150,000 hynospores (Fig. 2d) at 10°C and 10 ppt, 
tcrif is again &60 days, whereas at 30°C and 25 ppt, t^n, is <1 day. 

DISCUSSION 

The relationship of temperature and salinity to levels of infec- 
tion of P. inarinus in the eastern oyster is well documented (e.g., 
see Mackin et al. 1950, Ray and Mackin 1955, Mackin 1962, 
Soniat 1985). These two factors, however important, do not ex- 
plain most of the variation in levels of infection observed in the 
field. Soniat (1985). for example, showed that the interaction of 
temperature and salinity explained only about 49% of the variation 
in weighted incidence of infection. Clearly other factors are im- 
portant. These include, but may not be limited to, pollution and 
other human influences, host nutrition and growth, spawning and 
reproduction, age and resistance, oyster density and distribution, 
and disease vectors (Soniat 1996) — factors that comprise part of 
the oysters' history (Powell et al. 1996). However, it is impractical, 
if not intractable, to ascertain and measure all of the factors that 
determine levels of P. inariiuis in oysters. Nevertheless, determin- 
ing T and S. considering their interaction in a model, measuring 
WI at reasonable intervals, and iteratively calculating a time to a 
critical level of parasitism (tf-^,) should be useful to oyster man- 
agement. 

The data to support the model, namely, environmental data (T 
and S) and WI of P. iiuiriiiiis. have considerably distinct attributes. 
The collection of environmental data is easily automated, rela- 
tively inexpensive, and in some cases instantaneous. In contrast, 
obtaining WI data is relatively expensive and time consuming, and 
not amenable to automation. Applications of the model should 
therefore make best use of limited WI data and abundant environ- 
mental data. This might best be achieved by a stratified sampling 
program in which oysters are sampled for P. nuirimis in a few 
broad salinity zones. Decisions could therefore be made on a zone- 
by-zone basis, as t<--n, changes from season to season. The model 
justifies a broad delineation of zones in which the levels of para- 
sitism could be characterized as (I) of "no immediate concern" 
(e.g.. tcnt > 30 days), (2) of "current concern" (e.g, l^^^ < 30 
days), and (3) of "immediate concern" (t^-r,, approaching days). 
Management decisions that would be aided by a better estimate of 
t(-ni include early harvesting oysters in the "zone of immediate 
concern," transplanting of oysters to the "zones of no immediate 
concern" (Andrews and Ray 1988), and timing and positioning 
freshwater inputs into estuaries (Chatry et al. 1983). Sampling 
decisions would also benefit since when t^ni becomes less than the 
sample interval (in this case 1 mo), more fine-scaled measurements 
might be initiated. 

The model behaves reasonably in that as T, S, and initial Cj_ 
increase, the t^-^, progressively and predictably decreases (Fig. 2). 
Calculations of tc^, using the data set (Table 2) also show the 
expected decrease in the late summer and early fall (Ray and 
Mackin 1955, Mackin 1962). The model does not predict future 
values of WI because it cannot predict future values of T and S. It 
can be used to determine trends in tcn, and help reduce the risks 
associated with oyster cultivation. Thus, a single low value of t^n, 



Time to Critical Levels of P. marinus 



1073 



TABLE 1. 

A Visual-Basic program used to calculate time to critical level of P. marinus (tent), using temperature (T), salinity (S), and parasite number 

(C,,) as input values. 

Const normal density = 300000 
Const alpha = 0.0693 1 
Const beta = 3.454 * (10'* 8) 
Const delta = -0.08153 
Const gamma = -1.5 
Const SO = 20 
Const TO = 20 
Const crit = 194800 

Function rd (T As Double, S As Double, Ck As Double, Wj As Double) As Double 
Const RdO = 0.25 

If S < 10 Then 

mult = S / 10 
Else 

mull = 1 
End If 

rdt = RdO * mult * Exp (alpha * (T - TO)) 
If (Ck < = normal density ) Then 

rd = rdt 
Else 

rd = beta * rdt * |(Ck / Wj) ^ gamma] 
End If 

End Function 

Function rm (T As Double, S As Double, Ck As Double, Wj As Double) As Double 
Const rmO = 0.3 

If ((T- 10) >= 10) Then 

e = T- 10 
Else 

e = 10 
End If 



rmt 



rmO * Exp (delta * (S / SO) * e) * E.xp (delta * [(TO - 10) / SO) * (S - SO)) 



If (Ck <= normal density) Then 

rm = rmt 
Else 

rm = beta * rmt * f(Ck / Wj) ^ gamma] 
End If 

End Function 

Function tCrit (T As Double. S As Double, Ck As Double, Wj As Double) As Double 

Tm = 

r = 

While (Ck <= crit) 

Tm = Tm + 1 

rdt = rd (T, S, Ck, Wj) 

rmt = rm (T, S, Ck. Wj) 

r = rdt - rmt 

Ck = Ck * (1 +r) 

If Tm > 999 Then Ck = crit + 1 
Wend 
tCrit = Tm 

End Function 



is not necessarily cause for alarm. For example, in 1992. 1993. and 
1995, tcn, was often <7 days, but never reached days the fol- 
lowing week. In contrast, in 1994, 1996, and 1997, t(.^„ reached 
days on numerous occasions (Table 2). 

As constructed, the model errs on the side of caution for two 



reasons. First, although a Wl of 1 .5 in oysters is of concern it is not 
catastrophic but merely an indication of possible problems. Sec- 
ondly, the model does not consider recruitment and growth, in 
which young uninfected oysters replace the old, infected, and dy- 
ing ones. (The more complete and complex model of Hoffman et 



1074 



SONIAT AND KORTRIGHT 



T.\BLE 2. 

Sample date, weekly temperature (T, "O and salinity (S, pptl. weighted incidence (WD, and percent infection (PI) measured at mid-month. 

Values of initial parasite number (C|^) are converted from mid-monthly \VI values for each weekly combination of T and S. (Thus, WI and 
PI are repeated in the table.) A calculated time to critical level of P. marinus (t^ ,i,, days) is shown, with corresponding values of specific rate 

of parasite division (r^, day"'), specific rate of parasite mortality (r„, day"'), and the specific rate of change (r, day"'), where r = r j - r„. 



Date 


T 


S 


WI 


PI 


Ck 


t(>it 


rd 


rm 


r 


2/20/1992 


16.4 


2.0 
















2/27/1992 


14.7 


2.0 
















3/5/1992 


20.1 


15.0 
















3/12/1992 


15.5 


12.0 
















3/19/1992 


21.0 


14.0 


0.00 


0.00 


21,149 


37 


0.268 


0.205 


0.063 


3/26/1992 


19.2 


12.0 


0.00 


0.00 


21.149 


1000 


0.237 


0.255 


-0.018 


4/2/1992 


16.0 


7.0 


0.00 


0.00 


21,149 


1000 


0.133 


0.383 


-0.251 


4/9/1992 


20.8 


15.0 


0.00 


0.00 


21,149 


32 


0.264 


0.190 


0.074 


4/16/1992 


24.1 


19.0 


0.23 


40.00 


29.728 


10 


0.332 


0.105 


0.227 


4/23/1992 


22.9 


15,0 


0.23 


40.00 


29.728 


15 


0.306 


0.167 


0.139 


4/30/1992 


19.4 


11.0 


0.23 


40.00 


29,728 


1000 


0.240 


0.277 


-0.037 


5/7/1992 


19.8 


11.0 


0.23 


40.00 


29.728 


1000 


0.247 


0.277 


-0.030 


5/14/1992 


26.4 


20.0 


0.67 


50.00 


57,025 


5 


0.390 


0.079 


0.311 


5/21/1992 


26.8 


20.0 


0.67 


50.00 


57.025 


5 


0.401 


0.076 


0.324 


5/28/1992 


25,9 


18.0 


0.67 


50.00 


57.025 


6 


0.376 


0.101 


0.275 


6/4/1992 


27.1 


19.0 


0.67 


50.00 


57.025 


5 


0.409 


0.083 


0.326 


6/11/1992 


29.0 


22.0 


0.67 


50.00 


57.025 


4 


0.466 


0.050 


0.416 


6/18/1992 


31.2 


22.0 


0.70 


70.00 


59,614 


3 


0.543 


0.041 


0.502 


6/24/1992 


30.2 


16.0 


0.70 


70.00 


59.614 


4 


0.507 


0.095 


0.412 


7/2/1992 


28.6 


23.0 


0.70 


70.00 


59,614 


4 


0.454 


0.046 


0.407 


7/9/1992 


31.2 


20.0 


0.70 


70.00 


.59,614 


3 


0.543 


0.053 


0.490 


7/16/1992 


26.7 


22.0 


0.63 


80.00 


53,746 


5 


0.398 


0.062 


0.336 


7/23/1992 


30.0 


16.0 


0.63 


80.00 


53,746 


4 


0.500 


0.096 


0.404 


7/30/1992 


30.4 


18.5 


0.63 


80.00 


53,746 


4 


0.514 


0.068 


0.446 


8/6/1992 


31.0 


15.5 


0.63 


80.00 


53.746 


4 


0.536 


0,096 


0.440 


8/12/1992 


29.9 


18.0 


0.87 


70.00 


76,675 


3 


0.497 


0,076 


0.421 


8/20/1992 


28,0 


10.5 


0.87 


70.00 


76,675 


5 


0.435 


0.205 


0.231 


9/3/1992 


29.6 


14.0 


0.87 


70.00 


76,675 


4 


0.486 


0.125 


0.361 


9/10/1992 


30.0 


11.5 


0.87 


70.00 


76,675 


4 


0.500 


0.166 


0.334 


9/17/1992 


25.6 


15.0 


0.73 


70.00 


62,322 


6 


0.369 


0,142 


0.227 


9/24/1992 


26.0 


11.5 


0.73 


70.00 


62,322 


7 


0.379 


0.200 


0.179 


10/1/1992 


20.8 


15.0 


0.73 


70.00 


62.322 


16 


0.264 


0.190 


0.074 


10/8/1992 


23.0 


19.5 


0.73 


70.00 


62,322 


7 


0.308 


0.109 


0.199 


10/15/1992 


24.0 


17.5 


0.80 


90.00 


69.127 


6 


0.330 


0,122 


0.208 


10/22/1992 


TT -> 


22.0 


0.80 


90.00 


69.127 


6 


0.291 


0.093 


0.199 


10/29/1992 


25.0 


21.5 


0.80 


90.00 


69,127 


5 


0.354 


0.076 


0.278 


11/5/1992 


14.8 


12.5 


0.80 


90.00 


69,127 


1000 


0.174 


0.245 


-0.070 


11/12/1992 


20.0 


16.0 


1.30 


100.00 


144,920 


5 


0.250 


0.184 


0.066 


11/19/1992 


18.2 


19.5 


1.30 


100.00 


144,920 


4 


0.221 


0.138 


0.082 


11/25/1992 


17.6 


14.0 


1.30 


100.00 


144,920 


1000 


0.212 


0.217 


-0.005 


12/3/1992 


12.4 


12.0 


1.30 


100.00 


144.920 


1000 


0.148 


0.255 


-0.107 


12/10/1992 


13.8 


11.0 


1.30 


100.00 


144.920 


1000 


0.163 


0.277 


-0.114 


12/17/1992 


15.4 


7.5 


0.53 


90.00 


46,350 


1000 


0.136 


0.368 


-0.232 


12/23/1992 


20.8 


7.0 


0.53 


90.00 


46.350 


1000 


0.185 


0.374 


-0.190 


12/30/1992 


18.0 


9.0 


0.53 


90.00 


46,350 


1000 


0.196 


0,325 


-0.130 


1/6/1993 


11.4 


9.5 


0.53 


90.00 


46,350 


1000 


0.131 


0.312 


-0.182 


1/14/1993 


13.4 


2.5 


0.47 


70.00 


42,410 


1000 


0.040 


0.553 


-0.513 


1/21/1993 


18.8 


7.0 


0.47 


70.00 


42,410 


1000 


0.161 


0.383 


-0,222 


1/28/1993 


12.4 


7.0 


0.47 


70.00 


42.410 


1000 


0.103 


0,383 


-0.280 


2/4/1993 


14.4 


8.5 


0.47 


70.00 


42,410 


1000 


0.144 


0.339 


-0.195 


2/11/1993 


15.8 


14.5 


0.47 


70.00 


42,410 


1000 


0.187 


0.208 


-0.021 


2/18/1993 


13.0 


3.0 


0.07 


20.00 


23,458 


1000 


0.046 


0.531 


-0.485 


2/25/1993 


16.0 


17.0 


0.07 


20.00 


23,458 


108 


0.189 


0.170 


0.020 


3/4/1993 


15.0 


8.0 


0.07 


20.00 


23,458 


1000 


0.141 


0,353 


-0.212 


3/11/1993 


19.0 


14.0 


0.07 


20.00 


23.458 


128 


0.233 


0.217 


0.017 


3/18/1993 


13.5 


8.5 


0.27 


60.00 


31,541 


1000 


0.135 


0.339 


-0.204 



continued on next page 



Time to Critical Levels of P. marinus 



1075 



TABLE 2. 
continued 



Date 



\\l 



PI 



Ck 



rd 



3/25/1993 


19.7 


15.5 


0.27 


60.00 


31,541 


36 


0.245 


0.192 


0.053 


4/1/1993 


20.5 


10.5 


0.27 


60.00 


31,541 


1000 


0.259 


0.282 


-0.023 


4/9/1993 


16.9 


9.0 


0.27 


60.00 


31,541 


1000 


0.181 


0.325 


-0.144 


4/15/1993 


20.9 


11.5 


0.93 


100.00 


83,798 


74 


0.266 


0.254 


0.012 


4/22/1993 


19.0 


6.5 


0.93 


100.00 


83.798 


1000 


0.152 


0.399 


-0.247 


4/29/1993 


22.2 


15.5 


0.93 


100.00 


83.798 


8 


0.291 


0.167 


0.124 


5/6/1993 


24.5 


11.0 


0.93 


100.00 


83.798 


8 


0..342 


0.226 


0.116 


5/13/1993 


24.7 


5.0 


0.50 


70.00 


44.336 


1000 


0.173 


0.410 


-0.237 


5/20/1993 


25.4 


14.5 


0.50 


70.00 


44.336 


8 


0.363 


0.151 


0.212 


5/27/1993 


25.2 


1 1.0 


0.50 


70.00 


44.336 


12 


0.358 


0.219 


0.139 


6/4/1993 


28.0 


15.5 


0.50 


70.00 


44,336 


6 


0.435 


0.116 


0.320 


6/11/1993 


30.4 


14.0 


0.50 


70.00 


44.336 


5 


0.514 


0.120 


0.394 


6/17/1993 


29.3 


14.5 


0.63 


80.00 


53.746 


5 


0.476 


0.120 


0.356 


6/24/1993 


27.8 


14.5 


0.63 


80.00 


53.746 


5 


0.429 


0.131 


0.298 


7/2/1993 


30.6 


14.5 


0.63 


80.00 


53.746 


4 


0.521 


0.111 


0.410 


7/9/1993 


29.7 


9.0 


0.63 


80.00 


53.746 


7 


0.441 


0.228 


0.213 


7/16/1993 


29.2 


10.5 


0.43 


80.00 


39.972 


7 


0.473 


0.194 


0.279 


7/23/1993 


29.0 


5.5 


0.43 


80.00 


39.972 


1000 


0.257 


0.354 


-0.097 


7/30/1993 


31.9 


14.0 


0.43 


80.00 


39,972 


5 


0.570 


0.110 


0.461 


8/5/1993 


31.5 


13.5 


0.43 


80.00 


39.972 


5 


0.555 


0.120 


0.435 


8/12/1993 


31.4 


10.0 


0.07 


10.00 


23,458 


7 


0.551 


0.188 


0.362 


8/19/1993 


31.0 


5.5 


0.07 


10.00 


23,458 


1000 


0.295 


0.338 


-0.044 


8/26/1993 


29.0 


11.5 


0.07 


10.00 


23,458 


9 


0.466 


0.174 


0.292 


9/2/1993 


29.9 


10.0 


0.07 


10.00 


23,458 


9 


0.497 


0.200 


0.296 


9/9/1993 


30.1 


8.0 


0.07 


10.00 


23,458 


16 


0.403 


0.254 


0.149 


9/16/1993 


27.7 


7.0 


0.37 


70.00 


36,574 


1000 


0.298 


0.308 


-0.009 


9/23/1993 


30.0 


12.0 


0.37 


70.00 


36,574 


6 


0.500 


0.156 


0.344 


10/2/1993 


•24.0 


12.0 


0.37 


70.00 


36.574 


15 


0.330 


0.210 


0.120 


10/7/1993 


26.0 


12.0 


0.37 


70.00 


36.574 


10 


0.379 


0.190 


0.189 


10/14/1993 


23.0 


12.5 


0.77 


80.00 


66.124 


12 


0.308 


0.210 


0.098 


10/21/1993 


26.9 


14.0 


0.77 


80.00 


66,124 


5 


0.403 


0.146 


0.257 


10/28/1993 


20.9 


9.0 


0.77 


80.00 


66,124 


1000 


0.239 


0.315 


-0.075 


11/4/1993 


27.2 


8.0 


0.77 


80.00 


66,124 


23 


0.329 


0.279 


0.050 


11/11/1993 


15.2 


11.0 


0.77 


80.00 


66,124 


1000 


0.179 


0.277 


-0.097 


11/18/1993 


19.6 


8.0 


0.50 


50.00 


44,336 


1000 


0.195 


0.353 


-0.159 


11/26/1993 


17.6 


9.0 


0.50 


50.00 


44,336 


1000 


0.191 


0.325 


-0.1.35 


12/2/1993 


17.0 


15.0 


0.50 


50.00 


44,336 


424 


0.203 


0.200 


0.004 


12/9/1993 


17.2 


11.0 


0.50 


50.00 


44,336 


1000 


0.206 


0.277 


-0.071 


12/16/93 


11.8 


7.0 


0.27 


30.00 


31,541 


1000 


0.099 


0.383 


-0.284 


12/23/1993 


9.5 


1.0 


0.27 


30.00 


31,541 


1000 


0.012 


0.625 


-0.613 


12/30/1993 


9.9 


1.0 


0.27 


30.00 


31,541 


1000 


0.012 


0.625 


-0.612 


1/6/1994 


11.4 


9.5 


0.27 


30.00 


31,541 


1000 


0.131 


0.312 


-0.182 


1/13/1994 


12.2 


9.5 


0.13 


20.00 


25,637 


1000 


0.138 


0.312 


-0.174 


1/20/1994 


8.5 


13.0 


0.13 


20.00 


25,637 


1000 


0.113 


0.235 


-0.122 


1/27/1994 


17.8 


15.5 


0.13 


20.00 


25,637 


89 


0.215 


0.192 


0.023 


2/3/1994 


9.2 


9.0 


0.13 


20,00 


25.637 


1000 


0.106 


0.325 


-0.219 


2/10/1994 


21.6 


19.5 


0.13 


20.00 


25.637 


14 


0,279 


0.122 


0.158 


2/16/1994 


13.9 


14.0 


0.23 


30.00 


29.728 


1000 


0.164 


0.217 


-0.053 


2/24/1994 


14.9 


8.0 


0.23 


30.00 


29.728 


1000 


0.140 


0.353 


-0.213 


3/3/1994 


12.2 


5.5 


0.23 


30.00 


29.728 


1000 


0.080 


0.433 


-0.353 


3/10/1994 


13.3 


4.0 


0.23 


30.00 


29,728 


1000 


0.063 


0.489 


-0.426 


3/17/1994 


17.2 


16.0 


0.43 


70.00 


39,972 


73 


0.206 


0.184 


0.022 


3/24/1994 


23.7 


14.0 


0.43 


70.00 


39.972 


12 


0.323 


0.175 


0.148 


3/31/1994 


16.8 


9.0 


0.43 


70.00 


39,972 


1000 


0.180 


0.325 


-0.145 


4/7/1994 


16.0 


2.5 


0.43 


70.00 


39,972 


1000 


0.047 


0.553 


-0.506 


4/14/1994 


23.0 


16.0 


0.30 


50.00 


32,974 


13 


0.308 


0.151 


0.157 


4/21/1994 


22.4 


7.0 


0.30 


50.00 


32,974 


1000 


0.207 


0.358 


-0.151 


4/28/1994 


26.6 


14.5 


0.30 


50.00 


32,974 


8 


0.395 


0.141 


0.254 


5/5/1994 


23.2 


5.5 


0.30 


50.00 


32.974 


1000 


0.172 


0.403 


-0.231 


5/12/1994 


29.0 


11.5 


0.47 


70.00 


42,410 


6 


0.466 


0.174 


0.292 


5/19/1994 


25.0 


5.0 


0.47 


70.00 


42.410 


1000 


0.177 


0.407 
continued on 


-0.231 
next page 



1076 



SONIAT AND KORTRIGHT 











TABLE 2. 


















continued 










Date 


T 


S 


WI 


PI 


Ck 


ten, 


rd 


rm 


r 


5/26/1994 


26.9 


13.5 


0.47 


70.01) 


42,410 


7 


0.403 


0.154 


0.249 


6/3/1994 


27.6 


7.5 


0.47 


70.00 


42,410 


60 


0.318 


0.292 


0.026 


6/8/1994 


29.8 


10.5 


0.47 


70.00 


42,410 


6 


0.493 


0.189 


0.304 


6/17/1994 


31.6 


8.0 


0.27 


60.00 


31,541 


10 


0.447 


0.242 


0.205 


6/23/1994 


28.0 


15.0 


0.27 


60.00 


31,541 


7 


0.435 


0.122 


0.313 


6/29/1994 


29.5 


8.0 


0.27 


60.00 


31,541 


16 


0.386 


0.259 


0.127 


7/8/1994 


27.9 


14.5 


0.27 


60.00 


31,541 


7 


0.432 


0.130 


0.302 


7/15/1994 


28.2 


7.5 


0.63 


90.00 


53,746 


30 


0.331 


0.286 


0.045 


7/21/1994 


29.2 


12.0 


0.63 


90.00 


53.746 


5 


0.473 


0.162 


0.311 


7/28/1994 


27.0 


5.0 


0.63 


90.00 


53,746 


1000 


0.203 


0.391 


-0.188 


8/4/1994 


28.4 


13.0 


0.63 


90.00 


53,746 


5 


0.447 


0. 1 5 1 


0.297 


8/11/1994 


28.9 


11.5 


0.63 


90.00 


53,746 


6 


0.463 


0.175 


0.288 


8/18/1994 


29.2 


16.5 


0.50 


70.00 


44,336 


5 


0.473 


0.095 


0.378 


8/25/1994 


29.7 


7.5 


0.50 


70.00 


44,336 


17 


0.367 


0.273 


0.094 


9/1/1994 


29.7 


16.5 


0.50 


70.00 


44.336 


5 


0.490 


0.092 


0.398 


9/8/1994 


30.1 


13.0 


0.50 


70.00 


44,336 


5 


0.503 


0.138 


0.366 


9/15/1994 


26.5 


16.0 


1.47 


80.00 


186,394 


1 


0.392 


0.120 


0.272 


9/22/1994 


27.7 


14.0 


1.47 


80.00 


186,394 


1 


0.426 


0.140 


0.287 


9/29/1994 


26.0 


19.0 


1.47 


80.00 


186,394 


1 


0.379 


0.090 


0.288 


10/6/1994 


26.2 


16.0 


1.47 


80.00 


186.394 


1 


0.384 


0.123 


0.261 


10/13/1994 


20.9 


15.5 


1.50 


100.00 


194.859 





0.266 


0.181 


0.085 


10/18/1994 


25.8 


23.0 


1.50 


100.00 


194,859 





0.374 


0.060 


0.313 


10/27/1994 


20.4 


13.0 


1.50 


100.00 


194,859 





0,257 


0.230 


0.027 


11/3/1994 


22.6 


19.5 


1.50 


100.00 


194,859 





0.299 


0.112 


0.187 


11/10/1994 


24.8 


18.5 


1.50 


100.00 


194,859 





0.349 


0.104 


0.244 


11/17/1994 


19.9 


16.0 


1.07 


100.00 


103,097 


11 


0.248 


0.184 


0.064 


11/22/1994 


20.2 


18.0 


1.07 


100.00 


103,097 


7 


0.253 


0.154 


0.100 


12/1/1994 


13.8 


18.0 


1.07 


100.00 


103,097 


100 


0.163 


0.156 


0.006 


12/9/1994 


20.2 


11.5 


1.07 


100.00 


103,097 


1000 


0.253 


0.263 


-0.009 


12/15/1994 


14.5 


13.0 


0.80 


100.00 


69,127 


1000 


0.171 


0.235 


-0.064 


12/22/1994 


15.7 


17.5 


0.80 


100.00 


69,127 


46 


0.186 


0.163 


0.023 


12/29/1994 


15.2 


16.0 


0.80 


100.00 


69,127 


1000 


0.179 


0.184 


-0.005 


1/5/1995 


17.3 


6.0 


0.80 


100.00 


69.127 


1000 


0.124 


0.416 


-0.291 


1/12/1995 


18.3 


15.5 


0.43 


70.00 


39,972 


53 


0.222 


0.192 


0.031 


1/19/1995 


13.2 


9.5 


0.43 


70.00 


39,972 


1000 


0.148 


0.312 


-0.164 


1/26/1995 


12.0 


7.5 


0.43 


70.00 


39,972 


1000 


0.108 


0.368 


-0.260 


2/2/1995 


15.8 


9.5 


0.43 


70.00 


39,972 


1000 


0.178 


0.312 


-0.135 


2/9/1995 


10.0 


5.5 


0.43 


70.00 


39.972 


1000 


0.069 


0.433 


-0.364 


2/16/1995 


17.2 


13.5 


0.23 


50.00 


29,728 


1000 


0.206 


0.226 


-0.020 


2/23/1995 


17.4 


14.0 


0.23 


50.00 


29,728 


1000 


0.209 


0.217 


-0.008 


3/2/1995 


14.0 


4.5 


0.23 


50.00 


29,728 


1000 


0.074 


0.470 


-0.396 


3/9/1995 


10.7 


5.5 


0.23 


50.00 


29,728 


1000 


0.072 


0.433 


-0.361 


3/16/1995 


21.0 


20.5 


0.43 


80.00 


39,972 


12 


0.268 


0.117 


0.151 


3/23/1995 


23.3 


22.0 


0.43 


80.00 


39.972 


8 


0.314 


0.084 


0.230 


3/30/1995 


17.4 


18.0 


0.43 


80.00 


39,972 


31 


0.209 


0.156 


0.053 


4/6/1995 


19.2 


17.0 


0.43 


80.00 


39,972 


25 


0.237 


0.170 


0.067 


4/13/1995 


22.0 


13.0 


0.77 


90.00 


66,124 


15 


0.287 


0.211 


0.076 


4/20/1995 


25.4 


20.0 


0.77 


90.00 


66,124 


5 


0.363 


0.085 


0.278 


4/27/1995 


21.5 


16.5 


0.77 


90.00 


66,124 


10 


0.277 


0.160 


0.118 


5/4/1995 


25.0 


20.0 


0.77 


90.00 


66,124 


5 


0.354 


0.088 


0.265 


5/11/1995 


27.0 


17.0 


0.77 


90.00 


66,124 


5 


0.406 


0.104 


0.302 


5/18/1995 


27.6 


25.0 


0.93 


90.00 


83,798 


3 


0.423 


0.041 


0.383 


5/26/1995 


28.4 


20.5 


0.93 


90.00 


83,798 


3 


0.447 


0.063 


0.384 


6/2/1995 


27.5 


14.0 


0.93 


90.00 


83,798 


4 


0.420 


0.141 


0.279 


6/9/1995 


29.2 


15.5 


0.93 


90.00 


83,798 


3 


0.473 


0.107 


0.366 


6/16/1995 


25.9 


9.0 


0.40 


70.00 


38,235 


23 


0.339 


0.262 


0.077 


6/23/1995 


29.4 


14.0 


0.40 


70.00 


38,235 


6 


0.480 


0.127 


0.353 


6/30/1995 


26.4 


6.0 


0.40 


70.00 


38,235 


1000 


0.234 


0.355 


-0.122 


7/6/1995 


29.2 


13.0 


0.40 


70.00 


38.235 


6 


0.473 


0.144 


0.329 


7/13/1995 


30.6 


14.0 


0.17 


40.00 


27,201 


6 


0.521 


0.118 
continued on 


0.403 
next page 



Time to Critical Levels of P. marinus 



1077 











TABLE 2. 


















