PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION OFFICIAL PUBLICATION OF THE NATIONAL SHELLFISHERIES ASSOCIATION: AN ANNUAL JOURNAL DEVOTED TO SHELLFISHERY BIOLOGY VOLUME 64 Published for the National Shellfisheries Association. Inc. by Economy Printing Co.. Inc. Easton. Maryland JUNE 1974 THIS VOLUME IS DEDICATED TO THE MEMORY OF DR. LESLIE ALFRED STAUBER He will be missed by all who ivere fortunate enough to have knoivn him. M. R. Tripv University of Delaware u PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION CONTENTS Volume 64 - June 1974 Dedication to Dr. Leslie Alfred Stauber ii List of Abstracts by Author of Technical Papers Presented at the 1973 NBA Convention v Abstracts: NSA Convention 1 NSA Pacific Coast Section 10 H. J. Squires, G. P. Ennis and G. E. Tucker Lobsters of the Northwest Coast of Newfoundland, 1964-1967 16 Kenneth K. Chew, David Holland, John W. Wells, Daniel H. McKenzie and Colin K. Harris Depth Distribution and Size of Spot Shrimp, Pandalus platyceros, Trawled in Dabob Bay of Hood Canal, Washington from 1966-1971 28 Edwin B. May The Distribution of Mud Crabs (Xanthidae) in Alabama Estuaries 33 William N. Shaw and Frank Hamons The Present Status of the Soft-Shell Clam in Maryland 38 Howard M. Feder and A. J. Paul Age, Growth and Size-Weight Relationships of the Soft-Shell Clam, Mya arenaria, in Prince William Sound, Alaska 45 David A. Holland and Kenneth K. Chew Reproductive Cycle of the Manila Clam (Venerupis japonica), from Hood Canal, Washington 53 Richard Keck, Don Maurer and Robert Malouf Factors Influencing the Setting Behavior of Larval Hard Clams, Mercenana mercenmia 59 David A. Armstrong and Janet L. Armstrong A Haplosporidan Infection in Gaper Clams, TYesus capax (Gould), from Yaquina Bay, Oregon 68 R. G. Lough A Re-Evaluation of the Combined Effects of Temperature and Salinity on Survival and Growth oiMytilus edulis Larvae Using Response Surface Techniques 73 Philip A. Butler Trends in Pesticide Residues in Shellfish "^^ Roger S. Grischkowsky and John Listen Bacterial Pathogenicity in Laboratory-Induced Mortality of the Pacific Oyster (Crassostrea gigas. Thunbergj 82 Walter J. Conzonier Tissue Grafts in the American Oyster, Crassostrea virginica 92 Herbert Hidu, Willem H. Roosenburg, Klaus G. Drobeck, Andrew J. McErlean and Joseph A. Mihursky Thermal Tolerance of Oyster Larvae, Crassostrea virginica Gmelin, as Related to Power Plant Operation 1^2 iii Ramesh C. Dwivedy A Proposed Method of Waste Management in Closed-Cycle Mariculture Systems Through Foam-Fractionation and Chlorination Ill F. W. Wheaton Design Constraints on an Oyster Shucking Machine 118 Gordon Gunter and Katherine A. McGraw Basic Studies on Oyster Culture I. How Do Single Oysters Land on the Bottom When Planted? 122 Association Affairs 124 Errata 125 IV LIST OF ABSTRACTS BY AUTHOR OF TECHNICAL PAPERS PRESENTED AT THE 1973 NSA CONVENTION George Abbe and C. W. Hart, Jr. Growth and Mortality of Tray-Held Oysters in the Patuxent River, Maryland 1 Roger D. Anderson and Jack W. Anderson Physiological Responses of the American Oyster, Crassostrea vuyinka Gmelin, to Salinity Changes 1 Roger D. Anderson and Jack W. Anderson Uptake and Depuration of Petroleum Hydrocarbons by the American Oyster, Crassostrea inrginica Gmelin 1 •/ W. Rudd Douglass and Harold H. Haskin Crassostrea virginica - MSX Interactions: Changes in Hemolymph Enzymes Activities with Minchinia nelsoni Lesion Development 2 Harold H. Haskin Comparison of Resistance to Disease in Native Delaware Bay Oysters and Selected Lab-Reared Oysters 2 Herbert Hidu, Kathleen Donnelly, Julian Haynes, William Valleau and Frank Ricker Factors in the Recruitment of European Oysters in Maine 3 Robert E. Hillman Effect of Salinity on Mucus in the Mantle of the Quahog, Mercenaria wercenaria ^ S. H. Hopkins, J. W. Anderson and K. Horvath Biology of the Clam Rangia cuneata: What We Now Know and What It Means 4 Richard S. LeGore A Preliminary Assessment of the Effects of Alaskan North Slope Crude Oil on Developing Larvae of the Pacific Oyster, Crassost7-ea. gigas 4 Edward J. Little, Jr. Summary of Florida's Pensacola Area Oyster Culture Program 4 Richard A. Lutz Annual Periodicity and Its Relation to the Internal Shell Morphology of Mijtilus edulis 5 B. L. Marsh, A. W. Morrison and F. A..Costello Systems Engineering of Oyster Production 5 S. G. Martin Status and Potential of Oyster Culture in Puerto Rico 6 J. M. Neff and J. W. Anderson Uptake and Depuration of Petroleum Hydrocarbons by the Estuarine Clam Rangia cuneata 6 "^ Patrick R. Parrish Arochlor® 1254, DDT and DDD, and Dieldrin: Accumulation and Loss by American Oysters (Crassostrea virginica) Exposed Continuously for 56 Weeks 7 V Sam R. Petrocelli, Jack W. Anderson and Alan R. Hanks Biological Magnification of Dieldrin in a Two Part Food Chain 7 Charles E. Rockwood A Management Program for the Oyster Resource of Apalachicola Bay, Florida '^ W. C. Tinklepaugh, J. J. Charbonneau and R. J. Marasco The U.S. Regional Oyster Product Flow 8 Keh Tung and John W. Zahradnik Submerged Plastic Net Structures for Oyster Propagation 8 Stewart M. Tweed Settlement and Survival of Crassostrea virginica on Delaware Bay Seed Oyster Efeds 8 P. N. Walker and J. W. Zahradnik A Model Relating Filter Feeder Food Uptake, Metabolic Wastes, and Water Flow, and an Apparatus to Test the Concept 9 ABSTRACTS OF THE NSA PACIFIC COAST SECTION N. Bourne and J. C. Lee Predation of Juvenile Bivalves by the Shore Crabs Hemigmpsus oregonensis and H. nudus 1" David V. Buchanan and Paul S. Tate Acute Toxicity of Spruce and Hemlock Bark to Some E^stuarine Organisms in Southeastern Alaska Itj William F. Engesser and Daniel Cheung Improving Productivity by Using Tanner Crab Models 10 John B. Glude Identification of Oysters of the South Pacific Islands 11 C. Lynn Goodwin Diver Observations on Disposal of Dredge Spoil at Dana Passage, Washington 12 Duane W. Kama Epizootiology of MargaritifeTa margaritifera (L.) (Mollusca: Margaritanidae) Infection in Salmonid Fishes 13 Vance P. Lipovsky and Kenneth K. Chew Why "Fat" Oysters Die: A Theory on Mortality in Southern Puget Sound 13 Phillip L. Lynch and Wilbur P. Breese An Oyster Hatchery Evaluation Method 13 Louis Messmer and John M. Smith Grays Harbor Shellfish Investigations 14 Michael C. Mix Diseases of Shellfish in Yaquina Bay, Oregon 14 Albert J. Scholz Relationship Between Number and Size of Pacific Oyster Spat and Subsequent Growth 15 Frieda B. Taub A Continuous Algal Culure System for Feeding Shellfish 15 vi ABSTRACTS OF TECHNICAL PAPERS PRESENTED AT THE 1973 NSA CONVENTION GROWTH AND MORTALITY OF TRAY-HELD OYSTERS IN THE PATUXENT RIVER, MARYLAND George Abbe and C. W. Hart, Jr. Department of Limnology Academy of Natural Sciences of Philadelphia Philadelphia. Pennsylvania Three size classes of oysters, Crassostrea virginica, were held in trays for 27 months on the Patuxent River to determine differences in growth and survival. For 24 months seed and market oysters showed similar growth, but after 27 months differences could be seen. Differences in grovrth of spat were apparent from the begin- ning. Meat condition was similar throughout the study. Two-year mortality was within a normal range. Following tropical storm Agnes in June 1972, a 69% mortality occurred in the Patuxent River. It is believed that low salinity during high ambient temperatures was responsible for the heavy mortality. These data were discussed in relation to changes in salinity of the Patuxent River noted since 1963. PHYSIOLOGICAL RESPONSES OF THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA GMELIN, TO SALINITY CHANGES Roger D. Anderson and Jack W. Anderson Department of Biology Texas A&M Univer-sity College Station. Texas Measurements of osmotic and chloride ion con- centration of the pericardial fluid from Crassostrea m-ginica Gmelin showed that the fluid conformed to the ambient medium throughout the non-lethal range of salinities studied. The pericardial fluid remained very slightly hyperosmotic to the environment over the salinity range. Oysters moved to salinities below 4 ppt died before reaching osmotic equilibrium. Those animals transferred to salinities between 4 and 8 ppt reached a new steady state of fluid concentration at a slower rate than those moved to higher salinities. Analyses of chloride ion concentrations after transfer demonstrated a similar pattern of delayed conformity, but the resulting con- centrations were slightly lower than the media. Changes in percent body water and percent ash as a result of salinity alterations occurred at slower rates than those of the pericardial fluid, but final values were proportional to the extent of sea water dilution. UPTAKE AND DEPURATION OF PETROLEUM HYDROCARBONS BY THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA GMELIN ^ Roger D. Anderson and Jack W. Anderson Department of Biology Texas A&M University College Station. Texas American oysters, Crassostrea virginica Gmelin, were exposed to oil-water emulsions of selected crude oils and petroleum fractions. The rate of uptake and depuration of petroleum hydrocarbons was determined by gas chromatographic and ultraviolet spec- trophotometric methods. Oysters rapidly accumulated saturated and aromatic hydrocarbons from oil-water mixtures. Aromatic hydrocarbons were accumulated to a greater extent than n-paraffins relative to their ABSTRACTS respective concentrations in the exposure water. Saturated hydrocarbons were accumulated in liigher amounts from crude versus petroleum fractions. Accumulation of oil-derived petroleum hydrocarbons was not consistent when uptake of oil by oysters was measured over a period of several days. Following return to oil-free seawater, oysters depurated the saturated chains and most aromatic fractions rapidly. Depuration was nearly completed within 21 days. Groups of oysters were exposed to oil-water mixtures then returned to bay waters for shell growth studies. Daily average growth of ex- perimental and control populations revealed nearly uniform results. Growth of oyster control groups averaged slightly below most of the ex- perimentals except for a slight difference in one test group. ' Supported by a contract from the American Petroleum Institute. CRASSOSTREA VIRGINICA - MSX INTERACTIONS: CHANGES IN HEMOLYMPH ENZYME ACTIVITIES WITH MINCHINIA NELSONI LESION DEVELOPMENT ' W. Rudd Douglass and Harold H. Haskin Dept. of Zoology and N. J. Agricultural ExTperiment Station. Rutgers University New Brunswick. New Jersey During the summer and early fall of 1971 an experiment was performed to determine the ef- fect of Minchinia nelsoni lesion development on hemolymph enzymes of oysters (Crassostrea virginica) undergoing their first exposure to this pathogen. The enzymes examined were phosphohexose isomerase, a glycolytic sequence enzyme, and aspartate aminotransferase, an im- portant enzyme in the metabolism of amino acids and Kreb's cycle intermediates. The data were grouped in four categories: (1) Normal; (2) Pre- patent lesions; (3) Gill lesions; and (4) General infections. Changes in enzyme activities were in- terpreted as a reflection of the oyster's metabolism. During the pre-patent stage there is a 50% decrease from normal in the activities of both en- zymes. In the gill lesion stage there is a 100 - 120% activity increase over that of normal oysters. In the general infection stage activity levels are not significantly different from those of normal oysters. The significance of depressed and elevated hemolymph enzyme activities is discussed with respect to host and parasite metabolism and also their relationship with possible host defense mechanisms. ' Supported under PL 88-:3D9 contract .3-3-R-7 with the National Marine Fisheries Service and by the N. J. De- partment of Environmental Protection. COMPARISON OF RESISTANCE TO DISEASE IN NATIVE DELAWARE BAY OYSTERS AND SELECTED LAB-REARED OYSTERS ' Harold H. Haskin Dept. of Zoology and N.J. Agricultural Experiment Station, Rutgers University New Enmsinck. New Jersey For more than a period of 10 years members of this Laboratory have been reporting to the National Shellfisheries Association on developing resistance to MSX kill in native Delaware Bay oyster stocks and in oysters reared in the laboratory from parents previously selected for resistance to MSX mortality. Questions now asked are: (1) how much more resistant to kill are the present Delaware Bay stocks than those at the onset of the epizootic in 1957; and (2) what is the limit to which resistance may be developed. Samples of newly-set lower Delaware Bay spat of the five year classes 1966 through 1970 have been exposed, in trays, on the Cape Shore tidal flats for test periods of approximately 3 years each. Mortalities in these stocks have been com- pared with those in (1) lab-reared spat of 9 susceptible progeny groups; (2) lab-reared spat of parents selected against MSX disease in trays at the Cape Shore for at least three years prior to spawning (first generation, selected); (3) lab- reared spat whose parents were first generation, selected, which in turn were selected against MSX disease in Cape Shore trays (second generation, selected) and (4) lab-reared spat whose parents were second generation, selected (designated 3rd generation, selected). PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION Results, after the approximately 3-year period of exposure to MSX, are in brief: (1) an average of 8% of the 9 susceptible progeny groups sur- vived. These susceptible groups are believed to be closely comparable to the native susceptible Delaware Bay stocks present at the onset of the MSX epizootic. (2) In comparison, an average of 22% of the five lower Delaware Bay native year classes sui- vived the 3-year MSX exposure. (3) An average of 31% of the seven first generation resistant progeny groups survived. (4) An average of 44% of the eight second generation resistants survived. (5) Only one of the third generation resistant groups has been exposed for the usual test period to date, so it is not clear that its survival (50%) is significantly greater than that of the second generation groups. Rephrasing the results enables us to answer our two questions as follows. Over a three-year exposure period, com- pared with susceptible, control stocks, lower Delaware Bay native oysters will have about 3 x as many survivors; first generation lab-reared resistants about 4 x; second generation lab-reared resistants about 5'/2 x; and third generation lab- reared resistants may, or may not, show a slight increased resistance (6 x). This suggests that in- creased resistance within oyster populations ex- posed to the selective pressure of the lower Delaware Bay may approach a limit at 5 to 6 times the level of resistance in the old susceptible jxipulations. 'Supported under PL 88-.309 contract 3-3-7 with the National Marine Fisheries Service and by the N. J. Department of Environmental Protection. FACTORS IN THE RECRUITMENT OF EUROPEAN OYSTERS IN MAINE Herbert Hidu, Kathleen Donnelly, Julian Haynes, William Valleau and Frank Ricker University of Maine Walpole, Maine and Maine Departm.ent of Marine Resources, Augusta, Maine Ostrea edulis populations in Maine, introduced in the 1940's, today maintain marginal populations levels. However, hatchery-reared progeny from these stocks may exhibit superior overwintering qualities when compared to California hatchery -reared stocks. Thus preser- vation of this Maine-adapted gene pool is essen- tial for use here in an intensive aquacultural development. A combined laboratory and field program is investigating factors important in recruitment. In the laboratory the gregarious setting re- sponse is much in evidence. Larvae can be "trig- gered" to set by exposure to adult exirapallial fluid prior to exposure to cultch shells, indicating ac- tion of a waterbome pheromone. This contradicts the British view of "surface chemistry" response of the setting larvae. Extrapallial fluid of the American oyster stimulates setting in European oysters indicating an interspecific response. Other laboratory studies are investigating the biochemical natural of the setting pheromone in addition to describing the role and ultrastructure of larval sense receptors, particularly the eyespot and apical sense organ. A Latin-square field plot has been initiated in Boothbay Harbor to deter- mine the importance of gregarious setting in field populations of European oysters. The presence of adult oysters appears to increase setting on near- by cultch shells but results are inconclusive at this point. EFFECT OF SALINITY ON MUCUS IN THE MANTLE OF THE QUAHOG, MERCENARIA MERCENARIA Robert E. Hillman Battelle Columbtis Laboratories William F. Clapp Laboratories, Inc. Duxbury, Massachusetts The purpose of the study was to determine whether salinity changes had any effect on mucus secretions in the quahog clam, Mercenaria mer- cenaria. The specific tissues studies were ocated in the first fold of the mantle edge. Six separate groups of clams were established. One group was held in seawater at 35 pats per thousand salinity, one at ambient salinit ' (ap- proximately 30 ppt), one at 25 ppt, one at 20 ppt, one at 15 ppt and one at 10 ppt. After a veek of exposure at the appropriate salinities, seel 'ons of ABSTRACTS the mantle edge were prepared for histochemical studies of the quality and quantity of mucus. There appeared to be a relationship between salinity and mucus production in the quahog in that as salinity increased so did the amount of reactive acid mucopolysaccharide. BIOLOGY OF THE CLAM RANGIA CUNEATA: WHAT WE NOW KNOW AND WHAT IT MEANS S. H. Hopkins, J. W. Anderson and K. Horvath Department of Biology Texas A&M University College Station, Texas Our laboratory studies have shown that Rangia cuneata juveniles and adults can live indefinitely (months or years) in salinities from near 0 to at least 32 ppt; can regulate internal salinity (in water of salinity below 10 ppt); feed on algae and detritus particles, and absorb glucose from dilute solutions, at rates unaffected by salinity; can live 2 weeks (at 22° C) anaerobically by using their large supply of stored glycogen and have other adaptations to extremely variable or otherwise adverse conditions. Nevertheless, they are ecologically almost entirely limited to the zone of 0.5 — 15 ppt salinity. The reason is their requirement for a change in salinity to stimulate spawning and requirement of eggs and early lar- vae for salinity between 2 and 10 (possibly 15) in order to survive and develop. After reaching set- ting stage, 6-7 days after fertilization of eggs, the juveniles can live and grow in salinities from 2 to 30 ppt, and perhaps in lower and higher salinities that were not tested. Adults can live for 15-20 years in salinities too low, too high, or too stable for reproduction. In such waters the entire population may be of one or two year classes. Presence of several to many year classes means that the conditions favoring reproduction and recruitment occur every year, or most years. This makes R. cuneata useful as an indicator of salinity climate, in addition to its commercial value for shell and meat and its ecological value as food for fishes, crustaceans, birds and mam- mals. A PRELIMINARY ASSESSMENT OF THE EFFECTS OF ALASKAN NORTH SLOPE CRUDE OIL ON DEVELOPING LARVAE OF THE PACIFIC OYSTER, CRASSOSTREA GIGAS Richard S. LeGore College of Fisheries University of Washington Seattle, Washington The discovery of oil under Alaska's North Slope and proposals to transport the oil to the Puget Sound area for processing have precipitated public concern for the durability of the local marine biota. To help assess the poten- tial danger of accidental oil spills, the toxicity of Prudhoe Bay crude oil to larvae of the Pacific oyster (Crassostrea gigas) is under investigation. Oyster larvae were selected as the test organisms for this study because their use is rapidly becoming a standard for the evaluation of en- vironmental degradation. Fertilized oyster eggs were subjected to graded doses of whole crude oil and to doses of two different kinds of seawater extracts of the oil. The extracts are subsequently being analysed for their content of small hydrocarbon compounds consisting of fewer than nine carbon atoms. Dif- ferences in the larval developmental responses to the various toxicants were discussed, and some potential biological repercussions of oil im- portation into Puget Sound were considered. SUMMARY OF FLORIDA'S PENSACOLA AREA OYSTER CULTURE PROGRAM Edward J. Little, Jr. Florida Department of Natural Resources Marine Research Laboratory St. Petersburg, Florida To offset effects of extensive September 1971 kills of Crassostrea myinica, the Florida Depart- ment of Natural Resources used a National Marine Fisheries Service grant to conduct public oyster culture programs in the Pensacola estuarine area. Hydrographic and biological sampling during October 1971 through February 1972, led to selec- tion of five oyster restoration sites in East and Escambia Bays. Beginning in April 1972, 50-100 yd '^ mounds of clam shells and oyster shells were PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION planted on firm mud bottoms. In addition, ap- proximately 5,725 bu of live seed oysters were relocated to planting areas in Escambia Bay. At two areas spa tf all on 100 cm- asbestos tiles was monitored and temperatures and salinities were recorded. Spatfall on cultch plantings and on asbestos tiles was negligible except in September and Oc- tober 1972. The effects of siltation, predators and fouling organisms were generally slight. Spatfall was better on East Bay plantings than on those in Escambia Bay and commercial hai-vesting during the 1973-1974 oyster season appears feasible. ANNUAL PERIODICITY AND ITS REALTION TO THE INTERNAL SHELL MORPHOLOGY OF MYTILUS EDULIS Richard A. Lutz Ira C. Darling Center University of Maine Walpole, Maine Although daily or tidal periodicity structures are generally poorly preserved or lacking within the shell of the blue mussel, annual cycles are reflected in growth increment sequences in the innermost shell layer, facilitating age deter- mination. Each valve of 24 specimens of Mytihis edulis, all of known or assumed ages, was longitudinally sectioned along the antero- posterior axis. Individuals having survived one, three and five winters show, respectively, one, three and five dark bands in inner nacreous layer of the shell. Spawning and disturbance lines, if present, are readily distinguished from annual bands and, therefore, present no problems similar to those encountered in classical age determination studies based upon surface shell morphologj'. Careful examination of growth patterns in the inner shell layer of other bivalves may facilitate age and growth rate determinations of many recent and fossil mollusks. SYSTEMS ENGINEERING OF OYSTER PRODUCTION B. L. Marsh, A. W. Morrison and F. A. Costello University of Delaware Department of Mechanical and Aerospace Engineering Newark, Delaware The possibility of producing oysters in a closed environment, away from the hazards of nature, has been discussed among people working in mariculture for a number of years. However, oysters produced in their natural environment still cost less than those that might be produced in the currently envisioned closed systems. The purpose of this paper is to indicate the important cost factors in the closed system, show some of the important developments needed and, finally, indicate where research effort might be expended in making the closed system competitive. The base system analyzed is that proposed in a study performed by the American Cyanamid Company for the Connecticut Research Commission in 1968. The dominant system cost results from the pumping and heating of the mixture of salt and fresh water being delivered to the oysters. A recycle system, with at least 85% recycle, is necessary to bring the costs within range of the naturally produced oysters. Developments beyond that of the partially recycled water are required to make the system economically competitive. A sensitivity analysis shows areas where major gains might be realized. An analysis of research costs, probability of suc- cess and relationship to the cost-sensitive areas shows that effort is justified for research in the following areas, listed in order of decreasing im- portance: heat recovery, improved growth rates in the hatchery, growing algae and the oysters in the same tanks, cross-breeding for more rapid growth, developing less costly tank designs such as PVC-lined artificial ponds and better definition of water requirements for the growing oysters. With the expected degree of success in each research task, the cost of oysters produced in a closed environment would be less than that for naturally grown oysters. ABSTRACTS STATUS AND POTENTIAL OF OYSTER CULTURE IN PUERTO RICO S. G. Martin Puerto Rico Nuclear Center Maycuguez, Puerto Rico Current production of the mangrove oyster, Crassostrea rhizophorae, in lagoons and small bays of Puerto Rico is limited. Historically, the greatest harvest area, Laguna Rincon, produced only 20,900 pounds of oyster, including shell, during 1972. Several factors are responsible for this condition, including primitive harvesting techniques, overexploitation, predation, lack of appreciable setting areas, competition for space on mangrove aerial roots and little knowledge of modern oyster growing techniques. Approximately 776 acres are potentially available for raft and shoreline culture methods in four prime oyster-producing areas on the island. If one or more mariculture methods prove feasible and these areas are extensively utilized for oyster culture, oyster farming in Puerto Rico can be greatly enhanced. To study the possibilities of augmenting production, oyster mariculture experiments are in progress. First, the growth, survival, seasonal sexual pattern and histologic condition of mangrove oysters transplanted from Laguna Rin- con to several prime growing areas are being studied. Second, raft culture is being attempted in two suitable areas involving experimentation with various cultch materials such as rubber, as- bestos and wood and varying the horizontal dis- tances between strings in order to obtain optimum growth and maximum production. Growth in lagoons versus gi'owth in open water areas are being compared. Third, disease-free seed of the Pacific oyster, C. gigas. and the eastern oyster, C. virginica. are being raised to planting size in a running seawater system and will be planted in key areas, protected from gastropod predation, and monitored closely for growth, survival and presence or absence of disease organisms. Although all oyster imports into Puerto Rico have failed thus far, it is thought that by closely monitoring these factors mortalities can be con- trolled and the introduced species survive and eventually compete favorably with local species. Preliminary observations based on examination of 490 specimens show oysters from two typical growing areas, Puerto Real and Laguna Rincon, with an average length of 30.7 mm and a maximum length of 75.0 mm. Also, histologic analyses have revealed evidence of protandry in oysters from both areas and the presence of a gregarine parasite, Nematopsis sp. in the con- nective tissue surrounding the digestive tubules, gut and mantle, and in the gill epithelium. Also, a Lab ifrint horn yxa-\\ke organism has been ob- sei-ved in the stomach, gut, and collecting duct epithelium, often accompanied by increased hemacytic diapedesis. These organisms do not ap- pear to be harmful to the host. UPTAKE AND DEPURATION OF PETROLEUM HYDROCARBONS BY THE ESTUARINE CLAM RANGIA CUNEATA^ J. M. Neff and J. W. Anderson Department of Biology Texas A&M University College Station, Texas Clams, Rangia cuneata, were exjwsed to oil-in- water dispersions and water-soluble fractions of #2 fuel oil and South Louisiana crude oil or to sea water solutions of specific aromatic petroleum hydrocarbons. The rate of uptake of oil hydrocarbons by the tissues during exposure and rate of depuration when the clams were returned to oil-free sea water was determined by gas chromatographic and ultraviolet spec- trophotometric techniques. Clams rapidly accumulate oil-derived n-paraf- fins and aromatic hydrocarbons from oil in water dispersions and solutions. Aromatic hydrocarbons are accumulated to a greater extent than n-paraffins relative to their respective con- centrations in the exposure water. The alkylnaphthalenes, 2 methylnaphthalene and dimethylnaphthalenes were the hydrocarbons ac- cumulated to the greatest extent from the oil- in-water dispersions. Following return of the clams to oil-free sea water depuration of all classes of oil hydrocarbons was very rapid, though depuration rate was dependent on the hydrocarbon type. N-paraffins were depurated most rapidly followed by naphthalene and ' Supported by a contract from the American Petroleum In- stitute. