PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION OFFICIAL PUBLICATION OF THE NATIONAL SHELLFISHERIES ASSOCIATION; AN ANNUAL JOURNAL DEVOTED TO SHELLFISHERY BIOLOGY VOLUME 65 Published for the National Shell fisheries Association, Inc. by Waverly Press, Inc., Easton, Maryland JUNE 1975 PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION CONTENTS Volume 65 - June 1975 List of Abstracts by Author of Technical Papers Presented at the 1973 NSA Convention v Abstracts: NSA Convention 1 NSA Pacific Coast Section 7 J. C. Medcof Living Marine Animals in a Ship's Ballast Water 11 H. Hidu, S. Chapman and P. W. Soule Cultchless Setting of European Oysters, Ostrea edulis, Using Polished Marble 13 T. K. Sawyer, M. W. Newman and S. V. Otto A Gregarine-Like Parasite Associated with Pathology in the Digestive Tract of the American Oyster, Crassostrea virginica 15 CM. Langefoss and D. Maurer Energy Partitioning in the American Oyster, Crassostrea virginica (Gmelin) 20 T. J. Price, G. W. Thayer, M. W. LaCroix and G. P. Montgomery The Organic Content of Shells and Soft Tissues of Selected Estuarine Gastropods and Pelecypods 26 C. E. Epifanio and C. A. Mootz Growth of Oysters in a Recirculating Maricultural System 32 R. D. Anderson and J. W. Anderson Oil Bioassays with the American Oyster Crassostrea virginica (Gmelin) 38 J. B. Sunderlin, W. J. Tobias and O. A. Roels Growth of the European Oyster, Ostrea edulis Linne, in the St. Croix Artificial Upwelling Mariculture System and in Natural Waters 43 C. L. Goodwin Observations on Spawning and Growth of Subtidal Geoducks (Panope generosa, Gould) 49 R. W. Menzel, E. W. Cake, M. L. Haines, R. E. Martin and L. A. Olsen Clam Mariculture in Northwest Florida: Field Study on Predation 59 P. N. Walker and J. W. Zahradnik Scale-Up of Food Utilization by the American Oyster, Crassostrea Virginica Gmelin 63 W. P. Breese Out-Bay Culture of Bivalve Molluscs 76 B. Morton and K. F. Shortridge Coliform Bacteria Levels Correlated with the Tidal Cycle of Feeding and Digestion in the Pacific Oyster (Crassostrea gigas) Cultured in Deep Bay, Hong Kong 78 iii R. T. Riley Changes in the Total Protein, Lipid, Carbohydrate, and Extracellular Body Fluid Free Amino Acids of the Pacific Oyster, Crassostrea gigas during Starvation 84 IV LIST OF ABSTRACTS BY AUTHOR OF TECHNICAL PAPERS PRESENTED AT THE 1975 NSA CONVENTION H. Arnold Carr Culturing and Transplanting Hatchery-Spawned Quahogs 1 Melbourne R. Carriker and James G. Schaadt Motion Picture Film Entitled "Predatory Behavior of the Boring Snail Urosalpinx cinerea" 1 W. Rudd Douglass Host Response to Infection with Bucephalus in Crassostrea virgin ica 1 W. Rudd Douglass and H. H. Haskin MSX-Oyster Interactions: Leucocyte Response to Minchinia nelsoni Disease in Crassostrea virginica 2 W. Rudd Douglass and H. H. Haskin MSX-Oyster Interactions: Some New Observations on Minchinia nelsoni Disease Development in Stocks of Oysters Resistant and Susceptible to M. nelson /-caused Mortality 2 Klaus G. Drobeck and Donald W. Pritchard A Field Experiment to Determine the Role of Sediment Bound Heavy Metals and Salinity Regime in the Heavy Uptake of Oysters 3 Richard D. Glenn A Report on the Successful Utilization of Plastic Trays for the Large Scale Culture of C. Gigas in Baja California, Mexico 3 Gordon Gunter An Example of Oyster Production Decline with a Change in the Salinity Characteristics of an Estuary, Delaware Bay 1800-1973 3 Dexter S. Haven, Virginia and Elgin A. Dunnington and Klaus G. Drobeck Growing of Hatchery Reared Spat in the Potomac River 4 Gmae Loy and A. E. Eble Locomotion and Phagocytic Behavior of Amebocytes of the Hard Clam, Mercenaria mercenaria, as Revealed by Time-Lapse Cinemicrography 4 Clyde L. MacKenzie, Jr. Increasing Earnings and Production in the Oyster Industry of Prince Edward Island 4 Sara V. Otto, Janet B. Hammed and Aaron Rosenfield Decline of Minchinia nelsoni in Maryland Waters of Chesapeake Bay 5 Walter L. Smith Pharmacology and Chemistry of Natural Gums Used as Binders in Foods for Mariculture 5 G. U. Schaffer, F. W. Wheaton and A. J. Ingling v Cooling Methods for Soft-Shell Clams 5 H. S. Tubiash The Role oCSerratia marcescens in the Coloration of "Red" Oysters and Soft-Shell Clams 6 Ronald E. Westley A Lawsuit (Environmentally Oriented) Brought Against Mechanical Clam Harvest in Washington State with a Hanks-Type Harvester 6 ABSTRACTS OF THE NSA PACIFIC COAST SECTION Norman E. Buroker and William K. Hershberger Genetic Variation in the Pacific Oyster, Crassostrea gigas 7 Linda Chaves and Kenneth K. Chew Application of Spanish Mussel Culture Techniques in Puget Sound, Washington 7 Richard A. Eissinger Progress in Central California Shellfish Seed Production 7 Lynn Goodwin The Assessment of Subtidal Geoduck Clam Populations by Visual and Photographic Techniques 7 William Hershberger and Kenneth Chew Genetic Manipulation and Breeding in the Pacific Oyster, Crassostrea gigas ° Chris Jones and Kenneth K. Chew Planting Hatchery Spawned Manila Clams (Venerupis japonica) in Puget Sound Beaches 8 Earl E. Krygier Crangon nigricauda and Crangon franciscorum in Yaquina Bay, Oregon - 9 Gerald Lasser Amino-Acid Requirements mf the Dungeness Crab 9 Patsy R. Lipp, Bruce Brown, John Liston and Kenneth Chew Recent Findings on the Summer Diseases of Pacific Oysters 9 Mark B. Miller, Charles H. Hanson and Kenneth K. Chew Shell Growth of Butter Clams and Littleneck Clams Near Madrona Beach on Camano Island 10 Sally Nickelson and Stephen B. Matthews Energy Efficiency in the Pacific Oyster Industry 10 VI Proceedings of the National Shellfisheries Association Volume 65-1976 ABSTRACTS OF THE TECHNICAL PAPERS PRESENTED AT THE 1975 NSA CONVENTION CULTURING AND TRANSPLANTING HATCHERY-SPAWNED QUAHOGS H. Arnold Carr Massachusetts Maritime Academy Division of Marine Fisheries Buzzards Bay, Massachusetts Between May and October, 1973, 5.5 million quahogs, averaging 2.0 mm along their ante- rior-posterior axis, were placed in trays sus- pended in the water column. By November, the mean survival rate was 52% and the mean size of quahogs delivered in May and June was 6.4 mm. Quahogs subjected to freezing water tem- peratures and ice during the winter of 1973-74, had a survival rate of 99%. Other hatchery- spawned quahogs with a size range of 8-20 mm were transplanted onto natural bottom. Recov- ery and survival of shell stock planted at a water temperature of 6°C was greater than that planted at 20°C. MOTION PICTURE FILM ENTITLED "PREDATORY BEHAVIOR OF THE BORING SNAIL UROSALPINX CINEREA" Melbourne R. Carriker and James G. Schaadt Biological Science Center Boston University Boston, Massachusetts The film depicts the approach, mounting, penetration, and feeding of oysters and mussels by the oyster borer. After a short sequence on fossils, the film shows snail approaching a jar filled with live pumping oysters, climbing the outside against the flow of water, entering the jar and boring oysters. The control jar is ig- nored. The remainder of the film demonstrates mounting of oysters, selection of boring site, details of shell penetration magnified under an optical system, slow motion pictures of radular activity, and feeding on the flesh inside. HOST RESPONSE TO INFECTION WITH BUCEPHALUS IN CRASSOSTREA VIRGINICA W. Rudd Douglass Oyster Research Laboratory N. J. Agricultural Experiment Station Rutgers University New Brunswick, New Jersey Little or no host response to Bucephalus in- fections in Crassostrea virginica has been re- ported by Hopkins (1954) and Cheng and Burton (1965). Mackin and Loesch (1954) reported an intense cellular response to Bucephalus sporo- cysts infected with a haplosporidan hyperpara- site. Sprague ( 1962) mentions a similar response to Bucephalus sporocysts infected with a mi- crosporidan hyperparasite. Canzonier (per. comm.) reported leucocytic responses to mori- bund and dead Bucephalus sporocysts. During a year-long survey (1972-73) of Bu- cephalus infections in a natural population of oysters from the Navesink River (New Jersey), host response was noted in a number of varying infection conditions. One case is of special inter- est. Young sporocysts in presumably new infec- tions elicited an intense cellular response in 3 of 7 oysters with similar infection intensities. In one case phagocytes were observed within the sporocyst proper. This response may be due to previous experi- ence with the parasite. It may also be due to environmental conditions which alter the nor- mal host-parasite relationship in this biological system. ABSTRACTS MSX-OYSTER INTERACTIONS: LEUCOCYTE RESPONSE TO MINCHINIA NELSONI DISEASE IN CRASSOSTREA VIRGINICA W. Rudd Douglass and H. H. Haskin Oyster Research Laboratory, N. J. Agricultural Experiment Station Rutgers University New Brunswick , New Jersey Several investigators have reported an in- crease in leucocytes in oysters with the develop- ment of Minchinia nelsoni (MSX) disease (Myhre, 1967; Farley, 1968). However, there has been no attempt at quantification of this re- sponse to date. This presentation represents an attempt to measure leucocyte population changes during MSX disease development. Two stocks of oysters, one resistant and one susceptible to MSX, were sampled at regular intervals for one year (May 1972-June 1973). Alterations in the total leucocyte populations (TLP) were monitored by counts of leucocytes/ oil field (OF), (20 OF/oyster), number of hyaline leucocytes/OF, (10 OF/oyster), and the percent- age of MSX plasmodia phagocytized/200/MSX/ oyster. Both stocks showed an increase in the num- ber of leucocytes/OF during the summer of 1972. There was no significant difference between the two stocks. During the winter and spring the number of leucocytes/OF ranged from 10%-30%> higher in infected oysters when compared with uninfected oysters in the resistant stock. Dur- ing the same period the number of leucocytes/ OF in infected susceptible oysters ranged from 80%-140% higher than that of uninfected oys- ters. The number of leucocytes in infected sus- ceptible oysters was significantly higher than that of infected resistant oysters during this time interval. There was no significant difference in the percentage of MSX plasmodia phagocytized/ sample, between the stocks. In both stocks the average percentage of MSX phagocytized was usually less than 10%. Hyaline leucocytes constitute less than 10% of the TLP in uninfected oysters. Changes in the % hyaline leucocytes/TLP were similar in both stocks. In oysters with gill lesions in the sum- mer there is a slight increase in hyaline leuco- cytes (10%-20% of the TLP). Hyaline leucocytes total 15%-40% of the TLP in oysters sampled in the winter and spring. In oysters with general infections throughout the year, hyaline leuco- cytes ranged from 25%-50% of the TLP. The lack of significant differences in leucocy- tic responses to MSX between resistant and sus- ceptible oysters verifies earlier speculation from this laboratory that leucocytes probably play a minor role in the resistance mechanism. This suggests that unknown humoral responses may control MSX lesion development in resistant oysters. MSX-OYSTER INTERACTIONS: SOME NEW OBSERVATIONS ON MINCHINIA NELSONI DISEASE DEVELOPMENT IN STOCKS OF OYSTERS RESISTANT AND SUSCEPTIBLE TO M. Af£LSCW/-CAUSED MORTALITY W. Rudd Douglass and H. H. Haskin Oyster Research Laboratory N. J . Agricultural Experiment Station Rutgers University New Brunswick, New Jersey Two stocks of oysters (Crassostrea virginica), one resistant, one susceptible to Minchinia nel- soni (MSX) were sampled at regular intervals for one year (May 1972-June 1973) on the Cape Shore tidal flats, Cape May, New Jersey. Mor- tality rates were monitored and histological sec- tions of 250 oysters/stock were examined for MSX prevalence/sample, weighted MSX inten- sity/sample, MSX lesion type/oyster, and MSX Plasmodium type/sample. Patent MSX lesions were observed in both stocks by the first week of July 1972. Three weeks later the susceptible stock reached 100% prevalence and remained high (70% -100%) for the rest of the experimental period. The in- crease in prevalence in the resistant stock was much slower, reaching 90% six weeks after the initial patent infection was detected. Infection prevalence declined in the stock to 10% by mid- September 1972, rose to 90% over the winter- spring period, and then dropped to 50% in June 1973. The quantity of MSX lesions in susceptible PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION oysters was three times that of the resistant stock during the summer of 1972. No significant difference in numbers of lesions between the two stocks was observed during the fall 1972 to spring 1973 period. In June 1973 the numbers of MSX lesions in the resistant stock was signifi- cantly lower than that of the susceptible stock. Gill lesions outnumbered general infections for every sample except one (May, 1973) in the resistant stock. Only three general infections were observed in this stock during the three months following detection of the first patent infection. General infections developed within 4 weeks of the first patent infection detected in the susceptible stock and persisted for the re- mainder of the experiment. Within 3 weeks of the first patent infection in the resistant oysters, 70% of the MSX plasmodia observed were either uninucleated or binu- cleated. These plasmodial types declined to 10% throughout the winter and then increased to 35% in June 1973. Susceptible oysters had an average of 10% uninucleated and binucleated plasmodial types throughout the experiment. These observations are discussed relative to previous reports from this laboratory and other laboratories involved in monitoring MSX infec- tion in C. virginica. A FIELD EXPERIMENT TO DETERMINE THE ROLE OF SEDIMENT BOUND HEAVY METALS AND SALINITY REGIME IN THE HEAVY METALS UPTAKE OF OYSTERS Klaus G. Drobeck University of Maryland and Donald W. Pritchard The Johns Hopkins University Hatchery-reared oyster spat were set on panels and deployed to three field environments of varying salinity. Exposure levels at each sta- tion were 12", 36", and 72' from the bottom. The stations were sampled at approximately one- month intervals. Collected samples: oysters, settleable solids, suspended sediments and wa- ter were analyzed for heavy metals concentra- tions by A. A. spectrophotometry. The data was treated statistically by analysis of variance and a theoretical relationship of salinity, growth rate, and metal uptake is proposed. Design and fabrication of an apparatus for panel deploy- ment, exposure, and recovery was tested. A REPORT ON THE SUCCESSFUL UTILIZATION OF PLASTIC TRAYS FOR THE LARGE SCALE CULTURE OF C. GIGAS IN BAJA CALIFORNIA, MEXICO Richard D. Glenn Pan Aqua Incorporated and National University California Laboratory produced seed of C. gigas have been grown to marketable size in six months or less utilizing plastic trays in an off-bottom cul- ture. This report describes the general physical, chemical and biological factors of the growing area and the design of a continuous production oyster farm. AN EXAMPLE OF OYSTER PRODUCTION DECLINE WITH A CHANGE IN THE SALINITY CHARACTERISTICS OF AN ESTUARY, DELAWARE BAY 1800-1973 Gordon Gunter Gulf Coast Research Laboratory Ocean Springs, Mississippi The oyster production of Delaware Bay has declined in two vast steps so that three rather striking and declining levels have come to pass. From 1880-1931 inclusive, the average annual production was 14,247,000 pounds a year; from 1932-1957 it was 7,951,000 pounds a year; and from 1959-1970 the average has been 859,000 pounds or 5.9% of the 1880-1931 level. Each stepwise decline occurred 3 years following the diversions of Delaware River water to New York City in 1929 and 1953. These diversions were strongly opposed by many American oys- ter biologists, particularly Thurlow C. Nelson and his colleagues, acting for the State of New Jersey. The predicted effects of these diversions upon Delaware Bay have come to pass. Doubt- less, other fisheries production dependent upon low salinity estuarine waters, such as men- haden, have also been seriously diminished. Questions of equity could arise. ABSTRACTS GROWING OF HATCHERY REARED SPAT IN THE POTOMAC RIVER Dexter S. Haven Virginia Institute of Marine Science Gloucester Point, Virginia and Elgin A. Dunnington and Klaus G. Drobeck Chesapeake Biological Laboratory Solomons, Maryland In October and November, 1972, the Virginia Institute of Marine Science, Chesapeake Biolog- ical Laboratory and the Potomac River Fisher- ies Commission planted two acres in the upper Potomac River near Morgantown with cultch- less spat. Densities were about 765,000 to 405,000 per half acre. Size ranged from approxi- mately ll2-3W. To date about half of these spat have survived. Mortalities were associated with unusually low salinities. LOCOMOTION AND PHAGOCYTIC BEHAVIOR OF AMEBOCYTES OF THE HARD CLAM, MERCENARIA MERCENARIA , AS REVEALED BY TIME- LAPSE CINEMICROGRAPHY Gmae Loy and A. E. Eble Trenton State College Trenton, New Jersey Two principle types of amebocytes of the hard clam, the large and small granulocytes, were studied with phase-contrast and interference phase-contrast (Nomarski) optics. Cells were taken from blood sinuses of the posterior adduc- tor muscle and placed on clean cover slips in a moist chamber for five minutes. Then the cover slips were fastened to slides by a vaseline seal which prevented desiccation of the preparation during prolonged filming. Small granulocytes were relatively active and usually flowed in unidirectional patterns. Cells moved by rapid extensions of ectoplasm. Gran- ules of the endoplasm rapidly flowed in the di- rection of the advancing ectoplasm. Much move- ment of endoplasmic granules was evident even when cell locomotion temporarily ceased. Mito- chondria were highly plastic and usually took the form of large blunt granules and would stretch into long sausage-shaped structures for brief intervals. Cells adhered firmly to the cover slips. This was most evident in the posterior portion of the cell as strands of ectoplasm would stretch, then suddenly release contact with the cover slip in order to "catch up" with the main portion of the cell. Large granulocytes adhered to cover slips as large, flat cells. Granules appeared as distinct mounds and ridges when viewed with Nomarski interference phase optics. No motion of these cells could be detected by direct viewing. Time- lapse studies at 30 frames per minute (fpm) revealed an extremely active waving motion of the ectoplasmic border of the cell. Although large granulocytes had fewer granules than small granulocytes, all granule types were in constant motion in the endoplasm. Granules were always confined to a small area around the nucleus. This large cell does move at a very slow rate by a sliding motion. Boiled, washed yeast cells were added to prep- arations of granulocytes and ensuing phagocy- tosis was filmed at 40 fpm. Small granulocytes rapidly flowed into a cluster of yeast cells. Ecto- plasmic extensions of the granulocytes flowed rapidly in between and around until all the yeasts were incorporated as phagosomes. The latter consisted of a membrane wall that sur- rounded a clear area in which the yeast cell was contained. Film records revealed as many as eight to ten yeast cells engulfed within a few minutes. Large granulocytes phagocytized yeast cells by enveloping them with wave-like extensions of the outer ectoplasm. INCREASING EARNINGS AND PRODUCTION IN THE OYSTER INDUSTRY OF PRINCE EDWARD ISLAND Clyde L. MacKenzie, Jr. NOAA, Mid-Atlantic Coastal Fisheries Center Highland , New Jersey The oyster industry of Prince Edward Island has been an important part of that Province's tradition and economy since the mid-1800's. In recent years, annual oyster production has been 20,000 to 30,000 boxes (a box contains V-U stand- ard U. S. bushels), and, along with fishermen's earnings, is trending downward. About 200 fish- ermen use hand tongs to harvest most oysters from 100 to 150 acres of public grounds in var- ious estuaries. The biological and ecological potentials for PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION increasing both quantity and quality (based on shell shape) of oysters are enormous because (1) adequate spatfall occurs practically every year, (2) growth increments of oysters range from zW-\ll2 a year, (3) survival rates of oysters are high, (4) at least 200,000 boxes of unharvested stocks of small oysters grow either in deep water or on poor shallow grounds, (5) several million bushels of oyster shells lie in buried deposits, and (6) about 1,000 acres of otherwise barren grounds possess favorable environmental fea- tures for producing high quality oysters. Use of large vessels to transplant both oysters from unharvested stocks and shells from depos- its and spread these on barren public grounds is recommended. In 1973 the Province's first oyster vessel, a 43-foot catamaran, was constructed. It transplanted and spread 34,000 boxes of oysters on 68 acres of grounds. Earnings of each fisher- man are expected to rise steadily from an an- nual average of $2,300 and reach about $4,550 after the transplanting program has been un- derway a few years. Earnings of local buyers and mainland wholesalers should rise signifi- cantly. Overall oyster production should more than double. Use of the vessels may not always be required because the newly-established oys- ter beds will self-perpetuate themselves with regular annual spatfall, even under intense harvesting pressure. The expenditures to finance the oyster reha- bilitation program should be exceeded many times by monetary benefits to the fishermen. Cost-to-benefit ratios should eventually exceed 1:20, as costs involve only the transplanting of existing live oysters and shells. The author developed and implemented this program during a one-year period, 1972-73, while engaged as oyster consultant to the Pro- vincial Department of Fisheries. DECLINE OF MINCHINIA NELSONI IN MARYLAND WATERS OF CHESAPEAKE BAY Sara V. Otto, Janet B. Hammed, and Aaron Rosenfield NOAA-NMFS Oxford, Maryland Prevalence ofMinchinia nelsoni, a haplospor- idan pathogenic to oysters (Crassostrea virgin- ica), steadily decreased from its highest level in 1964 (average of 19.6% prevalence for all areas sampled) until the cessation of regular sampling in 1972. At this time M. nelsoni was not ob- served in oyster tissues from any of the areas sampled. After Bay-wide sampling was discon- tinued, only oyster tissues from 9 areas in the Manokin River, a southern Maryland, Eastern Shore tributary to Chesapeake Bay, were exam- ined with any regularity. In spite of the reduc- tion in sampling and histological examinations, there are sufficient data to indicate that M. nelsoni either disappeared or became inactive at least 2 years prior to Hurricane Agnes. This information does not support the common claims that the storm drove M. nelsoni out of the Maryland waters of the Chesapeake Bay. PHARMACOLOGY AND CHEMISTRY OF NATURAL GUMS USED AS BINDERS IN FOODS FOR MARICULTURE Walter L. Smith Suffolk County Community College Selden, New York The chemistry and pharmacology of extracts from marine plants such as agar, carrageenan, furcelleran, and alginates are briefly described, these extracts and other products derived from them have many pharmaceutical uses; for ex- ample, as anti-coagulants, connective tissue growth enhancers, bulk laxatives, and coagu- lants. The chemistry of these extracts, the structure of the polysaccharides, and the varia- tions, both physical and chemical, are depend- ent on the types of plants and the kinds of salts present. The possible effects of these com- pounds, in the form of binders and capsules, when fed to certain marine animals are dis- cussed. COOLING METHODS FOR SOFT-SHELL CLAMS G. U. Schaffer, F. W. Wheaton, and A. J. Ingling College of Agriculture, University of Maryland Maryland soft clams harvested during the warm summer months are subject to quality ABSTRACTS deterioration due to bacterial growth. Cooling of the clams has been suggested as a possible solu- tion. This investigation was designed to develop data from which rational comparisons of various cooling techniques could be made. Three sources of cooling were investigated: ice, dry ice and mechanical refrigeration. These sources were utilized in several different sizes and types of systems under conditions of both natural and forced circulation. Various types of containers were tested to determine their effect on cooling rate. Results of the experiments were used to de- rive cooling curves for various combinations of equipment and cooling source. Ice and dry ice quantities, equipment descriptions and power requirements are discussed. THE ROLE OF SERRATIA MARCESCENS IN THE COLORATION OF "RED" OYSTERS AND SOFT-SHELL CLAMS H. S. Tubiash National Marine Fisheries Service Oxford, Maryland A study was made to determine the role of a chromogenic strain of Serratia tnarcescens, iso- lated from shucked osyters, on seasonal red col- oration of shellfish. This event reduces the mar- ket acceptability of affected shellfish. Experi- ments were designed to test effects of the orga- nism in live and shucked oysters and soft-shell clams. Live clams and oysters were held over- night in water of varying temperatures contain- ing Serratia concentrations of 5.6 x 10" per ml. The mollusks were then opened and examined for coloration. The live clams were tinged pink in the gills and superficial tissues, but the digestive glands and shell liquor did not differ from unexposed controls. After 5 days refrigera- tion the shucked clam meats showed a pinkish- orange liquor, had a putrid odor and were of unacceptable quality. Oysters similarly treated showed no coloration and after 5 days refrigera- tion the meats were still in good condition. It was concluded that S. marceseens plays no sig- nificant role in the coloration of marketable quality shellfish. A LAWSUIT (ENVIRONMENTALLY ORIENTED) BROUGHT AGAINST MECHANICAL CLAM HARVEST IN WASHINGTON STATE WITH A HANKS- TYPE HARVESTER Ronald E. Westley Shellfish Laboratory Brin non , Wash ington Harvest of Eastern soft-shell clams on the inter-tidal flats of Skagit Bay by a Hanks-type harvester was halted by an injunction issued against the clam harvester and the Washington State Department of Fisheries. Initial basis for the injunction was fear of damage to a high intertidal rush used for food by wild fowl located above the clam beds. Trial was held and the Court ruled that the clam harvest operator had failed to comply with the terms of the Washing- ton State Shorelines Management Act, and that mechanical clam harvest was both a substantial development and dredging in terms of the Washington law. He further ruled that the De- partment of Fisheries had failed to consider terms of the Washington State Environmental Policy Act of 1971 and that the clam harvest permit was not valid. This trial and action have potential for major impact on the Washington shellfish industry and it currently appears that it may greatly complicate the management regulation and conduct of shellfish harvest in Washington State. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION NSA PACIFIC COAST SECTION GENETIC VARIATION IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS Norman E. Buroker and William K. Hershberger College of Fisheries University of Washington The genetic variation that has been investi- gated is shown by electrophoretic separation of proteins (enzymes) on starch gels. This proce- dure allows an analysis of single gene differ- ences between individuals and will give an indi- cation of the genetic diversity within and be- tween populations. Eleven enzyme (protein) systems have been examined at our laboratory reflecting twenty loci; seven loci were monomorphic, eight were polymorphic, and five remain unresolved. The eight polymorphic loci found in the fifteen re- solved loci (53.3%) indicates that C. gigas is a highly polymorphic species. This high degree of genetic variation within a population should provide material for genetic improvement of the species. APPLICATION OF SPANISH MUSSEL CULTURE TECHNIQUES IN PUGET SOUND, WASHINGTON Linda Chaves and Kenneth K. Chew College of Fisheries University of Washington Mussel culture in Spain is of the floating raft type which has a high yield per unit area. Man- ual labor plays a large role though in the growth and preparation of the finished product. Ropes are thinned and harvested manually. Prior to marketing, the mussels must undergo depuration or canning which is not yet fully automated there. Without modification this would not be economically sound in the United States. A pilot study is being conducted in Puget Sound to determine the biological possibility of a commercial industry. Natural setting, sub- strate for seed collection, and growth are sev- eral of the factors being observed. During the past eight months of the study a major set has occurred at only one station and prior foulding of the ropes has been observed to be a deterrent to successful setting. Of the three types of sub- strates being used, manila, sinclove, and oyster strings, the sinclove appears to be the most promising. PROGRESS IN CENTRAL CALIFORNIA SHELLFISH SEED PRODUCTION Richard A. Eissinger, Production Manager International Shellfish Enterprises Moss Landing, California International Shellfish Enterprises, a four year old California mariculture company, is presently expanding facilities to present for sale large quantities of oyster and clam seed to be available in the early spring, 1975. Using hatch- ery reared seed, International is growing oyster seed, primarily C . gigas, to a size of 1" for use by shellfish growers throughout the world. Clam seed of 3-7 mm will also be available for plant- ing. Production of these large quantities is pos- sible by use of a hatchery and nursery tank farm using the warm water from a Moss Land- ing power plant. THE ASSESSMENT OF SUBTIDAL GEODUCK CLAM POPULATIONS BY VISUAL AND PHOTOGRAPHIC TECHNIQUES Lynn Goodwin Washington Department of Fisheries Brinnon, Washington Since 1967 the Washington State Department of Fisheries has been surveying subtidal clam stocks in Puget Sound with SCUBA divers. Geo- duck (Panope generosa) stocks are evalutated by visual counts of siphons or siphon holes (shows). The number of geoducks detected by divers using the visual method varies consider- ably depending on how well the clams "show" when the surveys are made. Preliminary obser- ABSTRACTS vations from nine plots showed that the percent- age of the true population detected in our visual surveys could vary from as low as 26 to a high of 92. In more detailed studies, the percentage de- tected was low during the cold winter months and high during spring, summer, and fall, and averaged approximately 40% from year-around monthly samples. The percentage detected in underwater photos is also highly variable, and is subject to similar errors. The montly "show- ing factors" have been used to refine our diver survey counts and the total Puget Sound subti- dal geoduck population estimate. The estimate for the 33,992 acres surveyed thus far is 114,700,000 clams. Surveys with underwater TV or cameras mounted on sleds, tripods, or other devices which come in contact with the bottom should be done with a knowledge of the effects of me- chanical disturbance on the "showing factor". Geoducks at certain times of the year are ex- tremely sensitive to mechanical disturbance and will withdraw their siphons from the sub- strate surface at the slightest contact of divers or equipment with the bottom. We have success- fully surveyed geoducks with underwater TV and have observed large numbers of these clams in water as deep as 240 feet. Mechanical dis- turbance was kept to a minimum by keeping the camera and all other equipment suspended off bottom on cables. This study demonstrated that useful survey information on the populations of marine benthic organisms can be obtained with visual or photographic techniques, but the surveys should be done with knwoledge of the percent- age which can be detected and the effects of equipment or divers on that percentage. GENETIC MANIPULATION AND BREEDING IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS William Hershberger and Kenneth Chew College of Fisheries University of Washington As part of the Sea Grant program to investi- gate the summer mortality in Pacific oysters apparently associated with Vibrio species of bacteria, a genetics study has been initated to investigate the selective breeding of the oyster for resistance to the disease. Results on single gene differences demonstrate a high degree of genetic variation on which breeding can be based, but no genetic differences have been shown between the various populations which can be correlated to their historical mortality level. However, laboratory experiments in which oysters were challenged with a mortality-induc- ing situation and a large portion killed indi- cated that some individuals with a particular phenotype survived better. Although these re- sults are preliminary, there is an indication that at least one gene can be used to "mark" slightly more resistant oysters. With markers such as this, more resistant individuals can be chosen for a breeding stock to improve the re- sistance of the total population used for market- ing. Other characteristics desirable to the oyster grower that can also be improved through breeding and the types of genetic manipulation necessary will be discussed. PLANTING HATCHERY SPAWNED MANILA CLAMS (VENERUPIS JAPONIC A) IN PUGET SOUND BEACHES Chris Jones and Kenneth K. Chew College of Fisheries University of Washington Hatchery spawned Manila clams averaging 3.7 mm in length were planted in four Puget Sound locations in the spring of 1973 to deter- mine the feasibility of rehabilitating potential clam producing beaches that do not currently have commercial densities of clams. Heavily dug public beaches may also be repopulated in this way. The variables of planting density and tidal height were examined with regard to growth rate and mortality. Plots were planted with densities of 300, 600, 1200, and 2000 clam seed per square meter with an unplanted con- trol plot. Samples were taken at two weeks, thriteen weeks and twenty-five weeks subse- quent to planting. Two of the four plots were unsuccessful in that there was almost no recov- ery of planted clams after thirteen weeks. It is unknown whether the failure was due to move- ment of the clams or to mortality caused by predation and physical factors. The other two PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION plots were more promising with recovery rang- ing up to 140 clams per square meter in Decem- ber with a size averaging 23 mm. Recovery was higher and the clams were faster growing at the + 2 level than at the +4 level. Absolute numbers of clams recovered increased with increasing planting density but the percentage recovered dropped sharply. The possible reasons for this outcome were discussed. CRANGON NIGRICAUDA AND CRANGON FRANCISCORUM IN YAQUINA BAY, OREGON Earl E. Krygier Oregon State University School of Oceanography Corvallis, Oregon The distribution, reproduction, and growth of Crangon nigricauda and Crangon francisco- rum in Yaquina Bay, Oregon are described from the examination of 8,244 C. nigricauda and 4,568 C. franciscorum collected by beam trawl at bimonthly intervals during Dec. 1970 through Feb. 1972. The distribution of C. nigricauda and C. franciscorum is generally related to water tem- perature and salinity. Both species exhibit a wide tolerance for temperature (5.2-16.5 C for C. nigricauda; 5.3-21.5 C for C. franciscorum) and salinity (>19pptforC. nigricauda; 0.2-34.4 ppt for C. franciscorum). Between species, C. nigricauda displays a preference for cooler tem- perature and higher salinity than does C. fran- ciscorum. Within species, variations in re- sponse to temperature and salinity changes were observed between size and sex groupings. The spawning season for both species is from Dec. to mid-Aug. C. franciscorum and C. ni- graieauda ovigerous females disappear from the Bay in August and September, respectively. Berried females were collected in waters rang- ing from 6.8-12.9 C for C. nigricauda and 6.8- 19.2 C for C. franciscorum , and with salinity of >25.4 ppt for C. nigricauda and > 14.6 ppt for C. franciscorum. Both species exhibit bimodal spawning periods with larger females initiating the spawning season. Fecundity is correlated (R2 > .91) with total length (TL), with the mean and range being 4,016 and 2,393-7,000 for C. nigricauda and 3,528 and 1,923-4,764 for C. franciscorum . Growth of young was defined by the regres- sion equations: Y = -6.04 + 0.76 (TL) summer and Y = +7.79 + 0.95 (TL) winter for C. nigri- cauda. Only a summer growth rate estimate was attainable for C. franciscorum: Y = -25.44 + 1.37 (TL). Both species exhibited differential growth after attaining sexually recognizable sizes, with females being 8-10 mm TL larger than males at maturity. Females apparently live a maximum of IV2 yr and males not more than 1 yr. AMINO-ACID REQUIREMENTS OF THE DUNGENESS CRAB Gerald Lasser Humboldt State University Areata. California The amino acid requirements of the Dunge- ness crab, Cancer magister, were investigated with radiometric techniques. Seven crabs were tested in all. Five were injected with glucose- HC(U), one with glutamic acid-MC(U), and one with phenylalanine-' 4C(U). The following amino acids were determined to be nonessen- tial; alanine, aspartic acid, cystine, glutamic acid, glycine, hydroxyproline, proline, serine, tyrosine (with phenylalanine present). The glutamic acid injected crab showed no labeled proline though all others expected to be labeled were. The phenylalanine injected crab showed labeling on glutamic acid, alanine, and tyrosine. These results are similar to those reported for other arthropods. RECENT FINDINGS ON THE SUMMER DISEASES OF PACIFIC OYSTERS Patsy R. Lipp, Bruce Brown, John Liston, and Kenneth Chew College of Fisheries University of Washington Although the 1960's was a decade in which significant Pacific oyster mortalities occurred in specific bays along the Pacific Coast, the first four years in 1970 did not demonstrate a similar pattern. Some mortalities did take place during these years, but they were very low in incidence and were considered to be the normal back- ground mortalities due to predation or other 10 ABSTRACTS natural causes. During the summer of 1974, there was a general mortality of up to 15-20% in specific areas of Willapa Bay and Rocky Bay in central Puget Sound in late July and August. Laboratory studies and evaluation of field data have shown that high temperature and high nutrient levels in water are associated with oyster mortality. Under experimental con- ditions, oysters dying in high temperature wa- ter have been shown to carry large numbers of bacteria in the heart, blood, and pericardial fluid. Vibrios (particularly V. anguillarum ) iso- lated from dying oysters have been shown to cause mortalities when introduced into healthy oysters under high temperature conditions by either active or passive inoculation. Detectable ammonia levels are present in the dying and dead oysters. Field studies have shown that total bacterial counts and mesophilic vibrio counts rise with the onset of summer water temperatures. Per- cent incidence of V. anguillarum is higher dur- ing summer months also. The Willapa Bay and Rocky Bay mortalities porvided the first opportunity to test the hy- pothesis based on laboratory findings that dis- ease and death were due to bacterial infection. Dead or moribund oysters were found to contain bacteria in the heart blood. Apparently healthy oysters from the mortality area were also found to contain bacteria, though healthy oysters usu- ally have sterile heart fluids. NH:! was also detected. Vibrios from these oysters were shown by inoculation experiments to be virulent to- wards healthy oysters and could be recovered from the heart fluids. The initial findings thus support the bacterial infection hypothesis, and work is continuing to confirm or refute its valid- ity. SHELL GROWTH OF BUTTER CLAMS AND LITTLENECK CLAMS NEAR MADRONA BEACH ON CAMANO ISLAND Mark B. Miller, Charles H. Hanson and Kenneth K. Chew College of Fisheries University of Washington A possibly unique abnormality of unknown origin in butter clams (Saxidomus giganteus) and littleneck clams (Protothaea staminea) from a localized area on Camano Island, Wash- ington is being investigated at the College of Fisheries, University of Washington. The na- ture of the abnormality is a scalloping or inden- tation along the ventral margins of the valves which sometimes extend as grooves from the ventral margins of the valves which sometimes extend as grooves from the ventral shell margin to the umbo. Outside of a several hundred foot horizontal distribution of the abnormal clams, few traces of the shell defect are found. The same general area which is inhabited by high- est percentages of abnormal clams is also washed by a freshwater seepage which origi- nates at about the two foot tide mark and below. The problem of determining the cause of the abnormality is being approached by comparing the affected area with unaffected areas, map- ping the abnormal clam distribution and see- page range, histological examination of clam tissue, examination of clams for trace metals, analysis of properties and contents of the see- page, and a literature search. ENERGY EFFICIENCY IN THE PACIFIC OYSTER INDUSTRY1 Sally Nickelson and Stephen B. Mathews College of Fisheries University of Washington Seattle, Washington The energy efficiency of the Pacific oyster industry in Willipa Harbor was estimated by converting all manpower, fuel, vessel, machin- ery and other inputs of production of a standard firm into kcal and comparing this to kcal in the oyster meat. On an annual basis the ratio of kcal output to kcal of input was estimated to be .21. This level of efficiency was similar to that of other fishery industries of Washington and to other U. S. agricultural protein production sys- tems, which was surprising since oyster culture requires neither artificial feeding nor tradi- tional fishing. The diesel fuel used in planting, transplanting and harvesting accounted for 85% of the input energy. ' Research supported by a National Science Foundation Grant for Undergraduate Research, GY-11242. Proceedings of the National Shellfisheries Association Volume 65 - 1975 LIVING MARINE ANIMALS IN A SHIP'S BALLAST WATER J. C. MedcoP DEPARTMENT OF THE ENVIRONMENT FISHERIES AND MARINE SERVICE BIOLOGICAL STATION, ST. ANDREWS, N.B. ABSTRACT Seawater ballast taken aboard in Japan, 1 May, 1973, was sampled when the ship berthed in Australia two weeks later. It contained living copepods, amphi- pods, ostracods, unidentified crustacean larvae , polychaetes (larvae and adults) and chaetographs . No mollusc larvae were found although most physical- chemical conditions of the ballast water were optimal for larvae of Pacific oysters. INTRODUCTION Samplings of ballast water in two holds of a ship were made on 15 May, 1973, eight hours after it berthed at Eden in Twofold Bay, New South Wales, Australia (S. Lat. 37°05'; E. Long. 149°55'). It had just completed a 14.5-day pas- sage without cargo from Tagonoura, Japan (N. Lat 35°12'; E. Long. 138°42'). The ship's officers stated that the ballast water was boarded partly in Tagonoura Harbour and partly at sea during the first 4 days of the passage. CHARACTERISTICS OF BALLAST WATER Results of water sampling at the surface and bottom of both ballast holds and at the surface of Twofold Bay adjacent to the ship, are tabulated below (Personal Communication, 1973, Mr. E. A. Scribner, chemist). PLANKTON OF BALLAST WATER At the same time, two bottom-to-surface verti- cal hauls through the ballast water were made in each hold with a standard cone-type plankton net (length, 1.8 m; mouth diameter, 0.5 m; mesh apertures, 88 u). One 10 m-long, horizontal haul at a depth of 0-3 m was also made in Hold No. 4. Pocket-magnifier inspection of the fresh catches revealed numerous, swimming orga- 1 Retired. Present address: St. Andrews, N.B., Canada nisms; crustaceans being the most conspicuous. Microscopic examination after formalin preser- vation showed that the crustaceans included planktonic copepods of several species (0.5-3 mm long), amphipods (5 mm long), an ostracod and juvenile stages of various unidentified groups. The most abundant organisms were eyed polychaete larvae that had contracted into spherical forms (0.2 mm diam.). Adult benthic polychaetes (whole and fragments, 1-5 mm long) and chaetognaths (5-8 mm long) were also present in small numbers. The hauls were re- markably uniform as regards total numbers of animals caught and relative abundance of the various types composing the catches. DISCUSSION It is assumed that all these organisms were pumped aboard with the ballast water 10-14 days previous to the samplings. On that assump- tion the results indicate that conditions in the ballast water (Table 1) were favourable to sur- vival and that the plankters did survive an inter-hemisphere transport through more than 70 degrees of latitude before they were dis- charged with the ballast water into a new envi- ronment. Presumably most of these organisms could accommodate to the small salinity change (maxi- mum, 1.6 0/00) when discharged into Twofold Bay on 15 May (see Table 1). But the drastic temperature change (approximately 10°) would 11 12 J. C. MEDCOF TABLE 1. Physical characteristics of water samples. Tem- pera- ture CO Sa- linity ( ) Dissol ved Oxygen Sample Point (mg/1) {% sat'n.) (pH) Ballast Water: Hold No. 2 surface bottom (15 m) 25.6 25.4 35.1 35.3 6.5 6.5 100 100 8.1 8.0 Hold No. 4 surface bottom (12 m) 26.0 25.8 34.3 35.1 6.3 6.5 97 100 8.1 8.0 Twofold Bay: surface 16.7 35.9 7.8 103 8.1 reduce their metabolic rates and could well be directly or indirectly lethal to most or all of them. At other seasons and in other harbours the discharge of ballast water might be less shocking to organisms it contains. The Twofold Bay study supports the often- quoted conjecture of Peters and Panning (1933) that ballast water is a vector for long-dustance dissemination of aquatic organisms and may be responsible for mysterious appearances of exotic species. Pacific oysters appeared mysteriously in New Zealand recently (Dinamani, 1974) and may have been introduced in this way because, ex- cept for salinities, the tabulated data show that ballast water conditions were close to optimum for larvae of that species (Fujiya, 1970; Quayle, 1969). ACKNOWLEDGMENTS This study was assigned jointly to me and my then colleague, Mr. E. A. Scribner (chemist), by the Director of the Fisheries Branch of the New South Wales Chief Secretary's Department in which I was employed in 1972-73. I thank Mr. Scribner for personal communication of data and for helpful discussions of the draft of this paper. The plankton collections are deposited with Dr. W. B. Malcom, Chief Biologist, New South Wales State Fisheries, Sydney, N.S.W., Aus- tralia. LITERATURE CITED Dinamani, P. 1974. Pacific oyster may pose threat to rock oyster. New Zealand Min. Agric. and Fish., Catch '74, July. p. 5-9. Fujiya, M. 1970. Oyster farming in Japan. Hel- golander wiss. Meeresunter. 20: 464^479. Peters, N. and A. Panning. 1933. Die chine- sische Wollhandkrabbe (Eriocheir sinensis, H. Milne-Edwards) in Deutschland. Zool. Anz. 104: 1-180. Quayle, D. B. 1969. Pacific oyster culture in British Columbia. Bull. Fish. Res. Board Can. No. 169. 192 p. Proceedings of the National Shellfisheries Association Volume 65 - 1975 CULTCHLESS SETTING OF EUROPEAN OYSTERS, OSTREA EDULIS, USING POLISHED MARBLE1 Herbert Hidu, Samuel Chapman and Paul W. Soule IRA C. DARLING CENTER UNIVERSITY OF MAINE WALPOLE, MAINE AND READING MEMORIAL HIGH SCHOOL READING, MASSACHUSETTS ABSTRACT Early experiments indicate that polished marble may have ideal characteris- tics as a substrate in cultchless setting of oysters. It is highly attractive to setting larvae and is hard and smooth enough to permit removal of juveniles without excessive damage. Techniques are described for the setting process. Highly polished marble appears to possess ideal characteristics for use in producing cultch- less oysters. It is not only attractive to setting larvae but is hard and smooth enough to permit easy removal of spat after metamorphosis. This note reports some encouraging early results us- ing polished marble as a setting substrate. The idea of using polished marble as a setting substrate occurred to us after observing the be- havior of larvae while testing a variety of sub- strates for use in the cultchless process. First, we observed that apparently mature eyed lar- vae in 100-gal. polyethylene tanks would delay metamorphosis for several days if no suitable substrate was presented. Only occasionally have we observed a mass setting of larvae on the sides of polyethylene tanks. If, however, a molluscan shell (oyster, sea or bay scallop) was added to the culture, the shell would be black- ened with set very rapidly. Molluscan shells, however, are a poor choice of setting substrate because the irregular surfaces make it difficult to remove the juveniles and obtain the cultch- less form. Other substrates were tried, i.e., glass, var- Ira C. Darling Center Contribution No. 90, and was supported by NOAA Sea Grant Project No. 04-5-158-39. ious plastics, and "Mylar" sheets (Dupuy, 1972). Occasionally, we have obtained some set on "My- lar", but neither "Mylar" or the others are highly attractive to setting of European oysters. It appears that some property associated with a molluscan shell (possibly calcium carbonate) is highly stimulatory to setting larvae. Thus, the ideal setting substrate in the cultchless process would appear to be a calcium carbonate-derived material that is hard and smooth, permitting easy removal of metamorphosed oysters. Mar- ble, which is limestone recrystallized under heat and pressure, would appear to have these characteristics and when mature larvae are ex- posed to smooth marble, the response is dra- matic with heavy sets being achieved in short order. Setting procedure with European oysters. Larvae (1 x 106) are reared to maturity in large (100-gal) polyethylene vats. After the ma- ture larvae have achieved eyespots, a small scal- lop or oyster shell tied to a string is introduced to monitor the ability of the larvae to set. As soon as the small shell is heavily blackened with set, the entire batch of larvae is trans- ferred to a setting bath. Setting baths should be sufficiently shallow to permit easy addition and removal of cultch surface. 13 14 H. HIDU, S. CHAPMAN AND P. W. SOULE In the setting baths, we attempt to manipu- late conditions to obtain a massive set in as short a time as possible. This allows an efficient manipulation of cultch surfaces and maximizes the percentage conversion of larvae to spat. Since it has been demonstrated that adult oys- ter metabolites and increased temperatures may stimulate setting in American oysters (Veitch and Hidu, 1971; Lutz, et al., 1969), we raise the temperature to 24-26°C and add sev- eral gallons of sea water from adult European oyster conditioning baths. Cultured algae are added in liberal amounts. With a young vigor- ous brood of larvae which have delayed setting, these conditions have produced a heavy set on the marble surfaces within one to several hours. Intensity of setting on the marble slabs is closely monitored to avoid a total blackening of the marble surface which might result in the smothering of metamorphosing spat. The marble slabs are then moved to separate culture baths at 24°C with adequate algal foods for a 24 to 48 hour period to allow the spat to produce a fan of new juvenile shell growth. Spat are then scraped off with a double-edged razor blade to produce cultchless juveniles. If spat are scraped off too early, before they achieve a small band of juvenile growth, they then appear to have difficulty in metamorphosing in the cultch- less form. If they are scraped off too late, then excessive shell damage may result from re- moval. We are now using polished marble exclu- sively in our procurement of cultchless oysters and have much to learn about optimal methods of presentation and removal of oysters. How- ever, with our favorable early results, we feel that others should be aware of marble as a setting substrate and should experiment with it themselves. LITERATURE CITED Dupuy, J. L. and S. Rivkin. 1972. The develop- ment of laboratory techniques for the produc- tion of cultch-free spat of the oyster, Crassos- trea virginica. Chesapeake Sci. 13: 45-52. Lutz, R. A., H. Hidu and K. G. Drobeck. 1969. Acute temperature increase as a stimulus to setting in the American oyster, Crossostrea virginica (Gmelin). Proc. Nat. Shellfish As- soc. 60: 68-71. Veitch, F. P. and H. Hidu. 1971. Gregarious setting in the American oyster Crassostrea virginica Gmelin: 1. Properties of a partially purified "Setting Factor". Chesapeake Sci. 12: 173-178. Proceedings of the National Shellfisheries Association Volume 65 - 1975 A GREGARINE-LIKE PARASITE ASSOCIATED WITH PATHOLOGY IN THE DIGESTIVE TRACT OF THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA Thomas K. Sawyer, Martin W. Newman and Sara V. Otto NATIONAL MARINE FISHERIES SERVICE, U.S. DEPARTMENT OF COMMERCE, OXFORD, MARYLAND AND MARYLAND DEPARTMENT OF NATIONAL RESOURCES, OXFORD, MARYLAND ABSTRACT Histological examination of oysters, Crassostrea virginica, from Connecticut and Maryland waters showed that an amoeboid or gregarine-like parasite was associated with seasonal pathology in the digestive tract. Focal sloughing of cells of the columnar epithelium and clear zones, or halos, around individual parasites were observed only in the spring months. The specific pathological response was found during 1967 and 1968 in Connecticut, and in 1970 and 1973 in Maryland. Comparative studies with oysters from each location provided certain immature growth stages which resembled developmental forms of gre- garines of the Nematopsis-Porospora group. It is tentatively suggested, that the parasite overwinters in hibernating oysters, undergoes vegetative growth in the spring when oysters resume feeding, and is cleared from the digestive tract in association with a transient host tissue response. INTRODUCTION Crustacea. Further observations on the orga- The seasonal occurrence of an amoeboid orga- nisms found in C. virginica are presented in nism in the digestive tract of the American this report to illustrate probable life-cycle oyster, Crassostrea virginica, was first reported stages other than the amoeboid vegetative form, by Newman (1971), who briefly described the Pathological conditions recognized in commer- parasite in shellfish collected from New Haven cially valuable shellfish include two broad cate- Harbor, Connecticut. Subsequently, similar or gories: those which are attributable to a specific closely related organisms were discovered in pathogen and those which are characterized by oysters collected in April 1970 and 1973 from a specific pathological response for which the several tributaries of Chesapeake Bay, Mary- causative organism is unknown. Sprague (1971) land (Sawyer, Newman and Otto, 1973). Com- reviewed the principal pathological conditions parative studies on oysters from those two recognized in oysters which, although widely sources suggest that the amoeboid organisms recognized, often were characterized by the lack are vegetative stages of an unknown species of of an identifiable etiologic agent, viz., "Denman gregarine. The severe focal cellular response Island Disease," "Amber Disease" and others, and sloughing of columnar epithelial cells in the Sindermann and Rosenfield (1967) published a infected oysters appears to be similar to patho- comprehensive review which summarized the logical conditions discussed by Ball (1951), who principal shellfish diseases of known etiology, described new species of gregarines from marine and Sawyer (1966) briefly summarized the taxo- 15 16 T. K. SAWYER AND M. W. NEWMAN nomic status of amoeboid organisms from tissue and mantle fluid of C. virginica. The present report concerns both a specific pathological re- sponse and the documentation of the probable etiologic agent. METHODS Histological sections of 1,337 oysters, C. vir- ginica, collected from New Haven Harbor, Con- necticut were examined during 1966 and 1967 (Newman, 1971). Monthly samples from rivers and tributaries of Chesapeake Bay, Maryland were similarly examined during 1970 and 1973. Tissue sections were stained with Harris hema- toxylin and eosin, Fuelgen reaction with fast- green counter-stain, or the PAS (Schiff) proce- dure. Microscopic examinations were made of all specimens in each monthly sample and data were analyzed to determine whether seasonal variations were associated with histopathologi- cal findings. Water temperature and salinity were recorded at the time of all but one of the collections from Maryland (Table 1). RESULTS Histological examination of oysters collected in Connecticut showed that an amoeboid para- site was present in the gut epithelium of 14 of 1,337 oysters (1%) (Newman, 1971). All infec- tions in Connecticut oysters except for one in February and one in June were detected in the months of March and April. Subsequent obser- vations on oysters collected in Maryland showed that a similar parasite was present only in April 1970 and 1973. The number of infected oysters in Maryland ranged from 1 in 25 to 7 in 25 (Table 1). In all of the infected oysters the parasites were restricted to the columnar epithelium of the digestive tract, always in localized foci, and never beyond the limit of the basement mem- brane. Oysters sampled in the areas listed in Table 1 were studied throughout the year and their generally healthy condition indicated that significant mortalities were not associated with the seasonal appearance of the parasite. The smallest recognizable stage of the para- site was a spherical form found in localized areas of the intestinal epithelium (Fig. 1-2). Round forms had a distinct central sphere which stained deep red with the Feulgen reac- tion and was surrounded by a wide halo after staining with hematoxylin. Another stage re- sembled an emerging sporozoite (Fig. 3) which may have progressed to a pyriform body (Fig. 4) and developed to a trophozoite (Fig. 5). Tro- phonts, apparently in syzygy (Fig. 6), had an ovoid deutomerite and the elongate filamentous protomerite. Encysted forms, possibly young ga- metocytes (Fig. 7) were found infrequently. (See Newman, 1971, Fig. 7, for comparison.) Specific pathological response in infected oys- ters was evidenced by sloughing of degenerate cells of the columnar epithelium or the displace- ment of healthy host tissue by trophonts or spherical forms. Proliferating asexual stages of the parasites were not observed in any of the oysters examined. Life cycle stages of the para- site found in the oysters resembled forms which typically have been illustrated for gregarine par- asites of crustaceans and suggest that growth in molluscs possibly was atypical. DISCUSSION Further observations on the occurrence of an apparent gregarine parasite in C. virginica are presented to extend its geographical range to TABLE 1. Source and prevalence of a gregarine-like parasite in Maryland oysters, crassostrea virginica. Source Date Number Positive Condition T°/C Salinity* Chester River Apr 1970 7/25 Healthy 5.74 11.3 Choptank River Apr 1970 5/25** Healthy 7.26 13.6 Herring Bay Apr 1970 2/26*** Watery 7.94 5.5 Cedar Point Hollow Apr 1970 1/25**** Healthy 4.20 13.5 Manokin River Apr 1973 2/25 Healthy — — ppt * 2 with Nematopsis ostrearum ** 1 with Nematopsis ostrearum *** 19 with Nematopsis ostrearum •^RL7jc *? FIG. 1-7. Photomicrographs of gregarine-like parasites in columnar epithelium of digestive tract of Crassostrea virginica, hematoxylin-eosin stain, xl400. FIG. 1-2. Immature spores. Note host tissue response. FIG. 3. Sporozoite leaving spore. FIG. 4. Pyriform shape of sporozoite developing to trophozoite or sporont stage. FIG. 5. Mature trophont. Note absence of host response. FIG. 6. Paired trophonts in syzygy. apparently in process of sloughing into lumen. Note bulbous deutomerite and filamentous protomerite. FIG. 7. Encysted forms, probably a gametocyst. 