continued 










Datt 


T 


S 


\VI 


PI 


Ck 


to., 


rd 


rm 


r 


7/20/1995 


31.0 


10.0 


0.17 


40.00 


27,201 


7 


0.536 


0.192 


0.344 


7/27/1995 


31.2 


19.0 


0.17 


40.00 


27.201 


5 


0.543 


0.060 


0.483 


8/2/1995 


28.0 


16.0 


0.17 


40.00 


27.201 


7 


0.435 


0.109 


0.326 


8/10/1995 


31.0 


18.0 


0.17 


40.00 


27,201 


6 


0.536 


0.070 


0.466 


8/17/1995 


32.2 


14.0 


0.23 


40.00 


29,728 


5 


0.582 


0.108 


0.474 


8/24/1995 


30.8 


16.0 


0.23 


40.00 


29,728 


6 


0.528 


0.091 


0.438 


8/31/1995 


30.6 


15.5 


0.23 


40.00 


29.728 


6 


0.521 


0.098 


0.423 


9/7/1995 


27.7 


17.0 


0.23 


40.00 


29,728 


7 


0.426 


0.099 


0.327 


9/14/1995 


30.4 


15.5 


1,23 


100.00 


130,653 


2 


0.514 


0.099 


0.415 


9/21/1995 


30.0 


19.5 


1.23 


100.00 


130.653 


2 


0.500 


0.062 


0.438 


9/28/1995 


27.0 


19.0 


1.23 


100.00 


130.653 


2 


0.406 


0.084 


0.322 


10/5/1995 


23.8 


20.5 


1.23 


100.00 


130.653 


2 


0.325 


0.093 


0.233 


10/12/1995 


24.5 


19.0 


1.27 


90.00 


138.624 


2 


0.342 


0.102 


0.240 


10/19/1995 


23.0 


23.0 


1.27 


90.00 


138.624 


2 


0.308 


0.078 


0.229 


10/26/1995 


23.0 


23.0 


1.27 


90.00 


138.624 


2 


0.308 


0.078 


0.229 


11/2/1995 


24.6 


24.0 


1.27 


90.00 


138.624 


2 


0.344 


0.061 


0.283 


1 1/9/1995 


16.4 


19.0 


1.27 


90.00 


138.624 


7 


0.195 


0.144 


0.051 


11/16/1995 


17.4 


18.0 


0.73 


80.00 


62,322 


23 


0.209 


0.156 


0.053 


11/24/1995 


18.0 


18.0 


0.73 


80.00 


62.322 


20 


0.218 


0.156 


0.061 


11/30/1995 


15.0 


13.5 


0.73 


80.00 


62,322 


1000 


0.177 


0.226 


-0.049 


12/7/1995 


22 ~' 


16.0 


0.73 


80.00 


62,322 


10 


0.291 


0.159 


0.132 


12/12/1995 


10.4 


17.5 


0.70 


90.00 


59,614 


1000 


0.129 


0.163 


-0.034 


12/21/1995 


9.0 


12.5 


0.70 


90.00 


59,614 


1000 


0.117 


0.245 


-0.128 


12/29/1995 


6.6 


12.0 


0.70 


90.00 


59,614 


1000 


0.099 


0.255 


-0.156 


1/3/1996 


9.4 


11.5 


0.70 


90.00 


59,614 


1000 


0. 1 20 


0.265 


-0.146 


1/11/1996 


11.0 


14.5 


0.70 


90.00 


59,614 


1000 


0. 1 34 


0.208 


-0.074 


1/18/1996 


17.6 


20.0 


1.27 


100.00 


138,624 


5 


0.212 


0.133 


0.079 


1/25/1996 


il.6 


11.0 


1.27 


100.00 


138,624 


1000 


0.140 


0.277 


-0.137 


2/1/1996 


13.4 


12.0 


1.27 


100.00 


138,624 


1000 


0.158 


0.255 


-0.097 


2/8/1996 


10.0 


16.0 


1.27 


100.00 


138.624 


1000 


0.125 


0.184 


-0.059 


2/15/1996 


15.9 


15.5 


0.33 


50.00 


34,471 


1000 


0.188 


0.192 


-0.003 


2/22/1996 


20.0 


18.0 


0.33 


50.00 


34,471 


20 


0.250 


0.156 


0.094 


2/29/1996 


15.7 


4.0 


0.33 


50.00 


34,471 


1000 


0.074 


0.489 


-0.415 


3/7/1996 


17.0 


14.0 


0.33 


50.00 


34,471 


1000 


0.203 


0.217 


-0.013 


3/14/1996 


19.0 


22.0 


1.27 


80.00 


138,624 


3 


0.233 


0.113 


0.120 


3/21/1996 


12.2 


15.0 


1.27 


80.00 


138,624 


1000 


0.146 


0.200 


-0.054 


3/28/1996 


16.9 


18.5 


1.27 


80.00 


138.624 


7 


0.202 


0.150 


0.052 


4/4/1996 


19.2 


18.0 


1.27 


80.00 


138.624 


5 


0.237 


0.156 


0.080 


4/11/1996 


19.0 


21.5 


1.27 


80.00 


138.624 


4 


0.233 


0.117 


0.116 


4/18/1996 


20.7 


21.0 


1.90 


100.00 


352.293 





0.262 


0.115 


0.147 


4/25/1996 


20.6 


15.5 


1.90 


100.00 


352.293 





0.261 


0.184 


0.076 


5/2/1996 


21.1 


19.0 


1.90 


100.00 


352.293 





0.270 


0.132 


0.138 


5/9/1996 


26.7 


21.0 


1.90 


100.00 


352,293 





0.398 


0.069 


0.329 


5/16/1996 


26.0 


19.5 


2.53 


100.00 


895,300 





0.154 


0.035 


0.120 


5/23/1996 


28.6 


19.5 


2.53 


100.00 


895,300 





0.185 


0.028 


0.157 


5/29/1996 


29.2 


21.5 


2.53 


100.00 


895,300 





0.193 


0.021 


0.171 


6/4/1996 


29.0 


15.5 


2.53 


100.00 


895,300 





0.190 


0.044 


0.146 


6/14/1996 


27.3 


16.5 


0.97 


100.00 


88,910 


3 


0.415 


0.108 


0.307 


6/20/1996 


29.7 


15.0 


0.97 


100.00 


88,910 


3 


0.490 


0.110 


0.379 


6/28/1996 


28.6 


7.5 


0.97 


100.00 


88,910 


15 


0.340 


0.283 


0.058 


7/5/1996 


30.6 


8.5 


0.97 


100.00 


88,910 


5 


0.443 


0.235 


0.208 


7/12/1996 


31.1 


9.0 


0.97 


100.00 


88,910 


4 


0.486 


0.217 


0.269 


7/19/1996 


30.0 


15.5 


0.33 


70.00 


34,471 


6 


0.500 


0.102 


0.398 


7/25/1996 


29.1 


10.5 


0.33 


70.00 


34,471 


8 


0.470 


0.195 


0.275 


8/2/1996 


28.5 


9.5 


0.33 


70.00 


34,471 


10 


0.428 


0.225 


0.203 


8/9/1996 


30.0 


9.5 


0.33 


70.00 


34,471 


8 


0.475 


0.212 


0.263 


8/16/1996 


29.7 


6.5 


0.20 


50.00 


28,436 


200 


0.318 


0.309 


0.010 


8/22/1996 


27.4 


17.0 


0.20 


50.00 


28,436 


8 


0.418 


0.102 


0.316 


8/29/1996 


30.0 


12.5 


0.20 


50.00 


28,436 


7 


0.500 


0.147 


0.353 


9/5/1996 


29.3 


13.5 


0.20 


50.00 


28,436 


7 


0.476 


0.135 
continued on 


0.341 
next page 



1078 



SONIAT AND KORTRIGHT 



TABLE 2. 
continued 



Date 


T 


S 


\VI 


PI 


Ck 


t.H, 


rd 


rm 


r 


9/12/1996 


29.7 


15.5 


1.33 


100.00 


151.501 


1 


0.490 


0,104 


0,386 


9/19/1996 


28.4 


14.5 


1.33 


100.00 


151.501 


1 


0,447 


0,127 


0,321 


9/26/1996 


27,0 


17.5 


1.33 


100.00 


151.501 


1 


0.406 


0,099 


0,307 


10/3/1996 


25.1 


17.0 


1.33 


100.00 


151.501 


2 


0.356 


0,119 


0,237 


10/10/1996 


22.8 


19.5 


1.33 


100.00 


151.501 


-) 


0.304 


0.111 


0.193 


10/17/1996 


26.4 


20.5 


2.30 


90.00 


636.924 





0.265 


0.051 


0.214 


10/24/1996 


18.4 


20.0 


2.30 


90.00 


636.924 





0.152 


0.090 


0.062 


10/31/1996 


26.8 


21.5 


2,30 


90.00 


636.924 





0.272 


0.044 


0.228 


11/7/1996 


23.3 


24.5 


2.30 


90.00 


636.924 





0.214 


0.045 


0.169 


11/14/1996 


18.8 


15.0 


2.66 


90.00 


1.085.308 





0.070 


0.061 


0.009 


11/21/1996 


24.0 


16.5 


2.66 


90.00 


1.085.308 





0.101 


0.041 


0.060 


1 1/29/1996 


14.0 


16.5 


2.66 


90.00 


1,085.308 





0.050 


0.054 


-0.004 


12/5/1996 


15.1 


13.5 


2.66 


90.00 


1.085,308 





0.054 


0.069 


-0.015 


12/12/1996 


20.1 


15.0 


2,00 


100.00 


408.507 





0.252 


0.198 


0.053 


12/19/1996 


6.5 


2.5 


2.00 


100.00 


408,507 





0.025 


0.553 


-0.528 


12/26/1996 


11.8 


11.0 


2,00 


100.00 


408.507 





0.142 


0.277 


-0.135 


1/4/1997 


20.6 


12.0 


2,00 


100.00 


408.507 





0.261 


0.247 


0.013 


1/9/1997 


11.2 


6.5 


2,00 


100.00 


408,507 





0.088 


0.399 


-0.311 


1/16/1997 


9.4 


6.0 


1,60 


100.00 


225,952 





0.072 


0.416 


-0.344 


1/23/1997 


18.0 


14.0 


1,60 


100.00 


225,952 





0.218 


0.217 


0.001 


1/30/1997 


12.3 


6.0 


1,60 


100.00 


225.952 





0.088 


0.416 


-0.328 


2/6/1997 


14.9 


7.5 


1.60 


100.00 


225,952 





0.132 


0.368 


-0.236 


2/13/1997 


13.0 


16.5 


0.77 


90.00 


66,124 


1000 


0.154 


0.177 


-0.023 


2/20/1997 


18.0 


13.5 


0.77 


90.00 


66,124 


1000 


0.218 


0.226 


-0.008 


2/27/1997 


18.9 


18.5 


0.77 


90.00 


66,124 


14 


0.232 


0.150 


0.082 


3/6/1997 


18.0 


6.0 


0.77 


90.00 


66,124 


1000 


0.131 


0.416 


-0.285 


3/13/1997 


21.5 


18.5 


0.57 


90.00 


49.178 


11 


0.277 


0.134 


0.143 


3/20/1997 


18.2 


4.0 


0.57 


90.00 


49,178 


1000 


0.088 


0.489 


-0.401 


3/27/1997 


23.0 


10.5 


0.57 


90.00 


49.178 


26 


0.308 


0.253 


0.054 


4/3/1997 


19.0 


11.5 


0.57 


90.00 


49,178 


1000 


0.233 


0.265 


-0.032 


4/10/1997 


19.5 


15.0 


0.57 


90.00 


49,178 


34 


0.241 


0.200 


0.042 


4/17/1997 


19.5 


5.5 


0.80 


100.00 


69,127 


1000 


0.133 


0.433 


-0.300 


4/25/1997 


19.6 


12.0 


0.80 


100.00 


69.127 


1000 


0.243 


0.255 


-0.012 


5/1/1997 


23.4 


9.5 


0.80 


100.00 


69.127 


40 


0.301 


0.274 


0.027 


5/8/1997 


24.5 


14.5 


0.80 


100.00 


69.127 


7 


0.342 


0.159 


0.182 


5/16/1997 


25.8 


6.0 


0.53 


80.00 


46.350 


1000 


0.224 


0.361 


-0.136 


5/22/1997 


27.9 


12.0 


0.53 


80.00 


46.350 


7 


0.432 


0.173 


0.259 


5/29/1997 


26.9 


6.0 


0.53 


80.00 


46.350 


1000 


0.242 


0.351 


-0.109 


6/5/1997 


27.0 


11.5 


0.53 


80.00 


46.350 


8 


0.406 


0.191 


0.215 


6/12/1997 


28.6 


13.5 


0.80 


100.00 


69.127 


4 


0.454 


0.140 


0.313 


6/19/1997 


26.7 


10.0 


0.80 


100.00 


69.127 


7 


0,398 


0.228 


0.169 


6/26/1997 


28.0 


9.5 


0.80 


100.00 


69.127 


7 


0,413 


0.229 


0.184 


7/3/1997 


31.0 


9.5 


0.80 


100.00 


69.127 


4 


0,509 


0.204 


0.305 


7/10/1997 


29.7 


9.5 


0.80 


100.00 


69.127 


5 


0,465 


0.215 


0.251 


7/24/1997 


29.0 


7.5 


0.80 


100.00 


69.127 


16 


0,350 


0.279 


0.071 


7/31/1997 


29.7 


17.0 


0.80 


100.00 


69.127 


4 


0,490 


0.087 


0.403 


8/7/1997 


29.4 


12.5 


0.80 


100.00 


69.127 


4 


0,480 


0.152 


0.328 


8/14/1997 


30.8 


16.0 


0.63 


70.00 


53.746 


4 


0,528 


0.091 


0.438 


8/21/1997 


30.4 


14.5 


0.63 


70.00 


53.745 


4 


0,514 


0.112 


0.402 


8/28/1997 


29.0 


19.5 


0.63 


70.00 


53.746 


4 


0,466 


0.068 


0.399 


9/4/1997 


29.7 


14,5 


0.63 


70.00 


53.746 


5 


0,490 


0.117 


0.373 


9/11/1997 


29.0 


19.5 


0.63 


70.00 


53,746 


4 


0,466 


0.068 


0.399 


9/18/1997 


29.9 


16.0 


1.73 


100.00 


273,905 





0,497 


0.096 


0.400 


9/25/1997 


26.6 


19.0 


1.73 


100.00 


273.905 





0,395 


0.086 


0.309 


10/2/1997 


26.3 


13.5 


1.73 


100.00 


273.905 





0,387 


0.159 


0.227 


10/8/1997 


27.2 


22.5 


1.73 


100.00 


273.905 





0,412 


0,056 


0.356 


10/16/1997 


17.9 


19,0 


1.50 


100.00 


194.859 





0,216 


0,144 


0.072 


10/23/1997 


21.2 


21.5 


1.50 


100.00 


194.859 





0.272 


0,106 


0.166 


10/30/1997 


16.3 


19.5 


1.50 


100.00 


194.859 





0,193 


0,138 


0.055 


11/6/1997 


19.0 


19.0 


1.50 


100.00 


194.859 





0,233 


0,144 


0.089 


11/14/1997 


16.3 


14.5 


1.84 


100.00 


322.349 





0.193 


0,208 


-0.014 



Time to Critical Levels of P. marinus 



1079 




Figure 1. Changes in water temperature (a), salinity (b), VVI (weighted 
incidence of P. marinus, cl. r (specific rate of change of P. marinus. A). 
and t,^|, (time to critical level of P. marinus, el. Temperature ( C). and 
salinity IpptI are mid-month values taken when \\T was determined, 
whereas r (day 't and Ij^^i, (daysl are calculations for each month 
generated from the model. Monthly values are plotted for all param- 
eters from March 1992 to November 1997. Complete sample dates and 
data are given in Table 2. 



4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 
Figure 2. Time to critical level of P. marinus ^i^^„) in days calculated 
for various combinations of T (temperature in C), S (salinity in ppt) 
for an initial parasite number C,,. The scale on the y-axis is used for T, 
S, and ti ,1,. The x-axis is simply an iteration number for a T and S 
combination input into the model which generates a t^rir Values for 
initial C^ are 10.()(M) a), 50.000 b). 100.000 c), and 150,000 hypnospores 
d). Calculations are based on an oyster of 3.0 g ash-free dry weight. 



al. 1995, Powell et al. 1996 does.) Thus, a self-sustaining popula- time of year from which it was (determined. For example, a ten, of 

tion of oysters should be less susceptible to catastrophic decline a few days in early spring is of much greater concern than the 

than one that is not self-sustaining (e.g.. bedded oysters). Consid- same value in the late fall when water temperatures are rapidly 

eration must also be given not only to the \.Q„^ value, but also to the declining. 



1080 



SONIAT AND KORTRIGHT 



The model is used to present as a hypothesis which relates 
temperature, salinity, and initial disease level to time to a critical 
level of infection. With more frequent measurements of T and S. 
more frequent estimates of tc, could be made, improving its re- 
liability. It is our hope that the simple format in which the model 
is presented, and its modest data requirement, will encourage its 
testing and application in numerous estuaries. 



ACKNOWLEDGMENTS 

This project was funded by the Nicholls State University Re- 
search Council. Randy Robichaux helped with data management. 
The manuscript was greatly improved by comments from Eric N. 
Powell and an anonymous reviewer. We appreciate this support 
and assistance. 



LITERATURE CITED 



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in Chesapeake Bay. Chesap. Sci. 6:60-67. 
Andrews, J. D. & S. M. Ray. 1988. Management strategies to control the 

disease caused by Perkinsus marinus. Amer. Fish. Soc. Spec. Publ. 

18:257-264. 
Behems, E. W. 1965. Use of the Goldberg refractometer as a salinometer 

for biological and geological field work. J. Mar. Res. 2:165-171. 
Burreson. E. M.. R. S. Alverez. V. V. Martinez & L. A. Macedo. 1994. 

Perkinsus marinus (Apiconiplexa) as a potential source of oyster Cras- 

sostrea virginica mortality in the coastal lagoons of Tabasco. Mexico. 

Dis. Aquat. Org. 20:77-82. 
Chatry. M., R. J. Dugas & K. A. Easley. 1983. Optimum salinity regime for 

oyster production on Louisiana's state seed grounds. Conlrib. Mar. Sci. 

26:81-94. 
Choi. K.-S.. E. A. Wilson. D. H. Lewis, E. N. Powell & S. M. Ray. 1989. 

The energetic cost of Perkinsus marinus parasitism in oysters: quanti- 
fication of the thioglycollate method. J. Shellfisli Res. 8:125-131. 
Craig. A.. E. N. Powell. R. R. Fay & J. M. Brooks. 1989. Distnbution of 

Perkinsus marinus in Gulf coast oyster populations. Estuaries 12:82- 

91. 
Ford, S. E. 1996. Range extension by the oyster parasite Perkinsus marinus 

into the northeastern United States: response to climatic change? J. 

Shellfish Res. 15:45-56. 
Hofmann, E. E., E. N. Powell, J. M. Klinck & G. Saunders. 1995. Model- 



ing diseased oyster populations. 1. Modeling Perkinsus marinus in oys- 
ters. J. Shellfish Res. 14:121-151. 

Krantz, G. E. & S. J. Jordan. 1996. Management alternatives for protecting 
Crassostrea virginica fisheries in Perkinsus marinus enzootic and epi- 
zootic areas. J. Shellfish Res. 15:167-176. 

Mackin, J. G. 1962. Oyster disease caused by Dermocystidium nuirinum 
and other microorganisms in Louisiana. Publ. Inst. Mar. Sci. Univ. Te.x. 
7:132-229. 

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

Powell, E. N., J. M. Klinck & E. E. Hofmann. 1996. Modeling diseased 
oyster populations. II. Triggering mechanisms for Perkinsus marinus 
epizootics. J. Shellfish Res. 15:141-165. 

Ray. S. M. 1966. A review of the culture method for detecting Dermocys- 
tidium marinum with suggested modifications and precautions. Proc. 
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Ray. S. M. & J. G. Mackin. 1955. Studies of pathogenesis of Dermocys- 
tidium marinum. Proc. Natl. Shellfiish. Assoc. 45:164—167. 

Soniat. T. M. 1985. Changes in levels of infection of oysters by Perkinsus 
marinus. with special reference to the interaction of temperature and 
salinity on parasitism. Northeast Gidf Sci. 7:171-174. 

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oysters in the Gulf of Mexico. J. Shellfish Res. 15:35—13. 



J„i(rncil of Shcllfiih Research. Vol. 17. No. 4. 1081-1083. 1998. 

RESPONSE OF SETTLING OYSTER LARVAE, CRASSOSTREA VIRGINICA, TO SPECIFIC 
PORTIONS OF THE VISIBLE LIGHT SPECTRUM' 



PATRICK BAKER* AND ROGER MANN 

Virginia Institute of Marine Science 
Gloucester Point. Virginia 23062 

ABSTRACT Settlement site choice was used to lest the ability of conipetent-to-settle oyster [Crassostrea virginica) larvae to detect 
specitlc portions of the visible light spectrum. Larvae were permitted to settle on illuminated or shaded sides of vertically oriented 
settlement substrates. Five light treatments were used, including white light (400-700 nm), three fractions of white light: red light 
(600-700 nm), green light (450-575 nm. peak at 525 nm), blue light (400-500 nm. peak at 425 nm); and total darkness. In total 
darkness, no settlement preference for either side of the substrates was detected. In all light treatments, larvae settled in significantly 
higher numbers onto shaded surfaces than illuminated surfaces. Crassostrea virginica larvae respond to most portions of the visible 
light spectrum, unlike many previously studied marine invertebrate lar\'ae. This ability may reflect the diverse light conditions in the 
largely estuarine habitat of this species. 

KEY WORDS: Crassostrea virginica. larvae, settlement, light 



INTRODUCTION 

Phototactic behavior in marine larvae can contribute to site 
selection during the settlement process even though the presence 
or absence of light is not required for settlement (Baker 1997). 
Pediveliger (competent-to-settle) larvae of the American oyster. 
Crassostrea virginica (Gmelin). settled primarily on shaded sur- 
faces of substrates in laboratory trials (Ritchie and Menzel 1969). 
The pediveliger larva of some bivalve mollusks, including oysters 
(Ostreidae). has a distinct pigmented region termed the eyespot. 
and believed to be photosensitive. Cole ( 1938) initially described 
the eyespot and its presumed function for the larvae of the oyster 
Ostrea edulis. although Thompson et al. (1996) point out that 
research clarifying "eyespot"" function is still needed. 

Coastal planktonic invertebrates are usually unresponsive, or 
only weakly responsive, to long \'isible light wavelengths (orange 
and red). Serpulid polychaete larvae, which are negatively photo- 
tactic when swimming, are responsive to blue and green light 
(400-550 nm), but are indifferent or respond weakly to orange and 
red light O600 nm) (Young and Chia 1982. Marsden 1986. 1988, 
1990). Barnacle (Balamis impnnisus Darwin) nauplii. which are 
positively phototactic. respond strongly to blue and green light, 
and also to long- wave ultraviolet (350 nm). but show a marked 
decrease in response to wavelengths above 600 nm (Lang et al. 
1979). The larvae of some estuarine brachyuran crabs, including 
Sesanna reticulatuin and Uca minax, appear to be most sensitive 
to light wavelengths of 500-600 nm (green to orange), but sensi- 
tivity of most species declines sharply above 600 nm (Forward and 
Cronin 1979). This phenomenon is not limited to lai^ae: coastal 
planktonic adult arrow worms. Sagitra hispida Conant. are most 
sensitive to blue and green light (400-540 nm) but much less 
sensitive to wavelengths above 520 nm (Sweatt and Forward 
1985). The above phototactic responses correlate with spectral 
attenuation pattern in coastal oceanic waters in which red light 
(>6()0 nm) is strongly attenuated, whereas violet to yellow light 
(400-600 nm) is attenuated the least (Austin and Petzold 1984). 

Crassostrea virginica is not primarily an oceanic species, like 



'Contribution 2181 of the Virginia Institute of Marine Science. 
*Present address; Dept. Ecology and Evolution, State University of New 
York, Stonv Brook, NY 11794-5245. 



most of the above examples, but occurs in greatest abundance in 
estuaries. The adult and lar\ al life history of C virginica has been 
reviewed by Stanley and Sellers ( 1986). In the estuarine environ- 
ment of the Chesapeake Bay. Virginia, the attenuation of visible 
light is greatest for short wavelengths (<500 nm). and greatest 
during the summer when C. virginica larvae are most abundant. 
Yellow and orange light (550-650 nm) generally have the greatest 
irradiance (transmission) through the water column (Champ et al. 
1980, van Tine 1987). The attenuation coefficient (a natural log 
scale of light reduction with depth) in the Chesapeake Bay during 
summer is about 3.1 at 400 nm. 1.6 at 500 nm. 1.1 at 600 nm. and 
1.4 at 700 nm (van Tine 1987). At a depth of 1 meter, less than 5% 
of the surface violet light (400 nm) penetrates, but about 30% of 
the yellow and orange light (550-650 nm) is still present. Water 
quality, including inorganic and organic particulate matter, 
strongly affects light quality in estuarine waters (Pierce et al. 
1986). It is probable, therefore, that C. virginica larvae experience 
a wide range of light quality within a single estuary. 

If oysters are unresponsive to certain portions of the visible 
light spectrum, as are the larval species discussed above, then 
water quality parameters that affect light quality (Pierce et al. 
1986) could affect the ability of oyster larvae to use light as a 
settlement cue. If oyster larvae do not respond to red and orange 
light wavelengths, then much of the Chesapeake Bay would be a 
light-poor environment to larval oysters (van Tine 1987). This 
study addresses the question: Do larvae of oysters (C virginica) 
respond to different portions of the visible light spectrum, as ob- 
served by settlement site choice? 