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION alkynaphthalenes. Alkyl benzenes and polycyclic aromatics appear to be depurated most slowly. Depuration is essentially complete within 1-2 weeks after exposure to oil. AROCLOR® 1254, DDT AND DDD, AND DIELDRIN; ACCUMULATION AND LOSS BY AMERICAN OYSTERS (CRASSOSTREA VIRGINICA) EXPOSED CONTINUOUSLY FOR 56 WEEKS ' Patrick R. Parrish U.S. Eninronmental Protection Agency Oiilf Breeze Environmental Research Laboratory Sabine Island, Gulf Breeze. Florida Separate populations of oysters were exposed continuously for 56 weeks to 0.01 iig/\ of Aroclor® 1254, p,p' -DDT and DDD, or dieldrin and sampled at 8-week intervals for residues. Maximum concentrations based on body weight (/ig/g) occurred after 8 weeks of exposure, but maximum concentrations based on absolute amount of toxicant accumulated ((ig) occurred af- ter 56 weeks of exposure. After 8 weeks, average whole-body residues (wet weight) from five oysters analyzed individually were: Aroclor 1254, 1.65jLig/g, 4.0 Mg; DDT (and metabolites DDD and DDE), 0.46 iig/g, 1.0 Mg; and dieldrin, 0.08 (ig/g, 0.2 (jg. After 56 weeks, residues were: Aroclor 1254, 0.89 iig/g. 25.7 Mg; DDT and metabolites, 0.37 Mg/g, 7.0 iug; and dieldrin, 0.03 jig/g, 0.6 Mg. Seasonal patterns of accumulation and loss of the three toxicants were similar. Residues based on body weight {^ig/g) decreased 45%-81% in early July and late October, apparently as the result of spawning, and increased following these periods. This shows that the life history of oysters must be considered when evaluating residue data from monitoring programs. Growth rate (height and in-water weight) of exposed oysters was not dif- ferent from that of control oysters (Student's t- test; a = 0.01) Mortality was not significant in any group. ' Contribution No. 174, Gulf Breeze Environmental Research Laboratory. ® Registered trademark, Monsanto Company, St. Louis. MO. Mention of commercial products or trade names does not constitute endorsement by the Environmental Protec- tion Agency. BIOLOGICAL MAGNIFICATION OF DIELDRIN IN A TWO PART FOOD CHAIN Sam R. Petrocelli, Jack W. Anderson and Alan R. Hanks Department of Biology and Deparim.ent of Biochemistry and Biophysics Texas A&M University College Station, Texas This study explored the possibility of biological magnification of the chlorinated hydrocarbon in- secticide dieldrin in a two member food chain consisting of the bivalved mollusk Rangia cuneata and the decapod crustacean Callinectes sapidus. Clams were exposed to dilute solutions of dieldrin in seawater for 36 hours. At the end of the exposure time sub-samples of clam tissues were analyzed for residues of dieldrin and remaining contaminated tissues fed to blue crabs in a specially designed feeding apparatus. Results of analyses of tissues by gas-liquid chromatography indicate a magnification factor of 33-35 times ambient water concentration in clam tissues and 3.9 — 6.8 times clam tissue residue levels in crabs. Thus it is shown that dieldrin can be ac- cumulated from water by bivalves and con- centrated in predator tissues as a result of feeding. A MANAGEMENT PROGRAM FOR THE OYSTER RESOURCE OF APALACHICOLA BAY, FLORIDA Charles E. Rockwood Florida State University Tallahassee, Florida This study develops cost-benefit data on management practices suitable for protection and enhancement of the oyster producing en- vironment of Apalachicola Bay as one aspect of development of an overall management plan for the oyster resource. Detailed data have not yet 8 ABSTRACTS been released but should be available to research- ers very soon. Some of it may be of interest to marine biologists. Another product of this research of possible in- terest to biologists is the development, from com- mercial sources, of a statistical series showing monthly averages of oyster meat yields in gallons per Florida barrel. This series is continuous from 1959 to the present. Recent analysis of this data by the author and a colleague. Dr. Warren F. Mazek of Florida State University, indicates that changes in atmospheric temperature and fresh water inflow into Apalachicola Bay explain as much of the variation in yield of oyster meats to the Florida Barrel as is explained by seasonal fluctuations. Work on establishing the reliability of this statistical series and further efforts to explain the observed variation in meat yields are continuing. A third aspect of the research effort of possible interest to biologists is the diminished need for estimates of the maximum sustainable yield for various fisheries and the heightened need for estimates of optimum harvestable size by species and area. The unreliability of maximum sustainable yield estimates makes these calculations of lessened value to economists. By contrast, since minimum legal harvestable size is a principal element of much of shellfish regulation economists are in great need of ad- ditional biological data which would help establish the minimum appropriate. Biological calculations needed are life cycle growth rates by geographical area and incidence of loss through predation and disease at various life cycle stages, again by geographical area. THE U. S. REGIONAL OYSTER PRODUCT FLOW William C. Tinklepaugh, Joseph J. Charbonneau, and Richard J. Marasco Dept. Agricultural and Resource Economics College of Agriculture University of Maryland College Park, Maryland Two aspects of the inter-regional marketing problem of particular interest in this study were the regional destination of the oyster product after it leaves the processor, and the volume marketed between regions. To facilitate the study, the United States was divided into the nine geographic regions used by the Census Bureau. The data required for investigation were generated by stratified random sampling. Each of the selected firms were contacted either by telephone or personal interview. SUBMERGED PLASTIC NET STRUCTURES FOR OYSTER PROPAGATION Keh Tung and John W. Zahradnik Aquacultural Engineering Laboratory University of Massachusetts Wareham, Massachusetts ITie work presented in this report is an ex- perimental investigation of the performance of submerged plastic net structures (S.P.N.S.) for oysters. The experimental model is described. There are three variables involved in this study: net mesh size, population density and oyster initial length. Floatation was added to the struc- ture so the unit floated off the bottom and below the surface to avoid both bottom predation and surface freezing. Data gathered from the ex- perimental models were used to make cost/benefit comparisons. Graphs were presented which showed various relations between growth rate, mesh size, population density, initial size, final size and total cost-benefit relations, the S.P.N.S. system appears to be an attractive possibility for a future low investment shellfish aquafaiTn. However, other biological aspects of the concept require investigation before the feasibility of this system is firmly established. SETTLEMENT AND SURVIVAL OF CRASSOSTREA VIRGINICA ON DELAWARE BAY SEED OYSTER BEDS ' Stewart M. Tweed Dept. of Zoology and N. J. Agricultural Experiment Station. Rutgers University New Brunswick, New Jersey The seed oyster beds of Delaware Bay are located in low salinity areas. These beds have long been considered a sanctuary for young oysters because oyster drill populations are restricted by the lowered salinity. Since 1952 ex- tensive data has been collected on the spat poten- tial and seed production of these beds. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION During the past three years two of these beds were studied to determine the factors which are important in settlement and early spat survival. Cohansey Bed, with an average salinity of 12 %o , has been a commercially producing seed bed, but New Beds, with an average salinity of 16 %o , had not been in production during the preceding 20 years. These beds were chosen for study because of the presence of a reproducing drill population on New Beds and the absence of drills on Cohansey. The three years of study were anomalous years. The spat potentials as determined by clean test shells were some of the highest record- ed in 20 years. Total seed bed production has in- creased and New Beds has begun producing seed oysters. Spat potentials recorded with clean test shells over 20 years have been the same for these beds, but tests in 1972 with fouled bottom shells have shown that only a fraction of these spat poten- tials are realized on each bed. The realized poten- tial is greater on the less fouled Cohansey shells than on the New Beds shells. Lab-reared spat have been used to monitor early mortalities on the beds. The mortality from August to December was 10 - 20% greater on the lower salinity bed. Large initial mortalities of 50 - 70% occur on both beds during the first two weeks of spat exposure but can be reduced to 30 - 40% if the spat are protected from bottom predators. Quantitative estimates of predators have been made with a diver-operated suction dredge. Though drill populations in the bay have declined over the period of the study, the Xanthid crab populations on both beds are prominent at 150-250 crabs per square meter. Seed oyster production of these beds appears to be related to the differential setting pattern of the lai-vae as influenced by space competitors, and the number of predators present. Supported jointly by a grant from the Water Resources Research Institute and by the State of New Jersey. A MODEL RELATING MOLLUSK FOOD UP- TAKE, METABOLIC WASTES, AND WATER FLOW, AND AN APPARATUS TO TEST THE CONCEPT Paul N. Walker and John W. Zahradnik Aqiuwultural Engineenng Laboratory University of Massachusetts One major problem in the design of commer- cial shellfish raising systems is the lack of feeding information which is useful to the design engineer. In commercial systems economics dic- tate that some of the shellfish in the system will receive the water before others. The first in- dividuals to receive the water will remove some of the food and add their own body wastes. Therefore, later individuals will receive water which has a lower food concentration and a higher waste concentration. These later animals will not grow as fast. Optimum utilization of the food supply can be enhanced by a knowledge of the effect of these feeding parameters. Chemical kinetic techniques are used to deter- mine the relationship of food and waste con- centrations in the water, water flow rate, and various water distribution patterns to the rate of shellfish growth. Included are a theoretical analysis of these feeding parameters, and the design of an experimental apparatus, to test the technique. 10 ABSTRACTS NSA PACIFIC COAST SECTION PREDATION OF JUVENILE BIVALVES BY THE SHORE CRABS HEMIGRAPSUS OREGONENSIS AND H. NUDUS N. Bourne and J. C. Lee Environment Canada Pacific Biologi<:ul Station Nanaimo, British Columbia Predation by two species of shore crabs, Hemigrapsus oregonensis and H. nudus on juveniles of three bivalve species, Saxidomus giganteus, Veneriipis japonica and Mytilus edidis. was studied under experimental con- ditions. Crabs ranging in carapace width from 5- 20 mm were fed four size-groups of bivalves; 0-2, 2-4, 4-6 and 6-8 mm shell length. Predation rate was measured at two temperatures, 10 and 15 C and when bivalves were exposed in experimental dishes and buried. Both crab species ate juvenile bivalves; H. oregonensis was a greater predator than H. midus. Smaller size bivalves were consumed in larger numbers by all sizes of both crab species and larger crabs ate more bivalves than smaller crabs. V. japonica and M. edulis were eaten in greater numbers than 5. giganteus by both crab species. Whether S giganteus and V. japonica were buried in substrate or exposed in experimental dishes had little effect on predation rate of the two crab species, and predation was similar at the two temperatures. ACUTE TOXICITY OF SPRUCE AND HEMLOCK BARK TO SOME ESTUARINE ORGANISMS IN SOUTHEASTERN ALASKA David V. Buchanan and Paul S. Tate Oregon State University Newport, Oregon The acute toxicity of Sitka spruce and western hemlock bark to pink salmon fry (Oncorhipichus gorbuscful), pink shrimp adults and larvae (Pan- dalus borealis). and Dungeness crab larvae (Can- cer mnyister) was investigated. For salmon fry, toxic effects were observed as soon as 3 hr after exposure to hemlock bark leachates. After a 96 hr exposure, a concentration of 56 mg/liter killed 50% of the test fry (96-hr EC 50 for death). The 96 hr EC 50 using death for spruce bark leachates was 100-120 mg/liter. Although hemlock had little effect on the in- vertebrates tested, spruce bark leachates were consistently toxic to both vertebrates and in- vertebrates. The 96 hr EC 50s for spruce bark leachates to larval shrimp, adult shrimp, and lar- val crabs, with death as the criterion, were 415, 205, and 530 mg/liter, respectively. Using loss of swimming as the criterion of toxic effect, the 96 hr EC -,y^ for larval shrimp and larval crabs were 155 and 225 mg/liter, respectively. Spruce bark particles were found to be 2-6 times more toxic than leachates to shrimp larvae. The significance of these findings relative to log dumping and storage in southeastern Alaska is discussed. IMPROVING PRODUCTIVITY BY USING TANNER CRAB MODELS William F. Engesser and Daniel Cheung Oregon State University, Corvallis, Oregon A processor can conduct better long-range planning and quickly can get more, substantial short-range benefits by using pictorial and mathematical models. The authors demonstrated how motion-picture model-building techniques were used to define and measure current tanner- crab operations. In addition, simulated improved models were constructed from data of actual production runs and special equipment in-plant test runs. Preliminary potential economic benefits showed a direct labor savings of over 3 man hr./lOO lbs. of extracted meat (plus other benefits in quality, sanitation and profitability). Process charts and layout diagrams illustrated the difference between current and potential tan- ner-crab processing systems. The most important aim of our presentation was to seek both respon- ses and future model building participation from NSA members and/or other interested people. Lord Kelvin once said, "When you can measure what you are talking about, you begin to know something about it." Perhaps Lord Kelvin could have extended that truism to include: "and given this knowledge, you have the basis for im- proving and controlling the phenomenon in question." PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 11 For a written report on shrimp, crab and bottom-fish processing standards by Wm. Engesser write for Bulletin #47, Oregon State University Engineering Experiment Station, Corvallis. Oregon 97331. IDENTIFICATION OF OYSTERS OF THE SOUTH PACIFIC ISLANDS John B. Glude National Marine Fisheries Service Seattle. Washington An assignment with the South Pacific Islands Fishery Development Agency in 1971 to in- vestigate opportunities for shellfish aquaculture provided an opportunity for collection of oysters from Palau Islands, Yap Islands, Truk, New Caledonia, Ponape, Fiji Islands, Cook Islands, French Polynesia and American Samoa. In ad- dition, oysters and oyster culture of Australia and New Zealand were observed. This report covers a comparative study of oysters from various parts of the Pacific, specimens at the U.S. National Museum and a study of the published literature on the taxonomy of oysters. Oysters of the three genera Ostrea. Crassostrea and Pycnodonte described by Thomson (1954) were found in the Pacific Islands. Stenzel's (1971) description of genus Lopha was accepted instead of Thomson's genus Ostrea for the tropical cockscomb oyster. The giant or black-rimmed oyster found in Palau, Truk, Fiji and New Caledonia was iden- tified as Crassostrea echinata (Quoy and Gaimard), 1835, following Thomson's (1954) description. Thomson's use of spiny protuberances on the upper valve of juveniles as an identifying characteristic was questioned since my ob- servations in Palau Islands and American Samoa indicate that similar spiny processes occur on the shells of Crassostrea glomerata. In Stenzel's (1971) classification system these oysters would fall in genus Striostrea Vyalov, 1936. The most common oyster in the South Pacific Islands is the small mangrove oyster which was identified as Crassostrea glomerata (Gould), 1850. This species was found in Palau, Yap, Ponape, Fiji, New Caledonia, French Polynesia, American Samoa and New Zealand. The Sydney rock oyster of Australia, known as Crassostrea commercialis (Iredale and Roughley), 1933, is believed to be the same as C glome-mta (Gould), 1850, and the earlier name glomerata is preferred. Ecomorphic variations of these oysters among the islands may warrant designation as varieties, but further study is needed to decide this point. Stenzel (1971) would place these oysters in genus Sac- costrea Dollfus and Dautzenberg, 1920. The intertidal pink or coral rock oyster, which I collected in Palau Islands, Yap Islands, Fiji Islands and New Caledonia, is attached to the substrate by nearly all of the surface of the left valve which is thin in comparison with the upper or right valve. As a result it is difficult to remove these oysters fi'om the substrate without cracking the shell. This oyster is triangular in cross sec- tion with crescent-shaped meats and usually with bright yellow mantle rim. Since the pink or coral oyster is a marine species occurring in full oceanic salinity it may have potential for culture in the low atolls. The pink or coral oyster was identified as Crassostrea mordax (Gould), 1850, although some authors have used the names tuberculata. amasa and cucullata for this species. Under Stenzel's (1971) classification system this oyster would be placed in genus Saccostrea Dollfus and Dautzenberg, 1920. The green oyster occurs subtidally in various places in the South Pacific Islands and I have examined samples ft'om Palau, Truk, American Samoa, French Polynesia and Cook Islands. These oysters, which are generally less than 50 mm in height, occur attached to coral or other substrate and are characterized by greenish interior, and by sharp crenulations which are apparent on both upper and lower valves. The green oyster of the tropics is identified as Ostrea nomades Iredale, 1939, although there is a possibility that further study will indicate that 0. crenulifera Sowerby, 1871, may describe this same oyster, in whicli case that name would be preferred. Further study will be heeded to determine if 0. plicatula. 0. sandmchensis. and 0. ^AaawMW? -describe dif- ferent species of oysters. Stenzel (1971) would place the green oysters in genus Alectryo^iella Sacco, 1897. The hyotid, or subtidal rock oyster, is an ex- tremely large subtidal oyster usually attached to coral heads or other hard "substrate. I have ob- served specimens in Palau, TrUk, Ponape, Fiji 12 ABSTRACTS Islands, American Samoa and New Caledonia. The most obvious characteristics of this oyster are its extremely large size, heavy shell and sharp crenulations along the lip. In some younger specimens the folds on the surface are more ap- parent and may be produced into tubular spines. This large oyster is identified as Pijcnodonte hyotis (Linn^), 1758. although Stenzel (1971) proposes a new genus Hyotissa for this oyster. The cockscomb or bronze oyster is an unusual subtidal form of the South Pacific Islands, which has 6-12 deep, sharp radial folds extending to the margin. The exterior coloration is usually bronze-red, sometimes tinged with purple, and the interior is bronze or brown, as are the oyster meats. The name Lopha cristagalli (Linne), 1758. is used for the cockscomb oysters which I collect- ed in the South Pacific Islands of Palau, Truk and Fiji, although Thomson (1954) considers this species as an ecomorph of a group including Ostrea bresia and Ostrea folium for which he uses the name Ostrea folium Linne, 1758. Stenzel (1971) places the cockscomb oyster in genus Lopha and subgenus (Lopha). The paper included photographs and descrip- tions of these oysters. DIVER OBSERVATIONS ON DISPOSAL OF DREDGE SPOIL AT DANA PASSAGE, WASHINGTON C. Lynn Goodwin Washington State Department of Fisheries Brinnon, Washington Dredge spoil from Olympia Harbor navigation channel, composed primarily of soft mud, was barge-hauled to Dana Passage for disposal. This channel connects Case Inlet with various bays of southern Puget Sound and is about 2 miles long and V2 mile wide, with an average depth of 100 ft, ^nd tidal currents up to 3 knots. Substrates in the disposal area before dumping were primarily firm sand with scattered shell and localized patch- es of gravel mixed with sand. The sand bottom supported populations of geoducks, Panope generosa; sea pens, Ptilosarcus quadrangularis: various species of sea anemonies, crabs, sculpins, and starfish. 21,000 cubic yards of clamshell dredged spoil was dumped on the disposal area. The majority of the barge loads were dumped within 100 ft of a marker buoy. Obsei-vations made 3 days after disposal was finished, indicated the spoil was about 3 ft thick at the marker buoy, '2 ft 180 ft from the buoy, and beyond 245 ft only a trace of .spoil could be found. Using 212 ft. as a radius of a cir- cle upon which measureable quantities of .spoil were found, an estimated 3.2 acres were affected. Assuming an average depth of l'/2 ft, the volume of the spoil was estimated to be 7,880 cubic yards or 38% of the total disposed. Geoducks were buried and presumed killed within an 80 ft radius centered around the buoy, but were visible and appeared normal in areas where the spoil layer was 1 ft or less. Sea pens, sea anemonies, and other benthic organisms were buried and presumed killed under the thicker portions of the spoils. Observations completed 4 months after disposal showed that the location of the spoil had not changed but the thickness had decreased due to scouring and settling. The volume of the spoil was estimated to be reduced to 25% of the total deposited. Geoducks were alive and apparently normal at every station where they were present before dumping, even at the marker buoy where the spoil was about Wt ft thick. Geoducks were able to reach the surface with their siphons. Large sea whips, Stylata eUmgata, sea pens, and sea anemonies were found in the spoil area. They were observed in the dredge site and may have been brought in with the spoil. Large mounds of spoil were not observed as might be expected from barge dumping. These ob- servations plus water turbidity measurements in- dicate that the barge loads remained intact as a discreet mass as they fell to the bottom. Upon contact with the bottom, the material spread out laterally resulting in a relatively flat layer. Thick deposits of spoil built up in the disposal zone, changing the substrate from a firm sandy bottom to a soft muddy one. Undoubtedly, benthic animals which could not escape the area were covered and killed by the thick layer of spoil. Some geoducks survived the disposal even where the spoil layer was thickest. Sea anemonies, and other organisms either survived the disposal operation or repopulated the disposal area after dumping was completed. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 13 EPIZOOTIOLOGY OF MARGARITIFERA MARGARITIFERA (L.) (MOLLUSCA: MARGARITANIDAE) INFECTION IN SALMONID FISHES Duane W. Kama Environmental Protection Agency Seattle, Waslmigton Glochidial development in freshwater mussels (Margaritifera margarififera) located in the Siletz River, Oregon, was < . pleted in 13 days at an average water temperature of 12.8 C. Glochidia were released by these mussels for 33 days, 13 May to 15 June, 1971. The comparative susceptibility of four species of salmonid fishes, 30.5 to 87.0 mm in fork length, to glochidiosis was determined by examination of 343 caged and 177 free-swimming (native) fish for infection. Of the caged fish, 99% of the chinook salmon (Oncorhynchus tshawytscha), 75% of the coho salmon (0. kisutch), 88% of the cutthroat trout (Salmo clarki), and 95% of the steelhead trout (S. gairdneri) were infected. There was a similar relationship in infection incidence in the native fish species. Mean infection intensities in the caged and native fish were: 446 and 399 for chinook salmon, 8 and 24 for coho salmon, and 72 and 88 for steelhead trout, respectively, and 212 for caged cutthroat trout (native trout were not captured). This is the first detailed description of the metamorphosis of M. margaritifera glochidia in fish and the associated histopathology. Invading glochidia, 70 by 75 ju in size, increased in length by 500% during metamorphosis. Encysted glochidia occurred on the gill filaments, arches, rakers, and occasionally on the pseudobranchs of all fish species; however, most were on the lamellae of the filaments. Initially, the encysted glochidia have uneven walls approximately 15 i:i in thickness, but as the parasite increases in size the outer wall, i.e., the part of the cyst that is not embedded in and surrounded by gill lamellae, becomes thinner. Also, approximately 15 gill lamellae may become fused to the wall. Except for the lamella grasped by the glochidium, blood apparently continues to flow through the capillaries of the fused lamellae. However, these lamellae apparently can no longer function in gas exchange, except for the outermost lamella. Parasites encysted on the side of the gill filament restrict blood flow by pinching the filamental ar- teries. Large cysts on the lamellae increase the physiological dead space in the water flow. Club- bing of the filaments results when large cysts are located near the distal end of the filament. These pathological changes in heavy infections may result in immediate death of the fish by asphyxiation. In less heavy infections, delayed mortality occurs due to secondary infection with fungi, probably Saprolegnia sp. The invading or exiting glochidia may provide portals of entry for the fungi. WHY "FAT" OYSTERS DIE: A THEORY ON MORTALITY IN SOUTHERN PUGET SOUND Vance P. Lipovsky and Kenneth K. Chew College of Fisheries University of Washington Seattle, Washington Results from laboratory studies were compared with field data collected by the State of Washington, Department of Fisheries, to support the theory that oyster deaths in Puget Sound are a consequence of a bacterial disease. Water tem- perature, nutrient enrichment of the water, and the physiological condition of the oysters were shown to influence the outcome of the mortality. AN OYSTER HATCHERY EVALUATION METHOD Phillip L. Lynch and Wilbur P. Breese Department of Fisheries and Wildlife Oregon State University Marine Science Center Newport, Oregon We evaluated the Oregon State University oyster hatchery for procedural efficiency and biological reliability. Efficiency was determined by recording man-hours expended on various tasks. Reliability records consisted of algal cell concentration, larval growth and survival and setting success. These records were maintained while the technician adhered to a strict routine of hatchery operation. Periodically the records were reviewed and the routine revised, if necessary, to alleviate problems of efficiency or reliability. 14 ABSTRACTS The evaluation revealed that the algal culture system was unreliable, indicated by a rise in man-hours expended in maintaining algal cultures, without a concurrent rise in demand for algae. This case points out the necessity of biological records to aid in interpreting man- hour/task data. Importance of the evaluation lies in the fact that we have an effective evaluation method that is easily and inexpensively applied to a variety of situations. It is also significant that we have data to show how effort is expended in operating an oyster hatchery. ' Supported in part by the National Oceanic and Atmos- spheric Administration. U.S. Department of Commerce; Institutional Sea Grant No. 04-.3-158-4. GRAYS HARBOR SHELLFISH INVESTIGATIONS Louis Messmer and John M. Smith Grays Harbor College Aberdeen, Washington Population density and size range sampling of Mija arenaria in Grays Harbor were carried out in the years 1971 and 1972. Commercially im- portant quantities were not evident. A similar, less intensive investigation of Willapa Harbor yielded similar results. Gonad samples were collected and evaluated to determine spawning times for Mya. Ghost shrimp populations, Callianassa gigas, C. califomiensis. and Upogebia pugettensis. were surveyed in Grays Harbor. High population den- sities were recorded in many parts of the bay. Extensive ghost shrimp beds are present and in- dications are that the Callianassa species have greatly extended their range in recent years. Manila clam spat were planted at two gravelly locations in Grays Harbor in May 1973, to deter- mine feasibility of more extensive clam culture. Preliminary sampling indicates good growth rates at both locations. Survival at two months ranged from a low of 3.2% to a high of 80%. The optimum planting density appeared to be 300 clams per m^ . DISEASES OF SHELLFISH IN YAQUINA BAY, OREGON ^ Michael C. Mix Department of General Science Oregon State University Corvallis. Oregon A disease with several potentially serious im- plications and ramifications — the so-called "neoplastic disease" of bivalve mollusks — has been reported in several species of economically important shellfish in Yaquina Bay. According to published and unpublished reports, the disease found in oysters (Ostrea lurida, Crassostrea gigas), clams (Macoma nasuta, M. iris) and mussels (Mijtilus edulis) exhibits several of the general criteria recognized as indications of neoplasia in vertebrates: proliferation of a single cell line, unrestricted infiltration, nuclear and mitotic abnormalities, and morbid, gross and histologic changes indicative of fatal outcome. However, it has not been universally accepted that these clearly abnormal conditions are, in fact, neoplastic or that it is even possible for molluscan cells or tissues to undergo malignant or neoplastic alterations. A large sampling program is currently being conducted in an attempt to confirm the existence and authenticity of this disease. Information is also being accumulated which will hopefully provide clues relative to its etiology. Preliminary results, based on one year's sampling and tissue analysis of 700 oysters, were described and discussed. As a result of this research program, a second disease was found in the native oyster, 0. lurida, during the fall of 1972. The disease organism has been identified as a haplospwridan and con- stitutes the first report of a haplosporidan disease of 0. lurida and perhaps of any Ostrea species. Supported in part by the National Oceanic and Atmo- spheric Administration, U.S. Department of Commerce; Institutional Sea Grant 04-3-158-4. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 15 RELATIONSHIP BETWEEN NUMBER AND SIZE OF PACIFIC OYSTER SPAT AND SUBSEQUENT GROWTH Albert J. Scholz Washington State Department of Fisheries Bnnnon, Washington Fourteen different stocks from five areas of commercial seed production were grown at the North Bay Oyster Reserve. Plantings of two or more stocks were made each year from 1968 through 1972. Seed with high numbers of spat per shell grew more slowly than lower count seed for the first 6 months after planting. There was no difference in growth rate from 6-18 months after planting. There was no evidence of any dif- ference in potential growth among the stocks tested. A CONTINUOUS ALGAL CULTURE SYSTEM FOR FEEDING SHELLFISH Frieda B. Taub Univeysity of Washington College of Pishenes Seattle, Washington Our Sea Grant research has shown that con- tinuous cultures are a practical way to produce large amounts of algae in small amounts of space. Given a 8' x 6 floor space, 2.0 x 10" cells/day (,-(|ual to 1,000 1 of 2 X 10 "' cells/ml, in theory, enough to feed 200 cases of oyster larvae) can be produced on a daily basis with only a single line in operation. The space is adequate for a double line. The unit requires replenishment of pasteurized sea water and sterilized enrichment solution; a 2% CO^-air .source; and removal of the yield if it is stored. The unit may require cleaning at approximately monthly intervals due to wall growth, but this is highly variable and a unit may remain functional for three months or longer. Since there is space for two independent cultures, there is reasonable assurance of an uninterrupted supply of algae. The approximate cost of our unit can range from $1,000 to $2,500 depending on the use of a shaker, the brand of pump, the type of gas- mixing apparatus and refrigeration. The cost and complexity of the unit can almost certainly be reduced for hatchery use. The major labor requirement is for pasteurization of the sea water which we current- ly do in batches. If an in-line pasteurization procedure were used, the remaining labor needs would be small. Proceedings of the National Shellfisheries Association Volume 6Jf - 19Ti LOBSTERS OF THE NORTHWEST COAST OF NEWFOUNDLAND. 