JOURNAL OF SHELLFISH RESEARCH VOLUME 6, NUMBER 1 JUNE 1987 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 Bigelow Laboratory for Ocean Science and Department of Marine Resources West Boothbay Harbor Maine 94575 Publications Committee Mr. Michael Castagna, Chairman The College of William and Mary Virginia Institute of Marine Science Wachapreague. Virginia 23480 Dr. Melbourne R. Carriker University of Delaware College of Marine Studies Lewes, Delaware 19958 Dr. Robert E. Hillman Battelle Ocean Sciences Duxbury. Massachusetts 02332 Dr. Roger Mann The College of William and Mary Virginia Institute of Marine Science Gloucester Point. Virginia 23062 Journal of Shellfish Research Volume 6, Number 1 ISSN: 00775711 June 1987 Editorial Comment It is an honor and a privilege to assume the duties of Editor of the Journal of Shellfish Research. Taking on a journal with an already established reputation for quality makes the task all the more enjoyable. Beginning in 1987. readers will note a few changes. The editorial board will be expanded and members will be expected to take more responsibility for reviewing manuscripts, suggesting authors for review articles and suggesting outside reviewers. Individual issues devoted to special topics will be published. These topics can be conceptual, geo- graphic or commemorative/memorial. The topics should be unifying, but at the same time the papers should represent various and diverse elements of that topic. The organizers of special symposia or workshops should consider JSR as an avenue for publication of their presentations. Those who are interested should contact the editor directly. Authors are encouraged to submit manuscripts dealing with any aspect of shellfish research. More papers on crustaceans will be most welcome. Review articles are also invited. The review process will continue as in the past. At least two outside reviews will be invited. Should those reviewers disagree, a third opinion will be solicited. I intend to keep the time element at a minimum. Reviewers will be asked to submit their opinions within two weeks of receipt of manuscripts. This will alleviate the frustrations often experienced by authors and will also serve to keep production of the JSR on schedule. It is my intention to see that the JSR is produced twice a year and on schedule. As many of you are aware, the JSR had experienced a number of problems over the past few years. Despite the difficulties, frustrations and problems of this time. Roger Mann has managed to produce a respected journal. Bob Hillman handled the production of Volume 6 and eased the transition period for me. Their efforts have been noticed and are greatly appreciated. We are grateful for their past, and future, guidance. I anticipate your criticisms and comments with enthusi- astic acceptance of the continuing need for improvement. Let us begin. — Sandra E. Shumway Marine Biological Laboratory LIBRARY JUL 2 9 1987 Woods Hole, Mass. Journal of Shellfish Research, Vol 6, No 1, 1-5. 1987. HOST-TO-HOST TRANSMISSION OF PERKINSUS MARINUS IN OYSTER (CRASSOSTREA VIRGIN 1C A) POPULATIONS BY THE ECTOPARASITIC SNAIL BOONEA IMPRESSA (PYRAMIDELLIDAE) M. E. WHITE,' 2 E. N. POWELL,1 S. M. RAY,3 & E. A. WILSON1 1 Department of Oceanography Texas A&M University College Station, TX 77843 USA; 2 Present address National Center for Atmospheric Research Advanced Studies Program Boulder, Colorado 80307 3 Department of Marine Biology Texas A&M University at Galveston Galveston, TX 77550 ABSTRACT Perkinsus ( = Dermocystidium) marinus is a primary cause of oyster mortality in the Gulf of Mexico. In a laboratory study, the ectoparasttic snail Boonea impressa was capable of transmitting P. marinus from one oyster to another. Thirty-six percent of oysters previously free of P. marinus contracted the disease when parasitized by B. impressa which had previously fed on infected oysters. P. marinus was subsequently found in the tissue of these snails. Feeding by B. impressa also resulted in the intensification of P. marinus infection in previously infected oysters. Consequently, B impressa may be a determining factor in the impact off. marinus in oyster populations. KEY WORDS: transfer Boonea, Odostomia, Perkinsus, Dermocystidium, Crassostrea, ectoparasitism, oyster. Pyramidellidae, disease INTRODUCTION Perkinsus (= Dermocystidium) marinus is a primary cause of oyster (Crassostrea virginica) mortality in the Gulf of Mexico and along the southeastern coast of the United States. P. marinus characteristically disseminates slowly from centers of infection within an oyster reef com- plex and spreads even more slowly from reef to reef (An- drews and Hewatt 1957; Mackin 1962). Consequently, in- fection is patchily distributed on many reefs, often with un- infected oysters immediately adjacent to infected oysters. This slow, sporadic pattern of dissemination suggests that host-to-host transfer through the water, now considered the primary mechanism (Ray 1954; Mackin 1962; Andrews 1965), is inefficient, perhaps because dilution reduces the number of infective elements below the dosage required to initiate new infections (Andrews 1979). The dissemination pattern suggests that a vector of low mobility also may be important in spreading the infection within oyster popula- tions. Hoese (1962) considered oyster predators and scav- engers as possible vectors and isolated P. marinus from several of them, but transmission to uninfected oysters seemed unlikely. Boonea impressa, a gastropod ectoparasite of oysters, is a potential candidate as a vector for P. marinus. Crassos- trea virginica is the primary host of B. impressa. Concen- trations of up to 100 snails per oyster have been observed (Hopkins 1956). with concentrations of 5-20 snails per oyster frequently found (White et al. 1984). Feeding of B. impressa, as in other pyramidellids. is accomplished by the attachment of a sucker, located at the end of the proboscis. Following attachment, the host's body wall is pierced by the snail's buccal stylet and the host's body fluids sucked by means of a buccal pump (Fretter and Graham 1949; Allen 1958; Maas 1965). The presence of large salivary glands and the location of duct openings near the tip of the proboscis suggest that B. impressa may inject saliva into the host. A similar method of feeding is employed by mos- quitos which are known to transmit diseases, such as ma- laria, from one host to another. Leeches feeding on alli- gators also are able to transmit disease (Glassman et al. 1979). Like leeches and mosquitos. B. impressa can change hosts frequently (White et al. 1984) but normally excursions are limited to relatively small areas within the host population. Consequently, the dissemination pattern of P. marinus would correlate with the feeding pattern of B. impressa, if B. impressa was a vector. Boonea impressa significantly depresses growth rates in oysters (White et al. 1984). Thus, besides direct transmis- sion, the added stress of parasitism by B. impressa may exacerbate infections already present or lower the oyster's resistance to new infection by P. marinus. Here, we report the results of experiments designed to ascertain the role of B. impressa in the transmission and intensification of in- fection by P. marinus in oysters. White et al. MATERIALS AND METHODS Transmission of Perkinsus by Boonea Approximately 80 market-sized oysters uninfected with P. marinus were obtained from Lake Borgne, Louisiana and transferred to a flowing seawater system at the Port Aransas Marine Laboratory of the University of Texas. In- fected oysters were collected from Confederate Reef, West Bay, Texas. At the time of collection, 100% of the Confed- erate Reef oysters were infected with an average intensity of 3.5 (n = 25) on the 5 point scale commonly used to quantify infection (Mackin 1962). To demonstrate host-to-host transmission, uninfected oysters were exposed to snails which previously had fed on oysters infected with P. marinus. These snails were col- lected from Confederate Reef and allowed to feed on Con- federate Reef oysters until use. At the start of the experi- ment, some of these snails were placed on individual, unin- fected oysters from Lake Borgne. To ensure that Lake Borgne oysters were continually exposed to P. marinus, every 2 weeks the snails were replaced by new snails ob- tained from the same stock feeding on highly infected Con- federate Reef oysters. Two types of controls were needed to determine the method of Perkinsus transmission: oysters without snails and oysters parasitized by uninfected snails. The latter group was necessary to distinguish increased susceptibility to P. marinus in the water from actual host-to-host trans- mission, both of which could be produced by snail para- sitism. Snails were not present at Lake Borgne, nor were disease-free populations present in any of about 20 reefs examined in the Aransas Bay-Copano Bay and Galveston Bay-West Bay area of the Texas Coast. Because snails could not be obtained from a P. marinus-free oyster popu- lation, snails were obtained from an oyster population with a lower infection intensity. The collection site was a reef along Morris and Cummings Cut near Shellbank Island, Harbor Island, Texas. Seventy-five percent of oysters from this site were infected with Perkinsus marinus with an in- tensity of 2.25 (n = 12). We then assumed that frequent snail contact with infected oysters was necessary for P. marinus transmission. (This, in fact, was the case — see Results). Consequently, we maintained these snails on un- infected oysters from Lake Borgne for several weeks prior to the experiment and then transferred them to new Lake Borgne oysters at the beginning of the experiment. To min- imize further exposure of these oysters to Perkinsus, these snails were never replaced during the experiment. The transmission experiment began on November 23, 1984. Oysters were placed individually in PVC containers covered with coarse cloth and closed with rubber bands to prevent snail immigration or emigration. Unfiltered sea- water was fed by gravity into each container from a central reservoir receiving seawater from the laboratory's flowing seawater system. All containers were located in parallel rather than in series to isolate each container from all others during the experiment. A set of 15 oysters in individual containers was used for each of the three groups, two con- trol groups and the experimental group. Each group was placed in a tray elevated above the water level in the sea table to isolate them from the common outflow. Seven snails were placed on each oyster in the experimental group and the one control group. The experiment was terminated at the end of 2 months. Snail and oyster mortality was insignificant in any group. Oysters and snails were assayed for P. marinus using the thioglycollate method of Ray ( 1966). The entire mantle and rectum of each oyster was examined. Snails were crushed and the entire organism placed in the thioglycollate me- dium. Effect of B. impressa on infection intensity Experiments began on October 29, 1983 and April 8, 1984 and ran for 1 month. In both experiments, market- sized oysters were collected from Confederate Reef and di- vided into 3 groups with 0. 10 and 30 snails per oyster, respectively. Snails were collected from Mud Island Reef, Aransas Bay, Texas and were not replaced for the duration of the experiment. Conditions of the experiment were as described previously, except no effort was made to isolate groups by using elevated trays. At the end of 1 month, the oysters were sacrificed and P. marinus assayed as pre- viously described. RESULTS Transmission of Perkinsus by Boonea Boonea impressa was capable of transmitting Perkinsus marinus from one oyster to another (Table 1 ). No oysters in either control group became infected during the 2-month experiment. Thirty-six percent of the experimental oysters were infected. That is, 100% of all infected oysters had been fed upon by infected snails. If the occurrence of dis- ease in oysters was unaffected by snail parasitism, an equal distribution of Perkinsus infection among the three groups of oysters would be expected. The results are significantly different from this expectation (P = 0.007). If snail feeding merely increased the oysters' susceptibility to Per- kinsus infection, an equal distribution of infected oysters among the two groups of oysters parasitized by B. im- pressa, the one control group and the experimentals, would be expected. The results are also significantly different from this expectation (P = 0.06) (both tests were two- sided Kolmogorov one-sample tests with exact critical levels for discontinuous distributions calculated from Con- over 1972). All infected oysters had a very light intensity of infection (0.5 on the 5 point scale of Mackin, 1962). Infec- tions were localized in discrete portions of the mantle; only the most heavily infected oyster (still a very light infection) had P. marinus in the rectal tissue. The entire mantle of Transmission of Perkinsus marinus in Oyster TABLE 1. Percent of originally disease-free oysters which contracted Perkinsus marinus after two months. (Experimental oysters were exposed to P. marinus in infected snails, w hereas control oysters with snails were fed upon by uninfected snails. ) The percent of snails infected w ith P. marinus after feeding on control and experimental oysters. Crassostrea rirginica Controls Experimentals without snails with snails Percent infected with Perkinsus 0% 0% 15 15 36% 14 Boonea impressa Snails on Control Oysters 0% Snails on Experimental Oysters 39% 18 each oyster was searched for P. marinus. In all cases most of the mantle of infected oysters remained uninfected, as if new infections were localized near the point of B. impressa feeding. Thirty-nine percent of the snails on the experimental oysters were infected with P. marinus at the end of the experiment. Infections ranged from very light to moderate (0.5-3.0) in these snails. In contrast, snails removed from the control group were all uninfected. This suggests that snails retain the infection for only a short time after feeding on infected oysters, at least during the winter months. Effect of B. impressa on infection intensity Boonea impressa also increased the intensity of infec- tion in oysters. In the Fall 1983 experiment, oysters with 30 snails per oyster had significantly higher intensities of in- fection than controls or oysters with only 10 snails per oyster (chi-square test of medians; 0.05 < P < 0.10). Me- dian infection intensity in oysters with 30 snails was as high as theoretically possible [5 on Mackin's scale (1962)]. The experiment was repeated in Spring 1984 with similar re- sults. The infection intensity of the control group was sig- nificantly lower than either the group with 10 or 30 snails per oyster (0.05 < P < 0. 10). The highest infection inten- sity (5) was found only in oysters parasitized by snails (Table 2). The possibility that both significant effects oc- curred by random chance is small (P < 0.01, binomial test). We checked these results using an alternative test, the Mann-Whitney test modified for samples with ties of con- siderable extent (Conover 1980). Results were comparable. Oysters parasitized by 30 snails had significantly higher in- fection intensities than oysters with no snails (one-sided test; P < 0.05 for Fall 1983; P < 0.01 for Spring 1984). In both experiments, the proportion of oysters with an infec- tion intensity of 5 was highest in the group with 30 snails and lowest in the controls. This hierarchy of groups based on the proportions of oysters with an infection intensity of 5 (controls « 10 snails per oyster =£ 30 snails per oyster) is significant (Page's test for ordered alternatives. P = 0.05, see Carman and Thistle 1985 for discussion of this test). DISCUSSION Transmission of Perkinsus by Boonea Unquestionably, Boonea impressa can transmit Per- kinsus marinus from infected to uninfected oysters. Only oysters fed upon by B. impressa, which were previously exposed to infected oysters, became infected. Moreover, P. marinus was found in the tissues of B. impressa that had fed on infected oysters (Table 1). Infection intensity in the snails used in this experiment was light to moderate, but we have observed even heavier infections in other snails from other experiments (Figure 1). If snail feeding merely in- creased the oyster's susceptibility to P. marinus, the pro- portion of infected oysters would be expected to be the same in the control group parasitized by snails and the ex- perimental group because both groups were fed upon by the same number of snails. None of the control oysters became infected, however. Consequently, infected snails must have transmitted P. Marinus Snail parasitism resulted in infection in the mantle. Elimination of P. marinus by oysters is primarily by phago- TABLE 2. Infection intensity in Confederate Reef oysters after 1 month parasitism by Boonea impressa 0 10 30 Snails Snails Snails FALL. 1983 Percent with infect on intensity of 5 37% 53% 63% median 4 4 5 mean ± standard deviation 4.03 ±1.12 4.18 ± 1 19 4.32 ± 1 10 number 19 19 19 SPRING, 1984 Percent with infection intensity of 5 0% 10% 38% median 3 4 4 mean ± standard deviation 2.95 ± 1.34 3.60 ± 1.07 4.00 ± 1 .31 number 10 10 8 White et al. Figure 1. A specimen of Boonea impressa, heavily infected with Perkinsus marinus; after 2 weeks culture in thioglycollate medium. cytosis and egression of phagocytes, probably through the gut epithelium because egression through the mantle epi- thelium apparently is precluded (Cheng 1967). Conse- quently, if B. impressa transmitted P. minimis, P. minimis should be present initially in the mantle tissue and only later in the rectum as phagocytes attempted to remove the cells. Significantly, all infected oysters had localized infec- tions in the mantle tissue, but only the most highly infected oyster showed any evidence of P. marinus in the rectal tissue. Thus, the distribution of P. marinus is consistent with the observed method of transmission by B. impressa during feeding. Transmission could have occurred in one of two ways. Either P. marinus was directly injected into the oyster from infected snails during feeding or P. marinus was trans- ferred through the water the short distance from parasite to host during feeding, perhaps entering the wound made by the snail. In either case. B. impressa was required for the spread of P. marinus to uninfected oysters in this experi- ment. Temperature is an important factor limiting transmission and intensification of P. marinus infection (Hewatt and Andrews 1956; Andrews 1965). Low temperatures usually reduce intensity and percent infection. The transmission experiment was run during the cooler part of the year, yet P. marinus transmission occurred in 36% of the available oysters. Low temperatures probably were also responsible for infections remaining localized in the mantle tissue. Transmission and intensification of P. marinus infections by B. impressa may be even more successful during the warmer summer months when temperatures are more con- ducive to the growth of P. marinus. For B. impressa to be an effective vector, the snails must change hosts from time to time. White et al. (1984) demonstrated that B. impressa migrated from one oyster clump to another frequently, covering distances of several meters in 1 week. Proximity is an important factor in trans- mission of P. marinus between oysters (Andrews 1967. 1979). The patchy distribution of P. marinus and the slow rate of dissemination from centers of infection in oyster populations are consistent with the known behavior of B. impressa, if it were important in P. marinus transmission in the field. Effect of B. impressa on infection intensity The intensity of P. marinus infection was increased by B. impressa parasitism. One month of parasitism by 30 snails was sufficient to increase infection intensity in market-sized oysters an average of 1 point on the 5 point scale of Mackin (1962), from moderate, 3.0, to moderately heavy, 4.0, for example. Infection intensity increased more in the spring experiment probably because initial infection Transmission of Perk/nsus marinus in Oyster intensities were lowest, allowing a greater span of possible increase to occur, and because rising temperatures in the spring are conducive to rapid increases in infection inten- sity. Clearly, if B. impressa occurs in sufficient numbers, it may significantly increase the infection intensity of P. marinus in oysters and, thus, may be an important contrib- uting factor in oyster mortality. CONCLUSIONS White et al. ( 1984) showed that feeding by B. impressa decreased oyster growth rates significantly. To this can now be added the interaction of B. impressa with one of the most important disease-producing organisms in oysters, P. marinus. The life cycle of P. marinus and previous studies on the transmission of P. marinus have demonstrated the importance of transmission through the water (Mackin and Boswell 1955; Andrews 1965; Perkins and Menzel 1966). Nevertheless, transmission by this method is obviously slow because dilution of infective elements occurs. Boonea impressa offers a potentially significant method of trans- mission because this dilution step is bypassed. Even in winter, the least favorable time for P. marinus transmis- sion, 36% of the experimental oysters exhibited localized, low intensity infections. Although the importance of B. im- pressa in the life cycle of P. marinus remains to be shown in the field, laboratory studies, demonstrating that B. im- pressa can affect the transmission and intensity of an im- portant disease in oysters, indicate that B. impressa may play an important role in the health of oyster populations. ACKNOWLEDGMENTS We thank M. Mysing-Gubala and T. Soniat for their help in obtaining P. marinus-free oysters from Lake Borgne. M. Mysing-Gubala generously shared her data on P. marinus incidence. We thank the Port Aransas Marine Laboratory of the University of Texas for providing space and C. Kitting for helping maintain the experiments in the flowing seawater system. Dr. R. E. Hillman, Dr. J. D. Andrews. Mr. J. Parrack, and an anonymous reviewer pro- vided helpful suggestions that improved the manuscript. The research was funded by an institutional grant #NA83AA-D-00061 to Texas A&M University by the Na- tional Sea Grant College Program, National Oceanic and Atmospheric Administration, U.S. Department of Com- merce to EP and SR and by a Sea Grant Marine Fellowship and a National Center for Atmospheric Research Postdoc- toral Fellowship to MW. The National Center for Atmo- spheric Research is sponsored by the National Science Foundation. Allen, J. 1958. Feeding habits of two species of Odostomia 72:11-15. Andrews, J. D. 1965. Infection experiments in nature with Dermocysti- dium marinum in Chesapeake Bay. Chesapeake Sci. 6:60-67. Andrews. J. D. 1967. Interaction of two diseases of oysters in natural waters. Proc. Nati. Shellfish. Assoc. 57:38-49. Andrews, J. D. 1979. Oyster diseases in Chesapeake Bay. U.S. Nat. Mar. Fish. Sen\ Mar. Fish. Rev. 41(1-21:45-53. Andrews, J. D. & W. G. Hewatt. 1957. Oyster mortality studies in Vir- ginia. II. the fungus disease caused by Dermocystidium marinum in oysters of Chesapeake Bay. Ecol. Monogr. 27:1-25. Carman, K. R. & D. Thistle. 1985. Microbial food partitioning by three species of benthic copepods. Mar. Biol. [Berl.) 88:143-148. Cheng, T. C. 1967. Marine molluscs as hosts for symbioses with a review of known parasites of commercially important species. Adv. Mar. Biol. 5:1-424. Conover. W. J. 1972. A Kolmogorov goodness-of-fit test for discon- tinuous distributions. J. Am. Stat. Assoc. 67:591-596. Conover, W. J. 1980. Practical nonparametric statistics. 2nd edition. New York, NY: John Wiley & Sons, 493 p. Fretter, V. & A. Graham. 1949. The structure and mode of life of the Pyramidellidae, parasitic opisthobranchs. J. Mar. Biol. Assoc. U.K. 28:493-532. Glassman, A. B.. T. W. Holbrook & C. E. Bennett. 1979. Correlation of leech infestation and eosinophilia in alligators. J. Parasitol. 65:323- 324. Hewatt, W. G. & J. D. Andrews. 1956. Temperature control experiments REFERENCES CITED . Nautilus. on the fungus disease, Dermocystidium marinum, of oysters 1. Proc. Natl. Shellfish. Assoc. 46:129-133. Hoese, H. D. 1962. Studies on oyster scavengers and their relation to the fungus Dermocystidium marinum. Proc. Natl. Shellfish. Assoc. 53:161-174. Hopkins, S. 1956. Odostomia impressa parasitizing southern oysters. Science (Wash. DC). 124:628-629. Maas, D. 1965. Anatomische und histologische Untersuchungen am Mundapparat der Pyramidelliden. Z. Morphol. Oekol. Tiere. 54:566- 642. Mackin, J. G. 1962. Oyster disease caused by Dermocystidium marinum and other microorganisms in Louisiana. Publ. Inst. Mar. Sci. Univ. Tex. 7:132-229. Mackin, J. G. & J. L. Boswell. 1955. The life cycle and relationships of Dermocystidium marinum. Proc. Natl. Shellfish. Assoc. 46:112-115. Perkins, R. O. & R. W. Menzel. 1966. Morphology and cultural studies of a motile stage in the life cycle of Dermocystidium marinum. Proc. Natl. Shellfish. Assoc. 56:23-30. Ray, S. M. 1954. Experimental studies on the transmission and pathoge- nicity of Dermocystidium marinum. a fungus parasite of oysters. J. Parasitol. 40:235. Ray. S. M. 1966. A review of the culture method for detecting Dermo- cystidium marinum. with suggested modifications and precautions. Proc. Natl. Shellfish. Assoc. 54:55-69. White. M. E., E. N. Powell & C. L. Kitting. 1984. The ectoparasitic gastropod Boonea ( = Odostomia) impressa: population ecology and the influence of parasitism on oyster growth rates. P. S.Z.N. I.: Mar. Ecol. 5:283-299. Journal of Shellfish Research, Vol. 6, No. I, 7-15, 1987. A COMPARATIVE STUDY OF THE REPRODUCTIVE CYCLE OF THE SOFT-SHELL CLAM, MYA ARENAR1A IN LONG ISLAND SOUND DIANE J. BROUSSEAU Dept. of Biology Fairfield University Fairfield, CT 06430 ABSTRACT Three populations of Mya arenaria in Long Island Sound were studied during a 2.5-yr period to determine the se- quence of gametogenic development of gonadal tissue and the frequency and duration of spawning under natural conditions. Some individuals in the population at Stonington. CT spawned twice annually, while the two populations in Westport, CT exhibited a single spawning cycle. There was no evidence of hermaphroditism or protandry in any of the populations studied. Sex ratios of M. arenaria in each of the three populations did not differ significantly from 1:1 . Photomicrographs of the gametogenic cycles of both male and female clams are included. KEY WORDS: Reproduction; Spawning; Soft-shell clam; Long Island Sound INTRODUCTION Even though a considerable amount of information on aspects of the reproductive cycle of the soft-shell clam, Mya arenaria, is available in the literature (Landers 1954; Pfitzenmeyer 1962; Shaw 1962; Ropes and Stickney 1965; Munch-Petersen 1973; Porter 1974; Brousseau 1978; Began 1979) there is surprisingly little known about the spawning pattern of this species in Long Island Sound. In the only study reported, Coe and Turner (1938) indicated that M . arenaria from New Haven, Connecticut spawn in early June. These investigators did not. however, rule out the possibility that multiple spawnings may occur. Reported here are the results of a 2.5-yr study of spawning and gametogenic patterns in three populations of Mya arenaria, which was carried out as part of a broader study of the population dynamics of this species along the Connecticut shore of Long Island Sound. MATERIALS AND METHODS Monthly collections of Mya arenaria were made from three intertidal clamflats, one located at Barn Island in Stonington (lat. 41°20'N;long. 71°53'W). the second in the Saugatuck River in Westport (lat. 41°06'N;long. 73°23'W) and the third at Old Mill Beach in Wesport (lat. Figure 1. Map showing locations of the three study sites: Barn Island in Stonington, CT (A), Saugatuck River in Westport, CT (Bl and Old M Beach in Westport, CT (C). Brousseau Figure 2. Photomicrographs of gonadal stages of male and female Mya arenaria at 125 x magnification, al Indifferent male, 13 July 1985, Westport 1, hi Developing male, 29 May 1984, Westport 1, c) Ripe male, 7 May 1985, Westport 2, dl Spawning male, 3 June 1985, Stonington, e) Spent male, 6 June 1985, Westport 2, f) Indifferent female, 3 June 1985, Stonington, g) Developing female, 15 August 1985, Stonington, h) Ripe female, 7 May 1985, Westport 2, i) Spawning female, 4 June 1985, Westport 1, j) Spent female, 6 June 1985, Westport 2. (o = oocyte; p = pycnotic cell; s = spermatozoan). 41°07'N;long. 73°21 'W) (Figure 1). Sampling at the Ston- shell length from Stonington; 25 to 228 clams, 22.3-82.35 ington site began in June 1983 and at the Westport sites in mm shell length from Saugatuck River, Westport (Westport March 1984. The study was completed in December 1985. 1); and 11 to 193 clams, 21.3-90.0 mm shell length from Sample sizes varied from 22 to 199 clams, 19.0-94.45 mm Old Mill Beach, Westport (Westport 2). A total of 3,854 Reproductive Cycle of Mya j • v v Ik. _ ^ O * t. t ?•>■' > « ♦> v V * i V* "V " * * • •* O 7m Figure 2. Continued clams were examined ( 1 ,583 from Stonington; 1 ,243 from Saugatuck River; 1,028 from Old Mill Beach) and used in the analysis of the reproductive cycles in these three popu- lations. In the laboratory, each clam was numbered and its max- imum shell length determined to the nearest 0.1 mm. The visceral mass (gonad, liver and gastrointestinal tract) was removed and fixed in 10% buffered formalin. The M. are- naria tissues were then prepared histologically for exami- nation according to the method described by Brousseau (1978). A microscopic examination was made of the vis- ceral mass gonadal tissue before assigning each individual to one of five categories of gonadal condition: indifferent, developing, ripe, partially spawned and spent. Photomicrographs of representative stages of the male and female reproductive cycle were taken with a light mi- croscope at 125 x magnification using a 35 mm camera. High contrast Panatomic X ASA 32 film was used. Categories of Gonad Condition The following descriptions of the male and female de- velopmental stages represents an attempt to divide the re- productive process (either spermatogenesis or oogenesis) into distinct phases. The criteria used are based solely on morphological observations. Categories used previously for Mya arenaria (Ropes and Stickney 1965; Brousseau 1978) and for other species of bivalves (Porter 1974; Keck et al. 1975, for Mercenaria mercenaria; Brousseau 1981, for Petricola pholadiformis; Brousseau 1982, for Geukensia demissa; Brousseau 1984, for Anomia simplex) were used in this study where appropriate. Developmental Stages of the Male. During the indif- ferent stage the follicles contain the aberrant forms, multi- nucleated non-pycnotic cysts and pycnotic cells, as de- scribed by Coe and Turner (1938). The basal membrane and follicle cells are the dominant structural elements. A few primary spermatocytes or spermatogonia are visible at the periphery of the lumen (Figure 2a). In the developing stage, the maturation and proliferation of the spermatocytes takes place. Early development is characterized by the expansion of the follicle, increase of the number of primary spermatocytes at the basal mem- brane of the follicles and the appearance of some sper- matids. As development proceeds, the spermatids begin 10 Brousseau STONINGTON u J FMAMJJASOND 1983 WESTPORT 1 WESTPORT 2 z LU o 111 Q. 80- 60- { 1 40- In 1 40- 20- 1 .1 . . . lull J FMAMJJASOND 1984 FMAMJJASOND 1984 J FMAMJJASOND 1984 ! ] | '™ " 80- 80- I "... y i 1 1 1 1 . -■-■ ■ L....iiiul J1..H11L J FMAMJJASOND 1985 J FMAMJJASOND 1985 J FMAMJJASOND 1985 MONTHS Figure 3. Proportions of Mya arenaria populations with active or inactive gonads during 1983, 1984 and 1985. Open portions of each bar represent inactive gonads (indifferent, no gametogenesis or spent); solid portions represent active gonads (developing, ripe or partially spawned). Observations on males and females are combined. migrating toward the center of the follicle where they ar- range themselves in radial columns. Proliferation of sper- matids and the differentiation into spermatozoa follows (Figure 2b). In the ripe male clam the follicle is filled with dense radiating bands of spermatozoa, the tails of which project into the central lumen (Figure 2c). In the partially spawned stage the follicle is character- ized by fewer spermatozoa than the ripe clam. A few sper- matozoa remain in the radiating bands but the rows of fol- licle cells gradually increase to replace the spawned sper- matozoa (Figure 2d). In the spent male, the follicles are almost completely filled with follicle cells and the reduced lumen contains a few sex cells (Figure 2e). Developmental Stages of the Female. In the indifferent stage, the distinctive female inclusions (Coe and Turner, 1938) are visible in the follicle cells. Extremely small pri- mary oocytes are present in the alveolar membrane (Figure 2f). The developing stage is characterized by an increase in the number and the size of oocytes. The elongation of the primary oocytes on stalks is accompanied by a decreased number of follicle cells and inclusions. A central lumen is present in each follicle, into which protrude the stalked oo- cytes. There is the gradual appearance of a nucleolus and amphinucleolus in the maturing oocytes (Figure 2g). In the ripe female there are many mature, spherical oo- cytes, 65-70 um in diameter, that appear to be free within the follicular lumen (Figure 2h). Reproductive Cycle of Mya II STONINGTON INDIFFERENT ^ DEVELOPING = RIPE PART SPAWN SPENT 100 60 II II ! I I I J F M A M J J A S 1983 0 N D ~Z_ 60- UJ O DC LU 40- Q. I T i T ^ J F M A 1 II I t ' i 'i M J J A S 1984 111 0 N D 60 11, "IP S S = 20 I I I ' I J F M A Iff § § I § i i in ■ ■■OS 1) III i I S fc 8 11 M J J A S 1985 MONTHS 0 N D Figure 4. Proportion of Mya arenaria from Stonington with gonads in each developmental phase during 1983-1985. In the partially spawned stage, the mature oocytes are gradually discharged. Very small oocytes are embedded in the follicle cells at the periphery of the empty alveoli. Nu- tritive inclusions are visible in many of the follicle cells. Emptying follicles and the cessation of oogenesis in all fol- licles is characteristic of this stage (Figure 2i). In the spent stage, unspent oocytes in the early phases of cytolysis are present. These appear in the lumen as large. WESTPORT 1 INDIFFERENT ^ RIPE DEVELOPING = PART SPAWN SPENT l 100 20- 0-1 1 ^ i ^ 1 LU o cc LU 0. 100 J FMAMJ JASOND 1984 fill ■*■ i «!!!§ 1 Ifjlf U g m* g J FMAMJ JASOND 1985 MONTHS Figure 5. Proportions of Mya arenaria from Saugatuck River, West- port (Westport 1) with gonads in each developmental phase during 1984-1985. Brousseau WESTPORT 2 INDIFFERENT ^ RIPE DEVELOPING = PART SPAWN o DC III Dl 100 H 0-1— , r— ^ Z J F M o 1 MX '■' ■■■ '■' Y T A S 0 N D 60 40 IfflF Hi II is ■ ■ ■i"i n ^ ^ ~r J FMAMJ JASOND 1985 MONTHS Figure 6. Proportions of Mya arenaria from Old Mill Beach, West- port (Westport 2) with gonads in each developmental phase during 1984-1985. darkly staining bodies with obscure nuclei. Follicle cells begin to reinvade the follicles from the basal membrane (Figure 2j). RESULTS Reproductively active individuals were encountered at the Stonington study site throughout the 2.5-yr study period with the exception of October 1984 (Figure 3). Low levels of gametogenic activity were observed in both sexes in No- vember of each year, but by mid-May 40-60% of the indi- viduals in the population were ripe (Figure 4). In 1985 spawning began as early as April, however, the majority of individuals released gametes during the month of June. A second ripening of gonads occurred during the late summer-early fall. This event was most pronounced during the 1983 breeding season. Fewer clams participated in the second spawning cycle during 1984 and 1985 than during the previous year. There is some indication that the larger (older) clams in the population are more likely to undergo multiple spawnings than are the smaller (younger) ones (Brousseau unpubl.). Reproductively active individuals were found in all samples taken at the Saugatuck River (Westport 1) study site except those collected during August and October of 1984 (Figure 3). Gametogenic activity resumed in the fall in both sexes and by mid-May of both years, 40-50% of the individuals in the population were ripe (Figure 5). Ga- mete release was completed in July. Reproductively active clams were encountered at the Old Mill Beach (Westport 2) study site throughout the 2-yr study period except during the months of October and No- vember 1984 and November 1985 (Figure 3). Gametogenic activity was again evident through the fall and winter months and ripe individuals were first encountered in April (Figure 6). Spawning began in late April and was com- pleted in July. The proportion of females in all size-classes at Ston- ington (n = 1,583), Saugatuck River, Westport (n = 1,243) and Old Mill Beach, Westport (n = 1,028) did not differ significantly from one-half (Table 1). Male and fe- male gonads were usually indistinguishable in clams <20.0mm shell length since they were immature. No evi- dence of hermaphroditism or protandry was observed. DISCUSSION Mya arenaria is a dioecious pelecypod, the sexes of which are distinguishable only after examination of the gonads. The extremely low levels of hermaphroditism in this species suggest that M. arenaria is a strictly gonocho- ristic species. Only four hermaphrodites were reported in a combined total of over 10,000 clams examined in five studies (Coe and Turner 1938; Shaw 1965; Porter 1974; Brousseau 1978; this study). Indirect evidence from sex ratios also reveals no evidence that M. arenaria undergoes protandric development. In the five studies containing in- formation on the proportion of males to females, sex ratios of 1:1 were reported in all populations (Coe and Turner 1938; Shaw 1965; Porter 1974; Brousseau 1978; this study). The body of literature dealing with reproductive patterns of Mya arenaria is extensive when compared with other species of marine bivalves. This is largely due to the species' commercial value. The results of gonad examina- tions completed in this study indicate that M. arenaria from Westport, Connecticut spawn only once, during the summer months, whereas there is evidence those from Reproductive Cycle of My a 13 TABLE 1. Proportion of females in eaeh size class in populations of Mya arenaria from Barn Island, Stonington, Saugatuck River, Westport (Westport 1 1 and Old Mill Beach, Westport (Westport 2). (Number of individuals per sample is given in parentheses). Stonington Saugatuck R. Old Mill Beach Size Class Proportion Confidence Proportion Confidence Proportion Confidence (mm) Females Limits (95%) Females Limits (95%) Females Limits (95%) 20.0-24.9 .50(2) .50 (4) .50(2) 25.0-29.9 .43 (23) 24 -.63 .39(31) 21-56 .41 (39) .25-. 57 30.0-34.9 .60 (30) 41-. 77 .35 (66) 23-. 47 .37 (49) .24-. 52 35.0-39.9 .54(84) 43 -.65 .47(133) 39-. 55 .54 (61) .41 -.67 40.0-44.9 .53 (85) 42-. 64 .48 (245) 42 -.54 .45 (87) .34 -.56 45.0-49.9 .51 (110) 41-61 .52(256) 46-. 58 .50(130) .41-. 59 50.0-54.9 .52 (166) 44-. 60 .56 (232) 50-. 62 .51 (193) .44-. 58 55.0-59.9 .48 (251) 42-. 52 .57(153) 49-. 65 .56(183) .48-. 63 60.0-64.9 .50 (268) 44-.56 .46(71) 34-. 58 .51 (144) .42-. 59 65.0-69.9 .55 (234) 48-. 61 .53 (32) 30-. 64 .52(76) .41-. 64 70.0-74.9 .53 (165) 46-. 61 .43 (14) 17-. 69 .58 (48) .28-. 56 75.0-79.9 .60 (91) 49-.70 .25 (4) 00-. 67 .62(21) .41-83 80.0-84.9 .57(53) 43-. 70 1.00(2) .40(5) .00-. 83 85.0-89.9 .60 (15) 32-. 84 — — 90.0-94.9 .67 (6) 29-96 — .oo m Total .52 (1583) 50-. 55 .50(1243) 47-. 53 .51 (1028) .48-. 54 Stonington, Connecticut may spawn twice each year. The second spawning could be a facultative event which occurs only when environmental conditions are favorable. In their review of reproductive cycles of marine bi- valves, Giese and Pearse (1974) concluded that the repro- ductive cycles of bivalves tend to be extended in the southern portions of a species geographic range (Orton's Rule). Information available on M. arenaria, however, in- dicates that spawning in widely separated populations of this species occurs at different times and with varying fre- quency (Figure 7). Even in this study there is general dis- agreement with Orton's Rule. The population from Ston- ington, Connecticut may exhibit a biannual spawning cycle in some years, but clams from the two Westport pop- ulations which are located slightly to the south, spawn once annually. This finding for M. arenaria supports the argu- ment made by Newell et al. (1982) in work on Long Island populations of Mytilus edulis, namely that latitudinal ef- fects on the reproductive cycle of marine bivalves are sec- ondary to the effects of habitat-specific exogenous factors such as temperature and food supply. As more information on bivalves is gathered, it becomes clear that the traditional view of fixed patterns of spawning based on latitudinal range is inadequate. ACKNOWLEDGMENTS I wish to thank K. Gahwyler, A. Gomes, G. Micalizzi, G. Pfund, S. Prunk, J. Smeriglio. J. Trautman and J. Wachter for technical assistance in the field and the histo- logical preparation of some of the material used in this study. Photomicrographs were taken by A. Gomes, G. Mi- calizzi and S. Prunk. Financial support for this study was provided under grant number NA82AA-D-00018 of the Connecticut Sea Grant Program. 14 Brousseau NO. AMERICA EAST COAST NO. AMERICA ■ WEST COAST EiifiC^E MALPEQUE BAY do. ST. ANDREWS do. EASTERN MAINE BOOTHBAY HARBOR ROBINHOOD COVE do. GLOUCESTER PLUM ISLAND SOUND N. of CAPE COD N. ol BOSTON IPSWICH PLYMOUTH SOUTHERN CAPE COD do. CHATHAM MARTHA'S VINEYARD WOODS HOLE RHODE ISLAND WICKFORD do. do. STONINGTON NEW HAVEN WESTPORT NEW JERSEY do. CHESAPEAKE BAY do. do. do. do. PUGET SOUND ROSKILDE FJORD BLACK SEA STAFFORD 1912 SULLIVAN 1948 STAFFORD 1912 BATTLE 1932 ROPES & STICKNEY BROUSSEAU 1978 ROPES & STICKNEY BELDING 1930 STEVENSON 1907 1907 1930 STEVENSON 1907 DEEVEY 1948 BUMPUS 1898 MEAD & BARNES 1904 LANDERS 1954 do BROUSSEAU COE & TURNER 1938 ROUSSEAU BELDING 1930 NELSON & PERKINS 1931 ROGERS 1959 1956 PFITZENMEYER 1962 ■ do. |1958 do. ■ 1959 do. MUNCH-PETERSEN 1973 BEGAN 1979 Figure 7. The duration of the spawning season of Mya arenaria reported in the literature. Reproductive Cycle of Mya 15 REFERENCES CITED Battle, H. I. 1932. Rhythmic sexual maturity and spawning of certain bivalve mollusks. Contrib. Can. Biol. Fish.. New Series 7:255-276. Began. Yu. P. 1979. Reproduction and growth of Mya arenaria in the Black Sea. Sov. J. Mar. Biol. 5:521-523. Belding. D. L. 1907. Report on the shellfisheries of Massachusetts. In Rcpt. Comm. Fish, and Game 1906, Commonwealth of Massachu- setts, Public Doc. 25:46-67. Belding. D. L. 1930. The soft-shell clam fishery of Massachusetts. Mass. Dept. Conserv., Div. Fish Game, Mar. Fish. Sen1. 1. 65p. Brousseau. D. J. 1978. Spawning cycle, fecundity and recruitment in a population of soft-shell clam, Afya arenaria from Cape Ann, Massa- chusetts. Fish. Bull. U.S. 76:155-166. Brousseau. D. J. 1981. Spawning cycle and fecundity in a population of Petricola pholadiformis (Pelecypoda:Petricolidae) from Milford, Con- necticut. Veliger 24:56-61. Brousseau. D J 1982. Gametogenesis and spawning in a population of Geukensia demissa (Pelecypoda:Mytilidae) from Westport, Connect- icut. Veliger 24:247 '-251. Brousseau. D. J. 1984. Reproductive cycle of Anomia simplex (Pelecy- poda:Anomiidae) from Cape Cod. Massachusetts. Veliger 26:299- 304. Bumpus. H. C. 1898. The breeding of animals at Woods Hole during the months of June. July and August. Science. New Ser. 8:850-858. Coe, W. R. & H. J. Turner. Jr. 1938. Development of the gonads and gametes in the soft-shell clam (Mya arenaria). J. Morphol. 62:91 — 111. Deevey. G. B. 1948. The zooplankton of Tisbury Great Pond. Bull. Bingham Oceanogr. Collection 12:1-44. Giese, A. C. & J. S. Pearse. 1974. Introduction — General Principles (Chapt. 1). Reproduction in marine invertebrates (A. C. Giese and J. S. Pearse. eds.) 1:1-49. Academic Press, New York. Keck. R. T.. D. Maurer & H. Lind. 1975. A comparative study of the hard clam gonad developmental cycle. Biol. Bull. (Woods Hole) 148:243-258. Landers. W. S. 1954. Seasonal abundance of clam larvae in Rhode Island waters. 1950-52. U.S. Fish. Wildl. Serv. Spec. Set. Rep. Fish. 117. 29 p. Mead, A. D. & E. W. Bames. 1904. Observations on the soft-shell clam. R.I. Comm. Inland Fisheries. 34th Ann. Repl. 29-68. Munch-Petersen, S. 1973. An investigation of a population of the soft- shell clam (Mya arenaria L.) in a Danish estuary. Medd. Dan. Fisk. Havunders., New Ser. 7:47-73. Nelson, T. C. & E. B. Perkins. 1931. Annual Report of the Dept. of Biology, July 1. 1929-June 30. 1930. N.J. Agric. Expt. Stat. Bull. No. 522:1-47. Newell. R. E. I.. T. J. Hilbish. R. K. Koehn & C. J. Newell. 1982. Temporal variation in the reproductive cycle of Mytilus edulis (Bi- valvia. Mytilidae) from localities on the east coast of the United States. Biol. Bull. Woods Hole) 162:299-310. Pfitzenmeyer. H. T. 1962. Periods of spawning and setting of the soft- shelled clam, Mya arenaria at Solomons, Maryland. Ches. Sci. 3:114-120. Pfitzenmeyer. H. T. 1965. Annual cycle of gametogenesis of the soft- shell clam. Mya arenaria at Solomons. Maryland. Ches. Sci. 6:52-59. Porter, R. G. 1974. Reproductive cycle of the soft-shell clam, Mya are- naria at Skagit Bay, Washington. Fish. Bull. U.S. 72:648-656. Rogers, W. E. 1959. Gonad development and spawning of the soft clam. Maty land Tidewater News 15:9-10. Ropes. J. W. and A. P. Stickney. 1965. Reproductive cycle of Mya are- naria in New England. Biol. Bull. (Woods Hole) 128:315-327. Shaw, W. N. 1962. Seasonal gonadal changes in female soft-shell clams. Mya arenaria in the Tred Avon River, Maryland. Proc. Natl. Shellfish Assoc. 53:121-132. Shaw, W. N. 1965. Seasonal gonadal cycle of the male soft-shell clam, Mya arenaria in Maryland. U.S. Fish. Wildl. Serv. Spec. Sc. Rep. Fish. 508, 5pp. Stafford. J. 1912. On the recognition of bivalve larvae in plankton collec- tions. Contrib. Can. Biol. Fish. 1906-1910:221-242. Stevenson. J. R. 1907. Report of J. R. Stevenson upon observations and experiments on mollusks in Essex County during 1906. In Rept. Comm. Fish and Game 1906. Commonwealth of Massachusetts, Public Doc. 25:68-96. Sullivan, C. M. 1948. Bivalve larvae of Malpeque Bay, P.E.I. Fish. Res. Bd. Can.. Bull. 77. 36p. Welch. W. R. 1953. Seasonal abundance of bivalve larvae in Robinhood Cove, Maine. Fourth Ann. Conf. on Clam Res.. U.S. Fish and Wildl. Sen-.. Clam Investigations. Boothbay Harbor. Maine. Journal of Shellfish Research, Vol. 6, No. 1. 17-24. 