MATERIALS AND METHODS 

Oyster larval settlement chambers were constructed from 1/8 
inch (3 mm) thick black acrylic Plexiglas. One side was made of 
clear acrylic to permit light entry. Each chamber had internal di- 
mensions of 7.5 cm in height. 7.5 cm in width, and 2.5 cm in depth, 
and a volume of about 60 ml. A clear acrylic bracket was placed 
in the bottom of each chamber, at least 2 cm from the sides of the 
chamber (Fig. 1 ). to hold a settlement substrate plate in a vertical 
position. The vertical orientation of the settlement plate was used 
to eliminate geotaxis (Pires and Woollacott 1983. Baker 1997) and 
barokinesis (Crisp and Ghobashy 1971. Mann and Wolf 1983) as 
confounding settlement cues. 



1081 



1082 



Baker and Mann 



1cm 




SHELL 
SUBSTRATE CHIP 



PLEXIGLAS 
CHAMBER 



-*^ii\^INCOMING LIGHT 



BRACKET 



^CLEAR PANEL 
Figure 1. Diagram of a settlement chamber. 

Settlement substrate plates were cut from adult oyster iCras- 
sostrea virginica) shells, 3x3 cm square by 2 mm thick, and 
ground to equal roughness on both sides. Settlement plates were 
placed in seawater two days prior to use. to allow bacterial growth 
on the shell surfaces; water-soluble compounds from oyster shell 
and some levels of bacterial colonization induce settlement in 
Cmssostrea (Hidu et al. 1978, Fitt et al. 1990). The tops of each 
chamber were covered with a black acrylic plate to reduce evapo- 
ration, but were not airtight. 

The light source used for this study was a pair of new 48-inch 
Philips cool white fluorescent tubes. About 9.'S'7r of the light output 
of the fluorescent lamp was between 400 and 700 nm, based on 
specifications provided by the manufacturer. The light source was 
fixed at 25 cm from the settlement chambers (chambers were 
arranged in rows facing the tluorescent tubes). Total visible light 
flux from the fluorescent lamp was measured with a Li-Cor radi- 
ometer-photometer. Model LI-185A, using an on-deck light sen- 
sor. Neutral density black scrim was used to regulate light inten- 
sity. Light entering the settlement chambers was 2.4-2.6 microE- 
insteins ((xE) m"" • m'", after all filters were in place. This range 
is equivalent to midsummer light intensity at about a 5 m depth in 
the lower Chesapeake Bay, using a light attenuation coefficient (k) 
of 1.1, and assuming surface insolation of 500 |jiE • m"" • s'" (Wet- 
zel and Neckles 1 986). Light levels used in similar trials by Ritchie 
and Menzel (1969) were 25-50 foot candles (=5.2-10.4 
(xE • m"" • s'"). 

Five light treatments were used: white, red, green, blue, and 
total darkness. White light was unfiltered from the fluorescent 
lamp. The total darkness treatment consisted of settlement cham- 
bers made entirely of black acrylic, which blocked all light, with 
no clear acrylic panel. For red, green, and blue light, theatrical 
light filters (Rosco and Lee brands) were used. Transmittance 
spectra of filters were measured with a Shimadzu spectrophotom- 
eter. Model UV 160. The red filter was Roscolux Medium Red 
overlaid with Lee Orange; this combination transmitted wave- 
lengths only above 600 nm. The green filter was Roscolux Dark 
Yellow Green (470-600 nm, peak at 525 nm), and the blue filter 
was Lee Deep Blue (380-525 nm, peak at 425 nm). Both green and 
blue filters transmitted some far red (above 740 nm) light, but 
these wavelengths were mostly above the fluorescent lamp output. 
Light filters and black scrim were attached directly to the outside 
of the settlement chambers. 



Competent-to-settle pediveliger larvae of Crassostrea virginica 
were reared at 20 ppt salinity in the Virginia Institute of Marine 
Science oyster hatchery. Approximately 500 larvae, in 55 ml of 20 
micron-filtered seawater (20 ppt). were pipetted into each settle- 
ment chamber. A fan was used to circulate air past the settlement 
chambers, and temperature within the chambers remained at 25- 
28°C throughout the trial. 

All settlement trials ran simultaneously for 24 h at constant 
light conditions. There were six settlement chambers for each color 
treatment, and each series of trials (five treatments) was run twice, 
for a total of 12 replicates per treatment. At the end of each trial, 
settled juveniles (spat) on the illuminated ("front") and shaded 
("back") surfaces of each substrate plate were recorded. Settle- 
ment on each side (illuminated and shaded, or front and back in the 
case of the no-light treatment), was expressed as a proportion of 
total settlement for each substrate plate. The difference between 
proportional settlement onto shaded and illuminated surfaces was 
calculated for each replicate substrate. 

Paired-sample Mests were used to test the null hypothesis that 
the mean proportional difference between illuminated and shaded 
(front and back) for each light treatment was equal to zero (Zar 
1996). Prior to analysis, the absolute of each difference was trans- 
formed using the arcsine-square root transformation (Zar 1996), 
and then converted back to its original sign. 

RESULTS 

In total darkness, no significant settlement difference between 
front and back surfaces of the settlement substrate plates was de- 
tected. In all other treatments, proportional settlement of Crassos- 
trea virginica was significantly higher onto shaded sides of settle- 
ment plates. Results are summarized in Table 1 . 

Larval mortality was less than 1% in any treatment, and larvae 
that had nol settled were still swimming. About 10-12% of the 
larvae in each trial settled, which was typical for hatchery-reared 
C. virginica larvae al that time (Baker 1994). 

DISCUSSION 

Competent-to-settle larvae of Crassostrea virginica respond to 
red, green, and blue portions of the visible light spectrum in the 
same manner as they do to white light. In this regard, they are 
unlike many previously studied coastal larvae, which appear to be 
unable to use orange or red light (Lang et al. 1979, Young and 

TABLE 1. 

Summary of differences of settled Crassostrea viginica between back 

(shaded) and front (illuminated) surfaces. Values are given for mean 

proportion of larvae settled on shaded (Back) surfaces; the means 

(A Mean) and standard deviations (A STDS) of the differences 

between proportions on shaded and illuminated surfaces, and type I 

error probabilities (p) from one-sample /-tests (based on 

arcsine-square root data transformations). 





Total 


Blue 


Green 


Red 


White 


Treatment 


Darkness 


Light 


Light 


Light 


Light 


Back 


0.483 


0.783 


0.792 


0.711 


0.743 


A Mean 


-0.0.^5 


0.565 


0.583 


0.422 


0.485 


A STDS 


0.513 


0.265 


0.346 


0.267 


0.309 


P 


0.9467 


<0.0001 


0.0001 


<0.000l 


0.0001 



Oyster Settlement Response to Light Spectrum 



1083 



Chia 1982, Marsden 1986. 1988, 1990). Coastal waters that trans- 
mit little usable light to other species still transmit sufficient light 
in the longer wavelengths to be a settlement cue for C. viri;iincci 
larvae. 

The breadth of the spectral responsivity of C. vii\^iiiica larvae 
reflects the range of water column conditions this species encoun- 
ters. The spectral transmittance of sea water is strongly modified 
by dissolved and particulate terriginous matter, and thus varies 
between and within estuaries (Pierce et al. 1986). The adult habitat 
of C. virginica, and thus the habitat of competent-to-settle larvae, 
ranges from oceanic to seasonal salinities as low as 5%6 (Wells and 
Gray 1960, Stanley and Sellers 1986). A broad spectral respon- 
sivity would be a beneficial adaptation for a species occupying this 
range of habitats. The brine shrimp. Anemia sulinu, also has a 
broad spectral sensitivity, and also occupies highly variable water 
column conditions (temporary ponds), although peak sensitivity 
appears to be below 600 nm (Aiken and Hailman 1978). 

On the other hand, light is not a required settlement cue for C. 
virginica. In both this study and others (Richie and Menzel 1969, 
Baker 1997), C. virginica larvae settled in the total absence of 
light. Gravity appears to be a strong settlement cue by itself Either 
light avoidance or geota.\is could permit larvae to settle on lower 



surfaces of adult oyster shells in the field, but the settlement pat- 
terns that result are equally marked in darkness (Baker 1997), 
indicating gravity as a sufficient cue. Gravity is a constant, light is 
not. Furthermore, other cues are also available: C. virginica has 
been shown to settle in response to water-bom chemicals from 
conspecifics (Hidu et al. 1978), and C. gigas. a similar species, 
settles in response to chemicals produced by certain bacterial con- 
ditions on the substrate (Fitt et al. 1990). 

The question then arises: Why has C. virginica evolved a pho- 
totactic response during settlement? Assuming that the eyespot is, 
in fact, a photosensory organ (Thompson et al. 1996), why does it 
develop only in the competent-to-settle larvae? 

One possibility is that phototaxis permits a 'Tine-tuning" of 
the settlement response. Oysters, unlike most bivalve mollusks, 
cement permanently to the substrate immediately upon settlement, 
and cannot subsequently adjust their habitat choice (Kennedy 
1996). Laboratory studies deliberately reduce variables and pro- 
vide larvae with clear choices (up-down, light-dark), but natural 
ecosystems are likely to be more complex. It is. therefore, probably 
of selective advantage to invest in additional sensory systems to 
gain as much information as possible about a potential permanent 
home. 



LITERATURE CITED 



Aiken. R. B. & J. P. Hailman. 1978. Positive phototaxis of the brine shrimp 
Anemia saliiia li) monochromatic light. Can. J. Zool. 56:7(^8-71 1. 

Austin. R. W. & T. J. Petzold. 1984. Spectral dependence of the diffuse 
attenuation coefficient of light in ocean waters. Proc. Soc. Pliolo-Opt. 
Inslnim. Eiig. 489:168-178. 

Baker, P. 1994. Competency to settle in oyster larvae. Cra.ssosrrea vir- 
ginica: wild vs hatchery-reared larvae. Aquaculture 122:161-169. 

Baker, P. 1997. Settlement site selection by oyster larvae, Crassostrea 
virginica: evidence for geotaxis. J. Shellfi.':li Res. 16:125-128. 

Champ. M. A., G. A. Gould III. W. E. Bozzo, S. G. Ackleson & K. C. 
Vierra. 1980. Characterization of light extinction and attenuation in 
Chesapeake Bay. August. 1977. pp. 26-V277. In: V. S. Kennedy (ed.). 
Eshiurine Perspectives. Academic Press. New York. NY. 

Cole. H, A. 1938. The fate of the larval organs in the metamorphosis of 
Ostrea edidis. J. Mar. Biol. Assoc. U.K. 22:469^89. 

Crisp, D. J. & A. P. A. A. Ghobashy. 1971. Responses of the larvae of 
Diplosoma lislerianiim to light and gravity. 4rh Eiirop. Mar. Biol. 
Svm/>.;433^65. 

Fitt. W. K., S. L. Coon, M. Walch. R. M. Weiner, R. R. Colwell & D. B. 
Sonar. 1990. Setdement behavior and metamorphosis of oyster larvae 
[Crassostrea gigas) in response to bacterial supernatants. Mar. Biol. 
l(»:389-394. 

Forward. R. B.. Jr. & T. W. Cronin. 1979. Spectral sensitivity of larvae 
from intertidal crustaceans. J. Comp. Physiol. 133:311-315. 

Hidu. H., W. G. Valleau & F. P. Vietch. 1978. Gregarious setting in Eu- 
ropean and American oysters — response to surface chemistry versus 
waterbome chemicals. Proc. Natl. Shellfish. Assoc. 68:11-16. 

Kennedy. V. S. 1996. Biology of larvae and spat. pp. 371^21. In: V. S. 
Kennedy. R. I. E. Newell. & A. F. Eble (eds.). The Eastern Oyster, 
Crassostrea virginica. Maryland Sea Grant. College Park. Maryland. 

Lang. W. H.. R. B. Forward. Jr. & D. C. Miller. 1979. Behavioral re- 
sponses of Balanus improvisus nauplii to light intensity and spectrum. 
Biol. Bull. 157:166-181. 

Mann, R. & C. C. Wolf 1983. Swimming behavior of larvae of the ocean 
quahog Arctica islandica in response to pressure and temperature. Mar. 
Ecol. Prog. Ser. 13:211-218. 

Marsden, J. R. 1986. Response to light by trochophore larvae of Spiro- 
branchus giganteus. Effects of level of irradiance, dark adaptation, and 
spectral distribution. Mar. Biol. 93:13-16. 



Marsden, J. R. 1988. Light response of the larva of the serpulid polychaete 
Galeolaria caespitosa. Mar. Biol. 99:yil^ni . 

Marsden. J. R. 1990. Light response of the planktotrophic larva of the 
serpulid polychaete SpirolTranchics polyceriis. Mar. Ecol. Prog. Ser 
58:225-233. 

Pierce, J. W., D. L. Correll, B. Goldberg, M. A. Faust & W. H. Klein. 
1986. Response of underwater light transmittance in the Rhode River 
estuary to changes in water-quality parameters. Estuaries 9:169-178. 

Pires, A. & R. M. Woollacott. 1983, A direct and active influence of 
gravity on the behavior of a marine invertebrate larva. Science 220: 
731-732. 

Ritchie. T. P. & R. W. Menzel. 1969. Influence of light on larval setdement 
of American oysters. Proc. Natl. Shellf. Assoc. 59:116-120. 

Stanley. J. G. & M. A. Sellers. 1986. Species profiles: life histories and 
environmental requirements of coastal fishes and invertebrates (Mid- 
Atlantic) — American oyster. LIS. Fish Wildl. Serv. Biol. Rep. 
82(11.65). 25 pp. 

Sweatt, A. J. & R. B. Forward, Jr. 1985. Spectral sensitivity of the chae- 
tognath Sagitta hispida Conant. Biol. Bull. 168:32-38. 

Thompson, R. J.. R. I. E. Newell, V. S. Kennedy & R. Mann. 1996. Re- 
producuve processes and early development, pp. 335-370. In: V. S. 
Kennedy. R. I. E. Newell. & A. F. Eble (eds.). 7"/ic Eastern Oysters. 
Crassostrea virginica. Maryland Sea Grant, College Park, Maryland. 

van Tine, R. F. 1987. Aspects of the ecology of estuarine light, with special 
reference to seagrasses of the Chesapeake Bay: measurements and 
models. Ph.D. Thesis. College of William and Mary. Williamsburg, 
Virginia. 139 pp. 

Wells. H. W. & 1. E. Gray. I960. Some oceanic sub-tidal oyster popula- 
tions. Nautilus 73:139-146. 

Wetzel. R. L. & H. A. Neckles. 1986. A model of Zostera marina L. 
photosynthesis and growth: simulated effects of physical-chemical 
variables and biological interactions. Aquatic Botany 26:307-323. 

Young. C. M. & F.-S. Chia. 1982. Ontogeny of phototaxis during larval 
development of the sedentary polychaete. Serpida vermicidaris (L.). 
Bwl. Bull. 162:457-468. 

Zar. J. H. 1996. Biostatistical Analysis. 3rd ed. Prentice-Hall. Upper 
Saddle River. NJ. 918 pp. 



Joiiniul ol Shfllfish Research. Vol, 17. No. 4. 1()S5-I()91. 1998. 

RECRUITMENT PATTERNS OF THE EASTERN OYSTER, CRASSOSTREA VIRGINICA, ALONG 
A CREEK GRADIENT IN HOUSE CREEK, LITTLE TYBEE ISLAND, GEORGIA 



DEBORAH A. MORONEY AND RANDAL L. WALKER 

Shellfish Aquaculture Laboratory 

University of Georgia Marine Extension Senice 

20 Ocean Science Circle 

Saviinndh. Georgia 3141 1 

ABSTRACT Our goal i,s to produce large, deep-cupped, single oysters, Crassoslrea rirginicu, for the halt-shell and steamer market. 
However, oysters in Georgia are small and grow in clusters because of high recruitment rates and lack of appropriate substrate upon 
which oyster spat can settle. It was hypothesized that oyster recruitment would be lower at sites farther back into a tidal creek 
presumably because of high tidal flushing rates (40% e.xchange per tide). To test this hypothesis, spat collectors were deployed monthly 
at 10 sites along two branches of House Creek. Little Tybee Island, Georgia from April to November. 1996. The total number of oysters 
on each collector was enumerated and 30 oysters per site were measured for shell length. Significant differences in recruitment rates 
were observed between sites (p < .001). Generally, oyster spat recruitment was lowest at the most remote sites in a tidal creek and 
increased seaward. The recruitment rate at one site was higher than expected based on our hypothesis. However, the percent cover by 
adult oyster reefs at this site was also higher and may explain the higher recruitment rate. Therefore, our data supports the hypothesis. 
Based upon the results of this study, the most remote sites in a tidal creek with relatively low percent cover by adult oyster reefs should 
exhibit low recruitment rates. It may therefore be possible to reduce oyster clustering by culturing oysters in these areas for grow-out 
to market size. 

KEY WORDS: Oysters, aquaculture, fouling, recruitment, Crassoslrea virgiiuca. spatfall 



INTRODUCTION 

Georgia has 450.000 acres of salt marsh, most of which is 
undeveloped and relatively unpolluted. Given this, and the ideal 
environmental conditions of salinity (10 ppt-30 ppt) and water 
temperature (8-30°C). the Georgia coast has the potential to be a 
prime location for Eastern oyster. Crassoslrea riri;iiika (Gmelin). 
aquaculture (Heffeman and Walker 1988). Due in part to a large 
tidal amplitude (2.4-3 m), oyster population dynamics are unique 
in that oysters in Georgia occur mostly in the intertidal zone (Har- 
ris 1980). Therefore, oyster culturing techniques used successfully 
in areas where oysters occur subtidally might not be as effective in 
Georgia, Specific culturing techniques need to be developed for 
Georgia that account for the unique environmental characteristics. 

Dame et al. (1984) noted that oyster reefs reach their greatest 
biomass and density in the southeastern United States, in particular 
South Carolina and Georgia. However, the oysters are relatively 
small and tend to grow in clusters, making harvesting and subse- 
quent handling difficult. Also, high recruitment rates and the lack 
of appropriate substrate for setting cause oysters to settle upon and 
compete with each other. This results in overcrowding such that 
oysters only have room for lateral growth. Oysters grown in such 
conditions are long, narrow, and shallow, and the meat is of little 
commercial value (Adams et al. 1991). 

Eighteen months of growth are required for an oyster to attain 
legal market size in Georgia (Heffeman and Walker 1988). Thus, 
oysters must grow through at least one spawning cycle. May 
through October (Heffeman et al. 1989). where oyster spat (juve- 
nile oysters that have successfully recruited) may settle upon cul- 
tured oysters. O'Beirn et al. ( 1995) determined that oyster recruit- 
ment in Georgia can be higher than recruitment rates observed in 
other parts of the southeastern United States. Mean monthly re- 
cruitment rates as high as 35.000 spat/m" have been documented 
(O'Beirn et al. 1994). The degree of fouling by oyster spat presents 
a serious problem for oyster aquaculture in this region. 

It may be possible to control the degree of fouling by growing 



oysters to market size in areas of low oyster recruitment. There- 
fore, identifying areas of low recruitment would be useful infor- 
mation in developing aquacultural techniques. We hypothesized 
that oyster recruitment would be lower at sites farther back into a 
tidal creek presumably because of high tidal flushing rates. The 
hypothesis of this experiment was based upon the results of a 
two-yr study of oyster recruitment conducted by 0"Beim et al. 
(1997) in the Duplin River. Sapelo Island. Georgia. They found 
that areas farther back in this tidal river had lower recruitment rates 
which may be attributed to the larvae being flushed out of these 
areas during tidal changes. This is a possibility because Ragotzkie 
and Bryson (1955) showed that a 40% water exchange occurred 
with each tidal cycle for the Duplin River. Therefore, the most 
remote locations in tidal creeks might be the best for oyster grow- 
out because oyster spat fouling might be reduced by larvae being 
washed out with the tide. 

This study monitored the temporal and spatial patterns of re- 
cruitment, spat size, and water temperature at 10 sites in House 
Creek. Identifying spatial recmilment patterns will enable us to test 
the hypothesis. Studying temporal and spatial patterns of recruit- 
ment, spat size, and water temperature will provide important in- 
formation in developing oyster aquacultural techniques. 

Sile Description 

This study was carried out in House Creek which is part of the 
Little Tybee Island complex located just outside Savannah. Geor- 
gia (Fig. 1 ). The area is predominated by salt marsh and small tidal 
creeks, Wassaw Sound is the primary source of water for both 
branches of House Creek utilized in this experiment. One branch 
of the creek is connected to another water source, the Bull River, 
at high tide and the other branch is a dead end. Under our hypoth- 
esis, sites closest to Wassaw Sound and the Bull River should have 
higher recruitment rates than the sites farthest back into the creek. 
In addition, we expected to observe a gradient of recruitment rates 
from sites farthest back in the creeks seaward. In this case, cul- 



1085 



1086 



MORONEY AND WaLKER 




35- 37' 35- 36' 35- 35' 35' 34' 35" 33' 

Figure 1. Oyster spat collection sites in House Creek, Little Tvbee Island, Wassaw Sound, Georgia. 



turing methods could be employed so that spat could be collected 
from areas of highest recruitment and transplanted to areas of low 
recruitment for growout to market size. 

METHODS 

To test our hypothesis. 10 sites were chosen along both 
branches of House Creek: one where the creek branches, five along 
the larger branch that is influenced by the Bull River, and four 
along the second branch (Fig. 1 ). Rebar frames were permanently 
installed at each site to ensure that the collectors were positioned 
at mean low water, near a living oyster reef, and approximately 15 
cm off the shell bottom. The collectors were gray PVC pipes (2 cm 
in diameter, cut in 15-cm segments, of which 12.5 cm was used in 
the analysis, which resulted in a total surface area of 0.01 m") with 
longitudinal grooves and embedded calcium chips. The four col- 
lectors were placed on PVC frames designed to fit on the rebar 
frames (Fig. 2). Prior to deployment, the collectors were condi- 
tioned in a raceway with running sea water for several days. 

The study was initiated on 27 March 1996 at which time the 
first set of collectors was deployed at each site. The collectors were 
replaced with a new set on a monthly basis and the study was 
terminated on 24 November 1996. After the deployment period, 
the collectors were lightly rinsed with fresh water to remove any 
loose debris and oyster larvae that were not fully cemented to the 
substrate. For the purpose of this study, we defined oyster spat as 
oysters that had metamorphosed, cemented, and exhibited a fan of 
shell growth. Therefore, larvae that were washed away because 
they had not fully cemented were not considered spat. 

On a monthly basis, all the oysters on each collector were 
counted using a dissecting microscope (lOx). In addition. 30 oys- 
ters were measured for shell length (umbo to lip) per site. When 
less than 30 spat settled per site, all oyster spat were measured. 
Oysters smaller than 10 mm were measured with an ocular mi- 



crometer in the microscope. Oysters larger than 10 mm were mea- 
sured using Vernier calipers. 

Optic Stow Away Temperature Loggers from Onset Computer 
Corporation were deployed at each site. The probes were placed in 
plastic bags with holes and then placed inside PVC pipes with 
holes to mhibit biofouling. These units were weighted down with 
a brick and attached to the rebar frame using heavy fishing line. 
The water temperature loggers were programmed to take measure- 
ments every six min. The data were downloaded to the Optic 
Shuttle and then directly into the computer via the Optic Base 
Station on a monthly basis. 

The areas occupied by the adult oyster reefs were estimated by 
multiplying the longest length by the longest perpendicular width 
of each reef within a 30.5 m radius of the collectors. The resulting 
number was divided by the area of a circle with a 30.5 m radius 
and multiplied by 1009?- to get the percent cover by the adult oyster 
reefs at each site. The oyster reefs included shell as well as living 
oysters. These data were collected in March 1998. It is possible 
that the areas occupied by the oyster reefs changed during the 
interval between the end of the recruitment study and the collec- 
tion of this data. However, this was intended to provide an estimate 
only, and based upon personal observation it is unlikely that the 
areas changed enough to substantially effect the estimate. 

The raw monthly recniitment data were converted to the aver- 
age number of spat per collector (n = 4) at each site and then 
analyzed using Friedman's Method for Randomized Blocks. First, 
months were used as blocks (b = 6) and then sites were used as 
blocks (b = 10) to determine if there were differences in recruit- 
ment between sites and months, respectively. The raw monthly 
size data were converted to the average spat size (n = 30, when 
available) at each site and then analyzed using Friedman's Test, 
again blocking months (b = 6) and sites (b = 10). independently 
(Conover 1980). The purpose for deploying collectors during the 



Recruitment of the Eastern Oyster 



1087 



Gray, grooved PVC collectors 
Reinforcement bar home 




Figure 2. Spat collectors attached to an exchangeable PVC frame mounted on a permanent frame on site made of rebar. 



months of April and November was to ensure that we did not miss 
the beginning and end of the recruitment period. We did not expect 
to observe recruitment during those months, although it is possible. 
Therefore, only those months in which recruitment was actually 
observed were used in the analysis of the recruitment and size data. 
In October, although no recruitment was observed at sites 2. 4, and 
8. they were still used in the analysis and assigned the lowest rank 
in their blocks. If significant differences were detected, the mul- 
tiple comparisons test suggested by Conover (1980) was used to 
determine which sites or months were significantly different from 
each other. 

Daily (n = 225) and biweekly In = 3100) water temperature 
averages were obtained from the raw data at each site. Monthly 
water teinperature averages were calculated as the mean of the 
daily water temperature averages (n = 30) at each site. The daily 
water temperature averages (n = 30) were analyzed using one-way 
analysis of variance (ANOVA) to detennine if there were signifi- 
cant differences in water temperature between sites for each 
month. The test was conducted with equal sample sizes for April, 
July, and August, and unequal sample sizes for the remaining 
months because the temperature probes failed at several sites. If 
differences were observed, a Tukey-Kramer test was used to de- 
termine which sites were significantly different from each other 
(Sokal and Rohlf 1995). The statistical package used for the 
ANOVA and Tukey-Kramer tests was JMP (SAS Institute 1989). 

RESULTS 

The average number of spat per collector (n = 4) at each site 
is plotted over time in Figure 3. Recruitment was first observed in 
May at all 10 sites. All sites demonstrated peak recruitment rates 
in June and another small peak was observed in September at most 
sites. Recruitment ended at sites 2, 4, and 8 in September, whereas 
the other seven sites last exhibited recruitment in October. Sites 1 
and 2 demonstrated the largest peaks, with means of 457 and 650 
spat/0.01 m". respectively, and had a total of more than 2.000 spat 



settle during the season. The lowest recruitment rates were ob- 
served at sites 4 and 10. neither of which exhibited a mean of more 
than 10 spat/0.01 m" during any time period or a total of more than 
100 spat settling during the entire season. 

There were significant differences in the average number of 
spat per collector (n = 4) between sites when months were 
blocked (Friedman, p < 0.001 ). Table 1 shows the results of the 
multiple comparisons test to determine which sites were signifi- 
cantly different. Sites 1. 7. and 9 were ranked the highest in re- 



o 
U 






3 




(irciner than 2000 
:>puf per season 

r — I Bcmeen 100 and 2000 
— spat per season 

^ Uss than 100 
spat per season 



Figure 3. Mean number of spat collected per month per site for the ten 
sites in House Creek, Little Tybee Island, Georgia. 



1088 



MORONEY AND WALKER 



TABLE 1. 