1964-67 H. J. SquiresK G- P- Emiis arid G. E. Tucker FISHERIES RESEARCH BOARD OF CANADA BIOLOGICAL STATION, ST. JOHN'S, NEWFOUNDLAND ABSTRACT Lobsters of Northwest Newfoundland were located in a narrow (1-2 km) hand along the largely unsheltered coast, nmong glacial boulder train or coarse tcdns and in potholes, joints and fractures in the limestone bedrock just offshore. The WO km coastline studied gave an area of lobster grounds no more than 600 km'^, and from this area about 500 tons of lobsters were taken each year. Since the fishery took about 60% of the commercial-size lobsters annually, the total stock in any year was probably 800 tons, and arerage density on the grounds was therefore about 1 per 108 m~. Temperatures were low: 0 C in winter, 5 C in early June aiul 16 C in late August, and salinities about 30% throughout the year. Females were first mature at 73 mm in carapace length at Sallys Cove and 67 mm at Port aux Choix. About 50% spawned arinually and numbers of eggs carried to hatching were 8,000 to 16,000 at lengths of 7i-97 mm. At these lengths the gain in weight af- ter one moult was U7% and 101% after two moults. A forced reduction in fishing because of a storm in 1966 showed up in a reduced proportion of 1st year recruits in 1967. INTRODUCTION Detailed studies of lobster populations just south of our study area were done by Squires (1970) and Squires et nl. (1971). Their data and those of the present study are comparable. Earlier data obtained by Templeman (1939) and Templeman and Tibbo (1945) are not. Although the latter used measurements of lobsters from the commercial catch to calculate proportions of small ones present in an area, they did not separate males from females in samples and em- phasized average lengths for comparisons between areas. Also, although they recognized that dif- ferent proportions of small animals could in- dicate different fishing rates between areas, they attributed the presence of large proportions of small ones to settlement of large numbers of lar- vae. Refutation of this thesis was proposed by ' Present address: Divi.sion of Fisheries, Box 3.58. Suva, Fiji Islands. Sciuires (1970) and Squires et ai (1971). Objectives of the present study were: to examine the grounds for topographical features giving shelter or which might contribute to distribution and abundance of lobsters; to deter- mine the temperature and salinity changes throughout the year which might affect size or maturity; to estimate rates of fishing on ad- joining fishing grounds and estimate stock size and density; to identify possibilities of increasing production by changes in fishing rates or im- proving grounds, and to compare lobsters from different areas. The following sections give an account of the predominant features of lobster grounds on this coast, the temperature regime, the fishery and fishing rates, an estimate of total stock and den- sity, and some aspects of the biology of the lob- sters. DESCRIPTION OF THE LOBSTER GROUNDS The following is modified from a description of 16 LOBSTERS OF THE NORTHWEST COAST OF NEWFOUNDLAND, 1964 - 1967 17 FIG. 1. Map of place names used in the text. Lobster grounds are in a narrow band quite near the coast. the grounds by the late Hugh Lilly, a geologist from the Memorial University of Newfoundland. His obsei-vations from SCUBA diving surveys (Lilly, 1965, unpublished) are supplemented by ours in some of the areas. Erosional processes which produced the land- scape of western Newfoundland also provided the material which now rests upon the submarine shelf. These influence the distribution of benthic animals, including lobsters. The effects of Pleistocene glaciation are recognized everywhere, for the glaciers scooped out the fjords such as Bay of Islands and Bonne Bay and deposited much of the resulting debris as terminal 18 H. J. SQUIRES, G. P. ENNIS AND G. E. TUCKER moraines at the mouths of the fjords or nearby. In many places the ice left the hills bare of residual soils and clays, adding this material to the shelf. The coastal lowlands from Bonne Bay to Cape Norman include the raised postglacial terraces of the Great Northern Peninsula. Several terrace levels reflect the development of temporary shorelines during periods of interrupted uplift. The abundance of glacial material derived from the uplifted terraces nearby provide cover for lobsters. Much of the sand and silt of the scallop environment is reworked outwash of glacial origin, refurbished now by attrition of the present shoreline. Much of the other coastal areas are subject to vigorous wave action, and where the waters are shallow, cliff talus appeai-s to become ground up or carried away as fast as it falls into the sea. The coastal marine shelves in some areas do not exceed 55 m in depth within 18 km from the coast. They are gently rolling, averaging 45 m in depth with numerous shallow areas less than 37 m. Gun Point Shoal and Whaleback Shoal are examples. West of Sallys Cove, south of Wliale- back Shoal and extending 16 km from the coa.st are the remains of an immense terminal moraine. Rock fragments representing the Precambrian crystalline complex of the northern Long Range Mountains, the St. Georges carbonate group and the Humber Arm group are found in this deposit. On the Whaleback Shoal are exposures of bedrock and long reaches of sand where the action of waves produce effects to depths of at least 36 m between the shoals. Talus is not abundant everywhere in the exposed zone. The lobsters seem to frequent numerous potholes, joints and fractures in bedrock in these areas and near cliffs such as at Table Point, and the coarse talus is found well away from the shore, often at distan- ces of several hundred metres. On some of the more sheltered lobster grounds, such as in St. John Bay, the bottom consists of shingle, talus derived from submarine and shore bedrock and glacial boulder train. AREA OF THE LOBSTER GROUNDS FROM CAPE GREGORY TO FLOWERS COVE AND AN ESTIMATE OF THE POTENTIAL STOCK AND DENSITY OF LOBSTERS The coastline from Cape Gregory to Flowers Cove is about 400 km long. Although lobsters were occasionally seen as deep as 35 m where diving was done along the coast, some areas just offshore such as the large shoal west of Sallys Cove were completely devoid of lobsters as shown by special fishing (Andrew Swim, personal com- munication). As a result of many years of fishing, trapping was done along the coast usually not more than 2 km from the shore and not deeper than 30 m. The lobster grounds, therefore, appear to be scarcely wider on the average than one and one-half km and the total area to be not more than 600 km- (Fig. 1). Since the total catch from the lobster grounds has been about 500 tons annually over several years and the fishing rate observed in 1966 and 1967 was little more than an average of 60% in most areas, the potential stock of commercial sizes on the grounds would be about 800 tons an- nually. From this available annual stock of ap- proximately 1.8 million lbs the first year recruits, averaging close to 1 lb each, would equal about 1.1 million lobsters (60% of the total). The post- recruits, averaging slightly more than 1.5 lbs each, would equal about 0.4 million lobsters. The pre-recruit groups (two size groups smaller than the legal sizes are usually seen on the grounds) would both approximately equal in number the recruit group, or about 2.2 million lobsters. In total, therefore, there would be about 3.7 million lobsters of near-commercial size on these grounds at the beginning of the fishing season in any year. The density of lobsters on these grounds can be calculated from: area of the grounds total number of lobsters = 600,000,000 m - 3,700,000 = 108 m- for each lobster. THE LOBSTER FISHERY FROM CAPE GREGORY TO FLOWERS COVE During 1955-1969 the annual landings of lob- sters in this area fluctuated between 320 and 510 tons (Fig. 2). They represented about one-quarter of the total Newfoundland landings each year. In the last five years the lowest catch in the area oc- curred in 1966, when about 370 tons were landed. LOBSTERS OF THE NORTHWEST COAST OF NEWFOUNDLAND, 1964 - 1967 19 o o o I 200 I 100 - .000 900 - BOO 70C 600 - 500- 400 - 300 - 20C. - lOO - NElAlFOUMDLAND TOTAL LANDINGS CAPE ST GREGORY TO CAPE NORMAN LANDINGS 953 1955 .957 i959 96 i963 965 967 969 YEAR FIG. 2. Lobster- landings far the northwest coast (Cape St. Gregory to Cape Norman) and for all of Newfoundland in 1953-69. In that year, near the middle of the fishing season, a strong gale from the southwest destroyed a large proportion of the lobster traps along the coast. The fishermen recovered as many traps as they could to resume fishing after the storm, but their fishing effort during the season was appreciably reduced. In 1967 size-samplings from the commercial catch, the proportions of small lobsters were less than they had been in 1966. The reduced fishing rate in 1966 had left a considerable number of commercial-sized lobsters on the grounds and these contributed to the larger than usual proportion of large lobsters in the catch in 1967. ESTIMATING FISHING RATES Fishing rates may be estimated from the proportion of first year recruits appearing in histograms of length composition of the com- mercial catch of uninjured male lobsters (Squires, 1%5: 1970; Squires et al. 1971). The five populations fi'om which length measurements had been obtained in both 1966 and 1967 (affected by the storm mentioned in the previous section) all had an appreciable decrease (4-10%) in the proportions of first year recruits present (Fig. 3). These comparisons within areas indicated that a change in fishing rate in one year would show up in the following year. Comparisons between areas along the coast showed that fishing rates were higher, in most in- stances, from Bonne Bay to Port aux Choix than further north. Areas to the north had less than 60% first year recruits in samples while to the south, almost all had more than 60% (Figs. 3 and 4). Pistolet Bay, the area of lobster fishing C^ RECRUIT MOLT 1966 ^_ 1 POST-RECRUlT MOLTS 1967 PORT S6UN0EHS BATEAU COVE JMk li>tf^ CARAPACE LENGTH FIG. 3 Percentage occurrence of first year recruits (first year at legal size) in histograms of length frequencies of commercial lobsters from, the same areas in consecutive years (1966 and 67). 20 H. J. SQUIRES, G. P. ENNIS AND G. E. TUCKER ..S67I P.STOLET Bflt (dij) IB RECRUIT MOLT C3 POST-HECfiUlT MOlTS (1967) FLOWERS cove l>390) (1966) ST MARGARET B0< ( 557 i u96r) euiiCK DUCK cove tazji 1 ,q66< ST JCHM ISLAND ' i'22l 11967) SRiG Bflf . 375 J II966J PORT Ay CMOiX ii639y CARAPACE LENGTH FIG. 4. Percentage occurrence of first year recndts in. length frequencies of commercial lob- sters from the northwest coast of Newfoundland. farthest north in America, had only 38% because the fishery had started only recently in this bay and only a few traps were used. COMPARATIVE BIOLOGY Lobsters for biological studies were obtained from fishermen trapping about 150 specimens monthly on charter at Sallys Cove, Cow Head and Port aux Choix from June to October, 1966. Data on 460 males and 650 females from the three locations were combined. Details on each specimen included: total live weight and claw weight (weight of 1st pereiopods) on a Mettler precision balance; carapace length and length of claws with a vernier calipers; total lengths and tail widths on a mm measuring board, and egg and ova diameters under stereomicroscope on a mm scale (see also Squires, 1970). An experiment on growth of lobsters held and fed in metal cages was conducted at Port aux Choix in 1966 (Stewart and Squires, 1967). From the data on weights, etc., the lobsters of the areas sampled were compared with those from Port au Port Bay, North Arm and York Harbour (Squires, 1970; Squires et ai. 1971). Relative lengths and weights. The graphs of chelae weight as a percent of total weight (Fig. 5) showed that in this respect the lobsters of the northwest coast were in- termediate between North Arm and Port au Port Bay lobsters. Size-for-size they were more at- tractive market lobsters than those from Port au Port Bay because of their larger claws. Regression equations of claw size in this paper (Table 1) and in others (Squires, 1970; Squires et ai. 1971) show that the crusher claw contributes most to the differences between lob- sters from different areas. For example, in West Coast samples the slope of the cutter claw lengths varies only from 1.50-1.52 CL while the slope of the crusher claw lengths varies from 1.46- 1.68 CL in male lobsters. (But in Bonavista Bay (Ennis, 1971) the slope in both cutter and crusher claws is the same at 1.41 CL. The weights of the crusher claw show even more distinc differences between lobsters from different areas than lengths (the range in slope of r'-jression curves in the four West Coast areas was from 3.5566 log CL-4.3045 log CL). The comparative weights of cutter claws showed similar differences: greater in males than in females and different from area to area, but the range was less than in crusher claws. However, adding the weights of both claws together would make more distinct differences between areas, substantiating the use of this pai'ameter as a percentage of total weight to show differences between areas (Fig. 5). Growth increment. Data from the experiment with lobsters moulting in cages at Port aux Choix indicated that increments in the carapace length of males varied rom 7-13 mm and averaged about 11 mm, increasing with the size of the lobster (Fig. 6). In females increments were 6-10 mm with an average of about 9 mm and decreased with the size of the lobster. The average increase in carapace length was 16.5% in males and 11.5% in females. Length increments of male lobsters from Port au Port Bay, North Arm, York Harbour and Bonavista Bay were estimated to be on the average 10, 9, 10 and 12 mm, respectively. Hypothetical gain in weight on moulting. The method of calculating hypothetical gain in weight from regression equations was described LOBSTERS OF THE NORTHWEST COAST OF NEW^OLWDLAND, 1964 - 1%7 21 TABLE 1. Length a>id weight relationship equations for lobsters from the northwest const of Neufoundlaml. TL = total length, CL = carapace length. Cut = cutter claw, Cru. = crusher claw, Abd Wd = abdomen width, TW = total weight, W = weight. Logs are to base 10. Body weight = total weight less weight of Lst periopods. Relationship ii^'iX Equation Length/range C.c. No. of Specimens tl/cl tl/cl M F TL = 2.53 CL - 23.96 TL = 2.84 CL - 6.13 47-113 47-126 0.98 0.99 456 590 Cut L/CL Cut L/CL M F Cut L = 1.51 CL - 11.14 Cut L = 1.29 CL - 4.70 47-113 47-126 0.96 0.96 433 567 Cru L/CL Cru L/CL M F Cru L = 1.56 CL - 23.43 Cru L = 1.23 CL - 0.40 48-113 47-126 0.96 0.97 449 584 Abd Wd/CL F Abd Wd = 0.91 CL - 19.00 47-126 0.94 621 TW/CL TW/CL M F Log W = 3.1637 log CL - 3.4054 Log W = 2.9660 log CL - 3.0236 48-113 47-126 0.99 0.99 422 552 Body W/CL Body W/CL M F Log Body W = 2. *858 log CL - 3.O764 Log Body W = 3.0482 log CL - 3.3417 48-113 47-126 0.99 0.99 422 552 Cut W/CL Cut W/CL M Log Cut W = 3.3567 log CL - 4.5857 Log Cut W = 2.6886 log CL - 3.3469 47-113 47-126 0.97 0.97 437 562 Cru W/CL Cru W/CL M F Log Cru W = 3.7597 log CL - 5.2340 Log Cru W = 2.8793 log CL - 3.6291 48-113 47-126 0.97 0.97 446 580 Cut W/Cut Cut W/Cut L L M F Log Cut W = 3.0188 log Cut L - 4.3521 Log Cut W = 2.6700 log Gut L - 3.6584 61-166* 63-175 0.98 0.97 428 557 Cru W/Cru Cru w/Cru L L M F Log Cru W = 3.1215 log Cru L - 4.3409 Log Cru W = 2.8257 log Cru L - 3.7843 57-165* 59-165 0.99 0.98 442 575 * Range of length of cutter or crusher claw by Squires (1970). With a moult increment of 11 mm in males from this area, the length groups used were 70-80 mm, 81-91 mm and 92-102 mm. These groups were chosen to represent the pre- recruit, recruit and post-recruit sizes of lobsters. (The legal minimum size is 81 mm in carapace length.) From the data on weights at each carapace length the gain in weight on moulting was calculated to be 47% in one and 101% in two moults. This gain for northwest coast lobsters was less than for Port au Port Bay or York Har- bour lobsters: 49% and 117%, and 59% and 119%, respectively. However it was somewhat larger than for North Arm lobsters: 45% and lOO'o in one and two moults (Squires et cd.. IWl). Maturity. From the data for June and October (Table 4) the female lobsters of Sallys Cove and Cow Head were close to the expected 50% potentially ovigerous each year such as is found in these populations (Squires, 1970). The 70% ovigerous in the month of August was unusually large, but most of them had recently laid eggs (eggs were 100% yolky), and in this period when non-ovigerous females were moulting, the number of ovigerous animals entering traps to feed could be disproportionately high. The percentage poten- tially ovigerous at Port aux Choix in July (Table 2) was 44% substantially lower than the ex- pected 50%. However, the data for one year might not be enough to indicate even in this nor- 22 H. J. SQUIRES, G. P. ENNIS AND G. E. TUCKER 50 48 46- 44 42 40 38 Q: LU a 36- 34 32- 30- 28 26 • NW COAST (422) ' PORT AU PORT BflV (483) u NORTH ARM ( 396) COAST NORTH PORT AU PORT — r r 1 r 1 r 1 1 1 1 1 r 1 ~i 1 1 1 t 1 1 1 1 1 1 ' ' ' 62 64 66 68 70 72 70 76 76 80 82 81 86 88 90 92 94 96 98 100 102 104 106 108 MO (12 il4 ll6 ilB CARAPACE LENGTH -mm FIG. 5. Gaw (chelae) weight as a percentage of total weight in lobsters from North Arm. Port an Port Bay and the northwest coast of Newfoundland. therly area that the rate of egg-laying could be as low as was seen in lobsters at York Harbour, Bay of Islands (Squires ef al. 1971). Size at first maturity. Female lobsters from Sallys Cove and Cow Head were first mature at 73 mm in carapace length. This was the smallest size seen with large ova (Table 2). The smallest with large ova in Port au.x Choix, however, was only 67 mm. These samples were obtained from the partly enclosed area of St. John Bay, resembling North Arm in this way, although the summer temperatures were lower. They were lower than off Cow Head and Sallys Cove so that temperatures could not account for the earlier maturity. Combined data from lobster sampling on the coast for abdomen width as a percentage of carapace length showed an acceleration in the rate of increase in ab- domen width through the sizes of 64-76 mm (Fig. 7), representing the range of sizes when maturity first occurred in female lobsters from these areas, areas. LOBSTERS OF THE NORTHWEST COAST OF NEWFOUNDLAND. 1964 - 1967 23 TABLE 2. First mature sizes and ovigerous or potentially ovigerous female adult lobsters from the northwest coast of Neufoundland. in 1966. Month No, of Carapace Percent Percent Minimum Minimum Maximum adult length oviger- potenti- size size size specimens range of ous ally oviger- vd.th large juvenile specimens oviger- ous ova ous mm % io mm mm mm Area June 170 52-107 15 52 75 73 71 Sallys Cove & Cow Head August 119 67-116 70 23 - - - It M October 64 67-102 22 53 81 74 70 11 ft July 50 61- 96 10 Uh 80 67 61 Port aux Choix August 65 50-102 28 37 72 68 73 It tt FecuyiAity. Lobsters from Sallys Cove and Cow Head had average counts of 8.200-16,300 eggs ready to hatch, according to carapace lengths of 74-97 mm (Fig. 8). These counts were higher than those from lobsters of the same length from Port au Port Bay: 6,200 - 15,000 (Squires, 1970). This finding supports the assumption that fecundities of lobsters from different areas may differ. Counts of recently laid eggs were higher than of eggs ready to be hatched in lobsters larger than 80 mm (Fig. 8) in agreement with findings of Saila et al. (1969). DISCUSSION Pishing conditions on the northwest coast. In Lilly's (1965) report the reference to intense wave action suggests that the lobster grounds on this coast can be fished only with difficulty. At- trition of the shore and destruction of talus blocks, etc., are evidence of occasional heavy winds and waves on this open coast. Occasionally, such as in 1966, a storm destroys almost all the lobster traps. There are few harbours and fewer villages where boats can be berthed without having to be hauled up on the beach, out of reach of the waves, or where floating lobster crates can be anchored in safety. Harbour improvement, such as breakwaters that might be built at some of the villages, would be of value if they were adequate for protecting the lobster-holding crates under storm conditions. However, the lobster fishery can be carried on with the small boats presently in use because the grounds are located close to shore. The use of plastic traps in addition to, or instead of, wooden ones would help to sustain fishing effort through stormy periods. •MALES (14) "FEMALES (17) • 12 - • • • ^/^t • • 1 1 - •^..'^ ^ • 10 - t-.-''''^* 0 0 • 9 - 0 0 8- 0 -jL_o^ 8 7 - • 0 • 08 0 6 - 0 0 1 ! 1 1 T 56 58 60 62 6^ 66 68 70 72 74 76 78 80 82 84 CARAPACE LENGTH- mm FIG. 6. Moult increment in carapace length of male and female lobsters from the northwest coast of Newfoundland. 24 H. J. SQUIRES, G. P. ENNIS AND G. E. TUCKER 50 54 58 62 66 TO 74 78 82 86 90 94 98 t02 106 110 CARAPACE LENGTH -mm FIG. 7. Abdomen widths as a percent of carapace lengths in female lobsters from the northwest coast of Newfoundland. The characteristics of the exposed shoreUne, as described by Lilly, leave little possibility of im- proving the grounds by blasting cliffs, for exam- ple, to provide more sheltering rubble for lob- sters. Only in partly enclosed areas, such as Port aux Choix, could this be effective (Lilly, 1965, and pei-sonal communication). Fishing rates. An estimate of a comparative fishing rate for lobsters based on the proportion of first year recruits in the commercial landings (Squires, 1965; 1970) may be more reliable than one based on fishing effort, which is difficult to define with accuracy. Fishing effort is not only the number of traps used or the number of times they are hauled; it is also the skill of fishing experience, which can put traps in advantageous positions on the grounds, and the frequent changes in position and replenishment of fresh bait each time a trap is hauled. Results can be dependant also on den- sity of lobsters on the grounds, scarcity of natural food for the lobsters, etc.. all of which are difficult or impossible to measure. An estimate independant of these and based only on the com- position of the commercial catch has a distinct advantage. A representative sample of the catch can be secured by sufficient measurements of specimens before the moulting period. About 1,000 obtained from each fishing ground appears adequate for comparisons between grounds. With first year recruits comprising 60-70% of the lobster landings on the coast south of Port aux Choix, an increase in fishing could not be recommended. Tlie fishing rate apparently made best use of the resource as shown by only slight fluctuations in annual landings. The accidental change in rate in 1966 was clearly apparent in 1967 indicating that the method of estimating fishing rates was applicable and also that its sensitivity might be due to a maximum level of fishing in most years. Low proportions of first year recruits, such as the 38% in Pistolet B;iy, suggested that more fishing could be done to catch the large lobsters present but that these would soon decrease under fishing pressures. Recovery from moulting and egg-laijing. The high percentage of ovigerous females cap- tured in August (Table 2) indicated that they had laid eggs recently and were searching for food. Although the number of non-ovigerous females and males had been reduced on the grounds as a result of fishing, many of them could have been in the process of moulting and not feeding. This was shown in later months when the proportions of ovigerous ones were much less. The search for food by lobsters entering traps is an indication of the general paucity of natural lobster food on the grounds. The appropriateness of supplemental feeding is worth considering in the period after 30 28 26 24 - ?22 ETED EGGS • NEW EGG5 O No = 360 CL - 18 279 (63) No. = 648 CL- 40 965 (19) 70 72 74 76 78 80 82 84 86 88 90 92 94 96 98 lOO 102 104 106 108 CARAPACE LENGTH -mm FIG. 8. Numbers of eggs (recently laid or eyed and ready to hatch) at each carapace length in lobsters from the northwest coast of Newfound- land. LOBSTERS OF THE NORTHWEST COAST OF NEWFOUNDLAND, 1964 - 1967 25 moulting or egg-laying, to assist in the recovery of body proteins lost as a result of cessation of feeding (Squires et ai. 1971). Shore fisheries that leave offal or dead fish on the lobster grounds may be important to production. Dying-out of shore fisheries may be detrimental to lobster populations, and may have caused the decrease in lobster production in such areas as Conception Bay. Comparing lobster populations. Given sufficient data, comparisons may be made using the following characteristics: relative sizes of claws in males, percentage increase in weight as moulting in uninjured males, sizes of females when first mature, numbers of eggs carried, patterns of behaviour in nature, etc. Some of these may be attributed to conditions of the environment such as prevailing temperatures (phenotypical differences) or evolved attributes, as a result of long isolation (genotypical dif- ferences). Perhaps the most important, from the point of view of the fishery, are the patterns of behaviour such as those that keep the lobsters a relatively constant distance from the noise of surf at the shore. The lobsters of the northwest coast kept to a narrow band of seabed near the coast, not venturing any deeper than 35 m for the most part. Although there were extensive shallow banks not far from the coast with temperatures similar to the nearshore grounds, no lobsters could be trapped there. Yet, lobsters are found many miles from shore off southwestern Nova Scotia, for example (Wilder, 1947). If the lobsters of the northwest coast were genotypical in their behaviour pattern, it might be said that they would make ideal transplants to exposed coasts, where they would not be likely to wander into deep water nor to come too near the shore, but to build up a po]5ulation within trapping di.stance. Maturitij and estimates of potential spawning. Oogenesis, in these lobsters of northern low temperatures, was unmistakeably slow but ac- celerated during the summer, when temperatures became relatively high for a short period. Tlie dif- ferences in sizes of ova would, therefore, have to be carefully defined in order to indicate whether they would be spawned in the current year. For example, ova less than 1.0 mm in diameter in June and July could not be expected to be spawned in August; and ova of 1.3 mm in diameter in September would not be spawned un- til the following year. The percentages of poten- tially ovigerous females observed by Ennis (1971) were based on a definition of " ova larger than the ova of ovigerous females" (about 0.5 mm or less) and were, therefore, unusually high. Generally, the potential spawners are about 50% or less in these areas. Prevailing tem.peratures ayid salinities. The general hydrography of the Gulf of St. Lawrence has been described by Lauzier et al. (1957) and Lauzier and Bailey (1957). With reference to the northwest coast of Newfoundland TABLE 3. Temperatures (C) at stations on the lobster grounds off the northwest coast of Neufoundland 1966. Station 4 10 J U N 18 E 25 28 J U 19 L Y 27 9 AUG 17 U S T 23 31 Depth Green Point 7.3 4.8 - - - 10.6 8.3 - 14.0 13.5 - - 16.8 16.3 - Surface 15 m Cow Head 7.4 - - - 10.8 - 14.8 - — 16.4 - Surface 5.0 - - - 8.6 - 14.5 - - 15.8 - 15 m Port aux Choix — 7.1 6.4 8.6 7.0 8.1 7.6 — 11.8 11.6 14.3 13.0 15.4 14.9 16.2 14.9 15.8 14.7 15.0 14.8 Surface 15 m 26 H. J. SQUIRES, G. P. ENNIS AND G. E. TUCKER TABLE 4. Salinitks ( '/„ ) at stations on the lobster groumis off the northwest coast. 196i>. Station J U N E J u L Y AUG U S T Depth 4 10 18 25 28 19 27 9 17 23 31 Green 30.8 - 29.8 30.0 30.2 Surface Point 30.6 - - - 30.4 - 30.3 - - 29.8 - 15 m Cow Head 27.8 - - - 30.3 - 29.6 - - 29.8 - Surface 30.3 - - - 30.3 - 29.7 - - 29.8 - 15 m Port aux - 30.0 29.6 30.1 — 30.0 29.8 30.1 29.7 29.9 29.7 Surface Choix - 30.5 30.1 30.1 — 30.3 29.9 30.1 29.7 29.9 29.9 15 m they showed late spring and early autumn tem- peratures to be about 5 C at 20 m. During the present investigations, tem- peratures on the lobster grounds (at about 15 m) increased from nearly 5 C to about 16 C from June to August. Surface temperatures were somewhat higher but began to decrease late in August when the peak had been passed. This peak was later off Green Point then off Port aux Choix, about 200 km farther north. From previous work in Port au Port Bay and Bay of Islands (Squires, 1970; Squires, et ai. 1971) and personal experience of the senior author, temperatures were seen to decrease to somewhat lower than 0 C on the lobster grounds from January to March and there was ice cover at some time during the winter. Although the prevailing set of the current is toward the coast and northward, as described by various authors, offshore winds were seen to carry ice away from the coast, leaving clear water while they lasted. With the return of onshore winds, the ice soon re-appeared on the horizon and grounded again on the shore in less than a day. The ice cover of- ten persisted into May but the prevailing currents brought warm water into the area and by early June the temperatures reached 5 C on the lobster grounds (Table 3). Salinities did not vary much and were close to 30%. throughout the year (Table 4). Some slight variations were a low reading of 27.8%. off Cow Head in June, and a few high readings slightly over 32%o in St. John Bay, also in June. ACKNOWLEDGEMENTS We acknowledge with thanks the assistance of summer students: Messrs. G. Lilly, F. Fifield and G. Parsons; also Capt. G. Brown of the CGS PARR and Mr. J. Paine of Cow Head and Mr. H. Robeits of Sallys Cove, fishermen. LITERATURE CITED Ennis, G. P. 1971. Lobster (Homanis anierwanus) fishery and biology in Bonavista Bay, New- foundland 1966-70. Fish. Res. Board Can., Tech. Rep. 289, 46 p. Lauzier, L. and W. B. Bailey. 1957. Features of the deeper waters of the Gulf of St. Lawrence. Fish. Res. Board Can., Bull. 11; 213-2.50. Lauzier, L., R. W. Trites and H. B. Hachey. 1957. Some features of the surface layer of the Gulf of St. Lawrence. Fish. Res. Board Can., Bull. 11: 195-212. Lilly, H. D. 1965. Unpublished MS. Marine inven- tory West Newfoundland. ARDA Project 20007. Report 74 p. Saila, S. B., J. M. Flowers and J. T. Hughes. 1969. Fecundity of the American lobster, Homaiiis amerkayius. Trans. Am. Fish. Soc. 98: 537-539. Squires, H. J. 1965. Decapod crustaceans of New- foundland, Labrador and the Canadian eastern Artie. Fish. Res. Board Can., MS Rep. Ser. (Biol.) No. 810, 212 p. Squires, H. J. 1970. Lobster (Homanis amerwamis) fishery and ecology in Port au Port Bay, Newfoundland, 1960-65. Proc. Natl. Shellfish. Assoc. 60: 22-39. LOBSTERS OF THE NORTHWEST COAST OF NEWFOUNDLAND, 1964 - 1967 27 Squires, H. J., G. E. Tucker and G. P. Ennis. 1971. Lobsters (Homarus americanus) in Bay of Islands. Newfoundland 1963-65. Fish. Res. Board Can. Manuscript Rep. 1151, 58 p. Stewart, J. E. and H. J. Squires. 1968. Adverse conditions as inhibitors of ecdysis in the lobster Homants americanus. J. Fish. Res. Board Can. 25: 1763-1774. Templeman, W. 1939. Investigations into the life history of the lobster (Homarus americanus) on the west coast of Newfoundland 1938. Res. Bull. Div. Fish. Res. Newfoundland, No. 7, 52 p. Templeman, W. and S. N. Tibbo. 1945. Lobster investigations in Newfoundland, 1938-1941. Res. Bull. Div. Fish. Res. Nevirfoundland, No. 16, 98 p. Wilder, D. G. 1947. The effect of fishing on lobster populations as detennined by tagging e.xperiments. Fish. Res. Board Can., Prog. Rep., Atl. Coast Sta., No. 37: 10-13. Proceedings of the National Shellfisheries Association Vohime 64 - 1974 DEPTH DISTRIBUTION AND SIZE OF SPOT SHRIMP, PANDALUS PLATYCEROS, TRAWLED IN DABOB BAY OF HOOD CANAL, WASHINGTON FROM 1966 to 1971 1 Kenneth K. Cheiv, David Holland. John W. Wells, Daniel H. McKenzie and Colin K. Harris COLLEGE OF FISHERIES, UNWERSITY OF WASHINGTON SEATTLE, WASHINGTON ABSTRACT The January size frequency distribution of male and female spot shrimp (Pandalus platyceros) was presented and discussed. The females revealed more variability from year to year. There appeared to be two size groups of females in some years. The male size distribution was more consistent. The 1966. 