1987. A COMPARATIVE STUDY OF AGE AND GROWTH IN MYA ARENAR1A (SOFT-SHELL CLAM) FROM THREE POPULATIONS IN LONG ISLAND SOUND DIANE J. BROUSSEAU1 AND JENNY A. BAGLIVO2 xDept. of Biology Fairfield University Fairfield, Connecticut 06430 2Dept. of Mathematics & Computer Science Fairfield University Fairfield, Connecticut 06430 ABSTRACT Three populations of Mya arenaria in Long Island Sound were studied during a 2.5 year period to determine age and shell growth characteristics. Internal growth bands deposited annually in the shell during the spring, were used to age individuals Age-size relationships were computed using a two-parameter von Bertalanffy growth curve. Growth rates were compared with those reported for the same species from other locations. No clear-cut geographical pattern in growth could be established forM. arenaria Growth allometry analysis indicates slow-growing clams from coarse sediments have heavier shells and larger shell length-width regression slopes than fast-growing clams from finer sediments. KEY WORDS: Age; Growth; Soft-shell clam; Long Island Sound; von Bertalanffy curve. INTRODUCTION Age and growth rate information is critical in under- standing the population dynamics of any species. Due in large part to the commercial value of Mya arenaria, a con- siderable body of literature has accumulated on the growth rates of this species from a number of geographic localities (Mead & Barnes 1903; Kellogg 1905; wllton and Wilton 1929; Belding 1930; Newcombe 1935; Smith et al. 1955; Matthiessen 1960; Swan 1952a. 1952b; Munch-Petersen 1973; Warwick and Price 1975; Brousseau 1979). The ex- treme variability in growth patterns evident in this species (Brousseau. 1979). however, makes it impossible to gener- alize. Recent advances in aging techniques based on internal growth lines present in thin sections of the shell of various bivalve molluscs, including M. arenaria (MacDonald and Thomas 1980), have facilitated studies of age and growth. The purposes of this investigation were first, to determine if the shells of Mya arenaria from Long Island Sound pos- sess growth lines suitable for use in aging studies; second, if so, to determine when these lines are formed; and third, to ascertain the growth patterns of M. arenaria from three intertidal populations in Connecticut. A two-parameter von Bertalanffy growth curve, expressed by the formula y = tjl - exp( -b(x + 0.5))), was used to analyze and com- pare available information on the growth rate of this species. MATERIALS AND METHODS From 1983-1985, Mya arenaria were dug from the mid-intertidal zones at three localities along the Connect- icut shore of Long Island Sound: Barn Island in Stonington (lat. 41°20'N; long. 71°53'W), Saugatuck River in West- port (lat. 41°06'N; long. 73°23'W) and Old Mill Beach in Westport (lat. 41°07'N; long. 73°21°W) (Figure 1). In the laboratory the clams were shucked and the shells were weighed to the nearest 0.01 gm. and measured anteropos- teriorly and dorsoventrally to the nearest 0. 1 mm. The left valve of each individual was cut from the umbo through the chondrophore to the ventral margin of the shell using a diamond saw. The valves were then mounted with epoxy cement on a glass slide and cut again to produce a thin section of the shell. The cross-sections were then pol- ished to a high glass on a vibrating lapidary machine using medium and fine grit powder for 1-2 hours. Growth lines were counted under a dissecting microscope. The ages of the clams were estimated by counting the number of growth bands between successive dark lines in the chondrophore and umbo region of the shell (Figure 2a). For aging pur- poses it was assumed that growth line formation (Figure 2c) occurred in May in all individuals (Table 1 ). A total of 491, 585 and 411 clams from Stonington, Saugatuck River. Westport and Old Mill Beach, Westport, respectively were aged using this method. Substrate analysis, using the method of Buchanan ( 1971 ), was performed on samples taken seasonally at each site. Two cores were excavated at each site on each sam- pling. Date and analysis of each core was done in triplicate. The Wentworth scale of particle classification was used. The age and growth data were analyzed using linear and non-linear regression analysis and analysis of variance (SPSSX 1983; Daniel and Wood 1980).' Comparison of substrates was done using the Kruskal-Wallis test (SPSSX 1983). RESULTS Internal growth lines were present in the chondrophore and umbo regions of the shells of clams collected from each 17 18 Brousseau and Baglivo Figure 1. Map showing locations of the three study sites: Barn Island in Stonington. CT (A). Saugatuck River in Westport, CT (B) and Old Mill Beach in Westport, CT (C). of the three study sites (Figure 2a). Growth lines in clams from Saugatuck river, Westport site (WP1 ). however, were better defined and more easily counted than those in indi- viduals from the other two sites. Variation in line thickness was apparent in all indi- viduals, but in the larger clams the growth bands (GB) were narrow and the growth lines were close together (Figure 2b). The oldest animal found in the study was an 1 1 -year- old clam from the WP1 population. In the populations studied, annual growth lines are pro- duced in the inner shell layer of M. arenaria during the spring (Table 1). Growth line formation had begun in a small number of clams by mid-March and by early May a fully formed line (Fig. 2c) was observed in approximately 75% of the animals examined. The relationship between age and shell length (mm) in the three populations is shown in Figure 3. The von Berta- lanffy curve, y = Cjl - exp( -b(x - x0))), is a curve of the decaying exponential type, where y is shell length (mm) and x is the age of the animal in months. The parameter x0 represents the age of zero length. In the present study x0 was fixed at -0.5, the end of the veliger or free-swimming stage, a period of about fourteen days prior to settlement (Belding 1930). The two parameters t „ and b represent the asymptotic length and the growth rate, respectively. These parameters are population dependent and were estimated using a non-linear least squares program written by Wood (Daniel and Wood 1980). The estimated parameters for each of the three sites were as follows: STN: €„ = 81.1063 (1.13 s.e.), (3 = 0.05092 (0.0209 s.e.) WP1: 4, = 60.8927 (0.586 s.e.). P = 0.0482 (0.0015 s.e.) WP2: ix = 73.5594(1.72 s.e.), (3 = 0.0604 (0.0034 s.e.) In all cases, the fit was significant. However, since the range of the ages for the WP2 site was so narrow (Figure 4), the results for this site were judged unreliable. The al- tered population structure at the WP2 site is probably due to recreational harvesting at this site. Both WP1 and STN are "closed" to digging because of high levels of bacterial contamination. Statistical comparison of STN and WP1 curves were made using approximate 99% confidence regions for the two parameters (Cx,b) (Draper and Smith 1981). There is a clear difference in the growth rates between the two popu- lations. Clams from STN show the most rapid growth when young, and reach a higher estimated asymptotic size than those from WP1. There is some indication, however, that the slower growing clams from WP1 are longer lived than those from STN. Figures 5 and 6 show allometric relationships in M. are- naria. The relationship between shell length (y-axis) and shell width (x-axis) is shown in Figure 5. Linear regression models of the form y = Bx + a were developed for each of the three sites. The estimated parameters were as follows: STN: (3 = 1.66 (.018 s.e.), d = 0.93 (.708 s.e.) WP1: (3 = 1.41 (.019 s.e.), a = 4.69 (.652 s.e.) Age and Growth of Mya 19 GL3 GL2 \GL4 GL1-^K\ / GL5 ^^^^^^^ A\ 191 [bJ m ^ ^ GLF wV # GL2 « ^ /_ a GL1 Figure 2. Thin sections of the chondrophore and umbo region of the shells of . I/, arenaria. (A) Internal growth line (GL) appearing between two white growth bands (GB) in a 1 + yr. old from Saugatuck River, VVestport. (B) Growth lines numbered from first formed to last formed in a 7 yr. old from Saugatuck River, Westport. (C) Newly-formed growth line (GLF) in a 5 yr. old collected in May 29, 1984 from Saugatuck River, VVestport. 20 Brousseau and Baglivo TABLE 1. Percentage of M . arenaria in the three populations showing a newly-formed growth line in samples taken during the year. Number of individuals per sample is given in parentheses. Percentages Sampling Site March April Mav June Bam Island, Stonington 19.5(41) 60.0(50) 66.7(117) 46.3(95) Saugatuck River, Westport 2.3(44) 34.5(29) 71.8(167) 14.9(127) Old Mill Beach. Westport 7.8(26) 21.2(165) 75.6(82) 50.0(16) WP2: B = 1.57 (.022 s.e.). a = 3.03 (.742 s.e.) These relationships are significantly different (p < .001). and indicate that the slowest growing clams (Figure 3) were the most globose in shape. Similarly, shell length-weight relationships are plotted in Figure 6. Linear regression models of the form y = Bx + a. where y is shell length and x is the natural logarithm of shell weight, were developed for each of the three sites, with parameter estimates: STN: p = 15.31 (.254 s.e.), d = 30.39 (.627 s.e.) WP1: B = 12.79 (.216 s.e.), d = 24.39 (.485 s.e.) WP2: |3 = 14.67 (.259 s.e.), d = 27.53 (.530 s.e.) These relationships are significantly different (p < .001), indicating that the slow-growing WP1 clams also had the heaviest shells. The difference in growth rates and shell form may be attributed in part to the effects of substrate. Substrate com- position differences at the three sites (Figure 7) suggest that heavier more distorted shells of M. arenaria from WP1 may be due to the coarse sediments found at the site. DISCUSSION MacDonald and Thomas (1980) described annual in- ternal shell growth patterns in Mya arenaria from Prince Edward Island. Similar patterns are evident in thin sections of the chondrophore and umbo region of the Long Island Sound clams used in this study. MacDonald and Thomas (1980), however, had difficulty aging clams older than 7 years because of crowding of growth lines. This was not a severe problem in this study; clams 10 years and older could be aged easily using this method. Even though in- ternal growth lines were present in clams from each of the populations studied, some variability in line definition was encountered. Clams from Saugatuck River, Westport (WP1) showed the most clearly defined lines, whereas 80 70 | 60 S50 hi 40 u 30 20 10 Stn Wp 2 Wp 1 20 30 40 50 60 70 80 90 100 110 120 130 140 AGE (MONTHS) Figure 3. Von Bertalanffy growth curves for clams from Stonington (STN), Saugatuck River, Westport (WP1) and Old Mill Beach, Westport (WP2). Age and Growth of Mya 21 Stn 2 s z i- o z ; Wp 2 ij;7«j;ii3 i 80 60- 40- 20 100 STN WP 1 WP 2 SHELL WEIGHT (GM) ;ure 6. Shell length (mm) plotted against shell weight (gm) for dams from Stonington (STN), Saugatuck River, Westport (VVP1) and Old Mill Beach, Westport (WP2). PARTICLE SIZE (MM) Figure 7. Particle size composition of sediment at the three sites: Stonington (STN), Saugatuck River, Westport (WP1) and Old Mill Beach, Westport (WP2). Values represent percent dry weight frac- tions. Substrate compositions are significantly different (p = .012). those from Old Mill Beach (WP2) were least easily distin- guished. Whether this is due to differences in growth rate or variation in environmental factors is yet to be deter- mined. Age and Growth of Mya 23 Annual growth line formation has been linked to low water temperatures by some investigators (Weymouth et al. 1931; Green 1957; Feder and Paul 1974: MacDonald and Thomas 1980) and to spawning events by others (Jones et al. 1978; Thompson et al. 1980). For jW. arenaria the evi- dence suggests the latter explanation. MacDonald and Thomas (1980) reported that the lines are formed by May of each year, prior to spawning, in Prince Edward Island clams. This corresponds to the timing of line formation found for Long Island Sound clams (Table I ). MacDonald and Thomas (1980), however, interpret this event as a re- sponse to decreased winter growth. If this were the case, one would expect to see evidence of line formation much earlier in the year, when water temperatures are at a min- imum. It seems more likely that growth lines are formed in response to slowed shell growth brought on by gamete buildup prior to spawning. This hypothesis is further sup- ported by evidence for Spisula solidissima (Jones et al. 1978) and Arctica islandica (Thompson et al. 1980), both of which deposit lines during the late summer, a time coin- cident with spawning period rather than decreased tempera- tures. An inverse relationship between growth rate and age has been described for M . arenaria, as well as for a number of other bivalves, including Cardinal edule (Orton 1926; Kristensen 1957), Crassostrea virginica (Ingle and Dawson 1952), Siliqua patula (Weymouth et al. 1931), Pecten maximus (Mason 1957). Mercenaria mercenaria (Haskin 1954), Scrobicularia plana (Hughes 1970) and Macoma balthica (Gilbert 1970). One of the simplest methods of analyzing growth when such a relationship exists is to apply the von Bertalanffy equation. Although the usual form of the equation involves three parameters, we chose a two-parameter version for several reasons. First, a value x0 = —0.5 is a reasonable estimate given our knowledge of the species. Second, fixing x0 facilitated the comparison of the populations. Third, and most important, since the min- imum age for individuals in each of the three populations was much greater than zero, the value of x0 could not be estimated reliably from the data. The values of b and €„ were fairly stable in all of our calculations. As more detailed information becomes available, it be- comes increasingly clear that local environmental condi- tions may have a larger effect on growth patterns in M. arenaria than geographical ones. In this study, clams from the more southerly site (WP1) grew appreciably slower than those from farther north (STN). Similarly, a review of the literature reveals that although a general trend of in- creased growth rates with decreasing latitude exists, the ef- fects of local environmental conditions can be strong (Table 2). For example in Newcombe's (1935) study, clams living at mid-tide level reached harvestable size in 5-6 years, whereas those living higher in the intertidal zone had not reached harvestable size after 7 years of growth. Geographical considerations alone are poor pre- dictors of growth patterns in M. arenaria. TABLE 2. The time needed for Mya arenaria to reach harvestable size (51 mm) reported in the literature. Site Latitude Age at 51 mm (yrs) Reference Prince William Sound, Alaska 60C,34'N Roskilde Fjord. Denmark 55°34'N Lynher River. England 50°23'N Economy Pt., Nova Scotia (8 ft. above chart datum) 45°20'N St. Andrews. New Brunswick (8 ft. above chart datum) 45°10'N Clam Cove, New Brunswick (16 ft. above chart datum) 44°45'N Clam Cove, New Brunswick (8 ft. above chart datum) 44°45'N Sissiboo River. Nova Scotia (8 ft. above chart datum) 44°30'N Bedroom Cove (Georgetown Is.). Maine 43°35'N Sagndahoc Bay (Georgetown Is.). Maine 43°35'N Rowley. Mass. 42°26'N Quincy, Mass. 42°09'N Gloucester, Mass. 41°39'N Monomoy Pt., Mass. 41°30'N West Falmouth, Mass. 41°30'N Narragansett Bay, Rhode Island 41°24'N Stonlngton. CT 41°20'N Old Mill Beach. Westport, CT 41°07'N Saugatuck River. Westport, CT 41°06'N 6-7 6-7 3-4 5-6 5 >7 5-6 5-6 5-6 3-4 2-3 2-3 2-3 2 2 1-2 1.5 1.5 3 Feder & Paul, 1974 Munch-Petersen. 1973 Warwick & Price, 1975 Newcombe. 1935 Newcombe. 1935 Newcombe. 1935 Newcombe. 1935 Newcombe, 1935 Spear & Glude. 1957 Spear & Glude. 1957 Belding. 1930 Turner. 1949 Brousseau. 1979 Belding, 1930 Kellogg, 1905 Mead & Barnes, 1903 present study present study present study 24 Brousseau and Baglivo One easily measured local environmental factor shown to be important in controlling growth rate and shell allom- etry in infaunal bivalves in sediment type. Results of various field studies have shown that M. arenaria grows fastest in sand or sandy mud substrates (Belding 1930; Newcombe 1935; Swan 1952b; Smith et al. 1955). Belding (1930) and Swan ( 1952b) also found that the dimensions of the shells varied in different types of sediment. Under field conditions, however, substrate composition is associated with other factors, such as current, elevation on shore and wave exposure, factors which also influence growth pat- terns. It was not until Newell (1982) used a laboratory ap- proach to isolate the effects of sediment type from other environmental variables that it was shown conclusively that growth rates and shell form are dependent on the physical properties of the substrate. Examinations of the clams from the Saugatuck River (WP1) site, where the coarsest sediment was found, re- vealed thick, irregular growth and damaged shells. This is consistent with observations made by Belding (1930) and Swan (1952b) on clams collected from coarse intertidal sediments. Damaged shell and mantle tissue sustained during digging in coarse environments would require en- ergy for repairs, resulting in the reduced growth and more globose shell shape found in clams from the WP1 popula- tion. ACKNOWLEDGMENTS We wish to thank K. Schellinkhout, J. Smeriglio, J. Trautman and J. Wachter for technical assistance in the field and the preparation of the shell material. Photomicro- graphs were taken by B. Machler. Financial support for this study was provided under grant number NA82AA-D-00018 of the Connecticut Sea Grant Program. LITERATURE CITED Belding. D. L. 1930. The soft-shelled clam fishery of Massachusetts. Mar. Fish. Ser. Div. Fish. Game Mass. 1-65. Brousseau, D. J. 1979. Analysis of growth rate in Mya arenaria using the von Bertalanffy equation. Mar. Biol. 51:221-227. Buchanan, J. B. 1971. Measurement of the Physical and Chemical Envi- ronment: Sediments. In: Methods for the Study of Marine Benthos (N. A. Holme and A. D. Mclntyre, eds.) p. 30-58. DaJiiel, C. & F. S. Wood. 1980. Fitting Equations lo Data. John Wiley & Sons. New York. N.Y. Draper, N. & H. Smith. 1981 . Applied Regression Analysis. 2nd ed., John Wiley & Sons, New York, N.Y. Feder, H. M. & A. J. Paul. 1974. Age, growth and size-weight relation- ships of the soft-shelled clam Mya arenaria in Prince William Sound. Alaska. Proc. Nat. Shellfish. Assoc. 64:45-52. Gilbert. M. 1970. Growth rate, longevity and maximum size of Macoma balthica. L. Biol. Bull. 145:119-126. Green, J. 1957. The growth of Scrobicularia plana in the Gwendraeth estuary. J. Mar. Biol. Assoc. U.K. 36:41-47 Haskin. H. H. 1954. Age determination in molluscs. Trans. N.Y. Acad. Sci. 16:300-304. Hughes, R. N. 1970. Population dynamics of the bivalve Scrobicularia plana (da Costa) on an intertidal mudflat in northern Wales. J. Anim. Ecol. 39:333-356. Ingle, R. M. & C. E. Dawson. 1952. Growth of the American oyster. Crassostrea virginica (Gmelin) in Florida waters. Bull. Mar. Sci. Gulf Caribb. 2:393-404. Jones, D. S., I. Thompson & W. Ambrose. 1978. Age and growth rate determinations for the Atlantic surf clam, Spisula solidissima (Bi- valvia; Mactracea) based on internal growth lines in shell cross-sec- tions. Mar. Biol. 47:63-70. Kellogg, J. L. 1905. Report of the special commission for the investiga- tion of the lobster and the shoft-shell clam IV. Conditions governing existence and growth of the soft-shell clam (Mya arenaria). Rep. U.S. Comm. Fish. 29:195-224. Kristensen, I. 1957. Differences in the density and growth in the cockle population in the Dutch Wadden Sea. Archs. Neerl. Zool. 12:351- 453. MacDonald, B. A. & M. L. H. Thomas. 1980. Age determination of the soft-shell clam Mya arenaria using shell internal growth lines. Mar. Biol. 58:105-109. Mason, J. 1957. The age and growth of the scallop, Pecten maximus in Manx Waters. J. Mar. Biol. Assoc. U.K. 36:473-492. Matthieassen. G. C. 1960. Observations on the ecology of the soft clam, Mya arenaria in a salt pond. Limnol. Oceanogr. 5:291-300. Mead. A. D. & E. W. Barnes. 1903. Observations on the soft-shell clam. Mya arenaria. Rep. Rhode Island Comm. Inland Fish. 22:29-46. Munch-Petersen, S. 1973. An investigation of a population of the soft clam (Mya arenaria L.) in a Danish estuary. Meddr. Kommn. Damn Fisk.-og Hanunders. (Ser. J) 7:47-73. Newcombe. C. L. 1935. Growth of Mya arenaria in the Bay of Fundy region. Can. J. Res. 13:97-137. Newell, C. 1982. The effects of sediment type on growth rate and shell allometry in the soft-shelled clam Mya arenaria. J. Exp. Mar. Biol. Ecol. 65:285-295. Orton. J. H. 1926. On the rate of growth of Cardium edule. Part I. Exper- imental observations. J. Mar. Biol. Assoc. U.K. 14:239-279. Srnith. O. R.. J. P. Baptist and E. Chin. 1955. Experimental farming of the soft-shell clam, Mya arenaria in Massachusetts. 1949-1953. Commer. Fish. Rev. 17:5-16. Spear, H. S. and J. B. Glude. 1957. Effects of environment and heredity on growth of the soft clam (Mya arenaria). Fish. Bull. 57:279-292. Statistical Package for the Social Sciences, Inc. 1983. User's guide to SPSSX. McGraw-Hill Co. New York. Swan, E. F. 1952a. Growth indices of the clam Mya arenaria. Ecology 33:365-374. Swan, E. F. 1952b. Growth of the clam Mya arenaria as affected by the substratum. Ecology 33:530-534. Thompson, I., D. S. Jones & D. Dreibelbis. 1980. Annual internal growth banding and life history of the ocean quahog Arctica islandica (Mollusca: Bivalvia). Mar. Biol. 57:25-34. Turner. H J . Jr. 1949. The soft-shell clam industry of the east coast of the United States. Appendix I. Report on investigations of the propa- gation of the soft-shell clam, Mya arenaria. Woods Hole Oceanogr. Inst., Coll. Reprints 1948, Contrib. 462. p. 11-42. Warwick. R. M. & R. Price. 1975. Macrofauna production in an estuarine mudflat. J. Mar. Biol. Assoc. U.K. 55:1-18. Weymouth, F.. H. McMillan & W. H. Rich. 1931. Latitude and relative growth of the razor clam, Siliqua patula Dixon. J. Exp. Biol. 8:228- 249. Wilton. M. H. & H. I. Wilton. 1929. Conditions affecting the growth of the soft-shell clam Mxa arenaria L. Contr. Can. Biol. Fish. 4:81-93. Journal of Shellfish Research, Vol. 6, No. 1, 25-28, 1987. EFFECTS OF INBREEDING ON GROWTH IN THE PACIFIC OYSTER (CRASSOSTREA GIGAS) J. HAROLD BEATTIE, JAMES PERDUE, WILLIAM HERSHBERGER AND KENNETH CHEW College of Fisheries University of Washington Seattle, Washington 98195 ABSTRACT The Pacific oyster {Crassostrea gigas, Thunberg) industry on the West Coast of the United States relies heavily on hatcheries for its seed. As such, the possibility exists for developing of selective breeding programs which could be of economic value to the industry. In a selection program, inbreeding depression due to crossing of close relatives exists as a potential problem. On an experimental basis in four years of breeding with C. gigas. it was found that two year old progeny from first generation brother-sister matings were smaller in shell size and wet and dry meat weights than those from outbred or control stocks. Effects of inbreeding should be considered in designing any shellfish hatchery breeding scheme. KEY WORDS: hatchery, selection, inbreeding, growth, oyster INTRODUCTION The oyster industry on the West Coast of the United States relies almost entirely on the culture of the Pacific oyster, Crassostrea gigas. A major change in the com- plexion of this industry has resulted from the establishment of commercial oyster hatcheries. These oyster larvae facto- ries have proven to be more reliable, consistent, and cost effective than natural sources of oyster seed. A potential benefit to oyster culture may lie in the em- ployment of genetic methods for stock improvement. Though the principles of genetics have been known since Menzel, the use of modern genetics in agriculture has been practiced for only a little more than 50 years (Marshall 1977), and has aptly demonstrated a potential for applica- tion in molluscan aquaculture (Newkirk 1980). Oysters have been cultured for centuries, however the uncontrolled nature of their reproduction has precluded any attempt to select or breed them. This situation is changing drastically for three reasons: Recent developments in shellfish hatchery techniques allow for designed breeding practices. Electrophoretic analysis of oyster proteins and enzymes has revealed a high degree of genetic variation (Buroker 1975; Fugio 1979), suggesting potential for improvements through genetic manipulation. The institution of commer- cial oyster hatcheries has added major economic impetus to oyster genetic research due to demands for improvement in certain traits by the oyster growers. The control of reproduction is inherent in any hatchery. Such control provides an opportunity to study and imple- ment selective breeding for the improvement of desirable traits (Hershberger et al. 1984). Selective breeding using sibling crosses makes inbreeding inevitable (Falconer 1982). Inbreeding can be expressed in the depression of expressed traits such as fecundity, larval viability, physical stunting or malformation, and physical anomalies. While the effects of inbreeding are well documented among agricultural species (Wright 1977), they have only recently been reported with oysters. In the American oyster {Crassostrea virginica Gmelin) inbreeding was found to in- hibit growth (Mallet and Haley 1983). Zouros et al. (1980) found a direct association between growth and the level of heterozygosity among enzyme loci as determined by elec- trophoresis. These findings suggest that heterozy- gosity reduction, possibly due to inbreeding, might be ac- companied by a reduction in growth. In regard to the early life history of C. virginica, Longwell and Stiles (1972) ob- served that progeny of first generation full sib matings ex- hibited reduced larval growth and decreased larval survival. With the Pacific oyster (C. gigas), researchers in Japan re- ported no reduction in growth after four generations of in- breeding but did report a loss of larval viability (Imai and Sakai 1961). Lannan (1980) found that inbreeding among experimental stocks of C. gigas did not produce reduction in larval survival. As indicated by these studies with Amer- ican and Japanese oysters, the effects of inbreeding are not clearly defined and are somewhat contradictory. Since 1975 the University of Washington School of Fisheries has conducted a selective breeding project with the Pacific oyster. The main emphasis of this study was to develop strains of oysters with high survival during summer mortality conditions (Beattie et al. 1978; Beattie et al. 1980; Hershberger et al. 1984). The breeding methods consisted of single pair matings of oysters, thus producing sets of full sib families, and subsequent breeding within and among these various families. The families were moni- tored for summer survival rate and after two growing seasons, individuals were measured for size, including meat wet weight, meat dry weight, shell height and length. Since growth has been identified as the trait which oyster growers worldwide like to see improved (Mahon 1983), the size differences among these experimental families were of particular interest. This paper examines several years of hatchery production and compares oyster sizes (at 2 + years) of first generation full-sib crosses to sizes of out- 25 26 Chew et al. crossed groups and to controls from naturally reproducing populations. METHODS AND MATERIALS Oysters for this study were spawned and initially cul- tured at the University of Washington Experimental Shell- fish Hatchery at the National Marine Fisheries Service Aquaculture Station, Manchester. Washington. Full sib families of oysters were produced from single pair matings of adults from selected family lines. Inbred families were produced from brother sister matings. Outbred families were produced by crossing between families or between families and wild stocks. Beginning in 1982, replicate ex- perimental family lines were produced through rotational line crossing (Hershberger 1984; Kincaid 1977). Gametes were obtained by inducing spawning or by stripping. Polyethylene or fiberglass tanks (160 liter) were used for embryonic and larval culture. Just prior to larval metamorphosis, a setting substrate of oyster shells was in- troduced into the tanks. After setting, the new oyster seed was transferred to a raft for a subtidal nursery period. The seed was subsequently planted on commercial oyster beds in three bays in south Puget Sound, WA: Mud Bay, Oak- land Bay and Rocky Bay. Family integrity was maintained by the use of intertidal longlines or by plastic mesh pens. Oyster sizes were measured during the second autumn following planting. In 1980, meat wet weights were mea- sured for 100 animals from each family and from the con- trol (oysters grown from Japanese stock, or from the natu- rally reproducing stock from Dabob Bay). In all other years 20 animals from each family of stock were measured for meat wet weight and shell dimension, a subsample of 10 animals were measured for meat dry weight. The data for each year were grouped according to inbred (brother sister mating), outbred (crosses between families, or between families and wild stock), or control. The results were then compared using Student's "t" test for indepen- dence of means at a significance level of 0.01. RESULTS Meat weight. In each of the four year classes of oysters studied the mean meat weights of first generation full sib inbred oysters were smaller than most of the outbred groups and controls (Table 1 ). In 1980, the mean meat wet weight of inbred oysters grown in Oakland Bay was smaller than either the control mean or the grouped outbred mean. In 1981, sizes were compared in two different areas, Oakland Bay and Rocky Bay. The mean dry meat weight for the pooled inbred group was the smallest compared to both the pooled outbred group and the control. In 1982, families produced from rotational line crosses, and inbred families were compared in two bays, Oakland Bay and Mud Bay. The mean dry meat weight for the inbred group was the smallest in oysters from both bays; the mean wet meat weights reflect this same differential. In 1983, mean wet weight values of inbred oysters were smaller than those of either outbred groups or controls in all three bays. Dry weights for this year do not reflect this same differential however, with the inbred mean dry meat weight values from Oakland Bay actually greater than the mean dry meat weight values of the pooled outbred group. Shell area. Shell dimensions taken in 1982 and 1983 reflect the differential in size observed in mean meat weights. Oysters from the inbred groups were smaller than those from outbred groups and controls from all bays in both years with the exception of Oakland Bay in 1983. In this year, the shell size of oysters from the inbred group was actually larger than those from the outbred group. Statistical comparison. Although not all of the growth parameters considered were recorded in each of the years and bays, the statistical comparison of the data for mean dry meat weight, mean wet meat weight, and mean shell area indicate the effect of one generation of full sib in- breeding (Table 1). For mean dry meat weight, groups of inbred families were significantly (p = 0.01) smaller than groups of outbred families in 4 of 6 cases, and significantly smaller than controls in 3 of 5 cases. Compared to the con- trol group, the inbred group had 18 to 20% less dry meat weight in Oakland Bay, 18 to 40% less dry meat weight in Mud Bay and 36 to 40% less dry meat weight in Rocky Bay. For wet meat weight, grouped inbred means were sig- nificantly (p = 0.01) smaller than grouped outbred means in 5 of 6 cases and significantly (p = 0.01) smaller than controls in 4 of 5 cases; compared to controls the inbred groups were 9 to 27% smaller in Oakland Bay, 36 to 38% smaller in Mud Bay and 43% smaller in Rocky Bay. Com- parison of shell areas (Area = length x width x it/4, after Haley and Newkirk 1977) revealed that full sib Fl mean values were significantly (p = 0.01) smaller than other experimental groups in 3 of 5 cases, and in 3 of 4 cases compared to controls; the mean shell area for oysters from the inbred group was 4% less than the control in Oak- land Bay, 19 to 32% less than the control in Mud Bay and 33% less than the control in Rocky Bay. DISCUSSION In four hatchery years, mean values of grouped inbred families were smaller than values of either outbred or con- trol values for dry meat weight, wet meat weight and shell area. Size reduction had not been previously reported to be caused by inbreeding in Pacific Oysters, however, Fujio ( 1982) and Folz (1983) reported heterozygosity to be posi- tively correlated with weight in oysters, which would indi- cate that reduced growth among inbred stocks might be caused be reduced levels of heterozygosity. Mallet and Haley (1983) also reported increased spat growth rates among outbred families of C. virginica. Some oyster growers on the Pacific coast have noted smaller sized oysters grown from hatchery seed compared to seed from some naturally reproducing populations. Al- though inbreeding is a possible cause, it seems an unlikely Effects of Inbreeding on Growth in the Pacific Oyster 27 TABLE 1. Sample size (N), mean values (X), and standard deviation (S) of three parameters (meat wet weight, meat dry weight, and shell dimension) comparing values from grouped inbreds, grouped outbreds, and controls. Inbred Outbred )rv Weight (Grams) Year Bay Sig. N X S N X S N X S 1981 Oakland 10 3.73 1.21 80 4.19 1.33 10 4.58 1.37 Rocky *o 20 2.55 0.79 60 3.62 1 06 10 4.25 0.96 1982 Oakland * 40 2.62 0.94 220 3.69 1 21 Mud *o 40 3.09 1.07 220 3.89 1 26 10 4.76 1.24 1983 Oakland 40 3.20 1.25 100 3.13 1 15 10 4.20 1.44 Mud *o 40 2.62 1.12 100 3.73 1 49 10 4.39 1.17 Wet Weight (Grams) Inbred Outbred Control Year Bay Sig. X S N X S N X S 1980 Oakland * 101 19.82 6.59 194 25.85 7.01 100 21.88 8.01 1982 Oakland * 80 12.00 4.08 440 16.79 5.59 — — — Mud *o 80 13.11 4.52 440 16.55 5.45 20 21.95 4.34 1983 Oakland o 80 16.12 3.74 195 17.58 5.17 20 21.95 4.34 Mud *o 80 13.36 3.37 192 19.38 5.88 20 20.74 5.48 Rocky *o 80 8.82 2.20 180 10.67 3.95 20 15.39 3.68 Shell Area (cm2) Inbred Outbred Control Year Bay Sig. N X S N X S N X S 1982 Oakland * 80 26.22 5.40 440 30.70 7.56 — — — Mud *o 80 24.63 6.15 440 28.33 6.61 20 35.97 6.99 1983 Oakland 80 45.27 10.79 195 44.25 11.94 20 47.14 13.81 Mud *o 80 34.76 8.32 193 42.60 1 1 .65 20 42.98 13.61 Rocky *o 76 40.80 14.07 171 43.07 15.11 19 60.58 17.14 * Significant at 0.01 compared to the outbreds. ° Significant at 0.01 compared to the controls. candidate because of the random nature of the commercial hatcheries' brood stock selection process. Furthermore. Gosling (1982) found little difference in genetic variability in hatchery produced C. gigas compared to natural popula- tions of the species. Inadvertent selection due to domesti- cation may be in part responsible for this reduction in growth. Domestication, as defined by Doyle (1983) is the natural selection of traits which affect survival and repro- duction in a human-controlled environment. An oyster hatchery certainly qualifies as a human controlled environ- ment, and may thus require a new combination of traits for best larval performance. Unfortunately, those genes that contribute to the best larval hatchery performance may not be the ones that are most valuable for good growth in the adult phase. The timing of collection for size analysis is very impor- tant in that the occurrence of spawning can severely affect meat weight values. In August, 1981, spawning in Oakland Bay accounted for reductions in mean dry meat weights of from 25 to 40%. Propensity toward spawning and the rate of body weight recovery are variables which affect meat weight and which are probably independent of actual growth rate. Perdue (1982) reported variation in the com- pletion of gametogenesis of as much as six weeks in se- lected experimental stocks of C. gigas. The animals in this study were collected late enough after the spawning period to hopefully avoid the confounding influence of spawning on body weight. Genetic manipulation can be used to enhance desired traits such as disease resistance and improved growth rates. In addition to our work with summer mortality, selective breeding has been applied to developing disease resistant strains to MSX (Ford and Haskin 1986; Haskin and Ford 1986). Other genetic breeding schemes may have applica- tion. Incorporating the concept of combining ability (Griffing 1956). selective breeding might be used to de- velop lines which when crossed, would result in progeny which would exhibit higher growth. In using lines with spe- cific combining ability, it would be feasible to tailor stocks of oysters for good growth in specific environments. On the other hand, analyses of either population crosses or crosses among selected lines could result in the development of lines with general combining ability, whose progeny would then exhibit improved growth in a variety of environments. If lines of general combining ability can indeed be devel- oped, this would no doubt be the desired method for com- mercial application. The value of any breeding technique is only academic until it can be adapted on a commercial scale. Commercial hatcheries for Pacific oysters have only recently begun to 28 Chew et al. use genetics in a careful, controlled and consistent manner. Wescott Bay Seafarms, Friday Harbor, WA has incorpo- rated into its production line, a stock of high glycogen oysters which was developed at the University of Wash- ington shellfish hatchery. In addition, three oyster hat- cheries (Coast Oyster Company, Quilcene, WA, Wescott Bay Seafarms, Friday Harbor, WA, and Whisky Creek Oyster Co., Tillamook, OR), working with graduate stu- dents Stan Allen and Sandra Dowing from the University of Washington, have recently begun inducing triploidy in some of their production (Allen and Downing 1986; Downing and Allen 1986). The use of genetic techniques by these hatcheries marks a major step in the progress of Pacific oyster culture. These techniques require a careful manipulation of spawning stock and gametes, and represent a major departure from the comparatively sloppy tech- niques usually used. As genetic methods are used increas- ingly by commercial hatcheries, the demand for genetic studies by research hatcheries will increase and may be- come integral to the industry. ACKNOWLEDGEMENTS This research was supported by the Washington Sea Grant Program. 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Gamete cross incompatability and inbreeding in the commercial American oyster, Crassosirea virginica Gmelin. Cytologia 38:521-533. Mahon. G. A. T. 1983. Selection goals in oyster breeding. Aquaculture 33:141-148. Marshall, D. R. 1977. The advantages and hazards of genetic homoge- neity. The genetic basis of epidemics in agriculture. Annal N.Y. Acad. Aci. 287:1-20. Newkirk, G. F. 1980. A review of the genetics and the potential for selec- tive breeding of commercially important bivalves. Aquaculture 19:209-228. Perdue, J. A., J H. Beattie, & K. K. Chew. 1981. Some relationships between gametogenic cycle and the summer mortality phenomenon in the Pacific oyster, Crassosirea gigas in Washington State. J. Shell- fishenes Res. 1(11:9-16. Wright, S. 1977. Evolution and the genetics of population III. Experi- mental results and evolutionary deductions. Univ. of Chicago Press, Chicago, IL 613 pp. Zouros, E., S. M. Singh, & H. E. Miles. 1980. Growth rate in oysters; an overdominant phenotype and its possible explanation. Evolution 34:856-867. Journal of Shellfish Research, Vol. 6, No. 1, 29-36, 1987. THE REPRODUCTIVE AND ENERGY STORAGE CYCLES OF TWO POPULATIONS OF MYTILUS EDULIS (LINNE) FROM BRITISH COLUMBIA B. EMMETT1, K. THOMPSON2, AND J. D. POPHAM2 ^Archipelago Marine Research #10, 1140 Fort Street Victoria, BC, Canada V8V 3K8 2Seakem Oceanography Ltd. 2045 Mills Road Sidney, BC, Canada V8L 3S1 ABSTRACT The relationship between growth, mortality, reproductive and energy storage cycles was examined in two populations of cultured mussels (Mytilus edulis) situated in exposed (Mackenzie Anchorage) and sheltered (Departure Bay) habitats of the British Columbia coast. In both locations mussels grew to a mean length of approximately 50 mm within 14 to 16 months of settlement; however, mortalities during the second summer exceeded 90% in both populations. Growth during this summer period was negligible. In both populations glycogen stores decreased in fall but were not accompanied by increasing lipid content. This indicates that the carbohydrate stores were used to meet metabolic demands other than the synthesis of gonad. At Mackenzie Anchorage spawning activity and the rebuilding of glycogen stores occurred concurrently in May and June, and was followed by a second period of spawning in late August. In Departure Bay spawning occurred throughout the summer period and the rebuilding of glycogen stores did not occur until July. These patterns contrast with observations from European mussel populations in which the decline in carbohydrate levels in the fall is coupled with the synthesis of gonad and spawning generally occurs over a short period in spring. Summer is a time ot reproductive quiescence in most European mussel populations. It is suggested that the high mortalities observed in the present study are related to reproductive stress and that the reproductive strategy of Mytilus edulis on the west coast of North America emphasizes high reproductive output at an early age. KEY WORDS mussel, Mytilus edulis, spawning, glycogen, lipid, reproductive ecology. INTRODUCTION The seasonality of gametogenesis and the cyclic nature of energy reserves in marine mussels has been the subject of considerable research, particularly in Europe where ap- proximately 60% of the world"s cultured mussels are pro- duced. These studies (Pieters et al. 1979; Zandee et al. 1980a,b; Lowe et al. 1982; Bayne et al. 1983) demonstrate a complex relationship between reproductive activity and energy storage cycles (Gabbott 1983). In Mytilus edulis (Linne) periods of storage reserve accumulation and gamete production are usually temporally separated. Throughout the summer, reproductive activity is minimal ("rest pe- riod") and reserves of protein, lipid and carbohydrate are accumulated in both gonadal and non-gonadal tissues. The carbohydrate (glycogen) is utilized during autumn and winter for gonadal development. Glycogen reserves are also used during these seasons to meet energy requirements during periods of low food availability. There is some evi- dence that protein reserves are also used at this time (Pieters et al., 1979). Egg and sperm in bivalves are com- posed primarily of protein and lipid (Pieters et al. 1980) and, thus, the cyclic pattern of lipid and protein is corre- lated with the accumulation and shedding of gonadal products. In the spring, when glycogen levels are low, spawning occurs as a discrete event or as a series of successive spawns. (Pieters et al. 1980; Lowe et al. 1982). Local vari- ation in this pattern does occur. Lowe et al. (1982) describe a population of mussels (Lynher) where gametogenesis and the development of storage reserves occur concurrently during the summer. This is followed by a second spawning in autumn. In North America, data on reproductive and energy re- serve cycles have been restricted to Atlantic coast popula- tions of Mytilus edulis. In a subtidal population of mussels in Newfoundland, gametogenesis does not occur during the fall and winter (Thompson 1984a). Rather, carbohydrate levels increase in spring and are accompanied by the syn- thesis of gametes. Spawning then takes place in July. Ne- well et al. (1982) found considerable variation in the pat- tern of gametogenesis and timing of spawning activity in seven populations of mussels on the east coast of the United States. The authors conclude that temporal and quantitative differences in food supply had a greater influence on the reproductive cycles than water temperature or latitude. On the Pacific coast Mytilus edulis is found primarily in the intertidal zone of protected bays and does not occur in extensive subtidal beds as in Europe or the eastern coast of the United States. The California mussel, Mytilus califor- nianus (Conrad), is a larger species found both intertidally and subtidally on exposed rocky coasts. In these exposed communities the two species may overlap in distribution, as M. edulis quickly colonizes primary space in the inter- tidal zone and is later displaced by the larger, longer-lived M. californianus (Suchanek 1978). Hines (1979) reports a 29 30 Emmett. Thompson, and Popham sharp decrease in gonad index (indicative of spawning) during the spring for an experimental group of M. edulis in California. In contrast an adjacent group of M . califor- nianus showed a relatively constant gonad index throughout the year. In British Columbia, Quayle (1978) reports that M. edulis larvae are continuously present in the water from April to November, with a peak of abundance from June until September. At several sites in Puget Sound, Skidmore (1983) found peak larval settlement rates of M. edulis from early May through July, then a gradual de- crease in the rate until winter when settlement ceased com- pletely. These results indicate that M . edulis on the Pacific coast spawn most extensively in late spring and early summer, but that spawning may be prolonged until au- tumn. The concept of M. edulis as a colonizer of primary space (Suchanek 1978) suggests that this species may be adapted for high reproductive output in those areas where it is often displaced by the longer-lived M. calif ornianus. In recent years the production of cultured Mytilus edulis in British Columbia and Puget Sound has been increasing. Landings of cultured mussels exceeded 75 tons in 1984 (Skidmore and Chew 1985). An understanding of the spawning and energy storage cycles in this species will help the industry to develop appropriate methods and timetables for seed collection, spat handling and product harvesting. The present study was undertaken to determine the rela- tionship between growth, mortality and these cycles in two populations of M. edulis situated in exposed and sheltered habitats of the British Columbia coast. MATERIALS AND METHODS Study sites were established at Departure Bay, on the east coast of Vancouver Island, and at Mackenzie An- chorage in Barkley Sound on the west coast of Vancouver Island (Figure 1). The former site is situated in the pro- STRAIT OF GEORGIA Nrr* £••■ S& BARKLEY V5 SOUND -~^*^ MACKENZIE _ ANCHORAGE *** \0< /• DEPARTURE %. • BAY JUAN DE FUCA STRAIT Figure 1. Location of study sites at Departure Bay and Mackenzie Anchorage on the east and west coasts of Vancouver Island, British Columbia. tected waters of the Strait of Georgia while the latter site is exposed to the Pacific Ocean. Populations of mussels were established at each site by collecting spat on ropes during the summer of 1982. In September 1982, the ropes were suspended at a depth of one meter from a raft (Departure Bay) or longline (Mackenzie Anchorage). The mussels were protected from bird and fish predators by enclosing each rope in a cylinder of plastic mesh (4 cm mesh size). The protective cylinders were cleaned on a regular basis to minimize the impact of fouling organisms. Beginning in November 1982, the populations were sampled on a monthly basis until October 1983. At each site, one of the rope/cylinder modules (containing 400 mussels) was designated to monitor growth and mortality. On every sampling date the lengths of 50 mussels from each site were measured to the nearest 0.5 mm. The number of dead mussels was also noted, the shells removed and lengths measured. From the remaining rope/cylinder modules ten mussels were removed from each population for biochemical analysis and twelve for the determination of condition index. Beginning in April 1983, 25 additional mussels were also collected for the assessment of spawning stage. On all sampling dates temperature and salinity pro- files were taken at each