17 18 T. K. SAWYER AND M. W. NEWMAN include Maryland and to amplify the original observations of Newman (1971). At the same time, we propose to redesignate the organism as gregarine-like instead of amoeboid as reported in the earlier publication. It is noteworthy that the pathological condition in the digestive tract was identical in both Connecticut and Maryland oysters. Furthermore, the location of the para- sites among sloughing cells of the gut epithe- lium represented a host response which was similar, although not identical, to an earlier report (Ball, 1951) of intestinal pathology in crabs caused by several new species of Nematop- sis and Carcinoectes. Until new information on the host-parasite relationship in oysters is ob- tained, we propose tentatively to identify the parasites as gregarines, possibly of the Nema- topsis-Porospora type. Mature spores were not detected in tissue sections of gill and palp epithelium of the in- fected oysters. According to Leger & Duboscq (1913), and to Hatt (1927a, 1927b, 1928), the spores of certain species of Nematopsis and of Porospora mature in the gills of hosts such as oysters, mussels, chitons, etc., after infection by gymnospores from crustacean hosts. Typical life cycles have been elucidated for Nematopsis os- trearum Prytherch (1940), which develops in the oyster and mud crab, Porospora gigantea v. Beneden (1869), which develops in the lobster and the common mussel, and others. Typically, infection of the molluscan host is initiated by direct penetration of the gymnospores into cells of the gill epithelium and the pallial lobes of the palps, but not by penetration of the epithelium of the tegument or the digestive tract. In con- trast, development in the crustacean host does occur in the digestive tract, beginning with spor- ulation and progressive development of sporo- zoites to trophozoites, syngyns, and finally, ga- metocytes. Photomicrographs of the life-cycle stages present in tissue sections of oysters from Connecticut and Maryland show developing par- asites in stages of growth that usually are found in a crustacean host. We do not have a satisfac- tory explanation for our findings, but we pro- pose that such development is atypical and in- complete. The absence of parasites and degener- ative changes in the gut epithelium, except dur- ing March and April, suggest that the orga- nisms probably overwinter in hibernating oys- ters, causing transitory tissue pathology in early spring; then either perish or undergo fur- ther development in an unknown second host species. The present stage of our knowledge suggests that atypical growth of the parasites induces transient sloughing of gut epithelium with progressive repair and recovery of the oys- ter host. The apparent seasonality of the infection sum- marized in this report suggests that several in- terpretations may be proposed on a strictly spec- ulative basis. We have considered the possibil- ity that feeding oysters might have spores or sporocysts passing harmlessly through their digestive tracts during the months in which they feed and grow; such stages not necessarily belonging to species which normally parasitize oysters. Later, when oysters cease feeding dur- ing cold seasons, transient organisms might be trapped in the digestive tract when they would reside until feeding activity was resumed in the spring. During the winter interlude, such orga- nisms could be trapped between crypts or villi of the intestine and transported from the lumen to the columnar epithelium by phagocytes. Fi- nally, during the spring months of March and April, when we found active cell sloughing and growth stages of the parasites, transient host response would effectively remove the last ves- tiges of the overwintering protists. This hypo- thetical interpretation might account for the presence of immature stages of a gregarine which more appropriately would be expected to reside in the digestive tracts of crustacean hosts. Abortive growth of immature parasites in atypical host animals is well-documented in par- asitological literature. Future reports on the same or related host-parasite interactions should extend the geographic range of this dis- ease and perhaps yield new information on de- velopmental stages which we did not observe. It is likely that the tissue response illustrated here for Connecticut and Maryland oysters will be recognized as an entity in much the same way as "Malpeque Bay Disease," "Amber Dis- ease" and other pathologic conditions are diag- nosed although the precise nature of the causa- tive agent is uncertain. ACKNOWLEDGMENT The authors gratefully acknowledge the as- INTESTINAL PATHOLOGY IN THE AMERICAN OYSTER 19 sistance of Mrs. Janet Hammed, Maryland De- partment of Natural Resources, Oxford, Mary- land. LITERATURE CITED Ball, G. H. 1951. Gregarines from Bermuda ma- rine crustaceans. Univ. Calif. Publ. Zool. 47: 351-368. Hatt, P. 1927a. Spores de Porospora (Nematop- sis) chez les Gasteropodes. C. R. Seances Soc. Biol. Fil. 96: 90-91. Hatt, P. 1927b. Le debut de revolution des Poros- pora chez les Mollusques. Arch. Zool. Exp. Gen. 67: 1-7. Hatt, P. 1928. L'evolution de la Gregarine du Homard (Porospora gigantae E. V. Bened.) chez les Mollusques. C. R. Seances Soc. Biol. Fil. 98: 647-649. Leger, L. and O. Duboscq. 1913. Sur les pre- miers stades du developpment des Gregarines du genre Porospora (= Nematopsis). C. R. Seances Soc. Biol. Fil. 75: 95-98. Newman, M. W. 1971. A parasite and disease survey of Connecticut oysters. Proc. Natl. Shellfish. Assoc. 61: 59-63. Sawyer, T. K. 1966. Observations on the taxo- nomic status of amoeboid organisms from the American oyster, Crassostrea uirginica. J. Protozool. 13(Suppl.): 23 (Abstract). Sawyer, T. K., M. W. Newman, and S. V. Otto. 1973. Seasonal pathology in the American oys- ter associated with a gregarine-like intestinal parasite. J. Protozool. 20(Suppl): 511 (Ab- stract). Sindermann, C. J. and A. Rosenfield. 1967. Prin- cipal diseases of commercially important ma- rine bivalve Mollusca and Crustacea. U.S. Fish. Wildl. Serv., Fish. Bull. 66: 335-385. Sprague, V. 1971. Disease of oysters. Annu. Rev. Microbiol. 25: 211-230. Proceedings of the National Shellfisheries Association Volume 65 - 1975 ENERGY PARTITIONING IN THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA (GMELIN) Curt Michael Langefoss and Don Maurer FIELD STATION COLLEGE OF MARINE STUDIES UNIVERSITY OF DELAWARE LEWES, DELAWARE ABSTRACT Research was conducted to develop an energy budget for the American oyster, Crassostrea virginica (Gmelin), in culture. Caloric values ofpseudofeces, feces, food ingested, food cleared and food assimilated were determined at three levels of alga! {Phaeodactylum tricornutum Bohlin) concentration , 1 .0 x 205, 5.0 x 104 and 2.5 x 104 cells -ml-' at 20° and 25°C; approximately 74-148 calories per 12 hours were used by the oysters. Oysters at the lowest food concentration showed the greatest amount of filtration , while oysters at the medium food concentration cleared the most food. In addition, the greatest amount of feces and pseudofeces were produced at the medium food concentration . There was little difference between the amount of energy assimilated at the high and medium concentra- tion. The mean assimilation efficiency obtained from the three food concentra- tions was 67.6%, and the mean filtration rate (water pumped 6 2 mlhr~' mgdry tissue weight'') was consistent with other studies. INTRODUCTION This research was undertaken to develop an energy budget for the American oyster, Crassos- trea virginica (Gmelin). An energy budget re- lates the intake of food by an organism to its subsequent utilization. To develop an energy budget, it requires assessing food intake, rejecta and the amount of energy utilized by the ani- mal. The budget takes the form: C = P + R + F + U (Crisp, 1971) where C = Consumption, P = Production, R = Respiration, F = Feces, U = Urine. Some aspects of an energy budget for C. vir- ginica have been studied. Oxygen consumption of C. virginica was reviewed by Galtsoff (1964). Walne (1972) determined the influence of cur- rent speed, body size, and water temperature on the filtration (water pumped) rates of two spe- cies of oysters. Dame (1972) reported for the first time the quantitative examination of growth rates in intertidal oysters and a simultaneous examination of respiration in relation to size and temperature. Tenore and Dunstan (1973) investigated the effects of different concentra- tions of mixed phytoplankton on the feeding and biodeposition rate of the American oyster to understand bioenergetics of filter-feeding herbi- vores. The significance of this budget resides in its application to mariculture. MATERIALS AND METHODS Water Sea water for the oyster experiment was pumped from the Broadkill River, Delaware, at high slack water to assure salinity of 25%o and low turbidity. This water was filtered through a cloth bag (5 /j.) and then vacuum filtered through a "Whatman 40" filter and finally passed through a 0.45 fi Millipore filter. Back- ground count of particles per ml determined by a Coulter Counter was less than 500 per ml. The 20 ENERGY PARTITIONING IN OYSTERS 21 range of pH was 8.0-8.1. Water temperature (20°C ± 0.5) for all experiments was controlled by a constant temperature bath and circulator. Algae Sea water for culturing algae was pumped into a settling tank at high slack water. After a few days, water was filtered through a 5 ix bag and "Whatman 40" filter. The water was en- riched according to the method of Matthiessen and Toner (1966). After enrichment, a twenty liter carboy (filled to the 16 liter mark) was autoclaved at 18 pounds pressure and 125°C for two hours, cooled and inoculated with two liters of a 14 day old Phaeodactylum tricornutum (Bohlin) culture. Caloric content of the alga was determined by two methods: 1) bomb calorimeter (Parr auto- matic adiabatic calorimeter), 2) quantitative di- chromate oxidation (Standard Methods, 1965; Maciolek, 1962). The conversion of mg COD/L (as obtained by wet oxidation) to calories per gram of sample follows Maciolek (1962). The conversion factor used was 1 mg Oa/L = 3.4 calories/L. The dried alga was ashed to constant weight at 600° in a muffle furnace (Parsons, et al., 1961). Oysters Oysters were artificially spawned, reared and set in the laboratory. They were then held for 18 months in running sea water from the Broadkill River at ambient temperatures. Oysters were randomly selected from the labo- ratory-reared stock and were allowed to accli- mate and purge their guts at 20° C for 12 hours prior to each experiment. Each oyster was placed in a three-liter glass jar filled with two liters of filtered sea water. The oysters were placed on a pedestal so that feces would fall to one side of the jar and pseudofeces would fall on the other (Haven and Morales-Alamo, 1965). Four oysters (approximately 6.8-7.2 cm in height and 27.7-30.9 gm wet weight) were used in each 12-hour experiment. These were sup- plied one of three levels of algal concentration: 1.0 x 105 cells/ml (high), 5.0 x 104 cells/ml (me- dium), or 2.5 x 104 cells/ml (low). After an exper- iment, each oyster was sacrificed and dried to a constant weight in an analytical oven (at 80°C). Thirty experiments were performed. A Coulter Counter, Model B, was used to count the number of algal cells cleared by the oyster. A one hundred micron aperture tube was used. The method to obtain a specified amount of algal cells and to maintain a constant amount of cells throughout an experiment was described in Langefoss (1973). Feces and pseudofeces were collected by pipet- ting immediately upon production and placed in a collecting jar. Desalting was accomplished us- ing a Dow hollow fiber beaker dialyzer Model b HFD-1. Water was passed through the dialyzer for 40 minutes, after which the sample was dried to constant weight at 80° C. The sample was then placed in a Ca S04 filled dessicator and cooled to room temperature (22-23° C). This weight represents total feces or pseudofeces pro- duction for the animal for 12 hours. Caloric determinations of feces and pseudofeces were made by the quantitative dichromate oxidation method (Maciolek, 1962; Standard Methods, 1965). Pseudofeces production was subtracted from the amount of algal material cleared with the difference as a measure of ingestion. Assimila- tion was estimated by subtracting fecal produc- tion from ingestion estimates. All data were converted to energy units based on calorimetric analysis. The total number of cells removed was di- vided by the cells/ml (food concentration) to yield the number of milliliters of water filtered (water pumped). Cell counts were made with a Coulter Counter. RESULTS Oysters Energy partitioning by oysters at three levels of algal concentration, 1.0 x 105 cell/ml (high), 5.0 x 104 cell/ml (medium), and 2.5 x 104 cell/ml (low) is shown in Figure 1. Each mean repre- sents at least 24 oysters. Oysters at the medium food concentration cleared the most food (Fig. 1). In addition, the greatest amount of pseudo- feces and feces was produced at the medium food concentration. However, there was little or no difference between the energy assimilated at the high and medium concentration. The percentage assimilation and amount of filtration (L-hr_1-gram dry tissue weight-1) is 22 C. M. LANGEFOSS AND D. MAURER present in Figure 2. Determination of filtration rates was described in detail by Langefoss (1973). Oysters at low food concentration showed OF OYSTERS AT i . |0= CELL M OF Ot'STERS AT 5 0 - I04 C E L LS / ML OF OYSTERS AT 2 5 X I04 CELLS/ML o 1 T O o A B £c T1, i i (x/gm dry tissue weight / hour) FIG. 1. Plot of calories cleared, calories in- gested, calories of pseudofeces , calories of feces, and calories assimilated. Bars represent the 95% confidence interval. N = 24-30 oysters. the greatest amount of filtration. However, the rate of filtration and the percentage assimila- tion were inversely related between the me- dium and high algal concentration (Fig. 2). Table 1 summarizes the various aspects of energy partitioning in the oyster. The mean assimilation efficiency obtained from the three food concentrations was 67.6%. Students' 't test (Sokal and Rholf, 1969) was performed on data comparing the three food concentration levels with one another for each part of the energy budget (Table 2). The nota- tion A-B, A-C, and B-C was the mean of calories cleared; for example, at a concentration of 1.0 x 105 cell/ml (A) when compared with calories cleared at concentration 5.0 x 104 cell/ml (B), etc., there were significant differences for the 00 rlOO r T E) -i i. — — • 1\ ^ 9) FIG. 2. Growth of oysters, Crassostrea virgin- ica fed Phaeodactylum tricornutum. Bars around points are ± one standard deviation. DIETS FIG. 3. Size of oysters in each group after 46 weeks of growth . TABLE 3a. Analysis of variance table for mean shell height in Groups B to H after 46 weeks of growth. Asterik denotes significant F value at 0.01 probability level . Sum of Squares d.f. Mean Squares F Between 12666.96 6.00 2111.16 72.56* groups Within 34333.45 1180.00 29.10 groups Total 47000.41 1186.00 TABLE 3b. Duncan Multiple Range Test (a = 0.01) for differences in mean shell height. Letters refer to experimen- tal groups, and underscoring indicates overlapping ranges of shell height. C D F G B E H smaller larger smallest animals. Animals in Group B were significantly larger than those in Groups G and F and those in G and F significantly larger than those in Group D. The algae used in this study were chosen because of their wide taxonomic variety and because they had been shown to have some utility as foods for oysters in other studies (Goodrich et al., 1968). While it is impossible to make any statistical inference concerning the relative food-value of the individual algal spe- cies used in the experiment, it can be seen that Carteria chuii was a component of the diets fed to both of the fastest growing groups, and it makes up fully half (in terms of numbers of cells) of the composition of Diet B which pro- duced second fastest growth. Since Phaeodac- tylum tricornutum, which makes up the other half of Diet B, is undoubtedly a poor food, it can be inferred that Carteria chuii is one of the more important components of the diets tested. This is further substantiated by the fact that Isochrysis galbana. when combined with Phaeodactylum tricornutum, alone, promoted significantly slower growth than any of the diets containing Carteria chuii and that Croom- onas salina, when combined with Phaeodac- tylum tricornutum alone, produced slower growth than any diet other than the one-part Phaeodactylum tricornutum diet. Animals in the fastest growing group reached a mean shell height of slightly greater than 27 mm after 46 weeks of growth. This compares favorably with growth of oysters in natural wa- ters of the Middle Atlantic region during their first growing season. Shaw (1966) indicated that oysters which set in Broad Creek, Maryland, during the summer of 1960, had reached a mean shell height of about 25 mm by May, 1961, and Beaven (1952) reported that oysters from sev- eral Maryland locations in the Chesapeake Bay reached a shell height of about 30 mm by the beginning of their second growing season. Maurer and Aprill (1973) found that hatchery- reared spat grown in off-bottom culture in var- ious rivers in Delaware reached a mean shell height of between 10 and 20 mm (depending on location) by the beginning of their second grow- ing season. It must be pointed out, however, that those animals living in nature grew only during the warmer months of the year while 36 C. E. EPIFANO AND C. A. MOOTZ those in the present study grew at a rather constant rate for the entire 46 weeks. Therefore, while the yearly growth of the oysters in our system was comparable to or better than natu- ral growth, the daily growth during the respec- tive growing seasons was somewhat less than natural growth. Nevertheless, the results of the present study show that it is unquestionably possible to grow oysters in recirculating seawater systems on a diet consisting solely of cultured phytoplankton. There is no particular reason why this method of culturing oysters must be conducted adjacent to or even near a source of natural seawater. The possibilities for mariculture of oysters at inland sites is certainly not out of the question, and in the least, our techniques allow the main- tenance of filter-feeding marine bivalves at in- land research facilities. ACKNOWLEDGMENTS The authors wish to thank their colleagues Dr. Richard Srna for providing the water qual- ity analyses and Mr. Gary Pruder for oversee- ing the design and construction of the culture system. Mr. Dennis Logan, Mr. Andrew Marin- ucci, and Mr. Robert Flaak fed the animals on weekends while Ms. Christine Turk and Mr. Earl Greenhaugh were responsible for the daily maintenance of the shellfish and algae over the 46 weeks of the experiment. Dr. Ellis Bolton provided light intensity measurements. LITERATURE CITED Beaven, G. F. 1952. Some observations on rate of growth of oysters in the Maryland area. Proc. Nat. Shellf. Assoc. 43: 90-98. Collier, A. 1959. Some observations on the respi- ration of American oyster Crassostrea virgin- ica (Gmelin). Inst. Mar. Sci. 6: 92-108. Davis, H. C. 1950. On food requirements of lar- vae of Ostrea virginica. Anat. Rec. 108: 132- 133. Davis, H. C. 1953. On food and feeding of larvae of the American oyster, C. virginica. Biol. Bull. 104: 334-350. Davis, H. C. and P. E. Chanley. 1956. Effects of some dissolved substances on bivalve larvae. Proc. Nat. Shellf. Assoc. 46: 59-68. Davis, H. C. and R. R. Guillard. 1958. Relative value of ten genera of micro-organisms as foods for oyster and clam larvae. U. S. Fish. Wildl. Serv. Fish. Bull. 136 (58): 293-304. Dean, B. 1887. The food of the oyster: its condi- tions and variations. Second Rep. Oyster In- vest., State of New York, Albany. Dupuy, J. L. and S. Rivkin. 1972. The develop- ment of laboratory techniques for the produc- tion of clutch-free spat of the oyster. Crassos- trea virginica. Ches. Sci., 13: 45-52. Epifanio, C, G. Pruder, M. Hartman, and R. Srna. 1973. An interdisciplinary study on the feasibility of recirculating systems in mari- culture. Proc. 4th Ann. World Mariculture Society Workshop: 37-52. Epifanio, C. E., R. Srna, and G. Pruder. 1975. Mariculture of shellfish in controlled environ- ments: a prognosis. Aquaculture , 5: 227-241. Galtsoff, P. S. 1964. The American oyster Cras- sostrea virginica Gmelin. Fish. Wildl. Serv. Fish. Bull., U. S. 64: 1-480. Garvard, D. 1927. De quoi se nourrissent les huitres? Leur nourriture envisagee au point de vue "Ostreiculture." Bull. Trav. Stat. Aquic. Peche Castiglione Alger. 2: 237-254. Goodrich, D. R., R. B. Wainwright, L. J. Balbo, and A. Perlmutter. 1968. New Engineering approaches for the production of Connecticut oysters. I. Problem analysis. American Cy- anamid Co., Central Res. Div., Stanford, Connecticut. 140 pp. Guillard, R. R. L. and J. Ryther. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana and Detonula confervacea. Can. J. Mi- crobiol. 8: 229-239. Hartman, M., C. Epifanio, G. Pruder, and R. Srna. 1974. Farming the artificial sea: Growth of clams in a recirculating seawater system. Proc. Gulf Carib. Fish. Inst. 26: 59- 74. Imai, T. and M. Hatanaka. 1949. On the artifi- cial propagation of the Japanese common oys- ter, Ostrea gigas Thun. Res. Tokohu Univ. 1: 33-46. Loosanoff, V. L. and H. C. Davis. 1963. Rearing of bivalve mollusks. Adv. Mar. Biol. 1: 1-136. Loosanoff, V. L., H. C. Davis, and P. E. Chan- ley. 1955. Food requirements of some bivalve larvae. Proc. Natl. Shellf. Assoc. 45: 66-83. Loosanoff, V. L. and R. R. Marak. 1951. Cultur- ing lamellibranch larvae. Anat. Rec. Ill: 129-130. GROWTH OF OYSTERS IN A RECIRCULATING MARICULTURAL SYSTEM 37 Mattheissen, G. C. and R. C. Toner. 1966. Possi- ble methods for improving the shellfish indus- try of Martha's Vineyard. Marine Research Foundation, Inc., Edgartown, Massachusetts. 138 pp. Maurer, D. and G. Aprill. 1973. Feasibility study of raft culture of oysters in the Dela- ware Bay area. Report to Delaware River Ba- sin Commission. Maurer, D. and K. S. Price. 1967. Holding and spawning Delaware Bay osyters ( Crassastrea virginica) out of season. I. Laboratory facili- ties for retarding spawning. Proc. Natl. Shellf. Assoc. 158: 71-77. Nelson, T. C. 1947. Some contributions from the land in determining conditions of life in the sea. Ecol. Monogr. 17: 337-346. Petersen, C. G. J. and P. B. Jensen. 1911. Val- uation of the sea. Animal life of the sea-bot- tom, its food and quantity. Rep. Danish Biol. Sta. 20: 1-81. Putter, A. 1909. Die Ernahrung der Wassertiere und der Stoffhaushalt der Gewasser. Jena, Fisher. 168 pp. Shaw, W. N. 1966. The growth and mortality of seed oyster, Crassostrea virginica, from Broad Creek, Chesapeake Bay, Maryland, in highl- and low-salinity waters. Proc. Natl. Shellf. Assoc. 56: 59-63. Srna, R., C. Epifanio, G. Pruder, M. Hartman, and A. Stubbs. 1973. The use of ion specific electrodes for chemical monitoring of marine systems. Part I. The ammonia electrodes as a sensitive water quality indicator probe for re- circulating mariculture systems. University of Delaware Sea Grant Publication No. DEL- SG-14-73. Steel, R., G. D. and J. H. Torrie. 1960. Princi- ples and Procedures of Statistics. McGraw- Hill, New York. 471 pp. Ukeles, R. 1971. Nutritional requirements in shellfish culture. In Proceedings of the Con- ference on Artificial Propagation of Commer- cially Valuable Shellfish-Oysters. K. S. Price and D. L. Maurer (ed.). University of Dela- ware, Newark, Del. p. 43-64. Walne, P. R. 1956. Experimental rearing of the larvae of Ostrea edulis L. in the laboratory. Fish. Inves., Minist. Agric. Fish. Food Ser. 2 20 (9): 1-23. Walne, P. R. 1963. Observations on the food value of seven species of algae to the larvae of Ostrea edulis. I. Feeding experiments. J. Mar. Biol. Assoc. U. K. 43: 767-784. Walne, P. R. 1965. Observations on the influ- ence of food supply and temperature on the feeding and growth of the larvae of Ostrea edulis L. Fish. Invest., Lond. Ser. 2, 24: 1-45. Walne, P. R. 1970. Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Merce- naria, and Mytilus. Fishery Invest., Lond. Ser. 2, 25 (5): 62 pp. Proceedings of the National Shellfisheries Association Volume 65 - 1976 OIL BIOASSAYS WITH THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA (GMELIN)' Roger D. Anderson2 and Jack W. Anderson3 DEPARTMENT OF BIOLOGY TEXAS A&M UNIVERSITY COLLEGE STATION, TEXAS 77843 ABSTRACT Oyster bioassays were conducted to determine the relative toxicity of four test oils and a reference toxin. The oysters (Crassostrea virginica) were exposed to oil-water dispersions of two crude and two partially refined petroleum hydro- carbons. The partially refined oils #2 fuel and Venezuela bunker C were found to be more toxic than the two crude oils tested. South Louisiana and Kuwait. Oysters demonstrated greater resistance to test oils than to the reference toxin, dodecyl sodium sulfate. Valve closure by oysters made it difficult to determine percent mortality data in 96-hour or extended studies. Composition of test solutions is compared to calculated values of oil in water and referenced to the relative toxicity demonstrated. Behavior and condition of test animals is dis- cussed in relation to bioassay results. INTRODUCTION Considerable bioassay work on marine ani- mals has been conducted in the laboratory to determine the relative toxicities of petroleum hydrocarbons, dispersants and other potentially hazardous compounds. These investigations have focused on selected crude or refined petro- leum products tested on a wide variety of ani- mals. Many of the studies include discussions on the relative merits of static and flow-through bioassays, behavioral responses of test animals and use of suitable test animals for given re- gions. Much of this work is detailed and dis- cussed by Anderson (1973). In an effort to compare laboratory studies to field conditions, recent reviews attempt to re- late the composition and behavior of oil in water to both the animals tested and their ecosystems (Nelson-Smith, 1970; 1973; Wilber, 1969; Moore, 1 This work was partially supported by the American Pe- troleum Institute, through Letter of Agreement No. 0520-P to Texas A&M University and the Texas A&M Sea Grant College Program through the National Oceanic and At- mospheric Administration, Grant No. 04-3-158-18. - Present address: Virginia Institute of Marine Science, Gloucester Point, Virginia 23062. 3 Present address: Battelle Northwest Marine Laboratory, Sequim, Washington 98382. 19731. Little information, however, has been de- rived or cited in these studies pertaining to the actual concentration of the exposure medium, particularly in regard to the petroleum hydro- carbons in aqueous phase or the chemical com- position of oils tested. This present work emphasizes the additional need to reference behavior and condition of test animals. Rather than report calculated concen- trations of oil in seawater, actual concentra- tions, composition and relative toxicity of test solutions are cited and relationships between behavior, condition and relative toxicity of the oil-water dispersions discussed. This study was initiated to provide baseline information in a broad program to examine the sublethal effects on estuarine animals, brought about by both environmental alteration and introduction of potentially harmful compounds. MATERIALS AND METHODS Two crude oils. South Louisiana and Kuwait, and two partially refined oils, #2 fuel oil (con- taining approximately 40% aromatics) and Ven- ezuela bunker C oil (a residual), were utilized. Oils were supplied in 55-gallon barrels by the American Petroleum Institute. Contents were transferred to one-gallon amber glass jars. Be- 38 OIL BIOASSAYS WITH THE AMERICAN OYSTER 39 fore being sealed with aluminum foil liners or Teflon caps, jars were flushed with nitrogen. American osyters, Crassostrea virginica, were collected at natural and artificial reefs in the Galveston Bay system. Primary collection sites were intertidal reefs at Six Mile Road and Eight Mile Road in West Bay, and Morgan's Point Reef in Galveston Bay (Fig. 1). Oysters collected in West Bay ranged from approxi- mately 40-70 mm in shell length, while oysters collected at Morgan's Point ranged from 70-90 mm. West Bay oysters were collected as needed, while oysters from Morgan's Point were held in trays at the Seabrook Laboratory, Texas Parks and Wildlife Department, prior to shipment to the laboratory in College Station. After shell cleaning, oysters were placed in large, aerated aquaria and maintained at 20%o salinity and 20°C in the commercial seawater product, Instant Ocean (Aquarium Systems, Inc., Eastland, Ohio). The 10-, 20- and 30-gallon aquaria used as holding tanks were maintained in subdued light, gently aerated and main- tained at 20%o salinity by addition of distilled water. Oysters were used within 7 to 28 days after collection and were not fed. Preliminary experiments showed that in the laboratory oysters could acclimate within 72 hours to new salinities ranging from 10 to 30%° (Anderson, 1973). Animals having low condition indices or testing positively for fungal parasites, Labyrinthomyxa spp., were discarded. To deter- mine the comparative health of animals tested at various times and from different locations, a standard toxicant (dodecyl sodium sulfate: DSS) was used as a reference during toxicity studies. The basic procedure used in this bioassay was a modification of LaRoche, Eisler and Tarzwell (1970). One-quart, wide-mouth jars were used as assay containers. Measured amounts of the oils or reference toxin, DSS, were added to the arti- ficial seawater to make 500 ml test solutions. Solutions to be tested were tightly capped with new aluminum foil liners or Teflon liners and shaken for five minutes at approximately 200 cycles per minute on a shaker platform. One hour after mixing, one oyster was added per jar. Since extremely high concentrations of oil were employed, dispersions were character- ized by large numbers of droplets. Solutions were aerated by using disposable pipettes low- -Six Mile Road 2-Eight Mile Road 3-Morqan's Point 4-Seabrook Laboratory FIG. 1. Location of oyster sampling and hold- ing stations in the Galveston Bay system. ered into the test jars through holes in the lids. When impossible to count the number of bub- bles, aeration was decreased. For tests with the reference toxin, vessels were packed with towels around the holes in the lids to contain the foam from the detergent-like material. Test animals were examined every 12 hours. Oysters that were gaped or failed to close their valves after being soundly tapped by a pipet, were removed. Bioassays were considered for termination after 96 hours. However, this proved to be inappro- priate because of the oysters' great resistance to test oils and their ability to remain closed. Long-term bioassays, often extending over sev- eral months, were then conducted, and the time to first death (TL„), time to 50% mortality (TL,„) and time to 100% mortality (TL100) determined for each group of animals tested. Petroleum hydrocarbons present in the aqueous phase of the oil-water dispersions were prepared for analysis by infrared and gas chro- matographic methods. Aliquots were drawn carefully from below the surface of the oil-water dispersions, with care taken to avoid contami- nation of the water sample by any surface slick present. A 200 or 400 ml aliquot of each oil- water dispersion was taken for analysis. De- 40 R. D. ANDERSON