1968, 1969, 1970 and 1971 spot shrimp length-weight lines were compared and the stmilarities and differences from year to year were shown. Further, it was shown that this species of shrimp may have a diel migration pattern. Trawl hauls for 1970 and 1971 revealed they were found to be greatest in numbers at shallower depths during the nighttime and greatest in numbers at deeper depths during the daytime. INTRODUCTION The spot shrimp (Pandalus platyceros) is caught commercially by pot gear in Dabob Bay and other parts of Hood Canal. Approximately 67,000 pounds were landed in 1972 (pers. comm. Mr. Dale Ward, Washington Department of Fisheries) and sold locally in Hood Canal and throughout the Fuget Sound region. Small pot fisheries for this species also occur near Carmel in California; Cook Inlet and southeastern areas of Alaska; and off British Columbia. Several biological studies of the spot shrimp have been conducted by various researchers; Berkeley (1930), Butler (1964, 1970), Hynes (1930) and Price, and Chew (1972). This paper represents data collected in trawl-caught spot shrimp from 6 consecutive years (1966-1971) during the month of January in Dabob Bay in Hood Canal, Washington. The objective of the ' Contribution No. 399, College of Fisheries, University of Washington. study was to determine the size distribution, length-weight relationship and daily migration pattern for the local population of spot shrimp in Dabob Bay. MATERIALS AND METHODS The shrimp samples were taken with a 40 ft. semi-balloon, Gulf of Mexico shrimp trawl using the University of Washington research vessel M/y Commando. The stretch mesh size of the cod end was Vi inch and the outside linear V2 inch. Day and nighttime tows averaging 20 minutes in duration, were made at the four depths as shown in Fig. 1. Sampling for the respective years were either the second or third week of January. The shrimp were sorted, weighed and placed in heavy plastic bags for freezing on the vessel. They were later thawed in the laboratory for processing. The sexes were separated, and the carapace length (mm) and corresponding individual weight (g) were measured. The sexes were separated by examination of the inner ramus of the first 28 DEPTH DISTRIBUTION AND SIZE OF SPOT SHRIMP 29 TRAWL HAULS A 10-20 fathoms 18-27 C 28 40 D 42-60 Yards Length -Frequency Hlstogrom for PondQius plotyceros Oobob Soy FIG. 1. Location of trawl hauls taken in Dabob Bay. pleopod (Berkeley, 1930) or by the presence of eggs attached to the pleopods. The length was taken between the base of the eye socket to dorsoposterior point of the carapace. In- dividual weights were not taken if any part of the rostrum was broken off, or if the specimen was badly damaged. More than 8,000 individual measurements were obtained over the six years. Sub-sampling was necessary at times when large catches were made. RESULTS AND DISCUSSION Length Pr-equency Distribution Fig. 2 presents the carapace length frequency distribution of male and female spot shrimp collected from 1966 through 1971. The distribution was expressed in percentages and discussed briefly as follows: 1966: Only eighteen females were measured and the correct distribution may not be as shown in Fig. 2. However, it was interesting to note that the males and females were of the same size range, reflecting an overlap. The FIG. 2. Carapace length-frequency distribution of spot shrimp from 1966 through 197L 30 K. K. CHEW, D. HOLLAND, J. W. WELLS, D. H. MCKENZIE AND C. K. HARRIS I 28 I 12 Log (Corapace length) FIG. 3. Length-weight relationship of Dabob Bay spot shrimp for five different years. reason for this overlap was unclear. Com- parisons with subsequent years of male distribution would indicate we caught mostly 1+ or second year males. It has generally been recognized that spot shrimp in Dabob Bay can bear eggs as early as September-October through February and hatching could occur be- tween December and February. Egg bearing females have been noted as late as March in some years. 1967: No female measurements were taken this year. Nine-hundred twenty-nine males were measured, indicating the presence of 0-group or first year and 1+ or second year males. There was a higher percentage of the former group caught. 1968: The 0-group or first year males and 1+ or second year male shrimp were again evident, with more of the latter group caught this year. The females were not much bigger and revealed some overlap with the larger 1+ or second year males. In fact, there appears to be two sizes of females, one group with a mode at 35-36 mm and another at 40-41 mm carapace length. 1969: Again, the two groups of males were revealed from the data. This time, the overall female sizes were larger. Similar to 1966, inadequate numbers of females measured may have given an inaccurate indication of its size distribution. 1970: The two age groups of males were Depth Distribution tor Pondalus platyceros Dabob Bay I 6-| |l970 |l97l I 4 12- 1.0- 1.0- I 6 04 02 0 04 12 2400 10-20 Folhoms 42-60 Fothoms 0400 ->- 0800 1200 1600 Time of day 2000 2400 FIG. 4. 77(e 1970 and 1971 catch per minute haul during the day and night at four different depth ranges. again similar in size to those taken in previous years. Several small females (19-25 mm) were tabulated and included in Fig. 2, but it is assumed that this was an error in measurement or tabulation. DEPTH DISTRIBITTION AND SIZE OF SPOT SHRIMP TABLE 1. Analysis of Co-variance for Length-weight spot shrimp data. 31 Total Due to Regression Error Source d.f. ss d .f. SS d.f. SS F 1966 164 1.348 1 .773 163 .575 219 ** 1968 626 26.077 1 19.609 625 6,468 1890 ^ 1969 539 31.220 1 26.918 538 4.302 3370 ^ 1970 686 32.716 1 26.310 685 6.406 2810 *^ 1971 624 31.735 1 21.975 623 9.760 1400 *^ Within 2634 27.511 Reg. Coef 9 4 .358 8.57 ** Total 2643 131.376 1 103.317 2642 28.059 *^ Significant at the .01 level. 1971: As in previous samples, two age groups of males were sampled. The distribution of females were similar to 1968 in that two size groups were indicated. The smaller group of females between 29-35 mm range was essen- tially the same size as the 1+ or second year males. It should be recognized that sampling in a given month does not necessarily mean they should be the same size range each year. Depending on environmental conditions, they may moult earlier or later. Further, the availability of food and other conditions may cause the shrimp to increase in size more or less than the normal amount after each moult from one year to the next. It was interesting to note that some consistency was shovm for the male size frequency distribution between 1966 and 1971. The general size range in carapace length for the 0-group or first year males, was comparable to British Columbia spot shrimp sampled during the same time of year (Butler, 1964). Although there were considerably fewer females measured they were more variable from year to year. As a matter of fact, there ap- pears to be two size groups of females in some years such as 1968 and 1971, indicating the possibility of earlier transformation from males to females or little or no growth as they moult into the female stage. Length — Weight Relationship Fig. 3 shows the relationship between carapace length and weight of spot shrimp sam- pled for all years except 1967. Almost all data for the lines were taken from male shrimp and a few from females without eggs. Most females encountered were bearing eggs and thus were not weighed. The length-weight data collected during the years 1966, 1968, 1969, 1970 and 1971 was fitted to the equation W = B„ L^i, where W was weight, L was carapace length and B,, and B, coefficients. The equation was linearized by transforming by common logarithms and fit by least squares techniques. The analysis of variance Table 1 presents the results of the statistical analysis. The high 32 K. K. CHEW, D. HOLLAND, J. W. WELLS, D. H. MCKENZIE AND C. K. HARRLS degree of fit to the data by the regression lines is reflected in the large F ratios. For example, testing the hypothesis that Bj =0 for the 1966 data results in an F ratio of 219, which is significant at the .ol level (F, i.iw, .en = 3.44). Thus, we reject the hypothesis that Bi =0 and conclude that Bi / 0. Co-variance analysis of the data was per- formed to compare the means and slopes of the length weight regressions. The test of the hypothesis of equal means, B (kgi;) = B ms] = B ri((;9) = B 0(70) = B 1X71) , results in an F value of 8.57 (Table 1) and was rejected at the .01 level (F ,.,„;y. ,„ = 4.61). Thus we conclude that the relationship between weight and length was not the same for the five years. More detailed inspection indicated that the relationship during 1968 differed from the other years, in both B„ and B, (Table 2). Excluding the 1968 data, the analysis of covariance for the other four years, 1966, 1969, 1970 and 1971 indicate that the four slopes (B, ) were the same, F 3,2o„h = .16, (F :,.,„„, „i 3.79). The test of the hypothesis of a common mean, i.e. common intercept (B „ ) resulted in an F :,2„i: = 5.08. Hence, we reject the hypothesis of a common mean at the .01 level. The four years in question (Fig. 3 — 1966, 1969, 1970 and 1971) can be represented by four parallel lines, but not by a single common line. Tliis means that the 1966, 1%9, 1970 and 1971 lines show similarity in rate of weight increase over length, but that the lines were operating at different levels (Fig. 3). Further, a common line could not represent the four years. The 1968 line was not similar in that the amount of growth in weight over increased carapace length was significantly lower than the other four years, even though all lines appear to be similar (Fig. 3). Dppth Distribution Bathmetric distribution for spot shrimp was presented for the 1970 — 1971 catch data as shown in Fig. 4. Day and night tows at dif- ferent depths and on the basis of catch per minute trawl haul reveal that the shrimp moved to shallow depths during the nighttime hours and returned to deeper depths when it was daylight. Very few spot shrimp were cap- tured below 40 Fm for the area sampled. Although not presented, subsequent tows made in January, 1972 have shown similar results. These observations indicate a possible diel migration behavior of the spot shrimp. ACKNOWLEDGEMENT Special thanks go to the Washington Depart- ment of Fisheries for their support in this work by providing the necessary funding to have the data placed on computer cards and analyzed. Without such support, it would have been most difficult to analyze the data and give it proper treatment. The senior author wishes to extend his gratitude and appreciation to the former students, who helped gather information for this paper. Tlie crew of the M/V Cummatido. Skipper Tom Oswold, Jr. and Engineer Olaf Rockness deserve special thanks for their patience and un- derstanding. LITERATURE CITED Berkeley, A. A. 1930. Tlie post-embryonic de- velopment of the common pandalids of British Columbia. Contrib. Can. Biol. Fish. 6: 69-163. Butler, T. H. 1964. Growth, reproduction, and distribution of pandalid shrimps in British Columbia. J. Fish. Res. Board Can. 21: 1403-1451. Hynes, F. W. 1930. Shrimp fishery of southeast Alaska. Rep. U.S. Comm. Fish, "for 1929, App. 1, 18 p. Price, V. A. and K. K. Chew. 1972. Laboratory rearing of spot shrimp larvae (Pandalus platyceros} and descriptions of stages. J. Fish. Res. Board Can. 29: 413-422. Proceedings of the National Shellfisheries Association Volume 6i - 197J, THE DISTRIBUTION OF MUD CRABS (XANTHIDAE) IN ALABAMA ESTUARIES Edwin B. May ALABAMA MARINE RESOURCES DIVISION DAUPHIN ISLAND, ALABAMA ABSTRACT Xanthid mud crabs are abundant associates of Alaba7na oyster reefs. Their distribution is affected by salinity, substrate and water quality. The crabs appear to function as commensals and scavengers rather than predators of oysters. INTRODUCTION Mud crabs of the family Xanthidae are among the most abundant macroscopic motile animals associated with oyster reefs in Alabama and probably elsewhere. The distribution of common species in estuaries is influenced by salinity and is largely restricted to hard substrates, especially oyster reefs. Little information is available on the density of mud crabs although they are important associates in oyster communities as predators, scavengers and as hosts for oyster parasites and diseases. Data on the role of mud crabs in association with oyster reefs have been given by Mc- Dermott and Flower (1953), Menzel and Hopkins (1956), Menzel and Nichy (1958), Mc- Dermott (1960), Hoese (1964), Kenk (1967), Menzel, Hulings and Hathaway (1966), and others. Few of these studies considered geographical distribution in relation to salinity throughout an estuary. Menzel et al. (1966) ob- served that Menippe ynercenaria was limited by salinities below 12-15 ppt and that Neopanope texana texana was more common at stations with highest salinity. Wass (1955) reported A'^^ t. texana to prefer soft vegetated bottoms in shallows. Ryan (1956) found Panopeus herbstii rare on mud bottoms in Chesapeake Bay at a salinity range of 14-19 ppt. Schwartz and Cargo (1960) reported P. herbstii from 10-34 ppt in Chesapeake and Chincoteague bays but found them more abundant at higher salinities. Tlie species was common on oyster reefs in central and lower Delaware Bay (McDermott and Flower, 1953) and on oyster reefs in North and South Carolina (Lunz, 1937). Eurypanopeus depressus was collected at salinities of about 4- 20 ppt in Chesapeake Bay and its distribution was not thought to be limited by estuarine salinities above 4 ppt (Ryan, 1956). Tlie species was abundant in lower Delaware Bay but not in the middle and upper portions (McDermott and Flower, 1953). METHODS Crabs reported in this study were collected from oyster reefs using scuba and random quadrats in May and June, 1969 and numerical data were reported by May (1971). Data on xanthids collected by trawling and seining in Alabama were reported by Swingle (1971). Sam- ples were collected by picking up all material in square-yard grids by hand. The crabs make little effort to escape so even if a few were missed during sampling the relative abundance between different areas should be valid. No large stone crabs, M. mercenaria, which may have been in burrows were collected, however, the species is not abundant in Alabama and few burrows were seen. Small stone crabs ap- parently do not bun'ow (Powell and Gunter, 1968). 33 34 E. B. MAY RESULTS AND DISCUSSION Abundance per acre is reported by species in Table 1. Areas of collection are identified by numbers in Figure 1 where general salinity patterns are given during average river flows. On all reefs throughout the estuaiy, E. depressus (range 4-24 mm) was by far the most abundant species. Ryan (1956) found that this was the most abundant species of mud crabs on oyster reefs in Chesapeake Bay and showed a positive relationship between the presence of oysters or shells and this species. The majority of the specimens reported by Lunz (1937) from the Carolinas were collected from oyster reefs where they composed 25 percent of the population. Panopevs herbstn (15-29 mm) and M mercenaria (15-29 mm) were collected in smaller numbers from higher salinity reefs in Alabama. There were more crabs on reefs which had larger amounts of boxes (empty, joined. TABLE 1. - May and June, 1969. abundance of mud crabs, per acre, in the Mobile Bay region stations shown in Fig. 1. Eurrpanopeus Panopeus Menippe Station depressus herbstii mercenaria 1 3,983 246 32 2 25,601 1,578 208 3 32,900 2,028 268 4 12,937 797 105 5 42,784 2,637 348 6 57,598 3,550 468 7 474 8 1 8 2,262 140 18 9 15,871 978 129 10 1,748 108 14 11 8,975 553 73 12 8,806 817 642 13 830 77 60 14 9,921 193 581 15 6,012 0 90 16 0 0 0 17 2,470 0 37 18 1,987 0 29 19 381 0 7 20 1,097 0 16 21 2,430 0 37 22 1,505 0 23 23 0 0 0 24 197 20 10 25 3,034 0 0 26 285 0 0 27 605 0 0 28 1,953 0 0 29 1,096 0 0 MUD CRAB DISTRIBLTTIOiN IN ALABAMA 35 FIG. 1 Locations ivhere mud crabs were collected from oyster reefs and general salinity patterns during normal river flows. bivalve shells) and single shells indicating strong dependence on habitat provided by shells. All three species were more abundant in higher salinity areas and were lowest in abundance or absent in areas where salinity averages less than 15 ppt. Reefs in the upper bay where an- nual freshets are severe (May, 1972) had lower densities. Eurypanope'us depressus and to a 36 E. B. MAY lesser extent M. mercenaria were less limited by lower salinity than P. herbstii which was found only on one reef where salinity normally averages below 15 ppt. Ryan (1956) collected none at salinities below 14 ppt. There is a salinity difference of about 5 ppt between area 1 (15 ppt) and area 6 (20 ppt) (May and Bland, 1970). The increase in numbers of crabs of all species proceeding toward the higher salinity area is evident. No Rhithropanopeus hanisii were collected from the reefs in this study, but 49 specimens were collected in the Mobile River delta and from upper Mobile Bay by Swingle (1971). He found six specimens at salinities above 10 ppt but most were collected from freshwater. Similar distribution for this species is reported from Chesapeake Bay (Ryan, 1956) and upper Delaware Bay (McDermott and Flowers, 1953). Ryan (1956) collected this species from 3-19 ppt and stated salinity did not limit distribution. Costlow, Bookhout and Monroe (1966) found that larval R. harrisii developed at salinities from 1 to 40 ppt and no optimum salinity was detected. Swingle (1971) also reported one specimen of Lobopilumnus agassizii from Mobile Bay at 23 ppt, one Eurytium limosum from the usually highly saline Dauphin Island Bay, one A^. t. texana from Perdido Bay at 32 ppt and four specimens tentatively identified as P. occidentalis from Mobile Bay and Mississippi Sound at salinities above 25 ppt. With the possible exception of large stone crabs, which are not abundant here, xanthids appear to be mainly commensals or scavengers of oysters in Alabama rather than predators unless they prey on very small spat less than 5 mm. In the wild I have seldom observed spat or small oysters which were killed by small mud crabs even though spat are frequently numerous in joined bivalve boxes occupied by a crab. They more likely prey on other commensals on the reef since there is little direct relationship between the abundance of crabs and the abun- dance of oysters. Many reefs have more mud crabs than oysters and spat combined. Although there is no doubt these crabs will kill and eat oysters, Powell and Gunter (1968) believed that even stone crabs preferred barnacles to any other food item. Some of the field observations of xanthid predation on oysters could have been scavenging of moribund oysters killed or weakened by other causes. Although the destructiveness of mud crabs to very small spat is unknovTO, they apparently are not a serious predator of larger spat and oysters in Alabama. Bottom water in upper Mobile Bay and Bon Secour Bay have extremely low dissolved oxygen levels in the summers (May, 1973). This condition affects the abundance and distribution of blue crabs and oysters and it is apparently true of xanthid crabs as well since high salinity reefs in Bon Secour Bay had few crabs. The severe flood in Mobile Bay from December 1972 through June 1973 either killed or caused migration of most of the mud crabs since very few crabs were collected from any reef in mid- May 1973 after the salinity had been below 1 ppt for about six weeks. Low salinity is com- mon in late winter and spring throughout the estuary. Pearse (1929) found P. herbstii and E. depressus died after 4 to 5 "hours in freshwater. Salinity below 12 ppt arrests the development of P. herbstii larvae (Costlow, Bookhout and Monroe, 1962). The rate of development of M. mercenaria is slightly slower at 20 ppt than at 30-40 ppt and no larvae survive at 10 ppt. Op- timum salinity for lai-val development is in the range of 30-35 ppt at a temperature of 30 C (Ong and Costlow, 1970). Porter (1960) found that few stone crab larvae developed below 23 ppt at 27-30 C and felt that at 23-25 C larvae may not be able to survive below 27 ppt. There is a positive correlation between the density of xanthids and the incidence of fungus disease Labtjrinthomyxa marina (Beckert, Bland and May, 1972) but this is more likely due to the demonstrated response of both organisms to salinity variations rather than an actual in- terdependent relationship. LITERATURE CITED Beckert, H., D. G. Bland and E. B. May. 1972. The incidence of Labyrinthomyxa marina in Alabama. Alabama Mar. Resourc. Bull. 8:18-24. Costlow, J. D., Jr., C. G. Bookhout and R. Monroe. 1962. Salinity-temperature effects on the larval development of the crab, Panopeus MUD CRAB DISTRIBUTION IN ALABAMA 37 herbsti'- Milne-Edwards, reared in the labor- atory. Physiological Zool. 35(l):79-93. Costlow, J. D., Jr., C. G. Bookhout and R. Monroe. 1966. Studies on the larval develop- ment of the crab, Rhithropanopeus hanisii (Gould). 1. The effect of salinity and temper- ature on lan'al development. Physiological Zool. 39:81-100. Hoese, H. D. 1964. Studies on oyster scavengers and their relation to the fungus Dermocysti- dmm. mannum. Proc. Nat. Shellfish. Assoc. 53:161-174. Kenk, V. C. 1967. A new crab host of the gregarine Nematopsis ostrearum. Proc. Nat. Shellfish. Assoc. 55:87-88. Lunz, G. R., Jr. 1937. Xanthidae (mud crabs) of the Carolinas. Charleston Mus. Leafl. 9:9-27. May, E. B. 1971. A survey of the oyster and the oyster shell resources of Alabama. Alabama Mar. Resourc. Bull. 4.53 p. May, E. B. 1972. The effect of floodwater on oysters in Mobile Bay. Proc. Nat. Shellfish. Assoc. 62:67-71. May, E. B. 1973. Extensive oxygen depletion in Mobile Bay, Alabama. Limnol. Oceanogr. 18(3):353-366. May, E. B. and D. G. Bland. 1970. Sui-vival of young oysters in areas of different salinity in Mobile Bay. Proc. SE Assoc. Game Fish Comm. 23:519-521. McDermott, J. J. 1960. The predation of oysters and barnacles by crabs of the family Xanthidae. Proc. Pennsylvania Acad. Sci. 34:199-211. McDermott, J. J. and F. B. Glower. 1953. Pre- liminary studies of the common mud crabs on oyster beds of Delaware Bay. Nat. Shellfish. Assoc, 1952 Convention Address, pp. 47-50. Menzel, R. W., N. C. Hulings and R. R. Hathaway. 1966. Oyster abundance in Apala- chicola Bay, Florida, in relation to biotic associations influenced by salinity and other factors. Ocean Springs, Mississippi, Gulf Res. Rep. 2(2):73-96. Menzel, R. W. and S. H. Hopkins. 1956. Crabs as predators of oysters in Louisiana. Proc. Nat. Shellfish. Assoc. 46:117-184. Menzel, R. W. and F. W. Nichy. 1958. Studies of the distribution and feeding habits of some oyster predators in Alligator Harbor, Florida. Bull. Mar. Sci. Gulf Caribbean 8(2): 125-145. Ong, Kah-Sin and J. D. Costlow, Jr. 1970. The effect of salinity and temperature on the larval development of the stone crab, Menippe mercenatia (Say), reared in the laboratory. (Chesapeake Sci. 11(1): 16-29. Pearse, A. S. 1929. The ecolog)' of certain estuarine crabs at Beaufort, N. C. J. Elisha Mitchell Sci. Soc. 44(2):230-237. Porter, H. J. 1960. Zoeal stages of the stone crab, Menippe mercenaria Say. Chesapeake Sci. 1(3-4): 168-177. Powell, E. H., Jr. and G. Gunter. 1%8. Obser- vations on the stone crab Menippe mercen- aria Say, in the vicinity of Port Aransas, Texas. Ocean Springs, Mississippi, Gulf Res. Rep. 2(3):285-299. Ryan, E. P. 1956. Observations on the life his- tories and the distribution of the Xanthidae (mud crabs) of Chesapeake Bay. Amer. Mid- land Natur. 56(1): 138-162. Schwartz, F. J. and D. G. Cargo. 1960. Recent records of the xanthid crab, Panopeus Herbstii from Maryland and Virginia waters. Chesa- peake Sci. l(3-4):201-203 Swingle, H. A. 1971. Biology of Alabama es- tuarine areas — cooperative Gulf of Mexico estuarine inventory. Alabama Mar. Resourc. Bull. 5. 123 p. Wass, M. L. 1955. The decapod crustaceans of Alligator Harbor and adjacent inshore areas of northwestern Florida. Quai-teriy J. Florida Acad. Sci. 18(3): 129-176. Proceedings of the National Shellfisheries Association Volume 6i - 197^ THE PRESENT STATUS OF THE SOFT-SHELL CLAM IN MARYLAND William N. Shaw ^ and Frank Hamons NATIONAL MARINE FISHERIES SERVICE OXFORD, MARYLAND AND MARYLAND DEPARTMENT OF NATURAL RESOURCES ANNAPOLIS, MARYLAND ABSTRACT The soft-shell clam, Mya arenaria, has been a major commercial fishery in Maryland since the developynevt and use of the escalator dredge in the early 1950's. Between 1960-70. annual landings have fluctuated between Jf-8 million pounds of shucked meats, valued between 1-3 million dollars. As a result of tropical storm Agnes in June 1972, high m.ortalities occurred among the Chesapeake Bay soft-shell clams and all commercial fishing in the Bay was stopped. It was important to know the true extent of the mortcdities in the Bay and to determine if those clams that survived the storm would spawn and establish new year-classes. Several extensive surveys were conducted throughout Maryland following Agnes and the results indicated that enough clams survived in certain areas so that the fishery could be reopened on 1 June 1973. Unfortunately, the greatest commercial numbers of clams were located in one county. Talbot, and unless careful surveillance is maintained these could be overfished. From microscopic examinations, it was determined that the surviving clams had normal gonadal development and spaumed a7'07ind October of 1972. From this spawning, a set occurred in almost all major clam areas in Maryland, as indicated by special spat collectors used to monitor the Bay. Monitoring was done again in the spring of 1973 and setting was extremely light. At this time, it is too early to know if the soft-shell clam will fully recover from the effects of tropical storm Agnes. Findings to date have been encouraging. INTRODUCTION The soft-shell clam, Mya arenaria. has been a major commercial species in the New England and Chesapeake Bay areas (Fig. 1). Prior to 1950, the majority of clams were harTested in two states, Maine and Massachusetts. The highest catch for this area was in 1940 when over 15 million pounds of meats were landed. Production Present addres,s: National Sea Grant Program/NOAA, U.S. Department of Commerce. Rockville. MD 208.52. declined (Hanks 1963) thereafter, reaching a low of 2.3 million pounds in 1959. Since then, produc- tion in New England has been increasing and, in 1971, 6.4 million pounds were harvested. In 1950, the hydraulic escalator dredge was in- troduced into the Maryland portion of Chesapeake Bay. Tlie dredge enabled the clam in- dustry to use previously neglected subtidal stocks. Production increased rapidly and in 1964 8.1 million pounds were landed. Since 1969, when 7.9 million pounds were caught, production has 38 SOFT-SHELL CLAM STATUS 39 U- oL_ n u\ POUNDS MO (S3 NE m OOLLARS ffi FIG. L Soft-shell clam landings and values Manjland and New England from 1935-1970. for declined and in 1972 only L4 million pounds wei- landed. Part of the reason for the low 1972 figure was related to tropical storm Agnes and the closing of the fishery on 1 July 1972. The clam fishery was reopened on 1 June 1973, closed on 23 June, reopened for bait use only on 30 July, and reopened again on 27 August for human consumption. TROPICAL STORM AGNES On 22 June 1972, tropical storm Agne,-- hit Chesapeake Bay and brought as much as 11 in- ches of rain to some areas during the 3-day period, 21-23 June. Enormous amounts of fresh water entered the upper Bay, mainly from the Susquehanna River, and salinities dropped way below 5% in many areas where commercial num- bers of clams were found. Shortly after the storm, -SALINITY (%<.) -TEMPERATURE CO -10-5 MAR. APR. FIG. 2. Weekly temperature and salinity data for the Tred Avon River, Oxford. Md. from June 1972 to May 1973. 40 W. N. SHAW AND F. HAMONS the Bay was subjected to a period of warm weather. The combination of wann water tem- peratures and low salinities caused extensive soft-shell clam kills. Especially hard hit were areas on the Western Shore and in the Chester River where mortalities were as high as 90%. In these areas, the salinities were 2% or lower and temperatures were in the high 20 C's. Mortality rates appeared much lower along Kent Island, Tilghman Island, Miles, Wye and Choptank Rivers. Here, temperatures were also in the high 20's but salinities never dropped below .5 %„ (Fig. 9.) SURVEY OF CLAM POPULATIONS FOLLOWING AGNES Federal Survey Personnel from the National Marine Fisheries Service, Biological Laboratory, O.xford, Maryland, conducted a 12-day survey of soft-shell clam populations in the Choptank, Miles and Wye Rivers, and along Kent Island and Tilghman Island shores during a period from 29 August to 25 September 1972. A commercial hydraulic escalator dredge was utilized during the sui-vey. At each station, a 5- minute run was made. An anchor buoy was dropped at the beginning of each run and a line attached to the anchor was fed out until the 5- minute sampling period was completed. Tlie area covered was estimated by multiplying the length of line fed out during the 5-minute period times the width of the dredge (36 inches). All clams on the escalator were removed, counted, and the length of each clam measured to the nearest millimeter with vernier calipers. An estimate of the number of clams per square meter was deter- mined at each station (Fig. 3). Clams were found in almost all areas sampled and recent mortalities were low. Clams from the 1971 year-class were greatest in number, which indicated that there had been a successful set of clams in the fall of 1971. In the area surveyed, it appeared that the majority of these clams had survived the effects of the storm. It was also ap- parent that if these clams continued to survive throughout the following winter and spring there would be enough to support a limited commercial fishery. i>i (^ Twve 881/190 Kent Pi 1 0/»-' 2" 5 ,7 5 8 2 / . 10 1 EASTERN 9 li 2 2'^^^{ ^Ay^ ^W ^y4=^ NeLn P,\^ T^ /)C^ 69 ^^^ FIG. 3. Total number of clams per m^ found at each station during the 12-day NMFS survey. State Survey As a result of tropical storm Agnes, special Federal funds were allocated to Maryland to determine the extent of the soft-shell clam kill throughout the state. Forty-eight commercial clammers, each with a helper, were hired to sur- vey the clam beds. Sampling began on 8 January 1973 and was completed on 5 May. Each clammer collected 652 samples for a grand total of 31,296. Each sample equaled a r2-foot long distance times the width of the standard hydraulic escalator dredge. All market clams (2 Vt inches or greater) were placed in a 5% measuring cup, and the fullness of this cup was recorded. A 5% measuring cup is a container that holds 5% of a bushel. Thus 4% or more means 4% of a bushel or more. When the sample was 4% or more, the clammers were required to mark the station with a buoy. This area was considered to have enough clams for commercial fishing. Results of the survey are shown in Figure 4. The only areas on the Western Shore where com- mercial fishing would be profitable were a few locations in the Patuxent River and one 60-acre stretch opposite Horseshoe Point. Populations of soft-shell clams on the Eastern SOFT-SHELL CLAM STATUS 41 FIG. 4. The results of the State of Maryland suf- vey showing areas where dams were abundant enough to support commercial fishing. Shore were considerably greater. Although tne Chester River was found to be commercially un- workable, most other areas south of the Bay Bridge had harvestable populations, especially in the Miles River, Wye River, along the southern end of Kent Island, and off Tilghman and Poplar Islands. In these areas there were 100 bushels or more of marketable clams per acre. The State's survey on the Eastern Shore, then, verified the findings of the 1972 Fall Federal survey. MONITORING OF SOFT-SHELL CLAM SPAWNING AND SETTING, 1972-73 As stated earlier, soft-shell clam populations in many parts of the Bay were seriously depleted as a result of tropical storm Agnes. The Maryland Department of Natural Resources was concerned about the strength of the spawning stocks. In or- der for clams to fully recover from the effects of FIG. 5. Buttle collector nsed to collect metamor- phosing clams. the storm, new year-classes would have to be established through successful spawnings and set- tings. One method of monitoring clam setting is to place special bottle collectors (Fig. 5) out at the time when spawning and setting occurs (Shaw, 196.5a). With the cooperation of the Maryland Department of Natural Resources, collectors were placed throughout major clam areas in the Maryland portion of Chesapeake Bay. Since clams 42 W. N. SHAW AND F. HAMONS are known to set during the fall and again m the spring (Shaw, 1965a, b; Pfitzenmeyer, 1965), bot- tles were set out from October to December 1972 and from March to June 1973. Two collectors were placed at each station (Fig. 6) and were exchanged after about 30 days of ex- posure. The contents of each bottle were poured into a No. 80 (.0070 -inch opening) sieve and flushed with seawater to remove silt and detritus. The contents were then examined under a dissec- ting microscope and all metamorphosed bivalves were identified and counted. In addition, soft-shell clams were collected weekly from shallow water in the Ti'ed Avon River opposite the National Marine Fisheries Service Oxford Laboratory, during the above period and examined microscopically to deter- mine if normal gonadal development occurred during fall and spring months. The gonad from each clam was fixed in Davidson's fluid, dehydrated in alcohol, cleared in xylene, and mounted in paraffin. The gonad was then sec- tioned at 7-10 ^ with a standard rotary microtome. The sections were stained in Harris' hematoxylin and counterstained with eosin. ^^- Fall Survey of Set Bottlefi The contents of 52 bottles were examined during the fall survey and a total of 64 newly settled soft-shell clams were found. Clam setti, .