study site using a YSI Model 33 temperature/salinity meter. The soft tissues of mussels used for biochemical analysis were excised at the sampling site and frozen on dry ice or liquid nitrogen. The tissue was weighed and then homoge- nized in five volumes of ice cold perchloric acid using a Sorvall Omnimix tissue grinder. Duplicate assays for gly- cogen, protein and lipid were conducted on a Unicam SP/1800 spectrophotometer. Glycogen was assayed by incubating an aliquot of the homogenate with amylo-glucosidase at 40°C for 2 hours (Bergmeyer 1974). The glucose so formed was then mea- sured by monitoring the reduction of NADP to NADPH spectrophotometrically using hexokinase and glucose-6- phosphate dehydrogenase. Glycogen extracted from mussels (Sigma Chemicals, St. Louis, Mo.) was used as a standard. Protein was measured from an aliquot of the perchloric acid homogenate using a modified Biuret method (Gornall 1949) in which deoxycholate is included to ensure that all cell and mitochondrial membranes are completely dis- solved. It was necessary to run a tissue blank for each sample. The blank consisted of a homogenate aliquot dis- solved in Biuret reagent without the addition of copper sul- phate. This procedure ensures that any variation in colour of the tissue extract will not interfere with the absorption of the Biuret complex at 540 nm. Bovine serum albumin was used as a protein standard. Lipid was extracted from the perchloric acid homoge- nate using chloroform/methanol/water in a ratio of 2/2/1.8 V/V (Bligh and Dyer 1959). The lipid content of the chlo- roform layer was then assayed colorimetrically (Barnes and Reproductive and Energy Storage Cycles in Mussels 31 Blackstock 1973). Purified olive oil (Sigma Chemicals. St. Louis. Mo.) was used as a lipid standard. Dry weight conversion factors for each sampling date were obtained in order to express glycogen, lipid and pro- tein values on a dry weight basis. The condition index of mussels was measured using the following procedure: „ , weight of dry tissue ci. = , ^ , . , ; x 100 volume oi whole mussel The external volume of the mussel was determined by the displacement of water by intact mussels cleaned of fouling organisms. Animals used for histological examination were placed in running seawater overnight to clear the digestive system. Mussels were then shucked and fixed for 24 hours in Helly's fixative (Barszcz and Yevich 1975). Following fix- ation the mussels were thoroughly rinsed in running tap water and stored in 70% ethanol. The samples were then dehydrated, embedded and sectioned using standard tech- niques (Luna 1968). Tissue sections were stained with he- matoxylin and eosin (Humason 1969). The tissue sections were subsequently examined using a Zeiss microscope and each specimen assigned to one of three spawning stages using a classification procedure adapted from Quayle (1969): Stage 1 . Development of gametes in the reproductive fol- licles up to the time that the gametes are mature (Figure Stage a) Spawning: Gametes are present in the repro- ductive ducts; or b) Partially spawned out condi- tion: The reproductive follicles are partially empty of gametes (Figure 2b). Stage 3. Spawned out condition: Follicles are spent and the resorption of postspawn reproductive follicles and unspawned gametes by phagocytotic hemocytes is occurring (Figure 2c). RESULTS At Departure Bay surface water temperatures ranged from a minimum of 6°C in December to a maximum of 19°C in June (Figure 3a). The seasonal temperature range was less at the more exposed site (Mackenzie Anchorage), increasing from a minimum value of 8°C to 16°C by late June. Over the summer the surface water temperature was less stable at Departure Bay due to prevailing offshore winds which displace warm surface water and create up- wellings of cooler water. At Departure Bay salinities reached a maximum of 28% r in April and a minimum of \(f/- z DEPARTURE BAY N> 12 16 17 17 17 16 17 15 17 IOO '^22////z 80 L' Y\ 60 40 - • i • // • • • -Z/ • • -^z_ 20 • • • • 0 • . • . • • • • i i • • 1— i r— i — APR MAY MAY JUN JUL AUG AUG SEP OCT 18 9 30 20 II 2 22 13 II MACKENZIE ANCHORAGE ui rr 100 r N» 20 19 Figure 6. Condition index of mussel population at Departure Bay ( •( and Mackenzie Anchorage (O). Expressed as mean ± S.D. N = 12. I I 1 1 1 I I APR MAY MAY JUN JUL AUG AUG SEP OCT 19 10 31 21 12 3 23 14 13 1983 0 STAGE I □ STAGE 2 E2 STAGE 3 Figure 7. Proportion of the mussel population in each spawning stage from April to October, 1983. for mussels in suspended culture at nine sites in the Strait of Georgia (Heritage 1983). The winter growth rates (1-2 mm/month) recorded in the present study are greater than rates for mussels grown in cooler (0-4°C) Canadian At- lantic waters, where winter growth is negligible (MacLeod 1975). In British Columbia cultured Mytilus edulis rarely 34 Emmett. Thompson, and Popham NtahM / ^ J I L L NDJFMAMJJASO 1982/1983 NDJFMAMJJASO 1982/1983 Figure 8. Glycogen (A), lipid (B) and protein (C) content of mussel tissue at Departure Bay <•) and Mackenzie Anchorage (O), expressed as mean ± S.D. N = 10. grows to lengths greater than 60 mm (present study. Heri- tage 1983). In contrast east coast mussels commonly grow to 60-70 mm in length (Freeman and Dickie 1979). The relationship between spawning activity and seasonal changes in energy reserves observed in this study display some unique characteristics when compared to the results of studies from European and western Atlantic populations. As outlined previously, in European mussel populations, the depletion of carbohydrate (glycogen) reserves in winter is due either to the energetic demands of non-reproductive tissue during periods of low food supply or to the synthesis of gonadal tissue (primarily lipid and protein). In both the Departure Bay and Mackenzie Anchorage populations lipid levels and condition index decline in conjunction with gly- cogen during the winter, and lipid content does not increase until glycogen reaches a minimum in late January. This suggests that carbohydrate stores are used primarily for non-reproductive metabolic requirements during the winter and that gonad is synthesized in late winter and early spring, probably in conjugation with the seasonal renewal of food resources. This is also the period of most rapid growth in shell length (2-4 mm/month). This contrasts with the coupled pattern of glycogen depletion and gonad synthesis during the fall described by Pieters et al. (1979) for mussels in the Wadden Sea. and more closely repre- sents the reproductive pattern reported by Thompson (1984a) for a subtidal population of mussels from New- foundland, Canada. The decline in lipid and condition index from late April to June suggests that this was the major spawning period for both populations of mussels. Direct histological obser- vation of gonadal tissue confirms this hypothesis and dem- onstrates that spawning continued until late August in De- parture Bay. At Mackenzie Anchorage this period of spawning occurred concurrently with the renewal of gly- cogen levels, and a second peak of spawning activity oc- curred in late August. Lowe et al. ( 1982) have described a situation similar to Mackenzie Anchorage in a population of Mytilus cclulis from Plymouth. England. In these mussels, gametogenesis and the development of carbohydrate reserves also occurred concurrently during the summer, and the population spawned in both spring and early fall. The authors report that, during the summer, the measured scope for growth of this mussel population was less than would be expected from populations in which spawning and the building of energy stores occur during separate time periods. At De- parture Bay glycogen levels remained low (<5% of dry weight) for four months prior to rebuilding in late June. This is an unusually long period of time, for which no prec- edent could be found in the literature. At both sites, a high proportion of the population was spawning over a prolonged period of time during the late spring and summer. Seed (1975) conducted a histological assessment of spawning activity for mussels in several hab- itats on the coast of England. He describes a spawning pe- riod of four to six months in which over 25% of the popula- tion are spawning. But rarely do more than 50% of the pop- ulation spawn simultaneously. In the present study up to Reproductive and Energy Storage Cycles in Mussels 35 90% of the population spawned simultaneously and over 50% were spawning for a five month period between April and September at Departure Bay. Intense summer spawning activity in these populations coincides with the time of high mortality. Summer mortali- ties in populations of sexually mature mussels on the West Coast have been observed by several researchers and com- mercial growers (Heritage 1983: Skidmore and Chew 1985). These mortalities cannot be accounted for by preda- tion. Incze et al. (1980) suggested that mortalities of raft cultured Mytilus edulis in Maine may have been caused by reduced food ration at a time of metabolic stress; however, these mortalities occurred only at sites where the water temperature had exceeded 20°C. Recently Worrall and Widdows (1984) examined the relationship between spawning and mortality in a population of mussels at Lynher which had exhibited reduced "scope for growth" following spring spawning activity (Lowe et al. 1982). The authors report that non-predatory mortality peaked one month after spawning activity, at a time of high metabolic cost when nutrient reserves in the mantle were at a min- imum. The highest mortalities were recorded in the larger size classes (40-70 mm), which exhibited a higher repro- ductive effort. It is possible that the summer mortalities of Mytilus edulis observed in the present study and by others in British Columbia and Washington are caused by repro- ductive stress. Certainly the suggestion of Suchanek ( 1978) that Mytilus edulis is a colonizer of primary space in the intertidal zone and is often displaced by the larger M . cali- fornianus fits with a reproductive strategy which empha- sizes high reproductive output at an early age. It would be useful to make comparative assessments of reproductive ef- fort, reproductive value and reproductive cost (Bayne et al. 1983. Thompson 1984b) in adjacent populations of Mytilus edulis and M . califomianus to further understand the repro- ductive strategies of these species and assess their relative potential as candidates for aquaculture on the Pacific coast of North America. ACKNOWLEDGMENTS The authors thank Robert Baden of Ocean Wave Farms Ltd. for providing invaluable advice throughout this study. Lynn Buchanan and Gillian Roe for their technical assis- tance, and the staff of the Bamfield Marine Station. Barn- field. B.C. for the provision of laboratory facilities. This research was supported by the British Columbia Science Council (Grants 58RC-5 and 58RC-7). REFERENCES CITED Bayne. B. L.. P. N. Salkeld & C. M. Worrall 1983 Reproductive effort and value in different populations of the marine mussel. Mytilus edulis. Oecologia 59:18-26. Barnes, H. & J. Blaekstoek. 1973. Estimation of lipids in marine animals and tissues: detailed investigation of the sulphophosphovanillin method for total' lipids. J. E.xp. Mar. Biol. Ecol. 12:1(13- 1 IS. Barszcz. C. A. & P. P. Yevich. 1975. The use of Helly's fixative for marine invertebrate histopathology. Comp. Path. Bull. 7:4-5. Bergmeyer. H. U. 1974. Methods of Enzymatic Analysis, Vol. 2. Aca- demic Press. N.Y. Bligh. E. G. & W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 32:911-916 Freeman. K. R. & L. M. Dickie. 1979. Growth and mortality of the blue mussel (Mytilus edulis) in relation to environmental indexing. J. Fish. Res. Bd. Can. 36:1238-1249. Gahbott. P. A. 1983. Development of seasonal metabolic activities in ma- rine molluscs. Hochaehka. P. W.. ed; The Mollusca. Vol. 2 Environ- mental and Biochemical Physiology. Academic Press. N.Y. 362 p. Gomall, A. G.. C. J. Bardawill & M. M. David. 1949. Determination of serum protein by means of the Biuret reaction. J. Biol. Chem. 177:751-766. Heritage. G. D. 1983. A blue mussel (Mytilus edulis L.) culture pilot project in south coastal British Columbia. Can. Tech. Rep. Fish. Aquat. Sci. No. 1174. 27p. Hines. A. H. 1979. Effects of a thermal discharge on reproductive cycle in Mytilus edulis and Mytilus califomianus . Fish. Bull- 77:498-503. Humason. G. L. 1979. Animal Tissue Techniques. W. H. Freeman & Co. San Francisco. 661 p. Incze. L. S . R. A. Lutz & L. Watling. 1980. Relationships between ef- fects of environmental temperature and seston on growth and mortality of Mytilus edulis in a temperate northern estuary. Mar. Biol. 57:147- 156. Lowe. D. M.. M. N. Moore & B. L. Bayne. 1982. Aspects of gameto- .'1. Mytilus edulis. J. Mar. Biol. Assoc. genesis in the marine mussel U.K. 62:133-145. Luna. L. D. 1968. Manual of Histological Staining Methods of the Armed Forces Institute of Pathology. 3rd Ed. MeGraw Hill. N.Y. 258 p. MacLeod. L. L. 1975. Experimental blue mussel (Mytilus edulis) culture in Nova Scotian waters. Interim Report No. 1 . A Joint Project of Nova Scotia Dept. of Fisheries and Fisheries and Marine Service. Environ- ment Canada. Newell. R. I. E.. T. J. Hilbish. R K Koehn. & C. J. Newell. 1982. Temporal variation in the reproductive cycle of Mytilus edulis from localities on the east coast of the United States. Biol. Bull. 162:299- 310. Pieters. H . J H Kluytmans. W. Zurberg & D. I. Zandee. 1979. The influence of seasonal changes on energy metabolism in Mytilus edulis iL.l. I. Growth rate and biochemical composition in relation to envi- ronmental parameters and spawning. Naylor. E. and Haatwell. R. H.. eds. Cyclic Phenomena in Marine Plants and Animals. Pergamon Press. N.Y. p. 285-293. Pieters. H.. J. H. Kluytmans. D. 1. Zandee & G. C. Gadee. 1980. Tissue- composition and reproduction of Mytilus edulis in relation to food availability. Neth. J. Sea Res. 14:349-361 Quayle. D. B. 1969. Pacific Oyster Culture in British Columbia. Fish. Res. Bd. Can. Bull. 169. 192p. Quayle, D. B. 1978. A preliminary report on the possibilities of mussel culture in British Columbia. Fish. Mar. Serv. Tech. Rep. No. 815. 37p. Seed. R. 1975. Reproduction in Mytilus edulis (Mollusca: Bivalvia) in European waters. Publ. Del. Sta:. Zool. Di. Napoli. Vol 39. Suppl 1:317-334. Skidmore. D. A. 1983. Settlement. Growth and Survival of Mytilus edulis L. in Puget Sound and Assessment of Mytilus califomianus for aqua- culture. M. Sc. Thesis. U. of Washington. 99p, Skidmore, D. A. & K. K. Chew. 1985. Mussel Aquaculture in Puget 36 Emmett, Thompson, and Popham Sound Washington Sea Grant Rep. 85-4. University of Washington. Seattle, Wa. 56p. Suchanek. T. H. 1978. The ecology of Mytilits edulis L. in exposed rocky intertidal communities. J. Exp. Mar. Biol. Ecol. 31:105-120. Thompson, R. J. 1984a. The reproductive cycle and physiological ecology of the mussel Mytilus edulis in a subarctic, nonestuarine envi- ronment. Marine Biol. 79:277-288. Thompson, R. J. 19X4b. Production, reproductive effort, reproductive value and reproductive cost of a population of the blue mussel Mytilus edulis from a subarctic environment. Mar. Ecol. Prog. Ser. 16:249- 257. Worrall, C. M. & J. Widdows. 1984. Investigation of factors influencing mortality in Mytilus edulis L. Marine Biol. Let. 5:85-97. Zandee, D. I.. D. A. Holwerda, & A. de Zwann. 1980a. Energy metabo- lism in bivalves and cephalopods. Gilles. R.. ed. Animals and Envi- ronmental Fitness. Vol 1, p 185-206. Pergamon Press, Oxford Zandee, D. [., J. H. Kluytmans. W. Zurburg & H. Pieters. 1980b. Sea- sonal variations in biochemical composition of Mytilus edulis (L. ) with reference to energy metabolism and gametogenesis. Neth. J. Sea Res. 14:1-29. Journal of Shellfish Research, Vol. 6, No. 1, 37-40, 1987. PREDATION ON SINGLE SPAT OYSTERS CRASSOSTREA VIRG1NICA (GMELIN) BY BLUE CRABS CALL1NECTES SAPIDUS RATHBUN AND MUD CRABS PANOPEUS HERBSTIJ MILNE-EDWARDS ROBERT BISKER AND MICHAEL CASTAGNA Virginia Institute of Marine Science School of Marine Science College of William and Mary Wachapreague, Virginia 23480 (U.S.A.) ABSTRACT Single spat oysters Crassostrea virginica of four size classes (3.4-24.6 mm mean shell heights (SH]) were offered to six size classes of blue crabs Callinectes sapidus (9.3-85.5 mm mean carapace width [CW]) and five size classes of mud crabs Panopeus herbslii (7.1-34.4 mm mean CW) for 48 hr. Predation rate, recorded as the number of dead oyster spat/crab/day. was directly proportional to crab size and inversely proportional to oyster size. Mud crabs of 34.4 mm CW and blue crabs of 85.5 mm CW had predation rates of 22.5 and 16.7 spat/crab/day on oyster spat of 24.6 and 24.4 mm SH. respectively. Larger sized spat could be more readily preyed upon by mud crabs than by blue crabs of similar size. Mud crabs of 7. 1 and 25.2 mm CW caused significant mortalities to oyster spat of 8.1 and 24.6 mm SH. respectively. Blue crabs of 9.3, 24.5 and 85.5 mm CW caused significant mortalities to oyster spat of 3.4. 13.9 and 24.6 mm SH. respectively. KEY WORDS: Predation. single spat oysters. Crassostrea virginica, crabs, Callinectes sapidus, Panopeus herbstii INTRODUCTION Predation on juvenile American oysters Crassostrea vir- ginica by portunid and xanthid crabs is an important cause of oyster mortality (Luntz 1947, Menzel and Hopkins 1956. McDermott 1960, Krantz and Chamberlin 1978, MacKenzie 1981, Elner and Lavoie 1983). Blue crabs Cal- linectes sapidus prey on cultchless oysters up to 40 mm in shell length (Krantz and Chamberlin 1978). Mud crabs Panopeus herbstii with carapace widths of 28.0 to 35.5 mm will attack oysters 17 to 54 mm length (McDermott and Flower 1952). MacKenzie (1981) found that adult mud crabs consumed attached oyster spat 10 mm long or less and single oyster spat up to 25 mm long. Estimated preda- tion rates vary from 0.4 to 19.0 spat per crab per day (Menzel and Hopkins 1956, McDermott 1960, Dare et al. 1983, Elner and Lavoie 1983). Larger crabs exposed to smaller single or cultchless spat at near optimum tempera- tures typically cause higher oyster mortality. Nursery growout of spat to a larger size less susceptible to predators and the use of predator-exclusion devices such as netting are techniques used to reduce the effects of crab predation (Walne and Davies 1977). Determination of the effects of crab size and oyster spat size on survival is neces- sary before predator exclusion can be planned for in field growout. This study examines size interactions influencing predation rates of Callinectes sapidus and Panopeus herb- stii on single spat of Crassostrea virginica in the labora- tory. MATERIALS AND METHODS Laboratory experiments were conducted from August to December 1985 using single C. virginica spat, set on small Contribution No. 1369 from Virginia Institute of Marine Science. pieces of crushed oyster shell. The spat were graded into four size classes 1-5,6-10. 11 -20 and 21-30 mm shell height (SH), measured from umbo to greatest height (Galt- soff 1964), and held in flowing seawater. Callinectes sa- pidus and P. herbstii were collected locally, held in flowing seawater and starved for at least 24 hours prior to use. Six size classes of blue crab 7-10, 13-20, 21-26, 30-34. 35-45, 70- 100 mm carapace widths (CW) (Table 1), and five size classes of mud crab 6-8, 10-15. 16-20, 22-27, 31-40 mm CW (Table 2) were tested. All the ex- perimental crabs appeared to be healthy and intact. Crabs that lost appendages, molted or died during the experiment were replaced. Experiments were conducted at the Virginia Institute of Marine Science in Wachapreague, Virginia. Seawater was pumped onto a seawater table holding 24 experimental containers 29 x 18 x 12 cm arranged in two rows. Holes in the sides of each container permitted an exchange of sea- water. Water level was maintained at a depth of 7 cm and a cover was placed over the containers to prevent escape of crabs. Temperature and salinity were measured daily and ranged from 20 to 28°C and 24 to 35 ppt. respectively. Each test consisted of exposing one size class of crabs to four size classes of spat for 48 hr. A crab was placed in a container with fifty spat of one size. Control containers held only spat. Tests were run in triplicate, requiring six containers (3 test replicates -I- 3 control replicates) for each of the four spat sizes. Placement of spat and crabs was random. Oyster mortality was recorded after 24 hours. Dead spat were replaced with live spat of the same size class and the test continued for a second 24 hours. Fifty spat were randomly selected from each size class and shell heights measured to the nearest 0. 1 mm prior to each experiment. Carapace width of each crab was mea- 37 38 BlSKER AND CASTAGNA TABLE 1. Shell heights in the four size classes of oysters (Crassostrea virginica) exposed to blue crabs {Callinectes sapidus) and carapace widths in the six blue crab size classes. Oyster size class Mean Shell Height (mm) ± SD Range 1 3.4 ± 0.84 1.6-5.4 300 2 7.6 ± 1.56 4.8-11.0 300 3 13.9 ± 2.84 8.8-20.1 300 4 24.4 ± 2.93 19.3-31.4 300 Blue crab size class Carapace Width I mm) Mean ± SD Range 1 9.3 ± 0.65 7.8-9.8 12 2 17.2 ± 2.16 13.3-20.4 12 3 24.5 ± 1.00 23.1-26.0 12 4 31.7 ± 1.04 30.5-33.9 12 5 39.2 ± 3.20 34.9-45.7 12 6 85.5 ± 8.50 73.2-96.4 12 sured to the nearest 0. 1 mm at the beginning of each exper- iment. Shell height and CW data were analyzed using anal- ysis of variance. Predation rates were recorded as the number of dead spat per crab per day and compared using analysis of variance after log (x + 1) transformation of data(Sokal and Rohlf 1981). A maximum successful predator ratio (Whetstone and Eversole 1981) for crab size classes with mean CW less than or equal to 25.2 mm was determined by dividing the mean SH of the largest spat size class which was signifi- cantly (p < 0.05) preyed upon by the mean CW. A max- imum successful predator ratio for each crab species was also estimated by averaging the ratios. Although actual predator exclusion tests were not con- ducted in this study, the authors suggest proper net mesh sizes which should prevent crab entrance. This determina- tion was first made by estimating the carapace lengths (CL) TABLE 2. Shell heights in the four size classes of oysters {Crassostrea virginica) exposed to mud crabs (Panopeus herbstii) and carapace widths in the five mud crab size classes. Oyster size class Shell Height (mm) Mean ± SD Range 1 3.7 ±1.19 1 2-6.8 250 2 8.1 ± 1.60 4.2-13.7 250 3 15.1 ± 3.03 8.3-22.6 250 4 24.6 ± 3.07 194-39.9 250 Mud crab size class Carapace Width (mm) Mean ± SD Range 1 7.1 ± 0.48 6.3-7.9 12 2 13.0 ± 1.26 10.7-14.6 12 3 17.8 ± 1.20 16.4-19.9 12 4 25.2 ± 1.09 22.9-27.0 12 5 34.4 ± 2.18 31.3-39.1 12 according to the ratios described by Williams (1965). The proper net mesh sizes which should prevent crab entrance were then determined. RESULTS Mortality of spat in the controls averaged less than 0.3% (max. 0.8%) for all experiments. Crab mortality and shed- ding was normally less than 5.0% (max. 20.0%) for all experiments. Most oysters were crushed or chipped and tissues removed in the manner described by Krantz and Chamberlin (1978) and Elner and Lavoie (1983). Some spat were crushed but not consumed. Predation rates are shown in Table 3. Significant differ- ences (p < 0.01) in predation were associated with crab- spat size interactions, but there were no differences (p < 0.01) in predation rates between the two sampling periods. Predation rates increased as oyster size decreased or crab size increased. Mud crabs caused higher mortalities than blue crabs of similar size. Mud crabs with mean CW of 34.4 mm had a higher predation rate than blue crabs with mean CW of 85.5 mm. Both predators caused significant (p < 0.01 ) mortalities on spat with mean SH of 24 mm. Total mean predation including all spat and crab sizes for blue crabs and mud crabs were 17.4 and 21.5 spat/crab/day, re- spectively. Maximum successful predator ratios are shown in Table 4. Mean ratios for blue crabs and mud crabs were 0.42 and 1.03. respectively. Net mesh sizes with the diagonal di- mension less than the carapace length of a selected crab size are shown in Table 5. DISCUSSION The increase in predation found when spat size de- creases or crab size increases has been reported by other crab-bivalve interaction studies (Whetstone and Eversole 1981, Dare et al. 1983. Gibbons 1984). High predation rates of the larger crabs on even the smallest spat suggests an opportunistic feeding behavior. Although tests on prey size preference were not conducted, Seed (1980) concluded that the preferred size is below the maximum size the crabs can consume. Mean predation rates greater than 50 spat/ crab/day were probable as some crabs preyed on all fifty spat offered. Panopeus herbstii. having higher predation rates than C. sapidus of similar size, may be more efficient at spat pre- dation. Elner and Lavoie (1983) reported that mud crabs Neopanope sayi had predation rates on attached spat similar to that of rock crabs C. irroratus twice their size. Panopeous herbstii had a higher maximum successful predator ratio than did C. sapidus. McDermott (1960) found that P. herbstii of 30 mm CW could kill oysters of 33 to 35 mm length. This indicated a maximum successful predator ratio of 1.13 which is close to that of 1.03 found herein. Ogle ( 1978) estimated a maximum successful pred- ator ratio of 0.40 for C. sapidus — C. virginica interactions, which is very similar to 0.42 calculated in this study. Predation on Oysters by Blue Crabs and Mud Crabs 39 TABLE 3. Predation rate (arithmetic mean and range; n = 3), # dead spat/crab/day on four size classes of oysters (Crassostrea virginica) exposed to six size classes of blue crabs (Callinectes sapidus) and five size classes of mud crabs (Panopeus herbstii). Oyster Shell Height x predation for Carapace width (mm) 3.4 mm 7.6 mm 13.9 mm 24.4 mm each crab size Blue crab 9.3 mm 17.8(11-26)*, 0.8(0-3) 0.0 0.0 4.6 17.2 36.7(18-50)** 3.0(0-8)**** 0.3(0-1) 0.2(0-1) 10.0 24.5 49.8(49-50)** 10.3(3-15)*, 1.8(0-4)** 0.0 15.5 31.7 49.2(47-50)** 28.0(18-33)** 0.8(0-3)*** 0.3(0-1) 19.6 39.2 49.5(49-50) 39.2(11-50)**** 12.5(0-31)**** 0.8(0-4)* 25.5 85.5 41.5(6-50) 49.7(49-50)** 9.7(1-30) 16.7(3-39) 29.4 x predalion rate for each spat size 40.8 21.8 4.2 3.0 17.4 Total predalion 3.7 mm 8. 1 mm 15.1 mm 24.6 mm Mud crab 7.1 mm 19.7(8-30)** 3.0(0-6)**** 0.0 0.0 5.7 13.0 47.5(42-50) 21.0(0-36) 2.2(0-8)* 1.0(0-3) 17.9 17.8 47.8(47-50)** 22.8(6-43)*, 8.8(5-12)** 0.5(0-1)* 20.0 25.2 44.3(30-49) 43.5(29-50)** 10.3(5-21)** 7.8(5-12)* 26.5 34.4 46.8(43-49) 48.2(47-49)** 31.7(21-46) 22.5(10-38) 37.3 x predation rate for each spal size 41.2 27.7 10.6 6.4 21.5 Total predalion * = significantly ** = significantly *** = significantly **** = significantly I = significantly x = significantly different from predation below (p = 0.01). different from predation to the right (p = 0.01). different from predalion below (p = 0.05). different from predation to the right (p = 0.05). different from control (p = 0.01). different from control (p = 0.05). Whetstone and Eversole (1981) reported maximum suc- cessful predator ratios for P. herbstii — Mercenaria merce- naria interactions of 0.65. Predator ratios for C. sapidus. Cancer irroratus, Carcinus maenas, Neopanope sayi and Ovalipes ocellatus preying on M. mercenaria are estimated to be 0.30 (Walne 1974, Mackenzie 1977, Castagna and Kraeuter 1981, Whetstone and Eversole 1981, Gibbons 1984). The data of the authors and others suggest that P. herbstii can cause mortalities to juvenile clams and oysters which are more than twice the size of those affected by other crabs of similar size. The presence of a large molari- form tooth on the crushing edge of the dactyl of the major TABLE 4. Maximum successful predator ratios for blue crabs {Callinectes sapidus) and mud crabs (Panopeus herbstii) preying on single spat oysters {Crassostrea virginica). CW mm claw in P. herbstii gives it a distinct mechanical advantage at crushing bivalve shells (Vermeij 1977). Blue crabs support a major commercial and recreational fishery on the Atlantic and Gulf coasts but population den- sities are hard to determine. Wells (1961) estimated four blue crabs to every 6 m of reef edge, while Larson (1974) found blue crab densities ranging up to 13 m-2. Estimated population densities of P. herbstii on oyster reefs range up to 103 m-2 with highest densities during the summer (Bahr TABLE 5. Carapace widths (CW) and estimated carapace lengths (CD in mm of blue crabs (Callinectes sapidus) and mud crabs (Panopeus herbstii) and net mesh dimensions which should exclude crabs. CW CL Blue crab Mud crab 9.3 17.2 24.5 7.1 13.0 17.8 25.2 Ratios Blue crab 9.3 17.2 24.5 3.7 6.9 0.37 9.8 0.44 31.7 12.7 0.44 39.2 15.7 0.42 x 85.5 34.2 1 14 Mud crab 7.1 4.8 1.16 13.0 8.7 0.85 17.8 11.9 0.98 25.2 16.9 1.03 x 34 4 23.0 Net mesh square mm 2.6 4.9 6.9 9.0 111 24.8 3.4 6.2 8.4 12.0 16.3 40 BlSKER AND CASTAGNA 1974. Larson 1974. Dame 1979. Dame and Vernberg 1982). Panopeus herbstii is the largest (43 mm CW) xanthid crab present in Maryland and Virginia waters and is found in salinities of 10 to 34 ppt (Schwartz and Cargo 1960). McDermott and Flower (1952) also found it to be the largest and dominant xanthid species in the Delaware Bay, as well as the most destructive to oysters (McDermott 1960). Callinectes sapidus ceases activity at 13°C whereas P. herbstii is still active at temperatures below 12°C (Wil- liams 1965, Van Den Avyle 1984). Mud crabs, tending to be less migratory than blue crabs, are usually present within the oyster reef most of their life (Bahr and Lanier 1981). Thus. Panopeus herbstii potentially may be a more dangerous predator than C. sapidus of single oyster spat in high salinity waters. Oyster culturists may reduce crab predation on spat by using plastic mesh netting for predator exclusion. Although not tested, the net mesh sizes presented in Table 5 should prevent crab entrance. Crabs smaller than the net meshes can enter the nets and grow to sizes which may cause some spat mortality if not eliminated by constant monitoring or addition of a biological control such as the toadfish (Gibbons and Castagna 1985). ACKNOWLEDGMENTS The authors would like to thank B. Blaylock, M. Gibbons, R. Mann, and D. Stilwell for their critical review of the manuscript. The authors also would like to thank N. Lewis, J. Moore, J. Watkinson and T. Watkinson for their valuable assistance. REFERENCES CITED Bahr, L. M., Jr. 1974. Aspects of the structure and function of the inter- tidal oyster reef community in Georgia. Ph.D. Dissertation. University of Georgia. Athens. Bahr, L. M. and W. P. Lanier. 1981. The ecology of intertidal oyster reefs of the South Atlantic coast: a community profile. U.S. Fish and Wildlife Service, Office of Biological Services, Washington, D.C. FWS/OBS-81/15. 105 p. Castagna. M. and J. N. Kraeuter. 1981. Manual for growing the hard clam Mercenaria. Spec. Rep. Applied Mar. Sci. Ocean Engin. No. 249. Virginia Institute of Marine Science, Gloucester Point, VA. 1 10 p. Dame, R. F. 1979. The abundance, diversity and biomass of macro- benthos on North Inlet. South Carolina, intertidal oyster reefs. Proc. Nat. Shellfish. Assoc. 69:6-10. Dame, R. F. and F. J. Vernberg. 1982. Energetics of a population of the mud crab Panopeus herbstii (Milne Edwards) in the North Inlet es- tuary. South Carolina. J. Exp. Mar. Biol. Ecol. 63:183- 193. Dare, P. J., G. Davies and D. B. Edwards. 1983. Predation on juvenile Pacific oysters (Crassostrea gigas Thunberg) and mussels (Mytilus edulis L.) by shore crabs {Carcinus maenas [L.]). Fish. Res. Tech. Rep.. MAFF Direct. Fish. Res., Lowestoft No. 73. I? p. Elner, R. W. and R. E. Lavoie. 1983. Predation on American oysters (Crassostrea virginica [Gmelin]) by American lobsters (Homarus americamis Milne-Edwards), mud crabs (Neopanope sayi [Smith]). J . Shellfish Res. 3:129-134. Galtsoff, P. S. 1964. The American oyster Crassostrea virginica Gmelin. U.S. Fish Wildl. Serv. Fish. Bull. 64:1-480. Gibbons, M. C. 1984. Aspects of predation of the crabs Neopanope sayi. Ovalipes ocellatus. and Pagurus longicarpus on juvenile clams Mer- cenaria mercenaria. Ph.D. Dissertation. State University of New York, Stony Brook. Gibbons, M. C. and M. Castagna. 1985. Biological control of predation by crabs in bottom culture of hard clams using a combination of crushed stone aggregate, toadfish. and cages. Aquaculture 47:101- 104. Krantz, G. E. and J. V. Chamberlin 1978. Blue crab predation on cultchless oysterspat. Proc. Nat. Shellfish. Assoc. 68:38-41. Larson, P. 1974. Quantitative studies of the macrofauna associated with the mesohaline oyster reefs of the James River, Virginia. Ph.D. Dis- sertation. School of Marine Science, College of William and Mary. Virginia. Luntz, G. R., Jr. 1947. Callinectes versus Ostrea. J. Elisha Mitchell Sci. Soc. 63:81. MacKenzie, C. L.. Jr. 1977. Predation on hard clam (Mercenaria mer- cenaria) populations. Trans. Amer. Fish. Soc. 106:530-537. MacKenzie, C. L., Jr. 1981. Biotic potential and environmental resistance in the American oyster (Crassostrea virginica) in Long Island Sound. Aquaculture 22:229-268. McDermott. J. J. 1960. The predation of oysters and barnacles by crabs of the family Xanthidae. Penn. Acad. Set. 34:199-21 1 . McDermott. J. J. and F. B. Flower. 1952. Preliminary studies of the common mud crabs on oyster beds of Delaware Bay. Conv. Addr. Nat. Shellfish Assoc. 47-50. Menzel. R. W. and S. H. Hopkins. 1956. Crabs as predators of oysters in Louisiana. Proc. Nat. Shellfish. Assoc. 46:177-184. Ogle, J. T. 1978. Predator prey relationshp between blue crabs and cultchless oyster seed. Miss. Acad. Sci. 23:112. Schwartz, F. J. and D. G. Cargo. 1960. Recent records of the xanthid crab, Panopeus herhsti. from Maryland and Virginia waters. Chesa- peake Sci. 1 (3-4):201-202. Seed, R. 1980. Predator-prey relationships between the mud crab Pa- nopeus herbstii. the blue crab. Callinectes sapidus and the atlantic ribbed mussel Geukensia ( = Modiolus) demissa. Estur. Coast. Mar. Sci. 11:445-458. Sokal. R. R. and F. J. Rohlf. 1981. Biometry. 2nd edition. W. H. Freeman, San Francisco, CA, 859 p. Van Den Avyle, M. J. 1984. Species profiles: life histories and environ- mental requirements of coastal fishes and invertebrates (South At- lantic)—blue crab. U.S. Fish Wildl. Serv. FWS/OBS-82/1 1. 19. U.S. Army Corps of Engineers, TR EL-82-4. 16 p. Vermeij, G. J. 1977. Patterns in crab claw size: the geology of crushing. Sxst. Zool. 26:138-151. Walne. P. R. 1974. Culture of bivalve molluscs: 50 years' experience at Conwy. Fishing News (Books) Ltd.. Surry England. 173 p. Walne. P. R. and G. Davies. 1977. The effect of mesh covers on the survival and growth of Crassostrea gigas Thunberg grown on the sea bed. Aquaculture 11:313-321. Wells, H. W. 1961. The fauna of oyster beds, with special reference to the salinity factor. Ecol. Monogr. 31:239-266. Whetstone. J. M. and A. G. Eversole. 1981. Effects of size and tempera- ture on mud crab. Panopeus herbstii. predation on hard clams. Mer- cenaria mercenaria. Estuaries 4:153- 156 Williams. A. B. 1965. Marine decapods crustaceans of the Carolinas. U.S. Fish Wildl. Serv. Fish. Bull. 65:1-298. Journal of Shellfish Research, Vol. 6. No. 1. 41-44, 1987. ANOXIA INDUCED MORTALITY OF OYSTERS, CRASSOSTREA VIRGIN IC A, ASSOCIATED WITH A SPOIL BANK BISECTING A BAY H. DICKSON HOESE1 AND ROBERT ANCELET2 ^Department of Biology University of Southwestern Louisiana Lafayette 70507 ^■Louisiana Department of Wildlife and Fisheries 400 Royal New Orleans Louisiana 70130 ABSTRACT Large mortalities of oysters were found downcurrent from a spoil bank in a small bay. The bank seemed to produce anoxic conditions as evidenced by extensive blackening of shells. KEY WORDS: Oysters, mortality, anoxia, dredging INTRODUCTION Other than direct burying of environments there is little evidence of impacts of dredging projects that modify the morphology of bay bottoms, with subsequent biological ef- fects. Despite frequent statements that spoil modifies cur- rents, which could cause such effects, the literature indi- cates a preoccupation with more immediate smothering (May 1973a; Windon 1976; Morton 1977). We are presenting here a case where a channel was dredged for a drilling operation in a small bay. The spoil was deposited adjacent to the channel so as to effectively bisect much of the bay. Mortality of blackened oysters en- sued, the study of which produced a number of facts that could be constructed into a reasonable theoretical cause. Unfortunately, no proper before-and-after study could be conducted, but the situation appears unique enough to give insight into processes that might be modified by dredging causing biological results. METHODS The site is a small water body connecting Hackberry Bay with upper Barataria Bay, Louisiana (Figure 1 ). On the north it is connected with Bayou Defond and. although un- named on maps, it is usually called Bay of Bayou Defond. The bay averages 1.3 m in depth, and is about 5 km long and slightly less than 3 km wide. Besides the aforemen- tioned openings there are two bayous. Creole Pass in the southwest comer and one unnamed in the southeast corner, both opening to Creole Bay. and a smaller one in between connecting with Creole Pass. A small oyster reef existed near the center of the bay, and scattered oysters are found from there to the periphery. They were especially common around the periphery and in the deeper bayous and channels. Planting is practiced on some of the leases in the bay, although the area produces native oysters, Crassostrea virginica (Gmelin). In the summer of 1976 a channel was dredged to the northeast off a poorly defined channel that ran WNW-ESE down the middle of the bay. Spoil from this channel was placed on the southeast side, and on 7 September the spoil bank was observed to be above the water for most of its length. Dredge and square-meter samples were taken by the au- thors after the construction in an attempt to determine its effect. RESULTS On 11 November 1976 oyster samples were dredged at six stations (Figure 1, Table 1) which indicated especially high mortality near the spoil bank. At that time, mortality was noted but not measured in the outlet of the bay. Three subsequent samples obtained by dredge on 4-5 January. 1977 near the spoil bank in lease 20734 showed 52, 66, and 86% (avg. 68.3) mortality. Twelve square-meter samples obtained in the same lease yielded a range of 0 to 17 live oysters/meter2 (avg. 2.1) and those recently dead (as evi- dence by unfouled inner shells) ranged from 0-69.5 (avg. 21.5). This gave an estimate for the lease of 3034 live and 761 .660 recently dead, an estimated mortality of 99%, cal- culated from the area and the average numbers/area. Since the samples obtained on 4-5 January 1977 were not designed to show statistical significance, additional sampling was conducted on 13 January. Four samples ob- tained from the open bay outside of the affected area had mortalities ranging from 38-53% (avg. 44.5%). These samples were compared to three samples exhibiting mortal- ities of 52-86% (avg. 68.3%) within lease 20734. Uti- lizing Studenfs "t" test for equality of means, a signifi- cant difference, p < 0.05, was found between samples taken in lease 20734 compared to those taken in the open bay. Since oysters were concentrated on a reef on the center of lease 20734 and the samples were taken randomly over the whole lease, the figures should be conservative. At 41 42 HOESE AND ANCELET Bay of Bayou Def ond B a rata r ia Bay Figure 1 least, unusually high mortality was apparently associated with considerably blackened shells and in some cases cov- erage with a blue-gray clay. On 8 March 1978 the area was again examined and 13 samples were taken, 4 from lease 20734 (Table 3). again showing statistically significant mortalities within lease 20734. At the stations where mortality was high, shells of both live and dead oysters were completely, or near com- pletely, blackened, with several cases of decomposing meat running between the valves. Additionally, dead hooked mussels Ischadium recurvum, and barnacles, Balanus sp., also blackened, were recorded. Some oysters were covered with mud; however, there was insufficient covering to smother the oysters or account for the black color. The oysters outside the mortality area had normally brown (oxi- dized) shells, showing the reduced black color only rarely, and then usually near the base. Sometime in March, the spoil bank was removed, and on another visit on 6 October samples were taken at 4 sta- tions. No sign of blackened oysters was noted and although there was no evidence of recent mortality, the presence of an exceptionally large number of shells on the reef in lease 20734 confirmed that a large mortality had occurred some- time in the past. Again on 26 November 1978 the bay was visited, but no further evidence of mortality or blackened oysters was noted or had been reported as late as 1982. DISCUSSION While it would have been desirable to obtain many more measurements, especially before construction of the spoil Spoil Bank Anoxia Induced Oyster Mortality 43 TABLE 1. Mortalities of commercial size (>7 cm) oysters on II November 1976. Locations are shown in Fig. 1. Number dead determined by counting recently dead unfouled shells and dividing by 2. TABLE 3. Mortalities of commercial size ( >7 cm) oysters on 8 March 1977. Number dead determined by counting recent boxes {attached valves); locations as in Fig. I. Station No. Live No. Dead % Mortalitv Station No. Live No. Dead % Mortality 1 98 34 28 15 15 16 52 2 37 68 65 16 16 6 27 3 104 34 25 17 16 13 45 4 5 59 92 18 25 10 29 5 63 54 46 Total ( !0734) 72 45 39 6 77 28 26 19 13 28 68 Total 3X4 277 41 20 21 13 14 34 25 72 64 22-27 280 26 9 bank, there are still certain facts that can be constructed into probable causes. First, the blackened shells are indicative of sulfide re- duction, generally limited to stagnant areas covered by or- ganically rich sediments of varying degrees of consolida- tion. Second, the extent of coverage of this reduction, especially adjacent to the position of the mantle in the si- phon areas, the putrifying meats, and the deaths of the other associates are symptomatic of death from anoxia. Third, testimony under oath of oystermen familiar with the area maintained that such conditions were never seen until construction of the spoil bank, and the conditions rapidly disappeared after the bank was leveled and the original bay depth restored. Based on our own observations and the rarity of seeing such conditions in oyster growing areas, there is no reason to doubt these assertions. Fourth, we found no evidence of other possible causes of mortalities. This area is in water of low salinity (2.1-22.5%o; avg. 10.3 during observations), which generally produces low mortalities (Mackin and Hopkins 1962). The mechanism for production of the conditions is more problematical. Sediments in the area are predominately silts and clays, richly organic (organic carbon 3-5.5%) with a TABLE 2. Mortalities of commercial size (>7 cm) oysters on 13 January. Locations shown in Fig. I; counts as in Table I. 1977. Station No. Live No. Dead % Mortality 7 26 52 66 8 15 16 52 9 5 32 86 10 27 86 69 Total 20734 73 186 72 11 133 82 38 12 115 76 40 13 29 26 47 14 10 12 53 Total Rest of Bay 287 196 40 BOD of surface sediments of 1 120-1731 mg/02/l with ob- vious blackened high sulfide. The spoil bank linearly bisected about 40% of the bay width. Utilizing Price's (1947) theory relating width to depth, it is not unreasonable that even partial blockage of cross-sectional area, thereby wind fetch and tidal current, would decrease the energy level on the bottom allowing either accumulation of sediments or moving the oxygen/hy- drogen sulfide interface out of the bottom into the water column above the oysters. This could be especially impor- tant in the winter when northerly winds could erode the spoil bank, which was built out of these high BOD sedi- ments. The downcurrent direction was where the mortali- ties occurred. The mortalities were restricted to the area near the spoil bank and the two lower outlets of the bay, with smaller evidence of much effect in between. Sedi- ments do tend to follow shorelines and a decrease in tidal flushing could have allowed accumulation in the deeper channels, similar to the accumulation of unconsolidated, anoxic sediment that occurs in blind, stagnant canals in the area. There does not appear to be a comparable case reported elsewhere, perhaps because such exact conditions have never been produced. Mackin (1962) described a case where oystermen alleged that small spoil banks and a large channel system caused smothering over one mile away in Lake Grande Ecaille. Louisiana. Although he concluded that the sediment was from a closer source, Waldo ( 1958) believed that the spoil and channel system were respon- sible. Price (1947), Breuer (1962). Ryan (1969), and May (1973b) reported shoaling in larger bays due to channels and their spoil banks. In many Louisiana locations low ox- ygen conditions have been found (Poirrier 1975. 1978. Junot 1979. Junot et al. 1983. Ragan et al. 1978) so the potential for development of anoxic bottom waters clearly exists. The present observations and the few literature cases 44 HOESE AND ANCELET suggest that modification of the hydrographic regime may be as or more important than the immediate dredging, and these effects may cause shoaling of fine sediments and hyp- oxia. It would seem to be imperative to develop informa- tion about the sediment and hydrographic budgets of bay systems where such projects occur and to study such projects for insights into these budgets. ACKNOWLEDGMENTS We are especially grateful to the owner of lease 20734. Buster Kass and his attorney. Alvin LeBlanc for consider- able assistance. Sediment data was kindly provided by Rodney Adams and Ron DeLaune of the LSU Center for Wetland Resources. REFERENCES CITED Breuer. J. P. 1962. An ecological survey of the Lower Laguna Madre of Texas. 1953-1959. Publ. Inst. Mar. Set. Univ. Tex. 8:153-183. Mackin, J. G. 1962. Canal dredging and silting in Louisiana Bays. Publ. Inst. Mar. Sci. Univ. Tex. 7:262-314. Mackin, J. G. & S. H. Hopkins. 1962. Studies on oyster mortality in relation to natural environments and to oil fields in Louisiana. Publ. Inst. Mar. Sci. Univ. Tex. 7:1-131 May, E. B. 1973a. Environmental effects of hydraulic dredging in es- tuaries. Bull. Ala. Mar. Res. Lab. 9:1-85 May. E. B. 1973b. Extensive oxygen depletion in Mobile Bay, Alabama. Limnol. Oceanogr. 18(3):353-366. Junot, J. 1979. Effects of salinity stratification and adverse water quality on the benthic invertebrate community of southern Lake Pontchartrain. Louisiana. Nat. Tech. Inf. Serv.. NTIS No. PB 81-185290. 73 p. Junot, J. A., M. A. Poirrier, & Thomas M. Soniat. 1983. Effects of salt- water intrusion from the Interharbor Navigation Canal on the benthos of Lake Pontchartrain, Louisiana. Gulf Res. Repi. 7(31:247-254. Morton, J. W. 1977. Ecological effects of dredging and dredge spoil dis- posal: a literature review. Tech. Rep. U. S. Fish and Wildlife Serv. 94:1-33. Poirrier, M. A. 1975. Epifaunal invertebrates as indicators of water quality in southern Lake Pontchartrain. Louisiana Water Resources Research Inst, Tech. Rept. No. 5 LSU. Baton Rouge. 