The results of the multiple comparisons test on the average 

recruitment data to determine which sites were 

significantly different. 

Ranking of Recruitment by Site 
Multiple Comparisons p = 0.05 



High- 
Site: 1 



10 



> Low 

8 4 



This test was warranted because the Friedman test on the average number 
of spat per collector (n = 4), using months as blocks, resulted in significant 
differences (p < 0.001) between sites. Only months in which recruitment 
was observed were used in the Friedman test. Sites underlined by the same 
line are statistically the same. 



cruitment and were significantly higher than sites 8 and 4. which 
were ranked the lowest. However, there is a high degree of overlap 
of significance based on this test. 

A summary of the rankings of the total number of spat settling 
during the season, the results of the multiple comparisons test on 
the average recruitment data, and the percent cover by adult oyster 
reefs at each site is presented in Table 2. The ranking of the total 
number of spat settling during the sea.son is based upon Figure 3. 
Table 1 is used to rank the sites according to the results of the 
multiple comparisons test on the average recruitment data. Sites 
where the percent cover by adult oyster reefs was greater than 10% 
were ranked high, between 5 and lO'yJ' were ranked medium, and 
less than 5% were ranked low. The location of each site along both 
branches is also described. Site 1 is considered part of both 
branches because it is located where House Creek bifurcates. 

When sites were blocked, there were significant differences in 
the average number of spat per collector (n = 4) between months 
(Friedman, p < 0.001 ). The results of the multiple comparisons test 
to determine which months were significantly different are shown 
in Table 3. Each month was significantly different in recruitment 



from the rest, with the exception of May and September which 
were statistically the same. June was ranked the highest in recruit- 
ment and October was ranked the lowest. 

The average spat size (n = 30 when available) at each site is 
plotted over time in Figure 4; it ranged from 0.37 mm to 4.92 mm. 
Spat size was highly variable within sites. However, the data seem 
to exhibit a general trend: a peak in spat size occurred in June, 
followed by another peak in August or September. 

When sites were blocked, significant differences in the average 
spat size (n = 30 when available) were observed between months 
(Friedman, p < 0.001 ). The results of the multiple comparisons test 
to determine which months were significantly different are shown 
in Table 4. August and September were statistically the same in 
ranking of oyster size, as well as October and May. All other 
comparisons were significantly different. June was ranked the 
highest in spat size and October and May were ranked the lowest. 
When months were used as blocks on this data, there were no 
significant differences between sites (Friedman, p > 0.05). 

The average oyster recruitment versus water temperature is 
plotted in Figure 3. The average oyster recruitment is the average 
number of spat per collector (n = 4) per month and site. The water 
temperature is the monthly average at the corresponding site. 
There were several sites where the temperature probes failed to 
collect data during the entire month of collector deployment. These 
sites were not included in this plot. Oysters settled within a water 
temperature range of 23.5-29.0°C. Maximum recruitment oc- 
curred at a water temperature of 27.5°C. Heaviest recruitment 
occurred in June when water temperatures increased to 27°C. 
rather than in September when water temperatures decreased to 
27°C (Figs. 3 and 6). However, a small peak in oyster recruitment 
was noted in September. 

Figure 6 is a plot of the water temperature over time throughout 
the recruitment season. The water temperature is the average of the 
biweekly water temperature averages at all 10 sites. On 16 April, sites 
8 and 9 had significantly higher water temperatures than the other 
eight sites (Table 5): therefore, those two sites were plotted separately 
on that date. The water temperahire rose steadily between 16 April 
and 26 June 1996. Between 26 June and 20 August, the water tem- 
perature leveled off at about 28.5°C. Following 20 August, the water 
temperature decreased to a low of about 14.5°C in December. 



TABLE 2. 

A summary of the rankings of the total number of spat settling during the season, the results of the multiple comparisons test on the 
average recruitment data, and the percent cover by adult oyster reefs at each site along both branches. The location of each site along both 

branches is described. 



Branch 



Site 



Location Along 
Branch 



Number 
of Spat 



Multiple 
Comparisons 



Percent 
Cover 



Large 



Small 



1 
2 
3 
4 
5 
6 
1 
7 
g 
9 
10 



Most seaward 



Most remote 

Close to Bull River 

Close to Bull River 

Most seaward 



Most remote 



h 

h 

m 

1 

m 

m 

h 

m 

m 

m 

1 



h 
m 



1 



h 

m 

1 

1 

h 

h 

h 

h 

1 

h 

1 



Site 1 is considered part of both branches because il is located where House Creek bifurcates. 

Key: (h)igh: >2000 spat: Sites 1, 7 & 9: >10':/f: (mledium: 100-2000: 2. 5. 6. 3 & 10: 5-10; (Dow: <100: 8 & 4; <5, 



Recruitment of the Eastern Oyster 



1089 



TABLE 3. 

The results of the multiple comparisons test on the average 

recruitment data to determine which months were 

significantly different. 

Ranking of Recruitment hy Month 
Multiple Comparisons p = 0.05 



High- 
Month: June 



July 



May September 



August 



— » Low 
October 



This test was warranted because the Friedman test on the average number 
of spat per collector (n = 4), using sites as blocks, resulted in significant 
differences (p < .001) between months. Only months in which recruitment 
was observed were used in the Friedman test. Months underlined by the 
same line are statistically the same. 

The results of the ANOVA and Tukey-Kratiier tests pei^'otTiied 
on the daily water temperature averages (n = 30) are shown in 
Table 5. Significant differences in water temperature were ob- 
served between sites (ANOVA. p < 0.05) during the months of 
April. August, November, and Decetnber. Of those, the only 
month during which oyster recruitment was observed was August. 
However, the Tukey-Kramer test (p = 0.05) was unable to detect 
significant differences in water temperature between the sites dur- 
ing August. During April, sites 8 and 9 were significantly higher in 
water temperature than all the other sites (Tukey-Kramer, p = 
0.05). In November and December, the Tukey-Kramer test (p = 
0.05) was able to detect significant differences in water tempera- 
ture between sites, however, there is a high degree of overlap. 

DISCUSSION 

In general, oyster recruitment began in May. peaked in June, 
demonstrated another small peak in Septetnber. and tertninated in 
October (Fig. 3). When sites were blocked, there were significant 
differences in the average number of spat per collector between 
months (Friedman, p < 0.001, Table 3). June was ranked the high- 
est month which corresponds to the peak recruittnent period in 
June. Since another small peak was observed in September, it 
would be expected that September should be ranked second. How- 
ever, the recruitment rate in July was higher than September, but 
July was exhibiting a decreasing trend and was therefore not con- 
sidered a peak. These general seasonal patterns were similar to 
those observed in this region by other authors (Kenny et al. 1990, 
McNulty 1953, O'Beirn et al. 1996). 




TABLE 4. 

The results of the multiple comparisons test on the average spat size 
to determine which months were significantly different. 



Ranking of Spat Size by Month 
Multiple Comparisons p = 0.05 



Month: 



Large - 
June 



August September July 



> Small 

October May 



This test was warranted because the Friedman test on the average size of 
30 spat (when available), using sites as blocks, resulted in significant 
differences (p < 0.(X)1 ) between months. Only months in which recruitment 
was observed were used in the Friedman test. Months underlined by the 
same line are statistically the same. 

More than 8.000 spat settled in June (the sum of all sites and 
collectors), whereas less than 50 spat settled in October. These 
results support the findings of Kenny et al. ( 1990) in that the within 
year variability in recruitment numbers may be high. In addition, 
Kenny et al. (1990) noted that these temporal patterns are fairly 
consistent among years for each geographic location, and that the 
peak recruitment periods coincide with the spawning patterns of 
the local adults. Yet, O'Beirn et al. (1997) found that spawning 
events were not necessarily followed by peaks in recruitment. This 
emphasizes the point that recruitment is dependent upon many 
factors, which include rate of gametogenesis and spavining, water 
temperature, salinity, food availability, tidal flushing, etc. Perhaps, 
in the year O'Beirn et al. ( 1997) conducted the study, these other 
factors had more important roles in determining recruitment rates. 

Based upon our hypothesis, we expected to observe the highest 
recruitment rate at site 1 (the site closest to Wassaw Sound). Along 
the small branch of House Creek we expected a gradient of re- 
cruitment rates, the lowest at site 10 and increasing seaward. The 







■ 


600- 






u 






o 






■5 5»)- 






<u 

















■ 


U 






4; 400- 






a 






^^ 






cs 






0. 






^300- 













0) 




■ 


£ 






1 200- 




■■ 


Z 




■ 


100- 








■ 


■ ■ 




■ 


j|^ 




^^H T" 


^^ 



26 



28 



30 



Water Temperature (°C) 



Figure 4. Mean spat shell length in mm of oysters collected per site per 
month from House Creek, Little Tybee Island, Georgia. 



Figure 5. The relationship between water temperature and the mean 
number of spat collected from House Creek, Little Tybee Island, Geor- 
gia. 



1090 



MORONEY AND WALKER 




1996 



Figure 6. The mean water temperature in House Creek. Little Tybee 
Island. Georgia. Significant differences in water temperature was de- 
tected only at sites 8 and 9 on April 16, thus the separate data point 
graphed on that date. 

large branch is influenced by the Bull River at site 5 at high tide. 
Along the large branch we expected site 1 to be the highest, the 
sites closest to the Bull River (sites 5 and 6) to be the next highest, 
and a gradient between sites I and 5. Consistent with our hypoth- 
esis, both the number of spat settling and the multiple comparisons 
test revealed that recruitment at site 1 was significantly higher. In 
fact, the number of spat settling is consistent with our hypothesis 

TABLE 5. 

The results of the analysis of variance on the daily water 

temperature averages (n = 30) to determine if there were significant 

differences between sites for each month. 

Ranking of Temperature by Site per Month 
ANOVA Tukey-Kramer p = (] 



Month p-Value 



High 



Lo\( 



Apnl 


0.0001* 


May 


0.4926"^ 


June 


0.6700"- 


July 


0.2423"^ 


August 


0.0394* 


September 


0.0912"' 


October 


0.2138"" 


November 


0.0313* 


December 


0.0001* 



No differences 



6 


8 


7 


4 


1 


3 


5 


10 


2 




8 


7 














6 


4 


"> 


3 


1 


10 


5 





















































Where significant differences were observed, the Tukey-Kramer test was 
used to determine which sites were significantly different from each other. 
Sites connected by the same line are statistically the same. 
ns = not significant; * = significant; x = site 9 had no data points and 
was excluded from the analysis. 



at all sites. However, the multiple comparisons test indicates that 
site 9 is the only site that is inconsistent with our hypothesis (Table 
2). This anomaly may be explained by the percent cover by adult 
oyster reefs. Metamorphosed oysters are believed to emit phero- 
mones which stimulate larvae to set (Hidu and Haskin 1971). The 
percent cover by adult oyster reefs at site 9 ranked high at 12.7% 
(Table 2). With greater numbers of adult oysters at site 9. the 
concentration of pheromones may be high enough to cause a 
greater number of larvae to set. Therefore, our data support the 
hypothesis of oyster spat recruitment being the lowest at the most 
remote sites and increasing seaward. 

This experiment identified two areas of low recruitinent. sites 4 
and 8 (Table 1 ). It is possible that the factors that cause lower 
recruitment rates at these sites could also affect the growth and 
survival of the oysters placed in these areas for growout to market 
size. However, there were no significant differences between sites 
for spat size when months were blocked (Friedman, p > 0.05). 
Consequently, factors causing lower recruitment rates do not sig- 
nificantly affect the growth of the spat. In addition, it is likely that 
the larger spat transplanted to low recruitment areas for growout 
will be more resistant to factors causing lower recruitment rates 
than oyster larvae and newly settled spat. Therefore, the spat could 
be collected from high recruitment areas (sites 1. 7. and 9) and 
transplanted to low recruitment areas ( sites 4 and 8) for growout. 
The spat size data, as illustrated in Figure 4, appear to exhibit 
a general trend: a peak in spat size in June, followed by another 
peak in August or September. There were significant differences in 
average spat size between months when sites were blocked (Fried- 
man, p < 0.001, Table 4). which substantiates this observation. The 
largest spat were measured on the June, August, and September 
collectors in which case oysters from the June sample were sig- 
nificantly larger than those measured in August and September. 
These peaks may be explained by the time at which the larvae 
settle. For instance, if the larvae settled when the collectors were 
first deployed, they would have an entire month to grow. Identi- 
fying sites or time periods with the best spat growth would be very 
useful information from an aquacultural perspective. However, the 
temperature, salinity, quality of food, and many other factors make 
growth rate difficult to predict. Further studies would need to be 
conducted to determine if time of deployment affects the spat size. 
Water temperature has been shown to act as a stimulus that 
induces setting behavior in oysters (Hidu and Haskin 1971). The 
average oyster recruitment versus water temperature plot (Fig. 5) 
shows that the majority of setting occurred between 23.5 and 
29.0°C with a peak at 27.5 'C. The peak biweekly water tempera- 
ture average for House Creek in 1996 was 29.2°C, consistent with 
the findings of O'Beim et al. (1996) in 1991. 1992, and 1994. In 
1993, O'Beim et al. (1996) noted higher than usual water tem- 
peratures ranging from 29.2 to 31.5°C which coincided with lower 
than usual recruitment rates observed on the biweekly and monthly 
collectors. They suggested that these lower recruitment rates might 
be explained by the observed temperatures exceeding the upper 
temperature tolerance for oyster larvae. During the months in 
which oyster recruitment was observed, there were no significant 
differences in temperature between sites (Table 5). Therefore, the 
differences between sites in recruitment rates cannot be attributed 
to different water temperatures. 

An interesting observation about the recruitment (Fig. 3) and 
temperature (Fig. 6) data was that both peak recruitment periods 
occurred when the temperature was around 27°C. In June, the 
temperature was increasing to 27°C and in September it was de- 



Recruitment of the Eastern Oyster 



\m\ 



creasing to 27^C. This might suggest that 27°C is an optimum 
temperature for oyster recruitment. However, additional studies 
would need to be conducted to verify this. 

Our goal is to produce large, deep-cupped, single oysters for 
the half-shell and steamer market in Georgia. A quality product 
may be obtained by reducing the degree of clustering and provid- 
ing the spat with the potential for fast growth. By deploying spat 
collectors in July or August, after the peak recruitment period, 
fouling by additional spat would be considerably reduced because 
the majority of setting would have already occurred. This, coupled 
with the larger spat observed in August and September, would 
provide the newly settled spat with fast growth potential and re- 
duced crowding. 

Since 18 months of growth are required for oysters to reach 
market size in Georgia (Heffeman and Walker 1988). the cultured 
oysters would then be exposed to additional oyster spat fouling 
during the second recruitment season. However, culturing oysters 
in areas of low recruitment would further reduce such foulinti 



without adversely affecting the growth potential. Based upon the 
results of this study, the most remote sites in a tidal creek with 
relatively low percent cover by adult oyster reefs should exhibit 
low recruitment rates. In addition, biofouling during the recruit- 
ment season can further be reduced by culturing oysters in mesh 
bags placed intertidally on the river bottom (Adams et al. 1991, 
Moroney 1997). 

ACKNOWLEDGMENTS 

The authors wish to thank Dorset Hurley, Richard Kupfer, and 
Dodie Thompson for their assistance throughout the project. Drs. 
Charles Champ, Francis 0"Beim. and C. Ray Chandler provided 
statistical advice. Drs. Francis O'Beirn, Sophie George, Steve 
Vives, and three anonymous reviewers improved the quality of the 
manuscript. Anna Boyette provided the graphics. This project was 
funded by the Georgia Sea Grant College Program under grant 
number NA66RG0282. 



LITERATURE CITED 



Adams. M. P.. R. L. Walker. P. B. Heffeman & R. E. Reinert. 1991. Elimi- 
nating spat settlement on oysters cultured in coastal Georgia: a feasi- 
bility study. / Shellfish Res. 10:207-213. 

Conover, W.J. 1980. Practical Nonparainctric Stcitislics. 2nd ed., John 
Wiley & Sons, New York, NY. 

Dame, R. F.. R. G. Zingmark & E. Haskin. 1984. Oyster reefs as processors 
of estuarine materials. J. Exp. Mar. Biol. Ecol. 83:239-247. 

Harris, C. D. 1980. Survey of the intertidal and subtidal oyster resources of 
the Georgia coast. Georgia Department of Natural Resources Coastal 
Resources Division. Project No. 2-234-R. Brunswick. GA. -14 pp. 

Heffeman. P. B. & R. L. Walker. 1988. Preliminary observations on oyster 
pearl net cultivation in coastal Georgia. NE Gulf Sci. 10:33^3. 

Heffeman. P. B.. R. L. Walker & J. L. Carr. 1989. Gametogenic cycles of 
three marine bivalves in Wassaw Sound. Georgia II; Crassoslrea vir- 
ginica (Gmelin, 1791). / Shellfish Res. 8:61-70. 

Hidu. H. & H. H. Haskin. 1971. Setting of the American oyster related to 
environmental factors and larval behavior. Proc. Natl. Shellfish. Assoc. 
61:35-50. 

Kenny, P. D., W. K. Michener & D. M. Allen. 1990. Spatial and temporal 
patterns of oyster settlement in a high salinity estuary. J. Shellfish Res. 
9:329-339. 

McNulty. J. K. 1953. Seasonal and vertical patterns of oyster setting off 
Wadmalaw Island, S.C. Bears Bluff Laboratories No. 15, Wadmalaw 
Island. SC. 



Moroney, D. A. 1997. Recruitmenl palterns and culturing techniques of the 
eastern oyster Crassostreu virgiiiica (Gmelin) in a coastal Georgia tidal 
creek system. Masters thesis, Georgia Southern University. Statesboro. 

O'Beirn, F. X., P. B. Heffeman & R. L. Walker. 1994. Recruitment of 
Crassoslrea virginica: a tool for monitoring the aquatic health of the 
Sapelo Island National Estuarine Research Reserve. University of 
Georgia School of Marine Programs. Marine Technical Report No. 
94-2. Athens. GA. 42 pp. 

O'Beim. F. X., P. B. Heffeman & R. L. Walker. 1995. Preliminary recmit- 
ment studies of the eastern oyster. Crassoslrea virginica. and their 
potential applications in coastal Georgia. Acjuaculture 136:231-242. 

O'Beirn. F. X.. P. B. Heffeman & R. L. Walker. 1996. Recruitment of the 
eastern oyster in coastal Georgia: patterns and recommendations. N. 
Am. J. Fish. Muiiug. 16:413^26. 

O'Beim. F. X., R. L. Walker & M. L. Jansen. 1997. Reproductive biology 
and parasite {Perkinsus marinus) prevalence in the eastern oyster, 
Crassoslrea virginica, within a Georgia tidal river. J. Elisha Mitchell 
Sci. Soc. 113:22-36. 

Ragotzkie, R. A. & R. A. Bryson. 1955. Hydrography of the Duplin River. 
Sapelo Island. Georgia. Bull. Mar. Sci. Gulf Carrih. 5:297-314. 

JMP 3. 1989. SAS Institute. I.. Gary. NC. 

Sokal. R. R. & F. J. Rohlf 1995, Biometry: The principles and practice of 
slatistics in biological research. 3rd ed., W. H. Freeman and Company, 
New York. NY. 



Journal of Shellftsh Research. Vol. 17. No. 4. 1093-1099. 1998. 

POWER TO THE OYSTER: DO SPIONID-INDUCED SHELL BLISTERS AFFECT CONDITION 

IN SUBTIDAL OYSTERS? 

SEAN J. HANDLEY* 

Cuwthron Institute. Nelson, New Zealand: 
School of Biological Sciences 
University of Auckland. New Zealand: and 
NIWA. P.O. Bo.x 893. Nelson, New Zealand. 

ABSTRACT Two static allometric condition indices and two static histological indices were assessed in detennining whether oyster 
shell blistering induced by Boccardia knoxi infestations affected the health of infested oysters. Analysis of variance. Spearman's 
correlation analysis, and a-posteriori power analysis were used to assess the condition indices. The use of the dry weight condition 



inde.\ CIn 



I and the shell volume condition index CI,, 



were first validated after checking for confounding due to increased 
shell weight and loss of shell volume resulting from blistering. No significant differences in condition were detected between the sexes 
within the three subjective shell grades chosen to represent varying levels of shell blistenng. Loss of condition was detected by three 
out of the four indices; the CIne5h:sheii •^nd Cln^shcv both detected significant differences, but did not have the required level of power 
to reject the null hypothesis of no effect. However, for heavily blistered oysters the highly significant reduction in oocyte size (CI^oj^..^) 
supported rejection of the null hypothesis. The loss of condition was considered insignificant in terms of subtidal oysters production, 
but demonstrates the parasitic effect spionids can ha\'e even under ideal growing conditions. The only index not detecting an effect, 
CIgonad area ^ad thc lowcst power (<3%). illustrating the importance of performing a-posteriori power analysis. The resuUs of the 
analysis of variance (ANOVA). Spearman's rank nonparametnc correlation coefficients (SCC). and power analysis all indicated the 
Clneshsheii ^^^ '^c mosi Sensitive of the four indices compared. The negative effects of shell blistenng induced as a result of B. knoxi 
infestations rendered this species a parasite of Pacific oysters in Admiralty Bay. 

KEY WORDS: Spionid polychaete. Spionidae. Boccardia. Polydora. Pacific oyster. Crassostrea gigas. shell blisters, aquaculture, 
subtidal, power analysis 



INTRODUCTION 

The effects spionid polychaete worm infestations can have on 
oysters have been confusingly portrayed in the literature. Some 
studies have detected a parasitic effect resulting in decreases in 
condition due to infestations (Wargo and Ford 1993, Lunz 1941) 
whereas others imply commensalism with no significant effect 
(Stephen 1978, Medcof 1946) or even greater condition (Schleyer 
1991). Spionid worms can infest oysters by two means: boring 
through the shell structure from the e.xtemal suiface using meta- 
bolic acids (Haigler 1969), or enteiing the mantle cavity of the 
oyster as larvae and thus settling within the shell (Handley and 
Bergquist 1997, Skeel 1977, "Whitelegge 1890). The latter results 
almost immediately in oysters secreting a blister of either conchio- 
lin or shell material to wall out the intruder from the shell. In 
response to the former, shell blistering can result if the worms bore 
through the inside shell surface and penetrate the mantle cavity. 
Loss of condition is expected in oysters secreting shell blisters; as 
the oysters have to expend energy making the blisters (Blake et al. 
1996), they lose internal shell volume, and the blisters' irregular 
shape may interfere with the feeding currents within the mantle 
cavity (Korringa 1951 ). The presence of blisters inside the mantle 
cavity also requires that the oysters increase shell deposition to 
regain internal shell volume and shape. 

The cause of some of the confusion over the etiological status 
of spionids may be due to a combination of inappropriate methods 
used to detect spionid impacts and low statistical power when a 
null result is concluded. The relationship between environmental 
variables and the apparent "'health'" of oysters has been investi- 
gated using ecophysiological indices to summarize the physiologi- 
cal activity of the animals (Lucas and Beninger 1985). The most 



♦Present address: NIWA, P.O. Box 893 Nelson, New Zealand 



popular methods used to measure physiological effects of spionid 
infestations in bivalves have been ""static" condition indices 
which determine the health of the host at a single point in time 
using shell \olume as a parameter (Wargo and Ford 1993. Lunz 
1941). These indices may, however, be inappropriate if con- 
founded by the loss of shell volume and increased shell deposition 
resulting from infestations (Handley and Bergquist 1997). 

In New Zealand, shell blisters in Pacific oysters have been 
attributed to a number of spionid species, especially Polydora 
websteri in intertidal cultivations in the North Island (Handley and 
Bergquist 1997), and Boccardia knoxi infesting South Island sub- 
tidal cultivations (Handley 1995, 1997). In these studies a dry 
weight condition index recommended by Lucas & Beninger (1985) 
detected a biologically insignificant correlation with loss of con- 
dition in infested oysters in intertidal cultivations (Handley and 
Bergquist 1997). but no adverse impacts on condition in subtidal 
oyster populations (Handley 1997). 

Condition indices relate the tissue weight of the animal to some 
estimate of shell size, with reductions in condition implying con- 
sumption of stored energy reserves (Newell 1985). The primary 
energy reserve stored by marine bivalves is the metabolite glyco- 
gen (Barber et al. 1988) which is responsible for the creamy con- 
dition and sweet taste of "fat" oysters (Beaumont and Fairbrother 
1991). The gametogenic cycle of oysters is influenced strongly by 
the condition of oysters, with glycogen being used up during ga- 
metogenesis (Perdue and Erickson 1984, Mason and Nell 1995). 
One of the most accurate techniques reported to assess the meta- 
bolic costs of parasitism in oysters is the "gamete volume frac- 
tion" derived histologically from the ratio of the gonad to visceral 
area (Mori 1979, Perdue et al. 1981, Newell and Barber 1988). 
More recent histological techniques have been developed to com- 
pare the size of gametes to quantify any effect on gamete produc- 
tion (Barber et al. 1988). To quantify the true effects of a parasite. 



1093 



1094 



Handley 



host individuals with infestation ranging from nil to severe must be 
sampled from within the same population (Newell and Barber 
1988). 

The aim of this study was to test the null hypothesis (H„) that 
spionid-induced shell blistering did not affect the condition of 
subtidal Pacific oysters. This was done by using two static allo- 
metric condition indices and two static histological condition in- 
dices. The validity of the condition indices in terms of confounding 
were tested, and power and correlation analyses were used to 
compare the sensitivity of the condition indices. 



METHODS 



Experiment 



Oysters were collected from the experimental oyster growing 
site "B" in Admiralty Bay. Marlborough Sounds (Handley 1997). 
One hundred and twenty intact oysters of a similar size were 
selected from a population of oysters grown on plastic sticks after 
they had been separated and scrubbed clean of fouling organisms. 
These oysters had previously been shown to contain a proportion 
of blistered oysters and were sampled on the 2 December 1995 
which was the time during which B. knoxi had been previously 
recorded to induce shell blistering as gonad condition increased 
(Handley 1997). 

Analysis 

After cleaning, each oyster's shell volume was individually 
measured by displacement before being labelled with rubber bands 
and plastic tags (Roper etal. 1991, Medcof and Needier 1941). The 
oysters were carefully opened with a sharp knife and separated into 
three subjective categories: "0" containing no shell blisters: "1" 
containing 0-50% of the internal shell covered by blisters, and 
"2" containing <50% of the internal shell blistered. The oysters 
were then sexed by removal of gametes by a small scalpel incision 
of the gonad at the base of the adductor mussel. Eight oysters from 
each sex were then selected to best represent each category. The 
oyster meats were individually removed and weighed wet. before 
and after a standard transverse .section had been removed from 
behind the labial palps (Wilson et al. 1993, Perdue et al. 1981). 
The gonad sections were fixed in Davidson's solution for 24 h and 
then transferred to 70% ethanol in preparation for histological 
sectioning and staining (Barber et al. 1988). The remaining meat 
fractions and shells were dried to a constant weight at 80°C (Roper 
et al. 1990) after the shucked shell displacement volumes had been 
measured. 