^ occurred in almost all major tributaries of the Maryland portion of Chesapeake Bay and on many clam flats of the Bay proper (Fig. 6). For example, 23 clams were found in bottles placed in the Miles River, 12 in the Patuxent River, 13 in the Potomac River and 5 in the Choptank River. Only one young clam was found in the Chester River, an area where mortalities were e.xtremely high following Agnes. The highest number of newly settled clams (16) was found in bottles placed at Tilghman Point near the mouth of the Miles River. The clams had set between 3 Oc- tober and 3 November. Unfortunately, the bottles placed at this site to monitor setting in Novem- ber were lost. On 22 May 1973 a survey was made at Tilghman Point to see if a set had actually taken place on the tottom. A standard hydraulic escalator clam dredge with a ' 2-inch mesh belt FIG. 6. Stations where set coUertor bottles tvere located are designated by X's and the total num- bers of settiiKj Mya found in each area are en- closed i)i ( ). was used to sample the clam flats. Three size groups were found, one of which, because of their size, had to be a 1972 fall set (Fig. 7); the most numerous size group at this site. Spring Su?i^ey of Set Bottles The contents of 77 bottles were examined during the spring survey. Only six newly settled soft -shell clams were found: three at Kentmoor in the Bay proper, one at Chlora's Point in the Choptank River and two at Cole's Creek in the Patu.xent River. Based on these findings, it was apparent that the spring set was extremely light. Light spring sets have been found in the past by biologists at Chesapeake Biological Laboratory and at the National Marine Fisheries Service Ox- ford Laboratory (Shaw, 1965a,b; Pfitzenmeyer, 1965). SOFT-SHELL CLAM STATUS 43 s 10- 15 711 25 31 3S j in i so 5S SO ss ?0 J5 80 85 90 n - A f V H ff * 4 U 1 S!PI 8 OCl 20 OCT 27 1972 KOV 6 HOV ?2 HOV 29 DfC < IAN 4 H« 12 l«» 1! fiB 1 F[8 20 I!! 28 WAR 9 APU 1 AP« 12 A?« 21 AP« 30 MAV 9 1973 COllECTION DAIi FIG. 7. Mean length (horizontal line), standard deviatioTis (white bar above and beloiv horizontal line), and range (vertical line attanched to bar), for year classes of soft-shell clams collected in the Tred Avon River adjacent to the Oxford Laboratory ) the 29 November and 22 May samples were collected at Tilghman Point, Miles River and. the 28 February sample was collected at Nelson Point, Choptank River). All clams 57 mm or less, were placed in the smaller year classes while all clams 58 mm or greater were placed in the larger year cla,ss. Microscopic Examinations of Histological Preparations Gonadal development of some 394 soft-shell clams was examined microsopically from the Tred Avon River from 8 September 1972 to 30 April 1973 and the results were similar to those described by Shaw (1964, 1965b). In September, gametogenesis was in the early stage of develop- ment with small ovocytes at the base of the alveolar walls. By the third week in October, the clams were almost ripe to partially spawmed out. By 6 November, the majority of clams examined were spawned out and some showed early gonadal stages similar to that found in Sep- tember. The results of examinations indicated that major spawning occurred during October. Gametogenesis began almost immediately after the fall spawning was over. During the winter months, gonadal development was slight. By March, some fully developed eggs were found but their numbers were far less than found during the previous fall. Similar conditions occurred in males. Only the centers of the alveoli contained mature sperm. By the end of April, soft-shell clams were in their summer stage (alveoli con- tained follicular cells and many inclusions), and the remaining ovocytes were undergoing cytolysis. No evidence of spawming during this cycle was observed. Because of the small num- bers of clams examined during this period, it is the authors' opinion that some minor spawning had taken place since one newly settled clam was found in the set bottles placed in an area ad- jacent to the Tred Avon River. Growth Rates of 1971 Set Clams collected for microscopic examination in the Tred Avon River were all measured for total length to observe growth rate of the 1971 set (Fig. 7). This set would be the principal stock taken upon reopening the fishery. At the time of the first collection, 8 September 1972, the mean length was 35.8 mm and by 9 May, the last sam- ple taken, the clams averaged 51.8 mm. (57 mm, 2''4 inches, is the minimum legal commercial size in Maryland.) 44 W. N. SHAW AND F. HAMONS Two samples were also taken from a com- mercial clam bed in the Miles River, one on 29 November 1972 and the other on 22 May 1973, 9 days before the clam fishery was reopened. It is apparent from the mean sizes of these two sam- ples, 52.2 mm and 59.2 mm, respectively, that clams grow faster in the sampled areas of Miles River than from the shallow flats in the Tred Avon River. PRESENT AND FUTURE After an analysis of the State survey, the clam fishery was opened for harvesting on 1 -June 1973 but with the restriction of a 15-bushel daily limit. A total of 110 licenses were issued and most clammers easily caught the limits. The dockside value for clams averaged $10.00 per bushel, and the majority of clams caught were 57-63 mm (2 ' 4 - 2 ' 2 in.) in length and were from the 1971 set. Marketing of Maryland soft-shell clams has some future problems. Landings in New England have increased since 1965 (Fig. 1). In the past the landing values of Maryland clams were con- siderably less than New England clams but in recent years value of Maryland clams has risen. In addition, many establishments, because of the past shortage of soft-shell clams, now use surf clams and may not rely on soft-shell clams again. It can not be said with certainty that the present soft-shell clam populations in Maryland are great enough to withstand the fishing pressure. A 1972 set occurred in many areas and was especially heavy on the Western Shore in Anne Arundel County. After a year's growth these clams could reach market size and be available for harvesting. Many of the clammers will fish this area and take some of the pressure off the Eastern Shore. The State plans to continue monitoring chim populations and to open or close areas, based on the i-esults of such observations. Continued monitoi'ing of fall and spring setting will be a useful tool to indicate future stocks for the fishery. ACKNOWLEDGEMENTS Tlie authors wish to thank the following for their assistance in this study: Mr. Clyde Brown, Commercial Clammer; Mr. Carroll Smith, Maryland Department of Natural Resources; and Mr. Anthony Farmen and Mr. David McQuay, Fishery Aides, NMFS, Oxford, Maryland. LITERATURE CITED Hanks, R. W. 1963. Tlie soft-shell clam. U.S. Fish Wildl. Serv., Girc. 162; 16 p. Pfitzenmeyer, H. T. 1965. Annual cycle of gametogenesis of the soft-shelled clam, Miin (iiviinna. at Solomons, Maryland. Chesapeake Sci. 6: 52-59. Shaw. W. N. 1964. Seasonal gonadal changes in the female soft-shell clams, Mya arenarui. in the Tred Avon River, Maryland. Proc. Natl. Shellfish. Assoc. 53: 121-132. Shaw, W. N. 1965a. Seasonal setting patterns of five species of bivalves in the Tred Avon River, Maryland. Chesapeake Sci. 6: 33-37. Shaw, W. N. 1965b. Seasonal gonadal cycle of the male soft-.shell clam, Mija arennria. in Mary- land. U.S. Fish Wildl. Serv., Spec. Sci. Rep. Fish. 50S, 5 p. Proceedings of the National Shellfisheries Association Volume t>4 - lf>7U AGE, GROWTH AND SIZK-WEIGHT RELATIONSHIPS OF THE SOFT-SHELL CLAM. MY A ARENARIA. IN PRINCE WILLIAM SOUND. ALASKA ^^ Hinrairl M. Feder and A. J. Paid INSTITUTE OF MARINE SCIENCE UNIVERSITY OF ALASKA FAIRBANKS, ALASKA ABSTRACT Soft-shell clams. Mya arenaria, frotn Simpson Bai/, Prince Will in m Sonnd. Alaska, were examined. A single sample tf 178 specimefis iras used to (let ermine the growth history of twelve ijear-classes In/ the aiiiudar method, hi I'rimr William Sound soft-shell clams reach a harvestahle size of .W mm long in i; i>r 7 years. Length-weight relatiimnhips are considered. Dry meat ireiglit (solids) averaged 18.8%. INTRODUCTION Mya arermria, the soft-shell clam, is commonly encountered along the southeastern and south- central Alaska coast (Feder and Paul, 1973; Gross, 1967; Haven, 1971; Hubbard, 1971; Kirk- wood and Gross, 1967). Preliminary observations by the authors indicate that some areas in Prince William Sound, an extensive embayment along the coast of southcentral Alaska, have populations of M. arenaria that may be dense enough to support limited commercial harvesting (Feder and Paul, 1973; Tussing et at, 1972). Soft-shell clams are highly esteemed for their distinctive flavor and are eagerly sought in- tertidally by commercial and sport diggers in eastern Canada and New England (Dow and Wallace, 1961; Hanks, 1963). Subtidal commercial harvesting ' of this species also occurs in Chesapeake Bay (Suttor et al, 1968). The soft- shell clamc^was originally introduced into San Contribution No. 208, Institute of Marine Science, Univer- ity of Alaska. This project was conducted with funds provided by the Univer-sity of Alaska's Sea Grant Program (Grant No. 04-3-1.58-41), NOAA Office of Sea Grant, Department of Commerce. Francisco Bay in the late 1800's (Hanks, 1963), and has since spread northward to southcentral Alaska (Gross, 1967; Feder and Paul, 1973; Tussing et al. 1972). However, commercial har- vesting of this species along the Pacific coast has only been reported for Oregon (Amos, 1966; Marriage, 1954). In New England, Mya has proven to be a valuable renewable resource, and has been har- vested continuously in Maine since the mid-19t.h Century (Hanks, 1963). However, the New England fishery is becoming less productive because of unmanaged fishing pressures and closure of many beaches polluted by industrial and domestic wastes (Dow and Wallace, 1961). The Chesapeake Bay fishery is currently the major source for soft-shell clams (Hanks, 1963). Numerous papers deal with the basic biology and fishery potential of M. arenaria along the Atlantic coast of the United States: Dow and Wallace, 1961; Hanks, 1963; Newcombe, 1936; Suttor et ai, 1968; Turner, 1949; Wallace et al, 1965. However, with the exception of the preliminary work of Porter (1972), no published work is available on the biology of the soft-shell clam from the Pacific coast of North America. The purpose of this investigation was to examine 45 46 H. M. FEDER AND A. J. PAUL the age, growth and size-weight relationships of a population of M. arenaria from Prince William Sound. Alaska. METHODS Specimens of Mya were collected from a mud- flat in Simpson Bay, Prince William Sound (Fig. 1), May 18, 1973 by digging between the tidal heights of -0.43 m (-1.4 ft) and 0.0 m. All shells were e.xamined under a 2x lens and shells with badly abraded surfaces were discarded (7% of the 192 clams collected). Age was determined for the 178 remaining clams by counting annuli; a series of closely-spaced concentric growth lines which are the result of slow winter shell growth (Newcombe. 1936; Paul and Feder, 1973; Weymouth, 1923). Growth histoiy was deter- mined by measuring the shell length at each an- nulus. /^S" 147' The size-weight relationships of 100 of the specimens were e.xamined. The adductor muscles were severed, and the free water in the mantle cavities allowed to drain. The clams were then shucked, the shells weighed and the differences between the whole-live-weight (drained) and the shell-weight recorded as wet-meat-weight. In- dividual meats were dried to a constant weight at 80 C for dry-weight determinations. Weights were obtained with a Mettler balance type P 120, and plots, regression lines, and regression equations were detennined and plotted by an IBM 360 Computer. The Gauss-Jordan method was used in the solution of all normal equations (Cooley and Lohnes, 1962; Ostle, 1954). RESULTS Age and Growth The sjwcimens examined had recently-formed US' Ma° FIG. 1. Mav of Prince William Sound. Alaska; location of the Simpson Bay mudflat sampled for Mya arenaria. AGE, GROWTH AND SIZE-WEIGHT OF MYA ARENARIA 47 annul i and new growth was evident at the shell margins. The mean shell lengths for the various age classes of soft-shell clams from Simpson Bay are included in Table 1. The oldest clam examined was 12 years old and 87.0 mm long. A growth curve for M. arenaria from Simpson Bay is presented in Fig. 2. From 19(il through 1972, the mean shell length at any given annular age, as determined from the examinations of the growth histories of in- dividual shells, showed some variation (Table 2); however, the majority of the shell lengths at any given annular age falls within the standard deviations calculated for the various age classes in the collection (Tables 1,2). TABLE 1. Average size and age of 178 Mya arenaria collected on a mudflat in Simpson Bay, Prince William Sound. Alaska on May 18. 1973. N = Number of clams. ML = Mean Length if Clams. SD = Standard Deviation. R = Range. Year Class N ML SD R (Age of Clams) (mm) (mm) (mm) 0* 4 9.87 1.76 8. A - 10.9 1 33 13.41 1.10 12.0 - 15.2 2 13 17.73 2.83 16.0 - 21.0 3 9 26.04 2.38 21.0 - 30.0 4 13 30.87 2.39 25.1 - 33.2 5 24 39.01 3.22 33.8 - 49.2 6 21 48.15 3.78 40.1 - 55.3 7 7 57.50 4.68 50.5 - 60.1 8 16 64.96 3.38 58.0 - 70.0 9 19 73.42 3.11 69.0 - 81.0 10 11 77.95 4.37 70.0 - 84.0 11 5 76.88 3.31 73.2 - 80.0 12 3 85.07 1.90 83.2 - 87.0 * The 0 age group refers to those individuals of the settling year class that have undergone only one grovdng season (5 to 6 months) before forming their first winter annulus. Thus, individuals re- ferred to as 1 year are actually I7 or 18 months old and have lived through two growing seasons. Size- Weight Relationships The equations describing the relationships be tween length and total weight (drained), length and wet-meat-weight, and length and dry-meat- weight are: k.3,0278 Total Weight ' """^'^ Wet-Meat-Weight Dry-Meat-Weight (Length I 23,40 f _ f Length I -\^27,7,5 ; _| Length I 1 48..'^1 J :?70 .3.2524 See Fig. 3 See Fig. 4 The equation of the line describing the relation- ship of dry-meat-weight (solids) to wet-meat- weight is: L0676 Drv-Meat-Weight (Solids) = I Wet Meat (Wet Meat I ,S,4.5 / Dry-meat-weight (solids) was found to average 18.8% with a standard deviation of ± 1.5. DISCUSSION Intertidal beaches in Prince William Sound are subject to considerable environmental stress during January and February with observed water temperatures varying from -2.0 — + 1.0 C and air temperatures from -7 — +3 C. During this period 6 inches of ice were observed over the sampling area (Feder and Paul, unpublished). Under such conditions Myo arenaria forms a distinct winter annulus. (See Feder and Paul, 1973 and Paul and Feder, 1973 for data on winter annulus formation in the clam, Protothaca staminea, in Prince William Sound,) Newcombe (1936) also reported the formation of a single an- nulus for M. arenann in the Bay of Fundy, and was able to use the annular method to age his specimens. The time needed for M. arenaria to grow to harvestable size in Prince William Sound is slightly longer than that reported for other nor- thern populations of soft-shell clams (Maine: Hanks, 1963; New Brunswick, Canada: Turner. 1949). In these areas the soft-shell clam requires 5 or 6 years to reach a length of 50 mm (2 in), an acceptable commercial size, as com- pared to 6 or 7 years for the clam in Prince William Sound (Fig. 2). In Massachusetts M. 48 H. M. FEDER AND A. J. PAUL arenaiia reaches a harvestable length in nn\\ 8 years (Turner, 1949). Examination of growth history of M. arendnd (Table 2) suggests that the growth rates for the vai'ious age classes have been relatively stable for an 11 year period. The Alaska earthquake of 19fi4. which destroyed appro.ximately 36% of the clams in Prince William Sound (Ba.xter, 1971), had nil aiiparent effect on the growth of the specimens examined. The 18.8% solids determined for M. arfnarut in Prince William Sound is close to the 18.6% reported for this species in Maine (Harriman, 1954). The solids value of the Alaska soft-shell clam is also similar to that reported for other commercially important north Atlantic bivalves; Crassostfea virginica. Spisula solidissima and Arctica idanddica with values of 17.0, 21.4 and 18.5%, respectively (Ropes, 1970). The market for clam products in the Ihiited States is constantly expanding (Ropes, 1970; Sut- tor ct nl. 1968). Simultaneously, an increasing 90 80 70 60- 5 50 30- 20- 10- T r 0 "1 r ±. Simpson Bay, Alaska X X 0 2 3 4 5 6 Age (year class) 8 10 II 12 FIG. 2. The relationship between shell length (mm) and age of Mya arenaria from a mudflat in Siynp- aon Bay. Prince Williani. Sound., Alaska. AGE, GROWTH AND SIZE- WEIGHT OF MYA ARENARIA 49 number of areas inhabited by these m()llusi.,^ -i^ ^h 25 i .^^ !M Ql K yo -^ i 15 10 5 0 90 FIG. 3. 77i(? relatio7iship of clam, length to total ard wet-meat-weight for Mya arenaria collected from a mudjlat in Simpson Bay, Prince William Sound. 50 H. M. FEDER AND A. J. PAUL additional source of supply for clam products foi- commercial markets (Feder and Paul, 1973 and 1974; Kirkwood and Gross, 1967; R. Nickerson, personal communication; Nosho, 1972; Tussing ct ni. 1972). ACKNOWLEDGEMENTS We thank the following staff members of the University of Alaska: Emma R. Dieter for collec- tion of material; Captain Ken Turner- and the crew of the R/V Acona for support and general assistance; George J. Mueller, Marine Soi'ting Center, for taxonomic assi-stance in the final identification of Myu arpimrin and for review of the manuscript; Rosemary Hobson for comjiutei' progi'amming assistance, and Shirley Wilson for drafting all figures. S .'i^ 4.8 44 4.0 3.6 3.2 2.8 I § 2.4 ^2.0 1.6 1.2 0.8 0.4 0.0\ I 0 10 30 40 50 Length (mm) 90 FIG. 4. The relationship of dam length to dry weight for spedmem of Mya arenaria collected on a mudflat in Simpson Bay. Prince William Sound.. AGE. GROWTH AND SIZE- WEIGHT OF MYA ARENARIA 51 TABLE 2. Grvwth histories of Mya arenaria, ayes 1-12, collected from a mudflat in Simpson Bay, Prince William- Sound, Alaska on May 18 1973. See Table 1 for the number of individuals inclvded in each age class. Measurements are in millimeters. |6 1 ^^ -j"^ ^^ .^^^ U-) Ui -O 1 Lo 5 .^ 1^ |§ 1 lit?" « g, • & • o. • ■■ ^■^ ^^ ^'^ ~j^ ^ (O ^ ri . § § 1§ j5 1 / X :, -, \ I39\ I30\ izr\ ir.ry^ i^e\ i«X 25e\ \ 27A \ jiaX 1 s^X saX 57X X \ ■ eaX 6.«X \ J J 5 ..5X A ix J43\ 3-l}\ \ \ \ 3W\ J9.6\ 4i.a\ 4ia\ ■}l.8\ 4I.9\ \ 5ao\ 5Qe\ 500\ \ 7jX tvjX X 7,'X 6 7 3 l^6\ i?e\ i^e\ \ 9 10 II B5\ I9.S\ -X ■K5\ ^jX ■J7S\ .vX ^x 59X 63- \ 65.x X ^ssX ;!fX /^X ->'X ssX 12 /''-'f-'j \/96^ \' -f-'I' 1964 1965 1966 /96r 1968 1969 /pro i9ri «'" Year of Annu lu^. Format/on • Mean She Ungth LITERATURE CITED Amos, M. H. 1966. Commercial clams of the North American Pacific Coast. U.S. Fish Wildl. Serv., Circ. 237. 18 p. Baxter. R. 1971. Earthquake effects on clams of Prince William Sound. //( The Great Alaska Earthquake of 1964. Biology. Natl. Acad. Sci., Washington, D.C. 287 p. Cooley, W. W. and P. R. Lohnes. 1962. Multi- variate Procedures for the Behavioral Sciences. John Wiley and Sons, New York. 211 p. Dow. R. L. and D. E. Wallace. 1961. The soft- shell clam industry of Maine. U.S. Fish Wildl. Serv., Circ. 110. 36 p. Feder. H. M. and A. J. Paul. 1973. Abundance estimations and growth-rate comparisons for the clam Protothaca staminea from three beaches in Prince William Sound. Alaska, with additional comments on size-weight relation- ships, harvesting and marketing. Inst. Mar. Sci., Univ. Alaska, Tech. Rep. No. R73-3, 34 p. Feder, H. M. and A. J. Paul. 1974. Alaska clams: a resource for the future. Alaska Seas and Coasts (Sea Grant Newsletter) 2(1): 1, 6-7. Gross, J. B. 1967. Note on the northward spread- ing of Mya arenaria Linnaeus in Alaska. Veliger, 10: 203. Hanks, R. W. 1963. The soft-shell clam. U.S. Fish Wildl. Serv., Circ. 162. 16 p. Harriman, D. M. 1954. Variations in total solids of the soft clam (Mya arenaria). Maine Dep. Sea Shore Fish., Res. Bull. No. 23, 14 p. Haven, S. B. 1971. Effects of land-level changes on intei'tidal invertebrates, with discussion of post-earthquake ecological succession. In The Great Alaska Earthquake of 1964. Biology. Natl. Acad. Sci., Washington, D. C. 287 p. Hubbard, J. D. 1971. Distribution and abundance of intertidal invertebrates at Olsen Bay in Prince William Sound, Alaska, one year after the 1964 earthquake, hi The Great Alaska Earthquake of 1964. Natl. Acad. Sci., Wash- ington, D.C. 287 p. Kirkwood, J. and J. Gross. 1967. Need for research on the soft-shell clam (Mya sp.) of Alaska. In E. Haynes and J. McCrary. eds. Minutes of the First Alaskan Shellfish Con- ference. Juneau, Alaska, May 23-26, 1967. Alaska Dep. Fish Game Inf. Leafl. 106. p. 19-20. Marriage. L. D. 1954. The bay clams of Oregon, their economic importance, relative abundance, and general distribution. Fish Comm. Oreg. Contrib. 20, 47 p. Newcombe, C. L. 1936. Validity of concentric rings of Mya arenaria, L. for determining age. Nature, 137: 191-192. Nosho, T. Y. 1972. The clam fishery of the Gulf of Alaska. In A Review of the Oceanography and Renewable Resources of the Northern Gulf of Alaska. Inst. Mar. Sci., Univ. Alaska, R72-23, 690 p. Ostle, R. 1954. Statistics in Research. The Iowa State Univ. Press, Ames, Iowa, 487 p. 52 H. M. FEDER AND A. J. PAUL Paul, A. J. and H. M. Feder. 1973. Growth, re- cruitment, and distribution of the littleneck clam, Protothaca staminea, in Galena Bay, Prince William Sound, Alaska. U.S. Dep. Commer., Natl. Mar. Fish. Serv., Fish Bull. 71: 665-677. Porter, R. G. 1972. Preliminary report on growth rate and reproductive cycle of the soft-shell clam at Skagit Bay, Washington. Proc. Natl. Shellfish. Assoc. 63: 9-10. (Abstract). Ropes, J. W. 1970. Percentage of solids and length-weight relationship of the ocean quahog. Proc. Natl. Shellfish. Assoc. 61: 88-90. Suttor, R. E., T. D. Corrigan and R. H. Wuhrman. 1968. Tlie commercial fishing and seafood processing industries of the Chesapeake Bay area. Agric. Exp. Sta., Univ. Md., College Park, Md. MP-676, 81 p. Turner, H. J., Jr. 1949. The soft-shell clam industry of the east coast of the United States. Appendix I. Report on investigations of the propagation of the soft-shell clam, Mya arenatia. Woods Hole Oceanogr. Inst., Coll. Reprints 1948, Contrib. 462. p. 11-42. Tussing, A. R., T. A. Morehouse and J. D. Babb, eds. 1972. Alaska Fisheries Policy. Economics, Resources and Management. Inst. Soc, Econ. and Gov. Res., Univ. Alaska, 470 p. Wallace, D. E., R. W. Hanks, H. T. Pfitzenmeyer and W. R. Welch. 1965. Tlie soft-shell clam ... A resource with great potential. Atl. States Mar. Fish. Comm., Mar. Resnur. Atl. Cmst Leafl. No. 3. 4 p. Weymouth, F. W. 1923. The life-history and growth of the Pismo clam (Tivela stultmum (Mawe). Calif. Fish Game Comm., Fish. Bull. 7, 120 p. Proceedings of the National Shellfisheries Assorintion Volume 6!t -1974 REPRODUCTIVE CYCLE OF THE MANILA CLAM (VENERUPIS JAPONICAl FROM HOOD CANAL. WASHINGTON^- David A. Holla ml and Kenneth K. Chen' FISHERIES RESEARCH INSTITUTE COLLEGE OF FISHERIES, UNIVERSITY OF WASHINGTON SEATTLE, WASHINGTON ABSTRACT Secuional gonadal changes were observed histologically in samples of the Manila clam (Venerupis japonica, Deshayes) collected from Misery Point and Big Beef Harbin- in Hood Canal. Washington between October 1970 and November 1971. With few exceptions, npe clams first appeared in May-June and most active spawning occmrred in .My. Spanmi^ig was nearly completed by October. Sexual maturation began at a shell length of 5 mm and spawning at 20 mm and aver. INTRODUCTION Tlie Manila clam (Venerupis japonica) is one of the principal hardshell clam species commercially harvested in the State of Washington. Tlie clam, introduced with Pacific oysters (Crassastrca giijits) fi-om Japan, has also become one of the more imjaortant recreational-sports species along the Pacific coast. Tlie importance of the Manila clam in the com- mei'cial and sport fisheries has prompted in- vestigations into the feasibility of developing new clam grounds or to re-establish previous com- mercial Manila clam producing areas in Puget Sound. Tlie seed setting, growth rate and recruit- ment of select stocks of this species have been studied by Nosho and Chew (1972). This paper provides added information on the cyclical gonadal development and spawning of the Manila clam. Tliese investigations were conducted prior to actual field transplantation studies of seed clams to .select areas to determine its feasibility. ^Contribution No. 398 College of Fisheries. University of Wash. " This study was suppjrted in part by the Sea Grant Pro- gram under the National Oceanic and Atmospheric Ad- ministration, U.S. Department of Commerce. METHODS AND MATERIALS Description of Experimental Areas: Two areas were chosen for this study. Big Beef Harbor (BBH) and Misery Point (MP) located on Hood Canal, Washington (Fig. 1). Temperatures at MP during the spring and summer were higher than those at BBH (Fig. 2), and the substratum at MP consists primarily of pea gravel whereas the sub- stratum is unsorted at BBH. The sampling site at MP is located in a lagoon which never empties completely. Water stands in the lagoon at low tide to a maximum depth of six inches. BBH clam beds are exposed at ap- proximately the +2.5 feet tide level relative to mean low tide. Sampling Scheme: Samples of 300 clams for each site were taken bimonthly during active gametogenesis, and monthly during the remainder of the year, and 40-50 examined histologically. Five to seven clams from each 5 mm length group (ranging from 5 to 65 mm) for both beaches were sampled for gonad tissue and fixed with Davidson's solution. Tissues were sectioned at 6- S/i and stained with Mayer's hematoxylin and eosin. Definition of Gonadal Stages: Reproductive ]iatterns and gonadal stages have been deter- mined for clams and related bivalves by many 53 54 D. A. HOLLAND AND K. K. CHEW FIG. L Location of study areas. researchei-s; Coe and Turner (1938), Qiiayle (1942), Allen (1953), Tranter (1958a,b,c,d,e), Tiir- ner and Hank (1960), Merrill and Burch (1960). Sastry (1963), Kennedy and Battle (1964), Porter (1964), Shaw (1964, 1965), Lammens (1967), Ropes (1968), and Holland's Master's thesis.^ Five arbitrary gonadal stages (early active, late active, ripe, partially spent and spent) were chosen to describe the reproductive cycle of the Manila clam. The stages were determined by the presence and degree of maturation of ganietogenic cells in the follicles. Developmental Stages of Male Gonad Follicles Early Active: Many follicles with numerous follicle cells in foot tissue. Spermatogonia cen- tripetal to follicle walls. Spermatocytes numerous and move toward center of lumen. Nutritive phagocytes, as described by Loosanoff (1937) for Venus mercenaria. abundant. Late Active: Majority of follicle filled by sper- matids and spermatozoa. Spermatocytes and sper- matogonia located along inner periphery of follicle wall. Few follicle cells and nutritive phagocytes. ' Holland, D.A. 1972. Various aspects of the reproductive cycle of the Manila clam fVenenipis japonica). Master's Thesis, University of Washington, College of Fisheries, Seattle, Washington. 61p. Ripe: Gonad composed of darkly staining sper- matozoa with their tails pointing toward center of lumen forming concentric bands centripetal to spermatocytes. Spermatocytes, few in number, located along follicle wall. Follicles neat and or- derly in appearance. Stages of spawning and regression follow sexual maturity. These stages are characterized by the presence of phagocytes and residual gametes. PaHially Spent: Majority of follicles empty. Full follicles, if present, located near digestive gland. Follicles disorganized in appearance. Phagocytes abundant in center of shrunk follicles. Spent: Gonad spent with few residual sper- matozoa undergoing phagocytosis. Inner wall of follicle usually lined with spermatogonia. Development Stages of Female Gonad Follicles Early Active: Oogonia arise from stem cells (Tranter, 1958a) along follicle wall. Largest oocytes, attached to follicle wall, with nuclei darker than cytoplasm. Nutritive phagocytes abundant. Late Active: Oocytes have nuclei lighter than c^'toplasm. Free and attached oocytes equally abundant in follicle. Nutritive phagocytes few in number. Ripe: Majority of oocytes lie free in lumen of follicle. Nutritive phagocytes rare. 85n i< 75- 65 I 55 * 45 35 '■-''". i V 1 '/ \ •— /^ r\ ""'1 V " V "\y J^ Big Beet HorDor Misefjr Point Oobob Boy Jan ' Feb ' Mar Apr ' May Jun Ju 'Aug ' Sept ' Oct ' FIG. 2 . Temperature regimes at Big Beef Har- bor, Misery Point, and Dabob Bay for 1971. Un- connected lines indicate a malfunction of the thermograph. Temperature at Dabob Bay was attained from May, 1971 to September. 1971 through the Washington State Department of Fisheries. NATIONAL SHELLFISHERIES ASSOCIATION J< I/I null p. 55 BIG BEEF HARBOR - A. AH clams MISERY POINT - A. All clams 3d|j-'t Oct Nov Dec Jon Feb Mar Apr Ma/ Juo Jo' Aug Sep Ocl Nov B . Male clams Ocl Nov Dec Jon Feb Mof Apr Moy Jun Jul Aug Sep Od Nov B. Male clams OC Nov Dec Jon Feh Mof Apr Moy Jun Jul Aug Sep Oct Nov C. Female clams B Q Pofhollj sptnl H Ure ocioi Oct Nov Dec Jon Feb Moi Apr May Jun Jul Aug Sep Ocl Nov 1970 1971 F^IG. 3. Gormdal stages of nil Venerupis Japonica representing the breeding season (A) followed hij the separation of males (B) and females (C) for Big Beef Harbor. Tfie percentage of clams in each gonadal stage is represented by the length of each shaded area. FIG. 4. Gonadal stages of all Venerupis Japonica representing the breeding season (A), followed b>i the separation of males (B) and females (C) for Misery Point. The percentage of clams in each gonadal stage is represented by the length of each shaded area. MANILA CLAM REPRODUCTIVE CYCLE 55 Partially Spent: Few to over half of follicles empty. Many degenerate ova undergoing cytolysis. Follicle walls broken and disordered. Immature germ cells located on follicle walls. Spoit: Few residual oocytes in lumen of follicle. Developing oogonia on follicle walls. Phagocytes nuinerous. RESULTS AND DISCUSSION All Clams Early active gametogenic development for all sizes of clams occurred in every month of the year (Figs. 3A and 4A). The large percentage of clams in the early active stage during the sum- mer months resulted from smaller clams, less than 20 mm in shell length, which developed sexual products but apparently failed to attain sexual maturity. Tlie majority of clams were either spent or showing early active gonadal development stage during the autumn and winter months. Tlie ab- sence of partially spent clams in MP during the winter months may have been due to the fact that they were exposed to a higher water tem- perature (Fig. 2) than those at BBH throughout the summer months and, as a result, spawning was complete. Further, clams at MP were con- tinuously under water, which may have provided them more spawning time. Clams at BBH were exposed to air at least once a day during low tide. The partially spent clams found at BBH ■ IC BfCF „.