43 p. Poirrier. M. A. 1978. Studies of salinity stratification in southern Lake Pontchartrain near the inner harbor navigation canal. Proc. La. Acad. Sci. 41:26-35. Price. W. A. 1947. Equilibrium of form and forces in tidal basins of coast of Texas and Louisiana. Bull. Amer. Assoc. Petrol. Geo!. 31(9): 1619-1663. Ragan, J. T , A. H. Harris & H. H. Green. 1977. Temperature, salinity, and oxygen measurements of surface and bottom waters on the conti- nental shelf off Louisiana during portions of 1975 and 1976. Prof. Pap. Ser. (Biology! Nicholls State Univ. 3:1-29. Rose. C. D. 1972. Mortality of market sized oysters [Crassostrea vir- ginica) in the vicinity of a dredging operation. Ches. Sci. 14(2): 1 35 — 138. Ryan, J. J. 1969. A sedimentologic study of Mobile Bay. Alabama. Contr. Sediment. Res. Lab. Florida St. Univ. 30:1-110. Waldo, E. 1958. Report Biological Section. 7th Biennial Rept. La. Wildl. & Fish. Comm. (1956-57), p. 93-95. Windon, H. L. 1976. Environmental aspects of dredging in the coastal zone. CRCCril Rev. Environ. Control. 5(21:91-109. INFORMATION FOR CONTRIBUTORS TO THE JOURNAL OF SHELLFISH RESEARCH Original papers dealing with all aspects of shellfish re- search will be considered for publication. Manuscripts will be judged by the editors or other competent reviewers, or both, on the basis of originality, content, merit, clarity of presentation, and interpretations. Each paper should be carefully prepared in the style followed in Volume 3, Number 1 , of the Journal of Shellfish Research ( 1983) be- fore submission to the Editor. 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If illustrations and/or tables make up more than one third of the total number of pages, there will be a charge of $30.00 for each page of this material (calculated on the actual amount of page space taken up), regardless of the total length of the article. All page charges are subject to change without notice. Proofs: Page proofs are sent to the corresponding author and must be corrected and returned within seven days. Al- terations other than corrections of printer's errors may be charged to the author(s). Reprints: Reprints of published papers are available at cost to the authors. Information regarding ordering reprints will be available from The Sheridan Press at the time of printing. Cover Photographs: Particularly appropriate photo- graphs may be submitted for consideration for use on the cover of the Journal of Shellfish Research. Black and white photographs, if utilized, are printed at no cost. Color illus- trations may be submitted but all costs associated with re- production and printing of such illustrations must be cov- ered by the submitter. Corresponding: An original and two copies of each manuscript submitted for publication consideration should be sent to the Editor, Dr. Sandra E. Shumway, Bigelow Laboratory for Ocean Science and Department of Marine Resources, West Boothbay Harbor, Maine 04575. JOURNAL OF SHELLFISH RESEARCH Vol. 6, No. 1 June 1987 CONTENTS Editorial Comment i M. E. White, E. N. Powell, S. M. Ray, andE. A. Wilson Host-to-Host Transmission of Pefkinsus marinus in Oyster (Crassostrea virginica) Populations by the Ectoparasitic Snail Boonea impressa (Pyramellidae) 1 Diane J . Brousseau A Comparative Study of the Reproductive Cycle of the Soft-shell Clam Mya arenaria in Long Island Sound Diane J. Brousseau and Jenny A . Baglivo A Comparative Study of Age and Growth in Mya arenaria (Soft-shell Clam) from Three Populations in Long Island Sound 17 J. Harold Beattie, James Perdue, William Hershberger and Kenneth Chew Effects of Inbreeding on Growth of the Pacific Oyster {Crassostrea gigas) 25 B. Emrnett, K. Thompson, and J. D. Popham The Reproductive and Energy Storage Cycles of Two Populations of Mytilus edulis (Linne) from British Columbia ... 29 Robert Bisker and Michael Castagna Predation on Single Spat Oysters Crassostrea virginica (Gmelin) by Blue Crabs Callinectes sapidus Rathbun and Mud Crabs Panopeus herbstii Milne-Edwards 37 H. Dickson Hoese and Robert Ancelet Anoxia Induced Mortality of Oysters Crassostrea virginica Associated with a Spoil Bank Bisecting a Bay 41 ■ r* :'• COVER PHOTO: The soft-shell clam Mya arenaria. Studies indicate that spawning patterns of the clam may vary widely within its range. ■>? s.V'v* t *» T Editor K Dr. Sandra E. Shumway -■ < Bigelow Laboratory for Ocean Science and . w ; Department of Marine Resources West Boothbay Harbor Maine 94575 Publications Committee Mr. Michael Castagna, Chairman The College of William and Mary Virginia Institute of Marine Science Wachapreague, Virginia 23480 Dr. Melbourne R. Carriker University of Delaware College of Marine Studies Lewes, Delaware 19958 Dr. Robert E. Hillman Battelle Ocean Sciences Duxbury, Massachusetts 02332 Dr. Roger Mann The College of William and Mary Virginia Institute of Marine Science Gloucester Point, Virginia 23062 Journal of Shellfish Research Volume 6, Number 1 ISSN: 00775711 June 1987 Dr. Loosanoff al his desk as Director of Milford Laboratory circa 1962 IN MEMORIAM Marine Biological Laboratory LIBRARY FEB 24 1988 Woods Hole, Mass. Dr. Victor Lyon Loosanoff Dr. Victor L. Loosanoff died at his home in Greenbrae, California, on June 15 at the age of 87. He was recognized internationally for his research in the field of shellfish physiology and ecology with emphasis on the larval culture of oysters. Born in Kiev, Russia in 1899. son of a career officer in the Imperial Russian Army, Dr. Loosanoff received a military academy education at the Emperor Alexander First Cadet Corps, Osmk, graduating at age 17. He served 4 years as a cavalry officer in the White Army during the revolution, escaping into China in 1921 after fighting a long retreating action from the Volga River across Russia. Immigrating to the United States in 1922, he worked in lumber camps and as a commercial fisherman on the West Coast while learning English. He entered the University of Washington in 1924 graduating 3 years later with a B.S. in Fisheries Science. Following positions as a marine biologist with the states of Washington and Virginia, Dr. Loosanoff was appointed in 1931 as Aquatic Biologist with the U.S. Bureau of Fisheries and stationed at Milford, Connecticut to undertake studies of the commercial oyster fishery in Long Island Sound. In that capacity, he founded the Milford Laboratory and served as its Director for 31 years. It was during this period that Dr. Loosanoff and colleagues under his direction carried out the research that resulted in the development of shellfish aquaculture methods now practiced commercially in the U.S. and abroad. Early in his Milford career, he also undertook graduate studies at Yale University and completed a Ph.D. in Zoology in 1936 under Professor Wesley R. Coe. Dr. Loosanoff stepped down as Laboratory Director at Milford in 1962 to accept a position as Senior Scientist with the Bureau of Commercial Fisheries (now NMFS) stationed in Tiburon, California at the then Bureau of Sportfish and Wildlife Laboratory. He continued his research and writing there while holding a concurrent appointment as Adjunct Professor at the University of the Pacific's Pacific Marine Station allowing him to supervise students in marine biology. Retiring in 1965, he remained active serving as a consultant to BCF and the shellfish industry as well as conducting research under a National Science Foundation grant. Over his long career. Dr. Loosanoff authored some 200 scientific papers and popular articles. His work is cited frequently in the current scientific literature and constitutes a significant contribution to the field of shellfish physiology and molluscan aquaculture. He served as President of NSA in 1947-49 and was elected an Honorary Member in 1963. Among other honors, he received the Department of Interior's highest, the Distinguished Service Award, in 1965 for "exceptional contributions to the scientific programs of the Bureau of Commercial Fisheries". At the end age had taken its toll. With failed eyesight and general poor health, he became despondent and chose to take his own life. He is survived by his wife of 59 years, Tamara, and a brother living in Kiev, Russia. Dr. James E. Hanks Milford Laboratory November, 1987 Journal of Shellfish Research. Vol. 6, No. 2. 45-48, 1987. COMPARATIVE ATTACHMENT, GROWTH AND MORTALITIES OF OYSTER (CRASSOSTREA VIRGIN IC A) SPAT ON SLATE AND OYSTER SHELL IN THE JAMES RIVER, VIRGINIA D. S. HAVEN', J. M. ZEIGLER1, J. T. DEALTERIS2 & J. P. WHITCOMB1 ^Virginia Institute of Marine Science- School of Marine Science The College of William and Mary Gloucester Point, Virginia 23062 2Dept. of Fisheries, Aquaculture and Pathology University of Rhode Island Kingston, R.I. 02881 ABSTRACT Slate was investigated as a substitute for oyster shells which are used as a substrate for oyster spat [Crassostrea virginica) settlement in James River. Virginia oyster repletion programs. Oyster shells and slate fragments were planted on adjacent plots in two submerged locations about 825 m apart in July 1984. Quantitative .093 m2 (one ft2) samples were collected by a diver on seven occasions through July 1985. with additional samples collected from the natural oyster bottoms adjacent to the two areas. Percent mortality, growth and numbers of live spat and spat scars (dead spat) per unit area of bottom were determined. At the end of the study, the number of spat on shell was 4-5 times higher than on slate; however, slate had 5-6 times more spat per unit area of bottom than the shell on the natural bottom. During the July to October setting season mortalities were much higher on slate than on shell; during the remaining period they were high but about equal on both substances. INTRODUCTION Experiments have shown that oysters will attach to al- most any hard surface, including asbestos plates, frosted glass, wood, cement and marble (Dupuy and Rivkin 1972; Hidu et al. 1975; Kennedy and Breisch 1981). These studies were primarily designed to study setting intensity and patterns of set; none were large scale field studies de- signed to find a substitute for shell on a commercial scale. We investigated slate as a substitute since it offers a hard surface, low cost, and a plentiful and readily available supply in Virginia. Moreover, it has been used experimen- tally to study setting patterns of benthic invertebrates such as barnacles (Osman 1977). METHODS The study was conducted in the James River. Virginia, in the Wreck Shoals area, a location which receives a mod- erate to heavy set of oysters each year (Haven and Whit- comb 1983; Haven and Fritz 1985). Two locations about 825 m apart were selected and marked by wooden stakes: 1) Wreck Shoals Inshore and 2) Wreck Shoals Offshore. Water depths (MLW) averaged 2.7 m on each plot. At each location two plots of (37.2 m2) in size 2.7 m and about 3.0 m apart were selected and marked with stakes (Figure 1). Salinities in the area during the July through October set- ting season ranged from 8.9 to 19.0%e (x = 13.3) and 1 1.8 to 20.4%o (x = 15.4) from October to July. The naturally productive bottom on the inshore plot was a mixture of sand, shells, and oysters; on the offshore plot, the bottom Contribution Number 1422 from the Virginia Institute of Marine Science. The College of William and Mary, Gloucester Point. Virginia 23062. was very hard and was comprised largely of oysters, shells, and small shell fragments (Haven and Whitcomb 1983). From 16-24 July 1984 about 250 bushels (8.8 m3) of oyster shells obtained from a shucking house and an equal volume of slate were placed on adjacent plots in the inshore and offshore locations. Slate fragments were flat to suban- gular and ranged from 0.5 to 5.0 cm in length (x = 3.0 cm); oyster shells averaged 7-9 cm in length (x = 7.5 cm). Four or five samples of oyster shells and slate were col- lected at random by a diver on seven occasions from each of the four plots from 10 August 1984 to 15 July 1985 (Table 1 ). In addition, the natural oyster bottom adjacent to each area was sampled in the same manner on three occa- sions. Each random sample collected by a diver consisted of material collected inside a .093 m2 (one ft2) frame placed on the bottom. The initial sampling depth of the substrate on 10 August 1984 was about 6 cm on shell and about 4.3 cm on slate. However, an examination of these two sub- strates, and of the bottom by a diver, indicated that sedi- ments had filled most of the voids at and below these two depths. Consequently, subsequent samples were collected to about 3 cm on slate and 5-6 cm on shell. The volume of slate collected in each sample averaged about 1500 cm3; shell number ranged from 28-60 (x = 35). All samples of shell and slate were examined with a dis- secting scope at 15X magnification after washing sediments from the material. Spat were counted and then measured to the nearest mm. Spat scars (the white area left after the top valve of the spat had fallen off) were also counted and measured, but only during and immediately after the setting season (Table 1). From 10 August to 23 November. 1984 mean data on 45 46 Haven et al. Figure 1. Locations of sampling stations in the James River, Virginia where shell and slate was planted. spat and spat scar density and their lengths are based on randomly collected subsamples of the slate and shell; 25 to 50% of the total material collected was examined. This was necessitated by the large number of spat and spat scars in the samples. Subsequent counts are based on an examina- tion of all material collected. The percent mortality of spat during the setting season was not calculated because of the interaction between re- cruitment and mortality. While spat scar numbers were re- corded, t :y are considered as unreliable indicators of long term mortality due to the difficulty in recognizing them after !-4 weeks. Mortalities were calculated after setting ceased for the 23 November 1984 to 15 July 1985 period on the basis of changes (percent) in numbers of live oysters between the two dates. Statistical studies compared numbers of spat m2 and spat lengths in mm for various locations, dates, and substrate types. Comparisons of spat density were made for the post setting period for October, November, January and March, but not for July (low sample numbers). Lengths were com- pared for the final two sampling periods in March and July 1985. Data sets being compared were first tested for homo- geneity of variance (p = 0.05) by a variance ratio (F) test. Later, mean spat lengths and mean number of spat were tested for significant differences between the various vari- ances by a two-sample t-test with Cochrans t approxima- Comparative Attachment, Growth, and Mortalities of Oyster Spat 47 TABLE 1. Mean numbers of oyster spat and spat scars per .093 m2 (one ft2) and mean lengths of spat and spat scars on oyster shell, slate, at two location on Wreck Shoals in the James River, Virginia, and on adjacent natural bottoms. OYSTER SHELL Inshore - - X Offshore - - X no. length no. length no. length no. length Date spat spat scars scars spat spat scars scars 10 Aug 84 63.2 — — — 98.9 30 Aug 84 80.8 5.0 21.0 3.8 27.3 3.8 14.2 2.5 8 Oct 84 228.9 8.2 19.4 7.9 26. I1 7.7 3.4 8.3 23 Nov 84 185.2 10.8 33.5 8.7 73.3 7.9 25.8 6.7 8 Jan 85 99.9 10.8 — — 57.4 10.2 1 1 Mar 85 128.4 10.9 — — 15.0 7.4 15 Jul 85 35.0 18.7 — SLATE 11.6 21.9 — — 10 Aug 84 5.0 — — — 1.6 — — — 30 Aug 84 42.1 4.8 16.8 3.0 27.8 3.9 16.9 2.1 8 Oct 84 33.5 7.5 30.2 7.5 22.3 7.9 13.6 7.9 23 Nov 84 45.2 9.6 16.2 6.3 23.6 7.6 12.8 5.8 8 Jan 85 31.6 111 — — 26.0 9.2 11 Mar 85 17.2 9.1 — — 11.2 9.9 — 15 Jul 85 8.4 16.9 — NATURAL BOTTOM 2.2 16.3 — — 23 Nov 84 2.4 11.0 — — — — 8 Jan 85 3.4 12.6 — — 1.2 12.3 — 15 Jul 85 1.4 24.0 — — 0.4 — — — This low value may be anomalous. tion, which depends on the homogeneity of variance (Guenther 1964). All statistical tests were made at the 95% confidence level or p = 0.05. RESULTS An inspection of the planted areas by a diver showed that slate and shell had not been evenly distributed at planting. On the Inshore plot, the slate formed an area about 6.1 x 6.1 m in extent, and the adjacent shell plot, about 3 m away, covered an area about 6. 1 x 10 m in size. On the offshore plots, the slate had been deposited in the form of an oval about 3.0 x 5.0 m in extent, and the shelled area about 3 m away formed a 4.6 x 4.6 m square. On the slate plots the diver observed that sedimentation began shortly after planting to form a thin veneer of fine sediment 1-2 mm thick, and it covered an increasing per- centage of the clean surfaces with each monitoring period. By 8 October 1984 the slate was about 90- 100% covered with fine sediment; the voids between the particles were relatively small or completely filled, and only the upper 2-3 cm were exposed to the water. On areas where shell had been planted there was also the initial fine layer of sed- iment 1-2 mm thick on 80-90% of the shell, but the re- maining surfaces appeared relatively free of silt and bio- fouling. Moreover, there were still some voids between the shells to a depth of about 4-5 cm. On 1 1 March 1985 a slight reduction in sediment thickness on both plots was noted and conditions remained relatively similar to the end of the study. On the inshore plots, there were significantly more spat on shell substrate than on slate for October and November 1984 and March 1985 (P < 0.05). No difference was shown for January 1985. A similar comparison for the off- shore plots showed no significant difference in mean number of spat on the two substrate types for any month (Table 1). Spat density on shells on the inshore area was signifi- cantly higher than shells offshore for the months of October and November 1984 and for March 1985 (P < 0.05). On slate, spat density on the inshore plot was also significantly greater than offshore (P < 0.05) during October and No- vember 1985. During the setting season, which extended to early Oc- tober 1984, there was an increase in numbers of spat on the shell and slate. This increase was not always linear due to continuing recruitment and heavy but irregular mortalities as evidenced by the occurrence of numerous spat scars in all areas (Table 1). After the setting period, the following percent mortalities were calculated from Table 1 for the 23 November 1984 to 15 July 1985 period: Shell Inshore— 81%; Shell Offshore — 84%; Slate Inshore — 81%; and Slate Off shore— 91%. 48 Haven et al. At the end of the study on the inshore plots for March and July 1985, spat were longer (P < 0.05) on shell than on slate. On the offshore area, however, spat on shell were significantly larger (P < 0.05) only during July, but the differences cited were not large (Table 1). While slate was less effective than shell in collecting spat, slate consistantly had more spat per unit area than the oysters and oyster shells on natural bottoms (Table 1 ). Dif- ferences calculated from that source showed that the slate had from 5.5 to 6.0 times more spat per unit areas than the natural bottom on 15 July 1985. DISCUSSION The cause(s) of the high mortality observed during the study are unknown, but deaths due to xanthiid mud crabs, blue crabs (Callinectes sapidus) and Hat worms [Stylochus ellipticus) were most certainly involved. These predators often cause excessive oyster mortalities in Chesapeake Bay (Webster and Medford 1961, Krantz and Chamberlin 1978). Siltation was also involved and the fact that its ini- tial coverage was greater on the slate plots may be the cause of much of the observed difference in numbers of spat between slate and shell (Mackenzie 1970). The reason for the higher setting on shell and slate on the inshore areas in comparison to that observed offshore is not apparent. Depths of the two locations were the same and they were only 825 m apart. Differences in factors such as hydrography, the chemical differences between the two substrates, and available food and predator density were not studied. While our study favors oyster shell over slate as a setting medium, it is emphasized that at the end of the study, slate still had more spat than old shells and oysters growing on adjacent natural bottoms. It is suggested that accumulated biofouling on the latter substrate might have been responsible for the mortalities. ACKNOWLEDGEMENT The authors express their gratitude to Mary Sue Jab- lonsky for the statistical treatment of data in this paper. Also we thank the Solite Corporation, Fluvana County, Va. for donating the slate. REFERENCES CITED Dupuy. J. L. & S. Rivkin. 1972. The development of laboratory tech- niques for the production of cultch-free spat of the oyster {Crassostrea virginica). Ches. Sci. 13( I ):45 — 52. Guenther, W. C. 1964. Analysis of Variance. Prentice-Hall, Inc.. N.J. 199 pp. Haven, D. S. & L. W. Fritz. 1985. Setting of the American oyster Cras- sostrea virginica in the James River, Virginia. USA: temporal and spatial distribution. Mar. Biol. 86:271-282. Haven, D. S. & J. P. Whitcomb. 1983. The origin and extent of oyster reefs in the James River. Virginia. J. Shellfish Res. 3:141-151. Hidu, H. S.. S. Chapman & P. W. Soule. 1975. Cultchless setting of European oysters (Ostrea edulis) using polished marble. Proc. Natl. Shellfish. Assoc. 65:13-14. Kennedy. V. S. & L. L. Breisch. 1981. Maryland's Oysters: Research and Management. Univ. Maryland (Sea Grant) Publ. No. Am-SG-TS- 81-04:1-186. Krantz. G. E. & J. F. Chamberlin. 1978. Blue crab predation on cultch- less oyster spat. Proc. Natl. Shellfish. Assoc. 68:38-41. Mackenzie. C. L.. Jr. 1970. Causes of oyster spat mortality, conditions of oyster setting beds, and recommendations for oyster bed management. Proc. Natl. Shellfish. Assoc. 60:59-67. Osman, R. W. 1977. The establishment and development of a marsh epi- faunal community. Ecol. Monogr. 47:37-63. Webster, J. R. & R. Z. Medford. 1961. Flatworm distribution and asso- ciated oyster mortality in Chesapeake Bay. Proc. Natl. Shellfish. Assoc. 50:89-95. Journal of Shellfish Research, Vol. 6, No. 2, 49-53, 1987. ENVIRONMENTAL EFFECTS ON THE GROWTH OF SIBLING PACIFIC OYSTERS CRASSOSTREA GIG AS (THUNBERG) AND OVERWINTERED SPAT ANJA ROBINSON & HOWARD HORTON Department of Fisheries and Wildlife Mark O. Hatfield Marine Science Center Oregon State University Newport, Oregon 97365 ABSTRACT The growth of hatchery-reared Crassostrea gigas siblings and over-wintered spat was measured on commercial oyster grounds at three locations on Oregon and Washington coasts: Coos Bay, Oregon; Willapa Bay. Washington; and Oyster Bay, Shelton, Washington. Sampling was carried out at four month intervals at each location from November 1984 through June 1986. After two years on commercial grounds oysters showed similar growth measured by shell length and width although wet and dry weights of the meat varied considerably between locations. The spat, overwintered in a nursery, showed the lowest mortality on the commercial grounds, but did not show better yield of meat than year younger oysters. KEY WORDS: Pacific oyster, siblings, overwintered spat, commercial grounds. INTRODUCTION The Pacific oyster Crassostrea gigas (Miyagi variety) is native to semi-temperate areas of Japan and is the primary species of oyster cultured in the Pacific Northwest. Due to low water temperatures this introduced species seldom re- produces in the northwest. Oysters are in spawning condi- tion during the summer months but the gametes are re- sorbed in the fall (Lannan et al. 1980). As a consequence, oyster growers in the Pacific North- west are dependent on hatchery produced seed. Culture methods are well established and local hatcheries are a reli- able source of seed. As a common practice, oyster growers obtain seed from the hatchery as eyed larvae and setting is accomplished in setting tanks at the sites of individual growers (Henderson 1983). Hatcheries acquire their brood- stock from oyster growers and condition oysters by ele- vating the water temperature for a period of time to speed the rate of gametogenesis (Lannan et al. 1980). The selection that takes place during the cycle from oyster grower through hatchery and growing grounds is of great concern to biologists as well as oyster growers. Sib- ling oysters may be adapted to some commercial conditions better than others, and growth rates and meat production can vary considerably. This paper reports on a preliminary study of the performance of a brood of sibling animals ob- tained from a hatchery and distributed to various commer- cial oyster grounds. Also the performance of two different broods at one location is compared. MATERIALS AND METHODS Broodstock oysters came from Willapa Bay, Wash- ington. Conditioning, spawning, and rearing of the larvae was carried out in a hatchery at Netarts, Oregon. Eyed larvae were shipped to oyster growers in June 1984 and each facility provided their own cultch and method of set- ting. After setting, oyster growers transferred the cultch with spat to commercial growing grounds where they were left for two years before the clusters were broken and thinned. Monitoring the growth of this brood was carried out at three locations on the Oregon and Washington coasts: Coos Bay, Oregon, a sheltered estuary where nat- ural recruitment rarely occurs; Willapa Bay, Washington, a TABLE I. Mean and (S.D.) (N = 50) shell length in millimeters of C. gigas siblings spawned May, 1984, measured from three commercial oyster beds, and overwintered spat spawned May, 1983, measured from one commercial oyster bed. Means indicated by * are significantly different (p > .05; Tukey's Studentized Range Testl comparison made only on oysters at 25 months. Months Willapa Bay on beds over-wintered Willapa Bay Oyster Bay Coos Bay 5 43.40 (9.25) 25.94 (8.04) 33.64(10.02) 17.99 (3.61) 9 58.32(12.50) 38.50(10.32) 40.60(11.38) 37.70(10.30) 13 90.47 (21.10) 59.60(11.59) 58.10(16.88) 56.60(12.16) 17 96.44(16.79) 77.10(14.45) 88.50(11.91) 87.90(14.51) 21 119.66(22.46) 99.70(26.21) 99.60(18.12) 79.20(13.62) 25 100.44 (17.17)* 99.94(19.94)* 112.20(15.82) 118.75 (18.62) 49 50 Robinson & Horton TABLE 2. Mean and (S.D.) (N = 50) shell width in millimeters of C. gigas siblings spawned May, 1984, measured from three commercial oyster beds, and overwintered spat spawned May, 1983, measured from one commercial oyster bed. Means indicated by * are significantly different (p > .05; Tukey's Studentized Range Test) comparison made only on oysters at 25 months. Months Willapa Bay on beds over-wintered Willapa Bay Oyster Bay Coos Bay 5 29.10(6.17) 19.20(5.01) 25.97(7.79) 14.40 (4.08) 9 36.50(8.79) 28.60 (8.50) 31.00(9.06) 25.80(6.70) 13 44.80(8.16) 38.60 (8.94) 39.10(10.23) 38.10(8.61) 17 50.80(7.81) 41.70(6.69) 56.20(8.84) 46.80(7.21) 21 51.90(8.94) 50.80(10.44) 58.90(9.72) 50.50 (7.68) 25 53.60 (9.85)* 56.30(11.23)* 63.25 (11.02) 63.25 (8.47) sheltered estuary about 25 miles long and 3-4 miles wide and where salinity fluctuations are great and summertime water temperatures are high enough for occasional nat- ural recruitment; and Oyster Bay, Shelton, Washington, a relatively shallow, long, narrow bay at the southern end of Puget Sound inlet to the sea where salinity fluctuations are small and occasional natural recruitment occurs (Fig. 1). At Willapa Bay, a second group of oysters was sampled. Parents of these oysters also came from Willapa Bay, and the oysters were conditioned, spawned, and the larvae reared in Coast Oyster Company's hatchery at Quilcene, Washington. Eyed larvae were transferred to Ocean Park. Washington, where setting was accomplished in June 1983. Spat were held over winter on a nursery bed at the plus 2-foot tide level in the sheltered area of the bay until May 1984 when they were transferred to a commercial growing area at Willapa Bay. Sampling at each site was carried out during low tides at four-month intervals starting November 1984. At this time the spat had been on the commercial grounds at least five months. Sampling was completed in June 1986. Approxi- mately 50 oysters were randomly picked from each area. Oysters were brought to the Hatfield Marine Science Center, Oregon State University, at Newport, for measure- ments of shell length and width as well as wet and dry meat weights. For wet weight, meats were drained on newspaper for an hour. For dry weight meats were dried in a forced air oven at 50°C for 24 hours and then at 100°C for 18 hours, cooled in a desiccator and weighed. From the measure- ments of each sample, a ratio of shell length versus shell width was calculated. From data on wet weight and dry weight of meats, the percent water content of the tissue was determined. Mortalities of groups of oysters were deter- mined by measuring the proportion of empty shells per sample. From the June 1986 samples, a subsample of 10 oysters was taken randomly for determination of condition factor index by the volumetric method (Grave 1912): C.F. vol. meat (ml) vol. shell cavity (ml) x 100 Differences in oyster sibling and overwintered spat per- formance among oyster beds were examined by analysis of variance followed by the Tukey-Kramer method of multiple comparisons between pairs of beds (Sokal and Rohlf 1981 ). RESULTS This study demonstrates that sibling oysters perform dif- ferently in different environments. Oysters from all areas sampled showed increases in mean shell length and width (Tables 1 and 2). Although sibling oysters showed similar growth as measured by shell length and width in each ran- domly collected sample from three locations, there was a significant difference in shell lengths after two years on the TABLE 3. Mean and (S.D.) .05; Tukey's Studentized Range Test I comparison made only oysters at 25 months. Months Willapa Bay on beds over-wintered Willapa Bay Oyster Bay Coos Bay 5 1.20 (.42) 0.29 (.02) 1.33 (.18) 0.18 (.09) 9 1.76 1.98) 0.93 (.12) 1.33 (.16) 1.30 (.13) 13 6.28 (1.31) 2.94 (.57) 6.50(.68) 5.90 1.87) 17 15.04(2.09) 6.20 1.92) 18.30(1.06) 9.70(1.07) 21 12.17 (1.67) 8.90 (.41) 16.70 (.98) 9.40 (.99) 25 11.20 (.86) 14.66 (.34) 21.41 (1.36)* 15.44 (1.58) 52 Robinson & Horton TABLE 5. Mean and (S.D.) (N = 50) dry weight in grams of tissue of C. gigas siblings spawned May, 1984, measured from three commercial oyster beds, and overwintered spat spawned May, 1983, measured from one commercial oyster bed. Means indicated by * are significantly different (p > .05; Tukey's Studentized Range Test) comparison made only on oysters at 25 months. Months Willapa Bay on beds over-wintered Willapa Bay Oyster Bay Coos Bay 5 0.12 (.09) 0.03 (.01) 0.15 (.02) 0.02 (.008) 9 0.26 (.10) 0.13 (.06) 0.20 (.04) 0.23 (.07) 13 1.06 (.36) 0.60 (.09) 1.50 (.12) 1.21 (.12) 17 1.12 (.38) 0.50 (.08) 2.70 (.38) 1.24 (.12) 21 1.53 (.83) 1.40 1.13) 3.30 (.96) 1.90 (.11) 25 2.17 (.98) 2.40 (.22) 4.45 (.46)* 2.80 1.31) oyster growers and the personnel carrying out the field work for evaluation. Hatchery siblings which are to be set by oyster growers all have the same genetic origin. Since the oyster growers setting facilities and cultch differ from each other, it is possible that selection takes place before siblings reach actual commercial growing grounds. How- ever, it does not seem likely that selection at setting was the sole factor for better yield at Oyster Bay versus Willapa Bay. At both locations siblings were on the muddy bottoms of the bay and no overcrowding could be found in either place. Other environmental factors, for example, better nu- TABLE 6. Mean (N = 10) condition factor indexes calculated as the ratio of volume of meat to volume of shell cavity of C. gigas siblings collected from three commercial oyster beds and overwintered spat collected from one commercial ovster bed after 25 months at each location. Location and Mean condition factor time when spawned index Willapa Bay overwintered spawned May, 1983 53.33 Willapa Bay spawned May, 1984 31.48 Oyster Bay spawned May, 1984 65.79 Coos Bay spawned May. 1984 46.34 trition, will make a difference in yield. Furthermore, over- wintered spat which had a different genetic background from the siblings showed a similar growth pattern to that shown by siblings in Willapa Bay. Walne (1970) compared seven populations of Ostrea edulis and found one location that produced higher quality oysters. The better production was probably caused by better feeding conditions. Since this paper compares sibling oysters on commercial grounds, meat volume is important and it is used for calcu- lating condition factor indexes. The calculations can easily be carried out in the field if necessary, thus enabling the grower to evaluate the performance of the sibling oysters in various locations. In order to carry out a long-term brood- stock management program, close attention needs to be given to each group of oysters used for spawning in the hatcheries. The origin and performance in the field of these animals should be recorded. The use of closely related an- imals as a sole parental group for brood production should be avoided. The gene pool should be kept at a maximum to ensure the best possible growth on various oyster beds. ACKNOWLEDGMENTS We thank Lee Hansen who helped to locate oyster growers who received sibling oysters from his hatchery in May, 1984. We also thank Larry Qualman, Justin Taylor TABLE 7. Mean (N = 50) mortality of C. gigas measured as a percent of empty shells in the samples of siblings spawned May, 1984, collected from three commercial oyster beds, and overwintered spat spawned May, 1983, collected from one commercial oyster bed. TABLE 8. Mean (N = 50) percent of water calculated from wet weight and dry weight of the tissue of C. gigas siblings spawned May, 1984, collected from three commercial oyster beds, and overwintered spat spawned May, 1983, and collected from one commercial oyster bed. Months on beds Willapa Bay over-wintered Willapa Bay Oyster Bay Coos Bay Months on beds Willapa Bay over-wintered Willapa Bay Oyster Bay Coos Bay 5.5 27.0 14.1 14.1 5 90.0 89.7 88.7 88.9 9 9.7 38.0 29.7 29.9 9 85.2 86.0 85.0 82.3 13 5.7 15.0 11.8 12.6 13 83.1 80.0 76.9 79.5 17 3.8 10.0 5.7 6.1 17 92.6 91.9 85.2 87.2 21 6.0 12.0 10.3 10.8 21 87.4 84.3 80.8 79.8 25 3.8 5.6 3.1 6.3 25 80.6 83.6 85.0 84.8 Sibling Pacific Oysters and Over-Wintered Spat 53 and Lee Weigardt, who assisted us in finding the sibling oysters on their commercial beds and helped us with sam- pling. We thank Jim Donaldson and Bob Matthews from Coast Oyster Company for supplying samples of overwin- tered oysters. We thank John Faudskar for his assistance and Dr. Chris Langdon for his helpful comments and for reviewing the manuscript. This study was supported by funds from the Extension/ Sea Grant Program at Oregon State University under grant NA 81 AA-D-00086. LITERATURE CITED Grave. C. 1912. A manual of oyster culture of Maryland, fourth report. Board of Shellfish Commissioners of Maryland, 376 p. Henderson. B. A. 1983. Handling and remote setting techniques of Pa- cific oyster larvae Crassoslrea gigas. MS Thesis, Oregon State Uni- versity, Corvallis, 37 p. Lannan. J. E.. A. Robinson, & W. P. Breese. 1980. Broodstock manage- ment of Crassoslrea gigas II. Broodstock conditioning to maximize larval survival. Aquacuhure 21:337-345. Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nd Ed Freeman, San Francisco, 769 p. Walne, P. R. 1970. The seasonal variation of meat and glycogen content of seven populations of oysters Ostrea edulis L. and review of the literature. Ministry of Agriculture. Fisheries and Food, Fisheries In- vestigations Series II, Vol. XXVI, 3:1-35. Journal of Shellfish Research, Vol. 6, No. 2, 55-65, 1987. THE PHYSIOGRAPHY AND EXTENT OF PUBLIC OYSTER GROUNDS IN POCOMOKE SOUND, VIRGINIA JAMES P. WHITCOMB & DEXTER S. HAVEN Virginia Institute of Marine Science School of Marine Science The College of William and Man- Gloucester Point. Virginia 23062 ABSTRACT Public oyster grounds in Pocomoke Sound. Virginia, were charted in 1978 using an electronic positioning system to locate areas of oysters, shell, sand or mud. Over five thousand stations were occupied and 1 ,267 samples of the substrate were taken with hydraulically operated patent tongs. The information was used to draw large scale charts showing shorelines, depths, bottom types and outlines of public grounds. Substrates, elevations, slopes, oyster densities and spatfall levels were analyzed. INTRODUCTION This paper describes the location, extent, and bottom characteristics of the oyster-producing areas in Pocomoke Sound, a sub-estuary in Chesapeake Bay. Virginia. The data are related to data from similar observations made in the James River, Virginia and in other areas, and to the James River's geologic history during the recent Holocene. The present study utilized data obtained during an ex- tensive bay wide investigation lasting from 1976 to 1981 (Haven et al. 1981)1. A portion of this study dealing with the James River has been published (Haven and Whitcomb 1983) and reference may be made to the original report and the latter publication for additional details on sampling and survey techniques. Pocomoke Sound is a large embayment shared by Mary- land and Virginia on the eastern side of Chesapeake Bay. The portion discussed here is bounded on the north by the Maryland-Virginia border, on the east by the headlands of the Eastern Shore, and on the west by Watts Island. The southern boundary is slightly south of a line from Watts Island to Onancock Creek. In the past. Pocomoke Sound was said to be enor- mously productive for oysters but reliable data are unavail- able. During the mid 1860's the entire Pocomoke Sound area (Maryland and Virginia) supported combined efforts of hundreds of dredge boats but by 1879 intense harvest from both states had depleted the area to the point where dredging was not profitable (Ingersoll 1881 ). Other areas in Virginia were being overfished during the late 1800s by boats equipped with dredges and. as a remedial measure, all of the naturally productive oyster grounds in Virginia were set aside by legislative action in 1894 for public use. Dredging on these bottoms was prohibited except for a very few areas (Baylor 1894, Code of Va. 1950). Contribution No. 1331 from the Virginia Institute of Marine Science. The College of William and Mary. Gloucester Point, Virginia 23062. 1 Funded in part by a grant from the National Marine Fisheries Service through the Virginia Marine Resources Commission (Contract No. 3-265-R-3). In Pocomoke Sound approximately 27,142 acres (10,984 ha) were designated in 1894 as public bottom or Baylor Ground (after Lt. Baylor who directed the survey). The Baylor Survey, using straight lines, simply outlined the broad reaches of productive bottom. (Figure 1). Conse- quently, much unproductive bottom was included (Moore 1911, Haven et al. 1981). Hydrography The circulation of water masses, and their salinity and temperature characteristics have received much study in Chesapeake Bay and many of its sub-estuaries (Pritchard 1951,1954; Nichols 1972; Hass 1977; Kennedy 1980; Boi- court 1982; and others). Similar studies, however, are lacking for the Pocomoke Sound area, which is located just to the east of the bay's north-south transition zone (Prit- chard 1952). That is, Pritchard (op. cit.) considers the bay north of the mouth of the Potomac (38°1 1 ') as an estuary of the Susquehanna. To the south of this junction, the bay may be classed as a composite estuary based on the fresh water inflow of all systems. Salinity data collected in the Pocomoke Sound from 1949 to 1961 show average fall salinities ranging from about 20 to 22%c over the north-south range. In the spring, over a similar area they ranged from about 16 to 18% 0.001 Diff. Sig. at P < 0.001 Not Sig. atP = 0.10 Not Sig. atP = 0.10 Diff. Sig. at 0.05 > P < 0.10 Not Sig. atP = 0.10 Not Sig. at P = 0.10 P.G. No. 13 14 15 17 18 19 13 14 15 17 18 19 13 14 15 No. Samples Average % Shell Average % Surface Shell 44 84 27 70 43 24 4 1 1 33 Oyster Reef 50.3 35.0 57.9 37.0 22.9 29.0 76.6 62.5 76.1 45.1 Sand-Shell 30.8 19.1 7.3 4.8 11.5 16.0 Mud-Shell 19.5 15.3 12.9 8.3 2.3 5.3 17 1 18 1 19 0 13 41 14 11 15 1 17 9 18 14 19 7 13 10 14 4 15 0 17 6 18 15 19 13 Mud 9.0 24.3 0.0 0.0 35.0 61.0 1.7 0.9 0.1 2.6 50.6 Sand 4.0 24.5 0.0 0.0 28.8 11.4 0.0 0.0 0.0 0.0 15.4 % Samples with Surface Shell 61.4 94.1 51.8 87.5 62.5 42.9 11.6 25.0 33.3 12.2 5.5 17.1 45.5 0.0 0.0 35.7 14.3 0.0 0.0 0.0 0.0 23.0 near the channel was about 2. 1 m (7 ft) vertically per 30.5 m (100 ft) horizontally (slope 1:14). Along these two tran- sects oyster reefs were often elevated 0.6 to 1.5 m (2 to 5 ft) above the surrounding bottom but the slope was still very gradual (less than 1:30) (Figure 5). Areas of mud-shell and sand shell bottom, although slightly elevated, were largely located toward shoal water from the oyster reefs. Generally these latter bottom types showed fewer definite peaks above the surrounding bottom types. The four transects on Public Ground No. 15 (Figure 6) differed on either end by about 0.3 m (1 ft) in height with sand or sand-shell at the beginning and end of all transects. The transects pass laterally across the prominent reefs. Be- cause of the transverse path of the transects, elevations of oyster reefs are not as evident as those shown in Figure 5. However, along transects IV and V elevations of from 0.6 62 Whitcomb & Haven PUBLIC GROUND ft 14 o-i 10 20 30 ■ REEF §1 MUD -SHELL □ SAND -SHELL □ SAND Q MUD m 5000 T 10,000 DISTANCE IN FEET Figure 5. Longitudinal profile of the bottom on Public Ground No. 14 in Pocomoke Sound, Virginia 15,000 to 1.5 m (2 to 5 ft) were measured. Slopes were gradual and ranged from 0.001 to 0.003 m (0.004 to 0.001 ft) ver- tically for each 30.5 -m (100 ft) horizontally (slopes 1:30,500 to 1:10,167, respectively). Mud-shell and sand- shell bottoms were occasionally elevated but to a lesser de- gree than for reefs. Spatfall Spatfall rates in Pocomoke Sound were low during 1978 as shown by weekly spatfall data and by samples of bottom material. Total number of spat/bu of substrate averaged only 9.5 with a range of 0 to 10. The total spat/shell counts based on shellstrings were low in 1978 with the monitored areas receiving only 0.0 to 1.6 spat/shell for the season (Table 7). The total spat/shell counts for P.G. No. 9 for 1975-1977 were also low. These levels of spat set are re- garded as too low for sustained commercial production (Krantz and Meritt 1977). DISCUSSION torn type was investigated on about 27,000 acres (10,646.6 ha) of public oyster ground in Pocomoke Sound. Only 19.5% of that area was classed as oyster reef, sand- shell or mud shell bottom. The remaining areas were classed as mud or sand and regarded as nonproductive for oysters. Large portions of nonproductive bottom have been also found at other locations in the state (Moore 1911, Loosanoff 1931 ). In their recent statewide survey Haven et al. (1981) showed that out of 203,404 acres (82.316.5 ha) of Baylor Ground surveyed in Virginia only 21.9% was classed as having significant volumes of shell in the sub- strate. The location and configuration of oyster reefs and areas of sand-shell and mud-shell in Pocomoke Sound are similar to those observed elsewhere (Graves 1905; Price 1954; Scott 1968; and Bouma 1976). Most are located between 1.8 to 5.5 m. Oyster reefs also have surface configurations that can be classed as longitudinal, irregular, transverse and pancake. In the James River, longitudinal reefs were adja- cent to and parallel to channels, while the remaining types were uniformally distributed between the 1.8 to 5.5 m depth zones (Haven et al. 1981). Samples collected with patent tongs confirm observa- tions made with the sonic gear and bottom probe. Bottom classes as oyster reef had the highest oyster density and more shell than all other bottom types. Mud-shell and sand-shell had lesser numbers of oysters and smaller volume of shell. Sand bottom had few oysters or shell. Mud bottoms typically contained few oysters, but shell content was high on P.G. Nos. 18 and 19. Similar results Physiography and Extent of Public Oyster Grounds PUBLIC GROUND # 15 63 u- io- m | REEF J MUD -SHELL ^ SAND-SHELL □ SAND [[] MUD 20- ■V)- t m mm/mm mm* m o- 10- 3C 20- 30 J m u m i i -miw/M 10 20- 30 0 10 20 30 -1 H 331 "> 1 10,000 5000 DISTANCE IN FEET Figure 6. Longitudinal profile of the bottom on Public Ground No. 15 in Pocomoke Sound, Virginia. were found in the public oyster rocks of the James River (Haven and Whitcomb 1983). While bottoms classed as mud-shell and sand-shell often contain oysters and shell we do not consider them as per- manent a feature of the estuary as oyster reefs. It has been suggested that oyster reefs were originally sharply defined, and that fishing activity by dredging and tonging spread living oysters and shell on to the less favorable mud or sand areas, thereby establishing populations on these areas where growing conditions are less than optimal (Winslow 1880, Loosanoff 1931). Elevations of oyster reefs above the surrounding bottom in Pocomoke Sound typically is not great except for those areas located along the edge of channels. Except for these areas, slopes are gradual and range from about 1:769 to 1:30,500. Mud-shell and sand-shell areas had even fewer degrees of elevation. In this respect these elevations re- semble those observed previously in the James River, Vir- ginia (Haven and Whitcomb 1983). Origin of Oyster Reefs The extent of shell deposits below existing reefs in Po- comoke Sound has not been investigated, but evidence from other locations suggest that they may be of consider- able age and thickness. An early study of several lagoonal systems off the Texas coast showed that oyster reefs re- tained their narrow shape to at least 2.7 m below the sur- face (Norris 1953). Bouma (1976) related the vertical growth of oyster reefs to the world wide rise in sea level during the recent Holocene (Emery and Uchupi 1972). Bouma (op. cit.) concluded that in San Antonio Bay in the Gulf of Mexico many of the present day oyster reefs exist on the top of older shell formations. Carbon- 14 data showed ages of the buried shell ranged from 1500 to 9000 years . Chesapeake Bay and its principal contiguous estuaries also experienced a rise in sea level during the Holocene. In one Chesapeake Bay sub-estuary (the James River) the basin flooded with sea water between 9000 and 6500 years 64 Whitcomb & Haven TABLE 7. I-Numbers of 1978 spat in one bushel of dredged bottom material in Pocomoke Sound in 1978. H-Numbers of spat per shell (spat/shell), based on the weekly average, of ten shells on various Public Grounds in Pocomoke Sound. Period of exposure June 16 to September 19, 1978. II Seasonal total of Weekly spat/shell Public Ground spat/bushel Year No. & Location 1978 1975 1976 1977 1978 9 upper — — — — 1.6 9 lower — 1.6 6.2 2.3 0.1 10 0.0 13 upper 5 — — — 0.0 13 lower 7 — — — 0.0 14 15 — — — 0.2 15 16 17 0 — — — 0.1 0.1 10 — — 18 20 — — — — Av. 9.5 ago (Nichols 1972). Here, large deposits of buried shell- have been located in the lower section (Haven and Whit- comb 1983). Flooding by sea water of Pocomoke Sound must have occurred during the same period as for the James River. The oyster reef in Pocomoke Sound may, therefore, be underlain by older shell deposits, and the present day reefs evolved as they did in the lagoons along the Gulf of Mexico from old shore or bottom features as sea levels gradually rose. Studies widely separated in time suggest that the Poco- moke Sound area has often experienced low annual recruit- ment rates (Winslow 1880; Krantz and Meritt 1977). During the mid 1860's, it is reported, the entire Pocomoke Sound area (Maryland and Virginia) supported the com- bined fishing effort of hundreds of dredge boats; but by 1879 this intense fishing pressure had depleted the area to the level where dredging was no longer profitable (Winslow 1880; Ingersoll 1881). Studies on spatfall made in Pocomoke Sound by the Maryland Department of Natural Resources between 1939 and 1975 documented that set failures (less than 25 spat/bu of cultch) were recorded during 43% of the years (Krantz and Meritt 1977). Average spatfall during this entire period was rated as poor (25 to 100 spat/bu). The data presented here for 1975-78 based on bottom samples and shell strings supports the concept that spatfall rates are low in this area. Data on production (harvest) from this area are lacking. Statistics based on the Virginia tax (two cents a bushel) indicate that during the 1940's and 1950's annual landings ranged from 1,000 to 50,000 Virginia bushels (Va. Comm. Fish. 1969). Reliable data from 1962 to 1977 (op. cit.) in- dicated landings were still low and ranged from 307 to about 35.000 bushels each year. In 1978 dredging was de- clared legal during a short, designated late winter season each year. As a result landings increased to 208,130 bushels for Pocomoke and Tangier sounds combined during the 1978-79 season, but this level of production quickly decreased to only 23,800 bushels in Pocomoke Sound and 3,570 bushels in Tangier Sound during 1983-84. Ob- viously, the accumulated stocks were quickly exhausted, which is the expected result of overharvest in a region where annual recruitment is marginal or low. We conclude that while there are 2.079 ha in Pocomoke Sound classed as productive or potentially productive bottoms, these areas have not been capable of maintaining a sustained high level of natural production. Remedial mea- sures would include limited shell plantings with the most effort being placed in planting low cost seed oysters dredged from the James River where natural spatfall is higher. ACKNOWLEDGMENTS We are indebted to Mr. Paul Kendall and Mr. Kenneth Walker who assisted in all field studies. We also recognize Mr. Paul Kendall's assistance in calculating acreages of various bottom type and entering field data on work sheets. We acknowledge the invaluable and constructive criticisms from Dr. Roger Mann, Mr. Reinaldo Morales and Mr. David Stilwell. REFERENCES CITED Baylor, J. B. 1894. Method of defining and locating natural oyster beds, rocks, and shoals. Oyster Records (pamphlets, one for each Tidewater. VA. county, that listed the precise boundanes of the Baylor Survey). Board of Fisheries of Virginia: 1-777. Boicourt. W. C. 1982. Estuarine larval retention mechanisms on two scales. Kennedy. V. S., ed. Estuarine Comparisons. New York: Aca- demic Press, p 445-457 Bouma, A. H. 1976. Subbottom characteristics of San Antonio Bay. Bouma, A. H., ed. Shell Dredging and Its Influence in Gulf Coast Environments. Houston, TX: Gulf Publishing Co. 132-148. Code of Virginia. 