Static Condition Indices 

The dry weight (CInesh>heii) and shell cavity (Cln^hsheii) con- 
dition indices (Roper et al. 1991) were derived from the above 
measurements after the whole dry ineat weights had been deter- 
mined from the dry meat weight fraction, given the assumption 
that the two wet weight fractions of oyster had the same water 
content when sectioned: 

"> CIn^5„:sheii = dry nieat weight (mg)/dry shell weight (g) 
(2) Cln^^h,,^ = dry ineat weight (mg)/shell cavity volume 
(ml). 

Histology 

Histological sections 4-5 |j.m thick were obtained with a Jung 
1 130 rotary microtome and stained with a standard Ehrlich's he- 



matoxylin and counterstained with Eosin (cf. Myers Hematoxylin; 
Barber 1996, Perdue et al. 1981). 

Histological Indices and Image Analysis 

The gamete volume fraction or "relative fecundity" (Cl„„„jj.^^^) 
and oocyte areas (Cl„„^.y,j) were determined from the histological 
sections using OPTIMAS video image analysis software viewed and 
calibrated through a dissecting microscope and video camera (Barber 
1996, Lowe et al. 1982, Perdue et al. 1981. and Barber et al. 1988). 
The area of 50 oocytes was measured usuig OPTIMAS: 

(1) Clg„„3j ^^^^ = area of gonad (mm~)/area of entire visceral 
mass (mm") 

(2) CI„„^.y,^, = cross sectional area of 50 female gametes 
(|xnr). 

Statistics 

Fixed factor analysis of variance (ANOVA. PROC GLM; SAS 
1992) was used to test the null hypothesis (H„): spionid-induced 
shell blistering had no impact on oyster condition as measured for 
the balanced data for the CIn^,h:sheii. CInesh:cv- and CI„„,„j ,„,. 
Mixed model ANOVA was used to test the same H,, for the bal- 
anced data for CIo„^,^,j using the mean squares error of the nested 
replicate interaction term to test significance between shell grades. 
The data sets were first log,,, transformed as they were growth 
related (LaBarbera 1989) and checked for normality and homoge- 
neity of variances before ANOVA procedures. Spearman's rank 
nonparametric correlation coefficients (SCC. SAS; PROC CORR) 
were also produced to test the H„, and a-posteriori power analysis 
was performed for each ANOVA to determine the power of not 
committing a "Type 11" error or accepting a false H„ (Searcy- 
Bemal 1994. Koele 1982). 

RESULTS 

Oysters grown in the vicinity of this site have a history of 
spionid infestations with 60% containing shell blisters due to in- 
festations dominated by Boccardia knoxi (Handley 1995. 1997). 
Visually there appeared to be no differences in the condition of the 
oysters, with all oysters in a "fat" condition. Histologically, all 
gonads were approaching "stage 3", the females having teardrop- 
shaped eggs and follicular channels or "curtains" evident, the 
male follicles being dark (basophilic) with sperm heads, and the 
center consisting of sperm tails which were paler staining (Wilson 
et al. 1993, Dinamani 1974). 

Although a trend was evident in the CI,i^,^h:sheii and Cld^^h^v 
data suggesting that females had greater condition index values 
than males, the 95% confidence intervals showed there was no 
significant difference between sexes (Fig. 1) and the mixed model 
ANOVAs failed to detect a significant difference between sexes 
for each of the condition indices (Table lA-F). These data were 
subsequently lumped within shell grades and reanalyzed. ANOVA 
comparing ineans between shell grades using the CI^^.,,,, .,^5,11 and 
CInesh cv both detected a loss of condition due to shell blistering 
between shell grades (Table 2, Fig. 2). These indices were not 
confounded, with no significant differences detected between dry 
shell weights or shell voluines between shell grades (Table IB & 
D. Table 2). The dry meat weights were, however, significantly 
different between grades across both sexes (Table lA, Table 2). 
Decreasing shell quality was significantly correlated with a de- 
crease in condition measured by the Cln^shsheii but not by the 



Power to the Oyster 



1095 



8^ 

DO On 

■5 -H 



550 -, 



525 



500 - 



475 - 



450 



400 




60 



0—55 



o U 



1 


T 


— 1 ^T — ' 1^ r 



M2 



1.3 



1.1 - 



1.0 






S o 



36 - 



-H 34 



30 



T 1 1 T" 



FO Fl F2 MO Ml M2 FO Fl F2 

Ovster sex: Shell grade Oyster sex: Shell grade 

Figure 1. Condition index values for oysters separated by sex: F, females (clear bars); M, males (grey bars); shell blister grades: 0, 1. 2. 



Clfieshcv (Tuble 2). The statistical power of the Cln^.^,, ,,^511 'i^st for 
rejecting the H„ was also greater than for the latter CI,-|^sh:cv 



The fixed model ANOVA and SCC for CI, 



failed to 



detect a significant impact or correlation of shell blistering im- 
pacts, with this index having the lowest power (<3%) of all three 
indices, with the s'ame degrees of freedom. The mixed model 
ANOVA and SCC for CIpj,;.^,^, however, detected a significant 
negative impact and correlation of decreasing oocyte size with 
increased shell blistering (Table 2 1. The oysters with greater than 
50% of the shell affected by shell blisters produced significantly 
smaller oocytes (Fig. 1 ). A significant difference was also detected 
between oysters nested within shell grades, but this was not sur- 
prising given the high degrees of freedom of this test (Table 1). 

DISCUSSION 



Type II error, or accepting a false null hypothesis" (Searcy-Bemal 
1994). In basic research power analysis, the cost of Type I error 
most often greatly exceeds the cost of Type II error (Toft and Shea 
1983). Power analysis for the simple one-factor ANOVAs after the 
data had been lumped for CIf|esh:sheii snd Cln^j^tA did not reach the 
required level of precision accepted by many authors (80%, 
Searcy-Bemal 1994). However, given the highly significant 
negative SCC and the rejection of H,, by the ANOVA tests for 



CI, 



II- CI,icsh cv yf"J CI„„^ (^, all results support acceptance of 



a highly significant decrease in size of the oocytes from oysters 
with greater than 50% of their shell blistered. The power of the 



ANOVA test for CL 



was not calculated, as power analysis 



involving mixed model interaction terms is not advised as a num- 
ber of severe restrictions have to be made on mixed model covari- 
ance matrices; even then it is only possible to derive the distribu- 
tion under the null hypothesis. When in some instances power can 
be calculated for mixed models, this power value must be under- 
stood to represent the lower bound of the power against a class of 
alternatives, and not the exact power against a specific alternative 
(Koele 1982). The results for the CI,,,,^,^,^ were, however, highly 
significant and surpassed the other indices at the 95% confidence 
level. Given the same degrees of freedom, the Clf,^.,,, ^heii had 
greater statistical power over the CI^^,.,,, ^.,, and the significant cor- 
relations detected by Cln^..,,, ^., further support the conclusions of 
Lucas and Beninger ( 1985) that the Clneshsheii 's a more sensitive 
Confounding would occur in the case of the Cln^.^|,:shL-ii '^nd indicator of condition than CIncsh cv '" oysters. The CIf|j.5h cv has 



ecophysiological indices that summarize the apparent "health" or 
physiological activity of cultured bivalves (Lucas and Beninger 
1985). In this study both these indices detected significant impacts 
of shell blistering on oyster condition. It was previously thought 
that the presence of shell blisters within the mantle cavity of oys- 
ters may increase either the shell dry weights or the shell cavity 
volumes, thus confounding these indices (Handley and Bergquist 
1997). Two or more effects are said to be "confounded" in an 
experiment if it is impossible to separate the effects after the sta- 
tistical analysis has been performed (Ostle and Mensing 1975). 



CIr 



if either the shell dry weights or shell cavity volumes 



were affected significantly by shell blistering, thus allowing for 
more than one possible null hypothesis to be tested. By determin- 
ing no significant effect of shell blistering against the denominator 



values of CI,- 



the results of these two indices 



were validated. 

In this study, power analysis was used to compare the sensi- 
tivity of the static condition indices given the same degrees of 
freedom. "Statistical power is the probability of not committing a 



also been criticized as it is relatively insensitive to low signal-to- 
noise ratio and therefore significant changes to the index are only 
likely to be evident after long-term exposures (Widdows 1985). 

Power analysis can also be used to determine the power of not 
detecting an impact when one was present (Searcy-Bemal 1994). 
The danger of this was highlighted by the low power of the 
CIgonad area t^st (<3%). This iudcx has bccn described as one of the 
most accurate techniques for assessing parasitism (Newell and 
Barber 1988), however, in this study, the power of the test was too 



1096 



Handley 



TABLE 1. 

Analysis of variance for (A) oyster dry meat weights, (B) oyster dry shell weights, (C) oyster dry weight condition index, (D) oyster shell 
volume, (E) oyster volume condition index, (F) oyster gonad/visceral mass condition index, and (G) oyster oocyte area condition index 

December 1995. 



Test of hypotheses for fixed model ANOVA 
Dependent variable: Dry meat weights 
Source DF 

Shell Grade 2 

Sex 1 

ShellGrade*Sex 2 

B 

Test of hypotheses for fixed model ANOVA: 
Dependent variable: Dry shell weights 
Source DF 

Shell Grade 2 

Sex 1 

ShellGrade*Sex 2 



Type 111 SS 
0.138 
0.046 
0.005 



Type 111 SS 
0.023 

0.019 

D.0 1 5 



MS 
0.069 
0.046 
0.002 



MS 
0.011 

0.019 
0.008 



F 
4.89 
3.23 
0.16 



F 

1.11 
1.85 
0.75 



Pr>F 

* 

ns 

ns 



Pr> F 
ns 
ns 
ns 



Test of hypotheses for fixed model ANOVA: 
Dependent variable: Clneshsheii 

Source DF 

ShellGrade 2 

Sex 1 

ShellGrade*Sex 2 

D 

Test of hypotheses for fixed model ANOVA: 
Dependent variable: Shell volume 

Source DF 

ShellGrade 2 

Sex 1 

ShellGrade*Sex 2 



Type 111 SS 
28424.168 
6041.096 
2774.957 



Type III SS 
0.071 
0.045 
0.069 



MS 
14212.084 
6641.0957 
1387.478 



MS 
0.036 
0.045 
0.034 



F 
5.05 
2.36 
0.49 



3.60 

2.72 



Pr>F 



ns 
ns 



Pr > F 
ns 
ns 
ns 



Test of hypotheses for fixed model ANOVA: 
Dependent variable Cln^^^^^ 
Source DF 

ShellGrade 2 

Sex 1 

ShellGrade*Sex 2 



Type 111 SS 
232.112 
42.803 
33.689 



MS 

116.056 
42.S03 
16.844 



F 

3.. 30 
1.22 
0.48 



Pr>F 



ns 
ns 



Test of hypotheses for fixed model ANOVA: 
Dependent vanable: Clj„„^j ,,„ 
Source DF 

ShellGrade 2 

Sex 1 

ShellGrade*Sex 2 



Type 111 SS 
0.002 
0.053 
0.035 



MS 

0.001 
0.053 
0.018 



F 

0.03 
1.38 
0.45 



Pr>F 
ns 
ns 
ns 



Test of hypotheses for mixed model ANOVA: 

Dependent variable: CI;,„y,5 

Source DF 



ShellGrade 

Oyster (ShellGrade) 



2 
21 



Denominator 
Type 111 SS 

0.234 

0.052 



Denominator 
DF 
21 
576 



MS 

0.052 
0.006 



4.. 545 
9.042 



Pr> F 



DF, degrees of freedom; SS, sums of squares; MS. mean squares; F, f statistic; Pr > F; 



*Pr< 0.001. 



*Pr <0.01. *Pr<0.05. 



low to determine whether shell blistering affected relative fecun- 
dity without increasing the sample size. As a loss in condition was 
detected by the other indices, the results of the Clgo^ad area test 
provided a good example of the value of calculating the power of 
statistical tests before acceptitig the null hypothesis, given that the 



four condition indices used on the same oysters in this study pro- 
duced different results with highly variable statistical power, it is 
not surprising there is confusion in the literature as to the etiologi- 
cal status of spionids around the world. 

Despite a measurable decrease in condition detected in this 



Power to the Oyster 



1097 



TABLE 2. 

Analysis of variance. Spearman's correlation coefficients (SCO and power values for oyster dry meat vieights. oyster dry shell vieights, 
oyster dry vieight condition index, oyster shell volume, oyster volume condition index, oyster gonad/visceral mass condition index, and oyster 

oocvte area condition index. December 1995. 



Dependent variable 



Test of hypotheses for fixed model ANOVA and SCC: 

Source: Shell grade 



DF 



Type III SS 



MS 



Pr > F 



"Power" of 




ANOVA 


SCC 


73.3% 


-0.293 


22.8<7c 


-0.088 


75.4% 


-0.390 


25.3% 


-0.371 


57.8% 


-0.228 



Pr> IR 

I 



Dry meat weights 
Dry shell weights 

^Inesh shell 

Shell volume 

^^lleshcv 
^'c.m.^ii area 



2 0.138 

2 0.023 

2 28424.168 

2 0.071 

2 232.113 



0.069 


4.83 


0.011 


1.10 


14212.084 


5.01 


0.036 


2.49 


116.056 


3.36 



Dependent 

variable 



2 0.002 0.001 0.03 ns 

Test of hypotheses for mixed model ANOVA and SCC: 
Source: Shell grade 
Denominator Denominator 

DF Type III SS DF MS F Pr > F 



'Power* of 
ANOVA 



-0.061 



SCC 



ns 



ns 
ns 



Pr > IR 



I 



Ci,. 



0.234 



21 



0.05 1 



4.55 



-0.284 



DF; degrees of freedom. SS; sums of squares, MS; Mean .squares. F; f stalistic. Pr > F; ***Pr < 0.001. **Pr < 0.01, *Pr < 0.05. 



study, the slight loss of condition is unlikely to be biologically 
significant in terms of survival of the oysters in the subtidal envi- 
ronment where ideal condition.s for growth are found. The infested 
oysters seemed capable of easily sustaining the loss of internal 
shell volume, irregular shell shape, and the increased shell depo- 
sition associated with shell blistering. Likewise, in terms of aqua- 
culture production, the impacts of the spionid infestations on oys- 
ter meat production was negligible if one ignores the loss in value 
to the half-shell trade due to the presence of the blisters. In eco- 
logical terms, however, the heavily blistered oysters produced sig- 
nificantly smaller oocytes, thus infestations may retard oocyte de- 
velopment, spawning, or have implications for larval survival. In a 



study of the effects of the Haplosporidium parasite "MSX' on C. 



virginica, "relative fecundity" (CI 



.J was significantly re- 



gonad arc 

duced but no significant differences were detected in the size of the 
mature oocytes (Barber et al. 1988). Stress-induced reductions in 
oocyte size have been measured in the Blue mussel Mytilus ediilis 
and shown to decrease the nutritive reserves, thus significantly 
reducing the viability of the larvae produced (Bayne 1975. Bayne 
et al. 1978). Recruitment was therefore reduced even though the 
total number of gametes did not differ between stressed and non- 
stressed individuals (Newell and Barber 1988). These subtle ef- 
fects of parasitism may be more common than is generally thought 
and may have severe consequences for bivalves if they are stressed 



g -H 

















525 - 




500 - 




I 




475 - 
450 - 










T 












425 - 






400 - 










1 



















1.4 - 




IJ - 








1,2 -, 






I 




















1.1 - 






1,0 - 
















o A 




1 2 

Shell grade 



Shell grade 
Figure 2. Condition index values for oysters for combined sexes; shell blister grades: 0, 1, 2. 



1098 



Handley 



due to adverse environmental change, given tlie intuitively attrac- 
tive concept that stress lowers resistance, rendering individuals 
more prone to parasitism (Newell and Barber 1988). Thus, spio- 
nid-induced impacts may not be trivial for all conditions, for ex- 
ample, if the oysters are in a stressed state or in poor condition 
because of overcrowding, limiting food availability. Shell blister- 
ing under these circumstances could produce commercially and 
biologically significant effects. As gonad development is linked to 
reductions in nutritive reserves of glycogen (Perdue and Enckson 
1984). the state of gonad development would play an important 
role in the response of the oysters to the intrusions of spionid 
polychaetes within their shells. As there are reports of spionid 
infestations causing mortality in oyster populations (Nelson and 
Stauber 1940, Whitelegge 1890. Wisely et al. 1979). further in- 
vestigations are needed into their effects over varying stages of the 
gametogenic cycle and under different environmental stresses. 
These studies could help elucidate the stress thresholds that render 
the oysters susceptible to shell blistering which result from spionid 
infestations. 

Spionid polychaetes do not derive a nutritional benefit from 
their host; rather they are in loose association with their host 
(Rohde 1982. 1993). Parasitism has been defined as "a close as- 
sociation between two organisms, one of which, the parasite, 
depends on the other, the host, deriving some benefit from it" 
(Rohde 1982). Spionids species that have no obvious effect on the 
host can be classified as "latent parasites" which have no obvious 
effects on the host (Rohde 199,^). For example, the Boccurdia 
species found infesting the external shells of intertidal oysters in 



the Mahurangi Harbour had no apparent effect on the oysters 
(Handley and Bergquist 1997). Some researchers have implied 
commensalism by spionids whereby the spionids utilize food sup- 
plied in the external or internal environment of the host (Thomson 
1954). If the association damages the host, then the commensal 
becomes a parasite (Rohde 1982). The results of this, and previous 
studies detecting negative impacts of spionid worms on oysters, 
render them parasites, for example, if they induce the formation of 
shell blisters either during settlement or during the process of 
burrowing within the shell (Odening 1976). Further debate about 
the biological significance of spionid infestations will continue 
until experiments integrating a range of gametogenic conditions 
and environmental stresses are carried out to determine the thresh- 
olds of stress induced by these parasites. Such studies could have 
important practical applications in aquaculture pest management. 

ACKNOWLEDGMENTS 

This study was part of a University of Auckland PhD project 
based at the Cawthron Institute, and later compiled at NIWA Nel- 
son. The research was funded by the Foundation for Research, 
Science, and Technology through a Technology for Business 
Growth secondment. Cawthron, the University of Auckland, and 
published with the support of NIWA. I wish to thank Sanford 
South Island. Havelock. especially Vaughan Ellis and Don Mitch- 
ell; Okiwi Bay Oysters for allowing me to catch spat on their lease; 
Beryl Davy for histological sectioning; and Prof. Dame Patricia 
Bergquist. Drs. Henry Kaspar, Chris Glasby, and Ron Blackwell 
for comments. 



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condition of oysters. Crassostrea virginica. Estuaries 16:229-234. 

Whitelegge, T. 1890. Report on the worm disease affecting the oysters on 
the coast of New South Wales. Rec. Austral. Mus. 1:1-15. 

Widdows. J. 1985. Physiological procedures, pp. 161-172. In: E. Bayne, 
B. L. Brown, D. A. Burns, K. Dixon. D. R. Ivanovici, A. I. Living- 
stone, D. R. Lowe, D. M. Moore, M. N. Stebbing, A. R. D.. & J. Wid- 
dows (eds.). The effects of stress and pollution on marine animals. 
Prager Press, New York. 

Wilson, J., J. Handlinger & C. E. Sumner. 1993. The health status of 
Tasmania's bivalve shellfish. Tech Rep.. Tasm. Mar. Lab. 47:1-63. 

Wisely, B., J. E. Holliday & B. Ried. 1979. Experimental deepwater cul- 
ture of the Sydney rock oyster {Crassostrea commercialis) IV Pilot 
production of raft oysters. Aquaculture 17:77-83. 



Jniiriuil i>l Shfllfish Rcseanh. Vol. 17. No. 4. I lOI-l 114, 1998. 

OYSTER REEF BROODSTOCK ENHANCEMENT IN THE GREAT WICOMICO 

RIVER, VIRGINIA 



MELISSA SOUTHWORTH AND ROGER MANN 

Virginia Inslitiite of Marine Science 
College of William and Mary 
P.O. Bo.x 1346 
Gloucester Point, Virginia 23062 

ABSTRACT The Great Wicomico River is a small, irap-type estuary on the western shore of the Chesapeake Bay that once supported 
substantial oyster populations. These populations were es.sentially eliminated by the combined effects of Tropical Storm Agnes in 1972, 
and subsequent disease mortalities related to Perkinsus marimis and Haplosptiridium nelsoni. Oyster broodstock enhancement was 
initiated in June 1996 by the construction of a three-dimensional intertidal reef with oyster shell, followed by the "seeding," in 
December 1996, of that reef with high densities of large oysters from disease-challenged populations in Pocomoke and Tangier Sound. 
Calculations of estimated fecundity of the reef population suggest that oyster egg production from this source is within an order of 
magnitude of total egg production in the Great Wicomico River prior to Tropical Storm Agnes. Field studies in 1997 indicate spawning 
by reef oysters from July through September. P. marirms prevalence increased from 32% in June to 100% in July, whereas intensity 
increased from June to September; H. nelsoni was absent. Plankton tows recorded oyster larval concentrations as high of 37.362 ± 4.380 
m"' on June 23. Such values are orders of magnitude higher than those typically recorded in Virginia subestuaries of the Chesapeake 
Bay in the past three decades, and lend support to a premise that aggregating large oysters may increa.se fertilization efficiency. Drifter 
studies suggest strong local retention of larvae, a suggestion reinforced by marked increases in local oyster spatfall on both shellstring 
collectors and bottom substrate compared with years prior to 1997. In locations where local circulation promotes larval retention, the 
combination of reef construction with broodstock enhancement may provide an accelerated method for oyster population restoration. 

KFA' WORDS: Oyster. Crassostrea viri^inica. Great Wicomico River, reefs, fecundity, larvae, oyster settlement 



INTRODUCTION 

The Eastern oyster. Crassostrea virgiiiica (Gmeliii). plays an 
important ecological role in the Chesapeake Bay and its tributaries 
as well as being the focus of a substantial commercial fishery. 
Oyster reefs developed in recent geological time as the current 
Chesapeake Bay was inundated by rising sea level. By early Co- 
lonial times, oyster reefs had become significant geological and 
biological features of the Bay. Intensive exploitation since Colo- 
nial times, combined with more recent impacts of two protistan 
parasites. Perkinsus iiuiriiius ( " 'Dermo' " ) and Haplosporidiiim nel- 
soni ("MSX"'), have lead to the degradation of these reefs such 
that only two-dimensional ' "footprints" " of the.se former reefs re- 
main. Today, these '"footprints" maintain drastically reduced oys- 
ter populations. The Virginia Marine Resources Commission 
(VMRC) supports an extensive replenishinent program throughout 
most of its portion of the Bay. Traditional replenishment programs 
have focused on spreading thin veneers of shell substrate for larval 
settlement over coastal and estuarine bottoms. The purpose of this 
practice is to provide a suitable substrate for settlement at mini- 
mum cost. Ideally, the end product is the retrieval of seed or 
market-size oysters from these shell ""plants": however, these 
thin, two-dimensional carpets bear little resemblance to the intri- 
cate, three-dimensional reefs that once supported a large oyster 
population. 

More recent replenishment programs ha\e focused on the con- 
struction of three-dimensional reefs that resemble more closely 
what was found in Colonial times. Since 1993. reefs have been 
constructed in the Piankatank. Great Wicomico. Coan, Yeo- 
comico. and James Rivers in the Virginia portion of the Chesa- 
peake Bay. and Lynnhaven Bay on the southern side of the Bay 
mouth. These reefs are built on the ""footprints" of former reefs 
and consist of several mounds of shell that protrude out of the 
water at low tide (e.g.. Bartol and Mann. 1997). Reef communities 
have been allowed to mature naturally with no addition of brood- 



stock oysters to the reef based on the premise that oysters would 
recruit to the reef from the plankton, and because there was no 
resident population of disease-infected oysters, would develop as a 
predominantly disease-free population. This was not found to be 
the case on an artificial reef built in the Piankatank River. Virginia 
(Mann et al. 1996. Mann and Wesson 1996). Endemic diseases did 
become established in the reef populations; however, the vertical 
relief of the reefs enhanced growth to such an extent that the 
oysters grew larger and faster than on adjacent "flat" oyster reefs. 
From 1993 to 1996 oyster populations on the Piankatank River 
reef developed to densities of 50-70 oysters m~" (Mann and Wes- 
son unpublished data). This compares with densities of 200-350 
m~" on the most commercially productive reefs (flat) in the James 
River system (Mann and Wesson unpublished data. Mann and 
Evans 1998). The disparity in these values suggests that develop- 
ment of very dense and stable oyster communities on constructed 
reefs is a long-term event that is delayed in regions that suffer poor 
natural recruitment, and may be accelerated with an initial stocking 
of broodstock. 

The current study describes the impact on local plankton com- 
munities and oyster settlement of an artificial reef, constructed in 
1996-1997 in the Great Wicomico River (Fig. 1 ) that was initially 
"seeded" with reproductively capable oyster populations. Specifi- 
cally, the study sought to determine temporal spawning patterns of 
'"seeded" oysters, estiinate the fecundity and larval production 
from oysters on the reef and examine subsequent larval abun- 
dance, distribution, and settlement in relation to local circulation 
patterns. 

METHODS AND MATERIALS 

Data Collected as Part of Long-Term Monitoring 

In order to describe the ecological impact of the reef, it is 
necessary to present 1997 data in the context of a brief historical 



1102 



SOUTHWORTH AND MaNN 



description of oyster populations. Original surveys of the limits of 
oyster distribution in the Great Wicomico River were provided by 
Baylor (1896) and subsequently revised by Haven et al. (1981). 
Temporal (intra- and inter-annual) description of oyster settlement 
(spatfall), population density, and demographics are available from 
continuing Virginia Institute of Marine Science (VIMS) stock 
monitoring programs. 

The spatfall survey has been completed annually from 1965 to 
the present. The collectors used to monitor spatfall were oyster- 
shellstrings, which consist of 12 oyster shells of similar size (about 
76 mm, max. dimension) drilled through the center and strung 
(inside of shell down) on heavy gauge wire. Shellstrings were hung 
0.5 m off the bottom at each station. Up to 16 stations have been 
used at various times throughout the history of the spatfall surveys; 
however, for consistency between years, this study reports only the 
six stations (see Fig. I) that have been used yearly since 1965. 
Shellstrings were replaced after a I -week exposure (with occa- 
sional deviations) from June through September, and the number 
of spat that attached to the smooth underside of the middle 10 
shells were counted with the aid of a dissecting microscope. 

The fall dredge survey provides information about spatfall and 
recruitment, summer mortality, and inter-annual changes in abun- 
dance of seed and market-size oysters. This survey has been com- 
pleted yearly from 1971 to the present, excluding 1974-1976. 
Figure 1 shows the geographical locations of the bars sampled in 
the Great Wicomico River during this time. As with the shellstring 
data, only the most consistently sampled stations were used in the 
analysis. Three stations (Fleet Point, Whaley East, and Haynie 
Point) have been sampled since 1986. Analysis was limited to 



these three stations. Three to four 0.5 bushel samples of bottom 
material were taken at each bar using a 24-inch dredge having 
4-inch teeth. For each sample the following were determined: 
number of market-size oysters (>76 mm, max. dimension), number 
of small oysters (below market size and yearlings), and the number 
of spat (young of the year oysters). In the ca.se where only 0.5 
bushels were counted, they were standardized to one bushel by 
doubling the counts. In the fall of 1995 and 1997, a collaborative 
survey effort between VMRC and VIMS resulted in a formal stock 
assessment on the oysters in the Great Wicomico River using 
patent tongs (Chai et al. 1992). The five oyster reefs that were 
sampled in the Great Wicomico River in 1995 and 1997 are shown 
in Figure 1. For each reef a uniform grid was generated over a 
current reef boundary map. Each grid location had a reference that 
could be located electronically by LORAN from the research ves- 
sel. Grid references were assigned a sampling order from a random 
number table to generate a randomized sampling grid. Samples 
were collected using hydraulic patent tongs with an opening of I 
m". All of the retained material was washed, and counts of live 
oysters as spat (young of the year), small oysters (<76 mm, max. 
dimension), and market oysters (>76 mm, max. dimension) were 
taken. Adequacy of sampling was assured using guidelines of Bros 
and Cowell ( 1987), as described in Mann and Evans ( 1998). 