«.„» . . AM „._. "ISEBV POINT - * All tl.". i d f^^j-|P«DO[c i from October to April (Fig. 3A) failed to release all their gametes during the summer and early autumn and the sex products were being resorbed. Late active clams from BBH began to appear in mid-April and ripe clams became prominent in mid-June (Fig. 3A). A small percentage of the clams spawned as early as the latter part of May whereas half the population of clams were spavming by early August. The percentage of late active clams in late August at BBH was greater than during late July, perhaps indicating a second spawning. This increase in late active clams was actually due to partially spent male clams redeveloping and may have resulted from the sudden in- crease in temperature from late July to early August (Fig. 2). By the end of September the majority of BBH clams were spawning (39%), or spent (30%), and by October spawning was practically completed since 9% of all clams were partially spent and 61% spent (Fig. 3A) Late active clams at MP first appeared in early April (18%) and by early May constituted 52% of the population (Fig. 4A). Tlie first ripe clams occurred in late May and reached a peak (29%) in late June. Spawning began as early as April since one clam was found in a partially spent stage of spawning but the majority of the population was spawning by July. By early July 39% of all MP clams were partially spent and 11% spent. Ripe clams from MP decreased from a maximum value of 30% near the end of June to a constant 21% during July and increased to 26% during the early part of August indicating a second maturation from partially spawned clams (Fig. 4A). A small percentage of partially spent clams occurred in late June (2%). By the end of August 54% of the MP population was spent as temperature began to decrease. Spawning was nearly complete by November (13% partially spent and 56% spent). Spawning did not occur all at once. It appeared to be continuous throughout the summer and into the autumn months in Hood Canal, Washington. Quayle and Bourne (1972) indicated that this species spavms in late spring, although some spawning may continue throughout the summer in British Columbia. Yoshida (1935) and 56 D. A. HOLLAND AND K. K. CHEW Kinoshita (1939) believed spawning was con- tinuous from spring until fall with one major peak for V. japonica in northern Japan and other Japanese researchers indicate that V. japonica has two major spawnings a year in southern Japan: Fujimori (1929), Tanaka (1954). Yasuda, Hamai and Hotta (1954), Ko (1957) and Ohba (1959). Male vs Female Clamt^ Although the above discussions refer to the combined data of both sexes, percentages of male and female clams for each of the stages are also presented in Figs. 3 and 4 for BBH and MP. Trends at MP were similar to BBH for the corresponding se.xes. Tlie only difference was the timing of events or designated stages. As a result of this similarity, the reproductive stages of BBH male and female clams will be discussed with MP information presented only where appropriate. One of the major differences between male and female clams at BBH (Figs. 3B, 3C) was the per- centage of males remaining partially spent from October 1970 to April 1971, which were slowly resorbing large quantities of residual sper- matozoa. A small percentage of ripe male Manila clams were found in December 1970 which were suspected to have been ripe in late summer or early autumn. Quayle (1942) states that mature males of a similar endemic species of clam, Paphia (Venempis) staminea can be found year round in British Columbia waters. Another major difference between male and female clams in BBH was the abundance of females in the early active stage throughout the autumn and winter months. When females were sexually mature, it was possible that inhibiting enzymes prevented the development or growth of young or new oocytes. Tranter (1958a) was able to demonstrate this for Pinctada albina in that there was arrest of growth of new oocytes until spawning took place, freeing space in the once- packed follicles. Once spawning commences, inhibiting enzymes apparently become inac- tivated and new oocytes proliferate, predomi- nantly, around the digestive gland. The gonad then takes on the appeai'ance of being early active in a large percentage of the female Manila clams. During May clams from BBH were predominantly in a late active stage of gametogenesis. By mid-June, 26% of the males and 36% of the females were sexually mature. Active spawning began in late June and early July when 25% of the males and 14% of the females were in a partially spent or spent con- dition (Figs. 3B and 3C). Eighty-one percent of the females and 36% of the males were in a par- tially spent and spent condition near the end of August; 14% and 36% of the males in late August were in a late active and ripe stage of gametogenesis, respectively. Apparently, the males were able to release a portion of their sexual products and quickly redevelop to reattain maturity. By late September 67% of the BBH males were partially spent. Females spawned continuously at intermittent periods during sum- mer and into autumn. Female clams in early August to late Sep- tember at BBH were dominated by the partially spent stage of oogenesis indicating continuous spawning over this period and occasionally we observed a spent clam (Fig. 3C). By October 1971 the majority of females wei-e in the spent con- dition, which was similar to MP females (Fig. 4C). Early active gametogenesis was seen in all months sampled during the year for both areas: this again partially resulted from clams under 20 mm in length which developed sex products but failed to attain maturity. Size and Ai/e at First Matnritij Three males collected at BBH during Novem- ber and December 1971 in the length categoi-y of 5-10 mm had ripe sperm but were not suspected of spawning; the gonad was too tightly packed with no empty spaces. Lammens (1967) suggested that it was possible to determine which young clams have spawned by spaces in the gonad. Tlie most advanced developmental stages attained by clams 5-10 mm in shell length were also in an early active stage of gametogenesis. Late active clams were observed histologically in early June and early July, 1971 for 15-20 mm clams. Clams ranging from 20-25 mm in length were ripe in early June and spawned in early August at BBH. Three females in the 5-10 mm size range, two collected in the latter part of September, 1971. and one in October, 1971 had some ripe gametes. However, further histological study indicated that the clams had not spawned, but were I'esor- bing their sexual products. MANILA CLAM REPRODUCTIVE CYCLE 57 Similar results were found at MP. Size at maturity is much less confusing than age at maturity. Quayle (1952) states that if the breeding season were extended (as in Venerupis japotnca) there would be a large range in size, and the animals of one year class may be con- fused with the smaller animals of a succeeding year class. Neave (1949) indicates also that the attainment of maturity for butter clams (Saxidmnm giganteus). appears to depend upon size rather than age. Bivalves that spawn in late spring and early summer may produce young which will ripen and spawn later in the same season (Thorson. 1946). Information from Japan reveals that Manila clams developed mature gonads at a shell length of 12 mm, and many individuals about 15 mm in shell length spawned (Ko, 1957). Clams at BBH and MP developed sex products at 5-10 mm and some matured at a shell length of 15 mm or greater and spawned at 20 mm. Nosho and Chew (1972) determined the size of a Manila clam at the end of the first year at BBH to be ap- proximately 24 mm. Tlierefore. clams at BBH mature and spawn in their first year of life. The sex ratios from BBH and MP were 48% males to 52% females and 44% males to 56% females, respectively. It was of interest to note that one clam out of 937 and one clam out of 768 were found to be hermaphroditic from BBH and MP, respectively. The specimen from BBH was sampled in September 1971 (50-55 mm) and the one from MP was sampled in August 1971 (40-45 mm). Typically dioecious pelecypods produce few hermaphrodites. This is shown by several research- ers such as the following: 3 out of 1000 soft- shell clams, Mya arenaiia (Coe and Turner 1938); 0 out of 800 M. arenaria (Shaw, 1965); 0 out of 1400 M. arenaria. (Ropes and Stickney, 1965); 2 out of several hundred quahogs, Mer- cenaria merceimria (Loosanoff, 1936); one out of 2500 surf clams, Spisula solidissima (Ropes, 1967); 2 out of 3000 sea scallops, Placopecten magellanicus (Merrill and Burch, 1960). SUMMARY 1. Specimens of Manila clams (Venerupis japonica) were removed periodically from Big Beef Harbor and Misery Point to determine the annual reproductive cycle. 2. Various gametogenic changes of development and regression are described. 3. Active gametogenesis began in April for both sampling sites and males matured earlier than females. 4. The majority of clams spawned in July; fe- male spawning was continuous throughout the summer with no apparent second matura- tion of gametes but males released a large portion of their products and quickly re- developed to maturity during the same season. 5. Active gametogenesis began with an increase in temperature as did spawning. 6. Clams developed sexual products at a shell length of 5-10 mm and reached maturity at 15-20 mm; all clams over 20 mm spavmed and a small percentage of clams 15-20 mm also spawned. 7. Hermaphroditism for the Manila clam is described. LITERATURE CITED Allen, R. D. 1953. Fertilization and artificial acti- vation in the egg of the surf clam, Spisula soli- dissima. Biol. Bull. 105: 213-239. Coe, W. R. and H. J. Turner. 1938. Development of the gonads and gametes in the soft-shell clam (Mya arenaria). J. Morph. 62: 91-111. Fujimori, S. 1929. A study on the utilization of shallow waters of Ariake Sea. Fujuoka-ken Suisan Shekinjo Fukuoka Fish. Exp. Sta. 715 p. Kennedy, A. V. and H. I. Battle. 1964. Cyclic changes in the gonad of the American oyster Ci'assostrea virginica (Gmelin). Can. J. Zool. 42: 30.5-321. Kinoshita, T. 1939. On the species name and spawning season of Venerupis semidecussata in Hokkaido. Ten-day Rep. Hokkaido Fish. Exp. Sta. No. 410: 3-7. (In Japanese, English sum- mary.) Ko, Y. 1957. Some histological notes on the go- nads of Tapes japonica Deshayes. Bull. Jap. Soc. Sci. Fish. 23: 394-399. (In Japanese, English summary.) Lammens, J. J. 1967. Growth and reproduction in a tidal flat population of Macoma balthica (L.) Neth. J. Sea. Res. 3: 315-382. 58 D. A. HOLLAND AND K. K. CHEW Loosanoff, V. L. 1936. Sexual plvses in the qua- hog. Science, 83: 287-288. Loosanoff, V. L. 1937. Development of the pri- mary gonad and sexual phases in Venus mcr- cpuaria Linnaeus. Biol. Bull. 72: 389-405. Men-ill, A. S. and J. B. Burch. 1960. Herma- phroditism in the sea scallop, PJacopecteii mujeUawcH.^ (Gmelin). Biol. Bull. 119: 197-201 Neave, F. 1949. Tlie legal size limit in relation to the size at which butter clams mature. Fi.sh. Res. Board Can., Pi'og. Rep. No. 61, p. 4-5. Nosho, T. and K. K. Chew. 1972. Tlie setting and growth of the Manila clam, Venenipia japonica (Deshayes), in Hood Canal, Washington. Proc. Natl. Shellfish Assoc. 62: 50-58. Ohba, S. 1959. Ecological studies in the natural population of a clam, Tapi^ japiniica. with special reference to seasonal variation in the size and structure of population and to indivi- dual growth. Biol. ,J. Okayama Univ. 5: 13-42. Porter, H. J. 1964. Seasonal gonadal changes in adult clams, Mercenaria ynercenana (L.), in North Carolina. Proc. Natl. Shellfish Assoc. 55: 35-52. Quayle, D. B. 1942. Sex, gonad development and seasonal gonad changes in Paphia ^itojiiixca (Conrad). J. Fish. Res. Board Can. 6: 140-151. Quayle, D. B. 1952. Tlie rate of growth of Vene- iv/p/.s' pullaMm (Montagu) at Millport, Scot- land. Proc. Prog. Soc. Edin. 64: 384-406. Quayle, D. B. and N. Bourne. 1972. The clam fish- eries of British Columbia. Fish. Res. Board Can. Bull. 179, 70 p. Ropes, J. W. 1967. Hermaphroditism in the surf clam Spmda solidissima. Proc. Natl. Shellfish A.SS0C. 58: 63-65. Ropes, J. W. 1968. Reproductive cycle of the surf clam, Spmdn ftimn. in offshore New Jei-sey. Biol. Bull. 135:349-365. Ropes, J. W. and A. P. Stickney. 1965. Repro- ductive cycle of Mya arcenaria in New England. Biol. Bull. 128: 315-327. Sastry, A. N. 1963. Reproduction of the bay scallop, Aequipecten irmdiam (Lamarck); in- fluence of temperature on maturation and spawning. Biol. Bull. 125: 146-153. Shaw, W. N. 1964. Seasonal gonadal changes in female soft-shell clams, Mya (uctKirid. in the Tred Avon River, Maryland. Proc. Natl. Shell- fish Assoc. 53: 121-132. Shaw, W. N. 1965. Seasonal gonadal cycle of the male soft-shell clam, Mya nrenaiin. in Maryland. U.S. Fish. Wildl. Ser., Sci. Rep. Fish. No. 508: 5 p. Tanaka, Y. 1954. Spawning seasons of impoi'tant bivalves in Ariake Bay. III. Vcnnntpis s^eDiidi'- rv/.s.snfo — (Reeve). Bull. Jap. Soc. Sci. Fish. 19: 1165-1167. (In Japanese, English summary.) Thorson, G. 1946. Reproduction and larval de- velopment of Danish marine bottom inverte- brates. Medd. Komm. Danm. Fiskeri-og Havun- ders., ser. Plankton 4: 1-523. Ti-anter, D. J. 1958a. Reproduction in Australian pearl oysters (Lamellibranchia). I. Pinctadn albina (Lamarck): Primary gonad development. Aust. J. Mar. Freshw. Res. 9: 135-143. Tranter, D. J. 1958b. Reproduction in Australian pearl oysters (Lamellibranchia). II Puietndn albina (Lamarck): Gametogenesis. Aust. J. Mar. Freshw. Res. 9: 144-1.58. Tranter, D. J. 1958c. Reproduction in Australian pearl oysters (Lamellibranchia). Ill Piuctada albina: Breeding season and sexuality. Aust. J. Mar. Freshw. Res. 9: 191-216. Ti'anter, D. J. 1958d. Reproduction in Australian pearl oysters (Lamellibranchia). IV Pinrtada inaiyaritifcra (Linnaeus). Aust. J. Mar. Freshw. Res. 9: 509-525. Tranter, D. J. 1958e. Reproduction in Australian pearl oysters (Lamellibranchia). V Pinrtada fncata (Gould). Aust. J. Mar. Freshw. Res. 10: 4.5-66. Turner, H. J. Jr. and J. E. Hanks. 1960. E.xperi- mental stimulation of gametogenesis in Hijdri)idri< dinnthm and Pecten irradianft dur- ing the winter. Biol. Bull. 119: 14.5-1.52. Yoshida, H. 1935. The full grown veliger and ear- ly young shell stage of Venempis philli- pimmm. Venus 5: 264-273. (In Japanese, English summary.) Yasuda. J., I. Hamai and H. Hotta. 1954. A note on the spawning season in Vvncrnp)!^ plnllipinnnnn. Bull. Jap. Soc. Sci. Fish. 20: 277-279. (In Japanese, English summary.) Proceedings of the National Shellfisheries Association 1974 FACTORS INFLUENCING THE SETTING BEHAVIOR OF LARVAL HARD CLAMS. MERCENARIA MERCENARIA' Richard Keck. Don Maurer and Robert Malouf P^IELD STATION COLLEGE OF MARINE STUDIES UNIVERSITY OF DELAWARE LEWES, DELAWARE AND OREGON STATE UNIVERSITY CORVALLIS, OREGON ABSTRACT Laboratory setting experiments with larvae of the hard clam. Mercenaria mer- cenaria, showed that chemimi factors (phenmunies) ayid phi/sical factors influenced setting. Clams preferred to set in sand as compared to nvid and in sediment treated with clam liquor rather than untreated substrate. Chemical factors masked physical selection of sediment by larvnc Clinn Iniiuir //Y(.s a strong setting stim^flus for clam larvae, indicating a gregarimis setting behavior similar to that if nyster larvae. INTRODUCTION Survival of early stages of benthic organisms is a critical aspect in the establishment of benthic communities. Thorson (1966) postulated that ben- thic invertebrates are subjected to three major types of selection which determine the extent of survival and composition of a benthic com- munity: 1) food, temperature, and predation during the planktonic stage, 2) effect of hydrography on the distribution of larvae, 3) sub- strate selection, competition among juveniles, and predation after metamorphosis. A part of the third category forms the basis of this paper; that is. substratum (sediment) selec- tion. Sanders (1958) and Bloom. Simon and Hun- ter (1972), in studies of benthic communities, showed that filter feeders and deposit feeders are dominant in coarse and fine sediments, respec- tively. Rhoads and Young (1970) related bottom stability and competition between similar species ' Contribution No. 87, College of Marine Studies. as determining factors in species distribution. For example, juveniles of suspension feeders, such as hard clams, are subject to environmental stress by deposit feeders that rework the surface, and survival and settlement are decreased by clogging of gills and the resuspension or burial of recently set larvae. Wilsnn (1948, 19.52, 1954) showed that survival of the larval polychaete, Ophelia, was dependent upon settlement on a favorable substratum. Thor- son (1957) and Scheltema (1961) reported that lar- vae delayed metamorphosis and thereby increased the probability of encounter with a favorable sub- stratum. The mechanism of substratum selection is related to the physical, chemical and geological properties of the given sediment. Maurer (1969) found that sediment size limited the distribution of the tellinid pelecypods, Tellina butbini and Tellina salmonea. Tenore, Horton and Duke (1968) showed the harmful effects of clay-silt sediments containing high levels of organic material on Rangia cnneata. Bader (19.54) 59 60 R. KECK, D. MAURER AND R. MALOUF reported that pelecypod density initially in- creased with an increase in organic level. However, at higher levels, products of decom- position produced an increase in bacteria and a decrease in oxygen levels which resulted in a deci-eased pelecypod population. Gurin and Carr (1971) suggested that larvae select substrata by means of sensitive chemoreceptor organs. Crisp (1967), Bayne (1969), Keck, Maurer, Kauer and Sheppard'(1971), and Veitch and Hidu (1971) discussed the chemical basis of setting in the American oyster. Oppenheimer (1961) and ZoBell (1963) discussed the importance of bacteria in aggregation, nutrient and mineral cycles in sediments. Crisp and Meadows (1962) and Meadows and Anderson (1968) postulated the importance of microorganisms and organic layere on the set- tlement of marine larvae. Many investigations have been made of the hard clam, Mercenami mercenaria, and its sedi- ment preferences. Pratt (1953) reported a greater density of hard clams in fine sediments containing large particles, such as shell. Carriker (1961) in- dicated that juvenile clams favored fine sedi- ments with appreciable amounts of organic detri- tus. He further stated that it was not possible to determine the setting preference of juvenile clams to a graded series of grain sizes. Wells (1957), in a study of hard clam distribution in Chincoteague Bay, found clams more prevalent in shell bottom than sand, sand-mud and mud. Tliis distribution resulted from either selection of sub- stratum or a pattern of survival. Planting ex- periments with shell aggregate by Castagna (197(1) indicated that predation by crabs was a critical factor in natural distribution. Saila, Flowers and Cannario (1967) studied several en- vironmental parameters in areas of high and low density. The difference in population abundance could not be explained in terms of sediment properties alone. They concluded that current, vegetation, predation and organic constituents all affect distribution. The objective of our research was to determine the setting preference of hard clam larvae in the labfiratory. The experiments were designed to study substratum selection based on particle size, chemical composition and treatment with pheromones. The interaction of the above factors was examined to determine which exerted the strongest influence on metamorphosing larvae. METHODS M. mercenaria larvae of setting size were ob- tained from the University of Delaware's Sea Grant Mariculture facility (Pruder, Epifanio, and Malouf, 1973). Tlie larvae were raised by .standard laboratory techniques (Loosanoff and Davis, 1963; Maurer and Price, 1967) and were released in a 284 1 setting tank containing a grid with 36 ran- domly-placed sediment blocks and control blocks with no sediment (Fig. 1). Approximately 200,000 larval clams were used in each experiment to in- sure significant sets. Experimentation showed that the size and age of larvae are extremely critical variables. Carriker (1961) stated that pediveligers exhibit a searching behavior charac- terized by alternate periods of swimming and crawling. The capacity to swim widely extends the territory which the clam can examine, especially in the laboratory. Larvae must be released at smaller than setting size (150-170 fjt) to assure that the clams are still highly mobile and capable of exhibiting a searching behavior. To select larvae, 9-12 day old stocks were poured through 149 m and 125 m screens. Larvae retained on the 125 M screen were transferred to a large beaker, held for about two hours, and .swimming larvae were poured off and used in the ex- periment; larvae that settled to the bottom were rejected. Larvae were released randomly throughout the setting tank. After a 48-hour period, the experiments were terminated by draining the tank and exposing the grid. A plastic, one inch square, measuring device was placed on the bottom and all sediment and clams contained in this area were pipetted into small finger bowls. The samples were sieved and washed to separate juvenile clams from the sediment and to facilitate counting. The results were analyzed using analysis of variance, Kruskal-Wallis H test, or Mann-Whitney U test (Sokal and Rohlf. 1969). Experiments The experiments were conducted in the following series with the results presented in Tables 1 through 5. SETTING BEHAVIOR OF HARD CLAM LARVAE 61 TABLE 1. Oroup I. Preliminary hard clam setting experiments ivith natural and incinerated sedi- ments. Numbers represent sum nf clams setting in 6 experimental hlnrks. Experiment 1 Natural Sediments Experiment 2 Natural Sediments Experiment 3 Incinerated Sediments Experiment 4 Incinerated Sediments Sand (250 ]x) 854 209 817 452 Sand Clam Liquor 1138 371 3369 447 Sand Mud 859 164 600 278 Mud (50 p.) 431 78 394 333 Mud Clam Liquor 140 23 1503 291 Control 1173 123 221 261 F Value ■ 6.39 * 18.02 * 4.95 * 2.39 * Denotes Values Significant at 95% Confidence Level TABLE 2. Group II. Graded particle size experiments with natural sediments. Numbers represent sum of clams setting in 6 experimental blocks. Experiment 1 1844 Experiment 2 Experiment 3 Experiment 4 Experiment 5 1 mm 2541 476 914 1406 707 M 2724 2776 402 1409 1236 500 p 4230 3125 629 1015 1663 250 )i 2696 5469 1621 866 1313 50 ,1 3154 3060 405 470 1293 Control 2478 1483 332 341 1085 F Value 1.91 .642 1.17 1.07 2.73 H Value 8.05 6.86 4.77 4.66 1.43 U Value 27.5 * 28.0 * 26.0 27.5 * 21.0 * Denotes Values Significant at Group I. Preliminary experiments were set up with the following natural sediments and in- cinerated sediments: 250 p sand treated with clam liquor, 250 m sand, 50 p sand-mud mixed in a 1:1 ratio, 50 p mud, 50 p mud treated with clam liquor and a control (no sediment). Clam liquor was obtained by shucking the clam over a beaker and retaining the liquid freed by the shucking process. Approximately 100 ml of clam Yo Confidence Level liquor was used to treat 1 kg of sediment. Gnmp II. Larvae were exposed to natural sediment particles of different sizes: 1mm, 700 p. 500 n. 250 M, 50 M and control (no sediment). Group III Larvae were exposed to the same particle sizes as in Group II. However, the sediments were incinerated at 500 C for 1 hour prior to use. Crr(mp IV. Larvae exposed to incinerated 500 p 62 R. KECK, D. MAURER AND R, MALOUF TABLE 3. Group III. Graded particle size experiments mth incinerated sediments. Numbers represent sum of clams .netting in 6 experimental blocks. Experiment 1 Experiment 2 Experiment 3 Ei'iperiment 4 1 mm 242 378 523 255 707 u 225 368 936 425 500 u 364 724 355 193 250 u 412 752 301 345 50 u 248 322 345 259 Control 307 249 473 183 F Value .546 4.98 * 3.89 .517 H Value 4.27 13.4 * 11.79 ,828 U Value 24.0 31.5 * 32,0 * 19.0 * Denotes Values Significant at 95^ Confidence Level FIG. L Setting tank mth random arrangement of experiment blocks. SETTING BEHAVIOR OF HARD CLAM LARVAE 63 TABLE 4. Group IV. 500 fj sand vs. 50 ^ mud incinerated sediments. Experiment with numbers represents ranked pairing of obsen'ations. Sand 20 30 30 45 46 56 57 59 61 75 85 91 100 106 108 128 177 809 Total 2083 U = 254.5 * Mud 18 18 19 23 24 24 29 33 36 41 41 42 46 54 54 6? 93 in 781 * Denotes Value Significant at 95^ Confidence Level sand and 50 p mud. Group V. Larvae exposed to SOU p sand treated with clam liquor and untreated 500 /i sand. RESULTS Gr-oup I. In Experiments 1 and 2 there was a statistically higher set of clam larvae in sand than in mud (Table 1). Sand treated with clam liquor also yielded higher setting than untreated sand. In both experiments, treated mud had the lowest set. These experiments showed that sediment was unnecessary for successful setting of hard clam larvae. Experiments with in- cinerated sediment showed the same association between high setting and treated sand; however, treated mud also yielded a high set. There was no statistical difference between setting in treated and untreated sand for Experiment 4. Never- theless, setting was higher in sand-mud and treated mud. sand than mud, Group II. In general, results from Experiments 1 through 5 did not show any statistically significant associations between setting and a particular sediment size (Table 2). However, there was a definite trend between setting and 250 p and 500 p particle sizes. Moreover, for Experiments I. 2 and 4 when the maximum set in sand sediment was compared to the maximum set in 50 ft sediment, there was a statistically significant dif- ference (Mann-Whitney U test). Group III Although there were higher sets in coar^ef sediments, results were not significantly different in three of four experiments (F Value, Table 3), 64 R. KECK, D. MAURER AND R. MALOUF TABLE 5. Ofoiip V. Treated and untreated (500 n) sand. Numbers represent ranked assemblage of data. Experiment 1 Experiment 2 Experiment 3 Experiment 4 Untreated Treated Untreated Treated Untreated Treated Untreated Treated 0 82+ 17 38+ 11 18+ 5 17+ 29 86+ 17 48+ 13 46+ 7 29+ 37 111+ 26 49+ 14 48+ 8 40+ 47 119+ 36 60+ 16 51+ 12 kU+ 56 123+ 43 74+ 19 55+ 12 47+ 67 130+ 45 75+ 21 79+ 14 48+ 75 149+ 49 80+ 23 81+ 18 54+ 80 155+ 50 84+ 26 84+ 28 59+ 94 174+ 52 94+ 27 92+ 40 67+ 103 266+ 54 98+ 28 100+ 45 79+ 131 301+ 55 102+ 32 102+ 48 84+ 161 317+ 60 104+ 48 114+ 61 85+ 204 361+ 69 118+ 50 122+ 98 104+ 243 495+ 70 25 Of 53 142+ 119 116- 256 573+ 89 289+ 63 164+ 139 204+ 332 578+ 94 381+ 118 207+ 148 349+ 351 627+ 97 585+ 226 204- 287 365+ 1523 645- 1643 2000+ 247 227- 337 514+ Maim-Whitney U Test U = 237 ■X- u = 253. .5 * u = 255 .5 * U = 218 * * Denotes Values Significant at 9^% Confide: Any trends between set and sediment particle size included a range in the latter from 250 m to 707 M. which exceeded that recorded for Group II experiments. U values further compare maximum set in sand sediment with set in 50 ju mud. Omup IV. Results of the single experiment performed showed that there was a statistically significant difference in setting between incinerated sand (500 m) and incinerated mud (50 m)- In 18 pairs of plots, without exception, there were higher num- bers of set in the sand compared to the mud (Table 4). Group V. Results from Experiments 1 through 4 showed that sand (500 p ) treated with clam liquor yielded higher sets than untreated sand (Table 5). Sixty-eight of 72 paired plots (94%) contained higher numbers of set in treated sand. Ex- periments 1, 3 and 4 had 1, 2 and 1 reversals. ince Level respectively, of the above pattern (Table 5). All experiments were statistically significant. DISCUSSION Gregarious setting among invertebrates and the oyster, Crassostrea virginica, in particular, has been well documented (Wilson, 1948, 1952, 19.54; Thorson, 1966; Crisp, 1967; Bayne, 1969; Keck, et al, 1971; Veitch and Hidu, 1971). The present research has expanded this phenomenon to include hard clams. Gregarious setting behavior was influenced by the presence of water soluble pheromones. We use the term pheromone in a broad sense as representing a biologically ac- tive substance, possibly protein or glycoprotein, emitted by adults or other juveniles by diffusion between water and shell liquor. Setting was statistically higher in sand than mud (Tables 2 and 4). SETTING BEHAVIOR OF HARD CLAM LARVAE 65 Treatment of sand with clam liquor dramatically enhanced the setting value of sand (Tables 1 and 5). However, treatment of non-in- cinerated mud reduced setting. High levels of organic materials and bacteria in the mud may have exceeded the threshold discussed by Bader (1954). We suggest that the addition of organic material may be responsible for reduced setting because of increased bacteria levels, reduced dissolved oxygen, and mcr^ased production of H2S. Hard clam larvae also set on control plots with no initial sediment. Carriker (1961) reported that hard clam laiTae set well on a hard surface covered with a thin layer of detritus. We ob- served that during the 48-hour testing period a thin layer of detritus settled on the control blocks. There was a progression of setting from sand to mud in untreated incinerated sediments (Table 1). Nevertheless, sand and mud treated with clam liquor were the best substrata for settlement. Organic materials and bacteria normally oc- curring in natural sediment were destroyed in the incinerated sediments. The concentration of clam liquor acted mainly as an attractant for the lar- vae and did not increase organic levels or cause a bacterial bloom within 48 hours. Even though metamorphosing hard clam larvae had higher sets in sand compared to mud (Tables 1, 2 and 4), which suggests a preference for the size range of sand particles, they apparently did not show a specificity of selection within that size range (Tables 2 and 3). Clam larvae did not discern among the particle sizes offered (Table 3). Regardless, the difference between maximum set in sand and mud further supported the con- clusion that clams exhibited preferential setting behavior. Experiments with incinerated sediments of the same particle size (Table 3) showed similar results to natural sediments (Table 2). Data from Group II (Table 2) showed statistically significant differences between setting and par- ticle size and corresponding data from Group III (Table 3) were essentially insignificant for in- cinerated sediments, indicating that the natural chemical properties of sediments from Group II were the factors influencing setting behavior. As reported earlier, sand treated with clam liquor yielded higher sets than untreated sand (Table 5). We suggest that a chemical isolate or prepared concentration of the pheromone would probably increase setting over that recorded with natural clam liquoi-. Carriker (1961) described clumped distribution of pediveligers in laboratory setting experiments. The clumping was par- ticularly evident in pitted shells covered with detritus. This clumping may have represented gregarious setting caused by the release of pheromones by recently set larvae. The gregarious setting factor caused by pheromones is probably adaptive. Evidence for this behavior is available (Keck, Maurer and Watling, 1972). On the other hand, distribution of juveniles has not been correlated with sediment size (Saila, et al. 1967). However, our laboratory data are supported by field data (Wells, 1957; Pratt and Campbell, 1956; Sanders, 1958; and Bloom, et at, 1972). Research should be designed to correlate clam distribution to chemical factors in the field. Raw clam liquor should be purified and setting factors that may be exploited in the mariculture of this species should be isolated. We suggest that previous studies on the ability of larvae to metamorphose in various sediments (Scheltema, 1961; Wilson, 1948) are not analogous to this work on sediment selection by the hard clam because larval hard clams do not need sediment to metamorphose. The random block ex- periments reported here indicate that larval hard clams do actively engage in sediment selection. ACKNO WLEDGEM ENTS Tlie authors thank the staff of the University of Delaware Mariculture facility. Dr. Charles Epifanio, Chief Scientist, and especially Mr. Earl Greenhaugh for supplying setting-size larvae. Special thanks are due Bill Daisey, Wayne Leathem, Peter Kinner and Lee Sterling for their help in the laborious counting sessions. Dr. Melbourne R. Carriker critically reviewed a first draft. This study was authorized by PL 88-309 and was supported by the NOAA National Marine Fisheries Service, Project 3-135-R, Delaware Bay Hard Clam Survey. LITERATURE CITED Bader, R. G. 19.54. The role of organic matter in determining the distribution of pelecypods in marine sediments. J. Mar. Res. 13: 32-47. 