1950. Code of Virginia 1950 5. Michie Co., Charlot- tesville. Virginia: 1-589. Emery, K. O. & E. Uchupi. 1972. Western North Atlantic Ocean: Topog- raphy. Rocks, Structure. Water. Life and Sediments. Tulsa, OK: Am. Assoc. Pet. Geol. 532 p. Graves, C. 1905. Investigation for the promotion of the oyster industry in North Carolina. U.S. Comm. Fish. Rep. 1903:247-341. Haas, L. W. 1977. The effect of the spnng-neap tidal cycle on the vertical salinity structure of the James. York and Rappahannock Rivers. Vir- ginia, U.S.A. Estuarine Coastal Shelf Sci. 5:485-496. Haven, D. S.. J. P. Whitcomb, J. M. Zeigler & W. C. Hale. 1979. The use of sonic gear to chart locations of natural oyster bars in lower Chesapeake Bay. Proc. Natl. Shellfish. Assoc. 69:11-14. Haven, D. S., J. P. Whitcomb & P. Kendall. 1981. The present and po- Physiography and Extent of Public Oyster Grounds 65 tential productivity of the Baylor Grounds in Virginia. Vols. I (167 pp.) and II (154 pp. 52 charts). Va. Inst. Mar. Sci.. Spec. Rep. Appl. Mar. Sci. Ocean Eng. No. 243. Haven. D. S. & J. P. Whitcomb. 1983. The origin and extent of oyster reefs in the James River. Virginia. J. Shellfish Res. 3(2): 141 — 151. Ingersoll, E. 1881. The oyster industry. Washington, D.C.: Government Printing Office: 1-251. Kennedy. V. S. 1980. Comparison of recent and past patterns of oyster settlement and seasonal fouling in Broad Creek and Tred Avon River, Md. Proc. Natl. Shellfish Assoc . 70:36-46. Loosanoff, V. L. 1931. Observation on Propagation of Oysters in the James and Corrotoman Rivers and the Seaside of Virginia. Newport News. VA: Virginia Comm. Fish.: 46 p. Krantz. G. E. & D. W. Meritt. 1977. An analysis of trends in oyster spat set in the Maryland portion of the Chesapeake Bay. Proc. Nail. Shell- fish. Assoc. 67:53-59. Moore, H. F. 1911. Condition and extent of oyster beds in the James River. U.S. Bur. Fish. Doc. No. 729:83 p. Nichols, M. M. 1972. Sediments in the James River, Va. Nelson, B. W., ed. Environmental Framework of Coastal Plain Estuaries. Geol. Soc. Am. Mem. 133:169-212. Norris, R. M. 1953. Buned oyster reefs in some Texas Bays. J . Pa- leontol. 27:569-576. Price, W. A. 1954. Oyster reefs of the Gulf of Mexico. Galtsoff, P. S., coordinator. Gulf of Mexico, its origin, waters, and marine life. U.S. Fish Wildl. Sen-. Fish. Bull. 89:491. Pntchard. D. W. 1952. Salinity distribution and circulation in the Chesa- peake Bay estuarine system. J. Mar. Res. 11:106-123. Pritchard, D. W. 1951. The physical hydrography of estuaries and some applications to biological problems. Trans. 16th North American Wildlife Conference March 5, 6 and 7. 1951:368-376. Pritchard, D. W. 1954. A Study of the salt balance in a coastal plain estuary. Sears. Found. J. Mar. Research. 13(11:133-144. Schubel, J. R. 1972. Distribution and transport of suspended sediments in Upper Chesapeake Bay. B. W. Nelson, ed. Environmental Frame- work of Coastal Plain Estuaries. The Geological Society of America Inc. 133:151-167. Scott, A. J. 1968. Environmental factors controlling oyster shell deposits. Texas Coast. From Fourth Forum on Geology of Industrial Minerals. Austin TX: Univ. of Texas; 1968. 8:131-150. Sokal, R. R. and F. J. Rohlf. 1981. Biometry, the Principles and Prac- tices of Statistics in Biological Research. San Francisco, CA: W. H. Freeman and Co. 859 p Stroup, E. D. and R. J. Lynn. 1963. Atlas of salinity and temperature distributions in Chesapeake Bay 1952-1961 and seasonal averages 1949-1961. Baltimore. MD: Chesapeake Bay Inst.. John Hopkins Univ. Graph. Sum. Rep. 2. (Ref. 63-1:410 p.) Virginia Commission of Fisheries. 1969. Annual Report to the Governor of Virginia. Richmond, VA: Virginia Depl. of Fisheries. 129 p. Winslow, F. 1880. Extracts from Report to Carlile P. Patterson. Supt. Coast & Geo. Sur. Rep. of the Commissioners of Fisheries of Man. Jan. 1880. Annapolis. Md. pp. 105-219. Journal of Shellfish Research, Vol. 6, No. 2, 67-70. 1987. OYSTER SIZE, AGE, AND COPPER AND ZINC ACCUMULATION HARRIETTE L. PHELPS1 & ERICH W. HETZEL2 'University of the District of Columbia 4200 Connecticut Ave. N.W. Washington, D.C. 20008 2 P.O. Box HM2091 Hamilton #5 Bermuda ABSTRACT Copper and zinc concentrations and growth parameters were measured in two artificially spawned sets of oysters, ages one through five years, raised in the mid Chesapeake Bay. The Shadyside, MD oyster set had yearly increases in shell size and body weight and decreasing tissue concentrations of copper and zinc. The Horn Point, MD oyster set had little increase in shell size or body weight after the first year and increasing tissue concentrations of copper and zinc. The Horn Point (stunted) oysters had significantly more metal than Shadyside oysters of the same age and preferentially accumulated copper, with twice the copper-zinc ratio. Sizes and metal concentrations in the two sets of oysters were comparable to mid-Chesapeake Bay field populations of normal and stunted oysters. Regression equations of age with size and metals were derived for the Shadyside and Horn Point oyster sets. INTRODUCTION Adult bivalve molluscs have been selected as aquatic biomonitoring organisms because of their fixed location and ability to concentrate and tolerate toxics (Goldberg, et. al. 1978). Estuaries are an important and highly impacted aquatic ecosystem but estuarine oysters present a problem when used as metal biomonitoring agents because the rela- tionships of size to age and metal accumulation have not been determined. A biomonitoring field study of oysters (Crassostrea virginica, Gmelin) in the mid-Chesapeake Bay reported a unique site having a population of small oysters with abnormally high concentrations of metals (copper, zinc, cadmium and silver) and no known source of contamination (Phelps 1984). High metal levels in a popu- lation of small estuarine oysters also has been reported from a South Carolina site (Burrell 1981 ). Field oysters are difficult to age even though there have been attempts to use internal shell "growth rings" (Downes 1957). The study reported here used two sets of oysters of known ages that had been spawned artificially and raised in the Chesapeake Bay under conditions causing normal and stunted growth, to examine zinc and copper accumulation with age and size. METHODS Two sets of artificially spawned oysters, showing both normal and stunted growth, were available for this study in June 1983. These sets were cohorts, oysters of identical age spawned artificially from a few normal parents each June over a period of several years. Since the artificial spawning process was destructive, different parents were used each year. One set had cohorts of one, two. three and five years, spawned and raised by Mr. Frank Wilde, Chesapeake Bay Oyster Culture, Shadyside, MD. The Shadyside set was raised in field trays anchored near the location of the cul- ture facility in Shadyside. MD. on the western shore in the upper third of the Chesapeake Bay just below Annapolis. MD. The second set had cohorts of one, two, three and four years, spawned and raised by Dr. George Krantz of the University of Maryland Horn Point Laboratory on the eastern shore near Cambridge, MD near in the mouth of the Choptank River in the middle of the Chesapeake Bay. The Horn Point set was raised in field trays anchored near Horn Point, MD during the summer and transferred to laboratory troughs with flowing estuarine water during the winter. These experimental oyster cohorts were compared with field oysters collected from two mid-Bay sites. Cornfield Harbor, MD, an unpolluted site at the mouth of the Po- tomac River near the middle of the western shore of the Chesapeake Bay. was selected for salinity similar to the Shadyside site because estuarine oyster metal concentra- tions are related to salinity (Phelps, et. al. 1985). Deep Neck Broad Creek, MD. an unpolluted site near the Horn Point Laboratory was previously reported to have the rare natural population of small oysters with high metal levels. Thirty to thirty-five oysters from each group were mea- sured for shell length and width. Oyster tissues were re- moved, rinsed, blotted, weighed, dried at 100°C for 5 days, and reweighed. Individual oyster tissues were dissolved by warming at 80°C in 20 ml concentrated HN03 (Fisher, Re- agent Grade), filtered through pre-acid- washed glass fiber filters, diluted to 100 ml and analyzed for copper and zinc by flame atomic absorption spectroscopy (Perkin-Elmer Model 460 Atomic Absorption Spectrophotometer), cali- brated by the method of standard additions. Calculation of means, standard errors, significant differences and linear regression was by SPSS/SCSS with the UDC DEC2060 computer. RESULTS Shadyside oyster cohorts had yearly significant (p < .05) increases in shell length and width and wet and dry 67 68 Phelps & Hetzel tissue weights (Table 1). Except for the first year, Horn Point oyster cohorts were significantly (p < .05) smaller than Shadyside cohorts (Table 1). Horn Point oysters of all ages appeared the size of one-year-old oysters and were termed stunted in growth. Cornfield Harbor oysters were nearly the size of three-year-old Shadyside oysters. Deep Neck Broad Creek oysters resembled the Horn Point oysters in size (Table 3). Total copper and zinc in oysters remained the same or increased with age in both sets of experimental oysters (Table 1). However, copper and zinc concentrations in oyster tissues (u,g/gm) decreased with age and increasing weight in the Shadyside oyster set, while increasing with age in the stunted Horn Point oyster set (Table 1 . Figure 1). Copper and zinc accumulation by the two oyster sets was compared by normalization of total copper and zinc in Sha- dyside oysters to the weights of Horn Point oysters of the same age (Figure 2). The linear regression equations calculated for shell length and width, tissue wet and dry weight, and copper and zinc wet tissue concentrations with age were different for the two experimental oyster sets (Table 2). Because the Horn Point third year age cohort had significantly lower wet and dry tissue weights which might indicate a signifi- cant genetic difference from the other age cohorts it was not used in the calculation of the regression equations. DISCUSSION Copper concentrations in Cornfield Harbor oysters were similar to Shadyside oysters of comparable size, although zinc concentrations were larger. Copper and zinc concen- trations in Horn Point (stunted) oysters were elevated, as were those of the field population of small oysters from Deep Neck Broad Creek (Table 3). Assuming a copper ac- cumulation history similar to that calculated for Horn Point oysters from the linear regression equations (9.8 |xg copper/gm oyster wet weight/year and 167 (xg zinc/gm oyster wet weight/year. Table 2) the Deep Neck Broad Creek oysters were estimated to be 5 to 11 years old. This corresponds to estimates of oyster age at that location based on periodicity of recruitment determined from field surveys (Krantz and Merrit 1977; G. Krantz, pers. comm.) The causes of stunted growth in these oyster populations could be inherent (genetic) or due to environmental factors. Natural stunted oyster populations such as reported by Bur- rell (1981) from the Wando estuary. South Carolina, and from Deep Neck Broad Creek in the Chesapeake Bay are found to be associated with high numbers of setting oyster spat. They have been traditionally used by oystermen as sources of seed oysters for growth when transplanted. Thus, they are genetically competent for growth. Environ- mental factors that have been suggested as responsible for the stunted oyster growth in these locations include the high infestation of boring sponge at Broad Creek (not at Wando estuary), differences in food availability (Nowell and Jones pers. comm.). the high concentrations of copper and other metals characteristic of such oyster populations (Burrell 1971), or the highly variable low salinities at both locations placing osmoregulatory demands on amino acids normally employed for growth. TABLE 1. Oyster Cohort Data Shell L Shell W Wet Wt Dry Wt Cu Zn CuConc ZnConc Cu/Zn Age cm cm gm gm total |i i; total p.g ixg/gm* (JLg/gm* xlO-2 years n (Standard Error) SHADYSIDE OYSTERS 1 30 33 2.6 .96 .135 18.3 240. 20.2 250. 8.08 (.72) (.44) (.058) (.0090) (1.10) (15.) (.65) (9.0) (.24) 2 30 8.5 6.4 8.4 1.38 60.4 2140. 7.19 327. 2.20 (2.0) (1.7) (.41) (.076) (4.2) (60.) (.66) (15.5) (.20) 3 30 10.6 6.7 14.0 2.29 52.7 1970. 3.76 141. 2.67 (1.9) (1.1) (.64) (.130) (5.8) (90.) (.61) (18.3) (.31) 5 29 12.5 8.7 26.7 4.4 62.1 1880. 2.33 70.4 3.31 (2.5) (2.0) (.82) (.146) (5.0) (98.) (.20) (4.13) (.32) HORN POINT OYSTERS 1 33 3.6 2.3 1.09 .297 24.5 400. 22.5 367. 6.13 (.14) (.11) (109) (.045) (1.7) (30.0) (2.84) (33.5) (.45) 2 36 7.1 4.3 3.80 .61 101. 2380. 26.6 626. 4.25 (.21) (10) (.212) (.048) (6.4) (190.) (1.66) (54.9) (.24) 3 35 6.6 4.4 3.04 .363 178. 3480. 58.6 1140. 5.12 (19) (.13) (.275) (.034) (15.4) (350.) (5.23) (113.) (.37) 4 35 7.2 5.0 4.22 .59 208. 3400. 49.3 806. 6.12 (.18) (.13) (.281) (.055) (18.8) (290.) (3.15) (45.) (.26) * Wet weight Oyster Size, Age, Copper and Zinc 69 Ol o 70u 60 50 40- 30 20 10- & stunted oysters. Cu ~ A stunted oysters, Zn O normal oysters, Cu - - • normal oysters, Zn - - / / - / ,■ a — *-■& M 0 \ ■-••-0......:::-::^ 1400 -1200 -1000 800 600 400 200 12 3 4 5 Age, years Figure 1. Copper and Zinc Wei Tissue Concentrations in Normal and Stunted Ovster Cohorts. 60 50 40 30- AGE CLASS AVERAGES A stunted oysters, Cu A stunted oysters, Zn O normal oysters, Cu • normal oysters, Zn !D) Cu values if normal oysters were stunted (■ Zn values if normal oysters were stunted 1400 1200 1000 800 -600 400 200 8 0 10 20 30 Wet Weight, g Figure 2. Copper and Zinc Totals Normalized to Weight in Normal and Stunted Ovster Cohorts. The normalization procedure showed Horn Point (stunted) oysters accumulated nearly twice as much total copper and zinc as Shadyside (normal) oysters of the same age (Figure 2). Deep Neck Broad Creek oysters also had abnormally high cadmium and silver concentrations for the mid Chesapeake Bay (Phelps 1984). There is no explana- tion currently available for this hyper-accumulation of metals by oysters under stunting growth conditions. It may be related to a variable salinity-stressed environment causing high mobility of osmoregulatory free amino acids, which are known to complex with metals. The copper-zinc ratio of 2-4 year Horn Point oysters and Deep Neck Broad Creek oysters was twice that of Sha- dyside and Cornfield Habor oysters (Table 3). Since this high copper-zinc ratio appears characteristic of stunted oyster populations it cannot be considered indicative of es- tuarine copper enrichment, as with normal oysters (Huggett 1973; Phelps 1984). A high copper-zinc ratio may be re- lated to differences in accumulation, storage or excretion mechanisms for the two metals. Ikuta (1968) reported oysters lost zinc more rapidly than copper when transferred to a metal-free environment. Boyden (1977) found zinc equilibration in oysters more rapid than copper equilibra- tion. In oysters, zinc accumulates in membrane-bound ves- icles that can be excreted in the urine (George et al. 1978). Copper in molluscs is found tightly bound to subcelluar fractions (Roesijadi 1983) and excretion may involve whole-cell loss at the body surface which may account for a lower rate of loss in stunted oysters and a preferential in- crease in copper retention. Boyden (1977) reported logarithmic slopes of total zinc and copper with body size of Crassostrea gigas and Ostrea edulis from clean environments were near unity. Slopes for oysters from contaminated environments were lower be- cause smaller oysters with higher surface/volume ratios TABLE 2. Oyster Cohort Regression Equations Shadyside Oysters Horn Point Oysters Shell Length (cm) Shell Width (cm) Wet Weight (gm) Dry Weight (gm) Log Copper Cone, (ug/gm) Log Zinc Cone, (ug/gm) = 2.89 + (2.13 x Age) = 2.33 + (1.37 x Age) = -4.96 + (6.35 x Age) = -.840 + (1.05 x Age) = 1.35 - (.16 x Age) = 2.62 - (.15 x Age) .764 .742 .907 .882 .698 .690 4.07 + (.89 x Age) 2.32 + (.73 x Age) .96 + (.89 x Age) .31 + (.082 x Age) 1.24 + (.10 x Age) 1.61 + (.070 x Age) .370 .579 .360 .360 .518 .212 * All correlations significant at the 95% confidence level 70 Phelps & Hetzel TABLE 3. Field Oyster Population Data Salinity Wet Wt CuConc ZnConc Cu/Zn ppt gm ug/gm ug/mg X10~2 Site n (Standard Error) Cornfield 34 15.0 11.3 11.2 631. 1.8 Harbor (.68) (1.44) (78.4) Deep Neck 35 14.3 2.8 82. 1430. 5.8 Broad Creek (.47) (4.9) (55.) concentrated bioavailable metals more rapidly than larger oysters. Boyden proposed lower slopes as indicators of en- vironmental enrichment. Logarithmic slopes of total copper and zinc with body size for Shadyside and Horn Point oysters were 0.5-0.8. Although these locations have no history of metal enrichment. Estuarine oysters normally show increased metal concentrations at lower salinities (Phelps, et al. 1985), which may be related to the higher relative concentration of ionic (e.g., bioavailable) metals with decreased salt complexation (Sunda and Guillard, 1976). At the time of collection in June. Bay salinities were decreasing normally due to spring runoff. Therefore these lower slopes relating oyster metal concentrations to size may be a normal response to an annual salinity change af- fecting metal bioavailability rather than indicative of envi- ronmental enrichment. The regression equations derived for growth and metal accumulation for these stunted and normal hatchery oyster cohorts can be used to calculate significant deviations. Thus if these Shadyside or Horn Point experimental oyster cohorts were transplanted to other sites, statistically signifi- cant changes in oyster growth rate or metal accumulation could be determined. These oyster cohorts might now be valuable tools for biomonitoring conditions affecting growth and metal accumulation by shellfish under estuarine conditions. ACKNOWLEDGMENTS Grateful acknowledgment is made to Dr. George Krantz, Mr. Frank Wilde, Dr. Joseph Mihursky, Dr. David Wright, and Dr. Roger Newell for helpful criticisms and suggestions. This work was funded by NASA grant NGR09-050-019. REFERENCES CITED Boyden, C. R. 1977. Effect of size upon metal content of shellfish. J. Mar. Biol. Ass. U. K. 57:675-714. Burrell, Jr. V. G . J. J. Manzi & W. Z. Carson. 1981. Growth and mor- tality of two types of seed oysters from the Wando River, South Caro- lina. J. Shellfish Res. 1:1-7. Downes, K. M. 1957. An investigation of manganese and zinc in the oyster. Crassostrea virginica (Gmelin). Ph.D. Thesis. University of Maryland, College Park, MD. 54pp. George. S. G . B. J. Pirie, A. R. Cheyne, T. L. Coombs & P. T. Grant. 1978. Detoxification of metals by marine bivalves: an ultrastructural study of the compartmentation of copper and zinc in the oyster Ostrea edulis. Mar. Biol. 45:147-156. Goldberg, E D.. V. T. Bowen, J. W. Farrington, G. Harvey, J. H. Martin, P. L. Parker, R. R. Risebrough, W. Robertson, E. Schneider, & E. Gamble. 1978. The Mussel Watch. Env. Conserv. 5(2): 101-125. Huggett. R. J.. M. E. Bender & H. D. Slone. 1973. Utilizing metal con- centration relalionships in the eastern oyster (Crassostrea virginica) to detect heavy metal pollution. Water Res. 7:451-460. Ikuta, K. 1968. Studies on accumulation of heavy metals in aquatic or- ganisms. IV. On disappearance of abnormally accumulated copper and zinc in oysters. Bull, of the Jap. Soc. ofSci. Fish. 34:482-487. Krantz, G. E. & D. W. Memt. 1977. An analysis of trends in oyster spat set in the Maryland portion of (he Chesapeake Bay. Proc. Nat. Shell- fish Assoc. 67:53-59 Phelps, H. L. 1984. A research program in determination of heavy metals in sedimenls and benthic species in relation to nuclear power plant operation. Final technical report to NASA, 1974-1982. 82pp. Phelps, H. L ., D. A Wright & J. A. Mihursky. 1985. Factors affecting trace metal accumulation by estuarine oysters (Crassostrea virginica). Mar. Ecol. Prog. Ser. 22:187-197. Roesijadi. G.. J. S. Young, A. S. Drum & J. M. Gurtisen. 1984. Be- havior of trace metals in Mytilus edulis during a reciprocal transplant field experiment. Mar. Ecol. Prog. Ser. 18:155-170 Sunda, W. & R. R. L. Guillard. 1976. The relationship between cupric ion activity and the toxicity of copper to phytoplankton. J. Mar. Res. 34:511-529. Journal of Shellfish Research, Vol. 6. No. 2, 71-77, 1987. MASS CULTURE OF SELECTED MARINE MICROALGAE FOR THE NURSERY PRODUCTION OF BIVALVE SEED DENNIS T. WALSH, CHRISTOPHER A. WITHSTANDLEY, RICHARD A. KRAUS & EUGENE J. PETROYITS Aquacultural Research Corporation, P.O. Box AC Dennis, Massachusetts 02638 ABSTRACT Techniques are described for the culture of selected marine phytoplankton in six 40 M-1 continuous flow cultures which serve as a year round source of food for the nursery seed production of the hard clam. Mercenaria mercenaria (Linne). Yield optimization of the algae was achieved in these nutrient saturated, light limited cultures by the proper selection of depth and dilution rate empirically determined for each month of the year. The cost of producing cultured algae was computed for the year 1983 and related both to the current cost of seed production and the expected gross revenue from the sale of cultured clams. KEY WORDS: Phytoplankton, continuous culture, nursery. Mercenaria mercenaria INTRODUCTION Bivalve aquaculture has long been plagued by an in- ability to culture economically the massive quantities of suitable algal species needed to grow the proper size seed for field planting (Loosanoof 1951; Walne 1974; DePauw 1981). The use of naturally occurring phytoplankton to feed millions of post set shellfish is frequently impractical in temperate climates because phytoplankton typically are abundant in the spring and fall when water temperatures may be too low for feeding and growth of bivalves, while during the summer, temperatures are optimal but phyto- plankton levels may be low due to nutrient limitations and intensive grazing by numerous filter feeding competitors (Spencer and Gough 1978; Lucas 1977). There is no con- trol of either the quantity or quality of algal species when only raw seawater is used; therefore, the carrying capacity of the nursery raceways or upwellers is limited by current velocities and ambient food levels. As part of our effort to develop a commercially suc- cessful aquaculture business for the hard clam, Mercenaria mercenaria (Linne), Aquacultural Research Corporation (ARC) has been experimenting for the past twenty years with continuous flow algal pond cultures. Our objectives in mass culture have been to create a selected community of algal species of proven nutritional value to shellfish and to optimize algal biomass yields. We report here results from three years of continuous operation of our mass culture fa- cility. The results clearly show that control, manipulation and yield optimization of selected marine microalgae is both feasible and reliable on a year round basis in the northern temperate zone. We also show that the cost of pro- ducing this algal biomass is low relative to the cost of seed clam production or the projected annual gross revenue of this aquaculture venture. MATERIALS AND METHODS Facilities This work was performed at Aquacultural Research Cor- poration (ARC) located at the mouth of Barnstable Harbor on the north shore of Cape Cod, Massachusetts (lat. 41°N). The culture units were six epoxy coated concrete circular ponds each of 40,000 liter capacity and 41 M3 surface area. The ponds were enclosed in a 18.3 M x 27.4 M fiberglass panelled greenhouse. The ponds were well mixed with a hydraulic rotor similar in design to that of Kanawawa et al. (1958) so that they resembled completed mixed reactors (Goldman et al. 1975). Influent for the ponds was obtained from a salt water well drawn from approximately 20 meters below the pond complex by a V/i h.p. cast iron pump. Inoculum for the pond complex was produced in a con- trolled environment room measuring 2.1 M x 4.6 M. Room temperature averaged 18°C (range 16-20°C). Con- tinuous fluorescent lighting was maintained at 0.02-0.03 cal • cm~2 • min-1. Cultures The clonal cultures used in this study are all maintained at ARC as well as the Bigelow Laboratory for Ocean Sciences, USA. The Thalassiosira pseudonana (clone 3H) was isolated by Dr. Robert R. L. Guillard. Details of its origin were given by Guillard and Ryther (1962). One of us (DTW) isolated a small Skeletonema-like diatom on \% agar from enriched water samples taken adjacent to ARC in September, 1979. A small single cell Chaetoceros diatom was isolated from enriched water samples obtained from Wellfleet Harbor. Wellfleet, Massachusetts in November 1982 using the extinction dilution method (Butcher 1952). The Skeletonema species superficially resembles Skeleto- nema menzelii (Guillard. et al. 1974) though it may well be 71 72 Walsh et al. a new species. Thalassiosira pseudonana and Skeletonema sp. were approximately the same size, measuring 4 to 10 u,m in diameter with a cellular volume of 25 to 35 p.m3. The Chaetoceros sp. was 2 to 4 |xm in diameter with a cellular volume of 10 to 15 |i,m3. Culture Procedures A simple three-stage batch culture process using 60 ml subcultures, 12-liter carboys, and 450-liter translucent fi- berglass tanks was developed to provide large inocula of selected microalgae for the continuous culture ponds. Media for all stages of the inocula production were based on saline well water. Salinity of the well water ranged from 26-28 ppt annually; pH ranged from 6.9-7. 1 and tempera- ture varied from 9.6 to 15.8°C. The well water was devoid of algae, algal predators or competitors. New stock cultures were started daily from two-day-old cultures by transferring 5 ml of actively growing culture to approximately 60 ml of sterile well water containing 2F silicate. IF nitrate, phosphate, trace metal mix and vitamin mix (Guillard 1974). The medium was buffered with 5 mM TR1ZMA and the pH adjusted to pH 7.0 with reagent grade HC1. Subcultures were monospecific but not axenic. Carboy cultures were started daily by the addition of the remaining 60 ml of the two-day-old subculture to 12 liters of sterile well water media containing 2F silicate and IF nitrate, phosphate, trace metal mix and vitamins (Guillard 1974). Carboys were sterilized by autoclaving at 20 lbs. pressure and 120°C for 30 minutes. Mixing and pH control were achieved by vigorous (10-20 liters/minute) aeration through 3 mm bore glass tubing. Growth of the algae was allowed to proceed for five or six days at which time the carboys were harvested at a density of 10 x 109 to 15 x 109 cells/liter. In the final stage of inoculum production, a volume of 1350 liters of algae with an average density of 4.7 x 109 cells/liter was produced daily by using a two-day batch cycle involving a bank of six 450-liter translucent fiber- glass tanks. Three tubes were harvested daily and each tube was hot rinsed, refilled with well water and nutrients added at the following levels: 2F silicate, F/2 nitrate, phosphate and vitamin mix (Guillard 1974). Two contents of five- or six-day-old carboys were added to each tank. The contents of each tank were mixed vigorously with 30-50 liters/ minute of air entering the bottom of the cultures through 3 mm bore glass tubing. The pH of two day batch cultures was maintained in the range of 7.5-8.5 by the periodic addition of HC1 or C02. The algal inoculum produced daily could be used to start a pond culture or to feed larvae and post set shellfish at the ARC hatchery. A pond was started by filling a concreted excavation with approximately 15,000 liters of well water, adding nutrients and inoculating with 450-1350 liters of a mixture of the three diatoms. During this batch growth phase lasting 24 to 96 hours, the pond's volume was in- creased with well water to approximately 26,000 liters; this volume provided a depth of 65 cm. The algal density at the end of the batch growth phase typically averaged 1 x 109 cells/liter at which time continuous culture was initiated by starting the inflow of well water to balance the outflow of the algal suspension pumped into the shellfish nursery system. The continuous cultures were enriched daily with nutrient concentrates to give an average nutrient level of 2F silicate and F/2 nitrate, phosphate and trace metal mix (Guillard 1974). No vitamins were added. The pH of the ponds was maintained in the range of 7.5 to 8.8 by the injection of CO\ once or twice daily. A pond's dilution rate (percentage of volume turnover per day) was adjusted daily after microscopic examination of the algae population and consideration of the daily temperature and solar irradiance levels. These continuous cultures lasted from one to six weeks. Manpower requirements for the six pond complex aver- aged four hours per day while inocula production averaged approximately five man-hours per day. Physical, Chemical and Biological Analysis Routine daily monitoring consisted of measuring pond depth, temperature and pH, and algal cell counts by species, as well as making a qualitative evaluation of heter- otrophic contaminants. Cell counts and heterotrophic eval- uation were made at 450 x under phase contrast using a Speirs Levy Eospinophil Counter. Algal yield for the con- tinuous cultures was computed as the product of biomass concentration and flow rate on a per unit area basis (Goldman et al. 1975). The average monthly cell density and fractional turnover of liquid in the culture per unit of time was assumed to represent the steady state algal bio- mass concentration and dilution rate respectively. Cell counts were converted to algal biomass by using an average cellular carbon content of 5.10 x 10" 12 g derived from simultaneous cell count and particulate carbon measure- ments. Particulate carbon measurements were made in a Perkin Elmer Model 240 carbon-hydrogen-nitrogen ana- lyzer. Samples from the various ponds were measured during a two week period in August. 1982 and for the same period in April, 1983. Thalassiosira pseudonana (3H) cells in log phase were found to weigh 18 x 10~l2g and had an ash content of 25% (Shifron and Chisholm 1981). As- suming a carbon content of 5 x 10"12gC per cell inferred that carbon comprised 37% of the total organic matter in this diatom. Therefore, dividing the particulate carbon con- centration of 0.37 gave an estimate of ash free dry weight concentration. Skeletonema sp. was found to have a carbon and ash content similar to those of Thalassiosira pseuod- nana (3H) (Walsh unpublished data). The caloric content of the product algae was calculated assuming a heat of combustion of 5.5 Kcal/g dry weight (Ryther 1959; Shelef et al. 1968) and solar energy conver- sion efficiencies were calculated based on this caloric con- MlCROALGAE FOR BIVALVE AOUACULTURE 73 600 □_ 2= 80 40 ■k=Chaet. ****.*< JFMAMJJASONDJFMAMJJASONDJFMAMJJASONDJ 1981 1982 1983 Figure 1. Total Solar Irradiance (A), Photosynthetic Efficiency (B), Dilution Rate (C), Algal Yield (D), and Species Composition (E). Ob- tained with cultured algae in six enriched saline 41 M2 continuous culture ponds located at latitude 41 "V during three years of operation (1981-1983). tent and the incident energy flux of visible light (Laws et al. 1983). Daily total solar irradiance was measured with Epply 6-90 pyranometers at Woods Hole, Massachusetts (Payne, personal communication 1981). Total solar irra- diance was multiplied by 0.45 to give the fraction of photo- synthetically active radiation (PAR) (Goldman 1979b); this value was multiplied by an empirically determined value of 0.85 to compensate for the loss of PAR due to the green- house structure and surface reflection and backscattering at the pond surface. Economic Analysis The economic analysis followed the format of Im and Langmo (1977). Fixed and variable cost components for 1983 were used to determine the cost of producing cultured algae at our facility. Direct labor consisted of four people. Algal department personnel assist in other hatchery proce- dures, research and development and the field program, so that wages for algal production were assumed to represent 60% of the total wages of the phycology department. Over- head includes costs of administration, security, insurance, travel, licenses, etc. Depreciation was calculated using the straight-line method, assuming no salvage value, on the basis of ten years for algal facilities initially valued at $150,000. Energy cost for lights, temperature control, pumps, aer- ation and sterilization were estimated from operating time for each item using a power charge of $0.08/KWH. Nutrient costs were computed from the annual con- sumption of the various commercial-grade chemicals used: sodium nitrate (16% N) at $0.44/kg; technical 75% phos- phoric acid (24% P) at $0.78/kg.. pentahydrate sodium metasilicate (12% Si) at $0.56/kg and sodium iron seques- trene (13% Fe) at $4.27/kg. The cost of reagent grade trace metals (Cu, Zn, Co, Mn and Mo) was included as well as vitamins (thiamine, biotin and B12) and compressed COz. Total cost of algae was expressed in three ways: cost per liter containing 109 cells, cost per gram algal carbon and cost per gram ash free dry weight (AFDW). RESULTS Average monthly total solar irradiance, photosynthetic efficiency, dilution rate, algal yield and species composi- tion data for the ARC continuous culture complex are plotted for the period 1981-1983 in Figure 1. Minimum total solar irradiance of less than 200 cal • cm-2 • day-1 occurred from November through January. Daily solar radi- ation increased sharply from March through May from ap- proximately 300 cal • cm-2 • day"1 to almost 500 cal ■ cm-2 • day"1. Yearly peak solar irradiance totals of 500-510 cal • cm"2 • day"1 occurred in June and July. Solar irradiance declined to approximately 430 cal • cm-2 • day"1 in August and 260 cal ■ cm "2 ■ day "' by October. Peak 1981 algal yields of 8-10 g dry wt • M"2 • day"1 occurred from May through September; corresponding di- lution rates ranged from 0.55-0.65 day1. Minimum yields of 3 g dry wt • M-2 • day-1 occurred in January and November- December when dilution rates averaged less than 0.30 day"1. In 1982 peak algal yields of 13-15 g dry wt • M"2 • day"1 occurred from May through August under dilution rates of 0.61-1.11 day"1 while minimum yields of 3 g dry wt • M"2 • day " ' occurred during January at an average dilution rate of 0.36 day"1. Maximum yields of 12-15 g dry wt • M~2 • day"1 occurred from May through September in 1983. Dilution rates during this five month period ranged from 1.04-1.18 day"1. Minimum values of 3 g dry wt • M"2 • day"1 occurred in January 1983 when dilution rates averaged 0.36 day"1. Photosynthetic efficiencies averaged 2.7% during 1981, 4.1% in 1982 and 4.4%- in 1983. Peak photosynthetic effi- 74 Walsh et al. TABLE 1. Estimates for the cost of production of microalgae at the ARC facility for 1983. Carbon content of algae assumed to average 5 x 10 ~12 g/cell representing 37% of ash free dry weight (AFDW). Fixed Costs Labor (4): Wages & Fringe Benefits $48,000 Maintenance: Labor & Materials 25,000 Overhead (administration, security. insurance, travel, licenses, etc.) 35,000 Depreciation 15,000 $123,000 Variable Costs Energy fa S0.08/KW $20,000 Nutrients 3.000 Fresh Water 1,000 Consumables 2,000 26,000 Total Cost of Production $149,000 Total Algal Production $ Cost/Production Unit 1) 4.46 x 107 liters (@ 109 cells/liter) $0.003/liter 2) 2.2 x 105gC 0.68/gC 3) 5.95 x 105gAFDW 0 25/gAFDW ciencies occurred during December 1981 (3.8%), De- cember 1982 (5.4%) and November 1983 (6.3%). Min- imum photosynthetic efficiencies were recorded in March 1981 (1.7%), January and March 1982 (2.4%) and Feb- ruary 1983 (2.7%). Species composition data indicated that Thalassiosira pseudonana (3H) tended to dominate from October through February while Skeletonema sp. dominated from March through September. Chaetoceros sp. was introduced in De- cember 1982 and averaged only 8% of the algal population in 1983. During the periods February -March and Oc- tober-November, Thalassiosira pseudonana (3H) and Skeletonema sp. were represented approximately equally in the ponds. Estimates of the cost of producing cultured algae at our facility for 1983 are shown in Table 1. Fixed costs were $123,000 with labor and associated costs for algal depart- ment personnel, administration, maintenance and security representing roughly 65% of this cost. Variable costs were $23,750. Energy consumption represented 77% of the vari- able costs. The cost of producing algae was estimated at $0,003 per liter (at 109 cells/liter), $0.68 per gram algal carbon or $0.25 per gram dry weight based on a 1983 total algal production of 4.46 x 1016 cells in 3.24 x 107 liters. The annual biomass production was equivalent to approxi- mately 223 kg algal carbon or 603 kg ash free dry weight. DISCUSSION Species Control The continuous mass culture of selected microalgae of proven nutritional value to bivalve spat has been suggested as highly desirable for the shellfish nursery stage, but it has been suggested that both technical and economic factors make this strategy impractical for the shellfish aquaculture industry (DePauw 1981: Persoone and Claus 1980; Ukeles 1980). Indeed, the culture of selected microalgae in contin- uous flow mass culture is reported to be the major unre- solved problem in mass culture technology (Goldman 1979a). Over the last twenty years ARC has developed a commercial process that currently provides a seasonally predictable supply of selected microalgae using continuous flow mass culture techniques. The ARC process was de- signed to minimize environmental variability and stress and maximize the productivity of the selected microalgae. Species control was assured by inoculating large volumes of selected microalgae into saline well water that varied minimally in salinity and pH and was essentially de- void of algae, algal predators and competitors. Pond har- vest could begin within 1 to 3 days in contrast to the 5 to 10 days necessary to develop a sufficiently dense culture when induced blooms of natural phytoplankton species were cul- tured. Use of saline well water eliminated macropredators such as copepods and barnacles as well as macroalgae such as Ulva and Enteromorpha which have plagued other mass algal culture facilities (DePauw et al. 1983). To provide sufficient inoculum of selected marine mi- croalgae for the continuous cultures, ARC upgraded labora- tory monospecific algal culture procedures to a commercial process. A daily volume of 1350 liters containing an average of 6. 3 x 10'2 algae cells was available for inocula- tion. Inoculum production was optimized by (a) mini- mizing the number of volume transfers, (b) keeping all cul- tures in or just past the exponential phase of growth and (c) most importantly, selecting microalgae that possessed an V -selected reproductive strategy characteristic of weedy species having high division rates, high productivity and few defense mechanisms, all factors increasing suitability to grazers such as shellfish (Kilham and Kilham 1980). The ARC algal production process for temperate zone shellfish hatcheries and nurseries is in marked contrast to those currently found in the literature. Dupuy et al. (1977) transferred algae from test tube to Fernbach flask, to 18- liter and 40-liter carboys and finally to 1000-liter tanks. The initial time needed to bring an algal volume from test tube to 1000-liter tank was 85 days. Lipschultz and Krantz (1980) reported a minimum of two weeks requirement for growth of algae prior to harvesting 100 liters per day from their algae tanks at an experimental oyster hatchery. Breese and Malouf (1975) reported that it took about a week at each of four steps in their batch algal culture procedure to reach an acceptable cell concentration for Pacific oysters at the Oregon State University Pilot Oyster Hatchery. In none of these examples have continuous flow algal culture tech- niques been employed and yet continuous cultures can pro- duce cells at a faster rate over an extended period of time than will a series of batch cultures of the same capacity over the same time period (Droop 1975). For example, in MlCROALGAE FOR BIVALVE AQUACULTURE 75 1983 the ARC pond complex produced an average daily volume of 24,295 liters in January with mean cell density of 9.50 x 108 cells/liter; this increased to 125,280 liters per day in July with an average cell density of 1.5 X 109 cells/liter. These continuous cultures of microalgae with an 'r'-se- lected reproductive strategy have a relatively short life cycle because the attributes of high division rates, high pro- ductivity and few defense mechanisms result in an algal population that is variable in time, and mortality is often catastrophic, non-directed and density independent (Guil- lard and Kilham 1977). Other possible reasons for the ab- breviated life cycle of these cultures are suboptimal light and temperature combinations which occur from November through March and C02-02 imbalance resulting in photo- respiration during the high light intensity period May through August (Pruder and Bolton 1980). When any of the above factors result in an unfavorable environment for the algae, bacteria and protozoans can bloom in the cultures; however, heterotrophic contamination would appear to be a secondary factor in the collapse of these algal cultures. Yield Optimization For optimizing biomass yields in outdoor algal mass cultures, it is imperative that all nutrients be added in ex- cess so that incident light energy is the main yield deter- minant. The key to optimizing the utilization of available light energy is the proper selection of growth rate (equal to the dilution rate under steady state conditions) and depth (Goldman 1979b). A depth of 65 cm was determined em- pirically to be best for our ponds. This depth, equivalent to 26,000 liters, resulted in the best compromise of minimum daily temperature variation and optimum light utilization in the cultures and provided a good bicarbonate buffer ca- pacity of C02 for the algae. C02 injection was not needed for the cultures from November through April because the dilution rate and bicarbonate content of the well water kept the pH of the algal cultures between 7.5-8.1. Inorganic carbon is sufficient in this pH range to sustain the growth of high-yield cultures (Pruder and Bolton 1980). Having determined the optimum depth for our cultures, the only other independent variable remaining to control yield was dilution rate. From 1981 to 1983 the dilution rate was increased from an average rate of 0.49 day-1 to 0.85 day-1. As a result, the 1983 annual algal yield averaged 9.6 g dry weight • M~2 • day"1 compared to 6.0 g dry weight ■ M~2 • day"1. It is believed the 1983 monthly average dilution rates, which range from 0.36 day-1 in January to 1.18 day"1 in September represent the optimal dilution rate for obtaining maximum potential yields from our continuous algal cultures at this northern latitude with the algal species cultured. The 1983 monthly average yields of 12-15 g dry weight • M"2 • day"1 that occurred from May through September compare favorably with the best average yields of 10-27 g dry weight • M"2 • day"1 reported for both fresh water and marine microalgae (Goldman 1979a). Production Costs The algal production process developed by ARC would be of academic interest only if the operational cost of this subsystem constituted a serious financial burden on the economic feasibility of the shellfish aquaculture venture. DePauw (1981) asserts that the well-established, but ex- pensive, indoor techniques used for monospecific algal pro- duction in hatcheries cannot, for economic reasons, be scaled up to the large volumes needed for feeding nursery bivalves. Species-controlled induced bloomings of natural phytoplankton for nursery rearing of juvenile bivalves is thus considered the only economical procedure despite nu- merous problems in controlling both the quantity and quality of microalgae (DePauw et al. 1983) as well as er- ratic growth of bivalve seed in the nursery (Claus et al. 1983). In point of fact, it can be shown that the continuous outdoor mass culture of selected microalgae using well es- tablished monospecific algal production techniques is eco- nomical for the nursery rearing of bivalve spat. The cost of providing food is but one of many costs as- sociated with a shellfish farming operation, but in order to provide that best economic perspective, food costs should be related either to the cost of shellfish seed produced with this algae or to the market price of the end product, i.e., littleneck clams in the case of ARC. The ARC partially recirculated indoor nursery system produced an average 14.6 X 106 seed (3.2-8.0 mm length) in 1982-1983 at a cost of $509,755 or $0.035/clam (Walsh, et.al. 1985a). Preliminary data indicate the survival in the field of this size range of seed to minimum legal size (47-52 mm length) has averaged 65% (Petrovits 1984). The algal pro- duction cost of $149,000 (Table 1) needed to produce this seed thus represents only 29% of the total seed product cost at ARC. ARC's production goal is to produce a minimum of 10 x 106 harvestable clams per year. The 1983 average wholesale price of littleneck clams on the New York Fulton Market was $75 per 500 count bushel, equivalent to $0. 