AND J. W. ANDERSON TABLE 1. Percent mortality of winter-collected oysters exposed for 96 hours to various concentrations of the four test oils. Percent oil concentration in solution is compared with duration of exposure in hours. #2 Fuel Venezuela Bun ker C South Louisiana Kuwait Crude E xposure Time (hours) E xposure Ti (hours) me Exposure Time (hours) E xposure Time (hours) <7c Oil 24 48 72 96 24 48 72 96 24 48 72 96 24 48 72 96 .001 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .005 0 0 10 10 0 0 0 0 0 0 0 0 0 0 0 0 .01 0 0 10 10 0 0 10 10 0 0 0 0 0 0 0 0 .05 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 .1 0 10 40 50 0 0 0 0 0 0 0 0 0 0 10 10 1 0 20 30 40 0 0 0 10 0 0 0 0 10 10 50 50 10 0 40 40 50 0 10 20 80 20 40 40 80 0 10 50 50 50 0 0 0 20 0 0 0 20 0 0 20 60 0 0 0 0 tailed petroleum hydrocarbon analyses were not conducted, since they were available in this lab- oratory through bioassay studies with other es- tuarine species (Neff and Anderson, 1973; An- derson, Neff, Cox, Tatem and Hightower, 1974). RESULTS AND DISCUSSION Reliable TL5„ data were difficult to obtain in these bioassay experiments because of the oys- ters' tendency to cease pumping and close their valves, as well as their ability to rapidly depur- ate petroleum fractions. Results of the 96-hour bioassays with the test oils and reference toxin show the oysters' low sensitivity to these mate- rials (Tables 1 and 2). The reference toxin, DSS, consistently resulted in lower TL50 values than the four test oils, while oysters collected in late fall showed greater resistance than summer- collected test animals. In all bioassays, includ- ing long-term studies (Table 3), #2 fuel and Venezuela bunker C oils were most toxic, with the two crude oils. South Louisiana and Ku- wait, being considerably less toxic. Kuwait crude demonstrated the lowest toxicity of the petroleum hydrocarbons tested (Table 3). Additional bioassays conducted with oysters collected in winter months revealed a pattern of increased resistance to test oils (Anderson, 1973). Again, this resistance was substantiated by testing with the reference toxin. Winter oys- ters also proved less sensitive to the standard toxicant than did summer-collected specimens. The water phase data for this study were generated by Anderson (1973), Neff and Ander- son (1973) and Anderson, Neff, Cox, Tatem and Hightower (1974). These investigators reported TABLE 2. Percent mortality of oysters collected during different seasons exposed to the reference toxicant, dodecyl sodium sulfate (DSS). % I )I)S DSS-October 1972 Exposure Time (hours) DSS-February 1973 Exposure Time (hours) 24 48 72 96 24 48 72 96 .001 0 0 0 0 0 0 0 0 .01 0 0 0 10 0 0 0 0 .05 0 0 10 20 0 0 0 0 .1 0 0 20 30 0 0 0 10 .5 0 0 20 20 0 0 10 20 1 60 80 80 80 0 20 60 60 5 60 80 100 100 10 10 60 90 TABLE 3. Results of static bioassays in which oysters were exposed to 1 7c oil-water dispersions, expressed as TL„ (time of first mortality in days): TL:M (time to 507c mortal- ity); TLW„ (time to 1007c mortality). TL„ TL,„ TL (Days) (Days) (Daysi Venezuela Bunker C 5 6 13 #2 Fuel 5 8 14 South Louisiana Crude 9 14 18 Kuwait Crude 20 35 70 that the concentration of total hydrocarbons in oil-water dispersions was a function of the amount of oil added to the bioassay container. They found that the concentration of total hy- drocarbons derived from South Louisiana and Kuwait crude oil increased linearly with the amount of oil added. For South Louisiana and Kuwait crude, the amount of oil in the water phase ranged from approximately 16 to 80 ppm for 0.01 to 10% oil added. For Kuwait crude, the range was approximately 19 to 42 ppm for 0.01 to 10% oil added. Using #2 fuel oil, they found OIL BIOASSAYS WITH THE AMERICAN OYSTER 41 that as the amount of oil added was increased, a corresponding rise did not occur in total hydro- carbon content in the water phase. At 0.1%, the maximum (51 ppm) was reached with slightly lower levels (47 ppm at 1%, 37 ppm at 10%) recorded with the addition of more oil. For #2 fuel oil, the range of oil in the water phase was approximately 14-15 ppm for 0.01 to 10% oil added. Anderson, Neff, Cox, Tatem and High- tower (1974) attributed this phenomenon to in- creased droplet coalescence at very high oil con- centrations. Similar data for the residual oil, Venezuela bunker C, were not available be- cause of the difficulty in working with this ex- tremely viscous product. Detailed composition of test solutions is discussed by Anderson, Neff, Cox, Tatem and Hightower (1974). As pointed out in literature already cited, the oil-water dispersions were unstable in the bioas- say containers. Concentrations of total oil hy- drocarbons in the aqueous phase of the oil-water dispersions dropped rapidly during gentle aera- tion. In analytical work provided to this study and cited in Anderson, Neff, Cox, Tatem and Hightower (1974), generally only 10% of the original hydrocarbons were present in the dis- persion after the first 24 hours of aeration. It is difficult then to assess over any length of time, the amount of petroleum hydrocarbons actually in solution. In addition, Anderson (1973) found that oysters depurate petroleum fractions dur- ing these static exposures, with amounts of oil in solution constantly changing. Anderson (1973) noted wide individual differences in the test exposures over time, along with the rapid rise of microorganisms in the various disper- sions. This study indicated an increased resistance to toxicants by oysters collected during the late fall and winter. A partial explanation is re- vealed in the Gulf oyster's life cycle. In summer and early fall, oyster meats are in poor condi- tion; i.e., watery, translucent, thin. Spring spawning, accompanied by loss of food reserves (glycogen), markedly affects quality. Rising summer temperatures increase incidence of in- fection by pathogens. Animals are further de- pleted by a second spawning in the fall. As a result, oysters collected in summer and early fall have poor condition indices and often are infected with the highly pathogenic Labyrintho- myxa spp. In this study, tests conducted by Dr. William Wardle at the Texas A&M Marine Lab- oratory in Galveston, indicated negative or very low incidence of ' Labyrinthomyxa spp. in oysters tested. "Experimentation conducted in late fall and winter was carried out with oysters in good to excellent condition (Anderson, 1973). The mol- luscs were fat with large glycogen reserves. Be- cause of the high quality of the meats and low incidence of disease, these oysters can be ex- pected to be more hardy and resistant. Results show the experimental animals used in winter months to be almost twice as resistant as those employed in the bioassay experiments con- ducted in summer and early fall. Observations by other investigators on loss of condition asso- ciated with summer months support these con- clusions (Fingerman and Fairbanks, 1956; Galt- soff, 1964; Roosenburg, 1969; Quick, 1971). As stated by Gardner, Barry and LaRoche (1973>, sufficient background data on the test animals must be available to assess bioassay results. Though static bioassays cannot be expected to replicate field conditions, good baseline data can be drawn, particularly when one references the animal's behavior and health in relation to actual concentrations of the test solutions. Though oysters are difficult to study in bioas- says, they are an important shellfish resource which reflect the problems of sessile animals. LITERATURE CITED Anderson, J. W., J. Neff, B. Cox, H. Tatem and G. M. Hightower. 1974. Characteristics of dis- persions and water-soluble extracts of crude and refined oils and their toxicity on estua- rine crustaceans and fish. Mar. Biol. 27: 75- 88. Anderson, R. D. 1973. Effects of petroleum hy- drocarbons on the physiology of the American oyster, Crassostrea uirginica Gmelin. Ph.D. Dissertation. Texas A&M Univ., College Sta- tion. 146 pp. Fingerman, M. and L. D. Fairbanks. 1956. Os- motic behavior and bleeding of the oyster, Crassostrea uirginica. Tulane Stud. Zool. 3(9): 149-168. Galtsoff, P. S. 1964. The American oyster: Cras- sostrea uirginica Gmelin. U.S. Fish Wildl. Serv., Fish. Bull. 64. 480 pp. 42 R. D. ANDERSON AND J. W. ANDERSON Gardner, G. R., M. Barry and G. LaRoche. 1973. Analytical approach in the evaluation of bio- logical damage resulting from spilled oil. Pa- per presented at National Academy of Science Workshop on Inputs, Fates and Effects of Pe- troleum in the Marine Environment. May 21- 25. Airlie, Virginia. LaRoche, G., R. Eisler and C. M. Tarzwell. 1970. Bioassay procedures for oil and oil dis- persant toxicity evaluation. J. Water Poll. Cont. Fed. 42(11): 1982-1989. 'Moore, S. F. 1973. Towards a model of the ef- fects of oil on marine organisms. Paper pre- sented at National Academy of Science Work- shop on Inputs, Fates and Effects of Petro- leum in the Marine Environment. May 21-25. Airlie, Virginia. Neff, J. M. and J. W. Anderson. 1973. Uptake and depuration of petroleum hydrocarbons by the estuarine clam, Rangia cuneata. Paper presented at 1973 annual meeting of the Na- tional Shellfisheries Association. June 24-28. New Orleans, Louisiana. Nelson-Smith, A. 1970. Effects of oil on marine plants and animals. Pp. 273-291. in P. Heppel (ed.) Water Pollution by Oil. Elsevier. New York. Nelson-Smith, A. 1973. Oil pollution and ma- rine ecology. Plenum Press. New York. 260 pp. Quick, J. A., Jr. 1971. A preliminary investiga- tion: The effect of elevated temperature on the American oyster, Crassostrea virginica (Gmelin). Florida Dept. Nat. Resources, Ma- rine Res. Lab, St. Petersburg, Fla. 190 pp. Roosenburg, W. H. 1969. Greening and copper accumulation in the American oyster, Cras- sostrea virginica, in the vicinity of a steam electric generating system. Chesapeake Sci. . 10(3): 241-252. Wilber, C. G. 1969. The biological aspects of water pollution. Charles C. Thomas Publish- ers. Springfield, 111. 296 pp. Proceedings of the National Shellfisheries Association Volume 65 - 1976 1Z- GROWTH OF THE EUROPEAN OYSTER, OSTREA EDULIS LINNE, IN THE ST. CROIX ARTIFICIAL UPWELLING MARICULTURE SYSTEM AND IN NATURAL WATERS' Judith B. Sunderlin,2 William J. Tobias LAMONT-DOHERTY GEOLOGICAL OBSERVATORY OF COLUMBIA UNIVERSITY PALISADES, NEW YORK 10964 and Oswald A . Roels2 LAMONT-DOHERTY GEOLOGICAL OBSERVATORY OF COLUMBIA UNIVERSITY PALISADES, NEW YORK 10964 AND THE CITY UNIVERSITY INSTITUTE OF OCEANOGRAPHY NEW YORK, NEW YORK 10031 ABSTRACT Three populations of the European oysters, Ostrea edulis Linne, were grown from 3-mm spat to marketable adults in 12 to 16 months in the artificial up- welling mariculture system on St. Croix, U.S. Virgin Islands. The spat were obtained from Pacific Mariculture, Inc., Pescadero, California. The shellfish were fed three species of diatoms, Bellerochea spinifera Harg. and Guill., Chaetoceros cf. simplex Ostf. and Thalassiosira pseudonana Hasle and Heim. Algal cultures, grown in 45,000-liter pools, were pumped continuously through the shellfish tanks; the salinity was 34.75 to 34.95%o and water temperature varied between 22° and 29°C during the experiments. Larvae produced and re- leased by one population were reared through setting, but the spat did not com- plete metamorphosis. For comparison , O. edulis were grown in Salt River, a natural inlet on St. Croix. After 82 days, mortality was 100% in the Salt River population. The salinity range was 33.7 to 37.6%0 and the temperature fluctuated between 25° and32°C. INTRODUCTION systems because the deep water is not diluted In the St. Croix artificial upwellmg maricul- with nutrient-poor surface water. Another ad- ture system, nutrient-rich deep water is vantaee of using deeP water in a mariculture pumped into ponds on shore, where planktonic system 1S that :t 1S free of Pollutants. parasites, diatoms are grown as food for filter-feeding dlseases and predators. The St. Croix site was shellfish. The productivity of this system is chosen because the ocean reaches a depth of much higher than that of natural upwelling l<°°0 meters approximately 1.6 kilometers off shore. Deep water is pumped continuously from 870 ' This work was supported by NOAA Sea Grant 04-3-158-66 meter depth through three 1,830 meter long, 7.5 from the U.S. Department of Commerce. Lamont-Doherty centimeter diameter polyethylene pipelines into Geological ^Observatory Contribution No 2381 City Urn- 45 ofj0 Uter concrete ls in which unialgal versitv institute of Oceanography Contribution No. 99. _ , , ... rrTi_ 2 Present address: University of Texas, Marine Science "inures of planktonic diatoms are grown. The Institute, Port Aransas Marine Laboratory, Port Aransas, pool Cultures are started by inoculating them Texas 78373. with starter cultures grown in 200-gal tanks. 43 44 J. B. SUNDERLIN, W. J. TOBIAS AND O. A. ROELS The pool cultures are operated continuously for up to 30 days. The growth rate of the algae is regulated by the rate at which nutrients are supplied by the incoming deep water, thus as- suring nearly complete utilization of the nutri- ents in the deep water. The algal cultures in the pools are pumped continuously to shellfish tanks, at metered rates, based on the feeding activity of the animals. The total flow pumped to the shellfish matches the flow of deep water into the algal pools, so that the pool volume remains constant. Ten species of shellfish have been screened for growth and survival in the St. Croix system. Eight species grew well and reached market size quickly: they are Ostrea edulis , Crassostrea gigas, Crassostrea gigas Kumomoto variety, Tapes semidecussata, Mercenaria campechien- sis, F, Clam (a cross M. campechiensis x M. mercenaria), Argopecten irradians, and Pinc- tada mertensi. Ostrea edulis Linne was first introduced in the artificial upwelling mariculture system in April 1972. This paper reports the growth of three populations of the European oyster in this system, and the results of comparative growth experiments in two natural environments. MATERIALS AND METHODS In the artificial upwelling mariculture sys- tem, sea water was pumped from 870 m depth in the Caribbean Sea through a one-mile-long pipeline into 45,000-liter pools on shore in which continuous unialgal cultures of planktonic dia- toms— Bellerochea spinifera (clone STX-114), Chaetoceros simplex (clone STX-105) or Thalas- siosira pseudonana (clone 3H) — were grown. The algal cultures (104 to 1011 cells ml-1) were pumped continuously into the shellfish tanks at metered rates. The temperature in the tanks varied between 22° and 29°C. Details of the sys- tem are given by Baab et al. (1973). In Salt River, a natural inlet on the North Shore of St. Croix, the oysters were grown in trays (positioned below the low tide mark) sus- pended from a dock. The water depth at the dock was about 2 m. The tidal range in Salt River is 38 cm. Cultchless, O. edulis used in all experiments were obtained from Pacific Mariculture, Inc., and grown in stacked Nestier trays (Division of Vanguard Industries, Inc., Cincinnati, Ohio). Until the spat were greater than 13 mm in diameter they were held in the trays by liners made from Vi6-inch mesh, plastic-covered fiber- glass window screening. At four-week intervals, wet weight and length (measured from umbo to posterior margin) were determined on a sample of 100 oysters randomly selected from each of the populations. RESULTS AND DISCUSSION In April 1972, 50,000 3-mm O. edulis spat were introduced to the mariculture system. These juveniles were part of an experiment de- signed to select suitable shellfish species for the artificial upwelling mariculture operations. Figure 1 shows the growth of O. edulis over a 16-month period. Total mortality of this popula- tion was 19%. After 13 months, the European oysters averaged 75 mm in length and their average weight was 40.7 gm. The oysters were nearly 100 mm in length and averaged 64.1 gm after 16 months. Bardach, Ryther and Mc- Larney (1972) report that O. edulis grow to market size of 75 mm in diameter or a total weight of 65 gm in four years in France. Our oysters attained market size diameter in 13 months and reached market size weight in 16 months. In April 1973, viable larvae (120-140 n in length) were collected with a 62-/x plankton net from the tank containing the O. edulis. The larvae were reared in 379-liter polyethylene tanks and fed the same algal diet as the adults. Water in the tanks was changed daily and no antibiotics were added. When the larvae began to set, cultch (O. edulis shells) was placed in the tanks. Even though the larvae set, they did not complete metamorphosis and died within a day. Possible causes of this failure of metamorphosis may be of nutritional, environmental or infec- tious origin or a combination of these factors. The larvae were fed only two species of diatoms; literature on larval feeding studies report that Isochrysis galbana, Monochrysis lutheri, Dun- aliella euchlora and Platymonas sp. in unialgal cultures and in mixtures are suitable food for bivalve larvae (Davis and Guillard, 1958; Loos- anoff and Davis, 1963; Walne, 1963; and Walne, 1966). The temperature in the larval rearing tanks reached 29°-30°C in the afternoons. Loos- GROWTH OF THE EUROPEAN OYSTER 45 or UJ h- > O 60 or UJ 0_ E o X o / o /°" ^^ 75mm in length 0X ^ ^0-o-«'0 _ — -— -©"" 0— r~ i 1 . .- 1 1 1 1 1 -t -t 1 -i 1— APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG 1972 1973 FIG. 1. Average weight per oyster (including shell) of the first population of Ostrea edulis in the artificial upwelling mariculture system. St. Croix, U.S. Virgin Islands. anoff and Davis (1963) report that 18°-20°C is optimal for the setting of O. edulis. Throughout the larval study, no antibiotics were added to the standing larval culture. In October 1972, an experiment designed to compare the growth of O. edulis (8-mm juve- niles) in a controlled environment (the artificial upwelling mariculture operation) to growth in an uncontrolled natural environment (Salt River Inlet, see Table 1) was started. The O. edulis in the mariculture operation reached 75 mm in length in 10 months; after 82 days no O. edulis survived in the Salt River Inlet popula- tion (Fig. 2). Land run-off caused by heavy rains increased the silt content of Salt River Inlet. The oysters were covered with 10-20 mm of silt in their trays; this was believed to be the cause of the 100% mortality. During these rains, the salinity in Salt River Inlet reached a low of 33.7%o . However, fouling by sponges and algae was heavy and predators (crabs and drills, Mu- rex pornum Gmelin and Murex brevifrons La- marck) were present. Mortality for the batch of oysters grown in the controlled environment was 81%. This in- creased mortality (first experiment was 19%) can be attributed to the saturated NaCl-dip given to this batch to remove infestations of the bryozoan, Bowerbankia gracilis Leidy. The shellfish were placed in a saturated salt solution (300 gm NaCl per liter of sea water) immedi- ately after they were removed from the shellfish tanks. After one minute in the vigorously aer- TABLE 1. Comparison of the environmental factors in the artificial upwelling mariculture operation and in Salt River Inlet. Environmental Mariculture Op- Conditions eration Salt River Inlet Temperature 22-29 25-32 (°C) Salinity (%o) 34.8-34.9 33.7-37.6 * Phytoplank- 22.4-54.0 0.56-1.14 ton chloro- phyll a (mg/ * Particulate Negligible Low during drought; matter (mg/ (<1) heavy during liter) rainy season Degree of foul- Light—Bow- Heavy— sponges, al- ing erbankia gae, bryozoans, gracilis tube worms, sea Leidy squirts Predators Absent Crabs and Murex brevifrons La- marck; Murex po- rnum Gmelin * Haines, K. C. (unpublished). ated salt dip, the shellfish were air-dried for one hour. On two occasions, however, the oysters were out of water almost an hour before the salt treatment and several days later high mortali- ties occurred. The total mortality for this exper- imental batch was 35% if the percent mortality reported is corrected for the deaths caused by the salt treatments. These oysters were checked for possible disease and/or pathogenic bacteria and nothing was found. In August 1973, an experiment using 3.2-mm 46 J. B. SUNDERLIN, W. J. TOBIAS AND O. A. ROELS spat was begun to substantiate the results of previous experiments on St. Croix and to com- pare growth in other natural environments — Long Island Sound, Virginia, Florida, and Salt River Inlet. Presently, there are O. edulis in only two of these locations — the St. Croix mari- culture system and Greenport, Long Island. Oysters sent to the Virginia Institute of Marine Sciences (Michael Castagna, Eastern Shore Laboratory) arrived in very poor condition and died within a week. From Florida State Univer- sity, R. Winston Menzel reported that the O. edulis were in satisfactory condition on arrival but died after being suspended in flowing sea water. After 55 days, none of the O. edulis in the Salt River Inlet population survived. Silta- >- o q: Ld E o O 20 0— O MARICULTURE OPERATION Hi— □ SALT RIVER INLET .0 _©-© 0' O o ,© ,0' © OT 972 ... gf NOV DEC -O' -O □ JAN 1973 FEB MAR APR MAY JUNE JULY AUG FIG. 2. Average weight per oyster (including shell) of the second population of Ostrea edulis in the artificial upwelling mariculture system compared to growth in Salt River Inlet, St. Croix. MAY JUN FIG. 3. Average length of the third population o/"Ostrea edulis in the artificial upwelling maricul- ture system. The average weight of this population is given in Figure 4. GROWTH OF THE EUROPEAN OYSTER 47 100. 80. 60. 40- .002- .001 A'" -A A .A OSTREA EPULIS POPULATIONS A A APRIL 1972 □ □ OCTOBER 1972 O O AUGUST 1973 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 TIME IN DAYS FIG. 4. Average weight per oyster ( including the shell) of three populations of Ostrea edulis in the artificial upwelling mariculture system. tion was suspected as the cause of death in the Florida and in the Salt River Inlet populations. In July 1974, the oysters in Greenport, New York (Paul Chanley, Shelter Island Oyster Company) averaged 44 mm; in June 1974, the O. edulis in the St. Croix mariculture operation averaged 69 mm in length (Fig. 3). Marketable adults were obtained after 12 months in the mariculture operation; mortality in this experi- ment was 24.3%. CONCLUSION Due to improved handling techniques for op- timization of growth, each successive batch of O. edulis grown in the St. Croix system at- tained market size in a shorter period of time (Fig. 4). The food and oxygen requirements and growth densities were established for the O. edulis grown in the St. Croix mariculture sys- tem. Densities varied with the size of the oys- 48 J. B. SUNDERLIN, W. J. TOBIAS AND O. A. ROELS ter — as juveniles, densities greater than 25/ft- were acceptable; at 40-mm length, 15 to 16/ft-, and at 75-mm length, 7 to 8/ft2. Feeding the oysters three species of diatoms on a rotating schedule appeared to be adequate. Oxygen con- centration in the shellfish tanks was kept at 5 ppt or greater. The excellent growth of O. edulis in the artifi- cial upwelling mariculture system can be attrib- uted to the constant food supply, the deep water source relatively free of particulate matter, and the extended growing season. Planned improvements in the supply of food and management of the oysters in the artificial upwelling system should further reduce the time they require to reach market size. LITERATURE CITED Baab, J. S., G. L. Hamm, K. C. Haines, A. Chu and O. A. Roels. 1973. Shellfish mariculture in an artificial upwelling system. Proc. Natl. Shellfish. Assoc. 63: 63-67. Bardach, J. E., J. H. Ryther and W. O. Mc- Larney. 1972. Aquaculture: the farming and husbandry of freshwater and marine orga- nisms. Wiley-Interscience, New York. Davis, H. C. and R. R. Guillard. 1958. Relative value often genera of microorganisms as food for oyster and clam larvae. U.S. Dept. of Inte- rior, Fishery Bulletin 136, 58: 293-304. Loosanoff, V. L. and H. C. Davis. 1963. Rearing of bivalve mollusks. In: Advances in marine biology, F. S. Russell (ed.), Academic Press, London 1: 1-136. Walne, P. R. 1963. Observations on the food value of seven species of algae to the larvae of Ostrea edulis. I. Feeding experiments. J. Mar. Bio. Assoc. U.K. 43: 767-784. Walne, P. R. 1966. Experiments in the large- scale culture of the larvae of Ostrea edulis L. Fishery Investigations Series II, 25(4): 1-53. Proceedings of the National Shellfisheries Association Volume 65 - 1976 OBSERVATIONS ON SPAWNING AND GROWTH OF SUBTIDAL GEODUCKS (Panope generosa, Gould)1 C. Lynn Goodwin WASHINGTON STATE DEPARTMENT OF FISHERIES BRINNON, WASHINGTON ABSTRACT Histological preparations of subtidal geoducks from Puget Sound were exam- ined to determine their annual reproductive cycle. One annual spawning season occurred in spring and early summer. Most clams were in a spawned-out conditions in late summer. Gametogenesis occurred rapidly in the fall and continued into the winter. By early spring, most were mature and ready to spawn . The growth rate of subtidal geoducks was estimated from mark and recovery studies in five locations throughout Puget Sound. These experiments showed that during the first 3 years of life, marked geoducks grew from 20 to 30 mm/ year in total shell length. Growth of older marked geoducks was less, and the shell length in the majority over 100 mm did not increase at all. Growth rate based upon length frequency distributions from two locations amounted to about 30 mm/year for the first 3 years. This figure more accurately estimated the true growth rate because of the setback in growth caused by handling in the mark and recovery studies. Geoducks are estimated to reach the average adult size of 158 mm in 10 years and, thereafter, growth is reduced. The average length of geoducks in separate populations varied from 123.8 mm to 171.3 mm in samples taken in 22 loca- tions. The largest clam from a sample of 2,037 was 206 mm. The growth rate probably varies considerably from one clam bed to another. The length-weight curve based on 1 £13 pairs of observations can be expressed by the equation: logw weight (in grams) = -3.42983 + 2.97281 logul length (in millimeters). The sigmoid age-weight curve shows that the average 10-year-old geoduck weighs about 1 £00 grams, and the greatest annual-weight gains occur between the third and seventh years. INTRODUCTION The author has observed them in Puget Sound from the lower intertidal zone to depths of over The geoduck is the largest clam in the Pacific 60 m. Puget Sound is hydrographically very Northwest. They range from Alaska to Califor- complex. Dabob Bay and other locations (Fig. 1) nia, and are very abundant in Puget Sound stratify during the summer and are warmed by (Goodwin, 1973), where they are an important soiar radiation to maximum temperatures of sport and commercial clam. They are normally 0ver 20 C at the surface. Minimum temperature found buried 50-60 cm in a sand-mud substrate. in tne winter can be as low as 6 C. In other areas of Puget Sound where waters are more thor- oughly mixed, summer maximums of 15 C or ' The work reported here was partially financed by the leSS are common. Geoducks living at 60 m are National Marine Fisheries Service, Fisheries Research probably never exposed to water Over 10 C. Be- and Development Act, PL 88-309. cause of this complexity and the wide vertical 49 50 C. L. GOODWIN distribution of geoducks, these clams in Puget Sound exist in many temperature regimes. SPAWNING Histological methods Spawning information was obtained from his- tological examination of gonads from 124 speci- mens collected by divers in several locations from July 1968 to August 1969. Sections from transverse slices from the gonads were pre- served in Davidsons' fixative and prepared by standard techniques of dehydrating in alcohol, embedding in paraffin, and staining with Har- ris' hematoxylin and eosin. Sex ratio Of the 124 geoducks studied, 58 were females and 66 were males, virtually a 1:1 ratio (Table 1; Fig. 1). None were observed with both sex prod- ucts. They ranged in size from 65 to 198 mm total shell length. Of the 29 which were less than 120 mm, 15 were females and 14 were males. This information suggests a lack of sex reversal. Anderson (1971) found a ratio of males to females of 51:3 in geoducks shorter than 100 mm, but concluded that they are gonochoristic Leo Reef • own Kilisut Harbor Bay 'V» o ' Bellingham '.. Foulweather Bluff 6. Port Gamble 7. lofall 10. B. Thorndik. Ba Dafcob Bay la 9. 10. : Creek Frenchmans Pt. 11. Fisherman 12. Dosewallips Fiver' 13. 111 1 !.:' in. Agate Passage IS. Rich Passage lb. Blake Tsland 17. Penrose Pt. 18. Pitt Passage '-VW?^2 11. Bay 2] .'■■. Herron 21. Herron Island 22. Peale P l: 23 — -j3f '$. land *~'£iSh 2U. Hunter Pt. OlympLa FIG. 1. Location of sampling stations. TABLE 1. Stage of gonadal development of geoducks in Puget Sound. 1968-1969. Sample location Water depth (me- ters ) Num- ber sample Gonadal condition Early ac- tive Late active Ripe Partially spent Spent Date Male Fe- male Male Fe- male Male Fe- male Male Fe- male Male Fe- male 1968 July 26 Rich Passage 11 1 0 0 0 0 0 0 0 0 0 1 July 29 Frenchmans Point 8-11 10 0 0 0 0 3 0 2 0 1 4 Aug. 23 Frenchmans Point 11 4 0 1 0 0 0 0 2 0 1 0 Sept. 20 Frenchmans Point 8-11 8 2 2 2 1 1 0 0 0 0 0 Oct. 15 Big Beef Creek 8 3 1 0 1 0 1 0 0 0 0 0 Oct. 24 Frenchmans Point 8-11 8 1 1 2 0 4 0 0 0 0 0 Nov. 22 Big Beef Creek 9 8 0 0 0 0 5 3 0 0 0 0 Nov. 27 Frenchmans Point 8-11 5 0 1 0 0 2 2 0 0 0 0 Dec. 19 Hope Island 3 7 0 0 1 3 2 1 0 0 0 0 Dec. 21 Agate Passage 5-6 2 0 0 1 1 0 0 0 0 0 0 Dec. 26 Frenchmans Point 8-11 6 0 0 0 0 1 5 0 0 0 0 1969 Feb. 28 Frenchmans Point 8-11 25 0 0 0 0 10 13 2 0 0 0 April 30 Frenchmans Point 8-11 8 0 0 0 0 4 4 0 0 0 0 May 19 Frenchmans Point 8-11 4 0 0 0 0 2 2 0 0 0 0 May 19 Big Beef Creek 5 8 0 0 0 0 5 3 0 0 0 0 July 3 Frenchmans Point 8-11 7 0 0 0 0 1 2 2 2 0 0 July 10 Leo Reef 18 2 0 0 0 0 0 0 0 1 0 1 Aug. 7 Frenchmans Point 8-11 8 0 0 0 0 1 0 2 2 1 2 SPAWNING AND GROWTH OF SUBTIDAL GEODUCKS 51 and the ratio was due to males maturing at a smaller size than females. STAGE OF GONADAL DEVELOPMENT The seasonal gonadal changes and spawning cycles have been studied and described for many of the important pelecypods of the East Coast of the United States, including the eastern oyster, Crassostrea virginica, Loosanoff (1942), Ken- nedy and Battle (1964); northern quahog, Mer- cenaria mercenaria, Loosanoff (1937), Porter (1964); soft shell clam, Mya arenaria. Ropes and Stickney (1965), Shaw (1962), Pfitzenmeyer (1965); coot clam, Mulinia lateralis. Calabrese (1970); and the surf clam, Spisula solidissima, Ropes (1968). Similar studies have been conducted on the West Coast pelecypods, such as littleneck clam, Paphnia staminea, Quayle (1943); jingle, Podo- desmus cepio, Leonard, Jr. (1969); mussel, My- tilus edulis, Moore and Reish (1969); native oyster, Ostrea lurida, Coe (1932); gaper clam, Tresus capax, Machell and DeMartini (1971); geoduck, Andersen (1971); manila clam, Vene- rupis japonica, Holland and Chew (1974); and the softshell clam, Porter (1974). Many different schemes have been used in these papers to describe the various stages of gametogenesis. For the present study, gameto- genesis was divided into five stages after Ropes (1968) except the stages "spent" and "resorbing" were combined into one called "spent". 