66 R. KECK. D. MAURER AND R. MALOUF Bayne, B. L. 1969. The gregarious behaviour of the larvae of Ostrea edulis L. at settlement. J. Mar. Biol. Assoc. U.K. 49: 327-356. Bloom, S. A., J. L. Simon and V. D. Hunter. 1972. Animal-sediment relations and community analysis of a Florida estuary. Mar. Biol. 13: 43-56. Carriker, M. R. 1961. Interrelation of functional morphology, behavior, and autecolog\' in early stages of the bivalve Mercenaria merce)inria. J. Elisha Mitchell Sci. Soc. 77: 168-241. Castagna, M. A. 1970. Hard clam culture method developed at VIMS. Va. Inst. Mar. Sci., Mar. Resour. Advisory Ser. No. 4, 3 p. Crisp, D. J. 1967. Chemical factors inducing settlement in Crassostrea virginica (Gmelin). J. Anim. Ecol. 36: 329-335. Crisp, D. J. and P. S. Meadows. 1962. Tlie chem- ical basis of gregariousness in cirripedes. Proc. R. Soc. Lond. B. 156: 500-520. Gurin. S. and W. E. Carr. 1971. Chemoreception in Nassarius obsoletus: "the role of specific stimulatory proteins." Science 174: 293-295. Keck, R., D. Maurer, J. C. Kauer and W. Shep- pard. 1971. Chemical stimulants affecting lar- val settlement in the American oyster. Proc. Natl. Shellfish. Assoc. 61: 24-28. Keck, R., D. Maurer and L. Watling. 1972. Sur- vey of Delaware's hard clam resources, Dela- ware Bay. Annual Report to the National Marine Fisheries Service. Loosanoff, V. L. and H. C. Davis. 1963. Rearing of bivalve mollusks. Adv. Mar. Biol. 1: 1-136. Maurer, D. 1969. Pelecypod-sediment association in Tomales Bay, California. Veliger 11: 243- 249. Maurer, D. and K. S. Price, Jr. 1967. Holding and spawning Delaware Bay oysters (Crasso- strea virgi)ika) out of season I. Laboratory fa- cilities for retarding spawning. Proc. Natl. Shellfish. Assoc. 58: 71-77. Meadows, P. S. and J. G. Anderson. 1968. Micro- organisms attached to marine sand grains. J. Mar. Biol. Assoc. U. K. 48: 161-175. Oppenheimer, C. H. 1961. Bacterial activity in sediment of shallow marine bays. Geochim. Cosmochin.Acta 19: 244-260. Pratt, D. M. 1953. Abundance and growth of Vf'ims mercenaria and Callncardia ttunrfmana in relation to the character of bottom sedi- ments. J. Mar. Res. 12: 60-74. Pratt, D. M. and D. A. Campbell. 1956. Environ- mental factors affecting growth in Venus mercennna. Limnol. Oceanogr. 1: 2-17. Pruder, G. D., C. Epifanio and R. Malouf. 1973. Tlie design and construction of the University of Delaware mariculture laboratory. DEL- SG-7-73. Coll. Mar. Stud., Univ. Del., Newark, Del., 96 p. Rhoads, D. C. and D. K. Young. 1970. The influ- ence of deposit-feeding organisms on sediment stability and community trophic structure. J. Mar. Res. 28: 1.50-178. Saila, S. B., J. M. Flowers and M. T. Cannario. 1967. Factors affecting the relative abundance of Mercenaria mercenaria in the Providence River, Rhode Island. Proc. Natl. Shellfish. Assoc. 57: 83-89. Sanders, H. L. 1958. Benthic studies in Buzzards Bay. I. Animal-sediment relationships. Limnol. Oceanogr. 3: 245-258. Scheltema, R. S. 1961. Metamorphosis of the veli- ger larvae of Nassarius obsoletus (Gastropoda) in response to bottom sediment. Biol. Bull. 120: 92-109. Sokal, R. R. and F. J. Rohlf. 1969. Biometry. San Francisco, W. H. Freeman, 776 p. Tenore, K. R., D. B. Horton and T. W. Duke. 1968. Effects of bottom substrate on the brack- ish water bivalve Rangia cuneata. Chesapeake Sci. 9: 238-248. Thorson, G. 1957. Bottom communities (sub- littoral or shallow shelf). //( J. W. Hedgpeth (ed.). Treatise on Marine Ecology and Paleo- ecolog\-. Vol. 1, Ecology. Geol. Soc. Am. Mem. 67, p. 461-534. Thorson, G. 1966. Some factors influencing the re- cruitment and establishment of marine benthic communities. Neth. J. Sea Res. 3: 267-293. Veitch, F. P. and H. Hidu. 1971. Gregarious setting in the American oyster Crassost)-ea virginica Gmelin: I. Properties of a partially purified "setting factor." Chesapeake Sci. 12: 173-178. Wells, H. W. 19.57. Abundance of the hard clam Mercenaria mercenaria in relation to environ- mental factors. Ecology 38: 123-128. Wilson, D. P. 1948. The relation of the sub- stratum to the metamorphosis of Ophelia larvae. J. Mar. Biol. ASsoc. U.D. 27: 723-760. SETTING BEHAVIOR OF HARD CLAM LARV.AE 67 Wilson, D. P. 1952. The influence of the nature of the substratum on the metamorphosis of the larvae of marine animals, especially the larvae of Ophelia bicornis Savigny. Ann. Inst. Oceanogr. Monaco 27: 49-156. Wilson, D. P. 1954. The attractive factor in the settlement of Ophelia hicorms Savigny. J. Mar. Biol. Assoc. U.K. 33: 361-380. ZoBell, C. E. 1963. Domain of the marine micro- biologist. In C. H. Oppenheimer (ed.). Sym- posium on Marine Microbiology. C. C. Thomas, Springfield, 111., p. 3-24. Proceedings of the National Shellfisheries Association Volume 6Jt - 197A A HAPLOSPORIDAN INFECTION IN GAPER CLAMS, TRESUS CAPAX (GOULD), FROM YAQUINA BAY, OREGON ^ David A. Armstrong and Janet L. Armstrong DEPARTMENT OF FISHERIES AND WILDLIFE MARINE SCIENCE CENTER, OREGON STATE UNIVERSITY NEWPORT, OREGON ABSTRACT A haplosporidan parasite was found in ^3% of 226 gaper clams, Tresus capax,. /?-om Yaqitina Bay. Oregon. Of these infections. 80% were rated as light and caused no apparent damage to the clams. Heavily infected clams were eynaciated. sluggish in response to prodding, were dark in color and had little gonad development even during the spawning season. Grossly, the most apparent sign of infection was numerous white cysts in the tissues. Cysts ranged up to 2.0 mm in diameter and were present predominantly in the mantle overlying the viscera and under the epithelium in the siphon. In heavy infections cysts also occurred in the gills, the digestive gland, gonadal tissue and the musculature of the body wall and foot. Histological examination of cyst-bearing tissue revealed stages of multinucleate Plasmodia 10-3.5 p. long, tvhich contained spherical nuclei l.i-3.0 M in diameter vnth light nuclear membranes. Many necrotic plasmodia were observed and some were phagocytized. Cysts were formed by host cellular response to the para.^He and were compDsnl of rruissive aggregations of hemocytes circumscribed, by fibntbla.^t-!ikc cilU mid light connective tissue depositions. INTRODUCTION Haplosporidans have been reported in several species of bivalves including oysters from the East coast of the United States (Sprague, 1970). Serious oyster epizootics have been attributed to haplosporidans of the genus Minchinia. Mirwhinia nelsoni was found to be the etiological agent in mass mortalities of Crassostrea virginica populations on the Atlantic coast (Andrews and Wood. 1967; Sprague, 1970), and M. costalis has caused heavy losses of C inrginica in the seaside bays of Virginia and Maryland (Wood and An- drews, 1962; Andrews et al, 1962). Subsequently M. costalis has been reported in C. viryinica held Technical Paper No ment Station. )3684, Oregon Agricultural Experi- in California bays (Katkansky and Warner, 1970a; Krantz et al.. 1972), and a single incidence of a haplosporidan in the Pacific oyster (C. gigns) was rejioited by Katkansky and Warner (1970b). Recently, Mix and Sprague (in press) reported plasmodial stages of a haplosporidan in Ostrrn lurida from Yaquina Bay, Oregon. Taylor (1966) described Haplospoyidium tumefacientis from the California sea mussel (Mytilus califomianus). but found no associated mortalities. This paper reports a haplosporidan infection in gaper clams, Tresxis capax. in Yaquina Bay, Oregon, and also discusses physiological trauma to infected animals. METHODS AND MATERIALS Clams were collected from Yaquina Bay, Oregon, predominantly from the Sally's Bend 68 HAPLOSPORIDAN INFECTION IN GAPER CLAMS 69 area on the north side of the bay. Collections were made sporadically from May through November 1972, and no clams were taken again until June 1973. Whole clams were removed from their shells, blotted with towels and weighed to the nearest g. The mean shell length was 112 mm (range: 76-135 mm) representing an average age ofabout 4 yr. (Marriage, 1954). The animals were then examined macroscopically for cysts and were dissected to inspect internal tissues and organs. Preliminary histological examination of in- fected tissues revealed that all parasites were present within host-formed cysts and no life stages were observed outside these areas of host cellular reaction. Therefore, haplosporidan in- fections were diagnosed solely by the presence or absence of such cysts. Infection was categorized as N, I (less than 30 cysts per clam) or II, denoting no infection, light and heavy infections respectively. Regression line anlysis of shell dimensions versus body weight were run on the three groups, and analysis of co-variance was performed to determine if there were differences in weight due to infection. Tissues were fixed 12-18 hr in either seawater- Bouin's solution or 10% formalin, dehydrated, embedded in Paraplast and sectioned at 7 ;u. Stains used were Mayer's hematoxylin and eosin, Geimsa, or Zeihl-Nielson acid fast, counter stained with Harris' hematoxylin and eosin (Farley, 1965). Sections of the mantle, siphon, gill, kidney, digestive gland and foot musculature were studied. RESULTS Gross signs of haplosporidan infection were white, spherical cysts ranging in diameter from 0.25-2.0 mm. In heavy infections, cysts were often so numerous that they appeared to fuse into larger masses with diameters up to 5 mm. Of 226 clams examined, 43% contained cysts and 80% of these infections were rated as light. Cysts were primarily located in the mantle overlying the viscera (Fig. 1) and in the siphon under the epithelium bordering the inhalent and exhalent channels. In more heavily infected clams, cysts were also present in the gills (Fig. 2), digestive gland, Leydig tissue and throughout the musculature of the body walls and foot. FIG. 1. An uninfected clam (above) and heavily infected clam (below). Note the transparent q^iality of the infected mantle revealing dark silt accumulations in the mantle cavity. Lightly infected clams appeared to be in good condition and were qualitatively in- distinguishable from uninfected animals. Clams with heavy infections were moribund. They ap- peared emaciated and were sluggish in response to prodding. The siphon was often flaccid and not retracted into the shell. The mantle was ab- normally transparent and watery in texture. The body hue was dark and contrasted markedly with the creamy white appearance of healthy clams (Fig. 1). Tliis color difference was partially at- tributable to sparse gonad development in heavily infected clams, even in mid-spring when healthy animals contained profuse gonads. Silt had ac- cumulated on the gills of several clams indicating impaired ciliary movement on this organ (Fig. 2). Histological examination of cyst-bearing tissue revealed the etiological agent to be a haplosporidan parasite (V. Sprague, personal communication). Only plasmodial stages were ob- served and these had none of the acid-fast qualities described by Farley (1965) for mature spores. Within a cyst there were numerous Plasmodia, often more than one hundred. Plasmodia were spherical to oval in shape and ranged in length from 10-35 m (Fig. 3). These organisms were often isolated in lacunae (Fig. 3) 70 D. A. ARMSTRONG AND J. L. ARMSTRONG FIG. 2. View ijj a heuvdij infected yaper clam- Tfie mantle has been cut and prilled back to show the cysts in the yilk and the accumulated silt. but sometimes had hemocytes directly in contact witli the Plasmodia! membrane. Plasmotomy of enlarged plasmodia, as described by Farley (1967) and depicted by C«uch ct al. (1966), was not ob- served. The supposition, based on our preliminary histological examination of clams, that stages of the haplosporidan were not found outside cysts was substantiated by this more extensive survey. There were variable numbers (2-60+) of spherical nuclei that ranged in diameter from 1.4-3.0 11 within plasmodia. Two stages of nuclear development, in what were judged to be viable pla.smodia, were observed. Nuclei were either small (1.5 /i) with large amounts of chromatin (Fig. 3) or were about 3.0 ii in diameter with lit- tle chromatin aggregated against the nuclear membrane, sometimes with a thin rod transecting the nucleus (Fig. 4). In several sections the larger nuclei appeared to be exiting from the Plasmodium into the lacuna and surrounding cellular material. Necrotic plasmodia were frequently observed. Their nuclei were pyknotic and the cytoplasm was more acidophilic than the cytoplasm of viable plasmodia. Many necrotic Plasmodia were in stages of dissolution and were being phagocytized. In several cases small Plasmodia were entirely within the membrane of a single phagocytic cell. Cysts were formed by host reaction to parasitic invasion of tissues and were composed of massive aggregrations of hemocytes circumscribed by fibroblast-like cells and connective tissue deposition (Fig. 5). Dr. Sprague noted that he had never seen a similar enclosure by cells resembling fibroblasts in sections of oysters bearing haplosporidans. Formation of cysts was ac- complished by displacement and destruction of normal tissues (Fig. 5, 6). Regression lines of weight vs shell length for groups N, I and II had r^ values of 75%, 53% and 61%, respectively. Analysis of co-variance showed the only significant difference (P = 0.05) was between the elevations of lines for groups N and **' ^•^ t K m%^ FIG. 3. Plasmodial stages of haplosporidans in lannme of a cyst in the mantle. 1600 X. HAPLOSPORIDAN INFECTION IN GAPER CLAMS 71 II. Tlie lower elevation value for group II re- presented a mean weight reduction of 14% from group N. FIG. 4. Plasmodium in a cyst in the mantle. Note less chromatin in these uclei than in Fig. 3. ami thin rod transecting them. 1000 X. DISCUSSION This is the first report of a haplosporidan in- fection in clams on the Pacific coast. Specific identification of this organism was not possible without observing more developmental stages and mature spores. If this haplosporidan is not a new species peculiar to T. capax, it may have been in- troduced into Yaquina Bay with eastern oysters, where it was able to survive and infect other bivalves. C virginica was cultivated in Yaquina Bay for about 50 years, sometimes with heavy mortalities of stocks (Sweetser, 1907; Dimick et ai. 1941). Recent reports of haplosporidan in- fections in C. virginica held in California bays (Katkansky and Warner, 1970a; Krantz et ai. 1972) and in indigenous oysters in bays where C. virginica have been reared (Katkansky and War- ner, 1970b; Mix and Sprague, in press) support the possibility of transmission in this case. Presently, the existence of this parasite in T. capax is knovra only from Yaquina Bay. In- fections as described here were noted in T. capax taken from this bay as long ago as 15 years, but they have not been seen in clams taken from other bays in the state (Dale Snow, Oregon Fish Commission, personal communication). This in- fection has not been seen in large samples of T. capax taken in British Columbia (Neil Bourne, FIG. 5. Section of a mantle showing several cysts about 1-2 mm in duimeter. Note the displacement and necrosis of mantle tissue and the connective tissue deposition around each cyst. BOX. Fisheries Research Board of Canada, personal communication) and northern California (John De Martini, Humboldt State University, personal communication) for growth and reproductive studies. The incidence of infection in our samples (43%) must be qualified by two observations. First, in- fection was diagnosed by macroscopic ob- servations of cysts. Undoubtedly, there is some period following the initial stages of infection in which cysts have not been formed. Also, non-resistant animals may not form cysts in response to the parasite, thus making our estimates low. Second, our samples were FIG. 6. Cyst. 1.5 mm in diameter, under the epithelium of a siphon. 100 X. 72 D. A. ARMSTRONG AND J. L. ARMSTRONG primarily composed of older clams. About 55% were 110-130 mm in shell length (4-5 years old, Marriage, 1954) and accounted for 70% of all in- fections, which precludes an accurate estimate of the incidence of infection based on all age groups of T. capax present in the bay. ACKNOWLEDGEMENTS We thank Dr. Victor Sprague who kindly examined slides of this material and shared his observations with us. Appreciation is also ex- pressed to Drs. Raymond Millemann, Michael Mix and Robert Olson who discussed this topic with us and reviewed the manuscript. LITERATURE CITED Andrews, J. D. and J. L. Wood. 1967. Oyster mor- tality studies in Virginia. VI. History and dis- tribution of Minchinia nelsoni, a pathogen of oysters in Virginia. Chesapeake Sci. 8: 1-13. Andrews, J. D., J. L. Wood and H. D. Hoese. 1962. Oyster mortality studies in Virginia. III. Epi- zootiology of a disease caused by Haplosporkiium costale Wood and Andrews. J. Insect Pathol. 4: 327-343. Couch, J. A., C. A. Farley and A. Rosenfield. 1966. Sporulation of Minchinia neboni (Haplosporida, Haplosporidiidae) in Crassostrea virginka (Gmelin). Science, 153: 1529-1531. Dimick, R. E., G. Egland and J. B. Long. 1941. Native oyster investigation of Yaquina Bay, Oregon. Progress Report II (for period covering July 1939 to September 1941). Oregon Agric. Exp. Sta., Corvallis, Oregon. Farley, C. A. 1965. Acid-fast staining of haplos- poridian spores in relation to oyster pathology. J. Invertebr. Pathol. 7: 144-147. Farley, C. A. 1967. A proposed life cycle of Min- chinia nehoni (Haplosporida, Haplosporidiidae) in the American oyster Cmssostrea virginica. J. Protozool. 14: 616-625. Katkansky. S. C. and R. W. Warner. 1970a. The occurrence of a haplosporidan in Tomales Bay, California. J. Invertebr. Pathol. 16: 144. Katkansky, S. C. and R. W. Warner. 1970b. Spor- ulation of a haplosporidan in a Pacific oyster (Crassostrea gigas) in Humboldt Bay, Cali- fornia. J. Fish. Res. Board Can. 27: 1320-1321. Krantz, G. E., L. R. Buchanan, C. A. Farley and H. A. Carr. 1972. Minchinia nelsoni in oysters fi'om Massachusetts waters. Proc. Natl. Shell- fish. Assoc. 62: 83-85. Marriage, L. D. 1954. The bay clams of Oregon. Fish. Comm. Oregon, Contrib. No. 20, 47 p. Mix, M. C. and V. Sprague. (In press) Occurrence of a haplosporidian in native oysters (Ostrea lurida) from Yaquina Bay and Alsea Bay, Oregon. J. -Invertebr. Pathol. Sprague, V. 1970. Some protozoan parasites and hyperparasites in marine bivalve molluscs. In S. F. Snieszko (ed.) A Symposium on Diseases of Fishes and Shellfishes. Spec. Publ. 5, Am. Fish. Soc, Washington, D.C., p. 511-526. Sweetser, A. R. 1907. 3rd biennial report of the state biologist to the 24th Legislative Assembly. State of Oregon. Taylor, R. L. 1966. Haplosporidium tumefacientis sp. n., the etiologic agent of a disease of the California sea mussel, Mytilus califomianus Conrad. J. Invertebr. Pathol. 8: 109-122. Wood, J. L. and J. D. Andrews. 1962. Haplo- spondium costale (Sporozoa) associated with a disease of Virginia oysters. Science, 136: 710-711. Proceedings of the National Shellfisheries Association Volume 6h - 197U A RE-EVALUATION OF THE COMBINED EFFECTS OF TEMPERATURE AND SALINITY ON SURVIVAL AND GROWTH OF MYTILVS EDULIS LARVAE USING RESPONSE SURFACE TECHNIQUES R. G. Lough SCHOOL OF OCEANOGRAPHY OREGON STATE UNIVERSITY CORVALLIS, OREGON ABSTRACT Response surface techniques were used to critically examine the combined ef- fects of temperature and salinity on late larval survival and growth of Mytilus edulis using experimental data reported in the literature. The range of con- ditions estimated for maximum survival ivas found to be significantly different than those for maximum growth. Temperature exerted a strong effect on both larval survival and growth, while a temperature-salinity interaction effect was not significant. INTRODUCTION Since Lough and Conor (1973a, b) have critically examined the combined effects of tem- perature and salinity on the larval life of Adula califomiensis by multiple regression analysis and the fitting of response surfaces to survival, growth, and respiration. Lough (1974) has undertaken further studies of bivalve larvae reported in the literature to re-evaluate their tolerances. Response surface techniques permit the estimation of an organisms response to a wide range of untested conditions and allows a visual interpretation of any change in its response at various stages of development. This relatively new and rigorous approach to the field of marine ecology has been reviewed by Alderdice (1972). The purpose of this paper is to re-evaluate the combined effects of tem- perature and salinity on the late larvae of M. edulis using the experimental data reported by Brenko and Calabrese (1969). METHODS Brenko and Calabrese (1969) reared the lar- vae of M. edulis at normal seawater conditions (18 ± 1 C and 27 ± 1 ppt salinity) to the straight-hinge stage and then transferred them to six levels each of temperature and salinity to determine the effects of these factors on larval development. Survival and growth were deter- mined when the first cultures reached setting size (16-17 days). The mathematical model used in the analysis was of the form: b,(S Y = b„+ b,(T) -I- b,(S) + b,(T') + + b.(T X S) where Y = percentage survival or growth, bg = a constant, T = linear effect of tem- perature, S = linear effect of salinity, T' = quadratic effect of temperature, S" = quadratic effect of salinity, T x S = interaction effect between temperature and salinity. 73 74 R. G. LOUGH 3 0^ =5 I se o 1^ o o S o H -H (D Ch 0) I CNJ W •H fn c (D o XH ■H p CO H (0 s S S O i-H CV U^ o^ r^ O CM r^ c^ r^ O to O • • • • • O ir\ C\i ;H r^ r^ UA CV O CNi -4- vO CV -J- O OJ CNi c^tX) O O CN CO CO S -H --H --I S C^ UA CNi O C^ iH T— { \0 vO C^ tXl lf\ "-H i-H 'H t^^ O UA O to O "-I r^O vo D- i-H !>- -4- <-l CV tH O u^ CO ^^^^ • ,-< T-i T-{ T-{ S !>- O O UA UA >-H u^ tr~ O r^ • • • • • to u> CV C\i CTv -d- CNi LA !>- -d- -3- I^O -4 ^ I uA cn; I I UAO I I CNi o 'd CO <-i .-H i-i iTN ; -4 r^CNi vH O r^ r^ r^ r^ r^ •k •» Ck an •« i-H CNi (V^ -4 U^ ■XN CO cn O CM r«-^ c^ to CNi Q i-H to LfN -4 O • • • • • to -4- --H I>- !>- CNi <-H r^ c^ r^ to O CN O O O U I CO CO CO S <-! S --H S -4- r^ CV --H O o^ r^ r^ CO c^ tH CNi ro -4 u^ -4- "-H CO -H o -J to CO CNi CO to vO to ro --H to CNi CO tH LPv tH to CN ITN -4- LTN O- O- o [^ ^-to to CO -p CO X C CNi CNi O E-i E-H CO CO E-t o CO -P CO X c i CNi o H H CO CO E-i CJ ro^ -4 >-( CO CNi LTN i-l CNi CO I OvO I I !>- ^ > CO • • CO CO vH .H S ^ S 1 I— 1 o o to ^-^o C^nO sO vO sO i-H CM CO -4 LO ^ 1^ o o to O O U^MD i-H ^ O u^ CO O • • • • • to CO "-f ^- CNi -4- T-l c^ t^ o o CNi UO ITNsO ^ CO CM EH t-i CM CO CO -p CO c o C3 <-i CM CO -4 u^ <-! CM CO -4 u^ CNi CO -4 LO TEMPERATURE-SALINITY EFFECTS ON MYTILUS EDULIS LARVAE 75 FIG. 1. Response stirface estimation of percent survival of Mytilus edulis veltger larvae after 16-17 days of development at experimental tem- perature and salinity combinatioTis given in Brenko and Calabrese (1969). The coefficients in the model (b's) were estimated by the Oregon State University Statistical Program Library, 'STEP, a stepwise multiple regression computer program. F-levels were set equal to zero to enter and remove variables. This allowed all variables to come in- to the equation by a forward selection process, their order of insertion determined by using the partial correlation coefficient as a measure of their importance. The contribution a variable makes in reducing the variance of the equation can also be considered by looking at the various values given as the program proceeds. One of the more useful is the square of the multiple correlation coefficient, R", defined as the sum of squares due to regression, b / total sum of squares, corrected for the mean. It is often stated as a percentage, 100R-. The larger R- is, the better the fitted equation e.xplains the variation in the data. Values of R- can be com- pared at each stage of the regression program. A t-test is also made indicating the equality of the individual regression coefficients of zero and their level of significance. The calculated regression coefficients from a 20 30 40 SflLINI TY (%, ) FIG. 2. Response surface estimation of percent growth of Mytilus edulis veliger larvae after 16- 17 days of development at experimental tem- perature and salinity combinations given in Brenko and Calabrese (1969). particular equation were fitted by computer to a full quadratic equation in temperature and salinity in order to print a contour diagram of the response surface. The computer program was instructed to print 20 percent contour in- tervals, wide enough to exclude the approximate ± 10 percent experimental error reported by the authors. Analysis of covariance methods, as used in Lough and Gonor (1973a, b), were used to test the significance of the difference between the estimated polynomials for larval sui-vival and growth. RESULTS Multiple regression analyses and response surfaces for the percentage survival and growth of M. edulis lai-vae at 16 to 17 days are given in Table 1 and Figures 1 and 2. Larval survival after 16-17 days of rearing was most affected by the quadratic and linear effects of tem- perature (T - , T). The orientation of the sur- vival response contours clearly shows this effect. Maximum survival to 16-17 days (80% contour) was estimated to occur between temperatures of 76 R. G. LOUGH 3.5 to 19 C and salinities above 14.5 ppt. Growth of the larvae after 16-17 days was most affected by the linear effect of tem- perature (T) followed by the quadratic effect of salinity (S-). Ma.ximum growth (80% contour) was estimated to occur between temperatures and salinities of 14.5 to 20.5 C and 24 to 33 ppt. Analysis of co-variance showed a significant difference (1% level) between the polynomials estimated for survival and growth indicating that the narrower temperature and salinity range for maximum growth is significantly dif- ferent than the range for maximum survival. Analysis of the combined survival and growth at 16-17 days indicated that the linear and quadratic effects of temperature (T, T-) were the two most important factors explaining the data. Optimum temperature and salinity con- ditions (80% contour) for maximizing both lar- val survival and growth was estimated to occur at 11 to 14 C and 22.5 to 36.5 ppt. DISCUSSION Bayne (1965) found that successful develop- ment of Talyfoel larvae from fertilization to trochophore stage occurred only in the range of 30 to 40 ppt salinity. The early embryos and larvae of M. edulis require a narrow range of salinity, near oceanic, despite the fact that the adults and late larvae (16-17 days of age) are euryhaline. Survival of the late larvae is delimited by temperature as noted for several other species of bivalve larvae studied (Lough and Gonor, 1973a, b; Lough, 1974). Maximum growth of late larvae required a much narrower range of temperature-salinity conditions than maximum survival. A true temperature-salinity interaction effect was not found for this species. The analysis of grovrth for all other species of bivalve larvae showed a pronounced in- teraction effect of temperature and salinity. This would indicate that within the suitable range of salinity, growth of M. edulis larvae is dependent only upon temperature. Natural sea water conditions (18 ±1 C, 27 ± 1 ppt) are found on the upper temperature limit of the 80% survival contour and within the area for maximum growth. These aie the conditions which were used for rearing the early larvae. Conditions under which the early larvae develop may influence the center and range of the contours for maximum survival and growth of late larvae. ACKNOWLEDGEMENT This research was supported in part by the National Oceanic and Atmospheric Ad- ministration (maintained by the U S. Depart- ment of Commerce) Institutional Sea Grant 2- 35187. LITERATURE CITED Alderdice, D. F. 1972. Factor combinations. Responses of marine poikilotherms to environ- mental factors acting in concert. In Marine Ecology, Vol. 1, Part 3, Ed. 0. Kinne, London, Wiley-Interscience, pp. 1659-1722. Bayne, B. L. 1965. Growth and the delay of meta- morphosis of the larvae of Mytilus edulis (L.). Ophelia 2:1-47. Brenko, M. Hrs. and A. Calabrese. 1969. The com- bined effects of salinity and temperature on larvae of the mussel Mytilus edulis. Mar. Biol. 4:224-226. Lough, R. G. 1974. A re-evaluation of the com- bined effects of temperature and salinity on survival and growth, of bivalve larvae using re- sponse surface techniques. Fish. Bull, (in press). Lough, R. G. and J. J. Gonor. 1973a. A response- surface approach to the combined effects of temperature and salinity on the larval de- velopment of Adula califomiensis (Pelecypoda, Mytilidae). I. Survival and growth of three and fifteen-day old larvae. Mar. Biol. 22:241-250. 