15/ clam (estimated from NMFS Market News Reports). A harvest of 10 x 106 clams would generate an annual gross revenue of $1,500,000. Therefore, algal production cost would represent only 9.9% of ARC's gross revenue. Clearly, the cost of producing selected marine microalgae for hard clam seed production at ARC's scale of production does not represent a serious financial burden. Current re- search and development efforts on the use of artificial light to enhance algal pond production at the ARC pond complex (Walsh et al. 1985a) and the development of a new algal harvest technology using tangential flow filters (Walsh et 76 Walsh et al. al. 1985b) should significantly improve algal, and conse- quently, seed production. The ARC mass algal culture techniques are neither site specific or species specific. Similar algal production tech- niques have been used for commercial oyster {Crassostrea virginica) production in Hawaii (Pryor 1978; Scura et al. 1979) and for experimental production of Japanese little- neck clams (Tapes semidecussata) in the U.S. Virgin Is- lands (Roels et al. 1976). ARC has successfully produced seed of the oyster {Crassostrea virginica), bay scallop (Ar- gopecten irradians), surf clam (Spisula solidissima) and the West Indian pearl oyster (Pinctada imbricata) in the same nursery system where hard clam seed are produced. The ARC algal protocol has also been used to mass produce other selected algal species such as the Bacillariophyceae Chaetoceros gracilis, Chaetoceros calcitrans, Thalassio- sira weissflogii, Thalassiosira nordenskioldii and Skeleto- nema costatum; the Prasinophyceae Tetraselmis suecica and the Prymnesiophyceae Isochrysis sp. (Clone T-Iso). ARC's techniques should be applicable to finfish. crusta- cean or other molluscan aquaculture ventures which rely on microalgae as an essential component in the animal's diet. ACKNOWLEDGMENT The authors wish to acknowledge L. Wuethrich, K. Wilson, S. Talin. J. Emerald, M. Mahieu, D. Ryan, R. Ryder, R. Tobin and G. Peterson for their valuable daily labor to the project. We also thank B. Colder (Marine Bio- logical Laboratory) for graphics and G. Hart for prepara- tion of the transcript. Special thanks to Dr. R. R. L. Guil- lard and Dr. Kenneth Chew and his graduate class on shell- fish research techniques for review and comments on this manuscript. REFERENCES Anderson, M. A.. F. M. M. Morel, & R. R. L. Guillard. 1978. Growth limitation of a coastal diatom by low zinc ion activity. Nature 276:70-71. Ansell, A. D., J. E. G. Raymont, K. F. Lander, E. Crowley, & P. Shackley. 1963. Studies on the mass culture of Phaeodactylum. II. The growth of Phaeodactylum and other species in outdoor tanks. Limnol. Oceanogr. 8:184-206. Barber, R T. 1973. Organic ligands and phytoplankton growth in nu- trient-rich seawater. In: Trace Metals ami Metal-Organic Interactions in Natural Waters. P. C. Singer (Ed.) Ann Arbor Science Publishers. Ann Arbor, Michigan, pp. 321-338. Baynes, J. C. 1981. Forced upwelling nurseries for oysters and clams using impounded water systems. 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The relationship between cupric iron activity and the toxicity of copper to phytoplankton. Mar. Res. 34:511-529. Ukeles, R. 1980. American experience in the mass culture of microalgae for feeding larvae of the American oyster, Crassostrea virginica. In: Algae Biomass, G. Shelef and C. J. Soeder (Eds.). Elsevier/North- Holland Biomedical Press. Amsterdam, pp. 287-306. Walne. P. R. 1974. Culturing of bivalve molluscs, 50 years experience at Conway. Fishing News (Books) Ltd.. West Byfleet. Great Bntain, 173 PP Walsh, D. T , R. A. Kraus, C. A. Withstandley, S. M. Talin. & E. J Petrovits. 1985a. Dimensioning of a mass algal culture facility for the temperate zone nursery culture of bivalve molluscs. J. World Maricul- ture Soc. 16:451-463. Walsh, D. T., E. J. Petrovits. R. A. Kraus. L. M. Wuethnch. C. A. Withstandley, & K. N. Wilson. 1985b. An evaluation of tangential flow filtration for microalgal cell harvesting. Final Project Report. NSF/SBIR #OCE-8460500, 48 pp. Journal of Shellfish Research, Vol. 6, No. 2, 79-83, 1987. EFFECT OF AIR-SUPERSATURATED SEAWATER ON ARGOPECTEN IRRADIANS CONCENTRICUS (SAY) AND CRASSOSTREA VIRGINICA (GMELIN) ROBERT BISKER & MICHAEL CASTAGNA Virginia Institute of Marine Science School of Marine Science College of William and Mary Wachapreague, Virginia 23480 ABSTRACT Argopecten irradians concentricus and Crassostrea virginica were exposed lo several different levels of supersaturated seawater at temperatures ranging from 10 to 21°C. Gas bubble trauma occurred at a total gas saturation level of 116%. causing mortality in juvenile A. i. concentricus and reduced growth in juvenile C. virginica. KEY WORDS: Gas bubble trauma, air-supersaturated seawater, Argopecten, Crassostrea, cultured bivalves. INTRODUCTION Gas bubble trauma, commonly referred to as gas bubble disease, is a noninfectious disorder of aquatic animals caused by the physical formation of gas bubbles in the tissues and vascular system due to the uncompensated hy- perbaric pressure of total dissolved gases (Bouck 1980; Colt 1986). Weitkamp and Katz (1980) reviewed the litera- ture on the effects of air-supersaturated water. Harvey ( 1975) summarized its cause and effect in fish, and Colt et al. ( 1984) reported gas bubble trauma in bullfrog tadpoles. A number of commercially important invertebrates are ad- versely affected by supersaturated seawater, including aba- lone, clams, oysters, scallops, lobsters, shrimp and crabs (Hughes 1968; Malouf et al. 1972; Lightner et al. 1974; Johnson 1976; Supplee and Lightner 1976; Goldberg 1978; Elston 1983: Brisson 1985). The effect of gas bubble trauma can be acute and ter- minal or chronic, often leading to secondary disease, re- duced growth and gradual mortality. In bivalves it is often characterized by formation of gas blisters in soft body tissues and buoyancy of the whole animal (Malouf et al. 1972; Goldberg 1978; Bisker and Castagna 1985). Supersaturation, created by heating ambient seawater during winter to temperatures about 20 C. caused gas bubble trauma in adult surf clams Spisula solidissima (Dillwyn), and adult and juvenile bay scallops Argopecten irradians (Lamarck) at gas saturation levels of 114% ox- ygen and 195% nitrogen and higher (Goldberg 1978). Ma- louf et al. (1972) reported gas bubble trauma in adult eastern oysters Crassostrea virginica (Gmelin) and adult hard clams Mercenaria mercenaria (Linne), but did not give saturation levels. Current research indicates that su- persaturation studies should report the total excess gas pressure (mm Hg) or percent total gas saturation. It is this difference between the total gas pressure and the baro- metric pressure or hyperbaric pressure which causes gas Contribution No. 1372 from Virginia Institute of Marine Science. bubble formation (Colt 1983). Gas bubble trauma has been reported at ambient seawater temperatures in juveniles of the coot clam Mulinia lateralis (Say), the soft shell clam Mya arenaria Linne and the hard clam M. mercenaria (Linne) at total gas saturation levels of 108%, 114% and 1 15%, respectively (Bisker and Castagna in 1985). Determination of dissolved gas concentrations which may affect various cultured bivalves either acutely or chronically would be useful, since procedures can be initi- ated to degas seawater to more tolerable levels. This study examined the effects of air-supersaturation on juvenile A. i. concentricus and C . virginica. MATERIALS AND METHODS This experiment was conducted from April to May 1986 using flowing seawater pumped from Finney's Creek, Wa- chapreague. VA. Compressed air was introduced through a needle valve installed on the intake (vacuum) side of the pump to supersaturate the seawater during delivery under normal pumping pressure. This supersaturated seawater was degassed by cascading down a stairstep arrangement to produce four different supersaturation levels as detailed in Bisker and Castagna (1985). The lowest saturation level, which was similar to that of the ambient seawater, was des- ignated the control. Each saturation level was replicated twice. Experimental animals were held in ambient flowing sea- water prior to the experiment. Each container received 100 A. i. concentricus of approximately 13 mm shell height (SH) held in mesh bags (15 x 12 cm, with 6 mm mesh), and 100 C. virginica of approximately 17 mm SH held on a sieve. Argopecten irradians concentricus were photocopied for convenient determination of initial SH measurements (Haines 1973), while SH of C. virginica was measured di- rectly due to their irregular shape. Final SH measurements were determined on the live animals at day 28. Dissolved gas levels of seawater in each experimental container and of ambient seawater were measured five times each week. Hyperbaric gas pressure was measured 79 80 BlSKER & CASTAGNA TABLE 1. Gas saturation levels (mean ± standard deviation and range). Gas Treatment Hyperbaric Gas Pressure mm HG % Total Gas % Oxygen % Nitrogen N 1 2 3 Control Ambient 122.8 ± 9.73 (98-137) 62.5 ± 5.12 (51.5-69.5) 29.9 ± 5.30(20.0-42.0) 6.3 ± 2.57 (1.5-12.0) 6.4 ± 9.96 (- 11.0-35.0) 116.2 ± 1.30(112.8-118.1) 108.2 ± 0.68 (106.8-109.2) 103.9 ± 0.69(102.6-105.5) 100.8 ± 0.34 (100.2-101.6) 100.8 ± 1.31 (98.6-104.6) 110.2 ± 2.94 (105.6-115.8) 105.1 ± 2.49(100.8-110,1) 102.5 ± 2.37 (98.7-107.6) 100.1 ± 2.44(95.2-104.4) 96.1 ± 5.25 (85.2-104.4) 118.1 109.3 104.4 101.0 102.1 ± 1.87 (113.9-121.4) ± 1.14 (106.6-110.6) ± 1.14 (102.6-106.8) ± 0.83(99.8-103.3) ± 1.75 (99.6-105.9) 21 21 21 21 20 with a gasometer (Bouck 1982). Concurrent dissolved ox- ygen (D.O.) measurements were taken using a YSI Model 58 oxygen meter with an oxygen probe Model 5775 (Yellow Springs Instrument Co., Yellow Springs. Ohio). The D.O. meter was air calibrated. Water temperature, sa- 120 115 110- 105- 100 25 20 15 10 5 10 20 25 Days lean percent total gas saturation for treatments 1 (O), 2 ', and control (■) and for ambient seawater (A), with mean water temperatures. linity, and barometric pressure were measured for determi- nation of total dissolved gas, percent oxygen saturation (% 02), and percent nitrogen saturation (% N2) as described by Bouck (1982). Experimental animals were monitored daily for flotation, noticeably visible air bubble formation in tissues and removeal of dead animals. Hyperbaric gas pressure (GP) and percent total gas satu- ration (ck TG) were compared between saturation levels using one-way analyses of variance (ANOVA) with repli- cation. The mean survival in days of animals at each treat- 200 150 a z UJ O < o cc 100- 80 60 40 20 0 o en f CO en LO en c\j in CO K HMP HMP HMP SERIES 1 SERIES 2 COMBINED Figure 3. Percentage of clams categorized as having high (H), me- dium (M), and poor (P) condition indexes prior to and after purging the digestive tract of sediment load. H = 100 + , M = 81-100, and poor §80. Cleansing and Condition Index in M. arenaric 87 The other categories were not well represented. Overall, more than 80% of clams showed greater than 10% differ- ence in condition index as a result of sediment load. Figure 2 also shows that there is little difference in the proportions of individuals in each sampling series which occupy the same percent difference category, even though the two series were conducted 27 mo apart. Figure 3 shows the distribution of clams with respect to a grading scale presented by Medcof ( 1961 ) for ranking the quality of oysters and subsequently adopted by Robert and Smith ( 1980), for ranking the quality of soft-shell clams. If we adopt this classification scheme for soft-shell clams the ranking is as follows: high quality, and index of 100 +; medium quality, 81-100; and poor quality ^80. It is evi- dent from each sample series that the condition index cal- culated for animals with sediment in the digestive tract sub- stantially overestimates the percentage of high and medium quality clams while underestimating the numbers of poor- quality individuals. A chi-square analysis of pooled samples supports this interpretation in that the quality level assigned to clams will be significantly influenced (P = 0.01) by the use of cleansed or uncleansed clams in the determination of condition indices. DISCUSSION The sediment load in non-cleansed soft-shell clams can have a significant effect on the determination of condition indices. We suggest that the results found here for soft- shell clams are likely to apply to other bivalve species which occupy similar habitats and exhibit similar feeding ecology. Consequently, researchers and aquaculturists who utilize this type of condition index as a means of moni- toring the quality or well-being of bivalves should be cog- nizant of the degree of error which may be introduced into the calculation if samples are not cleansed prior to the de- termination of the dry meat weights. Additionally, many ecological and biochemical studies on bivalves involve the determination of total solids to quantify condition index (Ansell et al. 1964, Shaw et al. 1967, Barker and Merrill 1967 and Jarzebski et al. 1986 among others). In the methods used by these researchers no consideration is given to the weight of sediment in the di- gestive tract. This we think is an oversight resulting from the adoption of a method for the determination of total solids in oysters, which have, to our knowledge, little or at least minimal inorganic content in the digestive tract. Fur- thermore, in studies by Ansell et al. (1964) on the seasonal cycle of biochemical composition in the hard-shell clam. Mercenaria mercenaria, weighed aliquots of tissue homog- enate from more than one animal were analyzed. Again, there is no clear indication in their methods that clams were cleansed prior to the preparation of tissue homogenate. Consequently, in those studies where weighed aliquots of homogenate are required for biochemical analysis, there is a substantial risk of overestimating the aliquot weight due to the presence of inorganics in the gut contents. In a review and comparison of morphometric, biochem- ical, and physiological indices of condition in marine mol- luscs, Mann (1978) makes reference to only two studies: de Wild (1975) on Macoma balthica (L.), and Walne and Mann (1975) on Crassostrea gigas (Thunberg) and Ostrea edulis (L.), where the ash-free dry weight is considered in the calculation of condition index. Using the latter method eliminates problems associated with the weight of sediment in the gut contents. Consequently, any other condition index which utilizes meat dry weight and is determined without cleansing may be suspect. In other disciplines, cleansing of the bivalve is a generally accepted practice (In- ternational Mussel Watch 1980). Specifically, in studies of polluting contaminants such as heavy metals and hydro- carbons, where researchers are concerned with the tissue contaminant loading, great care is taken to eliminate all possible sources of contamination other than that directly associated with tissues. From the ^bove, it is evident that some standardization must be in .-rporated into the method used to assess condition indexes or biochemical parameters of bivalves which ingest sediment actively or passively. Such standardization should also take into account the im- pact of the particular handling and holding methods on the level of sediment load in samples taken from the natural environment. For example, if bivalves are transported from the field in water and/or held in water in the laboratory prior to processing, they are likely to purge themselves of gut-borne sediments. Conversely, if transported and/or held without water, no purging is possible. In either case, some standard period of holding in water sufficient to allow purging of the gut should be adopted in any experiment or study in which gut sediment load is likely to influence re- sults. ACKNOWLEDGEMENTS We wish to thank R. B. Angus, P. Woo and F. G. Scat- tolon for their assistance in the field, laboratory, and manu- script preparation. We also thank G. Robert and R. E. Drinnan for their comments on an earlier draft of this manuscript. REFERENCES Ansell, A D.. F. A. Loosmore, & K. F. Lander. 1964. Studies on the hard-shell clam, Venus mercenaria, in British Waters. II. Seasonal cycle in condition and biochemical composition. J. Appl. Ecol. 1:83-95. Barker A. M. and A. S. Merrill. 1967. Total solids and length-weight relationship of the surf clam, Spisula solidissima. Proc. Natl. Shell- fish. Assoc. 57:90-94. Bavne, B. L.. D. A. Brown. K. Burns, D. R Dixon, A Ivanovici, D. R. Hawkins and Rowell Livingstone, D. M. Lowe, M. N. Moore, A. R D. Stebbing, and J. Widdows. 1985. The Effects of Stress and Pollution On Marine Animals. Praeger Scientfic Press. 384 pp. de Wilde, P. A. 1975. Influences of temperature on behavior, energy me- tabolism, and growth of Macoma balthica (L), pp. 239-256. In Pro- ceedings of the Ninth European Marine Biological Symposium. H. Barnes. Ed. Aberdeen Univ. Press, Aberdeen, Scotland. Drinnan, R. E. & E. B. Henderson 1959. An index of condition in oysters. Its Determination and Significance. Fish. Res. Board of Can., Biol. Sub-Sta., Ellerslie, PEL: 11 pp. Grave, C. 1912. A manual of oyster culture in Maryland. 4th report of the Board of Shellfish Commissioners of Maryland 1912:1-377. Havinga, B. 1922. Flora en fauna der Zuiderzee. Mariene Mollusken: 373-390. International Mussel Watch. 1980. Report of a workshop sponsored by the Environmental Studies Board Commission on Natural Resources. Nat. Res. Counc, Nat. Acad. Sci., Washington, D.C.: 248 pp. Jarzebski. A., L. Polak. R. Wenne and L. Falkowski. 1986. Microgeo- graphic differentiation in the lipid composition of the bivalve Macoma balthica from the Gulf of Gdansk (Southern Baltic). Marine Biology Vol. 91:27-31. Mann, R. 1978. A comparison of morphometric. biochemical, and physi- ological indexes of condition in marine bivalve molluscs, pp. 484-497. In Energy and environmental stress in aquatic systems. J. H. Thorp and J. W. Gibbons, Eds. Tech. Info. Center. U.S. Dept. Energy. Mann, R. & J. H. Ryther. 1977. Growth of six species of bivalve mol- luscs in a waste recycling-aquatic system. Aquaculture 1 1: 231-245. Medcof, J. C. 1961. Oyster farming in the Maritimes. Fish. Res. Board Can , Ottawa, Bull. 131 Robert, G., and D. W. Smith. 1980. Surveys of soft-shell clam (Mya arenaria) populations in some closed areas of Charlotte County, New Brunswick. Can. MS Rep. Fish. Aquat. Sci. 1567: 59 p. Shaw, W. N., H. S. Tubiash, & A. N. Barker. 1967. Freeze-drying for determining total solids in shellfish. J Rish. Res. Board Can. 24:1413-1417. Walne, P. R. & R. Mann. 1975. Growth and biochemical composition in Ostrea edulis and Crassostrea gigas, pp. 578-607. In Proceedings of the Ninth European Marine Biological Symposium. H. Barnes Ed. Aberdeen Univ. Press, Aberdeen. Scotland. Journal of Shellfish Research, Vol. 6. No. 2, 89-95, 1987. FOOD RESOURCES RELATED TO HABITAT IN THE SCALLOP PLACOPECTEN MAGELLANICUS (GMELIN, 1791): A QUALITATIVE STUDY SANDRA E. SHUMWAY' 2, RHONDA SELVIN2 & DANIEL F. SCHICK1 1 Department of Marine Resources West Boothbay Harbor, Maine 04575 2Bigelow Laboratory for Ocean Sciences West Boothbay Harbor, Maine 04575 ABSTRACT The gut contents of the scallop, Placopecten magellanicus, were analysed seasonally. Two populations were com- pared, one shallow water (approximate depth 20 m) and one deep water (approximate depth 180 m). A total of 27 species of algae, ranging from 10-350 u.m, were identified as well as a number of miscellaneous items including pollen grains, ciliates, zooplankton tests and considerable detntal material and bacteria. Benthic and pelagic food species were equally represented in shallow water scallops but benthic species outnumbered pelagic ones in the deep water population. Seasonal variations of food items occurred and coincided with bloom periods for the individual algal species. Gut contents generally reflected available organisms in the immediate habitat. It was concluded that P. magellanicus is an opportunistic filter feeder which takes advantage of both pelagic and benthic organisms as food. INTRODUCTION The sea scallop, Placopecten magellanicus, lives at the sediment-water interface in the Gulf of Maine at depths ranging from 2-180+ m and provides a valuable annual fishery along the Maine coast and on Georges Bank. In recent years, a substantial fishery has existed in the deeper waters of the Gulf of Maine. This fishery is supported by the survival at depth of a particularly strong year class in 1975 and of a few weaker year classes since then. These deep water scallops have been shown to differ from scallops found in shallower water in many of their allome- tric growth relationships (Schick et al. 1987a, b). Of partic- ular interest to the fishery is the smaller meat size for equivalent shell diameter in the offshore scallops and their apparent inability to form completely ripened gonads (Barber et al. 1986). The scallop is a semi-mobile filter feeder and utilizes available particulate matter for food. Several authors (Bayne and Widdow 1978; Newell and Bayne 1980; Ne- well et al. 1982; Berg and Newell 1986) have shown that the quality and quantity of food influence growth rate and fecundity in bivalve molluscs. Most recently, MacDonald and Thompson (1985 a,b 1986) demonstrated depth related differences in somatic growth and reproductive output in Placopecten magellanicus and attributed these differences to a combination of temperature and food availability. Un- doubtedly, the quantity and quality of the food utilized by the scallops is responsible, to a large extent, for many of the observed differences in the growth rate, gonad develop- ment and indeed the survival of our study populations. While there has been a multitude of laboratory studies on the feeding rates of bivalve molluscs (see Morton 1983; Bayne and Newell 1983 for reviews), little information is available on the specific food items utilized by these species in their natural habitats. It is generally assumed that these filter-feeding species rely on phytoplankton from the water column as their main source of energy. Primary pro- duction at depths >30m is negligible and the deep-water animals must rely on sedimenting food sources or benthic material for their survival. Previous authors (Davis and Marshall 1961; Hall (pers. comm.) and Mikulich and Tsikhon-Lukaniana 1981) have indicated that benthic or- ganisms plan an important role in the feeding ecology of other scallop species. There has been considerable recent interest in the near bottom nepheloid layer and its associated "fluff" layer of unconsolidated sediment on the bottom and on the impor- tance of sedimenting phytoplankton as a food source for benthic organisms (Christensen and Kanneworff 1985; Davies and Payne 1984; Graf et al. 1982, 1984). The ability of scallops to stir up the sediment surface places them in the unique position of being able to utilize both the suspended material in the near-bottom water and the re- cently deposited material to be found in the fluff layer. In the current study we compare the potential food or- ganisms of scallops from a "shallow water" population with those from a "deep-water" population on the basis of gut content analysis, in an effort to 1 ) characterize the na- ture of the food items consumed, 2) assess seasonal varia- tion of food items and 3) provide preliminary information regarding the value of the sediment surface "fluff" layer and nepheloid layer to the nutrition of bottom dwelling in- vertebrates. MATERIALS AND METHODS Specimens of the sea scallop, Placopecten magel- lanicus, were collected throughout the year from two loca- tions in the Gulf of Maine: Damariscotta River (43° 5 1 .26' , 69° 34.0'; depth approximately 20m) and 20 miles south of Boothbay (43° 26.5', 69° 33.3'; depth approximately 180m). Samples from the Damariscotta River were col- lected by divers and the offshore samples were collected by 89 90 Shumway et al. trawlers. In all cases, animals were immediately returned to the laboratory and digestive gland/stomach complex re- moved. After careful removal of the dorsal shell valve, the animal was washed to be sure that no debris remained to be confused with food items/gut contents. In one group of scallops, the gut contents were removed with a hypodermic syringe for comparison with other samples. Gut contents recorded from these samples were identical to those col- lected by removing the digestive glands and the possibility of contamination was eliminated. Guts were not homoge- nized. Samples were drawn from the gut by pipette. Samples of 0. 1 -0.2 ml were drawn from each gut. In ini- tial sampling trials, increasing the sample size to 1 ml did not yield additional species representation. In addition to the scallop samples, samples of the overlying water were collected from the Damariscotta River site and analysed for species content. Gut contents were identified using stan- dard light and phase contrast microscopic techniques at the Provosolli-Guillard Center for the Culture of Marine Phy- toplankton, Bigelow Laboratory for Ocean Sciences. A total of 78 fresh guts were analyzed from scallops ranging in size from 90- 140 mm shell height. TABLE 1. Gut contents of inshore scallops. Species Habitat Size lu.ml Nitzschia spp. B 25-150 Navicula spp. B 8-240 Pleurosigma sp. B 200 Thalassiothrix sp. B 50 (chain) Amphora sp. B 10-30 Licmophora spp. B 25-180 Acnanthes sp. B 40-90 Pinnularia sp. B 40-80 Surirella sp. B 15-25 Cylindrotheca closteriwn B 80-100 Protogonyaulax resting cyst B 35-40 unidentified cyst B 25-35 Melosira sp. B 30-55 (chain) Striatella sp. B 40-50 Coscinodiscus spp. B/P 40-180 Ditylum brightwellii P 50-150 Proloperidinium sp. P 60-70 Eucampia zoodiacus P 40-75 (chain) Peridinium sp. P 20-30 Prorocentrum micans P 45-55 Skeletonema costatum P 30-50 (chain) Dinophysis acuminata P 50-60 Dmophysis spp. P 32-60 Thalassiosira rotula P 20 (chain) Thalassiosira nordenskio Ida P 20 (chain) Thalassiosira spp. P 10-200 (chain) Miscellaneous: Silicoflagellate strew; pollen grains (30-40 |j.m); green filamentous alga (1000 > u,m); ciliates; zooplankton tests; bacteria; detritus; uniden- tified. unpigmented still active forms: (3 (xm) multiflagellate ( 10 u.m). ciliated mass (40-200 M-m) RESULTS The predominant food items identified from the guts of scallops from shallow water populations and deep water populations are summarized in Tables 1 and 2, respec- tively. A total of 27 species of algae ranging in size from approximately 10-350 u.m were identified from the diges- tive glands along with several miscellaneous items in- cluding pollen grains, ciliates, zooplankton tests, consider- able detrital material, and bacteria. Benthic and pelagic food species were equally represented in the shallow water scallops while, as might be expected, benthic species out- numbered pelagic species in the guts of deep water scallops in number of species but not necessarily in biomass. Resting cysts of the toxic dinoflagellate, Protogonyaulax tamarensis, were tru e prominent in the offshore popula- tion than in the shallow water animals. The theca of several Dinophysis spp. were a constant feature of both popula- tions, though not seen consistently or in great numbers in water samples. While we did not do a taxonomic study, different morphological variations, attributed in the litera- ture to different taxa, were observed. Seasonal variations in occurrence of the food items from both populations are summarized in Tables 3 and 4. In the offshore scallops the pelagic species Coscinodiscus spp., Prorocentrum micans, Dinophysis spp., Eucampia zoo- diacus and Ditylum brightwellii were prominent in the early fall (Oct/Nov), i.e. during the bloom period, and coincided TABLE 2. Gut contents of offshore scallops. Species Habitat Size (u.m) Melosira sp. B 50 (chain) Protogonyaulax resting cyst B 35-40 Navicula spp. B 60-350 Nitzschia spp. B 110 Thalassiothrix sp. B 50 (chain) Acnanthes sp. B 40-90 Amphora sp. B 10-30 Pleurosigma sp. B 280 Licmophora sp. B 120-180 Pinnularia sp. B 70-100 Surirella sp. B 15-25 unidentified dinoflagellate cyst B 25-35 Coscinodiscus spp. P/B 50-160 Prorocentrum micans P 45-55 Dinophysis spp. P 32-60 Thalassiosira sp. P 35-50 Eucampia zoodiacus P 100 (chain) Ditylum brightwellii P 150 Ditylum brightwellii resting spore P 40 Miscellaneous: Pollen grains (40-60 M-m) zc oplankton tests (100-250 u.m); bacteria; detritus; unidentified unp gmented. still active forms ; uniflagellate (3 u.m), multiflagellate 10 u,m) ciliated mass (70 -300 u.m) Food Resources Related to Habitat in Scallops 91 with the period of greatest mixing of the water column. The most prominent pelagic food items of the inshore scallops were Prorocentrum (Oct/Nov; Jan), Thalassiosira sp. (March; Jan), and Dinophysis (Oct) again, coinciding with bloom periods and the subsequent settlement of the algae cells. DISCUSSION The quality and quantity of food available is a major limiting resource for suspension feeding organisms in gen- eral and for Placopecten magellanicus in particular (Mac- Donald and Thompson 1985a, b; 1986a, b). As pointed out by Levinton ( 1972), not only is the food supply constantly fluctuating, it is unpredictable and these suspension feeding organisms must maintain an adaptive feeding strategy which maximizes the generality of their food requirement. Although the majority of lamellibranch bivalves have been divided into two groups, the suspension feeders and the de- posit feeders, there is no clear-cut distinction between these two food sources (Morton, 1983). In a recent survey, Tsikhon-Lukanina (1982) showed that most bivalved mol- luscs are detritus feeders and that their main sources of food are detritus, unicellular algae and protozoa. The surface deposits (fluff layer) can be stirred into sus- pension and thus made available to suspension feeding an- imals as seston (organic and inorganic suspended matter greater than 1 |xm). The quantity of this particulate matter and its quality as a food resource varies both temporally and spatially in response to physical and biological factors (Brut, 1955; Berg and Newell, 1986). The results reported here clearly indicate that the scallop, P. magellanicus is an opportunistic filter feeder which takes advantage of these resuspended particles. The gut contents generally reflect the available organisms in the immediate habitat (Tables 1-4). One notable exception is Chaetoceros spp., usually available in the water column but not observed in gut con- tent. This may be due to lack of resolution after spines have been altered or removed. It may also be an indication of TABLE 3. Seasonal variation in relative abundance of food items from inshore scallops. Oct/Nov Jan March July Oct/Nov Dec/Jan Species 1985 1986 1986 1986 1986 1987 Amphora sp. - - - + Cylindrotheca closterium - - + + Protogonyaulax tamarensis resting cyst + + - + + unidentified cyst + - + + Nitzschia spp. + + + + + + + + Navicula spp. + + + + + + + + + + + Pleurosigma sp. + + - + + + Thalassiothrix sp. + + - - + Licmophora spp. + + + + + + + Acnanthes sp. + + + + + + Pinnularia sp. - + - - - Surirella sp. + + - + + + + Siriaiella sp. - - + Ditylum brigktwellii + - + + Protoperidinium sp. + + - + + Eucampia zoodiacus + + - + + + Peridinium sp. + - + + Prorocentrum micans + + + + + + - + + + + + + Melosira sp. + + + + + + Coscinodiscus sp. + + + + + + + + Skeletonema costatum + - + Dinophysis acuminata - - + Dinophysis spp. + + + + + + + + + + Thalassiosira spp. + + + + + + + + + + Thalassiosira rotula - - + Thalassiosira nordenskioldii - - - + Miscellaneous: Silicoflagellates ( + I, green filamentous alga, bacteria (+ + +). pollen grains ( + ), zooplankton tests and spines ( + + + 1. diatom strew (+ +), detritus ( + + +) + + + common, very abundant + + common, abundant + common, not abundant 92 Shumway et al. TABLE 4. Seasonal variation in relative abundance of food items of scallops from 20 miles south of Boothbay. Sept Jan Nov Dec/Jan Species 1985 1986 1986 1987 Protogonyaulax tamarensis resting cyst + + + + + + + Navicula spp. + + + + + + + + Nitzschia spp. + + + + + Thalassiothrix sp. - + - - Amphora sp. - - - + Acnanthes sp. + + + + + Pleurosigma sp. + + + Pitmularia sp. + - + + unidentified dinoflagellate cyst + - + - Surirella sp. - - - + Coscinodiscus spp. + + + + + + Licmophora sp. - - - + Prorocentrum micans + + + + + + + Dinophysis spp. + + - + + Melosira sp. + + + + + Thalassiosira sp. - + + - + + Eucampia zoodiacus + + - + + + Ditylum brightwellii + + - + + + Ditylum brightwellii resting spore + - + - Miscellaneous: Pollen grains ( + ), woplar kton tests ( + ), bacteria (+ +) detritus (+ + ), motile forms 10 u.m colorless multiflagellate, 3 p,m colorless arrowhead, uniflagell ate 70- -300 u.m ciliated mass + + + common, very abundant + + common, abundant + common, not abundant selective feeding. In a previous laboratory study (Shumway et al. 1985). it was shown that P. magellanicus exhibited both pre-ingestive selection on the labial palps and differ- ential absorption in the gut. The gut contents varied both with depth and with season and these differences were primarily reflected in the con- sumption of species abundant during bloom conditions. In addition, a greater number of predominantly benthic species were identified from deep water scallops than from shallow water animals. These findings mirror those of other workers (see Table 5) and are not intended to be a compre- hensive or all inclusive listing of possible food items for this species. Some algal species, especially small forms (<10|xm) may be quickly digested and not seen or resolved in stomach contents. Conversely, many of the observed species may be difficult to digest and of little consequence to the diet. In analyses performed over several days, from the same guts, large pennates (>150u.m) remained intact and pigmented, while most other forms had degraded. During bloom concentrations of P. micans, many of these cells remained intact in untreated guts 5 days after the ini- tial sampling. Further, much of the food is undoubtedly comprised of the naked and minute nanoplankton which is probably too delicate to remain intact after ingestion. In addition to the differences noted between the shallow and deep water habitats, several other points emerged that raise a number of questions. It was noted in the present study that the food organisms ranged from 10-350 u.m. Mikulich and Tsikhon-Lukanina (1981) also found a wide range of food particles (9-950 |i.m). The majority of feeding studies on bivalve molluscs involve feeding pure cultures of rather small algae (4-20 p,m) to various species of molluscs. Several authors have demonstrated that scallops exhibit reduced efficiency of particle retention when fed particles less than 7 u,m (Chlamys opercularis, Vahl 1972; Pecten opercularis and P. septemradiatus, M0hlenberg and Riisgard 1978). Palmer and Williams ( 1980) demonstrated that Argopecten irradians was capable of adjusting the filtration efficiency in response to varying concentrations of particulate matter. The presence of zoo- plankton tests raises the question of whether or not these organisms are utilized as a food source. Crustacean larvae, tests and whole copepods have been identified from the guts of scallops and other bivalve molluscs; however, their role as a food resource is not clear. Wojtowicz (1972) studied a number of digestive enzymes in the digestive gland of P. magellanicus but found no chitinase. It seems most likely that these crustacean materials are taken inci- dently with other organisms and are probably not a major food source. Future feeding studies on scallops should in- clude species of algae in the larger size ranges and should investigate the possible role of zooplankton as a food source. The presence of Dinophysis in such large quantities is of particular interest. Only unpigmented, digested thecae were observed in the guts. Dinophysis spp. have been implicated in outbreaks of diarrhetic shellfish poisoning (DSP) world- wide (Campos et al. 1982; Kat 1979; 1985; Guzman and Campodonico 1975; Freudenthal 1985; Dahl-Lyndastad 1985; Krough et al. 1985) although no known cases have been reported from the Gulf of Maine. Species known to accumulate the Dinophysis cells include mussels {M. edulis and M. coruscum), scallops (Patinopecten yessoensis and Chlamys nipponensis akazara) and clams (Tapes (Ven- erupis) japonica and Gomphina melanaegis). Preliminary investigations (Yasumoto et al. 1985) have indicated that the DSP toxin is concentrated in the digestive gland and that intoxication could be avoided, as in the case of PSP. by eliminating the digestive glands. This method is feasible with the scallops since usually only the adductor muscle is marketed. A market does exist, however, for whole scallop meats and current legislation is pending with regard to re- stricting the taking of whole scallops meats for human con- sumption due to PSP infestation. Further, it has been dem- onstrated that mussels and scallops may become toxic beyond the regulation level in the presence of Dinophysis spp. at cell densities of 200/L or less, Yasumoto et al. Food Resources Related to Habitat in Scallops 93 TABLE 5. A summary of the feeding habits of various species of scallops. Species Feeding Habits Author Aequipecten irradians Chlamys opercularis Chlamys operacularis Chlamys tehuelchus Patinopeclen yessoensis Pecten septemradiatus P. opercularis P. varius Pecten varius Placopecten grandis ( = magellanicus) Placopecten grandis ( = magellanicus) Placopecten magellanicus Microflora, detritus, bacteria and organic matter common in water immediately adjacent to the bottom; identified 26 species of diatoms [ 17 benthic/tychopelagic: 9 planktonic); proposed that some selective feeding might be in effect Dinoflagellates, diatoms, crustacean larvae, sand grains, detritus Sedimenting phytoplankton; demonstrated that sedimentation of phytoplankton major factor regulating growth Identified over 100 food items; dominant algal species benthic; planktonic species comprising the spring bloom; 90% of ingested particles less than 100 lira; not all benthic algae of appropriate size range used: those attached to sand grains by gelatinous stalks very abundant but not found in guts presumably not easily resuspended 161 forms identified including algae and animals, spores, eggs, detritus and mineral particles (size range 9-950 um); detritus main food source; animal and plant material most important during reproductive period; Resuspended detritus "Unfailing occurrence of bottom, naviculoid diatoms and the frequency of sand-grains, spicules, and bottom living Foraminifera"; diatoms most important food organism; planktonic species important during their respective seasons of abundance Detritus, 'everything small that is to be found in the plankton', their own and other veliger larvae, copepods. Balanus nauplii: pine-tree pollen grains. Pleurosigma and Navicula not digested Identified 38 forms including algae, animals and eggs; diatoms comprised bulk of food, tintinnids and silicoflagellates Algae, pollen grains, silicoflagellate strew, ciliates, zooplankton tests, bacteria, detritus, 21 species of algae identified (10-350 u.m); some seasonal variation attributed to algal blooms; both benthic and pelagic species prominent Davis and Marshall (1961) Aravindakshan ( 1955) Christensen and Kanneworff (1985) deHall (personal communication) Mikulich and Tsikhon-Lukanina ( 1981 ) Blegvad (1915) Hunt (1925) Stevenson (1932. 1936) Borden (1928) Present study (1983). The concentrations found in the present investiga- tion could lead to cases of DSP if whole animals were in- gested. Further studies are needed on the occurrence of these dinoflagellates and their role in the food habits of not only the scallops, but also of other commercially important species of shellfish. Although the scallops are capable of some mobility, they remain essentially fixed in position and feed on par- ticles in the sea water that passes by them. These currents of water may be environnmentally induced or they may be created by the animals themselves. These factors take on special significance for scallops in that current orientation could be of real significance for maximizing the available food resources. Diver observations of scallops at various stages of the tide seem to indicate that scallops feed discon- tinuously and orientate with respect to current direction. Flume studies are currently underway to determine the sig- nificance of this orientation and/or feeding behavior. Some scallop species are also known to exhibit "clap- ping activity" which resuspends some of the surface sedi- ment (fluff layer) thus making the material available to the filter feeders. Davis and Marshall (1961) showed that the bay scallop, Aequipecten irradians, obtained a consider- able amount of its food through shell flapping i.e.. resus- pending the surface sediment materials. Divers have noted however, that this clapping activity is rarely seen in P. ma- gellanicus in shallow water (Schick, unpublished). This shell clapping activity could be of considerable signifi- cance, however, to the deeper water animals where pelagic algal species are not always readily available as a food source. Algal cells settling during spring and fall blooms are one of the main inputs of particulate organic matter from the pelagic to the benthic system. Downward mixing of plankton during certain times of the year undoubtedly plays a large part in making food organisms available to the deeper water scallops. The blooms (spring, summer) play an important role for shallow-water populations of P. ma- gellanicus as has already been demonstrated by several au- thors with regard to the gametogenic cycle (Ehinger 1978; Thompson 1979; Robinson et al. 1981). The deep water scallops live well below the photic zone and it might well be assumed that they do not utilize living algae from plank- tonic blooms as a food resource. Our results (Tables 2 and 94 Shumway et al. 4) indicate, however, that a relatively large number of in- tact planktonic algal species reach the scallops, probably due to vertical mixing of the water column during the fall (October/November). These sudden bursts of energy/food input as a result of the phytoplankton blooms (rain or mixing) may provide just enough energy to sustain the pop- ulation. Studies are currently underway to assess the extent of gonad production in the two populations. Preliminary indications are that the deep water scallops do not produce viable gonads (Barber et al. 1987). Davies and Payne (1984) suggested that the substantial increase in fresh organic carbon and nitrogen associated with the spring phytoplankton bloom might have a stimula- tory effect on the growth and reproduction of the benthic animals and might act as a triggering mechanism for their life cycle. It is possible in the offshore population of P. magellanicus studied here, that the influx of pelagic species after the fall bloom is the factor responsible for the survival of the population. This is the period of the year when the inshore animals have just spawned and their metabolic rate is at its lowest peak (Shumway et al.. in prep.). The exact extent of the energy input, however, is as yet not clear and still needs to be quantified. Our results indicate that the scallops are ingesting both pelagic and benthic organisms. The relative importance of these food organisms is not known, although it can be as- sumed that planktonic species such as Thalassiosira. Dino- physis and Prorocentrum must provide a significant portion of the energy intake of the scallops. In 1972, Levinton stated that the role of resuspended sediment from the fluff layer in the nutrition of suspension feeding animals still needed investigation and the statement is as true now as it was then. A better understanding of the feeding habits of "interfacing" species such as P. magel- lanicus may provide a unique opportunity to determine not only their role in the transformation of energy between the benthic and pelagic ecosystems, but also to study the rela- tionship between the fluff layer, the nepheloid layer and the benthic invertebrates. ACKNOWLEDGEMENTS The authors are indebted to the Department of Marine Resources dive team and K. Pinkham for collection of scallops. We would also like to thank M. Bricelj, M. Cas- tagna and J. 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Yasumoto, T., Murata, M., Oshima, Y , Matsumoto, G. K. & J. Clardy. 1984. Diurrhetic shellfish poisoning, in: Seafood Toxins, Ragelis. ed. American Chemical Society Symposium Series 262. 207-214 p. Yasumoto. T.. Yasukatsu. O. and M. Yamaguchi. 1978. Occurrence of a new type of shellfish poisoning in the Tohoku District. Bull. Jap. Soc. Sclent. Fish. 44:1249-1255. Journal of Shellfish Research, Vol. 6. No. 2, 97-102. 