1. Early active: Follicles are small and contain early stages of sex cells. Connective tissue is abundant. 2. Late active: Follicles are enlarged and con- tain sex cells of different stages of develop- ment. Male follicles contain spermatozoa and early spermatogenic stages. Female follicles contain primary oocytes, many of which are still attached to the follicle walls. Connective tissue is reduced in amount. 3. Ripe: Follicles are full size and filled with spermatozoa and oocytes. Connective tissue reduced to thin layer between the follicles. 4. Partially spent: Follicles are reduced in size and often partially empty. Connective tissue increasing in amount. 5. Spent: Follicles are greatly reduced in size and contain few if any mature sex cells. Con- nective tissue is greatly increased in amount. SEASONAL SEXUAL CYCLE OF FEMALES Spawning begins in the spring with the major release of ova occurring during June. The clams examined at the end of July 1968 and during the first of July 1969 were either partially or com- pletely spent. Most gonads in this stage con- tained a few residual ova in the contracted folli- cles. Connective tissue between the follicles was thickened. Females examined in September were beginning to show active gametogenesis. October and November samples revealed that gametogenesis was proceeding rapidly with the follicles proliferating and invading the connec- tive tissues and with oocytes enlarged and at- tached to the follicle walls. By January, all females contained follicles with oocytes of vary- ing stages of development, some of which were the mature size.2 All females appeared to be mature and ready to spawn in April. The go- nads were large, distended, and creamy in tex- ture. The follicles were large and filled with ova. The follicle wall was thin and most of the connective tissue between the follicles had dis- appeared. The connective tissue between the follicles contains inclusions (probably glycogen or lipid) which are abundant during early stages of gametogenesis and are gradually lost as the gonad ripens. These findings are in general agreements with those of Andersen (1971), who stated that geoducks spawn in a single mode from March to July. He found them partially spent as early as January, and completely spent in August. SEASONAL SEXUAL CYCLE OF MALES All males apparently do not follow as concise a seasonal pattern as do the females. Some males with sperm were observed in all months sampled. The majority, however, do follow a pattern similar to that described for females. Andersen (1971) found no difference between females and males in the timing of the annual gonadal cycle. The major release of sperm oc- curred during May and June, so that by July Oocytes stripped from clams sampled in June and mea- sured in wet smears were 75 /x. Newly released ova in natural spawnings averaged about 82 ix. 52 C. L. GOODWIN most males were partially or completely spent. The typical male sampled in August has con- tracted and partially empty follicles. Connec- tive tissue between the follicles is abundant. Six of the 15 males examined in September and October contained mature sperm, and the other nine were in active gametogenesis stages. By April and May, all males examined appeared ripe. LABORATORY SPAWNING OF GEODUCKS Additional information on the geoduck sexual cycle was obtained from 35 spawning experi- ments conducted in the laboratory. Thermal stimulation was used to initiate natural release of gonadal products in the majority of these experiments. The spawners were held in heated water pumped from Dabob Bay. The majority of these experiments were conducted within a few days after the clams were collected. The short time that the spawners were held in the labora- tory before spawning probably had little effect on the annual timing of spawning. Some spawn- ing was accomplished by mechanically stripping the gonads of sperm and ova. Natural labora- tory spawning, which produced normal larvae, has been initiated with thermal stimulation as early in the winter as January 10. Geoducks have spawned in water ranging from 8.5 C to 16.0 C, with the majority of successful spawn- ings occurring in water of 12 C to 14 C. Spawn- ing occurred in the laboratory as late as July 5. Mechanical stripping experiments, which have resulted in normal larval development, were completed as early as November 29, however, none were attempted later than June 1 1 . Spawning in nature was observed twice by our divers near Frenchmans Point. One oc- curred on April 20, 1969, in 9-12 m of water at 8- 10 C, and the other in 8-9 m of water on July 13, 1969, when the water temperature was not mea- sured. In each case, only a single clam was spawning. The sex products flowed from the excurrent siphon continuously over a period of several minutes. GROWTH Annual ring method Geoduck shells from many locations in Puget Sound were examined for annual rings. Diffi- culty was encountered in determining whether or not a ring was an annulus or not. After considerable effort, this method was judged to be unfeasible for geoduck age or growth esti- mation. Andersen (1971) came to the same con- clusion in his study of geoducks from Hood Canal. Tegelberg (1964) questioned the validity of the ring method in determining the growth of razor clams. Mark and recapture method Mark and recapture experiments were con- ducted at various locations in marked plots in substrates of sand and mud mixtures in water depths ranging from 5 m to 14 m calculated from zero tide (Table 2; Fig. 1). Geoducks were first removed from the plots by divers with small hand-held washout nozzles or venturi suction dredges. The same number of geoducks that were taken from a plot were marked, measured, and planted into the plot. Numbers were ground into the shell in large clams with thick shells, and small clams were marked with waterproof ink. Only the total length (greatest anterior-posterior distance) of the right valve was measured. Large geoducks were planted in individual holes excavated by a venturi dredge. They were planted in the sub- strate at a depth of about 60 cm, which is the depth at which most adult geoducks live. Small geoducks, being good diggers, were placed in small holes made by the diver's fingers and protected for a few minutes until the clams com- pletely buried themselves. Small wire stakes were placed near each planted geoduck to facili- tate future recapture. Four hundred-ninety geoducks were marked and planted in five separate plots. Of these, 202 were recovered: 107 alive and 95 dead (Table 2). The majority of the mortalities was due to han- dling. The geoducks in this table were divided into two groups, those that were less than 100 mm and those more than 100 mm total shell length at marking. The smaller geoducks showed substantial growth. The greatest rate of growth for any single group of clams was 2.8 mm/month, or 33.6 mm/year. These clams, which were planted at Fishermans Point, averaged 48 mm in shell length at marking. The average growth rate for all geoducks less than 100 mm was 1.8 mm/ SPAWNING AND GROWTH OF SUBTIDAL GEODUCKS 53 TABLE 2. Mark i md recapt lire data of five growth experiments (total shell length in mm). Number marked Number recovered Date planted Date re- covered Average length Aver- of geo- age ducks at length marking change Range of length change Greatest length change per month in any indi- vidual Average length change Experiment Alive Dead per month Clams more than 100 mm total shell length at mark ing Big Beef Creek 79 9 18 11/68 11/69 141.2 + 1.2 -3 to +5 + 0.4 + 0.1 Port Gamble A 81 10 30 12/69 2 in 1/71 141.0 -1.5 -2 to -1 -0.2 -0.1 8 in 5/72 133.5 -1.4 -3 toO -0.1 0.0 Agate Pass 86 25 27 1/70 6 in 11/70 131.7 -0.3 -1 to +4 + 0.4 0.0 19 in 5/72 133.9 -1.3 -7 to +1 -0.2 0.0 Herron Island 91 16 17 2/70 4 in 1/70 142.8 -0.7 -2 to 0 -0.2 -0.1 12 in 7/72 125.8 + 1.9 -5 to +15 + 0.5 + 0.1 Cla ms lest ; than 100 mm total shell length at marki ng Big Beef Creek 5 1 0 11/68 11/69 81.0 — - - - Port Gamble A 19 8 2 12/69 1 in 1/71 95.0 0 0 0 0 7 in 5/72 60.4 + 18.2 + 2 to +38 + 1.3 + 0.6 Port Gamble B 18 6 0 5/70 4 in 1/71 31.6 + 11.2 + 5.3 to +17.5 + 2.2 + 1.4 2 in 5/72 27.5 + 24.5 + 22 to +27 + 1.1 + 1.0 Agate Pass 11 3 0 1/70 5/72 54.3 + 43.0 + 5 to +62 + 2.2 + 1.5 Herron Island 9 2 1 2/70 6/72 85.5 + 25.5 + 15 to +36 + 1.2 + 0.9 Fishermans Point 91 27 0 2/71 10 in 9/71 39.6 + 20.2 + 11 to +39.5 + 5.0 + 2.5 3 in 2/72 47.7 + 27.3 + 16 to +33 + 2.8 + 2.3 5 in 12/72 48.0 + 61.4 + 54 to +73 + 3.3 + 2.8 2 in 12/73 51.0 + 87.0 + 84 to +90 + 2.6 + 2.6 7 in 10/74 41.8 + 95.6 + 80 to +115 + 2.7 + 2.2 Total 490 107 95 month, or 21.6 mm/year. The greatest rate of growth shown by an individual geoduck was 5 mm/month, or 60 mm/year. Significant growth did not occur in clams larger than 100 mm. Many of these larger clams showed a loss in total shell length from marking to recovery, apparently due to shell recession. Shell recession was also observed by Andersen, 1971. The marking process produced a significant TABLE 3. Relationship between size of geoducks and rate of growth (length in mm). Length at marking Num- ber of geo- ducks Regression intercept Slope Predicted increase in length after 52 weeks 16-39.9 20 + 9.4 + 0.28 23.8 40-59.9 10 + 7.0 + 0.46 30.8 60-79.9 4 + 18.2 + 0.04 20.2 80-99.9 7 -2.6 + 0.05 0.0 100-119.9 15 + 0.9 + 0.00 1.0 120-139.9 6 + 0.6 -0.01 -0.2 140-159.9 22 + 1.0 -0.02 0.0 160-179.9 4 + 0.6 -0.01 0.2 setback in growth. The setback caused pro- nounced checks in the rings of the shells. The shock of mark and recapture was apparently more severe in the larger clams since these clams collectively showed no growth after marking. The relationship between the size of the clams and growth is shown in more detail in Table 3. The data in the table are linear regressions of time from marking to recovery on change of length and include clams from all five locations listed in Table 2. The table shows an inverse relationship of size and rate of growth for marked clams and that small clams are capable of growing about 30 mm/year in shell length. Length frequency distributions Length frequency distributions were pre- pared from data collected from Fishermans Point and the mouth of the Dosewallips River (Fig. 2). The year classes were identified and the length differences between year classes used as a measurement of the yearly growth incre- ment. The samples from Fishermans Point were 54 C. L. GOODWIN obtained by divers who collected all geoducks observed regardless of size. Those from the Do- sewallips River flats may be biased for size be- cause they were obtained from sport clam dig- gers who normally select larger clams. Fig. 2-A, which depicts geoducks taken at Fishermans Point in December 1969, is typical of an unharvested subtidal population in that it is unimodal with the peak at 165 mm and in- cludes very few small geoducks. Many geoducks were removed from this population for various experiments between December 1969 and April 1970. In May 1970 another sample was taken and at this time a group of small geoducks was present which averaged 30 mm total shell length (Fig. 2-B). Whether or not sampling had stimulated setting was undetermined. The small clams were believed to have set in the spring of 1969 and to be 1 year old. The 1969 year class was still present in Janu- 40 30 20 10 0 40 30 20 10 0 40 30 20 10 0 30 20 10 0 30 20 10 n 30 20 10 0 50 40 L 30 • 20 • 10 0 50 40 30 1. 20 10 0 0 Fishermans Pt. Dec. 1969 Fishermans Pt. May 1970 1 year old average length v*-^ 30 mm (1969 set) 7 months old average length 21 ,im (1970 set) 1 year 7 months old average length 50 mm Fishermans Pt. Jan. 1971 1 year 3 months old average length 35 mm 2 years 3 months old average length 78 mm Fishermans Pt. Sept. 1971 (1971 set) A 2 years 9 months old average length 74 2 years 6 months (1972 set) old average a length 96 roru 3 years 6 months old average length 11 3 mny 2 years old average length 59 Fishermans Pt. Feb. 1972 Fi&£i™^2Pt- Dosewallips River June 1968 3 years old average lengthy 84 mm 2 years old average length 62 Dosewallips River June 1972 20 40 60 80 100 120 140 160 180 200 220 FIG. 2. Length frequency distributions of geoducks from Fishermans Pt. and Dosewallips (shell length in mm). SPAWNING AND GROWTH OF SUBTIDAL GEODUCKS 55 ary 1971 when another sample was taken (Fig. 2-C). They had increased to an average shell length of 50 mm. Some of these clams were marked at this time and replanted, thereby al- lowing positive identification of the year class at future sampling dates. Another year class was present which resulted from a set in the spring of 1970, and averaged 21 mm total shell length. The large adult clams were still present and unchanged at an average of 165 mm in total shell length. Both small year classes were present in Sep- tember 1971, February 1972, and December 1972, as well as the large adult clams which remained unchanged in average total shell length (Fig. 2-D, 2-E, and 2-F). The Dosewallips River flats were sampled on two occasions, and the geoducks are depicted in Fig. 2-G and 2-H. This population has been intensively harvested by recreational clam dig- TABLE 4. Shell length of geoducks based on length fre- quency distribution (length in mm). Ag 3 in years 1 2 3 Fishermans Point Average length 33.8 62.4 96.5 Sample size 65.0 61.0 17.0 Dosewallips River Average length - 61.0 84.0 Sample size - 26.0 11.0 Average length 33.8 62.0 91.6 gers for many years. The distance between the peaks in the curves of Fig. 2 indicates that for the first 2 years of life, geoducks grow about 30 mm/year. The average length of the various year classes is shown in Table 4. GROWTH DISCUSSION The mark and recapture experiments showed that growth in small geoducks was substantial but very slow, if at all, in large geoducks. Growth was found to be set back by the marking and planting procedure. Large clams seemed to be more affected than small ones. Due to the setback, the rate of growth estimated by this method is thought to be slightly less than the true rate. The growth estimate determined from length-frequency distributions probably gave the most accurate results and is a reliable method when enough is known about the popu- lation studied to be sure of making accurate judgments of the various year classes in the population. A growth curve based on the data of Table 4 is shown in Fig. 3. The end point of the curve (158 mm) is the average adult size for geoducks in Puget Sound. The average age was calculated from 22 locations where at least 20 geoducks were taken (Table 5). The largest one-half of the geoducks of each sample was selected and an average shell length calculated. These mean lengths varied from a high of 183.9 mm at Kili- sut Harbor to a low of 138.7 mm at Foulweather Bluff, and averaged 157.7 mm. FIG. 3. Relationship between shell length and age of geoducks. 56 C. L. GOODWIN TABLE 5. Average shell length ofgeoducks from 24 loca- tions in Puget Sound (length in mm). Area Sample size Total sample Largest one- half of geo- dueks from sample Jamestown 71 140.1 157.4 Kilisut Harbor 21 171.3 183.9 Useless Bay 85 134.1 146.9 Foulweather Bluff 95 126.8 138.7 Port Gamble 179 134.4 148.1 Lofall 25 129.8 142.9 Thorndike Bay 58 134.6 148.8 Big Beef Creek 77 147.6 156.9 Frenchmans Point 40 133.8 157.7 Fishermans Point 99 163.8 175.0 Dosewallips River 229 135.8 156.1 Indianola 89 123.8 142.5 Agate Passage 78 148.5 164.7 Blake Island 75 132.8 146.7 Penrose Point 39 154.7 167.7 Pitt Passage 48 146.8 160.0 Hogum Bay 32 145.5 157.1 Herron 33 144.7 158.9 Herron Island 233 146.1 155.9 Peale Passage 20 140.5 157.5 Hope Island 34 171.1 181.2 Hunter Point 32 156.4 165.4 Total 1,692 x = 143.8 x = 157.7 The time interval needed for the average geo- duck to reach 158 mm is unknown. However, an estimate is given in Fig. 3. The data for the first 3 years are mine; and for years 4 and 5, data from Andersen (1971) are used. The curve was extended beyond the fifth year and crosses 158 mm on the shell length axis between 8 and 10 years. The curve is extended beyond the data and is offered only as an estimate of the general growth pattern for subtidal Puget Sound geo- ducks based on present data. The average size of geoducks varies greatly from one population to another (Table 5), and I would expect that growth rates would also vary considerably from one bed to another. Andersen (1971) gives geoduck growth incre- ments based on length-frequency histograms for the first 5 years. He developed a von Bertalanffy growth curve which fits the data well for the first 5 years, and then extrapolated the curve beyond the data to 40 years. The curve is similar to Fig. 3 for the first few years, but goes above 200 mm at the end. I believe the curve is too high at the end of the growth phase. Of the 2,037 geoducks taken from numerous locations in Puget Sound from unexploited subtidal stocks, 3000 - , / 2800 - 2600 - '/ 2400 . , / 2200 - 1 / t / 1 2000 - 2 1 / 1 1 1800 . 1 1 1 2/ 1 1 31233/22 1 112 1 234 /2 1 1 1 2 1 412/ 1 1 1 I 1600 - ' 1 1 1 2 1 1 312/234 2 i t 242 JZM 1 1 1 I I 1 1 3 1232/2121 1 1 I 1400 - I 2I24I4M 421 1 1 2 1 1 217233/4615 III 1 1 2241 3 3SMS3 12 12 1 1 1 2 638 A/157515 1 1 1200 - 14232634*3723421 1 I 1 1 122 437S33/A44 2321 1 1 1 1316734*24984 1 1 1 1000 - ' 1 1 3236527946B422I2I 1 I4234353»4§<5877 5l 1 Z 1 142254*768^77453441 1 t 800 - I z 3 3476 46*466444121 1 221449436743 2111 1 21 1 1 l 73_B4l32l54l 2222 1 600 . 12342 fr/e«23222 ! ,613234111 12 314^ 351 231 21 1 1 i ,^2 36 1 1 1 1 111 400 - I 3 sy ■?2222233i 1 1 1 1 2 (211 1 l 1 1 1 <^ 1234] 2 1 1 200 - ,*- i**f32 2 2 2 ', i 1 1 o """111 ~&rTz 234 3Z247 12 1 1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 Total lenqth right valve 1n nm. FIG. 4. Relationship between total shell length and whole wet weight. SPAWNING AND GROWTH OF SUBTIDAL GEODUCKS 57 FIG. 5. Relationship between whole wet weight and age of geoducks. only four had been above 200 mm total shell length, the largest of which was 206 mm. The average length was only 142.9 mm. The relationship between shell length and whole wet weight is shown in Fig. 4. In obtain- ing the relationship of log,,, W = -3.42983 + 2.97281 log,,, L, 1,213 pairs of observations were used. Due to an oversight, the 66 geoducks of less than 50 mm shell length of Fig. 4 were not included in the equation. Data from the length- weight and age-length curves were combined in Figure 5. The curve shows that the greatest annual weight gain occurs between the third and seventh year of life. The average whole, freshly-dug geoduck con- sists of about 56% meat. Meat is defined as all portions of the clam minus the shell and water of the major body cavities. LITERATURE CITED Andersen, A. M., Jr. 1971. Spawning, growth, and spatial distribution of the geoduck clam, Panope generosa, Gould, in Hood Canal, Washington. Ph.D. Thesis, Univ. of Wash., Wash. Coop. Fish. Unit. 133 p. Calabrese, A. 1970. Reproductive cycle of the coot clam, Mulinia lateralis (Say), in Long Island Sound. Veliger, Vol. 12, No. 3, p. 265- 269. Coe, W. R. 1932. Development of the gonads and the sequence of the sexual phase in the Cali- fornia oyster (O. lurida). Bull. Scripps Inst. Oceanog. Tech. Ser. 3: 119-144. Goodwin, C. L. 1973. Subtidal geoducks of Puget Sound, Washington. Wash. Dept. Fish. Tech. Rept. No. 13, 64 p. Holland, D. A. and K. K. Chew 1974. Reproduc- tive cycle of the manila clam, Venerupis ja- ponica, from Hood Canal, Washington. Proc. of the Nat. Shellf. Assoc, Vol. 64. Kennedy, A. V. and H. I. Battle 1964. Cyclic changes in the gonad of the American oyster, Crassostrea uirginica (Gmelin). Canadian Journal of Zoology, Vol. 42, p. 305-321. Leonard, V. K., Jr. 1969. Seasonal gonadal changes in two bivalve mollusks in Tomales Bay, California. Veliger, Vol. 11, No. 4, p. 382-390. Loosanoff, V. L. 1937. Seasonal gonadal changes of adult clams, Venus mereenaria (L). Biol. Bull. 72: 406-416. Loosanoff, V. L. 1942. Seasonal gonadal changes in the adult oyster, Ostrea virgin ica, of Long Island Sound. Biol. Bull. 82. No. 2, p. 195-206. Machell, J. R. and J. D. DeMartini 1971. An annual reproductive cycle of the gaper clam, Tresus capax (Gould), in South Humboldt Bay, California. Calif. Dept. Fish, and Game. 57(4): 274-282. Moore, D. R. and D. J. Reish 1969. Studies on the Mytilus edulis community in Alamitos Bay, California — IV. Seasonal variation in gametes from different regions in the bay. Veliger 11:250-255. Pfitzenmeyer, H. T. 1965. Annual cycle of game- togenesis of the soft-shelled clam, Mya aren- aria, at Solomons, Maryland. Chesapeake Science, Vol. 6, No. 1. p. 52-59. Porter, H. J. 1964. Seasonal gonadal changes of adult clams, Mereenaria mereenaria (L), in North Carolina. Proc. of the Nat. Shellf. As- soc, Vol. 55. 58 C. L. GOODWIN Porter, R. G. 1974. Reproductive cycle of the soft-shell clam, Mya arenaria, at Skagit Bay, Washington. Fish. Bull. Vol. 72, No. 3., p. 648-656. Quayle, D. B. 1943. Sex, gonad development and seasonal gonad changes in Paphnia staminea (Conrad). J. Fish. Res. Bd. Can., Vol. 6, No. 2, p. 140-151. Ropes, J. W. 1968. Reproductive cycle of the surf clam, Spisula solidissima, in off-shore New Jersey. Biol. Bull. (Woods Hole) 135(2): 349- 365. Ropes, J. W. and A. P. Stickney 1965. Reproduc- tive cycle of Mya arenaria in New England. Biol. Bull., Vol. 128, No. 2. p. 315-327. Shaw, W. N. 1962. Seasonal gonadal changes in the female soft-shell clam, Mya arenaria, in the Tred Avon River, Maryland. Proc. of the Nat. Shellf. Assoc, Vol. 53. Tegelberg, H. C. 1964. Growth and ring forma- tion of Washington razor clams. Wash. Dept. Fish. Res. Pap., 2(3): 69-103. Proceedings of the National Shellfisheries Association Volume 65-1976 CLAM MARICULTURE IN NORTHWEST FLORIDA: FIELD STUDY ON PREDATION' R. W. Menzel, E. W. Cake-, M. L. Haines, R. E. Martin' and L. A. Olsen DEPARTMENT OF OCEANOGRAPHY FLORIDA STATE UNIVERSITY TALLAHASSEE, FLORIDA 32306 ABSTRACT Twenty thousand small hatchery reared Mercenaria clams were planted for a period of nine months in a predation experiment at two field locations in Northwest Florida. At each location four 4.5 M- plots were established , in each of which were planted 2500 clams. Before planting the clams, one plot was prepared with a substrate of pea gravel and one with crushed oyster shell at each location . Two plots at each location received no substrate additive and served as controls. One control plot at each location was covered with a wire cage to exclude predators. Survival was over 50% in the wire covered control plots; less than 1 % in the unprotected control plots; slightly more than 2% in the plots with shell and 10% in the plots with gravel. In this area of Florida gravel and crushed shell added to the substrate do not ensure satisfactory survival of small elatyis. INTRODUCTION Mariculture of quahog clams (Mercenaria ) has been advocated for some time (Menzel & Sims, 1961) as certain procedures were developed. Further refinements of techniques, as well as development of new ones, should enable such a venture to become even more attractive and result in clam mariculture becoming a viable reality. The advancements made so far have been experimental. Hybrids between the northern M. mercenaria (L.) and the southern M . canipe- chiensis (Gmelin) have better commercial traits than either of the parent species, at least in Sponsored by NOAA, Office of Sea Grant, Department of Commerce, under Grant #04-3-158-43. The U. S. Govern- ment is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notations that may appear hereon. 1 Present address: Gulf Coast Research Laboratory, Ocean Springs, Mississippi. ' Present address: Florida Board of Natural Resources, Marine Laboratory St. Petersburg, Florida. warmer southern waters. The northern quahog can remain closed, alive and in good condition for a considerable period, which permits the very valuable shell trade of cherrystones and littlenecks, whereas the southern species quickly gapes and spoils when removed from the water. Although the northern clam has an annual growth rate greater in Florida than in more northern waters, the growth rate is not as great as that of the native southern clam. The hybrid between the two species has the storage qualities of the northern parent and the fast growth rate of the southern parent (Menzel, 1962, 1968, 1971, 1974; Menzel and Sims, 1962). For the past two years we have been investi- gating F,, F2 and F3 hybrids as well as back- crosses of the two species to determine if certain combinations of crosses have even better growth rates, using original parent species from var- ious areas along the Atlantic and Gulf coasts of the United States. It is fairly easy to induce spawning and to rear the larvae through to setting stage, and up to 1-2 mm, under the controlled conditions of a 59 60 R. W. MENZEL, E. W. CAKE, M. L. HAINES, R. E. MARTIN AND L. A. OLSEN hatchery. At the present time it is more practi- cal to grow clams to marketable size in open waters where they can feed on naturally occur- ring food, than to rear them under the con- trolled conditions in a closed system, where the costs of space, equipment, food water quality and labor may be prohibitive.4 We have never been able to obtain growth rates in the laboratory comparable to those in the field. Since we are interested in the fastest growth rate and maturity possible, we plant clams in open water as soon as possible. We rear small clams (1-2 mm at planting) in the field, but encounter excessive mortalities, mainly from crab predation, unless protected. Experi- mentally we control predation by using sand- filled containers, covered with plastic window screening, over which we place open-ended wire cages (1.2 cm mesh), pressed into the bottom. We have successfully grown larger (15 mm at planting) clams within fences of wire and net- ting of 1.2 cm mesh), pressed into the bottom. We have successfully grown larger (15 mm at planting) clams within fences of wire and net- ting of 1.2 cm mesh. With our present tech- niques, the costs of rearing clams to 15 mm before planting in the field, are excessive from a commercial standpoint. We need a low-cost method whereby small clams (1-2 mm or less) can be planted, protected and reared in open water. Castagna, Mason and Briggs (1970) reported that "clams as small as sand grains" were planted in Virginia waters with excellent re- sults. They planted clams in a "protective" bot- tom prepared with a substrate (gravel, slag or shell). This paper reports on our attempts to use this technique in Florida waters. METHODS AND MATERIALS In the spring of 1972, in the course of our experiments on selection and hybridization, we reared excesses of clams. These clams were kept in holding tanks (1 x 1 x 0.65 M) supplied with continuously pumped seawater from the adja- cent bay at our Ed Ball Marine Laboratory, The school of Marine Science, University of Delaware is now operating a pilot closed system facility for rearing mollusks. Turkey Point, Florida. No substrate was pro- vided in the tanks and the accumulated silt was flushed out at intervals. The clams grew, but not at a spectacular rate. In the fall of 1972, the clams were used in a "protective substrate" ex- periment at Turkey Point and at Alligator Har- bor, the site of the former marine laboratory, Florida State University. In both locations clams were planted in a firm bottom, several centimeters below mean low water. Substrate samples from the two locations were analyzed for sand, silt, clay, organics and carbonate content. The range of salinities at both locations was 25-35%o, with an average ca. 30%o at Alligator Harbor and 28%o at Turkey Point. The clams had different parents for plantings in the several plots and the pedigrees were dif- ferent for the two locations. All were back- crosses. Those planted at Alligator Harbor were the progeny of an F, female (wild female M. campechiensis x wild male M. mercenaria tex- ana) crossed with a wild male M. campechien- sis. The pedigree for those planted at Turkey Point was the same for the male parent (wild M . campechiensis) but the female parent was an F, hybrid of wild female M. mercenaria x wild male M. campechiensis. The size range when planted (4-20 mm) ranged mostly from 7 to 10 mm long. The clams were removed from the tanks, allowed to dry and sprayed with red en- amel paint, prior to planting, to distinguish them from any that might be recruited from the wild population. After the paint had dried the clams were returned to the tanks for a period before planting. The treatment produced no mortality and the color is still readily visible after nearly two years on the remaining surviv- ing clams. Wooden frames of 5 cm x 10 cm lumber, having inside dimensions of 1.5 M x 3 M were constructed and four of these frames were placed at each location, parallel to each other about a meter apart. The frames were partially buried in the bottom and secured with stakes. At each location two of the plots were used as controls with no substrate added. One of the plots at each location was planted with ca. 5 cm of pea gravel and one with ca. 5 cm of crushed oyster shell. In each plot of 4.5 M'-, 2500 marked clams were hand-scattered as evenly as possible CLAM MARICULTURE IN NORTHWEST FLORIDA 61 at a low spring tide. At each location one of the control plots was covered with an open bottom, 1.2 cm mesh plastic-coated wire cage, which was pressed into the bottom 5-7.5 cm. The top sur- face of the cage was 7.5-10 cm above the bottom. The plantings at each location were examined at intervals and at the termination of the exper- iment the entire area of each plot was dug care- fully and the clams recovered. RESULTS The bottom substrate analysis showed no sig- nificant differences at the 5% level (Student t test) in sand and silt at the two locations. Per- centage sand ranged from 91-99. There were significant differences (Student t test at 5% level) in clay (mean 0.6% at Alligator Harbor, 3.5% at Turkey Point), organics (mean 0.5% Alligator Harbor, 1.5% Turkey Point) and car- bonates (mean 1.2% Alligator Harbor, 7.1% Turkey Point). These higher values at Turkey Point may have been due to the presence of a four-year old spoil deposit of sand, clay and limestone ca. 100 meters west and extensive seagrass beds ca. 25 meters south of the loca- tion. Table 1 summarizes the plantings at the two locations and the survival under each treat- ment. The clams at the time of termination were ca. 1.5 years old and had been exposed to the open environment ca. 0.8 years. The sur- vival was very low in the plots containing gravel and shell, only slightly better than the control plots with no protection at all. Pea gravel did provide better protection than shell. The best survival was under the cages. Various known predators were observed on the plots at the two locations, and on occasions were observed feeding on the planted clams. TABLE 1. Clam mortality field experiment. Plots of 4.5 square meters, each with 2500 marked clams. Planted November 1972; examined September 1973. Location I -Alligator Harbor; Location II -Turkey Point. LOCATION Substrate Type I Survival # % II Survival # % I&II Survival # % Control Shell 1 0 0.1 0.0 31 109 1.0 4.8 32 0.6 109 2.2 Gravel 18 0.6 487 19.5 505 10.1 Control (wire 901 36.0 2035 81.4 2936 58.7 cage) The predators appeared to be more numerous at Alligator Harbor, but no quantitative data are available. The predators include lightening whelks, Busycon contrarium (Conrad), moon snails, Polinices duplicatus (Say), blue crabs, Callinectes sapidus (Rathbun), and stone crabs, Menippe mercenaria (Say). Feeding depressions created by sting rays, Dasyatis spp. and butter- fly rays, Gymnura micrura (Bloch & Schnei- der), were seen in the plots. Many broken as well as intact, empty shells were recovered when the experiment was terminated, but their numbers were not recorded nor were attempts made to determine the probable cause of mortal- ity. DISCUSSION AND CONCLUSIONS Although clam survival under the wire cage control plots at Turkey Point is considered to be commercially satisfactory, the survival at Alli- gator Harbor was lower than usually obtained from other observations with caged enclosures. Perhaps the relatively low survival can be ex- plained by predators gaining entry. No routine inspections were made to be certain that the cage was firmly entrenched in the bottom. On one occasion a corner of the cage was found to be out of the bottom. Survival in protective substrates was several fold greater than in control plots with no preda- tor protection, but unacceptably low for com- mercial operations. It is assumed that the mor- tality resulted from predation and not from some other factor(s). There is little or no current at either location that might aid clam migra- tion, besides the abundance of dead marked shells many showing evidence of predation, be- lied this as a factor in the recovery of live clams. There is a remote possibility that gravel and shell hash caused mortality. A control for as- sessment of this possibility was not included in the tests, i.e. no graveled or shell planted plots were covered with wire cages. The different survival rates obtained in Vir- ginia and Florida, using similar substrate addi- tives, could be explained in several ways. First, particle size and amount of protective substrate may have been different. Second, we have a plethora of predators in Florida, more so than in Virginia, and our milder winter season permits activity of predators almost year round. 