Estimation of Historical Egg Production, Fertilization and 
Embryo Production 

To place in context the impact of adding broodstock, estimates 

of historical egg production when adult oysters were still abundant 



54'- 



37- 
50'" 



46'- 










l^' 



"■^^.^^"^^.n'^p^ 



Chesapeake Bay 



10 1 Km. 



1 

Figure 1. Map of the Great Wicomico River showing the locations of various stations used throughout this study (1-12) and the location of the 
artificial reef (R). Shellstring stations are represented by sites 1, i. 6-7, 9, and 11, fall dredge stations are represented by sites 6, 7, and 9, patent 
long stations are represented by sites 4-5, 7, and 1(1-11. Egg production was calculated using the area of shell bottom covering sites 2-12, Inset 
shows the location of the (Jreat Wicomico River within the Chesapeaite Bay system. The numerical key identifying stations corresponds to that 
used throughout the current text. 1: Glebe Point, 2: Rogue Point, 3: HudnalPs, 4: Shell Bar, 5: Sandy Point, 6: Haynie Point, 7: Cranes Creek, 
8: Whaley West, 9: Whaley East, 10: Dameron Marsh/Ingram, 11: Fleet Point. 12: Cockrell Creek. 



Oyster Reef Enhancement 



103 



in the river are required. Quantitative historical stock assessment 
data for the Great Wicomico are lacking, but there are current data 
for extant reefs in the James River in similar salinity regimes 
which have similar qualitative (based on dredge sur\ey data) popu- 
lation demographics. A combination of revised Baylor survey data 
(Haven etal. 1981) of reef area in the Great Wicomico (Fig. l)and 
current James River quantitative stock assessment data (Mann and 
Wesson unpublished. Mann and Evans 1998) were used to esti- 
mate historical oyster demographics in the Great Wicomico (see 
Table 1). Since salinity plays a role in reproductive success, it was 
necessary for the reefs being compared to have similar salinity 
regimes. Reefs in three salinity regimes (8.5. 10.5. and 1.3.5 ppt. 
see Mann and Evans 1998) in the James River were used in the 
calculation. Egg production per unit area for each reef in the James 
River, based on the appropriate size frequency distribution, was 
estimated by methods described in detail in Mann and Evans 
(1998) using size-specific fecundity taken from Thompson et al. 
( 1996). panty in sex ratio as suggested by Cox and Mann ( 1992). 
and density-dependent fertilization efficiency as described by 
Levitan (1991). 

1997 Field Studies 

The location selected for the study was Shell Bar Reef in the 
Great Wicomico River. Virginia (location R in Fig. 1). The reef 
was constructed in June of 1996 by deploying old oyster shells 
from a barge with a crane into a series of intertidal structures 
approximately 215 m long and 18 m wide. Broodstock oysters 
from the Tangier and Pocomoke Sound regions were planted on 
the reef in December of 1996. Oyster standing stock and density 
was obtained from VMRC records (Olsen and Wesson 1997). 
According to these records. 2.281 bushels of oysters were planted 
on the 3,900 m" reef in December of 1996. Estimating 500 oysters 
per bushel (Wesson, personal communication), density of brood- 
stock oysters on the reef was approximately 300 m"". Oysters 
surviving as .sparsely distributed individuals in many regions of the 



Bay are continually exposed to intense disease challenge and se- 
lection pressure. Consequently, they would be expected to have 
higher resistance to disease than low salinity populations where 
intermittent disease pressure fails to eradicate genetically suscep- 
tible individuals, which then continue to breed with more resistant 
individuals and thus fail to promote the process of developing 
uniformly high resistance. Tangier and Pocomoke Sounds are lo- 
cations where higher salinities (25-30 ppf) occur, but oyster den- 
sities are low (<1 m~-). thus failing to maximize the fertilization 
efficiency. The intent of aggregating the few remaining oysters 
from disease endemic areas was to increase fertilization efficiency 
of freely released gametes. 

Field studies were conducted biweekly from the 23rd of June 
through the 22nd of September 1997 (total of 8 field days). This 
time frame was chosen based on the historical timing of spat 
settlement in the Great Wicomico River system. To obtain a de- 
scription of tidal patterns of circulation and larval abundance in the 
system, all sampling was effected over one complete tidal cycle 
(approximately 12 h). Surface temperature for all field studies was 
measured near the reef (Station Nl; in Fig. 2) throughout the 
duration of the study. Temperature and salinity at the surface and 
bottom of the water column were obtained at three sites (Fig. 2) 
starting on July 28th (dates of collection coincided with the cir- 
culation study). Bottom water was collected using a Niskin bottle. 
Temperature was measured with an alcohol thermometer and sa- 
linity was measured with a refractometer. 

Oyster Reproductive Biology and Disease Status 

Initial broodstock oyster size frequency on the reef was ob- 
tained by measuring 150 oysters collected with the aid of hand 
tongs. Total egg production on Shell Bar reef, after broodstock 
enhancement, was calculated using density and size frequency 
data, as described in detail in Mann and Evans (1998). 

Temporal patterns of gametogenic development of the brood- 
stock oysters on the reef was examined by collection, with hand 



T.4BLE 1. 

Estimates of historical egg production in the Great Wicomico River using demographics obtained from analogous reefs in the James River 

in 1993. 







Egg 


Total # of 


Salinity to 






Corrected 


Corrected 




Reef Area 


Production 


Eggs 


Estimate 






Production 


Total # of Eggs 


Reef# 


m* 


10' m - 


» 10'= 


Fs 


Fs 


Ff 


10' m-- 


* 10'= 


1 


29.VS.'i 


1160 


34 


8.5 


0.09 


II 16 


16,8 


0.49 


2 


23475 


565 


13 


10.8 


0.51 


0.13 


38.5 


0.9 


3 


3636 


565 


2 


10.8 


0.51 


0.13 


38.5 


0.14 


4 


29462 


565 


17 


10.8 


0.51 


0.13 


38.5 


1.1 


5 


33261 


565 


19 


10.8 


0.51 


0,13 


38.5 


1.3 


6 


83452 


136.2 


11 


13.5 




0.04 


4,9 


0.41 


7 


137735 


136.2 


19 


13.5 




0,04 


4.9 


0.68 


8 


251573 


136.2 


34 


13.5 




0,04 


4.9 


1.2 


9 


82723 


136.2 


11 


13.5 




0.04 


4.9 


0.41 


10 


22047 


L%.2 


3 


13.5 




0.04 


4,9 


0.11 


II 


71929 


1.36.2 


10 


13.5 




0.04 


4.9 


0.35 


TOT.AL 






173 










7,1 



See Mann and Evans ( 1998) for explanation of calculations. 

Reef area — taken from tlie Great Wicomico Baylor survey data (Haven et al.. 1978). 

Egg production — calculated from size-specific fecundity of oysters in the James River (average of all reefs in the James River with the same salinity). 

Total # of eggs — reef area in the Great Wicomico multiplied hy egg production (based on James River demographics). 

Fs — correction for salinity, based on reefs sharing similar salinities in both rivers. 

Ff — correction for fertilization efficiency, based on densities found in the James River (also used for egg production calculations). 

Corrected production — egg production/m" corrected for salinity, fertilization efficiency, and disease (production * Fd * Fs * FO- 

Corrected total # of eggs — corrected egg production multiplied by reef area (in the Great Wicomico). 



1104 



SOUTHWORTH AND MaNN 




Figure 2. Location of zouplankton (GVV 1-3) samples and water samples (N 1-3) taken in the (Ireat Wicomico River. R denotes the location of 
the reef; 9 & 10 mark the main channel in the river. 



tongs, of 25 oysters per sampling day for a total of 200 oysters. 
Sections of the gonad and visceral mass were removed and fixed 
in Bouin's solution. Following fixation, specimens were dehy- 
drated in alcohol, cleared in xylene, and embedded in paraffin wax. 
Histological sections were cut at 7-10 (j,m. stained m Delafield's 
hematoxylin, and counterstained in eosin Y following the meth- 
odology of Humason (1962). Developmental stages were identi- 
fied based on those originally described for C. virgiiiica by 
Kennedy and Battle (1964) and for C. gigas by Mann (1979). 
Stages of gonadal development were defined as follows; 

( 1 ) Inactive: No evidence of the presence of follicles periph- 
eral to the digestive gland. Sex is essentially indetermin- 
able. 

(2) Early active: Male. Many follicles filled primarily with 
spermatogonia and spermatocytes. No spermatozoa. Fe- 
male. Eggs not well developed. A few nuclei in oocytes, 
but no nucleoli. Oocytes are still attached to the follicle 
wall. 

(3) Late active: Male. Follicles predominately filled with sper- 
matids. Characteristic swirling pattern of spermatozoa with 
tails oriented toward the center beginning to be evident, but 
follicle is not completely filled. Female. Some free oo- 
cytes. Most have distinct nuclei, with fewer than 509c hav- 
ing distinct nucleoli. 

(4) Rii^e: Male. Swirling of tails in the middle of the follicle. 
Female. Primarily free oocytes. Greater than SOVr have a 
distinct nuclei and nucleoli. All of the oocytes are about the 
same size. 

(3) Spaw)ting or spent: Male. Most follicles are empty or par- 
tially so. Some phagocytes present. Female. Granular look- 
ing eggs (ameobocyte activity). Eggs of varying sizes that 
appear to be breaking down. Follicles are empty or partial- 
ly so. 

Monthly assays to determine Perkin.siis iiiciriinis and Haplospo- 



ridium nelsoni (MSX) infections were effected using oysters col- 
lected for the reproductive development portion of the .study. Per- 
kinsus infection and prevalence were measured by Fluid Thiogly- 
collate assay (Ray 1963). MSX infections were detected using 
paraffin histology, as in Burreson et al. (1988). 

Plankton Studies 

A series of 36 zooplankton samples were taken on each sam- 
pling day (three replicates per site, per tidal stage). Samples were 
collected at three stations in the river (Fig. 2). Plankton samples at 
GW-1 describe larval abundance near the reef. GW-2 describes 
abundance in the main channel of the river, and GW-3 describes 
abundance near the sand spit at Sandy Point, a feature that affects 
and effects some local retention in the system. Samples were col- 
lected using a 0.3 m diameter. 3: 1 aspect ratio zooplankton net 
(Sea Gear Corporation. Melbourne, FL). The filtering surface con- 
sisted of an 80 fxm Nytex mesh cone attached to a PVC collection 
bucket lined with 80 )j.m mesh. The net was attached to a metal 
ring and towed by a three-point bridal system attached to the ring. 
The net was towed 0.05-0.10 ni below the water surface at ap- 
proximately 1.5 m sec"' for 3.25 min. The nets used were cali- 
brated in a separate study following the same protocol (Harding 
and Mann, in review). Samples were taken over a full tidal cycle. 
All samples were immediately preserved in 95% ethanol. 

Samples were split using a 0.5 L Folsom plankton splitter 
(Wilco Supply Company, Cass, MI). Final splits were filtered 
through a 400 |j.m Nytex mesh filter to remove large zooplankton 
that interfered with the counting. To ensure that no oyster larvae 
were lost in this process, samples were randomly chosen and 
counts were made before and after filtering. The difference be- 
tween these counts was less than 1%. Non-enumerated splits, as 
well as the filtrate from the final splits, were archived. Counts of 
umbo stage oyster veligers (larvae) in each subsample were made 



Oyster Reef Enhancement 



1105 



v\itli the aid of a dissecting scope. To verify adequate mixing (i.e., 
a homogenous mixture of larvae within the sample), both halves of 
the final split were counted, and coefficients of variation (CV) 
were calculated following Van Guelpin et al. (l')82). Acceptable 
CVs for invertebrate samples range from 5 to 20%. Counting error 
of the total abundance of organisms within a sample was kept to 
\07c or less by ensuring (when possible) that at lea.st 100 veligers 
were counted from each subsample. Total number of larvae per 
sample was obtained by multiplying the number of veligers in the 
.split by the split number. The number of larvae per m' was then 
obtained by dividing the total number per sample by the volume of 
water filtered. The mean volume of water filtered, was determined 
to be 1.054 m"" in a separate net calibration study (Harding and 
Mann, in review). 

Circulatiuii Studies 

Simple surface drogues (drifters) were constructed after the 
method of Davis et al. (1982) (Fig. 3). This design was used to 
ensure that the drifter was moved by the currents in the system 
with little input froiu the wind. The drifters were released at vari- 
ous sites around the reef and in the main channel of the river. The 
drifter locations were recorded approximately every hour using a 
hand held GPS system. The drifters were followed over one full 




Figure 3. Design of surface drogue (drifter) used in the circulation 
studies. 



tidal cycle. In the event that a drifter ran aground, it was reposi- 
tioned to another location, with exact location depending on the 
stage of the tide. Throughout the course of the sampling season, a 
total of 23 drifter paths were obtained on 5 separate days. Of the 
23 drifter tracks obtained, 6 were discarded because of multiple 
lost GPS points and 3 were discarded because of excessive stop- 
page (they ran aground at least three times). This left a total of 14 
drifter tracks to be analyzed. Drifter time and location information 
was loaded into the Geographical Information Systeni/ArcView 
computer program in the Coastal Inventory Program at VIMS. The 
drifter paths were then plotted in ArcView software and mean 
current speeds were estimated for each series of drifter recordings. 
These were then compared with predicted tidal flow for Sandy 
Point in the Great Wicomico River system (Tides and Currents for 
Windows, version 2.2. Nautical Software Inc.). 

RESULTS 

Data Collected as Part of Long- Term Monitoring 

Spatfall estimates obtained from the historical shellstring data 
are summarized in Table 2. From 1965 through 1971 spatfall in the 
Great Wicomico River was relatively high with mean weekly val- 
ues (number of larval oysters physically adhering to the substrate) 
ranging from 1 to 494 spat/shellstring per week. In 1970 nearly all 
stations received a moderate (20-50 spat/shellstring) to heavy (>50 
spat/shellstring) peak value and the settlement period extended 
over most of the summer-fall .season. In 1971. no significant 
settlement occurred until late fall. In 1972. due to Tropical Storm 
Agnes, oyster settlement was at or near zero at all stations. This 
year marked the beginning of a major decline in spatfall in the 
river. The years 1973 through 1979 were characterized by a very 
light settlement, usually less than one spat/shellstring per week. 
Starting in 1980. settlement events again became more consistent 
(lasting throughout most of the season) and heavier (2-38 spat/ 
shellstring per week). This increase in the number of spat in the 
1980s coincided with a heavy private "planting" of a large num- 
ber of small (seed) oysters on private lease grounds in the Great 
Wicomico; however, as these were harvested in the late 1980s and 
early 1990s, a further decline in the number of .spat was observed. 
The latest signal in the river occurred during the 1997 setting 
season, after the artificial reef was built and stocked with brood- 
stock oysters. In 1997, spatfall was recorded between the end of 
June and the beginning of September, with a peak set occurring in 
mid- to late July. During this peak settlement period, spatfall 
ranged from to 29.3 spat/shellstring per week, with the most 
intense sets occurring upstream or immediately adjacent to the reef 
(Glebe Point. Hudnall. Haynie Point, and on the reef itself; Fig. I). 
Mean spatfall estimates for 1997 from shellstring data ranged from 
1.4 to 43 spat/shellstring per week. 

The fall dredge data taken from the VIMS databa.se (VIMS 
archive) can be summari/ed as follows (Table 3): Between the 
years 1971 to 1987. the number of small oysters ranged from 90 to 
over 600 per bushel for all three stations (Fig. I ). During this time 
the number of spat per bushel ranged from a low of in 1972 (the 
year of Hunicane Agnes) to a high of 1.900 per bushel in 1987. 
This year marked the beginning of a slow decrease in the number 
of oy.sters in the system. For the past 3 ys. numbers of small 
oysters have ranged from 31 to 126 per bushel. 1987 also marked 
the beginning of essentially the absence of market-size oysters in 
the system. Before 1987 there were comparatively more market 
oysters (0-128) per bushel than after (0-22 oysters per bushel). 



1106 



SOUTHWORTH AND MANN 



TABLE 2. 
Spatfall survey data from shellstrings reported as average spat/shellstring per week. 





Dameron 


Fleet Point 


Cranes Creek 


Haynie Point 


Hudnall 


Glebe Point 


Year 


Marsh (10) 


(11) 


(7) 


(6) 


(3) 


(1) 


1965 


75.0 


NS 


524.0 


253.0 


300.0 


98.0 


1966 


NS 


NS 


NS 


NS 


155.0 


127.0 


1967 


8.5 


NS 


20.0 


61.0 


86.0 


174,0 


1968 


49.0 


NS 


257.0 


61.0 


227.0 


182.0 


1969 


8.7 


24,0 


45.0 


112.0 


89.0 


36.0 


1970 


36.0 


21.0 


100.0 


306.0 


302.0 


494,0 


1971 


2.6 


0.9 


2.4 


4.4 


9.6 


24,0 


1972 


0.0 


0.0 


0.0 


0.0 


0.2 


1,8 


1973 


2.2 


1.7 


0.5 


1.9 


0.1 


0.7 


1974 


O.I 


4.1 


0.8 


I.O 


1.9 


7.3 


1975 


O.I 


1.0 


0.4 


1.1 


0.1 


0.2 


1976 


O.I 


0.0 


0.1 


0.1 


0.6 


0.8 


1977 


0.6 


1.4 


1.6 


0.8 


2.7 


0.8 


1978 


0.3 


1.7 


0.1 


0.7 


0.9 


1.4 


1979 


0.6 


0.9 


0.1 


1.3 


8.3 


4.1 


1980 


9.1 


7.8 


9.9 


8.5 


38.0 


7.0 


1981 


2.3 


1.5 


3.8 


17.3 


21.0 


83.0 


1982 


23.0 


36.0 


46.0 


54.0 


89.0 


228.0 


1983 


6.8 


23.0 


3.8 


6.9 


8.6 


0.4 


1984 


0.5 


1.0 


0.7 


0.4 


1.9 


1.3 


1985 


5.4 


49.0 


4.5 


4.7 


8.9 


6.8 


1986 


25.0 


24.0 


71.0 


113.0 


139.0 


227.0 


1987 


17.0 


110.0 


17.0 


7.6 


26.0 


13,0 


1988 


22.0 


5.1 


8.6 


24.0 


32.0 


16,0 


1989 


3.2 


4.9 


5.2 


1 0.0 


16.0 


4,8 


1990 


18.0 


1 0.0 


23.0 


43.0 


59.0 


12,0 


1991 


8.5 


5.2 


6.6 


10.0 


4.0 


2,3 


1992 


0.5 


4.2 


0.2 


0.8 


0.8 


0.8 


1993 


0.6 


1.7 


0.1 


1.2 


0.7 


0.2 


1994 


0.0 


0.0 


0.0 


0.0 


0.0 


0.0 


1995 


0.0 


1.3 


0.2 


0.3 


0.1 


0.9 


1996 


2.9 


2.9 


2.3 


4.1 


0.2 


0,7 


1997 


1.9 


4.4 


1.4 


6.1 


43.0 


24.0 



Numbers in parenthesis correspond with the station IDs in Figure 1. NS means no samples were taken at that site during the corresponding year. 



The number of spat recorded per bushel also started to decrease in 
the late 1980s. For the 5 years prior to the building of the reef. 
mean spatfall values were about 55 spat per bushel, whereas a 
mean of 155 spat per bushel was recorded for the 1997 fall survey. 
Patent tong surveys revealed that in 1995 the number of market 
oysters ranged from 0,3 m"" at Fleet Point to 1.6 m"" at Sandy 
Point (see Fig. 1 for location of patent tong sites and Table 4 for 
summary of survey data). The number of small oysters ranged 
from 4 to 22 oysters m~" with the lowest densities at Shell Bar and 
Fleet Point and the highest at Sandy Point, The number of spat m"" 
ranged from 6,5 at Cranes Creek to 13,4 at Fleet Point, The overall 
density for the five reefs combined was 0,7 market oysters m"'^, 
10,2, small oysters ra"~, and 9,7 spat m"-. Whereas the mean 
number of market (1-3 m"") and small (9-37 m~") oysters re- 
corded in 1997 were similar to those recorded in 1995. the number 
of spat were considerably higher in 1997, Spatfall estimates ranged 
from 4,6 m"" at Ingram and Fleet Point to 103 m"~ at Shell Bar, 
The spatial pattern retlected that observed in shellstring studies 
with intense settlement upstreani of the sand spit, and near or 
adjacent to the artificial oyster reef, and a trend of decreasing 
settlement in a downstream diiection. Overall values represent a 
threefold increase in density of spat from 1995 to 1997, 



Collectively, these survey data can be summarized as follows: 
Oysters were present in relative abundance in the Great Wicomico 
River until about 1971, The combined effects of Hurricane Agnes 
in 1971 and disease decimated the natural broodstock population 
in the system. This in turn led to a decrease in larval production 
and spat recruitment. For a brief time during the 1980s, oysters 
appeared to be returning to the Great Wicomico, but this was 
probably related to a large private planting of seed oy.sters that 
grew and served as broodstock for the system. Once these oysters 
were harvested, recruitment once again plummeted. With the 
building of Shell Bar reef and the addition of broodstock on the 
reef, recruitment once again showed an increase from previous 
years. 

Fertilized Egg Production Estimates 

Historical production of fertilized eggs in the Great Wicomico 
River, after correcting for disease, salinity, and fertilization effi- 
ciency, is estimated at 7,1 x 10'~, Comparable estimates for fer- 
tilized egg production on Shell Bar reef were 5,4 x 10'" embryos 
using size frequency distribution data from Figure 4, a salinity 
value of 10,8 obtained from field observations, and a fertilization 



Oyster Reef Enhancement 



1107 



TABLE 3. 
Dredge survey data reported as number of market (>76 mm), small l<76 mm), and spat per hushel. 







Fleet Point |7| 






\Mialey East (9) 






Haynie Point (6) 




Year 


Market 


Small 


Spat 


Market 


Small 


Spat 


Market 


Small 


Spat 


1971 


NS 


NS 


NS 


NS 


NS 


NS 


0,0 


648,0 


68.0 


1972 


NS 


NS 


NS 


NS 


NS 


NS 


NS 


NS 


NS 


1973 


128.0 


260.0 


0.0 


NS 


NS 


NS 


64.0 


246.0 


2.0 


1977 


60.0 


88.0 


82.0 


48.0 


182.0 


24.0 


38.0 


112.0 


156.0 


1978 


88.0 


l.'iZ.O 


10.0 


58.0 


138.0 


46.0 


58,0 


214.0 


44.0 


1979 


32.0 


l-^S-O 


430,0 


NS 


NS 


NS 


32.0 


88.0 


220.0 


1980 


80.0 


344.0 


44S.0 


72.0 


368.0 


72.0 


64.0 


180.0 


98.0 


1981 


82.0 


502.0 


286.0 


116.0 


544.0 


306.0 


44,0 


356.0 


442.0 


1982 


30.0 


414.0 


1198.0 


36.0 


394.0 


432.0 


34,0 


292.0 


818.0 


1983 


28.0 


476.0 


124.0 


32.0 


188.0 


74.0 


10,0 


208.0 


78.0 


1984 


32.0 


544.0 


22.0 


24.0 


546.0 


24.0 


40.0 


178.0 


30.0 


1985 


76.0 


366.0 


1436.0 


126.0 


350.0 


566.0 


36.0 


584.0 


536.0 


1986 


9.5 


154.0 


1 1 14.0 


14.0 


212.0 


504.0 


15.0 


504.0 


638.0 


1987 


0.0 


107.0 


1911.0 


4.7 


281.0 


337.0 


0.7 


271.0 


501.0 


1988 


0.0 


145.0 


134.0 


0.0 


3.0 


179.0 


1.3 


228.0 


467.0 


1989 


4.0 


207.0 


300.0 


0.0 


174.0 


151.0 


1.3 


225.0 


182.0 


1990 


11.0 


297.0 


473.0 


0.7 


275.0 


44.0 


0.7 


141.0 


397.0 


1991 


9.3 


317.0 


217.0 


0.7 


229.0 


147.0 


6.0 


176.0 


328.0 


1992 


0.0 


33.0 


51.0 


0.0 


18.0 


45.0 


0.0 


21.0 


228.0 


1993 


2.7 


67.0 


62.0 


2.0 


189.0 


47,0 


0,0 


91.0 


147.0 


1994 


5.3 


150.0 


7.0 


2.5 


114.0 


6.0 


0.7 


153.0 


7.0 


1995 


5.3 


31.0 


51.0 


2.5 


48.0 


3.0 


1.0 


31.0 


113.0 


1996 


6.0 


123.0 


13.0 


8.0 


73.0 


21,0 


4,5 


126.0 


19.0 


1997 


ISd 


M6 


47.0 


22 


710 


ll)7() 


UK) 


115.0 


3120 


Numbers 


in parenthesis 


correspond with : 


station IDs in 


Figure one. NS 


means no samples were 


taken at that 


site during 


the corresponding year. 





efficiency of 29.8% based on Levitan et al's (1991) estimator. 
These calculations strongly suggest that by aggregating the brood- 
stock oysters into very dense populations, fertilization efficiency is 
greatly improved, and production of larvae on the reef is similar to 
that of the entire Great Wicomico system in predisease conditions. 

1997 Field Studies 

Surface temperature at the reef (station Nl; Fig. 2) reached a 
maximum of 29.5°C on July 28th (Fig. 5). The difference between 
the surface and bottom temperature increased in a down river 
direction (station Nl to N3) away from the reef (Fig. 6). The 
maximum temperature difference occurred on July 28th for all 
three stations. As with the temperature, the difference in salinity 
between the surface and bottom water increased downstream (from 
Nl to N3; Fig. 6). Salinity at the three stations ranged from 12 to 
18 ppt. The maximum difference encountered between surface and 
bottom samples was 3 ppt at station N2 and N3. 



Oyster Reproductive Biology and Disease Status 

At the beginning of the study, both males and females were 
either in the late active or ripe stage of development (Fig. 7). 
Evidence of spawning (spent specimens) were first seen in the July 
14th samples (Fig. 7). Most of the specimen sampled were com- 
pletely spawned out by early September, with a large majority of 
them returning to the inactive stage by the end of September. 

MSX was absent in all of the oysters examined. Perkinsus 
prevalence increased from 32% in June to 100% in July and con- 
tinued at that level for the remainder of the study (Fig. 8). Intensity 
of Perkinsus infection increased from June to September, with the 
highest percentage of highly infected oysters occurring toward the 
end of the study. 