1987. THE REPRODUCTIVE STRATEGY OF THE ATLANTIC SURF CLAM, SPISULA SOLIDISSIMA, IN PRINCE EDWARD ISLAND, CANADA THOMAS W. SEPHTON Canada Department of Fisheries & Oceans Science Branch, Gulf Region Moncton, New Brunswick E1A 9B6 CANADA ABSTRACT The reproductive cycle of Spisula solidissima from the Northumberland Strait and Gulf of St. Lawrence shores of Prince Edward Island. Canada was examined in 1984 and 1985. Annual cycles of gametogenesis were observed at both locations in both years and differences between study sites and years were closely related to water temperatures. Gonads developed quickly to the npe stage by June and July as water temperatures approached 15°C and a prolonged spawning period was observed during the warm water period from late July to October. Spawning terminated abruptly as water temperature decreased in October. The male to female sex ratio was 1:1 and no hermaphrodites were found. KEY WORDS: Reproductive cycle, gametogenesis. Surf clam. Bar clam, Spisula solidissima. INTRODUCTION The Atlantic surf or bar clam, Spisula solidissima (Dillwyn) is a dominant member of the bivalve molluscan fauna of clean sandy substrates from Cape Hatteras to the Gulf of St. Lawrence (Ropes 1980). Along the northeastern coast of the United States, the offshore (20-60 m) bar clam fishery is important. An extensive management plan is based on annual stock assessments (Serchuk et al. 1979. Murawski and Serchuk 1984) and studies of the biology of the species (see Ropes 1980). The bar clam fishery is smaller in the Gulf of St. Lawrence but has increased in the last few years (NAFO 1985). In the Gulf region, clams are fished intertidally by clam diggers and subtidally at depths of 3 to 10 m by commercial hydraulic harvesters. Biological information is scarce for S. solidissima within the northern range of its distribution. There are only a few studies on the biology of bar clams (Kerswill 1944. Caddy and Billard 1976. Robert 1981) and no studies on the reproductive cycle. A preliminary indication of the timing of bivalve spatfall is known from a study of bivalve larvae from Malpeque Bay. P.E.I, by Sullivan (1948). She identified larvae of S. solidissima in plankton samples from mid-June to mid-July and at the end of August. The repro- ductive cycle, and thus the timing of recruitment processes, is well known for bar clams found in the main fishing grounds off the coast of New Jersey. Ropes (1968) con- ducted a 3'/2 year study and found biannual and annual cycles for clams collected at depths of 18 to 32 m. Jones (1981) observed only annual cycles over a period of two years for clams collected at depths of 6-10 m. The objec- tive of the present study was to examine the reproductive cycle of bar clams from the Northumberland Strait and Gulf of St. Lawrence shores of Prince Edward Island to provide further life history information. MATERIALS AND METHODS Sampling sites were located in Hillsborough Bay on the Northumberland Strait shore of P.E.I, in 1984 and 1985; on the gulf of St. Lawrence shore, in Cascumpec Bay in 1984 and New London Bay in 1985 (Figure 1). Locations were unprotected beaches with clean sand substrate (<1.5% silt content), characteristic of typical bar clam habitat (Ropes 1980). At approximately two week intervals, twenty five clams (shell length range 95-150 mm) were collected from water depths of 1-2 m during mean low water at each site. The sampling period was during the ice-free season from late April to early December. Clams were hand picked while snorkling or collected with a hydraulic clam dredge towed from a Boston Whaler. Water temperature, salinity and dissolved oxygen were measured at the surface and near bottom at each site using YSI S-C-T and YSI Dis- solved Oxygen meters at the time of sampling. Clams were transported immediately to the laboratory in dry containers. This avoided premature aborting of ga- metes. Within three hours of collection, the entire visceral mass was excised, placed in seawater Bouin's fixative for 72 hours, and then stored in 109c ethanol. The gonad tissue of each individual was processed for microscopic examina- tion as described by Jones (1981) and summarized here. A portion of the gonad, along with underlying digestive gland, was removed from the visceral mass ventral to the heart, dehydrated and infiltrated with paraffin using stan- dard histological techniques (Humason 1979). Tissue sec- tions (7-9 (xm thick) were stained with Harris hematoxylin and counterstained with eosin Y (Humason 1979). During examinations of gonadal preparations, under a compound microscope at 100 x, each was sexed and assigned to a developmental stage as described by Ropes (1968): early active, late active, ripe, partially spawned and spent. Ropes 97 98 Sephton — i— 63 -T- 62 GULF OF ST. LAWRENCE NEW BRUNSWICK 0 15 30 45 km 1 I I I NOVA SCOTIA Figure 1. Location of study sites in Prince Edward Island, Canada. (1968) and Jones (1981) stated that these categories are convenient and not definitive because gonadal development is a continuous process and distinctions between stages are not clear. The gametogenic process and histological char- acteristics of both sexes were reviewed by Ropes (1968). and are summarized in Table 1 . RESULTS Gametogenesis for the Northumberland Strait study site is shown in Figures 2 and 3 for 1984 and 1985. respec- tively. Clams were in the late active phase when sampling commenced in mid-May, 1984, followed by the ripe stage from early June to late July. Partially spawned individuals dominated from early July through to mid-September. Fe- males appeared to have ripened before males but partially spawned males were detected before females (Figure 2). Spent individuals of both sexes were present from mid- September and spawning was completed by mid-October. All clams were in the early active stage when sampling ceased in mid-November. The pattern in 1985 was essentially the same as was de- termined in 1984 with minor differences in timing and du- ration. Males and females developed through all stages in similar time frames but spent individuals were encountered 2 to 3 weeks earlier than in 1984 (Figure 3). Gametogenesis for the Gulf of St. Lawrence study site is shown in Figures 4 and 5 for 1984 and 1985, respectively. Clams were in the late active stage when sampling com- menced in late May. 1984. The ripe phase predominated TABLE 1. Summary of the gonad condition and stage index used for female and male Spisula solidissima as described by Ropes (1968). Develop- mental Stage Female Male Early Active -Ovocytes irregularly -Spermatogonia shaped & attached to developing in follicle follicle wall by stalk. wall. -Lumina of gonad empty. - 1° spermatocytes in single cell columns close to wall. Late Active -Ovocytes rounded and -2° spermatocytes extend into follicle. numerous & cytoplasm -Many free with some irregularly shaped. still attached by stalks. -Spermatids small & in dense masses near centre of follicle. Ripe -Ovocytes free & fill -Mature sperm appear as lumina of follicle. dense swirling or -Vitelline membrane homogenous mass in contains cytoplasm & follicle. large nucleus. Partially -Some follicles void & -Mature sperm appear as Spawned walls appear flacid. less densely packed —Few large ovocytes free masses. in lumina of follicle. Spent -Follicles void of ripe -Follicles void of mature ovocytes & developing sperm & 1° ovocytes appear in spermatogonia appear in follicle walls. follicle walls. Reproductive Cycle of S. solidissima 99 1984 SPI Figure 2. Stages of gonad development of Spisula solidissima from the Northumberland Strait study site in 1984. Percent frequency of each stage is shown for combined sexes (total), females and males. The water temperature at the time of sampling is shown in the upper figure. M J A S O N D EARLY ACTIVE ^A .ATE ACTIVE RIPE PARTIALLY SPAWNED SPENT Figure i. Stages of gonad development of Spisula solidissima from the Northumberland Strait study site in 1985. Percent frequency of each stage is shown for combined sexes (total), females and males. The water temperature at the time of sampling is shown in the upper Figure. from late June to late July when some minor spawning ac- tivity was encountered. Partially spawned individuals dom- inated from early August to mid-September. The spent stage was encountered in early September and spawning had ceased by mid-October. Males spawned before females (partially spawned stage) but females were spent before males (Figure 4). Once again, the pattern in 1985 was similar to that ob- served in 1984 except that the cycle commenced 2-3 weeks earlier and the partially spawned stage was pro- longed by about 5 weeks. Both sexes exhibited a similar schedule of development in 1985 (Figure 5). Gametogenesis for both locations was similar in both years and appeared to be the result of exposure to similar thermal regimes at the respective inshore locations (Figures 2-5). Water temperatures at the Northumberland Strait study location (Figures 2 & 3) increased sooner than the Gulf location (Figures 3 & 4) and increased to seasonal maximums by mid-July (21-23°C) in both years. An early spring in 1984 resulted in warmer water temperatures 100 Sephton 1984 JASON EARLY ACTIVE LATE ACTIVE RIPE PARTIALLY SPAWNED SPENT Figure 4. Stages of gonad development of Spisula solidissima from the Gulf of St. Lawrence study site in 1984. Percent frequency of each stage is shown for combined sexes (total), females and males. The water temperature at the time of sampling is shown in the upper figure. (3-4°C) by late May compared with 1985. This was re- flected in the faster development from the early active to late active stage as observed in 1984. Water temperatures at the Gulf locations peaked in mid-August (23°C) and early July (19°C) in 1984 and 1985, respectively. The ripe stage coincided with about 15°C water temperatures at both loca- i while the partially spawned stage occurred after water temperatures had exceeded this temperature during July and August. The partially spawned stage was prolonged in 985 at the Gulf locations, probably resulting from stable July to September water temperatures. The cessation of spawning activity, as indicated by the spent stage, occurred SPENT PARTIALLY SPAWNED Figure 5. Stages of gonad development of Spisula solidissima from the Gulf of St. Lawrence study site in 1985. Percent frequency of each stage is shown for combined sexes (total), females and males. The water temperature at the time of sampling is shown in the upper figure. when temperatures decreased during September and Oc- tober at both locations. Fluctuations in salinity (Northum- berland Strait range: 20.1-25.5%P; Gulf of St. Lawrence range: 22.4-26. l%o) and dissolved oxygen (Northumber- land Strait: >100% saturation; Gulf of St. Lawrence: > 100% saturation) over the year probably had little influ- ence on the reproductive cycle. The male to female sex ratio was not significantly dif- ferent (p > 0.05) from 1:1 at either location in 1984 and 1985 (Table 2). No hermaphrodites were found in this study. There was no correlation between sex and colour of the foot. The colour of the gonad, noted while shucking was usually a pinkish-orangy colour in ripe females and creamy-yellow in males. No gonadal parasites were ob- served during the study. Reproductive Cycle of S. solidissima 101 TABLE 2. Results of \2 test to determine if male to female sex ratios were significantly different (p > 0.05) from 1:1 using Yates correction for continuity. Northumberland Strait Gulf of St. Lawrence 1984 1985 1984 1985 No. females 114 165 151 228 No. males 131 163 149 272 Expected 125 164 150 250 Xi 0.484 0.003 0.003 3.698 Significance NS NS NS NS DISCUSSION Spisula solidissima typically developed quickly to the ripe stage by June and July in both years, concurrently with the warming of water temperature to 15°C. A prolonged spawning period followed during the warm water period from late July to October. Spawning terminated abruptly when the water temperature decreased in October. The de- velopmental period through to the ripe stage is similar to those reported by Ropes (1968) and Jones (1981), but dura- tion of spawning observed in this study was shorter than reported for 5. solidissima from the northeastern coast of the United States where it extended to November and De- cember due to warmer water temperatures (Ropes 1968. Jones 1981). The differences observed in timing and duration of the stages of gametogenesis between years and study locations were primarily in response to different rates of warming and cooling of water (Ropes 1968. Jones 1981). Water temperature has an important influence on the reproductive cycle of many marine invertebrates (Giese 1959. Sastry 1979) and other bivalve molluscs such as Arctica islandica (Loosanoff 1953, Jones 1981. Mann 1982), Mercenaria mercenaria (Loosanoff 1937, Eversole et al. 1980. Manzi et al. 1985) and Mya arenaria (Ropes and Stickney 1965, Brousseau 1978). The rate of temperature change may evoke a more important spawning stimulus than physiolog- ical hormones and ambient temperature (Ropes 1968. Keck et al. 1975, Manzi et al. 1985) although they are closely interrelated. Salinity and dissolved oxygen appear to have little influence on the reproductive cycle of Spisida solidis- sima in the Gulf of St. Lawrence. The reproductive cycle of marine invertebrates has been observed to change with latitude (Giese 1959, Sastry 1979). Bivalve molluscs such as Mya arenaria, Mercen- aria mercenaria and to some extent Spisida solidissima, exhibit biannual cycles in the southern latitudes of their distribution and annual cycles in the northern regions (Shaw 1962. Porter 1964, Ropes & Stickney 1965, Brous- seau 1978. Manzi et al. 1985). The present study supports this hypothesis by documenting a single annual reproduc- tive cycle of Spisida solidissima in the northern most ex- tension of its range. Jones (1981) also suggested that the thermocline and the timing of turnover affects the repro- ductive cycle of S. solidissima such that stocks located above the thermocline exhibit annual cycles (Jones 1981, present study) and those below exhibit biannual cycles (Ropes 1968). Changes in water depth and circulation pat- terns affect the cycle of reproduction of Mercenaria mer- cenaria (Carriker 1961). Further experimental research is required to elucidate these effects for Spisula solidissima. The results of this study provide further evidence that Spisida solidissima has a male:female sex ratio of 1:1 and that the occurrence of hermaphrodites is a rare event (Ropes 1968. 1982, Jones 1981). Colour of the ripe gonad cannot consistently predict the sex of 5. solidissima (Ropes 1968) but the results of this study support the findings of Schechter (1941) that female gonads are generally pinkish while males' are cream coloured. Further research is re- quired to determine growth rates, fecundity, relative abun- dance of residual oocytes, and the age of sexual maturation of S. solidissima in the Gulf of St. Lawrence. ACKNOWLEDGMENTS I gratefully acknowledge the technical and field assis- tance provided by the following: Clair Bryan, Richard Gal- lant, Nancy Stavert, Cheryl Coulson, Jody Gamauf, Chris Maddocks, Paul Baker. Rickie MacKenzie and Melita MacDougall. I thank Drs. E. L. Drake, J. W. Ropes and J. M. Worms for critically reviewing early drafts of the manuscript. The Biology Department, University of Prince Edward Island, loaned us the histology equipment for this project, for which I am grateful. REFERENCES CITED Brousseau. D. J. 1978. Spawning cycle, fecundity and recruitment m a population of soft-shell clam. Mya arenaria. from Cape Ann, Massa- chusetts. Fish. Bull. 76:155-166. Caddy, J. F. & A. R. Billard. 1976. A first estimate of production from an unexploited population of the bar clam Spisula solidissima. Fish. Mar. Ser. Tech. Report 648. 13pp. Camker. M. R. 1961. Interrelation of functional morphology, behavior and auteeology in earyl stages of the bivalve Mercenaria mercenaria. J. Elisha Mitchell Sci. Soc. 77:168-241. Eversole. A. G , W. K. Michener & P. J. Eldridge. 1980. Reproductive cycle of Mercenaria mercenaria in a South Carolina U.S.A. estuary. Proc. Natl. Shellfish. Assoc. 70:22-30. Giese. A. C. 1959. Comparative physiology:annual reproductive cycles of marine invertebrates. Ann. Rev. Physiol. 21:547-576. Humason, G. L. 1979. Animal tissue techniques, fourth edition. W. H. Freeman. San Francisco. 661pp. Jones, D. S. 1981. Reproductive cycles of the Atlantic surf clam Spisula solidissima. and the ocean quahaug Artica islandica off New Jersey. J. Shellfish Res. 1:23-32. 102 Sephton Keck, R. T., D. Maurer & H. Lind. 1975. A comparative study of the hard clam gonad developmental cycle. Biol. Bull. 148:243-258. Kerswill. C. J. 1944. The growth rate of bar clams. Fish. Res. Bd. Canada Prog. Rep.. All. Coast Sta. 35:18-20. Loosanoff. V. L. 1937. Spawning of Venus mercenaria (L.). Ecology 18:506-515. Loosanoff, V. L. 1953. Reproductive cycle in Cyprina islandica. Biol. Bull 104:146-155. Mann, R. 1982. The seasonal cycle of gonadal development in Arctica islandica from the southern New England shelf. Fish. Bull. 80:315- 326. Manzi. J. J., M. Y. Bobo & V. G. Burrell Jr. 1985. Gametogenesis in a population of the hard clam. Mercenaria mercenaria (Linnaeus), in North Santee Bay. South Carolina. Veliger 28:186- 194. Murawski, S. A. & F. M. Serchuk. 1984. Assessment update for middle Atlantic offshore surf clam, Spisula solidissima. populations — autumn 1984. NMFS. NEFC. Woods Hole Lab. Ref. Doc. 84-32. 18pp. Northwest Atlantic Fisheries Organization. 1985. Tabular summaries of nominal catches. 1983. NAFO Stai. Bull. 33:56-58. Porter, H. J. 1964. Seasonal gonadal changes of adult clams. Mercenaria mercenaria (L.) in North Carolina. Proc. Null. Shellfish. Assoc. 55:35-52. Robert, G. 1981. Dynamics of an unexploited population of bar clam, Spisula solidissima. Can. Manuscr. Report Fish. Aquat. Sci. 1607. 12pp. Ropes, J. W. 1968. Reproductive cycle of the surf clam. Spisula solidis- sima. in offshore New Jersey. Biol. Bull. 135:349-365. Ropes, J. W. 1980. Biological and fisheries data on the Atlantic surf clam, Spisula solidissima (Dillwyn). Northeast fisheries Center. U.S. Nat. Mar. Fish. Sen-. Tech. Rep. Ser. 24:88pp. Ropes, J. W. 1982. Hermaphroditism, sexuality and sex ratio in the surf clam, Spisula solidissima. and the soft-shell clam. Mya arenaria. Nautilus 96:141-146. Ropes, J. W. & A. P. Stickney. 1965. Reproductive cycle of Mya aren- aria in New England. Biol. Bull. 128:315-327. Sastry, A. N. 1979. Pelecypoda (excluding Ostreidae). p. 113-292. In: Reproduction of marine invertebrates. Volume 5: Pelecypods and lesser classes. A. C. Giese & J. S. Pearse, ed. Academic Press, New York. Schechter, V. 1941. Experimental studies upon the egg cells of the clam, Mactra solidissima. with special reference to longevity. J. Exp. Zool. 86:461-477. Serchuk. F. M., S. A. Murawski, E. M. Henderson & B. E. Brown. 1979. The population dynamics basis for management of offshore surf clam populations in the Middle Atlantic, p. 83-101. In: Proceedings of Northeast clam industries: management for the future. U. Mass- MIT Sea Grant SP- 11 2: 157pp. Shaw, W. N. 1962. Seasonal gonadal changes in female soft-shell clams. Mya arenaria. in the Tred Avon River, Maryland. Proc. Natl. Shell- fish. Assoc. 53:121-132. Sullivan. C. M. 1948. Bivalve larvae of Malpeque Bay. P.E.I. Fish. Res. Bd. Canada Bull. 77:36pp. Journal of Shellfish Research. Vol. 6. No. 2, 103-108, 1987. THE EFFECTS OF CHELOTOMY ON MOLTING IN THE BLUE CRAB, CALLINECTES SAPIDUS ROY D. ARY JR., CLELMER K. BARTELL, & M. A. POIRRIER Department of Biological Sciences University of New Orleans New Orleans, Louisiana 70148 ABSTRACT Cheliped removal was studied as a possible means of inducing and synchronizing ecdysis in juvenile (40- 100 mm) blue crabs, Callinectes sapidus. Experiments were conducted in closed, recirculating seawater systems. In experiment I, chelotomy was induced in crabs for which the time period from the last ecdysis was unknown and assumed to be random. In this experiment, chelotomy did not significantly shorten the mean time to ecdysis, but it did reduce the variance of the individual times to ecdysis as compared to controls of a similar size. This synchronization effect was attributed to chelotomy and limb regeneration delaying molt in crabs which were approaching proecdysis and accelerating molt in crabs with some physiological preparation for ecdysis. In experi- ment II. chelotomy was induced in crabs five days after ecdysis. Results showed no significant difference in the molt interval between these crabs and the intact controls. The lack of molt acceleration was attributed to chelotomy not affecting very early intermolt crabs because they have minimal physiological preparation for ecdysis. Chelotomized crabs increased in carapace width an average of 16.9% in experiment I and 18.0% in experiment II, compared to a 21.6% and 20.5% increase for intact controls. The regenerated cheliped length in chelotomized crabs increased an average of 5.1% and 5.4% in the respective experiments, compared to a 23.1% and 21.5% increase in controls. KEY WORDS: Blue crab. Callinectes sapidus, chelotomy. autotomy, synchronization, molt cycle, soft shelled INTRODUCTION The status of the soft-shelled blue crab (Callinectes sa- pidus Rathbun) fishery has been discussed by Otwell and Cato (1982), Perry et al. (1982) and Oesterling (1984). One factor limiting the production of soft-shelled crabs in many areas is the lack of a reliable source of peeler crabs (Otwell and Cato 1982; Perry et al. 1982). Peeler crabs are premolt crabs that show signs which indicate that they are close to ecdysis. They are currently harvested from nature. Methods of producing peeler crabs from hard crabs should be explored. Holding and feeding intermolt-stage crabs until they molt is possible but at this time is not eco- nomically feasible. Mass culture of intermolt crabs to pre- molt status is not practical because all crabs have to be checked almost daily to remove crabs which have reached the peeler stage. Methods of inducing molt and synchro- nizing the molt cycle are needed. Various methods have been used to induce molting in crustaceans, including eyestalk removal (Zeleny 1905; Abramowitz and Abramowitz 1940; Passano 1960; Skinner 1985), treatment with hormones (Lowe et al. 1968; Flint 1972; Rao et al. 1972; Skinner 1985). and limb autotomy (Costlow 1963; Rao 1966; Skinner and Graham 1970, 1972; Fingerman and Fingerman 1974; Kuris and Mager 1975; Holland and Skinner 1976; Weis 1976; Hopkins 1982). Eyestalk removal has been used to induce molt in blue crabs; however, this method usually results in high blue crab mortality, either at the time of surgery or at ec- dysis (Skinner and Graham 1972; Otwell and Cato 1982). Treating blue crabs with molting hormones has been pro- posed (Swingle 1975), but the high cost of the hormones may limit commercial applications (Swingle 1975; Perry et al. 1982). Molting hormone treatment in other crustaceans often results in death shortly after injection or at ecdysis (Krishnakumaran and Schneiderman 1970; Flint 1972; Graf 1972; Hubschman and Armstrong 1972; Rao et al. 1972; Rao et al. 1973; Dall and Barclay 1977). Limb autotomy is known to induce premolt preparation in Decapods. Stoffel and Hubschman ( 1974) reported a de- crease in the intermolt period by as much as 40% with the removal of four limbs from the freshwater shrimp, Palae- monetes kadiakensis . Skinner and Graham (1970) demon- strated that the loss of eight walking legs or two chelipeds of the Bermuda land crab, Gecarcinus lateralis, causes the animals to undergo immediate preparation for molt with at- tendant limb regeneration. Other species of Brachyura in which multiple limb autotomy induces precocious molt in- clude the rock crab, Cancer paguris (Bennett 1973); the shore crabs. Hemigrapsus oregonensis and Pachygrapsus crassipes (Kuris and Mager 1975); and the fiddler crab, Uca pugilator (Weis 1976; Hopkins 1982). The number of limbs autotomized is important in initi- ating molt preparation. Skinner and Graham (1972) showed that the loss of 6 to 8 pereiopods or both chelipeds triggered limb development which they regarded as indicative of pre- cocious molts in the green crab, Carcinus maenas, the fid- dler crabs, Uca pugilator and U . pugnax, and the blue crab, Callinectes sapidus. Fingerman and Fingerman (1974) found that the removal of only two walking legs was sufficient to induce precocious molt in the fiddler crab, Uca pugilator. The loss of only one cheliped did not induce the Bermuda land crab, Gecarcinus lateralis, to initiate molt preparations (Skinner and Graham, 1972). Adiyodi (1972) described post-autotomy limb regenera- tion in the crab, Parathelphusa hydrodromous, during various stages of the molt cycle. Hopkins (1982) compared the rate of limb development in eyestalkless and multiple 103 104 Ary et al. limb autotomized Uca pugilator. Ary et al. (in press) de- scribed post-autotomy cheliped development in the blue crab. Callinectes sapidus, and described developmental stages in regenerating chelipeds which were compared to Regeneration Indices (Bliss 1956) and paddle molt stages. This study was conducted to determine the effects of cheliped autotomy on the molt cycle of juvenile blue crabs. We were interested in determining if chelotomy would reset the molt cycle and if limb bud growth would regulate the time to ecdysis. We also investigated the effects of che- lotomy upon the length of the molt interval. Our experi- ments were conducted to determine whether chelotomy could be used as a means of inducing and synchronizing molt in populations of hard, intermolt crabs held in ponds or other culture systems. Molt induction would reduce the time that it would take for crabs to reach premolt status. Molt synchronization would cause crabs to reach the peeler stage at approximately the same time and thereby eliminate the need for daily inspection of all crabs until they reach premolt. When they reach the premolt stage, the crabs would be sorted and moved to shedding systems. The ef- fects of chelotomy on cheliped size and carapace width were also studied. MATERIALS AND METHODS This study consisted of two experiments. In experiment I, chelotomy was induced in recently collected intermolt crabs that were assumed to have been in intermolt for random periods of time. These crabs were compared to similar crabs (controls) with intact chelipeds to determine whether the times to ecdysis differed. In experiment II. chelotomy was performed early in intermolt. Crabs were allowed to molt in the laboratory, and chelotomy was in- duced three to five days after ecdysis. The molt interval was compared to controls with intact chelipeds to deter- mine whether chelotomy affected the length of the molt in- terval. Crabs were obtained from the Rigolets Pass at U.S. Hwy 90, St. Tammany Parish, La. by using baited lines and dip nets. They were collected during the spring and fall of 1982. Only crabs with intact appendages and no overt signs of disease were retained. In the laboratory crabs were held and experiments con- ducted in closed, recirculating seawater systems. These systems consisted of circular plastic children's wading pools (120 cm in diameter and 20 cm high) which served as holding tanks and 161 plastic garbage cans (50 cm in height and diameter) filled to about 35 cm with medium grade crushed oyster shell which served as a biological filter. A 3.5 cm drain hose placed in the side of each pool hrovided gravity drainage into the filters and maintained the water depth at 6 cm. Pumps (1/40 h.p., Little Giant Model 8N) were used to move water from the bottom of the filter through a 1.5 cm hose to the holding tank. The flow rate into the tank was approximately 70 ml/sec. The total volume of the system was 450L. The tanks were aerated with pumps and air stones, and dissolved oxygen was maintained at saturation values. Salt water was prepared by adding synthetic sea salts (Carolina Biological Supply Co.) to tap water which was treated with sodium thiosulfate to remove chloramines. Sa- linity was maintained between 14.6-21 ppt. Thirty to forty percent of the water volume of the systems was removed and replaced every two weeks. Water temperature was maintained between 21 -24°C by controlling room temperature. Two 400 watt, cool white flourescent lamps (G.E. Corp.) located 1.4 meters above the tanks provided lighting. A 12 hour light/dark photo- period was established with the light period beginning at 6 A.M. Dissolved oxygen, salinity, ammonia, nitrite, nitrate, calcium, and total hardness were measured every week throughout the course of the experiments. Dissolved ox- ygen was measured with a Y.S.I, model 57 D.O. meter. Ammonia, pH and chloride were measured with an Orion 901 Ion Analyzer. Salinity was determined from chloride measurements. Nitrite, nitrate, calcium, and total hardness were analyzed according to the procedures of the American Public Health Association (1980). The diazotization method was used for nitrite, and the cadmium reduction methods was used for nitrate. Calcium and total hardness were measured by the EDTA titrimetric method. Two hundred fifty three crabs ranging in size from 40- 100 mm in carapace width were used in this study. The average number of crabs per tank was 23. Populations among the various tanks were matched according to size, sex. and number. Crabs were fed a sinking catfish ration (Purina Catfish Chow) which had a 30% crude protein content. Three to five pellets (0.9 to 1.6 gm) per crab were placed in each tank each day. Uneaten food and feces were removed with a fine mesh net four hours after each feeding. Experimental crabs were induced to autotomize che- lipeds by applying pressure to the merus with pliers. The chelae of control animals were immobilized by placing sec- tions of elastic tubing over the propodus and dactylus. In experiment II, chelotomy was induced three to five days after ecdysis when the exoskeleton became hard, presum- ably at stage C4. Carapace width and cheliped length were measured with metric rulers and vernier calipers to com- pare size before and after ecdysis. RESULTS Results of experiment I, investigating the effects of che- lotomy on crabs whose time from the last ecdysis was un- known, are shown in a scatter diagram (Figure 1). The number of days to ecdysis for individual crabs was plotted against their carapace widths. This diagram shows that the Chelotomy on Molting in the Blue Crab 105 80- 0 70- o 60- - CO CO > Q O LU 50- ° • °° °8 °° o " ° o 40- 0 • co > < a 30- 0 0 • • •• -»0 • • o 20- -•ap 0 ° „° ~, ° o oo 10- 0 0 oo 50 CARAPACE WDTH(mm) Figure 1. The relationship between carapace width and the number of days until ecdysis for each crab in experiment I. Symbols are: O = control (chelipeds intact), and • = experimental (chelipeds re- moved). time to ecdysis increased with increasing crab size, and il- lustrates differences between crabs with chelipeds intact (open circles) and chelipeds removed (dark circles). Be- cause the length of time to ecdysis varied with crab size, comparisons between experimental and control group were made in 10 mm size classes. (Figure 2). Differences in the mean time to ecdysis between the ex- perimental and the control group were not statistically sig- nificant (p > 0.05), except for the 51-60 mm size class. There was, however, a significant reduction (p < 0.05) in the ranges of the number of days to ecdysis in all size classes of the experimental group. Results of experiment II, which compared the length of the molt interval in crabs with chelipeds removed to con- trols, are presented in Figure 3. There were no significant differences (p > 0.05) between the means, standard devia- tions, and ranges between the experimental and control groups in the 10 mm size classes. With the exception of the 71-80 mm size class, the experimental groups had lower minimum values in the ranges than the control groups indi- cating possible molt acceleration in some of the crabs. Control crabs in Experiment I increased in size after ec- 52 CO > Q O LU CO < Q 40-50 SIZE RANGE (mm) Figure 2. The number of days until ecdysis for control and cheloto- mized groups in experiment I in 10 mm size classes. Horizontal lines are the means, vertical lines the ranges, open rectangles are ± 1 stan- dard deviation from the mean in the control groups, and dark rect- angles are ± I standard deviation from the mean in the experimental groups. The number in brackets indicate the number of individuals in each group. dysis an average of 2 1 .6% for all size classes. Crabs which had the chelipeds removed increased in size an average of 16.9% after ecdysis. The cheliped length for all size classes of control crabs increased an average of 23.1% following ecdysis. The chelotomized crabs had a 5.1% average in- crease in the size of the cheliped after ecdysis compared to the pre-autotomy cheliped length. The mortality of the crabs held in the closed system until the first molt was 7.5%. Of those that died, 4% belonged to the control group and 3.5% belonged to the experimental group. None died while in the process of molting. In Experiment II, the average percent increase in the carapace width following ecdysis for the control group was 20.5%. For the chelotomized group, the average increase in size was 18.0%. The chelipeds in the control group in- creased an average of 21.5% in all size classes. In the ex- perimental group, the cheliped length increased an average of 5.4%. Mortality was 17.4% of which 10.8% were in the control group, and 6.6% were in the experimental group. None died while in the process of molting. Chemical analysis of the water in the holding tanks re- vealed the following means and ranges: pH (mean = 7.4, range 6.9-7.9), salinity in ppt (mean = 17.6, range 106 Ary et al. CO >- 70 < a _l < > kH DC LU Y 18] |14j SIZE RANGE (mm) Figure 3. The molt intervals of control and chelotomized groups in experiment II in 10 mm size classes. Horizontal lines are the means, vertical lines are the ranges, open rectangles are ± I standard devia- tion from the mean in the control groups, and dark rectangles are ± 1 standard deviation from the mean in the chelotomized groups. The numher in brackets are the number of individuals in each group. 14.6-21.0), total ammonia ppm (mean = 0.46, range 0.1-0.8), nitrite ppm (mean = 0.15, range 0.02-0.99), nitrate ppm (mean = 462, range 290-679), calcium ppm (mean = 297, range 227-380), total hardness ppm (mean = 3045, range 1995-4225). DISCUSSION In experiment I in which the time from the last ecdysis was unknown when chelipeds were removed, there was no significant difference in the mean time to ecdysis between control and experimental groups. There was, however, a significant reduction in the variation in the time to ecdysis. In experiment II in which chelipeds were removed at the beginning of stage C4 (intermolt), there was no significant difference in the mean time to ecdysis or in the variation in the mean time to ecdysis between control and experimental groups. Skinner and Graham (1972) reported that the loss of both chelipeds triggered immediate limb regeneration in C. sapidus which they interpreted as an induction of pre- molt. In another study (Ary et al. in press), we demon- strated that much of the cheliped development in Callin- ectes sapidus occurred during intermolt. Adiyodi (1972) and Hopkins (1982) also reported basal limb growth during intermolt in other crabs. The limb bud growth after chelo- tomy in Callinectes sapidus reported by Skinner and Graham (1972) is not necessarily an indication of the in- duction of precocious molt. The reduction in the variation in the time to ecdysis in the first experiment indicated that cheliped removal can reset molting in Callinectes sapidus. The reduction of the variation in the days to ecdysis over controls was probably caused by cheliped removal delaying molt in crabs which were approaching premolt, and accelerating molt in stage C4 crabs which had some preparation for ecdysis but would have molted later without cheliped removal. The time to ecdysis in many crabs was apparently not affected. The overall effect of this delaying or accelerating of molt in crabs that have been in intermolt for random periods of time is to synchronize molt. Skinner and Graham (1972) obtained similar results with a land crab. They found that multiple limb autotomy synchronized molt in a randomly selected population of intermolt Gecarcinus (Skinner 1985). Variation in the molt interval did not differ from controls in experiment II because all crabs were at the be- ginning of stage C4 at the start of the experiment. The de- laying effect of cheliped regeneration on late intermolt crabs indicated by the lower end of the range being shorter in experimental over control groups present in experiment I (Figure 2) did not occur in experiment II (Figure 3). The acceleration effect indicated by a decrease in the upper end of the range of the number of days to ecdysis in experi- mental over control groups in experiment I was not present at significant levels in experiment II. However, mean values and the lower minimal values in ranges of variation were lower in the experimental groups than the controls with the exception of the 71-80 mm size class indicating that acceleration of molt in some crabs may have occurred (Figure 3). The presence of a greater acceleration effect in experi- ment I than in experiment II appears to be because the ef- fects of chelotomy on the molt cycle depend on the length of time a crab has been in intermolt. Intermolt is a period during which tissue growth and accumulation of metabolic reserves occurs (Passano, 1960). Chelotomy in early inter- molt may not have the same effect as chelotomy in late intermolt. This variable response would be caused by the degree of physiological preparation for molt. Charmantier- Daures (1976) reported that eyestalk removal in the crab Pachygrapsus induced proecdysial regeneration in only 50% of the crabs. Hopkins (1982) reported that 25% of destalked Uca did not respond to eyestalk removal, and suggested that the unresponsive crabs were physiologically inadequate to initiate the processes that lead to ecdysis. Dall and Barclay (1977) found a variable response in the treatment of intermolt Panulirus longipes to crustecdysone. Injections starting early in stage C4 produced no appre- ciable shortening of the molt cycle. Treatment after 35% of the molt cycle had elapsed shortened proecdysis as much as 42%. They attributed these results to lack of physiological Chelotomy on Molting in the Blue Crab 107 preparation in animals in early C4. In our study, the lack of acceleration in experiment II was probably due to all crabs being in very early intermolt and having minimal prepara- tion for ecdysis. Although crabs in experiment I were cap- tured from nature and used randomly, apparently no crabs in very early stages of intermolt used in experiment II were included in experiment I. This is shown by the molt interval for controls in experiment II always being longer for the some size classes than the time to ecdysis for controls in experiment I. If very early intermolt crabs were present in experiment I, these values should have been equal. The effects of the closed, recirculating water system must be considered in evaluating these data. In experiments on dietary effects on growth, Winget et al. (1976) obtained molt intervals in a closed system similar to those of this experiment. However molt intervals in our study and Winget et al. (1976) were longer than the molt intervals observed by Tagatz (1968) in crabs held in floats in the St. John River, Florida. Tagatz (1968) observed molt intervals less than a half of those found in this study and that of Winget et al. (1976). This indicates growth in closed systems is slower. This may be due to the unnatural habitat, higher concentration of nitrogenous compounds, inade- quate nutrition, more disease, or other unknown factors. Molt synchronization may be greater under more natural conditions and in larger crabs with a longer intermolt pe- riod. Cheliped removal only slightly reduces the percent in- crease in carapace width after molting. In the first experi- ment, there was a 4.7% difference in carapace width be- tween experimental and control groups, while in the second experiment the difference was only 2.5%. Cheliped regen- eration does not seem to place a heavy toll on general body growth. Hopkins (1982) suggests that since the new exo- skeleton of a post-ecdysial crab is initially expanded with water, this volume of water taken up during proecdysis is the same regardless of whether the crab is regenerating chelipeds or not. However, increasing the number of re- generating limbs reduces the size increase at molt addi- tively (Kuris and Mager 1975). Autotomy of the chelipeds produces a smaller cheliped than normal following ecdysis. There was a difference of 18.0% in experiment I and 16. 1% in the second experiment between experimental (chelipeds removed) and control (chelipeds intact) crabs. Other decapod crustaceans also are unable to immediately regenerate the appendages to normal sizes. Bennett (1973) reported a claw regenerate in Cancer paguris of only one half the size of a similar unregenerated claw. Skinner and Graham (1972) found that the size of the regenerated limbs in Gecarcious lateralis is reduced by a third of the normal size. Savage and Sullivan (1978) ob- served greater recovery (73.5% of pre-autotomy size) in minor chelae than in the regenerates of the major chelae (68.6%) in the stone crab, Menippe mercenaria. This study indicates that cheliped autotomy does syn- chronize molt in populations of blue crabs randomly ob- tained from nature by means of baited lines. Cheliped re- moval was not found to have a significant effect on the length of intermolt in our experiments involving chelotomy on very early intermolt crabs. In our experiment on chelo- tomy during random periods of intermolt. molt acceleration did occur in many crabs. This acceleration probably oc- curred in crabs which have some physiological preparation for ecdysis. Cheliped removal did result in slightly smaller crabs with slightly smaller chelipeds than controls. Cheliped removal could be used in large scale aquacul- ture of hard intermolt crabs to soft-shelled crabs. One ad- vantage would be a reduction in the variation in time to molt over untreated crabs. Other advantages would be that crabs without chelipeds would be easier to handle, would be less apt to become tangled in nets, limb bud develop- ment could be used to stage molt from intermolt to ecdysis (Ary et al. in press), and would result in reduced cannabi- lism among crabs. The disadvantages would be that induction of chelotomy is labor intensive unless easier methods of inducing au- totomy are devised, limb buds are sensitive to damage and drying, and soft-shelled crabs produced by this method are slightly smaller chelipeds than crabs of similar size with intact chelipeds. REFERENCES CITED Abramowitz, R. K. & A. A. Abramowitz. 1940. Moulting, growth and survival after eyestalk removal in ilea pugilator. Biol. Bull. 78:179- 188. Adiyodi, R. G. 1972. Wound healing and regeneration in the crab, Par- athelphusa hydrodromous . Int. Rev. Cytol. 32:257-289. 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EFFECT OF LARVAL DIET AND REARING TEMPERATURE ON METAMORPHOSIS AND JUVENILE SURVIVAL OF THE EDIBLE SEA URCHIN LOXECHINUS ALBUS (MOLINA, 1782) (ECHINOIDEA, ECHINIDAE) LAURA P. GONZALEZ, JUAN C. CASTILLA & CHITA GUISADO Estacion Costera de Investigaciones Marinas, Las Cruces Facultad de Ciencias Biologicas Porttificia Universidad Catolica de Chile Casilla 114-D. Santiago, Chile ABSTRACT The sea urchin Loxechinus albus (Molina. 1782) was reared to the 8-amied larval stage with a fully developed urchin rudiment. The plutei were induced to undergo metamorphosis on a solid substrate covered with a bacterial film. To ascertain the effect of diet and temperature on larval growth, larval survival, succes at metamorphosis and juvenile survival, two temperatures. 10°C-12°C and 16°C-19°C. and 2 diets were tested. Diets consisted of Dunaliella tertiolecta and Isochnsis aff. galbana. Larvae that were fed on a mixed diet of D. tertiolecta and /. aff. galbana underwent metamorphosis after about 33 days at 10°-12°C and after about 20 days at 16°-19°C. In either case, approx. 85% of the initial inoculated larvae underwent metamorphosis and reached the stage of healthy juvenile sea urchin. The best culture system for newly settled juveniles was a 2 1 plastic container with constant sea-water flow. The translucid container walls allowed the active growth of diatoms specially Navicula sp., Nitzchia sp. and Cocconeis sp. Water circula- tion from bottom to the top prevented the deposit of sediment on the urchins. The experimental conditions of this culture system resulted in about 90% survival of juvenile sea urchins. After 76 days they ranged in size between 500-2700 |xm. KEY WORDS: sea urchin; Loxechinus albus; larvae; diet; temperature; metamorphosis; juveniles; rearing. INTRODUCTION Loxechinus albus (Molina, 1782) is an edible sea urchin whose geographical distribution is restricted to the area be- tween Isla Lobos de Afuera in Peru (6°53'50"S) (Clark 1948) and the Estrecho de Magallanes, reaching the Isla de los Estados on the southern extremity (Bernasconi 1953). In Chile, the species has been harvested ever since the Pre- columbian Era when it was collected by coastal dwellers (Deppe and Viviani 1977). Exploitation of the species, which was restricted to local consumption up to the nine- teen-sixties, has now been increased owing to exports of this echinoderm especially to Japan and to other countries like France. United States and Italy. The total exportation value of L. albus between 1978 and 1981 ranged from about 2 to 3.8 million dollars. Since 1979, the annual landing of L. albus in Chile comprises over 10% of the total shellfish landings (SERNAP 1979-1983). The annual extraction increased from 1,206 metric tons in 1974 to 15,502 tons in 1981. The progressive decline in the annual landings of the species after 1981 (falling to 1 1 .826 metric tons in 1983). could reflect a fishing pressure that surpasses the maximum sustainable yield and an irreversible damage to this resource. To lessen the risk associated with this pos- sible overfishing, SERNAP (Servicio Nacional de Pesca) established a closed fishing season in 1979 which is in force for the whole Chilean coast, except the Xllth Region, from November 1st to January 15th. Furthermore, the min- imum collecting size was set at 7 cm of test diameter. In view of the declining stocks of this important re- source, strong measures, including rearing of juveniles for reseeding or complete mariculture systems may become necessary. Therefore, the main purpose of the present work was to rear L. albus larvae through metamorphosis. The basic factors that directly affect larvae survival, and spe- cially urchin rudiment formation, were studied. Namely, the adecuate diets and temperature for larvae development and metamorphosis. Furthermore, preliminary experiments were undertaken for laboratory rearing of early juvenile stages, which would serve as the basis for developing fu- ture rearing systems at a larger scale. The first rearings of sea urchin larvae throughout meta- morphosis were carried out in Great Britian by MacBride (1903) and then in Japan around 1932-1935 (see review of Matsui 1968). Nonetheless, it was only in 1969 that a com- prehensive technique for sea urchin larvae culture and metamorphosis was described by Hinegardner (1969) who studied several species of the North and East coast of North America: Arbacia punctulata (Lamarck). Lylechinus pictus (Verrill), Lytechinus variegatus (Lamarck), Strongylocen- trotus purpuratus (Stimpson). and Echinometra mathaei (Blainville). Cameron and Hinegardner (1974) determined some key factors that induced metamorphosis in sea ur- chins. In Chile, Arrau ( 1958) reared L. albus at small scale (2 1 flask), up to the one-year old juvenile stage, mostly in order to determine the systematic position of the species. The rearing methodology employed by Arrau (1958) though effective, lacks a complete and comprehensive de- scription and is not applicable to larger sea urchin larvae cultures. In order to develop an "echiniculture" in Chile. Buckle et al. (1976) carried out experiments in a larger scale, but failed to achieve metamorphosis in L. albus larvae. 