62 R. W. MENZEL, E. W. CAKE, M. L. HAINES, R. E. MARTIN AND L. A. OLSEN In our experiments the sizes of clams, when planted, was much greater than the sand-grain size reported in Virginia. This larger size should have lessened the clams' vulnerability to preda- tors and the survival rate should have been higher than that obtained for Virginia waters. In conclusion, for commercial mariculture, survival of planted clams must be at a profitable level, which we judge to be not less than 509c. Based on our observations in Florida, adequate survival cannot be obtained with protective sub- strate additives such as gravel and shell. Fenc- ing, with constant attention to make certain that predators do not gain entry, does give good survival. This method is expensive and cheaper and effective methods must be found before clam mariculture will become financially re- warding. LITERATURE CITED Castagna, M. A., L. W. Mason and F. C. Briggs. 1970. Hard clam culture method developed at VIMS. Va. Inst. Mar. Sci., Sea Grant Advi- sory Pgt., No. 4: 3 pp. Menzel, R. W. 1962. Seasonal growth of north- ern and southern quahogs, Mercenaria mer- cenaria and M. campechiensis, and their hy- brids in Florida. Proc. Natl. Shellfish. Assoc, 53, 111-119. Menzel, R. W. 1968. Cytotaxonomy of species of clams iMercenaria) and oysters (Crassos- trea). Symp. On Mollusca., Mar. Biol. Assoc. India. Part I: 75-84. Menzel, R. W. 1971. Quahog clams and their possible mariculture. Proc. World Maricul- ture Soc, II: 23-36. Menzel, R. W. 1974. Clams and oysters as ani- mals for mariculture in the United States. Proc. Marine Tech. Soc, 10th: 611-617. Menzel, R. W. and H. W. Sims. 1962. Experi- mental farming of hard clams, Mercenaria mercenaria, in Florida. Proc. Natl. Shellfish. Assoc. 53: 103-109. Proceedings of the National Shellfisheries Association Volume 65-1976 SCALE-UP OF FOOD UTILIZATION BY THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA (GMELIN) Paul N. Walker AGRICULTURAL ENGINEERING DEPARTMENT UNIVERSITY OF ILLINOIS URBANA, ILLINOIS and John W. Zahracinik UNIVERSITY OF MASSACHUSETTS AQUACULTURAL ENGINEERING LABORATORY WAREHAM, MASSACHUSETTS ABSTRACT An approach for studying scale-up of food utilization by shellfish is presented. The approach is based on the fact that food concentration is a primary variable controlling food removal and growth of filter- feeders. Twenty raceways were constructed each approximating a tubular reactor by a series often stirred-tank reactors. Each section contained ten second-year oys- ters. Each raceway received water at a different combination of flow rate and relative food concentration . The relative concentrations were formulated by mixing unfiltered seawater with filtered seawater. The oysters were allowed to grow relatively undisturbed for a period of 121 days. Analysis of the weight growth data indicate that scale-up of a filter feeding-system is possible using space time as a scaling factor. It is also demonstrated that higher conversions occur with lower concentrations. How- ever, a much larger raceway is required to obtain the same amount of growth . NOMENCLATURE a, b, c, d Molal ratios, dimensionless A Flow rate of food, grams/day A, Flow rate of food at beginning of a raceway, grams/day LA] Relative concentration of food, di- mensionless I A |(1 Relative concentration at begin- ning of a raceway, dimensionless B Cummulative rate growth, grams/day C Waste production rate, grams/day F Water flow rate, grams/day FCHT Filtered water constant head tower FCT Flow control tower k, k\ k,, k2 Rate constants, same units as r N PCHT r UCHT V a, fi, y, 8 Liter Number of animals Primary constant head tower Rate of reaction, grams/animal- day Time Unfiltered water constant head tower Volume of reactor Conversion ratio, grams growth/ grams food Order of reaction, dimensionless INTRODUCTION One problem common to all types of animal culture is that of finding a suitable food mate- rial and a satisfactory way of distributing this 63 64 P. N. WALKER AND J. W. ZAHRADNIK food to individual animals. Filter-feeders, oys- ters in particular, are not exempt from this problem. This paper does not concern itself with the oyster's specific needs of nutrition or the exact mechanisms by which the oyster uses this food. Rather, we are concerned with the engi- neering variables which must be used to design an oyster-growing system that will efficiently utilize the available food found in raw seawater. CONCEPTS OF FEEDING The first principle of food utilization is that the food leaving a system must be less than the food entering the system. In the case of filter- feeders, for which the food is suspended in wa- ter, this principle can be restated to say that the food concentration leaving the system must be less than that entering the system. Obviously, almost any feeding method for fil- ter-feeders will reduce the food concentration. However, for discussion purposes the methods can be categorized into three basic types, which were adapted from Smith (1970): (a) the batch system, (b) the stirred-tank system, and (c) the tubular-flow system or raceway. These types are schematically illustrated in Fig. 1. In types b and c the food concentration at any particular point within the filter-feeding system remains constant with time and therefore is in a steady state. In type a, food concentration varies with time and therefore is in an unsteady state. The batch system depends on unsteady state to lower the food concentration. With this method the filter-feeder chamber is filled with water-borne food. After a certain amount of time the chamber is drained and filled with another batch of water. The water, when drained, will have a lower food concentration than when added, thus adhering to the principle mentioned in the first paragraph above. There- fore, two basic engineering variables must be evaluated for the batch system. These are the initial food concentration and the time between water changes. The first of the steady state systems, 6, is the stirred-tank system. With this system water- borne food enters and leaves the chamber con- tinuously. Inside, the water is mixed perfectly so that all the filter-feeders receive the water at the same concentration. Notice that the animals OUT IN'TIAL BATCH Trnal IN — OUT STIRRED-TANK ■OUT RACEWAY FIG. 1. Food concentration profiles for the three basic types of feeding systems. will be feeding on water with the same concen- tration as that leaving the chamber and this concentration is lower than the concentration entering the chamber. The basic engineering variables which have to be considered with the stirred-tank system are flow rate and initial food concentration of the water entering the system. The disadvan- tage to this type of system is obvious. If the concentration of food in the system is at a level which can be utilized efficiently, then the food concentration leaving the system is also at this highly utilizable level and therefore food is being wasted. However, the food concentration may be lowered further by running the water through a series of stirred-tank reactors, and in this case the series of reactors approximate a raceway. The system selected for this study is the race- way system, c. In this type of system the water flows down a raceway containing filter-feeders. Ideally, each incremental unit of water flows at a constant rate through the raceway. The food concentration decreases as the water gets fur- SCALE-UP OF FOOD UTILIZATION 65 ther down the raceway. In the raceway system, as with the stirred-tank system, the basic engi- neering parameters to evaluate are food concen- tration and flow rate of water entering the sys- tem. This system has the advantage of being able to utilize food efficiently, but also has the disadvantage of nonuniform growth down the raceway. Nonuniform growth can be partially remedied by periodic reversal of flow direction. In comparing these three types of systems it becomes obvious that the question of food utili- zation is not an easy one. Given a specific supply of food to be used to feed a specific group of filter-feeders, this supply could be used in at least three very basic ways, each way giving different growth rates. Hence, when evaluating growth data, one must be careful to consider what feeding system was used. This necessity was also pointed out by Johnson et al. (1968). Among the first researchers to mathemati- cally relate food uptake to food concentration for bivalve mollusks were Fox et al. (1937). Fox hypothesized that the instantaneous rate of food uptake was directly proportional to the concen- tration of the food. For a batch system this theory yields a linear relationship between the natural logarithm of concentration and time. Fox's experiments with suspended CaCO:i and mussels substantiate the theory very well. Most bivalve feeding research has been done with batch-type systems. However, Matthiessen and Toner ( 1966) concluded that a more satisfac- tory arrangement would involve continually flowing water, and Ryther (1972) further sug- gested "a long rectilinear raceway" to remove food efficiently from the water. Pruder et al. (1974) studied this type of sys- tem using a vertical rector containing oysters. They concluded that the number of cells re- moved per unit time per oyster is a function of the quantity of cells already cleared and is inde- pendent of the algal cell concentration. One very serious objection to their work is that the experiment only lasted 24 hours. Their data indicate that 24 hours was not enough time for the system to come to equilibrium, much less obtain data at steady-state. As a result his ex- perimental apparatus was not a tubular flow reactor as proposed but some combination of a tubular flow reactor and a batch reactor. MODEL DEVELOPMENT The growth of filter-feeders is a very complex chemical process. This study will simplify the situation by approximating this process by two simple reactions: Food- Filter-Feeder Biomass Waste This is to say that food, in the presence of a filter-feeder, is converted to the biomass of that filter-feeder plus waste products. The use of this model derived from chemical kinetics can be partially justified by considering the following points: 1. The rate of a chemical reaction is affected by the concentration of the reactant. Smith (1970). la. The rate of food uptake by a filter-feeder is a function of the food concentration. Fox (1937). lb. The rate of growth of a filter-feeder is a function of the food concentration. Mat- thiessen and Toner (1966). 2. The presence of a catalyst is required for some chemical reactions to occur. 2a. The presence of a filter-feeder is required for the conversion of food to filter-feeder biomass. Because of the similarity of this model to a chemical reaction, it seem reasonable to sup- pose that chemical kinetics techniques could be used to model filter-feeder growth, food concen- tration, and waste concentration. The rate of reaction for a reaction which can occur only in the presence of a catalyst may be defined, for a raceway system, as: r = dP dN (1) where r = rate of reaction dP = incremental change in amount of a reac- tant or product per unit time dN = incremental amount of catalyst (in this case a pseudo-catalyst, the number of filter-feeders) Early workers in chemical kinetics found that simple relations existed between rates of reac- 66 P. N. WALKER AND J. W. ZAHRADNIK tion and concentration of reactants as cited in Smith. Consider the reaction k aA + bBficC + dD k' where the capital letters represent the reactants and products and the lower case letters repre- sent the appropriate molal ratios. The rate of reaction can be expressed as r = k[A]"|B] k'lCHDl where r is the rate of reaction, k and k' are the rate constants, and a, /3, y, and 8 are the orders of the reaction with respect to the concentra- tions [A], [B], [C|, and [D]. One type of reaction system which may be used to model filter-feeder growth is the simul- taneous complex system. A complex system is one in which more than one reaction occurs. The simultaneous complex reaction can be repre- sented: where A is the reactant and B and C are the products, the ratio of which depends on the rate constants k, and k2. In the filter-feeding system discussed in this paper, A represents the food in the water, B represents that portion which is used in growth by the filter-feeder, and C repre- sents that portion which is expelled from the filter-feeder as waste. The rates of reaction are given by dA dN k,[A]" - MA]*3 which is the rate of food consumption; dB , dN which is the rate of filter-feeder growth; dC dN k2[Af (2a) (2b) (2c) which is the rate of waste formation. However, this biological reaction is autocatal- ytic. In this case an autocatalytic reaction is one in which the product of the reaction serves as a catalyst to the reaction. In other words, the increase in size of the filter-feeder due to its growth increases its ability to remove food from the water and grow. The exact relationship between the size of the filter-feeder and its ability to convert food to biomass is not known. If it is assumed that over a short period of time this increase in ability is negligible, then Equations 2a, b, c may be used without modification. After the rate equations have been estab- lished it still remains to model the entire race- way to determine how well the model works. A mass balance over a differential mass of filter- feeder will provide the following relation: Biomass entering element + Biomass grown in element - Biomass leaving element = Accumulation of biomass (3) For non-motile filter-feeders this becomes: hence 0 + rB AN At - 0 = A„ AxB At (4a) rB AN = A, AxB (4b) where A. is the flow rate of food into the system (A, is the product of the water flow rate, F, and the initial relative concentration, [AJ„), and xB is the conversion fraction of food to growth. dxB = — rB dN 1 fN xB = — rB dN A, -'n (4c) <4d) B but xB = — and rB = k,[A]", B = k, LA]" dN (5) Because [A] is a complicated function of N, stepwise numerical integration is used to obtain the solution to this equation; that is at each step n, |AJn = [A],w - (k,[A]«_, AN F + k2[A]«_ 6) The cumulative growth rate B (Equation 5) can be plotted against N/F and the resulting SCALE-UP OF FOOD UTILIZATION 67 curves compared with experimental values. Stated in simple terms, Equations 5 and 6 show that N/F is the required ratio of the num- ber of fdter- feeders to water flow rate necessary to obtain B growth with an |A]„ initial concen- tration of food in the water, using a raceway. TESTING THE CONCEPT It is, of course, necessary to evaluate the pa- rameters a, /3, k,, and k, in the mathematical model so that it can be used to design a feeding system. These parameters were extracted from growth data taken from experimental race- ways. Each raceway was fed with water at a certain food concentration and flow rate, the two basic engineering variables for a tubular reactor. The system parameters will vary with the species and age of animals, the type of food utilized, the water temperature, salinity, pH, etc. Hence, it was necessary to choose a specific system in order to analyze the parameters. The system chosen employed second-year oysters, Crassostrea virginica (Gmelin), and natural wa- ter from the Wareham River in Southeastern Massachusetts. The water and food parameters were those natural to the river. For this study, natural water has advantages over alternate sources of food such as unialgal, mixed unialgal, enriched natural, or artificial foods. Two of these advantages are: 1. Natural water is the most widely used source of oyster food. No other source of food has been shown to permit normal growth for ex- tended periods of time. 2. It is a very reliable and easily obtainable source of food. The most disconcerting thing about using natural water is the difficulty of determining the exact amount of food in it. There is simply no practical way to compare size, number, and food value for the different types of plankton and arrive at a number representing the exact food concentration of a particular sample of sea- water for oysters. For this reason the food con- centrations were assigned values relative to the food concentrations of natural seawater, al- though it was recognized that the absolute con- centration did vary with time. Recall that two main engineering parameters control growth in a tubular reactor or raceway. These are flow rate and initial food concentra- tion. For the experiment, flow rate was varied by simply metering different amounts of water to each raceway. Food concentration must be varied between raceways in such a way that the quality of the food remains the same and only the concentration is lowered. The technique chosen was that of mixing filtered seawater with the unfiltered seawater. A stream contain- ing a 1.0 liter/minute flow of filtered water and and a 1.0 liter/minute flow of filtered water was taken to have a relative food concentration of 0.5. One additional complication is created by the use of natural water. Since the proposed model is based on food concentration as a primary independent variable, it is necessary to know the concentration profile down the raceway. But the concentration at various points down the raceway cannot be measured for basically the same reason that the food concentration in the original natural seawater cannot be measured. In addition, there are other problems. For ex- ample, what correction factor should be applied for plankton which are expired, partially di- gested, wrapped in the mucus-like pseudofeces, or clumped together in fecal material? These problems would seriously restrict accurate eval- uation of food concentration even for a unialgal culture. For these reasons, the concentration is mea- sured indirectly by first determining the growth-food concentration relationship. The concentration at any point can then be inferred by measuring the growth at that point down the raceway. The design of the tubular reactor or raceway is also an important consideration. In an experi- mental setup the water flow down the raceway may be small enough that the pumping motion of the oysters creates considerable local up- stream movement of the water. This upstream movement conflicts with the theory of a tubular reactor and must be avoided. To avoid this situ- ation in the experimental raceways, each race- way was divided by several sets of baffles. Hence, the raceway actually approximates a tubular reactor by a series of ten stirred-tank reactors, the oysters doing the stirring. A re- verse flow would probably not occur in a large operation because the increased flow rate neces- 68 P. N. WALKER AND J. W. ZAHRADNIK sary to feed a large number of oysters would dwarf the water flow caused by oyster pumping. THE EXPERIMENTAL APPARATUS The experiment was conducted at the Univer- sity of Massachusetts Aquacultural Engineer- ing Laboratory next to the Wareham River in Wareham, Massachusetts. Oysters were grown in 20 raceways, each with a different combina- tion of food concentration and flow rate. Each raceway contained 100 oysters for a total of 2000 oysters. The support system to maintain the raceways consisted of water supply, filtering, flow control, and drainage facilities. A line dia- gram of the entire experimental apparatus may be seen in Fig. 2. Water for the experiment was pumped into the laboratory from the Wareham River. That portion which was to be filtered flowed into a slow sand filter (Fig. 3). The sand filter was contained in a 2.4 x 2.4 x 1.2 meter deep stain- less steel tank coated with epoxy paint on the inside. Six meters of 3.8 cm. (IV2 in.) perforated FROM RIVER 700 fi FILTER FILTERED WATER TO RACEWAYS FIG. 2. Line diagram of the experimental ap- paratus. FIG. 3. Constant head towers and filtering sys- tems. polyethylene pipe was placed on the bottom to aid in water distribution. The tank was filled with pea gravel to a depth of 0.4 meter. Fine mortar sand was placed over the gravel to an additional depth of 0.4 meter. The filter was initially filled with water from the bottom to avoid air locks within the sand. After the water had reached a level higher than the sand, water was added from the top and drained from the bottom, which was the normal mode of opera- tion. In about one week, the top of the sand would become covered with so much material filtered out of the water that the sand would not filter the required 0.2 liter/second. At this time the water inlet would be stoppered and the water level allowed to fall to just below the sand level, so that the top one to two centimeters of sand could be shoveled off. The filter was then filled with water from the bottom and allowed to con- tinue normal operation. Additional sand was added to the filter every four to six weeks. To complete the filtering process, water was pumped from the sand filter through a nominal 1.0-micron filter (Fig. 3). The filter cartridge was orlon line wound around a perforated PVC center. This cartridge was not rechargeable and had to be replaced every three to five days. After water was filtered through this part of the sys- tem, it presumably contained no food. The water flowed from constant head towers to the distribution pipes, which were con- structed of CPVC. Each pipe had a hole drilled over the mixing chamber of each raceway. Short SCALE-UP OF FOOD UTILIZATION 69 lengths of Tygon tubing were fitted into the holes to carry the water into the raceways. Hoff- man clamps on the tubing were used to regulate the flow. This method of flow control worked satisfactorily on the filtered water but the tub- ing often clogged with the unfiltered water and had to be cleaned frequently. The raceway complex itself (Fig. 4) was con- structed of fir lumber, fir plywood, and glass. All 20 raceways were constructed on a single 102 x 204 cm. (4x8 ft.) piece of plywood. Each raceway was divided into 12 sections by small panes of glass. The water flowed from one sec- tion under a pane of glass, up between the two panes, and over the second pane into the next section. The first and last sections were used as mixing and discharge chambers, respectively. The raceways were covered with a piece of ply- wood with a black polyethylene film skirt to avoid algae growth within the raceways. OPERATING PARAMETERS The primary operating parameters for the system were the flow rate and food concentra- tion supplied to the system. The flow rate is simply the total of the unfiltered and filtered 8.9- GLASS PLATES 0.2 — \ 1 1 \ I TOP VIEW WOOD FRAME 9.1 SIDE VIEW CENTIMETERS I ' I ' I 0 5 10 40 8°. 12© IS© |70oib 10 30 <00 300 IOOO 3000 10000 FLOW [ liters /doy ) FIG. 5. Raceway flow rates and initial concen- trations. water flowing into each raceway. Taking the concentration of food in the unfiltered water to be 1.0 and the concentration of filtered water to be zero, the concentration of the water supplied to each raceway is unfiltered flow FIG. 4. Sketch of a section of the raceway com- plex. unfiltered flow + filtered flow The flow rates and food concentration for each raceway are indicated by the points on the graph in Fig. 5. The number beside each point represents the number assigned to each race- way. The points were so arranged that diagonal lines connecting the points would be lines of equal amounts of food. Raceways 19 and 20 re- ceived a zero concentration of food at flow rates of 982 and 2099 liters/day, respectively. These controls would help determine whether all the food was indeed removed from the filtered wa- ter. COLLECTION OF GROWTH DATA The oysters used for testing had been col- lected as spat in net bags on scallop shells in 1971. These animals were grown in net bags from March, 1972, until March, 1973, and were separated as individuals during the spring of 1973. On April 28, they were cleaned with a brush and barnacles were removed. The oysters were weighed and separated into size groups. On April 29, oysters were placed in the race- ways. Oysters were chosen from each size group so that each of ten raceway sections had ten oysters with approximately the same size distri- bution and with as small a size range as possi- ble. The oysters from each section were removed and weighed as a group. The weight range was 177 to 213 grams. 70 P. N. WALKER AND J. W. ZAHRADNIK On August 27, each group of ten oysters was removed from the raceway and cleaned with a brush. The glass plate baffles were also removed for cleaning, and the raceways were scraped clean and flushed out. Baffles and oysters were returned to the raceways. On August 28, 121 days after the start of the experiment, each group of ten oysters was re- moved and weighed. ANALYSIS OF DATA The weight growth rate of the oysters in each section of each raceway was determined by sub- tracting the initial weight of the group from the final weight and dividing by the number of elapsed days. Growth in raceways 1 through 7 was confined to the first very few sections. Under this circum- stance it is difficult to justify that the raceway was approximating a tubular reactor. For this reason raceways 1 through 7 were not used in the analysis of the data. Recall that raceways 19 and 20 received only filtered water with presumably all the food re- moved. Not surprisingly, the growth was nega- tive for nearly every section down the raceway. This indicates that the food level was indeed very low. However, the curves also show a very slight gradation in growth indicating that there was a very small amount of food in the water. The theory developed earlier mandates that a zero concentration yield a zero growth. The data indicate that a zero concentration yields a nega- tive growth. Presumably, this loss in weight provided the food necessary to maintain vital body functions. Since a similar amount of nutri- ents would be required by all the other groups of oysters in the experiment, they would also uti- lize a certain amount of food without exhibiting any growth. To correct for this condition, the weight loss per section was averaged for race- ways 19 and 20, yielding a loss of seven grams per section. This amount was added to the growth in every section of each raceway. The resulting growths were then numerically integrated down each raceway to yield the cu- mulative growth curves some of which are indi- cated by the data points in Fig. 6. In making the growth integral curve for each raceway, inte- gration was discontinued at the point on each curve where net negative growth occurred. This 40 60 NO OF ANIMALS FIG. 6. Cumulative growth (grams/day) of oysters versus number of animals. negative growth was usually concurrent with a large proportion of expired oysters in the race- way section. The data curves for each raceway were fitted to an equation of the form B b2 expl -b3N) (7) where, B = cumulative growth rate N = number of animals b,, b2, hj = constants. The least squares criterion was used. The re- sulting curve for each raceway is used to con- nect the data points in Fig. 6. The equation form was chosen because of its ability to fit the data points. By taking the first derivative of Equation (7) the following relationship is found: dB dN b>b, exp(-b:!N) (8) Recall from the earlier model development that dB dN k,[A]" (2b) hence, and. dB dN k,[A|" = bob, exp(-b;,N) (9a) SCALE-UP OF FOOD UTILIZATION 71 [A] Differentiating, d[A] b'b:> um*1"" -^exp(-b,N) k, b,b. dN a L k, Since dA = Fd|A], exp(-b.,N) dA _ -Fb3 dN " a b,b. exp(-b:,N) (9b) (10) 111) Recall again from earlier model development that (2a) dA dN = -k,[A]« - k2[A]" dA dN " -k,[A]Q - - k2[A}» -Fb3 rb'b-i , XT 1 — -^exp(-b:iN) a L k, (12) Equations (9a) and (12) must be solved to obtain values for the constants k,, k2, a, and (3. The only place where the food concentration, [A], is known is at the boundary, i.e. the begin- ning of the raceway before any food is removed, i.e. N = 0. Simplifying Equation (9a) to the case where N = 0, VdN/ = k,[AJ;; = b2b3. [13) Plotting the product of b2 and b:i versus the initial concentration for the respective raceways resulted in the points shown in Fig. 7. The points were fitted to a curve of the form k,[A]a using a weighted least squares criterion. The resultant values of k, and a were 0.1837 gram per animal per day and 0.8488 respectively. Equation (12) reduces to /dAN VdN/ k,[A];; k, [A]g -Fb3 b.b. k, (14) where N = 0. Again, a weighted least squares criterion was used in obtaining values for k, and /3. The values obtained for k, and /3 were 1.407 x 0 I 2 ooo OOO 0 20 0 40 0 60 0 80 I 00 CONCENTRATION FIG. 7. Growth rate (grams/animal-day) ver- sus relative food concentration. KIO" 20 0 O 16 0 - 12 0 - 0 o 80 o 0 yr o 4 0 / S I 1 1 1— 0 00 0 20 0 40 0 60 0 80 100 CONCENTRATION FIG. 8. Food removal rate versus relative food concentration . 