Plankton Studies 

The number of oyster larvae observed in plankton samples 
ranged from a high of 37,362 ± 4,380 m"' on June 23rd at station 



TABLE 4. 
Patent long survey data for 1995 and 1997 reported at number of market (>76 mm), small (<76 mm), and spat per square meter. 



Station 



Market 



1995 



Small 



Spat 



Market 



1997 



Small 



Spat 



Ingram ( lOl 
Fleet Point (II) 
Cranes Creek (7) 
Sandy Point (5) 
Shell Bar (4l 



0.4 
0.3 
0.8 

1.6 

0.1 



7.0 


8.3 


4.4 


13.0 


12.0 


6.5 


22.0 


10.0 


4,1 


11.0 



1.2 
3.0 
1.9 
0.9 

1.6 



8.8 


4.6 


9.8 


5.8 


14.0 


15.0 


37.0 


62.0 


27.0 


103.0 



Numbers in parenthesis correspond to the station IDs in Figure one. 



1108 



SOUTHWORTH AND MANN 




62.5 67.5 72.5 77.5 82.5 87.5 92.5 97.5 102.5 107.5 112.5 117.5 122.5 



Size Distribution (midpoint in mm) 

Figure 4. Size frequency distribution of broodstock oysters on Shell 
Bar reef (n = 150). 



GW 2 to a low of M all stations on several different sampling 
days. Larvae were most abundant at all stations on the 23rd and 
30th of June, and on the 14th of July (Fig. 9). From the 14th of July 
onward, there was a continuous decrease in the number of larvae 
seen in the water column. Coefficient of variation for most samples 
was within the accepted limits of between 5 and 20% (Van 
Guelpin et al. 1982). Higher CVs were observed when larval abun- 
dance was below 10 m"\ 

The total number of larvae m"^ was transformed to meet the 
assumptions of normality and homogeneity of variance. Differ- 
ences in larval abundance between tidal stage and station were 



U 



a, 
E 




6/30 



7/28 



8/25 



9/22 



Date 

Figure 5. Surface temperature measured at station Nl from .lune 23 to 
September 22, 1997. 



30 
29 
28 
27 
26 



— •— Surface T 
••••o- Bottom T 



-"=■— Surface S 
-* Bottom S 



- 


\ 






.A 




■■.. 


. \ 


"■■■ jT' 


>'^^^ 






S. 




- 


- 


^ 


^ 


Station Nl 


--^ 



20 
18 
16 
14 
12 
10 



u 
B 




28 
27 
26 

25 
24 







A. 






• — 


\ 




A*" 


_..A 


e> 


<3:- 


■ "V-G. 




" 


- 






Q^ 


-^ 


A 


-V 


Station N3 




'd 



18 
16 
14 
12 
10 



7/28 



8/11 



8/25 
Date 



9/8 



9/22 



Figure 6. Surface and bottom temperature and salinity for all three 
stations (N1-N3) measured from July 28 to September 22, 1997. 

then compared with analysis of covariance (ANCOVA) using day 
of the year as the covariate. The power transformation (X' = 
X" -"), recommended by Downing et al. (1987) for use in estimat- 
ing zooplankton populations, was used. The use of this transfor- 
mation met the assumptions of homogeneity of variance, but did 
not meet the assumptions of normality. Given that ANCOVAs are 
generally robust to non-normality (Underwood 1997). this trans- 
formation was considered valid and the resulting data were utilized 
in performing the ANCOVA. 

There was a significant difference in larval concentration be- 
tween tidal stages (p < .01) and stations (p < .05). with no inter- 
action between the two factors (p = .55). Student Newman Keuls 
(SNK) multiple comparison test for station effect showed that 
there were significantly more larvae at GW- 1 than at the other two 
stations (Table 5A). There was no difference in larval abundance 
between GW 2 and GW 3. The SNK for tidal stage showed there 
were significantly more larvae during the flood tidal stage than 
during the ebb or slack onto flood stages. (Table 5B). No differ- 
ences were found between any of the other tidal stages. 

Circulation Studies 

The direction, mean velocity, and distance traveled by the drift- 
ers on each sampling day, for each fix during the day, was re- 



Oyster Reef Enhancement 



1109 



6/23 6/30' 7/14 I 7/28 8/11 8/25 9/8 9/22 



50 



50 



o 

c 
o 
O 



e^ 100 




M EARLY ACTIVE 
n LATE ACTIVE 



RIPE 
SPENT 



Figure 7. Seasonal changes in gonadal development by sex in Crassos- 
trea virginica oysters collected on the reef from June 23 to September 
22, 1997. Number of male and female oysters sampled on each day are 
represented by the numbers above and beloH each bar. 



corded and/or estimated. Tidal cycle was recorded as the stage or 
stages of the tide occurring between a particular observation and 
the previous observation. For example, if the tide was ebbing for 
the first half of a drifter deployment, then changing to slack water 
for the second half, it was recorded as E-S. If the tide was flooding 
for the entire deployment, then tidal stage was recorded as F. Mean 



<^ 



•a 
o 

c 



5) 



Cm 

o 




Date 

Figure 8. Progression of Perkinsiis infections in broodstock oysters 
over the 1997 reproductive season (n = 25). 



> 



o 

E 

3 
C 

O 



-^— GWl 
■•••- GW 2 
•fl--GW3 




6/23 6/30 7/14 7/28 8/11 8/25 9/8 

Date 
Figure 9. Log number of larvae m" ' averaged over each sampling day 
(averaged over all four stages of the tide). 

velocities recorded by the drifters ranged between and 15.9 
cm/sec. Maximum predicted tidal cunent was between 10 and 20 
cm/sec on all sampling days. 

From the 14 drifter tracks analyzed, four patterns of movement 
were observed (Fig. lOA-D). Four of the 14 drifters followed the 
predicted tidal current, traveling downstream during the ebb tide, 
remaining approximately in the same place during slack water, 
then traveling upstream with the flooding tide. One drifter out of 
those four remained in the channel and drifted toward marker 9 
before turning with the tide (Fig. lOA). The other 3 drifters re- 
mained in the channel, further upstream from Sandy Point (Fig. 
lOB provides an example). 

Ten of the 14 drifters analyzed did not follow the predicted tide. 
Of these 10. 7 traveled downstream with the predicted tide, but 
turned westward, away from the channel, several hours before the 
predicted tide turned (see Fig. IOC for example). The other three 
drifters traveled downstream until they arrived in the shallow re- 
gion west of Sandy Point. Despite being repositioned, they essen- 
tially remained in the same area even during a flood tide (e.g., see 
Fig. lOD). These patterns of movement illustrated in Figures IOC 
and lOD indicate that some local retention of larvae is occurring 
upstream of Sandy Point. 

TABLE 5. 
SNK multiple comparisons test on larval abundance (p = .05). 

A. Ranking of Larval Abundance by Station 



Station 



High 
GW-1 



GW-: 



Low 
GW-3 





B. 


Ranking of Larval Abundance 


by Tidal Stage 


Tidal 


stage 


High 
Flood 


Slack-ehb 


Ebb 


Low 
Slack-Flood 











Stations/tidal stages underlined by the same line are statistically the same. 



1110 



SOUTHWORTH AND MANN 




Figure 10. Representative examples of the four patterns of drifter traclis (A-D) observed over the course of the study. Inset shows predicted tidal 
currents for Sandy Point, with arrows representing approximate times of deployment (in) and retrieval (out) of the drifters. Time is reported 
in Eastern Standard (E.S.) Military time. R denotes the location of the reef and 9 and 10 mark the main channel in the river. 



DISCUSSION 

Egg Production 

Egg production estimates for broodstock oysters planted on the 
reef were found to be similar to estimates of production seen 
throughout the entire system in historic times (when oysters were 
abundant in the river). Though the estimates are similar they 
should be viewed with caution because of our inability to offer 
better values for disease- and salinity-related modifiers of fecun- 
dity. These are both discussed in Mann and Evans ( 1998) and are 



widely acknowledged in the literature as having major effects on 
the bioenergetics of oysters, and yet they are still poorly described 
in a quantitative sense. 

The model used in the calculation of fertilization efficiency 
taken from Levitan's (1991) work on echinodenns, involves a 
series of assumptions concerning synchrony and completeness of 
spawning, half-life of gametes in the water column, dispersal or 
dilution, and probability of fertilization given absolute concentra- 
tions of sperm and eggs. There is a notable absence of models in 
the literature describing fertilization efficiency for sessile bivalves. 
The employment of the Levitan model in the current application 



Oyster Reef Enhancement 



nil 




Figure 10. Continued. 



was deemed reasonable based on the similarities in small scale 
hydrodynamic conditions seen in both Levitan's model and estua- 
rine oyster reefs. Other options for models are discussed by Levi- 
tan et al. (1991), Oliver and Babcock (1992), and Benzie et al. 
(1994). Based on the hydrodynamics of the Great Wicomico, con- 
trasting models such as the one for high-energy environinents seen 
in Denny and Shibata ( 1989) are inappropriate for u.se in the Great 
Wicomico system. The current model assumes synchrony in 
spawning throughout the entire oyster population; however, local 
synchrony is more appropriate when discussing these populations. 
The lack of synchrony throughout the population is demonstrated 
by the variation in developmental stage seen in this and other 
studies (e.g.. Haven and Fritz 1985) that observe spatial variation 



in settlement. Localized synchrony in spawning on the other hand 
is highly probable, so the cumulative effect of these localized 
events approximates in magnitude that of a single synchronous 
spawning in the entire population. In other words, the cumulative 
output from multiple spawnings that occurs throughout the re- 
productive season (typically a maximum of 2-3 per season based 
on historical spatfall observations) are within an order of magni- 
tude of the single synchronous spawning event estimate of pro- 
duction. 

Oyster Reproductive Biology and Disease Status 

Both histological evidence and the presence of significant num- 
ber of larvae in plankton samples indicate spawning of the trans- 



hi: 



SOUTHWORTH AND MANN 



planted bloodstock oysters on Shell Bar reef. Based on the larval 
abundance data, and an estimated 14 to 21 days spent in the water 
column by C. virginica larvae, inferences can be made about the 
timing of spawning. Oysters from the reef probably spawned con- 
tinuously from mid- June to the end of July, with a few late spawn- 
ings occurring in the beginning of August. C. virgiiuca populations 
in the Chesapeake Bay have been reported to spawn from June to 
August (Kennedy and Krantz 1982). Gonad data from broodstock 
oysters on the reef support this observation. Though histological 
evidence of spawning was not observed until mid- July, ripe males 
and females were observed by mid-June. Small sample numbers 
(25 oysters per sampling day compared with a resident population 
of approximately 1.1 x 10*' individuals) and spatial asynchrony of 
spawning on the reef inay have confounded the definition of the 
first spawning event. 

Pcikiiisus infections progressed through the broodstock popu- 
lation throughout the summer, but did not result in catastrophic 
mortalities. The effect of Perkinsus on adult oysters, mainly re- 
duced fecundity, increases as intensity of the disease increases 
(Choi et al. 1994). In the broodstock oysters, intensity was highest 
toward the end of the sampling season, after the majority of the 
spawning had already taken place, suggesting that disease was 
probably not a limiting factor in the production of larvae. 

Plankton Studies 

Larval concentrations at the surface were found to be signifi- 
cantly higher during the flood tidal stage, suggesting that the larvae 
are acting as more than just passive particles. Larvae are capable 
of depth regulating with changes such as density and salinity, 
associated with a change in tidal cycle, such that a net transport of 
larvae upriver is possible (Hidu and Haskin 1978, Mann 1988). On 
most of the sampling days in this study, there was some stratifi- 
cation occurring in the water column at stations N2 and N3 (in the 
channel). The lack of physical stratification at Nl may have been 
due to the shallower depths at this station. Despite the lack of 
stratification at Nl, the suggestion of larval depth regulation with 
the changes in tidal stage are still supported by the absence of a 
significant interaction between tidal stage and station. 

It has been proposed that larval retention in an estuarine system 
and subsequent movement upstream is brought about through a 
combination of passive transport and active larval swimming (Car- 
riker 1951. Kunkle 1957, Haskin 1964). Oyster larval concentra- 
tions reported in the literature over the past 75 years range from 1 2 
m"-' to 660,000 m'' (Table 6). Wood and Hargis (1971) report 
larval abundance at the surface during maximum flood tide in the 
James River, with concentrations of larvae ranging between 300 
and 800 m"\ They found that minimum concentrations (<100 
m""') were encountered during slack water, following the ebb tide. 
The highest larval concentrations reported in the literature were 
recorded in Delaware Bay as 660,000 m"' (Nelson and Perkins 
1931) and 125.500 nr' (Nel.son 1927). These numbers are ex- 
tremely high when compared with concentrations found in this 
study, but the date of the observations must be taken into account. 
In a more recent study by Mann ( 1988) in the James River, much 
lower concentrations of between 1 2 and 1 1 3 mT^ were reported. 
The concentration of larvae found, reported in this .study, is ex- 
tremely high when compared with other reports made for years 
after the onset of disease and decimation of broodstock oyster 
populations in the Chesapeake Bay subestuaries. Though not of the 
same order of magnitude seen in historical times, the present report 
of concentration of larvae in the Great Wicomico is still several 



TABLE 6. 

Oyster larval concentrations reported in the literature over the 
past century. 



Larval 








Concentration 








ni^-3 


Location 


Year 


Source 


1 25.000 


Barnegat Bay. NJ 


1927 


Nelson. 1927 


660,000 


Barnegat Bay. NJ 


1931 


Nelson and Perkins, 
1931 


16.680 


Lanoka Lagoon. NJ 


1938 


Carriker. 1951 


13,360-37,.WO 


Great Bay. NJ 


1939 


Carriker, 1951 


625-2400 


Delaware Bay, NJ 


1656 


Haskin, 1964 


300-800 


James River, VA 


1965 


Wood and Hargis, 
1971 


12-113 


James River, VA 


1985 


Mann. 1988 


17.000-37.500 


Great Wicomico 
River. VA 


1997 


This study 



orders of magnitude higher than that found in the James River, 
which is considered to be the most important oy.ster-producing 
river in the Chesapeake Bay. A few James River reefs have similar 
densities of broodstock oysters as that found on Shell Bar Reef 
(see Table 1 of Mann and Evans 1998). but the difference in larval 
abundance probably lies in the differences in size and fecundity 
between the two broodstocks. The typical size of oysters found in 
the James is between 45 and 60 mm, with only a few reaching 
above 85 mm. In contrast, typical oysters found on Shell Bar Reef 
are between 85 and 95 mm. Given that fecundity and size have a 
nonlinear relationship (Mann and Evans 1998). small differences 
in mean broodstock size, and hence fecundity, can lead to vast 
differences in larval production. 

Circulation Studies 

Estuarine circulation can be a critical component of larval re- 
tention (Pritchard 1953, Ruzecki and Hargis 1989). The general 
trend of the cuixent tracks in this study suggest that circulation in 
the system is favorable for the retention of larvae. The majority of 
the tracks had a tendency to turn away from the channel, prior to 
tidal current change, thus the drifters remained upstream of Sandy 
Point, in the general area of the reef. In several instances, initial 
drifter movement was downstream in the channel. On only one 
occasion, however, did the drifter continue traveling in the channel 
toward the mouth of the river. Settlement data were in concordance 
with circulation observations in that higher settlement was found 
upriver of the sand spit in both the patent tong and shellstring 
surveys, suggesting that some local retention of larvae produced by 
the broodstock oysters on the reef was occurring. The number of 
spat m~ on the bottom recorded from the patent tong survey were 
at least three times higher at stations upstream compared with the 
stations downstream. 

The Great Wicomico River has historically been termed a 
"trap-type estuary," along with the Piankatank River, also in Vir- 
ginia and the St. Mary's River in Maryland (Manning and Whaley 
1954). Andrews ( 1979), defines a trap-type estuary as one that has 
a low-tlushing rate, small tidal amplitudes, and restricted en- 
trances. Though these characteristics aie important, local circula- 
tion, dictated by both topography and tidal cuuents, has been 
shown to be an important component of larval retention (Carter 
1967). Larval retention is not, however, limited to trap-type estu- 
aries. The James River, for example, has proved to be a good 



Oyster Reef Enhancement 



1113 



seed-producing river, with larvae that are produced ni the lower 
reaches being moved upstream and subsequently settling on up- 
stream oyster beds (Ruzecki and Hargis 1989. Mann 1988). The 
important feature ot retention in the James River is the tidal-related 
gyre-like circulation in Hampton Roads. Thus, retention of larvae 
can occur in estuaries that have characteristics in direct contrast to 
the " "typical" trap-type estuary. 

Impact of Broodslock Seeding from a Management Perspective 

The demonstrated positive impact of broodstock addition to the 
Great Wicomico reef prompts the question as to the efficacy of this 
approach for widespread restoration efforts in the Chesapeake Bay 
and elsewhere. In comparison with prior reef construction efforts. 
the addition of broodstock to the reef in the Great Wicomico River 
approximately doubled the initial monetary investment, however, 
the ecological impact on replenishment was rapid in comparison. 
In just I y observed settlement of spat was comparable to historic 
conditions. Increased spatfall in 1997 has stimulated a resurgence 
of interest in deployment of substrate on private leased oyster 
grounds in the Great Wicomico in 1998 in anticipation of contin- 
ued high recruitment. A blanket endorsement for the construction 
and broodstock enhancement of reefs should still, however, be 
avoided. Location of any proposed reef construction is critical to 
its possible success. Though the typical definition of a trap-type 
estuary is u.seful in general location, knowledge of historic oyster 
reef topography can be critical at a finer spatial scale. Indeed, our 



study supports the use of both historical data sets and circulation 
studies of a target system as a common precursor to management 
decisions involving reef construction. Location definition should 
be followed by numerical estimation of optimal stocking density. 
The current study stands as an example of how a modest (com- 
pared with historical numbers) number of large oysters planted at 
a high density of 300 in"" can have an immediate impact in an 
effort to restore decimated oyster populations to historic condi- 
tions. 

ACKNOWLEDGMENTS 

This project was funded in part by the Virginia Council on the 
Environment's Coastal Resources Management Program through 
Grant #NA 570 Z 0561-01 (task #25) of the National Oceanic and 
Atmospheric Administration, Office of Ocean and Coastal Re- 
source Management Act of 1972 as amended, and forms part of the 
M.S. degree requirements of the senior author at the School of 
Marine Science. Virginia Institute of Marine Science, College of 
William and Mary. This project was also funded in part by the 
National Science Foundation Biological Oceanography Program 
under Grant #OCE-98 10624 to Roger Mann. This is Contribution 
Number 2170 from the School of Marine Science, Virginia Insti- 
tute of Marine Science. The assistance and productive comments 
of our colleagues James A. Wesson, Mark Luckenbach, David A. 
Evans, John Brubaker. Sandra Shumway, Juliana Harding, Ken- 
neth Walker, and Juanita Walker are gratefully acknowledged. 



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II 14 



SOUTHWORTH AND MaNN 



gametogenesis in Cnissostrea gigas and Oslrea edulis grown at sus- 
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Mann, R. & J. Wesson. 1996. Evaluation of oyster settlement and survival 
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Nat. Shellfish Assoc. 48:56-65. 

Nelson, T. C. 1927. Report of the Department of Biology, pp 103-1 13. In: 
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3^7. 

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Olsen. S. & J. A. Wesson. 1997. The creation of oyster reef sanctuanes in 



the Great Wicomico and Coan Rivers and in the Bayside Creek on 
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Joiirmil iif Slu'llflsh Rfsfanh. Vol, 17. No. 4. 1 1 l.'^-l 127. 1998. 

STUDIES ON TRIPLOID OYSTERS IN AUSTRALIA. X. GROWTH AND MORTALITY OF 
DIPLOID AND TRIPLOID SYDNEY ROCK OYSTERS SACCOSTREA COMMERCIALIS 

(IREDALE AND ROUGHLEY) 



ROSALIND E. HAND.' JOHN A. NELL.' AND 
GREG B. MAGUIRE-* 

^NSW Fisheries, Port Stephens Research Centre. 

Taylors Beach, NSW 2316. Australia 
'School of Aquaciiltnre. University of Tasmania, 

Laiinceston, Tas. 7250, Australia 

ABSTRACT Growth and mortality of triploid Sydney rock oysters, Saccostrea commerciaiis (Iredale and Roughley) were compared 
with those of sibling diploids at 13 oyster farms in 10 estuaries in New South Wales (NSW). Although results varied between farms, 
after 2-2'/2 years on commercial oyster leases, triploids were on average 30.7% heavier and 8.6% larger in shell height than sibling 
diploids. Seven of the 13 farms had triploids with a mean weight of at least market size (40 g) after 2-21 ; years while no farms had 
diploids of the same mean weight. The growth advantage of triploids began to be expressed at specific sizes rather than ages, 
specifically at a mean whole weight above 5-10 g or shell height of 30-40 mm. In general, tnploids appeared to grow faster compared 
with diploids at higher water temperatures. Although readily accepted by processors during cooler seasons, triploid Sydney rock oysters 
developed a patchy, discoloration of the gonad during warmer months that may affect marketability during summer. Mortality of 
tnploids was significantly (p < .01) lower than that of diploids at 6 of the 13 farms and did not differ (p > .05) at 6 of the remaining 
7 farms. The growth coefficients were lower and mortality higher for wild-caught diploids than both hatchery-reared diploids and 
triploids. Triploid Sydney rock oysters were found to offer significant commercial advantages over diploid oysters because ol faster 
growth rates and lower mortality when grown under commercial oyster farming conditions across a range of estuaries in NSW. 

KEY WORDS: Growth, farming, oysters, triploid, mortality 



INTRODUCTION 

Sydney rock oysters generally take from 3 to 4 years to reach 
marketable size (Nell 1993) compared with 1 '/2-3 years for Pacific 
oysters, Crassostrea gigas. Thunberg (Graham 1991 ) in Australia. 
Increasingly. Pacific oysters (predominantly from Tasmania and 
South Australia) are taking over traditional Sydney rock oyster 
markets. Adoption of tnploid technology may help New South 
Wales' (NSW) farmers to remain competitive against industries 
based on the faster growing Pacific oyster. Nell et al. (1994) com- 
pared the performance of triploid and diploid Sydney rock oysters 
in a small scale trial in Port Stephens, NSW over 2'/; years. At the 
end of the study, the triploids were on average 41% heavier and 
reached market size 6-18 months earlier than their diploid siblings. 
However, results varied among the four sites tested within the one 
e.stuary (range 32—48.8% difference in whole weights of diploids 
and triploids). The benefits of triploidy are known to be influenced 
by environmental factors such as food availability (Davis 1989a) 
and temperature (Davis 1989b, Shpigel et al. 1992), Oyster farm- 
ing in NSW is practiced throughout the coastal areas of the state, 
across a range of environmental conditions (particularly tempera- 
ture). At the conclusion of the preliminary study, there were no 
commercial bivalve hatcheries operating in NSW. To commercial- 
ize production and farming of triploid Sydney rock oysters, po- 
tential hatchery operators needed to be assured that the benefits of 
triploidy could be extended to other oyster-growing estuaries, us- 
ing commercial farming methods. In this study, to enable the com- 
mercialization of triploid technology, we compared the growth and 
mortality of diploid and triploid Sydney rock oysters grown on 
oyster farms throughout NSW by commercial oyster farmers. This 



*Present address; Fisheries WA, Research Division, PC Box 20, North 
Beach, WA 6020, Australia. 



study also provides a comparison of normal, diploid oyster growth 
across a range of NSW estuaries. 

METHODS 

Induction of Triploidy and Ploidy Determination 

Triploid (300 x 10") and diploid (60 x 10'') sibling larvae were 
produced in February 1994 from 48 female and 6 male oysters 
from Port Stephens, NSW. Triploidy was induced in zygotes using 
the method described in Nell et al. (1996) with a cytochalasin B 
(CB) concentration of 1.25 mg/L. Larvae were stocked directly 
into 20,000 I larval rearing tanks (three triploid and one diploid). 

Percentage triploidy was determined by direct chromosome 
counts at 4 h in embryos (Nell et al, 1996) and by flow cytometry 
for shelled larvae and spat. 

iMrvae and Spat Rearing 

Larvae were reared using standard hatchery techniques for Syd- 
ney rock oysters (Prankish et al. 1991 ). The three triploid treatment 
tanks were combined by day 5 due to low survival and similar 
percent triploid readings (78,3-81.5%). Screening at water 
changes was conservative to allow for initial slow growth of trip- 
loid larvae following exposure to CB (Wada et al. 1989). Larvae 
were settled on ground scallop shell between days 18 and 22 and 
reared in the hatchery for 5 weeks before being transferred to 
outdoor upwelling units. Spat were on-grown to a size of 7-10 
mm. 

Oyster Management 

Thirteen commercial oyster fanners in 1 1 different estuaries 
throughout NSW (Fig. 1) from Pambula Lake (36°58'S, 
149°54'E) to Hastings River (3I°25'S, 152°55'E) were each sold 
25,000 diploid and 25.000 sibling triploid spat between July and 



1115 



1116 



Hand et al. 



QUEENSUND \^ 




r XT-" J 


N 


/ 






200 i<m / 










1 / 


\,.,_jj' Hastings River 


NEW SOUTH WALES ? 


^^■^Kaiuah River 
f'''^TilIigerry Creek 


^Brisbane Waters 


,^-^*^J!/Hawkesbury River 

J SYDNEY PACIFIC OCEAN 


^Georges River/Woolooware Bay 


j^^^~Tf Shoalhavcn River 


C I J 

) i/CIyde River 


f^ AUSTRALIA \ 




/ Lake Merimbuia 
^v,^^ fl Lake Pambula 


V-A^ 




VICTORIA ^v/ 


o 















Figure 1. Estuaries in NSW where farming sites were located (adapted 
from Hollidav et al. 1988). 



November 1994. Rather than supplying oysters matched for initial 
size (i.e.. which would risk matching fast diploids with slow trip- 
loids), oysters were supplied to farmers as they reached a grow-out 
size of 7-10 mm (shell height). Mean whole weight of spat sup- 
plied to farmers ranged from 0.07 ± 0.02 to 0. 1 1 ± 0.05 g and 0.07 
± 0.02 to 0.10 ± 0.03 g (mean ± SD: n = 400) for diploids and 
triploids. respectively. Mean shell height ranged from 8.40 ± 0.82 
to 9.83 ± 1.68 mm and 8.22 ± 0.83 to 9.73 + 1.05 mm (mean ± SD; 
n = 400) for diploids and triploids. respectively. All farmers were 
encouraged to use standardized stocking densities throughout the 
experiment of 2 L for cylinders and SOVr coverage for tray culture 
(Nell 1993). However, in the colder South coast areas (i.e.. Lake 
Pambula and Lake Merimbuia, where conditions produce limited 
oyster growth in cylinders at these densities) they were reduced. 
Baskets (Maguire et al. 1994a) were stocked at between 1 and 2 L. 
Table 1 shows a summary of culture methods used at each farm. 
Oysters were grown by the farmers using standard commercial 
oyster farming techniques, and culture equipment was provided by 
farmers. For this reason culture trays, baskets, etc. could not be 
standardized across all estuaries, however, diploids and triploids at 
each farm were grown under the same conditions. The normal 
farming practice of grading oysters into several size grades, when 
there is a large range of sizes to allow growth of the smaller oysters 
without competition from the larger grades, was minimized where 
possible to make comparisons between ploidy levels and farms 
simpler. However, as this was a study based on commercial farm- 
ing methods, and large size ranges developed at least at some stage 
during the course of the experiment [at all sites except for Til- 
ligerry Creek. Port Stephens (farm 1 ) and Woolooware Bay), oys- 
ters were graded when necessary. Grades were recomhined when 
there was no longer a difference in size. 