109 no Gonzalez et al. MATERIALS AND METHODS Adult individuals of L. albus were collected in the Cen- tral Zone of Chile (El Quisco, 33°23'S;71°42'W) during September and October 1984. In the Central Zone L. albus spawning occurs from August through October (Buckle et al. 1978) and in the Southern Zone from October through December (Bay-Schmith 1982). To keep sea urchins alive during their transfer from the field to the laboratory, they were placed in styrofoam boxes immediatedly after collec- tion. The bottom of the boxes was lined with sea-water ice bags covered with a thick layer of algae, over which alter- nate layers of sea urchins and algae were placed, specially Lessonia sp. and Macrocystis pyrifera (Turn). In this way, the sea urchins survived at least for 36 hours out of the water. Cultures were carried out under controlled condi- tions in the Estacion Costera de Investigaciones Marinas of the Universidad Catolica de Chile at Las Cruces (33°30'S;71°38'W). Larvae Culture The methods for inducing spawning, as well as the larvae culture system in all experiments, were based on the method of Hinegardner (1969) as modified by Cameron and Hinegardner (1974). At hour 30 after fertilization, and once they had adopted the pyramidal shape the larvae were transferred to 5 1 glass flasks containing sea water filtered through a 0.45 (xm membrane (Sartorius) with a density of ca. 1 larva per 20 ml. To maintain these larvae in suspen- sion, each culture unit was stirred with a paddle attached to a 20 RPM clock motor (Herbach and Rademan Company. Philadelphia, USA). Sea water (33% salinity) was renewed once a week. Excessive amounts of food were provided (3.000 microalgae cells/ml every 2 days in the first stages, and daily once the larvae had reached the 8 arm stage). Temperature and Diet Experiments The effect of temperature on the development of larvae was tested on individuals that had been fertilized on Sep- tember 28, 1984 using two different temperatures ranges: 10°-12°C and 16°-19°C. Larvae were fed on mixed diet of Dunaliella tertiolecta (UTEX LB 999) and Isochrysis aff. galbana (UTEX LB 2307 "T-ISO" strain) in the same proportions. The effect of 2 diets on larvae development was studied in eggs of the same fertilization at 16°-19°C. These diets were as follows: (1) DI diet: a mixture of equal proportions of D. tertiolecta and /. aff. galbana; and (2) D diet: con- sisting of D. tertiolecta alone. For each diet and temperature treatment. 3 replicates were used, that is, 3 flasks with known number of initially healthy larvae. As the larvae developed they were mea- sured under the dissecting microscope. The following pa- rameters were measured in 10 larvae randomly selected from each flask: maximum body length in ventral view (not considering the arms) and diameter of the urchin rudiment measured in the same direction as the body length. In all cultures we determined, with respect to the initial in- oculum, the number of larvae alive, the number of larvae completing metamorphosis and of juvenile sea urchins with external healthy and normal appearance (henceforth: normal sea urchins). Metamorphosis and Early Juveniles Metamorphosis was induced in larvae as soon as the ur- chin rudiment had stopped growing and the 5 primary tube feet were well developed, active and emerging from the vestibule. Larvae metamorphosis and rearing of early juve- niles from each experiment of temperature and diet were carried out under two culture systems: a) Sea urchin from fertilizations carried out September 28, 1984 and reared as indicated above, were offered glass or polystyrene Petri dishes, previously conditioned in running sea water for at least one week to allow the development of a bacterial film (growth of blue-green algae was avoided by keeping illumi- nation low). The larvae attached readily by means of tube feet (Cameron and Hinegardner 1974). No food other than the scarce diatoms naturally collected off the Petri dishes was offered to the larvae or juveniles. Culture of urchins at 10°-12°C was kept under above conditions for 27 days, while those cultured at 16°-19°C were maintained for 41 days. Water filtered through a 0.45 fira Sartorius mem- brane from the flasks where the larvae had been raised, was daily changed in all cultures. After metamorphosis Petri dishes were covered with a plankton net and kept under running sea water. This system showed two problems. First, a low diatom collection and second, a high particle sedimentation on the bottom of the Petri dishes, b) Approx- imately 300 sea-urchin larvae from fertilizations carried out August 21, 1985 and cultured at 16°-19°C with diet DI were metamorphosed on undulated fiber-glass plates (UFGP) (units of 78 cm2). The larvae attached readily to this substratum. They remained for 19 days in this system with daily changes of filtered sea water as indicated in the previous case. They fed on the abundant diatom film. Out of the initial larvae, 149 survived and at day 20 they were transfered to a system specially designed for rearing juve- niles, the running sea water system (RSW System) shown in Figure 1 . It consisted in two translucid concentric plastic flasks in which the water enters through the open bottom, traverses a screen ( 1 mm gauge) and flows out by a supe- rior outlet which maintains the water level at 2 1. The un- dulated fiber glass plates (UFGP) with the urchins attached to them were hung in the inner flask, allowing the sea ur- chins circulation on the gravel and the walls. This system was set out doors, under cover, to attennuate daylight. Water temperature ranged from 9°C to 14°C. When urchins were about 2 months the macroalgae Ulva sp, Gelidium sp, Corallina sp, Schottera sp and Rhodymenia sp. were added to the diet. In all the cultures, the test diameter of surviving sea ur- Larval Diet and Rearing Temperature 111 Water Flow T ra n spa rent- Plastic Screen A Fiber Glass Pla te Gravel -i 5 cm Figure I. Running sea water system (RSW System) chins was measured using a dissecting microscope and the mortality was determined. RESULTS Figure 2 shows the growth of L. albus with diet Dl and under two different temperature regimes, 10°-12°C and 16°-19°C. In larvae reared at 10°-12°C the urchin rudiment appeared after 14 days, reaching a maximum diameter of 440 jj.m, while the larvae had a maximum mean length of 870-900 u,m and metamorphosed at 33.5 days. In larvae reared a 16°-19°C the urchin rudiment appeared after 7 days and showed a maximum diameter of 440 p.m, while the larvae reached a maximim mean length of 600-700 |xm and metamorphosed at 20.5 days. The Nested analysis of vari- ance (Sokal & Rohlf 1981) of larval body length data, just before metamorphosis, disclosed significant differences be- tween the two temperature regimes (p < 0.05; df = 4; F = 7.76). Larvae at 10°-12°C reached a larger size than the larvae at 16°-19°C. However, there was no difference in their urchin rudiment. The decrease in size of the larvae observed before metamorphosis (see Figure 2) was due to the contraction of the oral lobe when they were placed in the Petri dishes for measurements. Figure 3 shows the development of the larvae and their rudiment according to the 2 diets tested at 16°-19°C. Larvae fed with DI and D diets grow at the same rate until the 13.5 days. At 20.5 day-old the larvae showed a small difference in larval body length between treatments. This difference was not statistically significant (Nested analysis of variance p > 0.05; df = 4; F = 10.2). The maximum diameter of the urchin rudiment reached with both diets was 440 p.m. In spite of the above, at 13.5 days the rudiment of larvae fed on D diet presented a significantly smaller diameter than those fed on DI diet (p 0.05; df = 4; F = 0.16, F = 0.20 and F = 0.93 respectively). The one-way analysis of variance for larvae survival showed no significant differ- ences between DI and D diets. There was no significant difference either in the percentage of larvae completing metamorphosis between DI and D diets (p > 0.05; df = 6; F = 1.30). However the same statistical analysis of data from normal juvenile urchins showed that there were dif- ferences between the effects produced by DI and D diets with a level of significance slightly over 5% (df = 4; F = 6.98). All the treatments gave rise to juveniles after larvae had been induced to metamorphose. The mean diameter of the sea urchin 5 days after metamorphosis was 524 ± 24.4 u.m (based on the measurements of 20 individuals from larvae kept on DI diet at 16°-19°C). Figure 4 shows the size distribution of juvenile sea ur- chins 62 days old, from fertilization carried out September 28, 1984 (Figure 4a, 240 individuals cultered with DI diet and 4b. 66 individuals cultured with D diet). They were 112 Gonzalez et al. E 3. z UJ > Q O m < > < E a. OC 111 lu S < 900 800 5 600 a 500 I o cr 400- 300 200- M: Metamorphosis — i — 12 — I- 14 16 —I — 18 20 22 24 — 1 — 28 — J— 30 — I- 32 34 AGE (days) Figure 2. Loxeehinus albas. Larval and urchin rudiment growth at two temperatures with DI microalgae diet. Larval body length (mean and SD), Rudiment diameter (mode and 10th-90th percentiles). — O Larval body length at 16°-19°C; — O Rudiment diameter at 16°-19°C; — • Larval body length at 10°-12°C; — • Rudiment diameter at 10°-12°C. metamorphosed on glass or polystyrene Petri dishes, and two-sample test (Siegel 1956) showed that the population remained on this system for the whole period of time. The of urchins obtained from larvae fed on DI diet was larger mode of both populations was 550 \xm and 500 (i,m, re- than that larvae fed on D diet (p < 0.001; df = 2; Chi- spectively. The application of the Kolmogorov-Smirnov square = 22.8). OC LLI 1 t < x - O ,_ £. 7 ID 111 _l 2 > Q O CO o cc _l z < > X OC u < OC _l 3 900 800- 700- 6 00- 500- 400- 300 200- 100- M : Metamorphosis 1 1 1 1 1 1 1 1 1 1 1 r- 2 4 6 8 10 12 14 16 18 20 22 24 AGE (days) Figure 3. Loxeehinus albas. Larval and urchin rudiment growth with two diets of microalgae at 16°-19°C. Larval body length (mean and SD), Rudiment diameter (mode and 10th-90th percentiles). — O Larval body length with DI diet; — O Rudiment diameter with DI diet;— • Larval body length with D diet; — • Rudiment diameter with D diet. Larval Diet and Rearing Temperature 113 TABLE 1. Loxechinus albus. Surviving larvae, larvae completing metamorphosis and normal juvenile sea urchins in temperature and diet experiments. D: Dunaliella tertiolecta; I: Isochrysis aff. galbana. Diet DI DI I) Temperature (°C) Surviving Larvae (%) X(X + S, X - S) Larvae completing metamorphosis (%)X(X + S, X - S) Normal juvenile urchins (%) X (X + S, X - S) 10-1: 93.8 (91.8. 95.5) 81.5 (91.1, 83.6) 86.3 (91.8. 79.7) 16-19 93.0(91.1,95.6) 89.4 (94.5, 82.8) 88.6(93.6, 82.4) 16-19 84.2 (95.4, 68.1) 64.0 (87.3, 36.4) 50.0 (76.2, 23.8) Figure 5 shows the size range of 133 juvenile sea ur- chins 76 days old, from fertilizations carried out August 21. 1985. They were metamorphosed on UFGP and main- tained during the last 35 days in a RSW system. The sur- vival of juvenile sea urchins in this system was of 89.6%. The size of the above juveniles ranged between 500-2700 (jtm with a mode of 1 100 fxm. Juveniles on the UFGP had access to benthic diatoms once they had opened their mouth. Navicula cryptocephala var. veneta was predomi- nant on the plates, Nitzschia aff. acuminata, Fragilaria construens and F. breviestriata were also present. However in the RSW system N. aff. acuminata predominated first over Navicula cryptocephala var. veneta and Amphora per- pusilla were less abundant. DISCUSSION Arrau (1958) successfully reared larve of L. albus. The author obtained the metamorphosis and reported juvenile sea urchins of up to one year old. Afterwards, other experi- ments were carried out in Chile on this species though without reaching beyond the metamorphosis stage. This was probably due to some of the following factors: (a) the sea water used had a high level of bacteria and other or- ganisms, harmful to the larvae (Cameron and Hinegardner 1974); (b) too frequent changes of sea water and the tech- niques employed may have resulted in excessive manipula- tion of the larvae; and (c) inadequate diets. The success obtained by Arrau (1958) may be due to the fact that she used a diet consisting of phytoplankton with Chaetoceros sp. and Nitzschia sp. as the dominant species. These genera have been effectively used by Japanese re- searchers to feed larvae of the echinoderms Pseudocen- trotus depressus (Agassiz). Strongylocentrotus pulcher- rimus (Agassiz), Anthocidaris crassispina (Agassiz) and Mellita testudinata Klein (Matsui 1958). The experiments of Buckle et al. (1976) with culture units of over 300 1 in volume were less successful. They did not use sea water filtered to 0.45 \xm, the key condition for obtaining a high larvae survival (Cameron, pers. comm.). Further, they em- ployed air bubbling for stirring the larvae, which is known to produce malformations (Hinegardner and Rocha Tuzzi 1981). Buckle et al. (1976) tested diets ranging from mac- erated macroalgae to unimicroalgae, but in many cultures the urchin rudiment failed to appear; when it did appear, larvae did not reach the metamorphosis stage. The temperature of 16°-19°C used in our experiments of L. albus cultures, was based on that used by Cameron and Hinegardner (1974) for Lytechinus pictus and Arbacia punctulata. The results were positive, although the temper- ature of the Chilean Central Zone coastal waters fluctuates between 11°C and 14°C during August and November (B. Uceletti, pers. comm. based on the Annual and Monthly Statistics of the Instituto Hidrografico de la Armada be- tween 1948 and 1977). The temperature range of 16°-19°C accelerated growth and reduced the larval stage from 34 to 21 days without apparently affecting the larvae or the juve- niles. This could be due to the existence of a correlation between assimilation efficiency and temperature (Kirby- Smith and Barber 1974). Thus a higher temperature would allow the larvae to take better advantage of food. Nonethe- less, Arrau (1958) achieved induction of metamorphosis at 23 days with a temperature of only 13°C. This result sug- gest that the diet (phytoplankton) used by this author may have been best suited to L. albus than, for example, the mixed diet Dunaliella tertiolecta-Isochrysis aff. galbana (DI) used in our experiments. The microalgae selected for our experiments were small flagellates easily ingested by the larvae, which had been tested in other sea urchin and mollusc species rearing ex- periments (Hinegardner 1969; Guillard 1975; Cameron and Hinegardner 1974). In cultures where we used D. tertio- lecta alone (D diet) larvae survival was similar to that ob- tained with the mix of D. tertiolecta and / aff. galbana (DI diet). However, some differences were observed in the per- centage of normal juveniles, probably due to the differ- ences in the development of the urchin rudiment. The smaller size of the urchin rudiment in larvae fed on D diet might indicate an alteration of larval development and therefore of juveniles. Hence, D diet does not seem to have the optimal nutritional requirements forL. albus: D. tertio- lecta needs to be supplemented with /. aff. galbana. A newly metamorphosed L. albus individual (before the mouth opens) is an almost immobile organism of about 500 |xm. with an incomplete test, that adheres to the substrate 114 Gonzalez et al. 100 75 50 — 25 > o z LLI Z) o UJ cc LL 50- b 1 1 25 - I 11 i TEST DIAMETER ()im xio2 ) Figure 4. Loxechinus albus. Size distribution of juvenile sea urchins 62 days old from fertilizations carried out September 28, 1984 and cultures at 16°-19°C with two microalgae diets: 4a = DI diet (N = 240), 4b = D diet (N = 66». while developing its calcareous structures and digestive tract. During this stage these urchins are extremely sensi- tive to manipulation. Once the test is formed they become stronger, but the transfer from one substrate to another can always result in some injury. Also, the lack of food once the mouth is open may be lethal. This intial deficiency ob- served in sea urchins from the diet and temperature experi- ments and the subsequent lack of food resulted in a great difference in maximim size at 2 months of age, as com- pared with those that settled on the UFGP (800 p.m and 2700 |xm respectively) and also the former diet at 6 months of age. The running sea water system used for juvenile rearing prevents the deposit of sediment on the urchins, if the flow is abundant it allows a good oxygenation of the water. *" 30 O 3 O 20 UJ * 15 LL 50 5 Hh I I I 18 20 22 24 26 28 TEST DIAMETER (urn » 10^ ) Figure 5. Loxechinus albus. Size distribution of juvenile sea urchins 76 days old from fertilization carried out August 21, 1985 and cul- tured with DI microalgae diet at 16°-19°C (N = 133). In systems where water flows from the top, sediment accumulates on urchins, which are unable to get rid of it with their podia and pedicellaria. This sediment may affect the individual's respiration. The variable illumination of the substrate offered to the juveniles resulted in a different predominance in the popu- lation of benthic diatoms. The low illumination provided to the UFGP allowed the proliferation of Navicula crypto- cephala var. veneta and, to a lesser extent, of Nitzschia aff. acuminata. The translucid walls of the RSW System prob- ably incided in the predominance of Nitzschia over Navi- cula in the first phase of the succession of the benthic algae population. Hinegardner (1981) recommends the genus Nitzchia as the diet for Lythechinus pictus, L. variegatus, Strongylocentrotus purpuratus, S. franciscanus (Agassiz) and Arbacia punctulata. However Ito Yosinaba (pers. comm.) recommend the genus Navicula for the culture of japanesse sea urchin Hemicentrotus pulcherrimus (Agassiz) and Strongylocentrotus nudus (Agassiz). At approximately 3 months from the setting of the RSW System Cocconeis placentula predominates. Some blue- green algae (non identified) appear, which may be harmful to the urchins (Hinegardner 1981). The wide variation in the offer of diatoms suggests that it may be convenient to carry out monocultures of Nitzschia and Navicula to test for their alimentary efficiency. Despite the variation in the diet offered, the maximum size reached by sea urchins of 2700 ixm shows that the diet was adequate. In the natural environment L. albus recruits in intertidal crevices covered by broken shells and macroalgae (Ulva sp, Gelidium sp, Schottera sp, Corallina sp.). The urchins buried in the broken shells down to ca 6 mm first feed solely on benthic diatoms thereafter, when growing in size, they ingest the macroalgae (S. Contreras pers. comm.). When Ulva sp, Gelidium sp, Schottera sp, Corrallina sp and Rhodymenia sp where added to the food offer of juve- Larval Diet and Rearing Temperature 115 niles in the RSW System, the fronds of Ulva sp. and Schot- tera sp presented transparent grazed zones. This indicated that the urchins of ca. 2700 (xm already begin to ingest macroalgae which is the onset of a change in their diet. Summarizing, in the rearing of L. albus, filtered sea- water at a 0.45 |xm must be used. Water must be changed weekly in order to maintain a low bacterial density and to reduce stress produced by manipulation. Stirring is an es- sential factor for keeping the larvae in suspension and thus allowing an adequate filtration of food. A good diet con- sists of a mixture of equal proportions of D. tertiolecta and /. aff. galbana in excess (with a density of aprox. 3,000 cells per ml of culture). The temperature of 16°-19°C accel- erates larval growth. When the urchin rudiment reaches 440 |xm in diameter, considering the ventro-dorsal axis of the larvae, metamorphosis should be induced by providing a substrate covered with a bacterial film and with diatoms for juveniles. At first, recruits measure 524 ± 24 u,m and once the digestive tract is completed and the mouth has opened, they must be fed on pennate diatoms like Navicula sp, Nitz- schia sp. and Cocconeis sp, and be kept in a culture system for juveniles similar to that of RSW System. ACKNOWLEDGMENTS This work was financed by the International Develop- ment Research Center of Canada (1DRC). We wish to ex- press our sincere gratitude to Dr. R. A. Cameron for his generous contribution to the teaching of techniques for raising sea urchin larvae to LPG, and to Humberto Gon- zalez for the identification of diatoms. We are indebted to the Pontificia Universidad Catolica de Chile sede Talca- huano for providing us with adult specimens of sea urchins and to the personnel of the Estacion Costera de Investiga- ciones Marinas at Las Cruces for their valuable help. We are also thankful to Drs. B. Santelices, J. M. Cancino and R. A. Cameron for their critical review of this paper. REFERENCES Anau, L. 1958. Desarrollo del enzo comestible de Chile Loxechinus albus Mol.. Rev. Biol. Mar. 7(11:39-62 Bay-Schmith. E. 1982. Erizo. Loxechinus albus Molina. Echinoidea, Echinidae. Estado actual de las principales perquerias nacionales. Bases para el desarrollo pesquero. IFOP 9:52 pp. Bemasconi, I. 1953. Monografias de los Equinoideos argentinos. An. Mus. Hist. Nat. 2 Ser. VI(2):17- 18. Buckle, F., CH. Guisado, E. Tarifeno, A. Zuleta, L. Cordova, C. Serrano & R. Maldonado. 1976. Estudios biologicos del erizo Loxechinus albus Molina (Echinoidea; Echinodermata). I. Investigaciones preli- minares en cultivos masivos de larvas de erizo. Biologia Pesquera. Chile 8:31-64. Buckle. F.. CH. Guisado, E. Tarifeno, A. Zuleta, L. Cordova & C. Ser- rano. 1978. Biological studies on the Chilean sea-urchin Loxechinus albus (Molina) (Echinodermata; Echinoidea) IV. Maturation cycle and seasonal biochemical changes in the gonad. Ciencias Marinas (Mex.) 5(l):l-8. Cameron, R. A. & R. T. Hinegardner, 1974. Initiation of metamorphosis in laboratory cultured sea urchins. Biol. Bull. 146:335-342. Clark, H. L. 1948. A report on the Echini of the Warmer Eastern Pacific, based on the collections of the "Velero III". Allan Hancock Pac. Exp. Exp. 8(5):265. Deppe, R. & Viviani, C. A., 1977. La pesqueria artesanal del erizo co- mestible Loxechinus albus (Molina) (Echinodermata, Echinoidea, Echinidae) en la region de Iquique. Biol. Pesq. Chile 9:23-41. Guillard. R. L. 1975. Culture of Phytoplankton for Feeding Marine Inver- tebrates. In W. L. Smith and M. H. Chanley (Ed). Culture of Marine Invertebrate Animals. Plenum Publishing Corporation: 29-60. Hinegardner. R T. 1969. Growth and development of the laboratory cul- tured sea urchin. Biol. Bull. 137:465-475. Hinegardner, R & M. M. Rocha Tuzzi. 1981. Laboratory culture of the sea urchin Lytechinus pictus. In R T. Hinegardner (Ed.). Marine In- vertebrates. Laboratory Animal Management. National Academy Press. Kirby-Smith. W. W., R. T. Barber. 1974. Suspension-feeding aquacul- ture systems: Effects of phytoplankton concentration and temperature on growth of the bay scallop. Aquaculture 3:135- 145. MacBnde, E. W. 1903. The development of Echinus esculentus, together with some points on the development of E. miliaris and E. acutus. Phil. Trans. Ray. Soc. London. Series B 195:285-330. Matsui. 1968. The Propagation of Sea Urchins. Fisheries Research Board of Canada. Translation Series No 1063. Semap. 1979. Anuario Estadistico de Pesca. Servicio Nacional de Pesca. Ministerio de Economia, Fomento y Reconstruccion. Chile. Semap. 1980. Anuario Estadistico de Pesca. Servicio Nacional de Pesca. Ministerio de Economia, Fomento y Reconstruccion. Chile. Semap, 1981. Anuario Estadistico de Pesca. Servicio Nacional de Pesca. Ministerio de Economia, Fomento y Reconstruccion, Chile. Semap, 1982. Anuario Estadistico de Pesca. Servicio Nacional de Pesca. Ministerio de Economia, Fomento y Reconstruccion, Chile. Semap. 1983. Anuario Estadistico de Pesca. Servicio Nacional de Pesca, Ministerio de Economia, Fomento y Reconstruccion. Chile. Siegel. S. 1956. N onparametric Statistics for the Behavioral Sciences. Series in Psychology. McGraw-Hill Books Company. New York: 312 pp Sokal, R. R. & F. J. Rohlf, 1981. Biometry. The Principles and Practice of Statistics in Biological Research. Second Edition. W. H. Freeman and Company. San Francisco: 859 pp. Journal of Shellfish Research, Vol. 6, No. 2, 117-124, 1987. A CHECKLIST OF METAZOAN PARASITES FROM NATANTIA (EXCLUDING THE CRUSTACEAN PARASITES OF THE CARIDEA) LEIGH OWENS Graduate School of Tropical Veterinary- Science P.O. James Cook University of North Queensland Australia. 481 1 ABSTRACT A checklist of the metazoan parasites of natant Crustacea has been produced, excluding the crustacean parasites of the Caridea. The checklist is organised under prawns rather than under parasite species. Conclusions from the checklist show that records of the parasitic fauna of prawns from African waters is almost nonexistent. There is little overlap between the parasites of carids and penaeids. Two major families of trematodes (Microphallidae and Opecoelidae); two orders of cestodes (Trypanorhyncha and Lecani- cephalidea); two orders of nematodes (Spirurata and Ascarididea); and a single family of isopods (Bopyridae) commonly infect penaeid prawns. Only species of trypanorhynchs have a cosmopolitan distribution in prawns. Whilst Ascarophis has a pan-Pacific occurrence other nematodes appear to be regionalised. Bopyrids are restricted to the Indo-west Pacific. Whilst the mean number of parasites per host is approximately one. the more intensively studied penaeids have nine parasites per host showing the possible expansion of records in this field. INTRODUCTION Prawns are becoming an increasingly more valuable re- source every year. This includes wild caught prawns for which the sustainable yield is relatively constant and those prawns reared by aquaculture for which production is in- creasing every year (Lawrence 1985). Diseases have been seen to be a major limiting factor in production by both means. Therefore, in an attempt to increase preparedness, a search of the literature for disease agents was undertaken. Although somewhat outdated, a list of the protozoan para- sites of decapod crustaceans exists (Sprague and Couch 1971 ). Therefore, the need for a checklist of metazoan par- asites of the prawns was seen. This checklist attempts to fill that need. CHECKLIST OF METAZOAN PARASITES OF NATANTIA (excluding Crustacean Parasites of Caridea) Key to anatomical sites where parasites are found: (1) hepatopancreas (2) heart, gonads, proventriculus (3) cephalothoracic musculature (4) abdominal musculature (5) gills (6) subcutis (7) gut (8) exocuticle (9) antennal gland (10) nerve cord Host Parasite Site Locality References SECTION PENAEIDEA Amalopenaeus elegans Aristeomorpha foliacea Aristeus virilis Hymenopenaeus halli Hymenopenaeus lucasi Hymenopenaeus rohuslus Hymenopenaeus sibogai Hymenopenaeus triarthrus Hymenopenaeus triarthrus Hymenopenaeus triarthrus Metapenaeopsis Metapenaeopsis Metapenaeopsis Metapenaeopsis Metapenaeopsis Metapenaeopsis Metapenaeopsis andamanensis Metapenaeopsis andamanensis Metapenaeopsis andamanensis Metapenaeopsis stridulans Metapenaeus Metapenaeus afftnis Metapenaeus hennettae Metapenaeus brevirostris Nectonema agile Norway Nielson 1 1969 Orbione halipori New South Wales Dakin 1931, Bourdon 1979a Hadrothoe crosnieri 8 Madagascar Humes 1975 Orbione halipori 5 Madagascar Bourdon 1979a Orbione halipori 5 Louranco Marques Bourdon 1979b Microphallus pygmaeus Florida Hutton 1964 Orbione halipori 5 Madagascar Bourdon 1979a Cabirops orbionei 5 Natal Bourdon 1972 Orbione halipori 5 Louranco Marques Bourdon 1979b Orbione natalensis 5 Natal Bourdon 1972 Minieopenaeon apertum 5 Philippines Bourdon 1981 Minicopenaeon crosnieri 5 Philippines Bourdon 1981 Minieopenaeon intermedium 5 Philippines Bourdon 1981 Orbione thielmanni 5 Madagascar Bourdon 1979a Orbione thielmanni 5 Bay of Bengal Bourdon 1979b Parapenaeon coarctation 5 Philippines Bourdon 1981 Minicopenaeon crosnieri 5 Madagascar Bourdon 1979a Minieopenaeon apertum 5 Madagascar Bourdon 1979a Parapenaeon secundum 5 Madagascar Bourdon 1979a Orbione thielmanni 5 Gulf of Siam Bourdon 1979a Metacercariae 4 Gulf of Carpentaria Owens unpublished Eutetrarhynchus leucomelanum 1 India Chandra etal 1981 Polypocephalus 10 Moreton Bay Butler 1984 Eutetrarhynchus leucomelanum 1 India Chandra etal 1981 117 118 Owens CHECKLIST OF METAZOAN PARASITES OF NATANTIA (excluding Crustacean Parasites of Caridea) — {Continued) Host Parasite Site Locality References Metapenaeus ensis Metapenaeus ensis Metapenaeus ensis Metapenaeus ensis Metapenaeus ensis Metapenaeus lysianassa Metapenaeus macleayi Metapenaeus monoceros Metapenaeus monoceros Metapenaeus monoceros Metapenaeus monoceros Metapenaeus monoceros Parapenaeopsis sculptilis Parapenaeopsis stylifera Parapenaeus consolidata Parapenaeus fissurus Parapenaeus fissurus Parapenaeus fissurus Parapenaeus fissurus Parapenaeus longipes Parapenaeus longipes Parapenaeus stylifera penaeid penaeid penaeids Penaeopsis Penaeopsis akayebi Penaeopsis akayebi Penaeopsis rectacuta Penaeopsis rectacuta Penaeus Penaeus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus aztecus Penaeus braziliensis Penaeus braziliensis Penaeus braziliensis Penaeus braziliensis Penaeus braziliensis Penaeus braziliensis Penaeus braziliensis Penaeus californiensis Orbione halipori Orbione halipori Parapenaeonella lamellata Parapenaeonella lamellata Polypocephalus Parapenaeon richardsonae Polypocephalus Eutetrarhxnchus leucomelanum Orbione bonnieri Parapenaeon japonicum Parapenaeon japonicum Parapenaeonella lamellata Parapenaeon japonicum Epipenaeon quadrii Parapenaeon consolidation Epipenaeon fissurae Epipenaeon fissurae Epipenaeon fissurae Parapenaeon secundum Epipenaeon fissurae Parapenaeon tertium Eutelrurhynchus leucomelanum Anisorbione curxa Parapenaeonella distincta Metacercariae Teirarhynchus rubromaculatus Parapenaeon japonicum Parapenaeon japonicum Parapenaeon brevicoxale Parapenaeon coarctation Orbione penei Parapenaeon japonicum Croconema Hysterothylaciwn Hysterothylacium Hyslerothylacium Hysterothylacium habena Hysterothylacium relicpiens Leptolaimus Obelia bicuspidata Opecoeloides fimbriatus Parorchis Prochrisiianella hispida Prochristianella hispida Prochrisiianella hispida Hysterothylacium Parachristianella heteromegacanthu Parachristianella monomegacantha Polypocephalus Prochristianella hispida Renibulbus penaeus Unidentified nematode Hysterothylaciwn 5 Hong Kong 5 N Australia 5 Andaman Island 5 N Australia 10 Torres Strait Java 10 Moreton Bay I India 5 Singapore 5 Madagascar 5 S Africa 5 India 5 Gulf of Martapan 5 Pakistan 5 Japan 5 Natal 5 Madagascar 5 Philippines 5 Madagascar 5 Bay of Bengal 5 Timor Sea I India 5 Philippines 5 Philippines Philippines Japan 5 Hiroshima 5 Japan 5 Philippines 5 Philippines 5 Hong Kong 5 Japan 7 Mississippi Gulf of Mexico 1 Georgia to G. Mexico Mississippi 1.2,3 Florida. Texas 1 Georgia to G. Mexico 1.6,7 Mississippi 8 Alabama 2,6 Georgia 1 Texas 1 Louisiana 1 Florida Florida 1 Florida I Florida 1 Florida 1 Florida I Florida Mexican Pacific Mexican Pacific Markham 1982 Owens & Glazebrook 1985 Bourdon 1981 Owens & Glazebrook 1985 Owens unpublished Bourdon 1979b Butler 1984 Chandra era/ 1981 Bourdon 1979b Bourdon 1979a Bourdon 1979a, Kensley 1974 Bourdon 1981 Bourdon 1979a Ahmed 1978, Ahmed & Hakeem 1982 Bourdon 1979b Kensley 1974 Bourdon 1979a Bourdon 1981 Bourdon 1979a Bourdon 1979b Bourdon 1979a Chandra el al 1981 Bourdon 1981 Bourdon 1981 Overstreet 1986 Yamaguti 1934 Bourdon 1979a Hinawa 1933. 1934, 1936 Bourdon 1981 Bourdon 1981 Markham 1982 Markham 1982 Overstreet 1973 Hutton ei al 1959b; 1962; Overstreet 1973; Norris & Overstreet 1976; Deardoff & Overstreet 1981 Kruse 1959 Norris & Overstreet 1976 Corkem 1970, Hutton 1964 Deardorff & Overstreet 1981 Overstreet 1973 Overstreet 1973 Hutton ei al 1959a Hutton el al 1959b, Eldred et al 1961 Aldrich 1965 Ragan & Aldrich 1972 Kruse 1959 Feigenbaum 1975, Hutton el al 1962 Feigenbaum 1975. Feigenbaum & Carnuccio 1976 Feigenbaum 1975, Feigenbaum & Carnuccio 1976 Feigenbaum 1975 Feigenbaum 1975 Feigenbaum 1975. Feigenbaum & Carnuccio 1976 Feigenbaum 1975 Lamothe-Argumedo 1970 Checklist of Parasites from Natantia 19 CHECKLIST OF METAZOAN PARASITES OF NATANTIA (excluding Crustacean Parasites of Caridea) — (Continued) Host Parasite Site Locality References Penaeus carcinatus Pendens dalei Penaeus duorarum Penaeus duorarum Penaeus duorarum Penaeus duorarum Penaeus duorarum Penaeus duorarum Penaeus duorarum Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum duorarum esculentus esculentus esculentus esculentus esculentus esculentus indicus indicus indicus indicus indicus japonicus latisulcatus latisulcatus longistylus longistylus marginatus merguiensis merguiensis merguiensis merguiensis merguiensis merguiensis merguiensis merguiensis merguiensis merguiensis merguiensis monodon monodon plebejus plebejus Epipenaeon elegans Parapenaeon consolidatum Hysterothylacium Hysterothylacium Hysterothylacium habena Hysterothylacium reliquens Microphallus pygmaeus Opecoeloides fimbriatus Opecoeloides fimbriatus Opecoeloides fimbriatus Parachristianella heteromegacantha Parachrislianella dimegacantha Parachristianella dimegacantha Parachristianella monomegacantha Polypocephalus Polypocephalus Prochristianella Prochristianella Prochristianella hispida Prochristianella hispida Prochristianella hispida Renibulbus penaeus Unidentified nematode Cabriops orbionei Epipenaeon ingens Epipenaeon ingens Eutetrarhynchus Polypocephalus Prochristianella Epipenaeon ingens Eutetrarhynchus leucomelanum Gymnorhynchus malleus Parapenaeon expansus Trypanorhynch Parapenaeon japomcum Parapenaeon japonicum Parapenaeon japon icum Parapenaeon expansus Parapenaeon japonicum Parapenaeon expansus Ascarophis Bulbocephalus inglissi Contraceacum Epipenaeon ingens Epipenaeon ingens Parachristianella monomegacantha Parapenaeon expansus Polypocephalus Polypocephalus Prochristianella Rhadinorhynchid Caligus epidemicus Unidentified nematode Parapenaeon expansus Polypocephalus 5 Bengal Chopra 1923, Dawson 1958 5 Japan Bourdon 1979b 1 Florida Kruse 1959. Hutton et al 1959b; 1962: Deardorff & Overstreet 1981 2 Florida Martosubroto 1972 1.2.3 Florida Hutton 1964. Villella et al 1970 I Georgia to G. Deardorff & Overstreet 1981 Mexico 3.4 Florida Hutton el al 1962 4 Florida Kruse 1959 2.4,6 Georgia to G. Hutton c/ al 1959a. Sogandres-Bernal Mexico & Hutton 1959 Florida Hutton et al 1959b 1 Florida Feigenbaum & Camuccio 1976 1 Florida Kruse 1959 1 Texas Corkem 1970 1 Florida Kruse 1959, Villella et al 1970 7 Florida Hutton 1964, Villella et al 1970 7 Gulf of Mexico Overstreet 1973 Florida Hutton 1964 2 Florida Martosubroto 1972 1 Florida Hutton 1964, Villella et al 1970. Feigenbaum & Camuccio 1976 1 Louisiana Overstreet 1973 1 Florida Couch 1978 1 Florida Feigenbaum & Camuccio 1976 Florida Hutton et al 1962 5 N E Australia Owens unpublished 5 Darwin Bourdon 1979a 5 N E Australia Owens & Glazebrook 1985 1 Moreton Bay Paynter 1985 10 Gulf of Carpentaria Owens unpublished 1 Moreton Bay Paynter 1985 5 N Australia Owens & Glazebrook 1985 1 India Chandra et al 1981 1 India Chandra & Hanumantha Rao 1982 5 N Australia Owens & Glazebrook 1985 3.4 India Natarajan 1979 5 Hong Kong Markham 1982 5 Sudan Branford 1980 5 N E Australia Owens unpublished 5 N Australia Owens & Glazebrook 1985 5 N E Australia Owens unpublished 5 Madagascar Bourdon 1979a 1 Gulf of Carpentaria Owens 1986 1 Gulf of Carpentaria Owens 1986 1 Gulf of Carpentaria Owens 1986 5 Gulf of Carpentaria Tuma 1967, Nearhos 1980, Owens 1983, Owens & Glazebrook 1985 5 Rosslynn Bay Nearhos 1980 1 Gulf of Carpentaria Owens 1981 5 N Australia Owens & Glazebrook 1985 10 Gulf of Carpentaria Owens 1985 10 Moreton Bay Butler 1984 1 Gulf of Carpentaria Owens 1986 1 Gulf of Carpentaria Owens 1986 Thailand Ruangpan & Kabata 1984 Philippines Gacutan et al 1977 5 Moreton Bay Nearhos 1980 10 Moreton Bay Butler 1984 120 Owens CHECKLIST OF METAZOAN PARASITES OF NATANTIA (excluding Crustacean Parasites of Caridea) — {Continued) Host Parasite Site Locality References Penaeus semisulcatus Penaeus semisulcatus Penaeus semisulcatus Penaeus semisulcatus Penaeus semisulcatus Penaeus semisulcatus Penaeus semisulcatus Penaeus semisulcatus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus semisulcatus semisulcatus semisulcatus semisulcatus semisulcatus semisulcatus setiferus setiferus setiferus setiferus setiferus setiferus Penaeus setiferus Penaeus setiferus Penaeus setiferus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus Penaeus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus setiferus stylirostris stylirostris styliroslris trisulcatus vannamei vannamei Penaeus vannamei Penaeus vannamei Sergestes articus Sicyonia breviroslris Sicyonia dorsalis Sicyonia dorsalis Sicyonia dorsalis Cabirops Cabriops orbionei Epipenaeon elegans Epipenaeon elegans Epipenaeon ingens Epipenaeon ingens Epipenaeon ingens Epipenaeon ingens Epipenaeon ingens Epipenaeon ingens Epipenaeon ingens Eutetrarhynchus leucomelanum Parapenaeon expansus Parapenaeon japonicum Balanus amphitrite Balanus improvisus Balanus Croconema Hysterothylacium Hysterothylacium Hysterothylacium Hysterothylacium fortalezae Hysterothylacium reliquens Leptolaimus Microphallus Microphallus Microphallus Microphallus Microphallus turgidus Myzobdella lugubris Opecoeloides fimbriatus Opecoeloides fimbriatus Polypocephalus Prochristianella hispida Prochristianclla hispida Prochristianella hispida Spirocamallanus pereirai Ascarophis Epipenaeon ingens Hysterothylacium Eutetrarhynchus ruficollis Ascarophis Hysterothylacium Opecoelidae Spirocamallanus pereirai Nectonema munidae Balanus Balanus Hysterothylacium Polypocephalus 5 Sudan Branford 1980 5 5 N E Australia Persian Gulf Owens unpublished Dawson 1958, El-Musca et al 1981, Abu-Hakima 1984 5 5 Ganges Delta Sudan Chopra 1923 Branford 1980 5 5 5 Turkey. Suez. Darwin. Hong Kong Philippines Gulf of Carpentaria Bourdon 1979b Bourdon 1981 Nearhos & Lester 1984, Owens & Glazebrook 1985 5 5 Maryborough India Nearhos & Lester 1984 Thomas 1977 5 Hong Kong India Cheng & Tseng 1980 Chandrae; al 1981 5 Darwin Bourdon 1979b 5 Japan. Hong Kong South Carolina Markham 1982 Dawson 1957 South Carolina Dawson 1957 7 Mississippi Mississippi Dawson 1957, Joyce 1965 Overstreet 1973 1 Florida Kruse 1959 Florida Hutton e/a/ 1959a. 1959b, 1962. Woodbum et al 1957, Deardorff & Overstreet 1981 1.2.3 Mississippi Norris & Overstreet 1976 1 Gulf of Mexico Deardorff & Overstreet 1981 1 Georgia to G. Mexico Deardorff & Overstreet 1981 1.6.7 4 Mississippi N America Overstreet 1973 Hutton et al 1962 3.4 N America Hutton et al 1959a. Hutton et al 195S 4 Louisiana Overstreet 1973 N America Hutton et al 1959a 4.? N America Heard & Overstreet 1983 Mississippi Georgia to G. Overstreet 1973 Hutton et al 1959b Mexico 2.6 7 Georgia Gulf of Mexico Hutton et al 1959a Overstreet 1973 1 Louisiana Overstreet 1973 1 Texas Aldrich 1965 1 7 Florida Mississippi Kruse 1959, Feigenbaum 1975 Overstreet 1973 5 Mexican Pacific Israel Feigenbaum 1975 Overstreet 1986 1 El Salvador Hutton et al 1962 Tunis Heldt 1949 Mexican Pacific Mexican Pacific Feigenbaum 1975 Feigenbaum 1975, Lamothe- 2 7 Mexican Pacific Mexican Pacific Argumedo 1970 Feigenbaum 1975 Feigenbaum 1975 Norway Florida Florida Florida Greva 1972 Joyce 1965 Joyce 1965 Hutton et al 1962 Florida Hutton 1964 Checklist of Parasites from Natantia 121 checklist ok metazoan parasites of natantia (excluding Crustacean Parasites of Caridea) — (Continued) Host Parasite Site Locality References Sicyonia typica Sicyonia typica Solenocera atlantidis Solenocera atlantidis Trachypenaeus Trachypenaeus constrictus Trachypenaeus constrictus Trachypenaeus constrictus Trachypenaeus constrictus Trachypenaeus curvirostris Trachypenaeus similis Trachypenaeus similis Trachypenaeus similis Xiphopenaeus kroyeri Xiphopenaeus kroyeri Xiphopenaeus kroyeri SECTION CARIDEA Crangon Crangon allmani Crangon formosum Desmocaris trispinosa Eualus machilenta Heptacarpus kincaidi Leander squilla Lebbeus polaris Lebbeus polaris Lysmata intermedia Macrobrachium Macrobrachium Macrobrachium Macrobrachium Macrobranchium australiensis Macrobrachium nipponensis Macrobranchium ohione Neocaridina denticula Palaemon elegans Palaemon serratus Palaemon serratus Palaemonetes Palaemonetes kadiakensis Palaemonetes kadiakensis Palaemonetes paludosus Palaemonetes pugio Palaemonetes pugio Palaemonetes pugio Palaemonetes vulgaris Palaemonetes vulgaris Pandalis borealis Pandalis borealis Pandalis borealis Pandalis borealis Pandalis kessleri Pandalus montagui Pasiphaea tarda Pontophilus norvegicus Sclerocrangon boreas Sclerocrangon boreas Hysterothylacium Opecoeloides fimbriatus Hysterothylacium Polypocephalus Opecoeloides fimbriatus Hysterothylacium Microphallus pygmaeus Opecoeloides fimbriatus Polypocephalus Parapenaeonella dislincta Hysterothylacium Polypocephalus Unidentified nematode Hysterothylacium Opecoeloides fimbriatus Polypocephalus Microphallus Podoconle reflexa Helicometrina nimia Nesolecithus africanus Kronborgia caridicola Kronborgia pugettensis Neclonema agile Kronborgia caridicola Nectonema agile Helicometrina noma Angiostrongylus canlonensis Microphallus breviceaca Microphallus turgidis Phyllodistomum Opecoelus variabilis Paragonimus westermani Microphallus turgidis Maritrema caridiniae Nectonema agile Nectonema agile Fecampia erythrocephala Nectonema agile Alloglossidium Alloglossidium renale Microphallus turgidis Microphallus Microphallus turgidis Myzobdella lugubris Microphallus turgidis Nectonema agile Anisakis type I Ascarophis morrhuae Hysterothylacium aduncum Hysterothylacium aduncum Anisakis type I Podocotyle reflexa Kronborgia caridicola Nectonema munidae Crangonobdella murmanica Terranova decipiens Florida Hutton el al 1962 Florida Hutton 1964 Florida Hutton el al 1962 Florida Hutton 1964 Florida Woodburn et al 1957 Florida Hutton el al 1962 Florida Hutton 1964 Florida Kruse 1959 Florida Hutton 1964 5 Japan Bourdon 1979b Florida Hutton et al 1962 Florida Hutton 1964 Florida Hutton et al 1962 Florida Hutton et al 1962 Florida Hutton 1964 Florida Hutton 1964 Florida Hutton 1964 Oresund Koie 1981 Florida Manter 1934 Nigeria Gibson et al 1987 Greenland Kannerworff & Christiansen 1966 San Juan Is Shinn & Christiansen 1985 Nouvel & Nouvel 1938 Greenland Kannerworff & Christiansen 1966 Nouvel & Nouvel 1938 Florida Manter 1934 Tahaiti Alicata & Brown 1962 Philippines Velasquez 1975 4 Gulf of Mexico Johnson 1975 India Pandey 1967 Queensland Cribb 1985 Korea Soh et al 1964 4,5 N America Heard & Overstreet 1983 N America Shibue 1951 Arvy 1963 Ward 1893, Nouvel & Nouvel 1934 1938 4.5 Europe Bella-Humbert 1983 Ward 1893 9 Louisiana Font & Corkum 1975 9 Louisiana Font& Corkum 1975 4.5 N America Heard & Overstreet 1983 Georgia Sprague 1970 4,5 N America Heard & Overstreet 1983 Mississippi Overstreet 1973 4,5 N America Heard & Overstreet 1983 Born 1967 Japan Shiraki et al 1976 Barents Sea Uspenskaia 1953 British Columbia Margolis & Butler 1954 Barents Sea Uspenskaia 1963 Japan Shiraki et al 1976 Kattegat Koie 1981 Greenland Kannerworff & Christiansen 1966 Nielson 1969 Barents Sea Uspenskaia 1963 Barents Sea Uspenskaia 1963 122 Owens CONCLUSIONS A number of facts emerged from the compilation of the checklist. The parasitic fauna of penaeids in the Americas, especially in the Atlantic Ocean is well known, whilst the Indo-west Pacific is poorly known and parasitic fauna of penaeids from Africa is almost unknown. The helminth parasites of penaeids and carids show very little overlap with only the occasional trematode and nematode being shared between the two groups. Two major families of tre- matodes (Microphallidae and Opecoelidae); two orders of cestodes (Trypanorhyncha and Lecanicephalidea); two orders of nematodes (Spirurata and Ascarididea); and the single family of isopods (Bopyridae) commonly infect pen- aeid prawns. Of the parasites of penaeids, only one parasite has a def- inite world wide distribution, especially when the records are coupled with those in the ray definitive host (Owens 1986). That parasite is Parachristianella monomegacantha. Possibly the trypanorhynchs Prochristianella and Eutetra- rhynchus are cosmopolitan as well but the larval stages in prawns are not described well enough for positive identifi- cation and thence conclusions. Even though the genus Pol- ypocephalus is spread worldwide, at least three species are found in prawns in different geographical regions. Of the nematodes, Hysterothylacium and Spirocamal- lanus appear restricted to prawns in the Americas whilst Ascarophis appears pan-Pacific at least. Bulbocephalus inglissi is throughout the Indo-west Pacific (Owens 1986) and the genus has been described from fishes in west Africa where, unfortunately, the status of prawn parasites is poorly known. The bopyrids are conspicuously absent from the Americas and are restricted to the Indo-west Pacific and at least three species, Epipenaeon ingens, Parapenaeon ja- ponicum and Orbione halipori are widespread throughout that region. However, Epipenaeon ingens has been a suc- cessful Lessepian migrant through the Suez canal to infect prawns in Turkey and cultured prawns in Israel. After using only records that contained the identity of the species of both host and parasite, there was 56 penaeids represented which contained 55 parasite species. Likewise in the carids. there was 24 shrimp species which carried 21 parasite species. This gives a mean value of approximately one parasite species per host species. However, in those natant species which have been more thoroughly surveyed (P. aztecus, P. braziliensis , P. duorarum, P. merguiensis and P. semisulcatus), they have approximately nine para- sites per host species which may be a better estimate of the number of parasites that await discovery. The author would appreciate being notified of any defi- ciencies in this checklist being brought to his attention. REFERENCES CITED Abu-Hakima. R 1984. 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V /« j' >l RNAL OF SHELLFISH RESEARCH ■ Vol. 6, No. 2 December 1987 • j . * • ■ - -j • .-■•—■<» CONTENTS Obitu.ry: Victor L. Loosai.o.T (1899-19K , James E. Hanks fmi D. S. Haven, J. M. Zeigler, J. T. De Aliens, and J. P. Whitcomb Comparative Attachment, Growth, and Mortalities of Oyster (Crassostrea virginica) Spat on Slate and Oyster Shell in the . ames River. Virginia 45 A. Robinsor. >.nd H. Horton Environmental Effects on the G » th . Sibling Pa i'ic Oysters Crassotrea gigas (ThunU';:) and Over-Wintered Spat 49 J. P. Whitcomb and D. S. Haven The Physiography and Extent of Public Oyster Grounds in Pocomoke Sound, Virginia 55 H. L. Phelps and E. W. Hetzel Oyster Size, Age, and Copper and Zinc Accumulation 67 D. T. Walsh, C. A. Withstandley, R. A. Krause, and E. J Pelrovitz Mass Culture of Selected Marine Microalgae for the Nursery Production of Bivalve Seeu 71 R. Bisker and M. Castagna Effect of Air-Supersaturated Seawater on Argopecten irrm 'tans concentricus (Say) and Crassostrea virginica (Gmelin) 79 C. M. Hawkins, T. W. Rowell, and P. Woo The Importance of Cleansing in the Calculation of Condition Index in the Soft-Shell Clam, Mya arenaria (L.) 85 S. E. Shumway, R. Selvin, and D. F. Schick Food Resources Related to Habitat in the Scallop Placopecten magellanicus (Gmelin, 1791): A Qualitative Study .... 89 T. W. Sephton The Reproductive Strategy of the Atlantic Surf Clam, Spisula solidissima. in Prince Edward Island, Canada 97 R. D. Ary, Jr., C. A. Bartell and M . A. Poirrier The Effects of Chelotomy on Molting in the Blue Crab, Callinectes sapidus . . .■ 103 L. P. Gonzalez, J- C. Castillo, and C. Guisado Effect of Larval Diet and Rearing Temperature on Metamorphosis and Juvenile Survival of the Edible Sea Urchin Loxei hinus albus. (Molina, 1782) (Echinoidea, Echinidae) 109 L. Owens A Checklist of Metazoan Parasites from Natantia (Excluding the Crustacean Parasites of Caridt;a) 117 COVER PHOTO: Molting blue crab, Callinectes sapidus, emerging from its shell (see page xx). Photo courtesy of Warren Gravois, University of New Orleans. -V!i?m I*'11"1 UBHAB1 WH lAAfl Z