105 grams per animal per day and .9125 respec- tively. The experimental values of (dA/dN)N=0 were plotted against their initial concentration in Fig. 8. Note that the relationship between the rate of removal of food, dA/dN, and the concentration is nearly linear as shown in Fig. 8. This is in 72 P. N. WALKER AND J. W. ZAHRADNIK o > o -7 xlO 20 0 16 0 A A A00 X X X X X X XX 12 0 0 0» ,** 0 + + 8 0 - o * A Symbol Raceway Flou [A] (t/day) 40 n n + 0 ' A + O 1 12 15 17 18 1 983. 2230 4587. 5599. 1 1.0 1.0 1.0 1.0 log 5 0 - 4 5 N /F FIG. 9. Conversion versus logarithm of space time (animal-days/gram) at equal initial con- centrations. agreement with the work of ZoBell (1937) when he demonstrated that the relationship was lin- ear for a batch reactor system. A further comparison of the experimental data and the calculated values is given in the next section. RESULTS AND DISCUSSION OF RESULTS A basic question investigated was that of scale-up. That is, can space time, N/F, be used as the primary scaling parameter in this biolog- ical system? Recall that in the model N is the number of oysters over which the water has flowed and F is the flow rate. Systems which are directly scalable should have identical conver- sions at the same space time. Recall the conver- sion xB is the fraction of the food which has been converted to filter-feeder biomass. Directly scal- able systems in this study would be raceways with the same initial concentration of food, [A]„. Fig. 9 shows the data for the raceways where [A]n = 1.0. These data show that engineering scale-up can be accomplished on filter-feeders using space time as a scaling factor. This fact is independent of the question of whether the data fit the mathematical model proposed in this paper, and has much greater practical impor- tance. Fig. 9 also demonstrates that the growth rate is not a strong function of velocity at least in the velocity range studied. The arrangement of the points for a particular space time is not in the order of increasing or decreasing flow rates as would be expected if growth were a function of velocity. The next question to be answered is how well the chemical kinetics assumptions which were made approximate the actual growth in the raceways. To answer this the model was used to generate conversion data using a step by step numerical technique. The resulting curve is shown in Fig. 10. Note that the model best approximates raceway 18 (see Fig. 11). This would be expected since raceway 18 had the highest flow rate. With a higher flow rate, each section of the raceway represents a smaller por- tion of space time; hence the raceway more nearly resembles a tubular reactor. It is impor- tant to realize that the model curve was not made to fit the data points in Fig. 9, but rather was generated from constants obtained from Fig. 7 and 8. Fig. 10 is a check to determine if the model is reasonable. The implications of the kinetics of filter-feed- ing may be better understood by using the model to make a graphical parametric analysis z o (/) CE > o FIG. 10. Conversion versus logarithm of space time (animal-days /gram) at equal initial con- centrations. SCALE-UP OF FOOD UTILIZATION 73 en a. > z o Symbol Raceway Flow |A) (I/day) 5599. 1.0 Model - 1.0 0.0 ■ 6 0 5 -5 0 log N /F * 10 -4 0 A more dramatic characterization of the im- plications of this growth pattern may be seen in Fig. 14. These curves are simply integrations of the curves in Fig. 13. It may be seen that the total amount of growth in the raceway is higher for lower concentrations. The point is that lower concentrations require much longer raceways to achieve the same amount of growth. In an economic optimization situation where FIG. 11. Conversion versus logarithm of space time (animal-days /gram) for raceway 18. of the growth of the oysters. Fig. 12 contains the conversion versus space-time relationship taken at several concentrations. This graph could be used to design' a filter-feeding system for any food concentration (in the range studied) and amount of animals. It can be seen that at all space times the lower concentrations give slightly better food conversion. It might be mis- takenly inferred from Fig. 12 that it is best to feed lower concentrations. Fig. 12 shows that for a system with a specified N and F, a lower initial concentration of food results in a slightly higher percentage of the food, F[A]„, being con- verted to filter-feeder biomass. However, the absolute amount of food converted, xBF[A],„ will be much higher with a higher initial concentra- tion of food. Fig. 13 shows the calculated growth rate of the oysters down the raceway. In each curve the total amount of food is held constant and the flow rates and concentrations are varied to meet this criterion. Keep in mind that the low con- centration flow rate is ten times the high con- centration flow rate. Notice that much higher growth rates occur with the higher concentra- tions at the beginning of the raceway. The lower concentrations achieve a larger growth rate to- ward the end of the raceway. xlO 200 - [A]= = 0 093 ^^- 0 203 ^ ' 0 480 /^/^^^-~---~' ° '----£( i ! > i i i 150 N / F FIG. 12. Conversion versus space time (ani- mal-days I gram) at various initial concentra- tions. 0 20 0 00 FIG. 13. Growth rate (grams I animals-day) versus number of animals. 74 P. N. WALKER AND J. W. ZAHRADNIK food, raceway length, and flow rate all repre- sent capital outlays, the cost of the higher flow rate and longer raceways must be weighed against the benefits of higher conversion to ar- rive at an optimum design. The optimal race- way length for an extensive system in which a river provides the water flow would undoubt- edly be different from the optimal raceway length for an intensive system in which the water must be pumped out of the river. EXAMPLE Perhaps the best way to obtain a working understanding of some of the concepts presented herein is to work a simple example problem. The problem is to determine the flow parame- ters necessary to provide food for 25,000 oysters (100 bushels at market time) and the growth rate at these conditions. Assume that the oys- ters are second-year oysters weighing approxi- mately 20 grams each since the data are strictly applicable only to this size animal and to the seawater-food conditions of this study. The first step is to determine the initial rela- tive concentration of food in the water. Fig. 12 indicates that a slightly higher conversion is possible with lower concentrations. However, Fig. 14 demonstrates that a much larger and therefore more expensive system is required to obtain this conversion. Therefore, choose the higher relative concentration, [A],, = 1.0. 2 50 I- 40 60 80 NO. OF ANIMALS FIG. 14. Cumulative growth (grams/day) ver- sus number of animals. Assume that an economic analysis of the cost of the food, the cost of pumping water, the cost of the raceway, etc. and the value of the prod- uct, yields an optimum point on the space-time- conversion curve where N/F = 9.0 x 10 ,; ani- mal-days per gram and conversion = 10. x 10 " (see Fig. 12). The flow rate may then be determined. N 2.5 x 10' animals 9.0 x 10 K animal-days gram 2.5 x 101 9.0 x 10 - = 2.78 x 109 grams/day = 2.78 x 10" liters/day Finally, the growth of the oysters is calculated: Growth = Flow x initial concentration x con- version = (2.78 x 10H) (1.0) (10. x 1(F) = 2,780 grams/day, or an average of 2,780 ~ 25,000 .111 gram/animal/day. This amount represents gross growth and must be corrected to account for food used in meta- bolic processes. Recall that this correction is 7.0 grams per 10 oysters per 121 days. 7.0 (10.) (121.) = .0058 gram/animal/day then. Net growth 0.111 - 0.0058 mal/dav. .105 gram/ani- As a second sample, it is interesting to make a crude approximation of the pumping costs required to grow a bushel of oysters to adult size assuming that water-borne food is pumped from an estuary. It appears reasonable to assume that the oyster is able to maintain the same conversion ratio used in the previous example until it reaches market size. Also assume that a marketable oyster weighs 50 grams and that the seawater must be pumped against a three-me- ter head. SCALE-UP OF FOOD UTILIZATION 75 Growth = Flow x initial concentration x con- version 50 grams 250 oysters oyster bu. = (Flow) x (1.0) x (10. x 10-7) 1.25 x 1010 grams bu. 1.25 x 107 liters/bu. Flow 1.25(107)liters kg 3.0 m. bu. 1.0 liter 1 kwh $.03 3.67(105)kg-m. kwh LITERATURE CITED = $3.03/bu. Fox, D. L., H. Sverdrup and J. P. Cunningham (1937). The rate of water propulsion by the California mussel. Biological Bulletin, Ma- rine Biological Laboratory, Woods Hole, Mass. (72): pp. 417-438. Johnson, C. A., W. B. Pasko, J. W. Zahradnik (1968 1. Environmental control chambers for oyster propogation, Proceedings, Institute of Environmental Sciences. Matthiessen, G. C, and R. C. Toner (1966). Possible methods of improving the shellfish industry of Martha's Vineyard, Dukes County, Massachusetts. Marine Research Foundation, Inc., Edgartown, Mass. Pruder, Gary D., C. E. Epifanio, and R. F. Srna (1974). Engineering aspects of bivalve mollus- can mariculture. Presented at World Maricul- ture Society Meeting, Charleston, S.C., Janu- ary 21-24, 1974. Rytrier, J. H. (1972). Controlled eutrophica- tion — increasing food production from the sea by recycling human wastes. BioScience, (22): 3. Smith, J. M. (1970). Chemical Engineering Ki- netics. McGraw-Hill, New York. Proceedings of the National Shellfisheries Association Volume 65 - 1976 OUT-BAY CULTURE OF BIVALVE MOLLUSCS1 W.P. Breese FISHERIES & WILDLIFE DEPARTMENT OREGON STATE UNIVERSITY MARINE SCIENCE CENTER NEWPORT, OREGON ABSTRACT A relatively new technique in oyster fanning called "Out-bay Culture" is proposed. Its advantages and problems are discussed. Data are presented indicating the probable success of this technique. The technique provides neces- sary control over the operation. In the future oyster aquaculture will call for a formulated ration. Out-bay culture is speculative at the present but may develop into a commercial reality. Oyster culture has traditionally been accom- plished in estuaries. In the early stages, the bottom was used both intertidally and subti- dally. More recently, the entire water column has been used by hanging oysters from floating devices or by fixed off-bottom culture employing either strings or trays. Bottom culture is restricted to acceptable bot- tom types and current conditions as well as water quality. Many traditional oyster grounds are near cities or industry where the water may be polluted and unacceptable for culture. Water column cultures are also restricted by current conditions, storms and other water uses. Rafts or stationary devices may interfere with com- merce, boating, water skiing, fishing, etc. Good oyster sites are difficult to find and maintain. Oyster aquaculture, as practiced, is exten- sive. One has little control over environmental variables such as temperature, salinity, and water flow. Intensive culture brings large num- bers of animals confined in relatively close quarters with some control over environmental parameters. A ration is also involved. Out-bay 1 This work is a result of research sponsored by the Oregon State University Sea Grant College Program, supported by NOAA Office of Sea Grant, Dept. of Commerce, under Grant #04-5-158-2. Technical Paper No. 4108, Oregon Agricultural Experiment Station. culture is the term used in this report for con- trolled oyster culture in an intensive way. It is hardly more than an idea at present, but compe- tition for space in estuaries and water quality will make this concept progressively more at- tractive. Out-bay culture requires rearing oysters in tanks, ponds or some similar contained water volume with control of water flow, retention time, depth, and the opportunity to treat the water if necessary. The advantages are freedom from storm and tidal action, as well as salinity control during the rainy season. Some of the unresolved problems are stocking rate, water flow per unit of biomass, and exchange rate or retention time. The understanding and optimiz- ing of these variables will lead to maximizing the yield from a given sized container. The next step is the development of a ration. At present we can feed larvae and small spat with culture algae and get reasonably consist- ent results. However, we are a long way from "feed lot" oysters. To really get estuaries into oyster protein production a ration is needed to supplement or replace natural food. To illustrate what might happen in pond cul- ture we conducted a pilot exercise. This was not a controlled experiment. Only one tank was available and we sought to find out the water volume-oyster relationship under a precise set of conditions. 76 OUT-BAY CULTURE OF BI-VALVE MOLLUSCS 77 60 50 ^____y /T^' (LARGE) 40 . / iO- jf (SMALL) 20- l / / 1 0- ■ V 1 1 1 t 1 1 1 ! 1 1 1 \ TABLE 1 . Mean size in m m of large and smalt oyster seed (Crassostrea gigas I grown for one year in out-bay culture. 6-li 7-\2 8-B 9-10 10-15 11-12 FIG. 1 . Growth curve in mm of large and small oyster spat (Crassostrea gigas) from June 1974 to June 1975. The tank measured 17' by 30' with a water depth of 4 feet. The water volume was about 15,000 gal. We hung 112 strings of 7 shells each, or a total of 784 shells with a total of about 4,400 spat of Crassostrea gigas in the tank. The seed was of two sizes, one averaging 2 mm and the other 8.9 mm. The water flow was 10 gal per minute. This flow filled the tank in about 24 hr. At this flow rate, it was expected that the growth of the oysters would cease in 3 or 4 months, giving us an idea of the flow-oyster mass relationship under these conditions. Oysters were measured on a monthly basis. A total of 1,500 randomly selected oysters were measured, half from the large seed and half from the small seed. The tank was drained to facilitate measuring the oysters. All the shells were rinsed and the sides and bottom of the tank were cleaned before the tank was refilled. Fig. 1 shows the length of the two-sized oys- ters over a year's time. Inspection of the curve shows that growth proceeded through the fall months. A slow or non-growth period was noted during the winter. Growth resumed the follow- Date Small Large June 13, 1974 2.0 8.9 July 12. 1974 6.8 21.2 August 8, 1974 15.4 31.8 September 10, 1974 21.9 38.9 October 15, 1974 26.2 43.6 November 12, 1974 29.6 44.9 January 8, 1975 29.8 45.2 March 13, 1975 31.2 46.7 April 15. 1975 31.1 47.9 May 30, 1975 36.0 55.2 ing spring. Table 1 gives the mean size of spat. The 10 gal per minute flow allowed about 13 1 per oyster per day or about 0.5 1 per oyster per hour. It was felt that the incoming water could not provide enough food for the growth noted. Thus, there must have been significant production of phytoplankton in the pond which provided more food than the oysters required. It seems that the tank was not overstocked as anticipated. Although no rigorous conclusions can be drawn from this exercise and the results noted are only valid for this one set of conditions, the observations are encouraging. In the future we will examine some of the variables influencing the system's productivity. The contributions, if any, from the growth on the sides and bottom of the tank, and the flow-oyster-water retention relationship to achieve maximum protein pro- duction are being investigated. We also will use out-bay culture to test alternate rations, when they come along. Advantages of this culture method are me- chanical control, the opportunity to treat the water if necessary, and, in Oregon, to open new estuaries for oyster culture. Some of our estu- aries have too much fresh water in the winter months. With out-bay culture, pumping would cease at this time and acceptable salinities could be maintained. Finally, when rations are developed and "feed lot" culture becomes eco- nomical, out-bay culture could be the culture method for oyster protein production. Proceedings of the National Shellfisheries Association Volume 65 - 1976 COLIFORM BACTERIA LEVELS CORRELATED WITH THE TIDAL CYCLE OF FEEDING AND DIGESTION IN THE PACIFIC OYSTER (CRASSOSTREA GIGAS) CULTURED IN DEEP BAY, HONG KONG Brian Morton* and K. F. Shortridgef DEPARTMENT OF ZOOLOGY* AND DEPARTMENT OF MICROBIOLOGYt, THE UNIVERSITY OF HONG KONG ABSTRACT Over two 24 hour periods in the winter and summer of 1974 experiments have been undertaken upon the Pacific oyster (Crassostrea gigas) in Deep Bay, Hong Kong to determine the effect, if any, of the tide and the sequence of feeding and digestion in the oyster upon col i form bacteria levels. The results clearly show that the digestive process in the oyster, as revealed by the times of reformation and dissolution of the crystalline style is closely related to the tide; the animals feeding at the time of high tide and digestion being completed during low tide. Such a pattern fits in well with current concepts of the processes of feeding and digestion in the Bivalvia. In winter, pollution levels were generally low and no tidal pattern of contami- nation of the oysters was observable. In summer, however, when the monsoonal rains flush out the polluted streams of Hong Kong into Deep Bay and the Pearl River (into which Deep Bay empties) is in flood, pollution levels increase and a distinct tidal pattern of contamination is apparent resulting from the differen- tial feeding levels by the oysters at the times of low and high tide. These results show that isolated tests upon contaminated oysters have little or no value either on a tidal or a seasonal basis, and that only a comprehensive monitoring programme can explain wide ranging coliform counts. INTRODUCTION laying (Morton, 1975). Such oysters are typi- cally polluted especially in the summer months Hong Kong's rural New Territories compris- when the rain bearing S.E. Monsoon flushes out ing a land area of some 370 square miles sup- the streams and rivers into the bay and also ports a human population of over 1 million and causes a flooding of the Pearl river, draining a pig and poultry population estimated at some much of southern China and into which Deep 400,000 and 6 million respectively and for which Bay opens (Fig. 1) (Leung et a/., 1975). High on the agricultural side at least no means of coliform counts in the region of the Pearl River effluent disposal is available. Similarly human characteristically result from this flooding effluents receive only primary screening before (Watson and Watson, 1971). In Deep Bay too, being discharged into the sea. Streams and wa- pollution levels are high (Leung et al., 1975). tercourses passing through agricultural areas Wood (1969) showed how oysters and mussels are grossly polluted and Hong Kong's coastal collected at different states of the tide possessed waters are similarly degraded. variable numbers of faecal coliform bacteria To the north west, Deep Bay located on the (i.e. Escherischia coli) with a peak occurring at Hong Kong-Chinese border supports a Pacific the time of low tide. Recent research by Morton oyster (Crassostrea gigas Thunberg 1793) in- (1973) has shown how in a wide range of littoral dustry utilising a primitive method of bottom bivalves, noteably the European oyster Ostrea 78 COLIFORM BACTERIA LEVELS FIG. 1. A map of Hong Kong showing the posi- tion of the Pearl River and Deep Bay, where the commercial oyster beds are located and where these experiments were undertaken . edulis (Morton, 1971) the processes of feeding and digestion are closely correlated with the state of the tide. In Deep Bay where the oysters are known to be significantly polluted it was decided to repeat the experiments of Wood (1969) using Crassos- trea gigas and, if proven true, to correlate such findings with changes in water quality and the feeding behaviour of the oysters over two tidal cycles in both winter and summer. The results of these investigations are reported here. MATERIALS AND METHODS In February 1974 and in July 1974 four oysters were placed in each of 24 wire baskets and sus- pended in the water from the side of a pier at Tsim Bei Tsui that extends onto the mud flats of Deep Bay. The baskets were so arranged that they rested on the surface muds at approxi- mately mid tide level and were covered and exposed by the tide in time with the commercial oysters growing close by. The oysters were established 2 days prior to the experiments being carried out in order for them to acclimatise. During the experimental periods water sam- ples were collected every hour and the tempera- ture, salinity and pH recorded. Four oysters were also collected every hour and were scrubbed clean in distilled water, washed a sec- ond time and then opened, using a sterile Euro- pean oyster knife, through the ligament. The 79 pH of the mantle fluids was measured using a Radiometer pH meter 27 fitted with microcapil- lary electrodes. The crystalline style was then removed and measured along its greatest length and width to the nearest 0.5 mm. The volume of the style was calculated as the volume of a cone. Each oyster was then measured volumetrically, mixed with an equal volume of 0.2 M phosphate buffered saline (pH 7.0) and homogenised at top speed for 30 seconds in an MSE homogenizer. The samples were submitted sequentially to presumptive (PCC) and confirmed (CCC) coli- form counts for Escherischia coli according to the protocol of the American Public Health As- sociation (1970). Tubes showing acid and gas production after 24 or 48 hours were regarded as presumptively positive for faecal coliforms; the confirmed count was made by inoculating sam- ples from the positive tubes into brilliant green bile broth and tryptone water for acid/gas and indole production, respectively, at 44°C for 24 hours to exclude irregular and other coliform organisms. The coliform density of the oyster expressed as "Most Probable Number" (MPN) per 100 ml was computed from Standard tables (American Public Health Association, 1970) to give an average hourly MPN/ml of tissue. RESULTS Hydrology The tides. The tides in Hong Kong are semi- diurnal at the time of springs and diurnal at neaps. Moreover a diurnal inequality exists with respect to the seasons so that a higher high tide and a lower low tide occurs during the daylight hours in summer and during the night in winter (Fig. 2). Temperature. During February the water temperature was seen to remain relatively sta- ble at some 18-21°C with only a diurnal varia- tion of 1-2°C; the waters warming up in the afternoon and cooling at night. During the sum- mer, however, i.e. July, the water temperature was much higher (25-26°C) and with a similar diurnal variation. A dramatic fall in tempera- ture was seen at the time of the low tide which occurred in the late evening possibly resulting from a greater influence of cool fresh waters flowing over the muds and shallow bay waters that had been heated up during the day. 80 B. MORTON AND K. F. SHORTRIDGE II 12 13 14 15 16 17 1! 19 20 21 22 23 24 01 02 03 04 05 06 07 08 09 10 TIME ( hours 1 FIG. 2. Hydrological changes taking place in the waters of Deep Bay over the two, 24 hour experimental periods. Salinity. During February the salinity of the Deep Bay waters remained consistently high (between 25-29%o ) as the fresh water influence, created by the arrival of the S.E. Monsoon in summer, was seasonally reduced. At the time of the lower low tide in the early morning a num- ber of smaller salinity values were recorded, pre- sumably as the fresh water outflow temporarily exerted itself. During July, however, a different picture presented itself and the salinity was some 20%o lower as the seasonal rains flooded the rivers and streams draining into the bay. Only at the time of the higher high tide that occurred in the early morning were a number of higher salinity values recorded presumably as more oceanic water masses were pushed into the bay with the incursion of the tide. pH. In July the pH of the waters of Deep Bay remained relatively stable over the 24 hour pe- riod and were consistently lower than those seen in the winter. In February, moreover, marked fluctuations occurred correlated with the state of the tide. During both high tides higher pH values were recorded possibly indi- cating a greater incursion of more oceanic wa- ters at this time though this was only relatable to salinity with respect to the daylight high tide. The oysters pH of the mantle fluids. The pattern of changes seen in the pH of the mantle fluids closely followed that seen in the waters of Deep Bay. This trend was best seen in winter when peaks in the pH of the mantle fluids occurred typically one hour later than similar peaks seen in the waters of Deep Bay (Fig. 3). In winter also lower pH values were recorded especially at the time of the low tide which occurred during the evening. In summer much smaller varia- tions in pH occurred, though still following the trend seen in the waters of Deep Bay. The crystalline style. The crystalline style of intertidal bivalves e.g. Cardium edule and Os- trea edulis (Morton, 1970; 1971) has been shown to typically dissolve on the ebbing tide and re- form on the flowing tide. The dissolution of the style is primarily affected by the release of frag- mentation spherules into the stomach from the digestive diverticula (Morton, 1973) though, as it 3 1 1 is ^- 150 f- II BI3K15l6 17 1!Ba)2122 23 24O102(I3K[B0607l»CBlO IK hour- FIG. 3. A correlation between the state of the tide, the pH of the mantle fluids and style vol- ume of the oyster and the levels of presumptive and confirmed coliform counts in the oyster over the two 24 hour experimental periods. COLIFORM BACTERIA LEVELS 81 will be discussed later, the arrival of food in the stomach also causes an initial, but not com- plete, dissolution of the style. In winter, i.e. in February, the crystalline style of Crassostrea gigas retained its firm, rod like structure for a longer time over the period of the high water that occurred during the night. The style was rod-like for a much shorter period during the reduced tide of the daylight hours. During both periods of low tide, however, the style dissolved completely. The reverse situation was seen in summer with the style remaining firm longer during the extended period of high water that occurred during the daylight hours. At night it was a solid rod for some four hours only; possessing even then a volume only half that recorded at night. Again during both low tides the style dissolved completely. During the period in both winter and summer when the style was the largest i.e. during the time of the highest high tides, a slight dissolu- tion occurred in the middle of this period, but the presumably reforming style was able to overcome this minor disillutory effect caused, it is thought, by the arrival of food in the stomach, and to retain its structure until dissolved com- pletely later by the arrival in the stomach of fragmentation spherules from the digestive di- verticula. A similar pattern of formation and dissolution is seen in the European oyster Os- trea edulis (Morton, 1971). Microbiology Seasonal variations. A marked seasonal vari- ation was encountered in both the presumptive and confirmed coliform counts from the homoge- nised oysters. During the winter, i.e. in February, presump- tive and confirmed coliform counts never ex- ceeded 10 and 1 MPN/ml. respectively indicat- ing that at this time (if British hygiene stand- ards of less than 2 E. eoli/m\. of tissue are ap- plied) the oysters are relatively clean. In summer, however, the counts for presump- tive and confirmed coliforms rose to maxima of 285 and 240 MPN/ml. respectively. Estimates for the two series of tests are comparable and suggest that the majority of the faecal bacteria present in the oyster are E. coli and thus of human origin. Similar results have been ob- tained on a monthly basis by Leung et al (1975) who also correlated a rise in contamination lev- els with a flushing out of the watercourses and streams (typically serving as sewers) into Deep Bay. These results and the earlier results of Leung et al (1975) demonstrate that the Deep Bay oysters are contaminated, particularly in summer. Tidal variations. In winter given the gener- ally low levels of pollution no significant varia- tion in the levels of contamination could be detected with respect to tidal changes. This was especially true of the confirmed coliform counts. Presumptive coliform counts were generally high around the time of the low tide that oc- curred in the afternoon but not during the lower low tide in the early morning. In summer, however, a more obvious pattern was revealed especially with regard to the pre- sumptive coliform counts where a distinct tidal pattern emerged with peaks of bacterial abun- dance occurring, as first noted for Ostrea edulis and Mytilus edulis by Wood (1969), at the times of low tide. The pattern of changes in the con- firmed coliform counts (i.e. E. coli) of Crassos- trea gigas generally followed the trend seen with respect to presumptive coliform counts, but were much more erratic. DISCUSSION Deep Bay drains a number of small streams and rivers and in turn, forms an integral part of the much larger Pearl River. The hydrology of the bay reflects Hong Kong's compromised climate of cool dry