Oysters from Georges River (farm 2) and Lake Pambula were 



TABLE 1. 

Culture methods for diploid and triploid Sydney rock oysters Saccostrea commercialis grown by commercial oyster farmers in New South 

Wales from July 1994 to November/December 1996. 



Estuary 



Month 

Supplied 

(1994) 



Nursery Culture 



Growout 



Culture Method Stocking Density Culture Method 



Management 



Hastings River July 
Tilligerry Creek (t;inn 1) " 

Tilligerry Creek (farm 2) August 

Karuah River September 

Georges River ( farm 1 ) " 

Georges River (farm 2) " 



Hawkesbury River 
Woolooware Bay 
Clyde River 
Lake Merimbuia 
Lake Pambula 

Brisbane Waters 



Slioalhaven River 



October 



November 



Suhlidal trays 
Intertidal trays 
Intertidal trays 
Cylinders 

Cylinders 

Cylinders 

Cylinders 

Intertidal trays 

Intertidal trays 

Cylinders 

Cylinders 

Trays 

Cvlinders 



Cylinders 



50% coverage 

50% coverage 

50% coverage 

21 

21 

21 

21 
50% coverage 
50%' coverage 

1-21 
0.5-21 
50% coverage 

21 



0.5-2 1 



Suhtidal trays 
Intertidal trays 
Intertidal trays 
Baskets 
Intertidal trays 
Baskets 
Intertidal trays 
Baskets 
Intertidal trays 
Intertidal trays 
Intertidal trays 
Intertidal trays 
Intertidal trays 
Intertidal trays 

Baskets 
Intertidal trays 
Large cylinders" 
Intertidal trays 
Subtidal trays 



Oysters dried out 1-7 days every 4 weeks 
Mo\'ed during winter 
Moved during winter 



Moved during winter 

Moved during winter 
Divided between 3 sites 

Moved during winter 
Moved during winter 
Raised during winter 
Divided between 3 sites 

Large grades moved between large 
cylinders and trays 

After 9 months on intertidal trays 
Transferred to subtidal culture for 6 months 



' 200 I rotating cylinders with 1 2 mm mesh, similar to the smaller Stanway® cylinders. 



Triploid Oysters in Australia. X. 



III7 



60 
2 50 

5 30 
o 20 



Hastings River 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time {months) 



50 - 
















1 


II 


gerry 


c 


eeV 


(farm 1) , 














r^ 


2 40 - 












< 


oi 30 i 












y,^^-^ 


S 20 












yy 


01 












^ 


o 10 - 








tr 


:*=-» 




^ 0^ 






-^ 






— ■ — diploid 


•— • 




'" 






— *■ • triploid 



Jul-94 j3n-95 Jul-95 Jan-96 Jul-96 Jan-97 Jul-97 
Time (months) 



Tllligerry Creek (farm 2) 




Karuah River 



Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 





Jul- 



94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 




Jan-95 Jul-95 Jan-96 Jul-96 
Time (months) 



Hawkesbury River 




Woolooware Bay 



Jui-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Shoalhaven River 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Lake Merim bula 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Lake Pambula 




Jul-94 Jan-95 Jul-95 Jan-96 Jui-96 Jan-97 



Brisbane Waters 




Jan.95 Jul-95 Jan-96 Jul-96 Jan-97 



Time (months) Time (months) 

Figure 2. Whole oyster weight (g) of diploid and triploid Sydney rock oysters, Saccostrea commercialis. grown by commercial oyster farmers in 
NSW from July 1994 to December 1996. Mean ± 95'7f confidence intervals. 



1118 



Hand et al. 



TABLE 2. 

Time" taken (months) to reach a «hole weight of 10, 20, 30, and 40 g by diploid and triploid Sydney rock oysters Saccostrea commercialis 

grown by commercial oyster farmers in New South Wales. 





Site 




10 


g 




20 g 




30 g 




40 g 


Estuary 


Diploid 




Triploid 


Diploid 


Triploid 


Diploid 


Triploid 


Diploid 


Triploid 


Hastings River'^''^ 




15 




12 


28 


16 


_ 


22 


— 


27 


Tilligerry Creek (farm 1) 




12 




11 


18 


17 


23 


20 


- 


24 


Tilligerry Creek (farm 2) 




14 




13 


20 


17 


26 


22 


- 


26 


Karuah River 




11 




8 


15 


14 


23 


19 


- 


26 


Georges River (farm I) 




14 




11 


18 


16 


25 


19 


- 


25 


Georges River (farm 2) 


1 


12 




11 


17 


16 


26 


18 


- 


25 




2 


8 




7 


17 


16 


26 


18 


- 


25 


Hawkesbury River 




14 




15 


19 


18 


- 


27 


- 


- 


Woolooware Bay 




14 




15 


19 


18 


- 


25 


- 


- 


Clyde River" 




14 




13 


17 


14 


26 


19 


- 


26 


Lake Merimbula'' 




16 




17 


- 


26 


- 


- 


- 


- 


Lake Pambula'' 


1 


17 




16 


25 


20 


- 


- 


- 


- 




2 


16 




15 


27 


23 


- 


- 


- 


- 




3 


15 




15 


20 


19 


- 


- 


- 


- 


Brisbane Waters*' 




15 




16 


22 


20 


- 


24 


- 


- 


Slioalhaven River 




12 




11 


16 


14 


- 


22 


- 


- 


Average time'' 




14 




13 


(19) 


17 


(251 


(2()l 


- 


(26) 



" Actual time taken, estimated from grapii of whole weight against time; - represents oysters that have not reached set size. 

*" A weighted mean of more than 1 size grade was calculated. 

' Subtidal culture. 

■^ Mean time for all farms; months in brackets do not include data from slower growing sites where times are not available for that set size and for both 

ploidy types. 



divided among three sites when they reached a large enough vol- disease QX. After 12 months this site was no longer included in the 



ume (after 2 and 6 months, respectively). Oysters from Georges 
River (farm 2) were divided between one upriver lease and two 
downriver leases. During the first year, oysters from the upriver 
site suffered high mortality with symptoms consistent with the 



study because of very poor survival, and the remaining two down- 
river sites were combined. QX disease, which is caused by the 
protistan parasite Marteilia sydneyi. spread to the Georges River in 
1994 (Adlard and Ernst 1995). 



TABLE 3. 

Whole weight and shell height of diploid and triploid Sydney rock oysters Saccostrea commercialis grown by commercial oyster farmers in 

New South Wales from July 1994 to November/December 1996. 



Estuary 



Month 

Supplied 

(19941 



Whole Oyster Weight (g) 



Diploid 



Triploid 



Difference 

(%)" 



Shell Height (mm) 



Diploid 



Triploid 



Difference 

( % )>' 



Hastings River'"' 


July 


24.9 ± 0.5 


Tilligerry Creek (farm 1) 


" 


34.4 ± 0.3 


Tilligerry Creek (farm 2) 


August 


33.0 ± 0.3 


Karuah River 


September 


37.4 ± 0.4 


Georges River (farm 1) 


" 


33.5 ±1.1 


Georges River (farm 2) 


" 


30.3 ± 0.3 


Hawkesbury River 


October 


19.6 ±0.2 


Woolooware Bay 


" 


28.7 ±0.3 


Clyde River" 


" 


30.3 ± 0.4 


Lake Merimbula" 


It 


18.7 ±0.3 


Lake Pambula"" 


" 


21.9 ±0.5 


Brisbane Water" 


Noveinber 


26.7 ± 0.5 


Shoalhaven River 


" 


28.8 ±0.3 


Average 




28.3 



49.4 ± 0.8 
44.8 ± 0.5 
44.8 ± 0.5 

47.8 ± 0.5 
42.6 ± 0.5 

43.9 ± 0.5 
24.6 ± 0.3 
32.3 ± 0.4 
39.9 ±0.6 
20.9 ± 0.4 
26.9 ± 0.6 

28.2 ±0.7 

35.3 ± 0.5 
37.0 



98.9 
30.1 
35.8 
28.0 
27.1 
44.9 
25.3 
12.6 
32.0 
11.8 
23.0 
5.6 
22.6 
30.7 



60.4 ± 0.5 

68.0 ± 0.3 

69.6 ± 0.3 

66.1 ±0.3 

60.7 ±0.8 

60.5 ± 0.3 

52.2 ± 0.3 

58.8 ±0.3 

63.3 ± 0.4 
51.7 ±0.4 
57.0 ±0.6 
63.3 ± 0.5 
63.3 ± 0.3 

61.1 



69.6 ± 0.5 

74.0 ± 0.4 

75.1 ±0.4 

69.8 ± 0.3 

66.6 ± 0.3 

68.1 ±0.4 
59.0 ± 0.4 
62.4 ± 0.3 

68.7 ± 0.5 

56.6 + 0.4 

62.2 ± 0.6 

63.7 ± 0.7 

66.9 ± 0.4 
66.4 



15.2 
8.9 
7.9 
5.5 
9.7 
12.5 
13.1 
6.0 
8.6 
9.5 
8.4 
0.8 
5.6 
8.6 



" Difference (%) = (triploid - diploidi/diploid x 100. 

" A weighted mean ± SE of more than 1 size grade was calculated for mean whole weight and shell height. 

' Subtidal culture. 

** Oysters divided among three sites. 



Triploid Oysters in Australia. X. 



119 



70 - 
60 - 


H 


astings River 


^-^ 


— ^^^ 


50 - 
40 - 
30 - 






20 - 


^ 






— •— diploid 


10 1 

(1 n 


' 






— *- - tnploid 



Jan-95 Jul-95 Jan-96 Jul-96 
Time {months) 




1-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



80 -, 
70 
60 - 
50 - 
40 
30 
20 - 
10 



Jul-I 



Tilligerry Creek (farm 2) 




94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



30 
20 
10 



Georges River 


(farm 1) . .— ■ 


/^ 


^ 


/ 


— ■ — diploid 
— *- - tnploid 

—t>— control 



1-94 Jan.95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



80 

70 - 

60 

50 

40 

30 

20 

10 



Jul 



Karuah River 




60 
50 
40 
30 - 
20 - 
10 
Jul. 



34 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Georges River (farm 2) ^ ' 




Jan-95 Jul-95 Jan-96 Jul-96 
Time (months) 



E ?0 
E en 



0) 40 

X 

= 30 



Hawkesbury River 




10 


Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



80 T 
E 70 - 
E 60 
2 50 



30 
20 

10 




Wooloovk^are Bay 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Figure 3. Shell height (mm) of diploid and triploid Sydney rock oysters, Saccoslrea commercialis, grown by commercial oyster farmers in NSW 
from July. 1994 to December 1996. Mean ± 95'7f confidence intervals. 



As is commercial practice, oysters located in estuaries subject 
to the disease winter mortality were moved where possible (Til- 
ligerry Creek: fanns 1 and 2; Georges River: farms 1 and 2; Woo- 
looware Bay and Clyde River) to a lease further upstream or to a 
higher rack (Lake Merimbula) during winter. Winter mortality is 
caused by the protistan parasite Mikrocytos roiighleyi (Farley et al. 
1988). 

A further comparison of a cylinder of '"wild-caught" single- 
seed (Nell 1993) diploid oysters with the hatchery-produced dip- 
loids and triploids was set up at Georges River (fann 1). Lake 
Merimbula and Brisbane Waters. 

Sampling Strategy 

A subsample of 400 of each type of oyster was measured for 
whole weight and shell height before being supplied to fanners, 
then every 2 months thereafter at each farm until November 1995 
when the sampling frequency was changed to every 3 months. 



Where several grades were present, a smaller sample was mea- 
sured for each grade, i.e., 400-600 total per ploidy type and a 
weighted mean was determined. Samples for individual height and 
weight measurements were taken from a random 10% of stock 
brought in every 2-3 months. For this reason, mortalities were not 
removed during the course of the experiment (i.e.. to maintain 
similar densities of oyster shells and percentage dead between 
sampled trays and trays remaining on leases). Mortality was as- 
sessed from a subsample and returned to trays for a final count at 
the end of the experiment. 

Total weight of stock was measured every 2 months for the first 
6-9 months to assess losses and the proportion in each grade. As 
handling became more difficult with the large increase in volume 
during the first year (after March 1995), farmers were given the 
choice of bringing in either 25% of stock or everything every 6 
months to find the total weight of all oysters. Weights of dead 
oysters and overcatch were accounted for when total weights were 
measured. 



Hand et al. 



so 

70 
60 
50 - 
40 

30 
20 
10 



Clyde River 




Jul-94 Jan-95 



Jul-95 Jan-96 
Time (months) 



1-96 Jan-97 



ao 

70 - 

60 - 

50 

40 

30 

20 

10 



Lake Merimbula 




— " — diplord 
— *■ ■ tnploid 
— ©— control 



Jan-95 Jul-95 Jan-96 
Time (months) 



•c 50 
a> 4 



20 - 
10 



Jul-94 



Lalte Pambula 




Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



E 70 

1 50 

2 50 - 
o> 

Q) 40 

I 

= 30 



(fi 



E 70 - 

~ ^° 

S 50 
.? 

Z 40 

X 

= 30 

a> 

■c 20 

to 

10 



20 



Shoalhaven River 



Brisbane Waters 




— • — diploid 
— »- - tnploid 
^-<» — control 



Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Figure 3. Continued. 



Salinity and temperature data were measured weekly by farm- 
ers using a thermometer and hydrometer. 

Statistical Analyses 

All whole oyster weight and shell height data are presented in 
figures as means ± 95% confidence intervals. Weighted means of 
whole weight, shell height, and mortality data were calculated for 
farms with more than one size grade or site: 

- ^w,x, 
x„, = ^= 

where X„. is the weighted mean calculated from the mean. X, of 
each n grades, and each X, weighted by a factor W, (Sokal and 
Rohlf 1995). The total number in each grade was used as a weight- 
ing factor and was estimated from the total weight of all oysters for 
each grade divided by the mean weight of individual oysters for 
that grade. 

Growth coefficients (d,,,) were calculated from comparisons of 
hatchery stock with wild-caught diploids to account for differences 
in initial whole weights and in the duration of the experiment 
(Spencer and Gough 1978): 



90 
/-• 

''° Duration (days) 



xln 



Final weight (g) 



^ Initial weight (g) 

Mortality data were analyzed for significant differences (p = 
.05) by Chi-squared contingency tables (Sokal and Rohlf 1995). 
Initial and final height and weight data were analyzed for signifi- 



cant differences using ANOVA (Sokal and Rohlf 1995) after ho- 
mogeneity of variance was confirmed using Cochran's test (Winer 
1991). Size data were log,o transformed where necessary. Where 
several grades were present at the end of the experiment, a random 
subsample (the size of which was determined by the proportions in 
each grade) was used for ANOVA. 



RESULTS 



Induction of Triploidy 



The mean triploidy reading on day was 79.9 ± .92% (mean ± 
SE; n = 3 groups of 60 larvae) which increased to 87.8% by 10 
weeks' post-settlement. Flow cytometry of gill tissue biopsies 
from oysters from three farms (Hastings River, Kaniah River, and 
Clyde River) in January /February 1996 gave a mean triploid level 
of 88 ± 4.0% (mean ± SE. n = 3 groups of 183-292 oysters). 

iMnal and Spat Rearing 

Poor water quality during the larval run resulted in low survival 
to settlement (7.5% diploids and 2.0% triploids) with set rates of 
56.3% for diploids and 43.2% for triploids. Spat in upweller sys- 
tems were affected by "post-settlement mortality" (Frankish et al. 
1991 ) in April 1994. Survival from settlement in early March 1994 
to May 1994 was 26% for diploid and 31% for triploid spat, re- 
spectively. As spat were graded over the same mesh but not 
matched for size before being supplied to farmers (to avoid match- 
ing slow-growing triploids with fast diploids), initial sizes of dip- 
loids and triploids were sometimes different. However, this gen- 
erally favored the diploids with initially larger diploids (p < .05) at 



Triploid Oysters in Australia. X. 



1121 




Jul-95 Jan-96 
Time (months) 



Tjhigerry Creek (farm 1) 




salinity 
- temperature 



50 
45 
40 

^"^ 

25-1 
20 = 
15 « 

10 



1-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Tilligerry Creek (farm 2) 




50 
45 
40 

35 
30 
25 
20 - 
15 - 
10 - 
5 ■ 
- 
Jul 



Georges River (farm 1) 




40 
35 
30 
25 si 

20 I 
15 ■S 



Jul-94 j3n-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



-94 Jan-95 Jul-95 Jan-96 Jul-96 
Time (months) 



50 
45 

40 

35 ~ 

30 S 

25 ,= 

c 

20 = 

15 "> 

10 

5 





50 ■, 


Karuah River 




45 - 






40 ■ 






35 

30 


^y^j%/^ 


f^-r 


25 - 

?n ■ 


^"^L-a* »L. 


v"*° 


15-, 




10- 

I] 


^ 


salinity 
temperature 



50 
45 
40 

30 S 
25 .f 

20 1 
15 M 
10 
5 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 

Georges River (farm 2) 




30 ~ 
25-1 

15 M 
10 



Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



50 

45 

40 

35 

30 

25 

20 

15 ■ 

10 



Hawkesbury River 



salinity 
temperature 



40 
"J 

"1 
20 = 
15 « 

10 



Jul-94 Jan-95 



Jul-95 Jan-96 Jul-96 
Time (months) 



50 

45 

40 

35 - 

30 

25 

20 

15 

10 • 

5 



Jul-94 Jan-95 



Woolooware Bay 




50 
45 

40 

25-1 
20 = 
15 " 



Jul-95 Jan-96 Jul-96 
Time (months) 



Figure 4. Surface water salinity and temperature data for oyster farms in NSW from July 1994 to December 1996. 



8 of the 13 farms, larger triploids at two farms (Hastings River and 
Tilligerry Creek, 1). and no significant difference (p > .05) be- 
tween the initial size of diploids and triploids at Tilligerry Creek, 
2. Brisbane Waters and Shoalhaven. 

Appearance 

A brown to grey color appearing as distinct patches on the 
gonad was noted on triploid oysters. This coloration developed 
during the second year on leases and was most noticeable during 
summer irionths. 

Whole Oyster Weight 

Whole oyster weights over the 2'/2 year study are shown in 
Figure 2. For all farms except Lake Merimbula. mean triploid 
whole weight was greater than mean diploid weight from the time 
when oysters reached a whole weight of about 5-10 g. Triploids 
generally remained heavier until the end of the study. Average 
time taken to reach a mean whole weight of 10 g was 13 months 



for triploids and 14 months for diploids (Table 2). Largest in- 
creases in mean whole oyster weight occurred during spring 
through to autumn for northern and central NSW estuaries (Has- 
tings down to Shoalhaven Rivers). The growth season for oysters 
on the south coast occurred later, i.e., summer through to autumn/ 
early winter. Greatest relative growth of triploids compared with 
diploids generally occurred later in the growth season after the first 
year on leases. By the end of the 2'/2 year study the mean whole 
oyster weight from the 13 farms involved in the study was 28.3 g 
(range 18.7-37.4 g) for diploids and 37.0 (20.9-49.4 g) for trip- 
loids, a difference of 30.7% (Table 3). Final weights of triploids 
were significantly greater (p < .01) than diploid weights at all 13 
farms, with an apparent effect of temperature (Fig. 4) on the rela- 
tive growth of diploids and triploids. The seven sites with the 
greatest difference between diploid and triploid growth had mean 
water temperatures of 18°C or more, and four of the six remaining 
sites had mean water temperatures of less than 18°C. Time to 
market size could not be compared between the two ploidy types 
as diploids had not reached this size by the end of the study. 



1122 



50 
45 

40 
IT 35 - 
2 30 • 
E 25 
a. 20 

1 'M 
>- 10 



Clyde River 







salinity 
temperature 



Jan-95 Jul-95 Jan-96 Jul-96 
Time (months) 



Hanc 


) ET AL. 


50 


50 


45 


45 


40 


U 40 


^^J 


a, 35 


30 — 


5 30 


,,■■- 


S 25 


20 -5 


£. 20 


15 W 


E 15 



Lake Merimbula 



salinity 
temperature 



60 
45 
40 

- 35 J 

- 30 i; 

- 25 = 
I- 20;; 

15 " 

- 10 



1-94 Jan-95 



Jui-95 Jan-96 Jul-96 Jan-97 
Time (months) 




ul-95 


Jan-95 J 


Time 


[months) 




50 




45 




35 
30 - 



Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Shoalhaven River 




r 50 
45 
40 

35 -? 
30 S. 
25.? 

20 1 
15 « 
10 
5 




Jul-94 Jan-95 Jul-95 Jan-96 Ju|.96 Jan-97 
Time (months) 

Figure 4. Continued. 



instead, the time to reach a mean whole weight of 30 g was 
compared. Where data are available for both ploidy groups, trip- 
loids reached this size 20'7f faster than diploids. The average time 
taken for triploids to reach a mean whole weight of 40 g was 26 
months for the seven farms that had oysters at this size. There were 
no farms with diploids at a mean weight of 40 g. 

Shell Height 

Shell growth of diploids and triploids followed a similar pattern 
to whole weight (Fig. 3). In general, by the time oysters had 
reached a mean shell height of 30-40 mm. growth of triploids was 
greater than that of diploids. Triploids at most farms had reached 
a shell height of approximately 40 mm. after 8-12 months on 
leases. Fastest shell gi'owth occurred between winter/spring and 
autumn for northern and central sites and between spring/summer 
and autumn for sites south of the Shoalhaven River. Shell growth 
of triploids relative to diploids was greatest during spring and 
summer for northern and central sites and between spring and 
autumn for southern sites. After 2-2'/: years on oyster leases, mean 
shell height was 61.1 mm for diploids and 66.4 mm for triploids. 
a difference of 8.6% (Table 3) and was significantly larger (p < 
.001) for triploids at 12 of the 13 farms. 

Cumulative Mortality 

There was little difference between cumulative mortality of 
diploids and triploids at most sites during the first 12-18 months 
on leases (Fig. 5). However, cumulative mortality (Table 4) of 
triploids at the end of the study was significantly (p < .01) lower 
than that of diploids at 6 of the 1 3 farms and did not differ (p > .05) 



at 6 of the remaining 7 farms. Cumulative mortality was higher for 
triploids than diploids at only one site (Tilligerry Creek, farm 1; p 
< .05). 

Wild-Caught Diploid: Hatchery Diploid and Triploid Comparison 

Initial whole oyster weights and shell heights (Tables 5 and 6) 
of wild-caught diploids supplied by oyster farmers were signifi- 
cantly (p < .05) different from those of hatchery stock for all three 
sites (data were not homogeneous for the Georges River and Bris- 
bane Waters sites). There was also a significant difference (p < 
.05) between hatchery diploids and triploids at the Georges River 
site (for both height and weight) and Lake Merimbula (weights). 
For this reason growth was compared using growth coefficients 
(Table 7). Wild-caught diploids had lower growth coefficients than 
both diploid and triploid hatchery stock at all three sites, except for 
hatchery diploids at Lake Merimbula. High mortality (with symp- 
toms consistent with those of the disease winter mortality) of wild- 
caught diploids occurred at the Brisbane Waters site during the 
first year resulting in insufficient numbers to continue the com- 
parison. The cumulative mortality of wild-caught oysters after only 
1 1 months was 55.4% compared with only 6.0 and 2.7% for hatch- 
ery diploids and triploids. respectively. For Georges River and 
Brisbane Waters sites there was a significant effect (p < .05) of 
ploidy type on cumulative mortality (Table 7). 

DISCUSSION 

The variation in performance of triploid Sydney rock oysters 
between different sites in the preliminary study of Nell et al. 
(1994) was emphasized in the present commercial-scale study. 



Triploid Oy.st[:rs in Australia. X. 



1123 



Hastings River 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (inonths) 



£. 60 

= 50 



Ttlligerry Creek (farm 1) 



o 40 - 

E 

» 30 H ~*~ diploid 
> — *- - triploid 

™ 20 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



o 


40 


E 




o 


:^n 










n 


?n 


3 




E 





Tilligerry Creek (farm 2) 



diploid 
triploid 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Karuah River 



diploid 
triploid 



Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Georges River (farm 1) 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 

Time (months) 



r 15 
o 

E 



Hawkesbury River 



diploid 
triploid 



Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Woolooware Bay 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Figure 5. Cumulative inortality (%) of diploid and triploid Sydney rock oysters, Saccostrea commercialis, grown by commercial oyster farmers 
in NSW from .lulv 1994 to December 1996. 



Much of the variation in this study may be attributable to differ- 
ences in water temperatures. However, growth of both diploids and 
triploids at Brisbane Waters. Lake Merimbula, and Hawkesbury 
River appears to have been influenced by an additional factor, 
possibly food availability (L. Cooper pers. comm.). Davis ( 1989b) 
also measured faster growth rates of triploids relative to diploids at 
a site with maximum water temperatures (July and August) of 
20°C compared with a site with maximum water temperatures of 
16°C. This is most likely due to the relatively greater contribution 
by diploids of energy reserves to gametogenesis at higher tem- 
peratures (Shpigel et al. 1992). The unusually large difference 
(98.9%) between diploids and triploids at the Hastings River site is 
partly due to the poor growth of diploids. Oysters at this site were 
grown subtidally which may account for the faster growth of trip- 
loids at this site (Nell et al. 1994). However, growth of diploids 
appeared to be retarded even before they reached a size at which 



we would expect gametogenesis/spawning to affect their growth 
(Fig. 2). 

The growth seasons for both diploids and triploids were con- 
sistent with previous studies (Allen and Downing 1986. Nell et al. 
1994). i.e.. spring through to autumn with greater relative growth 
of triploids compared to diploids later in the growth season. The 
period of greater relative growth of triploids occurred prior to the 
normal spawning season (February to May) for Sydney rock oys- 
ters (Nell 1993). i.e., when diploids are diverting a large proportion 
of their energy stores to gametogenesis at the expense of somatic 
growth. Triploid Sydney rock oysters did not show an advantage 
over diploids until they reached a mean whole weight of between 
5-10 g or shell height of 30-40 mm. This corresponded to a 
growout time of between 8 and 14 months and is similar to results 
(12-13 months) obtained for Pacific oysters in Japan (Akashige 
and Fushimi 1992) and earlier results (6-9 months) for Sydney 



1124 



Hand et al. 



Clyde River 




Jul-94 Jan-95 Jul-95 Jan-96 JuI-96 
Time (months) 



Lake Merimbula 



diploid 
tnploid 
wild-caught diploid 




Jul-94 Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



Lake Pambula 




Jan-95 Jul-95 Jan-96 Jul-96 Jan-97 
Time (months) 



ss 


100 - 






B 


Isbane 


w 


ate 


rs 


>« 


90 - 
80 - 


. 


dipio 


d 








J 


r 


70 


— *- ^ 


Inplo 


id 








1 


F 


60 - 


© — 


wild- 


cau 


ght dipIo