winters and hot, wet summers so that the water has a low temperature and is of high salinity in winter and a high temperature and of low salinity in summer. A greater range of temperatures have been recorded from Deep Bay than elsewhere in Hong Kong (Morton and Wu, 1975) because the shallow waters of the bay are warmed up higher and cooled down lower. Tidal changes in the hydrology of the bay also influence water quality so that, for example, in winter the incursion of more oceanic waters into the bay at the time of high tide affected the pH. This was not so obvious in summer. The pH of the mantle fluids of Crassostrea gigas generally followed the changes seen in the pH of the water, though lower values were re- 82 B. MORTON AND K. F. SHORTRIDGE corded at the time of low tide, indicating that the shell valves were shut and that carbon diox- ide levels were built up in the mantle fluids at this time. The volume of the crystalline style of C. gigas also altered in accordance with the tide, becoming firm and rod like at high tide and dissolving with each low tide. Bernard (1973) has similarly shown that in Canada, the style of C. gigas dissolves every tidal cycle. Complete dissolution, affected by the release of fragmentation spherules from the digestive di- verticula when intracellular digestion had taken place occurred at the time of low tide though a slight dissolution mid way through the high tide period when the style was forming is thought to result from the arrival of food in the stomach. Similar patterns of feeding and diges- tion occur in the European oyster Ostrea edulis (Morton, 1971) and demonstrate the close rela- tionship that exists between the animal and the environment. High presumptive coliform counts from the oysters occurred in the summer and have been shown to coincide with the time of each low tide; winter values being considered too low to statis- tically demonstrate such a trend. A similar pat- tern has been reported upon by Wood (1969) for Ostrea edulis. With respect to the tide, the pH of the mantle fluids, the style volume and the coliform counts it can be seen that there is a time lag in, what is in effect, the sequence in the feeding and diges- tive processes of the oyster. Thus the pH of the mantle fluids rose a few hours after high tide, the style was well formed later still and a peak in coliform counts was recorded somewhat later at the time of low tide. The oysters, located at mid tide level, would still be feeding at half ebbing tide, indeed on the falling tide the con- centration of food in the oyster should be maxi- mal. These results do show that on the ebbing tide, the concentration of bacteria has risen to a high level and can only further be concentrated (but not added to) by the organs of feeding and digestion so that actual numbers can rise no further. Subsequent low levels of bacteria prob- ably result from the digestion of the bacteria by the oysters and the cleansing of the mantle fluids and intestine of pseudofaeces and faeces respectively with the returning tide. A number of important conclusions can be drawn from the results of this investigation. Oyster contamination is likely to be highest at the time of low tide and it would seem sensible to crop the oysters (at least in Hong Kong) at the time of high tide when contamination is likely to be less. The high levels of contamina- tion of the oysters in summer would also sug- gest that either the beds should be closed at this time or that the oysters should be cleansed prior to resale — a practice not yet undertaken in Hong Kong. Furthermore these results demon- strate that single or isolated tests for coliform bacteria in shellfish have little or no value. A comprehensive monitoring programme should test not only on a seasonal basis but also, as this study has shown, over a tidal regime. Finally this study has demonstrated the intimate rela- tionship that exists between the oyster and the environment, especially with regard to the ti- des, and how contamination levels (resulting from the oysters feeding in polluted waters) are similarly bound up with this pattern. ACKNOWLEDGEMENTS The authors are grateful to the Commissioner of Police, The Royal Hong Kong Police Force, for permission to use over the experimental pe- riods the facilities of Tsim Bei Tsui Police Post in Deep Bay and to Tim Garland and Rudolph Wu who helped with the running of the experi- ments. LITERATURE CITED American Public Health Association, 1970. Rec- ommended procedure for the examination of sea water and shellfish. American Public Health Association 4th Edition, 105 p. Bernard, F. R., 1973. Crystalline style forma- tion and function in the oyster Crassostrea gigas (Thunberg, 1795). Ophelia, 12: 159-170. Leung, C, Morton, B. S., Shortridge, K. F. and Wong, P. S., 1975. The seasonal incidence of bacteria in the tissues of the commercial oys- ter Crassostrea gigas Thunberg 1793 corre- lated with the hydrology of Deep Bay, Hong Kong. Proceedings of the Pacific Science As- sociation Special Symposium on Marine Sci- ences, Hong Kong 1973. 114-127. Morton, B. S., 1970. The tidal rhythm and rhythm of feeding and digestion in Cardium edu/e.J. mar. biol.Ass., U.K. 50:499-512. Morton, B. S., 1971. The diurnal rhythm and tidal rhythm of feeding of digestion in Ostrea COLIFORM BACTERIA LEVELS 83 eduhs. Biol. J. Linn. Soc. Lond. 3: 329-342. Morton, B. S., 1973. A new theory of feeding and digestion in the filter feeding Lamellibran- chia. Proceedings of the IV European Mala- cological Congress (In) Malacologia 14: 63- 79. Morton, B. S., 1975. Pollution of Hong Kong's commercial oyster beds. Marine Pollution Bulletin 6: 117-122. Morton, B. S., and Wu, R. S. S., 1976. The hydrology of the coastal waters of Hong Kong. Environmental Research . Watson, J. P. and Watson, D. M., 1971. Marine Investigation into sewage discharges; report and technical appendices. The Government Printer, Hong Kong. 72 p. Wood, P. C, 1969. The production of clean shell- fish. Ministry of Agriculture Fisheries and Food, Laboratory Leaflet (New Series). 20: 1- 16. Proceedings of the National Shellfisheries Association Volume 65-1976 CHANGES IN THE TOTAL PROTEIN, LIPID, CARBOHYDRATE, AND EXTRACELLULAR BODY FLUID FREE AMINO ACIDS OF THE PACIFIC OYSTER, CRASSOSTREA GIGAS, DURING STARVATION Ronald T. Riley DEPARTMENT OF GENERAL SCIENCE OREGON STATE UNIVERSITY CORVALLIS, OREGON A study was conducted concerning the response of the oyster Crassostrea gigas, to prolonged starvation. At the end of the starvation period there was a dry weight loss amounting to 61.0% of the pre-starved value. There was an increase in the % water content from 82.0 to 89.7. All major food reserves were extensively utilized. Lipid was found to be the most important energy reserve, assuming total oxidation. The digestive gland and mantle were the primary sources from which energy reserves were drawn during the early stages of starvation. The gills and adductor muscle exhibited the least depletion of reserves. The gonad exhibited a relatively low level of catabolic activity during the early stages of starvation. Signs of sexual maturity were evident at the middle of the starvation period. The degree of maturity was considerably less than that occurring under natural conditions during the same period. During the last 50 days of starvation the gonadal reserves were extensively catabolized. The free amino acid concentration in the hemolymph showed a considerable decrease. INTRODUCTION The ability of oysters to resist starvation is well known. Gillespie, Ingle, and Havens (1964) found that the oyster Crassostrea virginica could live up to 390 days without any apparent source of food. Pora, Wittenberger and Portilla (1969) found that individuals of the species C. rhizophorae , maintained under anaerobic con- ditions for two weeks, showed little change in their lipid, glycogen or nitrogenous constitu- ents, whereas individuals maintained under conditions of normal oxygenation consumed about 40% of their lipid and glycogen and 6% of their organic nitrogen. Studies by Millar and Scott ( 1967) with the larva ofOstrea edulis indi- cated that during periods of starvation the larva utilized lipid the most, carbohydrate the least, and protein utilization was intermediate. This reflected the fact that the lipid content of the larva was 2-4 times that of carbohydrate, whereas in the adult, carbohydrate content was 3-4 times that of lipid. Studies of starvation in other molluscs indicate a great variation in the extent of utilization of the various body constit- uents. The objectives of this study were to: 1) Determine the change in the total protein, lipid, carbohydrate, and body fluid-free amino acids, of Crassostrea gigas during starvation. 2) Develop some insights into the mechanisms by which the oyster survives prolonged pe- riods of starvation. MATERIAL AND METHODS Experimental Design 59 oysters of the 1970 year class were collected from North Humboldt Bay, California in mid- March 1972. The oysters were cleaned of all mud and fouling organisms and then placed in an insulated fiberglass tank containing 500 1 of sterilized and filtered sea water. Every two weeks the tank water was changed and the tank scrubbed with a dilute chlorine solution. Partic- 84 CHANGES IN THE PACIFIC OYSTER DURING STARVATION 85 ulate matter was removed by filtering the tank water to remove all matter of 2/x in diameter and greater. Prior to filtering, the water was passed through a UV sterilizer. The water was recirculated in the tank and continuously passed through two spun-glass and activated charcoal filters. Temperature control was ac- complished by use of a series of coiled 2 cm inner-diameter tubes placed off the bottom of the tank through which cool ocean water was continuously circulated. Temperature was maintained at 13. 5C ± 2C. Salinity was ad- justed to 25"/,„l-28"/(.(i by use of aged tap water. The tank water was maintained near oxygen saturation by use of compressed air. The following environmental factors were monitored: (i) temperature-daily; (ii) salinity- bi-weekly; (iii) pH-bi-monthly; (iv) oxygen satu- ration-bi-monthly. Analytical Procedure The initial weight was determined by insert- ing a wood wedge between the valves of venti- lating oysters, removing the oysters from the tank, draining the interval var fluid, air drying the valves, and then weighing the drained oys- ters to the nearest gram. The starvation period lasted 175 days. A sample of five oysters was removed from the tank every 25 days for the first 125 days. A sample of nine oysters was removed at the end of the starvation period. A total of seven samples were analyzed. Upon re- moval from the tank the hinge ligaments were cut, valves pried open sufficiently to drain the intervalvar fluid, the valves air dried, and then each oyster weighed. After weighing the drained oyster the adductor muscle was sev- ered, the body removed and placed in a petri dish, and the valves weighed. The oyster body was dissected into adductor muscle, mantle, gills and palps, digestive gland, and gonad (the style sac, intestines and rectum were pooled with the gonad). Care was taken to collect all the extracellular body fluid which was subse- quently filtered through Whatman no. 1 filter paper. Each body component was pooled, weighed, freeze-dried, and then reweighed. Pooling of the body components prevented any measurement of variation within the samples. The freeze-dried body components, including the body fluid, were homogenized and then stored in capped vials at -15C until ready for analysis. Each pooled body component was analyzed colorimetrically for total protein, total carbohy- drate and total lipid. The body fluids from the 25, 75, and 175 day samples were analyzed for their free amino acid composition. Total protein . A sample of each body compo- nent was homogenized with a glass tissue grinder in doubly distilled water. Cold 157c tri- chloroacetic acid was added to the homogenate. The homogenate was centrifuged, supernatant discarded, and the precipitate dissolved in IN NaOH. An aliquot was then treated following the method of Lowry, et al. (1951) using bovine serum albumin as a standard. Total carbohydrate . A sample of each body component was homogenized with a glass tissue grinder in a 2:1 (v/v) ehloroform-methanol solu- tion. The homogenate was centrifuged and the supernatant discarded. The precipitate was air dried, pulverized with a glass stirring rod, and then digested in 10% trichloroacetic acid at 95 C. An aliquot of the supernatant was diluted and then treated following the method of Du- bois, et al. ( 1956) using glucose as a standard. Total lipid. Lipids were extracted by the method of Folch, Lees, and Sloane-Stanley (1957) and subsequently analyzed by the method of Marsh and Weinstein (1966) using tripalmitin as a standard. Chromatography of body fluid free amino acids. Amino acid analysis was done by gas- liquid chromatography with a Varian Aero- graph model 1860 gas chromatograph. The freeze-dried body fluid was homogenized with a glass tissue grinder in doubly-distilled water followed by deproteinization and ion-exchange cleanup by the method of Gehrke, et al. (1968). Subsequently the samples were prepared by the method of Roach and Gehrke (1969) and then chromatographed on a column packed with sta- bilized grade ethylene glycol adipate (EGA). The body fluid chromatograms were compared with chromatograms of a standard amino acid mixture. RESULTS The first observed effect of starvation was the disappearance of the greenish-brown chloroform soluble pigment of the digestive gland. Its dis- 86 R. T. RILEY appearance was probably due to the fact that the oysters had stopped feeding on algae rich in carotenoids. At the end of the starvation period the oysters appeared extremely emaciated and were characterized by a change in body colora- tion from creamy-white to greyish-tan. The mantle, originally thick and creamy in appear- ance, became very watery and thin to the point of translucence. The interior of the valves ex- hibited regressive lines of shell layering which indicated a shrinkage of the mantle edges from the valve edges. The first sign of gonadal matu- ration appeared at 75 days. The sex was deter- mined by examining a gonadal smear. At 125 days four of the five oysters in the sample were sexed. At 175 days all of the oysters showed signs of gonadal atrophy. Only four of the oys- ters from the 175 day sample of nine oysters could be sexed. There was no indication in the tank or in the filters that the oysters had spawned during the starvation period. There was a total of 18 mortalities; all of which oc- curred during the first 100 days of starvation. The cause of the mortalities was uncertain, al- though excessive handling could have been a factor. Due to mortalities and difficulties in deter- mining the initial weight of some oysters it was impossible to determine the weight loss of all oysters. The wet weight loss was determined for the 25, 50 125 and 175 day samples (Fig. 1). At the end of the starvation period the wet weight was 68% of the original weight, a wet weight loss of 32%. There was an increase in the aver- age water content from 82.0%- to 89.7% (Table 1). The dry weight at the end of the starvation period was 39%- of the original dry weight (Fig. 1), a dry weight loss of 61%. The digestive gland, gonad, and mantle showed the greatest dry weight loss. The adductor muscle, and gills and palps showed the least (Table 2). In general the % protein of the whole body increased while carbohydrate decreased during starvation. The % lipid of the whole body ex- hibited little change (Table 1). By the end of the starvation period only the adductor muscle did not show a considerable increase in the % pro- tein. Similarly it was the only body component not showing a considerable decrease in % carbo- hydrate. The gonad and the adductor muscle were the only components showing considerable ioor 75 50 525 WET DRY i + and- one SE WET DRY WET DRY WET DRY 25 50 DAYS 125 175 FIG. 1 . Wet and dry weight expressed as a per- cent of the pre-starved wet and dry weight. increase in the % lipid. Considering the % pro- tein, carbohydrate and lipid of the time zero sample as the pre-starved values, I calculated the utilization of protein, carbohydrate and lipid of the whole body in terms of dry weight, expressed as % of the pre-starved value: U = 100 (Xf) (XJ (DW) U is the utilization expressed as a % of the pre- starved value; Xf is the % substrate in the starved sample; X,, is the % substrate in the pre- starved sample; DW is the dry weight of the 25, 50, 125 and 175 day samples expressed as a % of the original weight. Carbohydrate was utilized the most, protein the least, and lipid utilization was intermediate (Table 3). Following a similar procedure, the utilization of protein, carbohy- drate and lipid was calculated for each body component. These calculations were done exclu- sive of the weight of the body fluid. At the end of the starvation period all body components ex- cept the adductor muscle exhibited the same pattern as exhibited by the whole body with carbohydrate being the most utilized, protein CHANGES IN THE PACIFIC OYSTER DURING STARVATION 87 TABLE 1. Change in the gross biochemical constituents and water content of the oyster body components during starvation . TABLE 2. Dry Weight of each body component expressed as a per-cent of pre-starved value. Rela- tive Carbo- Per- compo- Pro- hv- cent Days Component sitiona tein1' drate Lipid water 25 50 75 100 125 175 Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole body' Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole Body Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole body Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole body Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole body Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole body Adductor Gonad Gills Mantle Dig. gld. Body fluid Whole body .12 .24 .10 .29 .25 .14 .29 .16 .29 .12 .16 .23 .19 .30 .14 .14 .23 .19 .30 .14 .12 .31 .14 .31 .11 .14 .30 .15 .31 .10 .16 .20 .18 .34 .13 668 284 358 291 300 343 651 332 378 286 341 372 615 363 344 320 358 381 617 363 344 320 358 381 638 446 370 297 401 403 649 451 396 307 440 425 671 402 428 366 414 443 62 346 219 284 299 269 76 319 236 358 304 281 60 211 183 261 215 200 60 211 183 261 215 200 67 179 168 292 200 200 76 158 182 281 176 190 69 155 153 227 165 168 49 186 150 162 187 159 52 219 164 180 174 170 55 218 162 171 185 166 55 218 162 171 185 166 52 203 163 183 186 169 64 241 158 183 183 178 65 235 155 177 174 168 76.4 73.8 86.6 77.9 60.8 95.6 82.0 75.5 73.4 79.9 77.3 72.9 96.4 84.5 76.6 77.6 79.9 81.5 74.2 96.0 86.2 76.6 77.6 79.9 81.5 74.2 96.0 86.2 78.9 79.4 83.5 82.3 77.7 96.8 86.8 79.4 79.4 82.4 81.3 78.2 96.2 85.4 79.7 81.9 84.3 83.8 80.0 96.5 89.7 " Less dry wt. of body fluids. b mg/g dry wt.; all samples analyzed in triplicate. ' Calculated by totaling the products of column 3, and columns 4, 5, and 6 respectively. Days Adduc- tor Gonad Gills Mantle Dig. gld. 0 25 50 125 175 89 90 66 46 91 84 70 29 117 108 84 62 75 62 60 41 36 36 23 18 TABLE 3. Utilization of protein , carbohydrate and lipid of the whole body expressed as a per-cent of the pre-starved value. Days Protein Carbohy- drate Lipid 0 25 50 125 175 19 20 30 55 21 37 60 78 20 30 36 63 the least, and lipid intermediate. In the adduc- tor muscle, protein was the most utilized, lipid the least and carbohydrate was intermediate (Table 4). Fifteen free amino acids were detected in the extracellular body fluid (Table 5). Glycine, ala- nine, proline, glutamic acid and aspartic acid comprised greater than 70% of each sample. With the exception of a high concentration of tyrosine in the 25 day sample, tyrosine, orni- thine, serine, leucine, threonine, and lysine were present in moderate concentrations while valine, phenylalanine, methionine and hydrox- yproline were present in amounts less than 0.01 mg/100 ml. All free amino acids in the body fluid decreased except aspartic acid, which showed an increase amounting to 9% of the 25 day value. DISCUSSION After the first 50 days of starvation the wet weight loss began to level off. However, the dry- weight loss continued to increase. The dry weight loss and a general increase in the state of hydration of the body components occurred con- comitantly. The hydration of the body fluid re- mained constant throughout the starvation pe- riod. This seems to indicate that cell volume was maintained. The adductor muscle and gills exhibited the least dry weight loss. These two organs are the most important in maintaining R. T. RILEY TABLE 4. Utilization of protein (P), carbohydrate (C). lipid (L) of each body component expressed as a per-cent of the pre-starved value. Adductor Gonad Gills Mantle Dig. gld. Days P C L P C L P C L P C L P C L 25 11 6 8 + 7 16 0 + 2 +20 + 33 26 0 10 59 65 70 50 19 16 7 + 17 33 0 + 11 +12 + 18 38 33 31 63 63 67 125 36 21 16 9 59 7 22 29 20 37 42 33 66 88 80 175 53 50 39 68 87 63 25 55 36 49 67 54 76 90 83 a Symbols: + = substrate concentration greater than pre-starved value. TABLE 5. Free amine acid composition of the extracellu- TABLE 6. Gonadal devel >pmen /; starved vs. non- lar body fluids. Concentration3 starved . Per-cent" Amino acid 25" 75 175 Ala 6.416 4.819 2.775 Val 0.161 0.073 0.044 Gly 6.833 3.552 2.802 Leu 1.035 0.433 0.515 Pro 5.921 4.000 1.918 Thr 0.951 0.690 0.320 Ser 1.263 1.611 0.746 Met + •' + + HyPro + + + Phe 0.095 + 0.036 Asp 2.359 2.425 2.570 Glu 4.700 4.150 3.357 Tyr 3.422 0.686 0.289 Orn 1.506 1.442 0.493 Lys 0.930 0.404 0.324 Total 35.592 24.285 16.189 ■' In mg/ 100ml. '' Davs of starvation. c Present in amounts less than 0.01 mg/100 ml the structural and biochemical integrity of the oyster. It is natural that they should show the least evidence of destructive metabolism. For the first 125 days, the mantle and digestive gland showed the greatest dry weight loss. These two organs are generally considered the major storage organs of the oyster. The diges- tive gland showed the most rapid dry weight loss. The early weight loss of the digestive gland was probably accentuated by the presence of undigested food in the stomach of the oysters taken as the pre-starved sample. There was considerable evidence of active shell layering in the starved oysters. As mentioned previously one of the visible effects of starvation was the shrinkage of the mantle from the valve periph- ery and the consequent layering of new shell material. The gonad showed relatively little weight loss during the early stages of starva- Days Starved Non-starved 0 75 175 23 20 24 49 52 " Per-cent gonad of total dry weight of sample ( less extra- cellular body fluid). tion. It was during this period that maturation of the gonad was occurring. Evidently the abil- ity of the gonad to ripen was not totally deterred by the extreme conditions of starvation. The degree to which gonadal maturation occurred was considerably less than that occurring under natural conditions at the same period. Oysters were taken from North Humboldt Bay from the same site from which the starved specimens were originally collected. These oysters were dissected and treated in the same manner as the starved oysters. Comparable data on gonadal development were thus obtained. Gonadal de- velopment was considerably retarded in the starved oysters (Table 6). During the early stages of starvation the pro- tein content of the gonad increased and the lipid content remained constant (Table 4). This was somewhat different from the response of the other body components, (with the exception of the gills) which exhibited decreases in all of their energy reserves. The large decrease in the carbohydrate reserve of the gonad concomitant with the increase in protein suggests that the material and energy for gonadal maturation was drawn directly from the carbohydrate re- serve of the gonad. Carbohydrate was the most metabolized re- serve in terms of grams utilized. If the utilized amounts of carbohydrate, protein, and lipid were completely oxidized to carbon dioxide and CHANGES IN THE PACIFIC OYSTER DURING STARVATION 89 water, then the energy provided by each sub- strate is derivable. Lipids were the most impor- tant substrate in terms of total energy (Table 7). Oysters are usually considered to have a carbo- hydrate-oriented metabolism because of their high glycogen content. The data presented indi- cates that during starvation lipid and protein are the major energy reserves. One of the physiological effects of starvation in molluscs is the decrease in oxygen consump- tion which implies a decrease in the metabolic activity of the starving organism with time. It is possible to calculate the change in energy re- quirements during starvation assuming com- plete oxidation of energy reserves. The total energy requirements decreased substantially for the first 125 days after which a dramatic increase in energy requirements occurred (Ta- ble 7). During the last 50 days there was a large increase in utilization of lipid; the major con- tributor being the gonad. It is known that the eggs of oysters are rich in lipid. The rate of dry weight loss was increased for every body compo- nent, except the digestive gland, during the last 50 days relative to the values for 125 days (Table 8). Possibly during the late stages of starvation there is a decrease in the efficiency of the meta- bolic pathways, accounting for the apparent in- crease in the energy requirements during the final sample period. It is quite possible that incomplete oxidation of protein and lipid is oc- curring. Decreases in the free amino acid content of the extracellular and intracellular body fluids of stressed bivalves seem to be common (Jeffries, 1972; Feng, Khairallah and Canzonier, 1970). Stressed bivalves show a decrease in the free amino acid pools of both intracellular and extra- cellular fluids. It is interesting to compare the response of the free amino acid pools of stressed bivalves with that of bivalves subjected to salin- TABLE 8. Weight loss of each body component between sample intervals." 0-25 25-50 50-125 125-175 TABLE 7. Energy mt ■tabolism of starved oysters. Days Kcal" protein Kcal car- bohy- drate Kcal lipid Kcal to- tal 0- -25 .011 .012 .013 .036 25- -50 .010 .001 .010 .021 50- -125 .005 .003 .001 .009 125- -175 .009 .015 .016 .040 Adductor muscle 60 + 60 100 Gonad 80 110 70 430 Gills +h 60 50 90 Mantle 330 220 10 380 Digestive gld. 760 - 60 60 a Kcal consumed/(T) (W,„); T = sample interval in days, W„, = median dry weight between interval in grams. a Weight loss is /^g/day/Wm between each interval; Wm = median dry weight between interval in grams. h Symbols: + = increase in dry weight. - = no weight change. ity changes. Lynch and Wood (1966) found that with decreasing environmental salinity there was a concomitant decrease in the free amino acid pool of the adductor muscle of C. virginica. Some of the free amino acids showed less re- sponse than others. Aspartic acid exhibited lit- tle response to change in salinity. The data of Feng et al. (1970) and Jeffries (1972) indicate that aspartic acid did not decrease with stress but actually increased. In my study aspartic acid was the only free amino acid to show an increase during starvation. Apparently whether oysters are exposed to stress by environmental pollution, parasitism, starvation or decreased salinity the free amino acid pools are generally affected in the same way with the major excep- tion of the non-protein amino acid taurine. I did not measure taurine, but in the studies by Jef- fries (1972) and Feng, et al. (1970) taurine was measured and found to increase with stress. In general, bivalves subjected to decreasing salin- ity show a decrease in taurine content (Schof- feniels and Gilles, 1972). It is possible to speculate on what the similar- ity between the salinity induced change and that due to starvation means. When a euryha- line mollusc is placed in a medium of low salin- ity, the extracellular fluid tends to adjust and to reflect the osmotic pressure of the external me- dium. The intracellular osmotic pressure re- flects that of the extracellular fluid (Schoffen- iels and Gilles, 1972). In marine molluscs free amino acids are present in high concentrations relative to vertebrates. These free amino acids serve as solutes for raising the osmotic pressure of body fluids and especially that of the intracel- lular fluids (Campbell and Bishop, 1970). When subjected to dilute media the tissues lose os- motic components, especially amino acids, and 90 R. T. RILEY inorganic ions, and assume osmotic pressures comparable to that of the media. In some cases the decreases in osmotic pressure of intracellu- lar fluids may be due to increased tissue hydra- tion (Campbell and Bishop, 1970), but normally changes in cell volume of euryhaline inverte- brates are slight (Gilles, 1969). In crustaceans the concentration of intracellular free amino acids is regulated partly by a mechanism in- volving changes in the permeability of the cell membrane and partly by modification of the pathways normally responsible for the metabo- lism of amino acids (Gilles, 1969). The modifica- tion of these pathways is believed to be due in part to changes in intracellular ionic concentra- tion. It is quite likely that this same mechanism is present in marine molluscs ( Schoffeniels and Gilles, 1972). In this study the decrease in the extracellular free amino acids probably reflected an intracel- lular decrease. This postulated intracellular de- crease could be due to the increased hydration of the cells due to the depletion of polymeric re- serves, the alterations in the pathways of amino acid metabolism. It is possible that this intracel- lular decrease could be partly compensated for by an increased concentration of taurine. A study to determine the effects of starvation on the extracellular and intracellular non-pro- tein amino acids would seem to be warranted. LITERATURE CITED Campbell, J. W., and S. H. Bishop. 1970. Nitro- gen metabolism in molluscs, p. 103 to 206. In J. W. Campbell (ed.) Comparative biochemis- try of nitrogen metabolism, Vol. 1. The inver- tebrates. Academic Press Ltd., New York. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and re- lated substances. Anal. Chem. 28 (3): 350- 356. Feng, S. Y., E. A. Khairallah, and W. J. Can- zonier. 1970. Hemolymph-free amino acids and related nitrogenous compounds of Cras- sostrea virginica infected with Bucephalus sp. and Minchina nelsoni. Comp. Biochem. Physiol. 34: 547-556. Folch, J., N. Lees, and C. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tis- sues. J. biol. Chem. 226: 497-509. Gehrke, C. W., D. Roach, R. 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