1973b. A response-surface approach to the combined effects of temperature and salin- ity on the larval development of Adula califor- iemis (Pelecypoda, Mytilidae). II. Long-term lai-val survival and growth in relation to respiration. Mar. Biol. 22:295-305. Proceedings of the National Shellfisheries Association Volume 64 - 197^ TRENDS IN PESTICIDE RESIDUES IN SHELLFISH Philip A. Butler ' U.S. ENVIROMENTAL PROTECTION AGENCY OFFICE OF PESTICIDES PROGRAMS GULF BREEZE, FLORIDA ABSTRACT The National Estnanne Monitoring Program, a cooperative effort between the State and Federal Governments, collected and analyzed shellfish samples for per- sistent synthetic pesticides at monthly intervals during the years 1965-1972 in 15 coastal states. The recently completed study of the 8000-plus analyses demonstrates that: (1) the residues found, primarily DDT and its metabolites, were universally too low to have human health significance, (2) areas of both high and. low residues were clearly defined geographically , (3) in some areas there has been a trend towards a wider distribution of smaller residues, and (It) there has been a marked decline generally in DDTresidues since 1968 when peak levels in molluscs were detected. INTRODUCTION During the period 1965-1972, samples of oysters and other bivalve molluscs were collected at month- ly intervals at about 180 estuarine locations to determine the incidence and magnitude of pesticide residues along the Atlantic, Pacific and Gulf of Mexico coasts. More than 8000 samples were screened for the presence of 12 of the more persistent chlorinated pesticides. In the later years, chlorinated pesticides. In the later yeai-s, chlorinated biphenyls or PCB's were in- cluded in the analytical procedures. This report briefly summarizes the implications of some of the principal findings. A detailed report of the sample collections and analyses has been published recently (Butler, 1973). BACKGROUND Oysters exposed to varying concentrations of pesticides under controlled conditions in the laboratory demonstrate their sensitivity to these pollutants. In aquaria with flowing unfiltered ' Contribution No. 176, Gulf Breeze Environmental Research LaboratoiT, U.S. Environmental Protection Agency. Gulf Breeze, FL .32561. seawater, for example, as little as l.O/ug/kg (ppb) of DDT inhibits oyster shell growth by about 20 percent in a 4-day period. One p g/g (ppm) inhibits shell deposition completely at water tem- peratures of about 17-20 C (62-68 F) (Butler, 1966). Concentrations as high as these were not an- ticipated in the natural environment and so it was of importance in the development of a proposed monitoring program to discover that oysters were sensitive to the presence of DDT in ambient water at levels as low as 10 x 10~'-(10 parts per trillion). Exposure of oysters for 7 days to this extremely low concentration led to the formation of DDT residues in the tissues of about 70 p g/kg, a biological magnification of 70,000x. DDT levels of this magnitude might be an- ticipated in the marine environment since it is less than the solubility of DDT in water. Further laboratory experiments demonstrated that oysters and other molluscs would be reliable as biological tools to monitor estuarine ecosystems because of this tendency to concentrate per- sistent chemicals (Table 1). Additional experimentation showed that con- taminated oysters cleansed themselves of resi- 77 78 P. A. BUTLER TABLE 1. Uptake of DDT by eastern oysters maintained in flowing seawater. Exposure period 7-15 days in different tests. (Butler 1968). Concentration in water Residue in oyster Biological magnification (ug/kg) or (ppb) {]ig/g) or (ppm 10.0 150.0 1.0 30.0 0.1 7.0 0.01 0.72 0.0001 0.07 control 0.06 (xlOOO) 15 30 70 70 0 dues when returned to clean water. The disap- pearance time or biological half-life of the resi- dues in molluscs was short; a matter of days as compared to months or years in fish and other vertebrates. Consequently, when oysters were sampled at about 30-day intervals, it was possible to estimate when pollution entered the estuary and tlius gain some insight as to its source. FINDINGS Analyses of monthly collections of oysters in an estuarine complex near Pensacola, Florida revealed a seasonal pattern of DDT residues later found to be typical of estuaries in many coastal areas. In the period February through May there was a gradual increase in residue magnitude to a seasonal high in late spring. This was followed by a decline to "background' levels typical of the remainder of the year. It seems reasonble to assume that this picture results primarily from the occurrence of sea- sonal rains and surface water run-off which carry soil eroded from agricultural lands through the river basin and into the estuary. In contrast to this picture, there was a second seasonal peak of DDT residues during the winter months in samples from the South Texas coast. This bimodal cycle probably reflected the double cropping of farm lands and the associated multiple applications of pesticides in this sub-tropical area. A more obvious result of the seasonal agricultural use of DDT was indicated by residues in oysters monitored in the Caloosahat- chee River Basin in southwest Florida. Here, peaks in DDT residues in oysters appeared soon after the seasonal application of DDT to maturing crops of sweetcorn and sugarcane. In 1967-68, the early spring residues were nearly ten times the level of residues found during the other months of the two-year monitor period (Fig. 1). In some instances, seasonal and annual patterns of pesticide accumulation in estuarine oysters could be associated with the dumping of industrial effluents or with the control of noxious insect populations. The declining use of DDT in stable-fly control in northeast Florida, for example, was clearly indicated by annual decreases in DDT residues in local oyesters in the period 1965-1968. DDT residues were no longer PESTICIDE RESIDUES IN SHELLFISH 79 J FMAMJ JASOND FIG. 1. DDT residues in the eastern oyster from the Caloosahatchee River Basin, Lee County, Fla., by month of collection (Butler, 1973). identified after the substitution of methoxy- chlor, a less persistent compound, for fly control in 1969. More importantly, methoxychlor was not detected in the monitor samples in suc- ceeding years. The significance of DDT residues in field samples may be judged to some extent by the magnitude of DDT residues observed in labora- tory experiments. Market-size eastern oysters were exposed to LO m g/kg of DDT in flowing seawater for a 10-day period and then 12 were individually analyzed. The sum of DDT and its metabolites found as residues ranged from a low of 3.9 to a high of 23.2 Mg/g with an arith- metic average of 10.1;ug/g (ppm) for the group. This value is about twice the largest DDT residue observed (5.39 ppm) in all of the moUuscan samples collected in the 7-year monitor- ing period. It should be noted further that DDT residues were less than l.OA/g/g in 99.5 percent of the 8000+ monitoring samples analyzed. It appears that despite the build-up of large resi- dues in higher carnivores DDT pollution of estuarine waters generally has been at levels below 1.0 /J g/kg (Fig. 2). It must be emphasized that the observed levels of DDT residues in molluscs were too low to have human health significance or to have demonstrable effects on the oysters themselves. Only in isolated area were DDT residues high enough to indicate that some elements of the estuarine fauna might have been damaged by DDT ppm INDIVIDUAL VARIATION EXPERIMENTAL AVERAGE MONITORED MAXIMUM MONITORED MAXIMUM IN 99 5 % n_ FIG. 2. DDT residues in experimental and field- collected oysters in the period 1965-1972. See text for explanation the magnification and accumulation of DDT residues in the food web. With these observations in mind, the overall findings of the monitoring data may be sum- marized geographically. The lowest average in- cidences of DDT positive samples were found, in order, in Washington, Georgia and Maine. Highest incidence rates were observed in New Jersey, Alabama, North Carolina and California. However, the largest residues of DDT and its metabolites were found in samples collected in the estuaries of Florida, California and Texas. There has been a well-defined but gradual decline in both the incidence and magnitude of DDT residues in oysters during the monitoring period in most areas. In some coastal estuaries this trend is obscured by the lack of uniformity in the timing of sample collections or by variations in the kind of mulluscs collected. Despite erratic fluctuations in magnitude and the fact that individual residues were never very high, it is clear that DDT pollution in estuaries was at peak levels in 1966-1967 and gradually declined thereafter. This 1966 peaking in the magnitude of residue data parallels, not unexpectedly, the findings of peak DDT levels in fresh water monitoring samples in 1966 followed by sharp declines in 1967 and 1968 (Lichtenberg, et al, 1970). Data demonstrating the overall decline in the magnitude of DDT residues in estuarine molluscs are summarized in Fig. 3. This dia- 80 P. A. BUTLER TRENDS IN PERCCNT OCCURRENCE OF DDT RESIDUES IN OYSTERS RESIDUE RANGE - ppm { 0 005-0 01 0 01 0 10 010-10 10-100 56 AVtRACI OF ALL STATIONS IN IS STAT IS 39 49 39 0 11 5 "' *n 64 51 CAllfORNIA STATIONS ONLY / \, 14 30 28 0 1 7 5 1965-70 ; 1971 IWS-TO ; 1971 1965-70 ; 1971 1965-70 ; 1971 FIG. 3. Percentage occurrence of DDT residues in estuarine bivalves in the period 1965-1970 as compared to 1971. Data summarize about 7000 analyses of more than 75,000 animals. See text for explanation. gram shows that, in the period 1965-1970, 39% of all samples contained negligible DDT resi- dues, less than 0.01 ppm. while in 1971 this value increased to 56%. Conversely, in these same years the percentage of samples contain- ing larger residues declined sharply. In Cali- fornia and a few other isolated locations there was an exception to this generalized picture in that the number of samples with DDT re- sidues in the 0.01-0.10 ppm range increased during the monitoring period but the per- centage of samples with high residues decreased sharply as in other coastal areas. Apparently in these drainage basins, there was an increased cycling of DDT in the trophic web accompanied by a diminution of the amount present in individual animals. In other words, DDT residues were distributed more thinly among more members of the biota. At ten monitoring stations in North Carolina, where the continuity of sample collections was especially good, the data provide a clear picture of annual trends in DDT pollution levels. Fig. 4 shows the decline in the percentage of sam- ples having measurable DDT residues as com- pared with the approximate percentage decline in the domestic use of DDT throughout the United States after 1965. DDT supplies in that year have been arbitarily designated as 100% for the basis of this comparison (USDA, 1967- PERCENTAGE DECLINE IN NORTH CAROLINA OYSTER SAMPLES PERCENTAGE DECLINE IN DDT USE IN U.S.A. (1965: 100*) FIG. 4. Percentage decline in DDT residues of more than 10 ju g/kg in North Carolina oysters as compared to the decline in the con- sumption of DDT in the entire United States in the period 196.5-1971. 72). These .data demonstrate the progressive loss of residual DDT from 'kt least one segment of an estuarine ecosystem following the general- ized curtailment in the agricultural use of DDT, and controvert the widespread belief that environmental problems with DDT would be longlasting regardless of how soon its use was terminated. SUMMARY These monitoring data show that the domes- tic use of DDT resulted in only nominal resi- dues in estuarine molluscs in the United States in the period 1965-1972. By extrapolation from laboratory data, we may infer that these re- sidues were too small to have a deleterious effect on the growth and productivity of estuarine bivalves. Despite the chemical stability of DDT, curtailment in its use was al- most immediately reflected by declines in the magnitude of residues in estuarine molluscs. The data establish a baseline for levels of DDT pollution in estuaries during the monitored period, and suggest that despite the stability biologically unavailable soon after its widespread use is discontinued. PESTICIDE RESIDUES IN SHELLFISH 81 LITERATURE CITED Butler, P. A. 1966. Pesticides in the marine en- vironment. J. Appl. Ecol. 3(Suppl.):253-259. Butler, P. A. 1968. Pesticide residues in marine molluscs. In Proc. Natl. Symp. Estuarine Pollut., Stanford Univ., Stanford, Calif. 1967. p. 107-121. Butler, P. A. 1973. Organochlorine residues in estuarine molluscs, 1965-1972 — National Pesticide Monitoring Program. Pestic. Monit. J. 6: 238-362. Lichtenberg, J. L., J. W. Eichelberger, R. C. Dressman and J. E. Longbottom, 1970. Pesticides in the surface waters of the United States — a 5-year summary, 1964-68. Pestic. Monit. J. 4:71-86. USDA, Agricultural Stabilization and Conserva- tion Service. The Pesticide Review. 1967-1972. Washington, D. C. Proceedings of the National Shellfisheries Association Volume 6i - 197i BACTERIAL PATHOGENICITY IN LABORATORY -INDUCED MORTALITY OF THE PACIFIC OYSTER (CRASSOSTREA GIGAS, THUNBERG) Roger S. Grischkowsky^ and John Liston COLLEGE OF FISHERIES UNIVERSITY OF WASHINGTON SEATTLE, WASHINGTON ABSTRACT Mortality of Pacific oysters (Crassostrea gigas) was monitored in four trials with loiv-tetnperature control (10 C), high-temperature control (20 C). high- temperature + UV (20 C). high-temperature + Vibrio spp. or Vibrio anguillarum (20 C), and high-temperature + oxytetracycline (TM-50) (20 C) treatment groups. Mortality was highest in the bacteria-inoculated treatments and lowest in low- temperature co7itrol troughs. Computer analysis, using contingency table analysis, substantiated observed mortality results by testing the independence of trial num- ber, treatment, time, and temperature. High temperature was a substantial con- tributing factor to mortalities. TM-50 successfidly decreased bacterial counts of water and oysters, as well as decreasing mortality. UV treatment decreased water counts but not mortality. Moribund or dead oysters previmisly held at elevated temperatures, compared uith normal low-temperature control, Puget Sound (Eld Inlet), and Humboldt Bay oysters, consistently had higher bacterial counts in all media tested. Bacteria associated with normal and moribund or dead oysters were isolated and identified as V. anguillarum, Vibrio alginolyticus. Vibrio parahaemolyticus. Vibrio spp., Pseudomonas spp., and Aeromonas spp. V. anguillarum and V. alginolyticus were implicated as facultative pathogens for Pacific oysters at elevated temperatures. INTRODUCTION Mass mortalities of Crassostrea gigas have oc- curred annually in recent years in Pacific waters, including Japan, Washington, and California. In studies at the University of Washington, ap- parently "similar" mortalities have been induced in the laboratory. In the laboratory systems, bac- teria, apparently vibrios, were involved. A com- prehensive review of this subject was presented by Grischkowsky (1973). Present address: Alaska Department of Fish and Game 333 Raspberry Road Anchorage, Alaska 99502 The etiology of these mortalities has not been determined despite the many theories proposed. Lipovsky and Chew (1972) produced mortalities of Pacific oysters under laboratory conditions at 14 and 21 C. They implicated unknown bacteria as the causative agents under enriched en- vironmental conditions. Breese (1971), working with C gigas spat at 12 and 2-5 C, found significantly higher mortalities at the elevated temperature. Colwell and Liston (1962) reported the normal microflora of C. gigas to be mainly Pseudomonas, Mavobacterium, Micrococcus spp. and gut group vibrios. Baross and Liston (1968, 1970) and Liston and Baross (1973) found V. alginolyticus, V. anguillarum, and V. parahaemolyticus in healthy 82 BACTERIAL PATHOGENS IN OYSTER MORTALITIES 83 Puget Sound oysters directly correlated with tem- perature in environmental ranges. V. ■parahaemolyticus and related halophilic vibrios were found by Thomson and Trenholm (1971) in unidentified species of clams, oysters (probably Crassostrea virginica), periwinkles, mussels (probably Mytilus edulis), and snails. Various species of bacteria, including vibrios, have been implicated as pathogens for pelecypod mollusks. Tubiash (1971) found the soft-shell clam (Mya arenaria) susceptible to V. alginolyticus, V. anguillarum, and Vibrio spp. when inoculated ac- tively into heart, incurrent or excurrent siphon tissue at 20 and 22 C. The same two species of Vibrio were isolated from dead and moribund larvae and juveniles of the hard clam (Mer- cenaria mercenaria) and oyster (C. virginica). They were identified as the etiologic agents of a disease called bacillary necrosis (Tubiash et. ai. 1970). These authors speculated that during mid- summer, conditions in Chesapeake Bay and Long Island Sound that favor both molluscan spawning and bacterial proliferation may be responsible for natural epizootics of bacillaiy necrosis in commercially valuable bivalve mollusks. Colwell and Sparks (1967) found the natural microflora of C. gigas to be composed of organisms representing the genera Psei/domonas. Achromobacter. Flavobacterium. Vibrio, and Micrococcus. The flora of dead or dying oysters included a greater incidence of Pseudomonas (particularly Pseudomonas encdia). P. enalia was thought to be pathogenic to C. gigas held in laboratory tanks when the body tissues were in- jected with viable suspensions. Histological examination suggested a bacterial invasion. The laboratory mortality was variable and sample sizes were small. Vibrios, including V. parahaemolyticus, have been shown by Colwell et ai. (1973) and Kaneko and Colwell (1973) to increase rapidly during summer temperatures of 14 to 19 C in Chesapeake Bay water and zooplankton. The authors determined that V. parahaemolyticus overwinters in sediment and shellfish or scavenger fish such as gobies, which occur on the bottom. Since little was known about bacterial diseases of oysters, a laboratory model system was developed for a study of oyster-bacterial in- teraction. An investigation of mortalities in- duced in the model was conducted and an at- tempt made to relate findings to the natural conditions of oysters in the field. METHODS AND MATERIALS Oysters were held in containers supplied with water at high (20 C) or low (10 C) temijerature, which was recycled through a filter and aeration system to maintain quality. Mortalities of the oysters in tanks held under various con- ditions with and without bacterial challenge were monitored, together with bacteriological factors. Seawater Seawater used in the experiments was collect- ed from Seabeck Bay, Washington and held in 55-gallon poly-drums with plastic inner barrels. Oysters Specimens of C. gigas (2+ years) collected from commercial beds in Eld Inlet (Mud Bay), Puget Sound near Olympia, Washington were used in all experiments. Eld Inlet is historically a high mortality area (Scholz et ai, 1971). Water temperature and salinity were monitored monthly at the collection site during 1972. Oysters were held in tanks with recycled, filtered seawater cooled to 12 C prior to use. Little or no natural mortality was experienced under these conditions for as long as six weeks. Before use, oysters were scrubbed under tap water and adherent large barnacles and mussels removed. Bacteria Strains of bacteria used in the mortality ex- periments were isolated by culture of dead or moribund oysters. The organisms were grown in a seawater (50%), peptone (2%), yeast extract (1%) and glucose (0.5%) broth (SWPYG) and the bacteria and medium were added directly to the water in the tanks. No indications of sterile broth toxicity to oysters was noted and the toxicity of bacterial metabolites introduced with the broth was not tested. Identification of the strains was made following the general characteristics described by Sakazaki et al. (1963); Sakazaki (1969 and 1971) and confirmed in the case of V. anguillarum, V. alginolyticus and V. parahaemolyticus by DNA homology analysis (performed by Dr. E. J. Ordal, 84 R. S. GRISCHKOWSKY AND J. LISTON Microbiology Department, University of Washington), using procedures described by Kiehn and Pacha (1969) and Anderson and Ordal (1972). Oyster Food Stock oysters were fed Monochnjsis lutheri at the rate of 20 ml of a culture containing 10'^- 10' cells/ml per oyster per day. The Monochnjsis was supplied by Dr. F. B. Taub from her continuous culture unit (Taub, 1971). Temperature Control and Aeration In general, seawater was added to the con- tainers (and to the reservoir system in the case of chilled water) and allowed to stabilize to the desired temperature. Scrubbed oysters were then added and permitted to acclimate in the tank for three days. Bacteria were added or other treatments initiated at this time. Cold water at 10 C was supplied to low- temperature group containers through a recycling unit. The reservoir consisted of a 30 gallon polyethylene cylinder fitted with refrigeration coils which were linked to a com- pressor (one '4 HP for trial 1 and two '4 HP for trials 2-4). Containers varied in different experiments, as indicated 'below. However, where tanks were used they were fitted with glass-wool/charcoal filters, aerators, and air pumps, following the general concept of Spotte (1970). This system maintained aerobic conditions and prevented ac- cumulation of toxic metabolites in the tanks. High-temperature groups were maintained at approximately 20 C. StatisticM Analysis Data from the four major experiments were analyzed using Biomedical Computer Program contingency table analysis, BMD02S (Dixon, 1968) on a Control Data 6400 computer. The variables tested at the 0.95 1-a significance level for independence were: trial number, treatment group, mortality, temperature, and time. These tests were first grouped to include all trials, then separately by trial number and by specific comparisons of treatment groups. The null hypothesis, Hq, for all of these tests was that there was independence between the selected variables. Preliminary Experiment A preliminary experiment, trial 1, was con- ducted with low-temperature control (LTC), high-temperature control (HTC), and high- temperature + Vibrio sp. (HT + Vibri(} sp.) Three, 20 gal. glass aquaria containing 29, 22, and 22 oysters were used. The HT + Vibrio sp. tank received lOVml unidentified vibrio isolated from pericardial fluid from a moribund oyster, in SWPYG Broth. Large Scale Expenments Three additional trials (2-4) utilized four salmonid rearing troughs, each containing 37 gal. of seawater from Seabeck Bay. Each trough was recirculated by a Teel chemical magnetic drive pump. Treatment groups included the previous three (LTC. HTC, and HT + V. anyuillarnm). plus a high -temperature trough (HT + UV) treated with a lOGPM AquaNomics ultraviolet sterilization unit and a high-temperature group in a 50 gal. fiberglass tank to which TM-50 (50 g oxytetracycline/lb of inert powder) was added at regular intervals (HT + TM-50). Ti-oughs held 100 oysters each from the same source as before, while the fiberglass tank contained 50 oysters. All con- tainers were aerated, filtered through sterilized glass wool/charcoal filters, and received 21./day of 10 cells/ml M. lutheri. To determine bacterial counts of normal and moribund experimental oysters and to recover introduced organisms, samples of oysters were washed and shucked by procedures recom- mended by the American Public Health Association (1970), and an equal weight by volume (g/ml) mixture of sample and dilution fluid (1.5% NaCl, 0.5% peptone in distilled water) was homogenized in a Waring blender for 60 sec. The mixture was serially diluted and plated on various media, including 5% blood agar (BA) at 25 C, bromothymol-blue Teepol agar (BTB) at 25 and 37 C, salt-starch agar (VPS) in an anaerobic jar (BBL gas pack) at 43 C and seawater starch agar (SWS) at 25 C. Suspensions of V anguillarum (culture num- ber 728), isolated from moribund oyster pericar- dial fluid, was added to troughs for a final con- centration of 10 '-10' bacteria/ml. The TM-50 treatment groups received approximately 20 mg BACTERIAL PATHOGENS IN OYSTER MORTALITIES 85 of the compound/day. To test the effectiveness of oxytetracycline, Terramycin sensitivity discs (10 meg), millipore filtered water from the HT + TM-50, and TM-50 powder were placed on blood agar plates previously streaked with V. anguiUanan or V. alginolyticus and incubated at 25 C. At the end of one of the trials, sur- viving low-temperature control oysters were transferred into high-temperature control waters, and in another LTC oysters were inoculated with V. anguiUariim at low tem- peratures. During all trials, close comparisons were made between microflora of treatment group oystere and regular monthly sample or LTC oysters from Puget Sound (Eld Inlet), Humboldt Bay, and the appropriate trials. RESULTS Field Data Temperature and salinity of the sampling area water for 1972 are depicted in Figure 1. The wide littoral temperature range from 1 to 27.5 C must resultantly place a large stress on resident oyster populations and may affect bac- terial invasiveness. Field studies included total heterotrophic bacterial counts at 25 C, mesophilic vibrio counts for water sediment, and monthly samples of oysters (Figure 2). Sediment samples had the highest counts, followed by oysters, and water. Total counts and mesophilic vibrio counts of oysters respond- ed directly to seasonal temperature increases (Fig. 1 and 2). Preliminary Investigations During preliminary investigations, various tissues and fluids of healthy oysters from month- ly samples and LTC treatments and heat- treated moribund oysters were examined under phase-contrast. Oyster fluids exhibited only amoebocytes, fat globules, and gonadal material in normal oysters. Moribund oyster heart and pericardial fluids consistently contained many actively motile curved rods with rounded ends easily visible under phase microscopy. Bacteria were not enumerated or isolated during the preliminary investigation. Mantle and gill tissue and shell liquor usually contained a wide range of mixed organisms. M J J A S Ti me ( months ) FIG. 1. Temperature ( C) and salinity ( °L ) for Eld Inlet, Puget Sound (Mud Bay) dming 1972. Mortality Results Trial 1, utilizing 20 gal. tanks and taking place in January, showed no mortality in the LTC, 46% for HTC, and 86% for HT + Vibrio sp. after 37 days (see Figure 3). The com- bination of an elevated temperature and vibrio inoculum significantly increased mortality. Dramatic mortality increases (of 46 and 86% respectively in HTC and HT + Vibrio sp. tanks) at the high temperature level occurred here and in successive trials. Total bacterial counts Mesophilic vibno counts JFMAMJJ ASOND T ime { months) FIG. 2. Total bacterial counts and mesophilic vibrio counts per gram of oyster meats from Eld Met, Puget Sound (Mud Bay) during 1972. 86 Trial 2, utilizing salmonid rearing troughs and taking place during May, represented the beginning of large scale experiments using 100 oyster sample sizes. A mortality of 2% occurred in the LTC, 100% in HT + UV and HT + V. anguillanim after 15 days, and 100% in HTC after 12 days (see Figure 4). The bacterial inoculation was apparently not virulent on this occasion. The mortality rate was greatly ac- celerated in this and subsequent trials during warmer months over that which took place during the winter. This phenomenon might be attributed to increased total counts, mesophilic vibrio counts, seawater temperatures, or am- bient high temperature levels. Surviving LTC oysters, when transferred to the HTC tank, exhibited a 94% mortality after 10 days as compared with 2% in the LTC tank. Mortality for trial 3 was 1% for LTC, 43% for HTC, 61% for HT + UV after 15 days, 100% for HT + V. anguillarum after 12 days, and 4% for HT + TM-50 (oxytetracycline) after 15 days (Figure 4). High temperatures and V. anguillarum increased oyster mortality, while the addition of oxytetracycline but not UV light decreased mortality significantly. Trial 4 caused the most rapid mortality. The usual 2% occurred in the LTC after 13 days, while the HTC mortality was 98% after 9 days. 100 -I 13 I- fe s ■fa ^; Low temperature control High temperoture control High temperature + Vi brio sp 17 21 25 Times ( doys) 29 33 37 FIG. 3. Rate of mortality, preliminary ex- periment (trial 1), for low temperature control, high temperature control, and high temperature + Vibrio sp. treatment groups. R. S. GRISCHKOWSKY AND J. LISTON lOO-i ^ § ■Cl 100 ^ 75 ^$ 50H 0-^3 Low temperature control • • Higti fernperoture control High temperoture + UV *— * Higti temperoture -t- Vtbrio onguillorum o— o Higti temperoture + TM-50 FIG. 4. Rate of mortality, large scale ex- periments (trials 2-Jt), for low temperature con- trol, high temperature control, high temperature + UV, high temperature + Vibrio anguillarum, and high temperature + TM-50 treatment grvups. HT + UV 97% after 9 days, HT + V. anguillarum 99% after 5 days, and HT + TM- 50 72% after 13 days (Figure 4). Of the high temperature treatment groups, the inoculated one showed the highest mortality, while the TM-50 treatment displayed the lowest. UV ap- parently was ineffective in reducing mortality. Surviving LTC oysters were inoculated with V. anguillanim and maintained at the low tem- perature for 15 days with 7.5% mortality. Bacteriological Analysis In trial 2 the number of bacteria as measured by MPN in SWPYG increased in all BACTERIAL PATHOGENS IN OYSTER MORTALITIES 87 troughs over the baseline, with a range of 1-log increase in the LTC and HT + UV to a 5- log increase in the HTC. Bacterial isolates were all gram-negative, motile, oxidase (Kovacs) positive, slightly curved rods, with rounded ends. Normal low-temperature oysters contained V. alginolyticus, Pseudomonas spp., and Aeromorms spp. The high-temperature moribund oysters contained large numbers of V. alginolyticus, some Vibrio spp., and few Pseudomonas and Aeromonas spp. Recovery of V. anguillarum from the inoculated tank oysters was frequent; of the 16 V. anguillarum isolates, 10 were recovered from the UT + V. anguillarum trough. Homogenate from moribund or dead oysters, when streaked on BA, yielded relatively pure cultures of V. anguillartim compared with mixed cultures usually obtained from low-temperature oysters. For trial 3, several MPN counts were made. The baseline seawater counts were 2 logs higher for trial 3 than trial 2. The LTC trough yielded surprisingly high numbers, lOVml, or 1-log higher than the inoculated trough and 2- logs higher than the HTC trough after 8 days. After 12 days, the highest counts were 10", 10*^, and lOVml respectively in the UV, V. anguillarym. and the TM-50 (oxytetracycline) treatment groups. The bacterial counts of oysters, using homogenate of grouped oyster samples for moribund or dead oysters, yielded in VPS, lO'-lO^ SWS, lO'-W; BTB, 10'- 10 Vg. For healthy oysters results were VPS, 0; SWS, 10^; and BTB, lO'-lOVg. Recovery of V. anguillarum from moribund or dead oysters was excellent in the inoculated trough. Of the 15 V. anguilla7-um isolates, 9 were from the HT + V. anguillarum trough. Most V. alginolyticus isolates were from the HTC and HT + UV troughs. Normal oysters contained low levels of all listed bacteria. MPN counts in SWPYG for trial 4 of treat- ment waters, indicated a 2-4 log increase after 5 days, from a basal level of lOVml. Bacterial counts of normal oysters held for 13 days in the LTC trough show the same ap- proximate levels as those sampled on the collec- tion day. Although water counts increased 2-4 times, oyster counts in the LTC trough remained constant. UV treatment usually reduced water counts but failed significantly to decrease mortality. TM-50 (oxytetracycline) reduced water counts, oyster counts, and mor- tality. Counts of moribund or dead HT + V. anguillarum^ oysters increased by 4-logs in VPS 43 C plates, probably denoting increased num- bers of vibrios. High-temperature oyster counts increased 1-4 logs over normal oyster counts. Recovery of V. anguillarum^ from moribund HT + V. ayiguillarum oysters was poor. Of the 13 V. anguillarum isolates, only one was re- covered from the HT + V. anguillarum tank. V. parahaemolyticus was isolated from a moribund oyster once in this tank. However, V. anguillarum was isolated from every oyster which died in the LT + V. anguillarum trough. In this case, 11 of the 13 V. ajiguillarum isolates were from the LT + V. anguillarum trough. Recovery of V. anguillarum, from mixed oyster microflora, including V. alginolyticus, is made more difficult by spreading characteristics of the latter vibrio. V. alginolyticus and Pseudomorwts spp. were well -represented in most trial 4 groups. Bacterial Identity The identification of 8 vibrios was confirmed by Dr. E. J. Ordal, using % homology and change of Tm. The organisms used for inoculation, culture no. 728, and bacteria recovered from the inoculated troughs of trials 2 and 3 (culture nos. 1445, 1491, 1800, and 1808), were 99.2, 100, 100 and 100% homologous (change of Tm = O-0.2 C) with V. anguillarum. V-2911. Cultures 1432, 1439, and 1823, isolated from HTC troughs of trials 2 and 3, were determined to be 94.5, 100 and 96.2% homologous (change of Tm = 0-1.3 C), with V. alginolyticus. ATCC 17749 and 65.9-68.0% homologous (change of Tm = 7.5-8.0 C) with V. parahaemolyticus ATCC 17802. Culture 4167 isolated from trial 4, HT + V. anguillarum was 100% homologous with ATCC 17802 (change of Tm = 0 C). G + C content of the DNA was 44.9-45.6 mole % for all of the tested isolates. All vibrios tested were found sensitive to oxytetracycline sensitivity discs, TM-50 powder and millipore-filtered water from HT + TM-50 treatment groups. Computer Analysis Computer analysis substantiated qualitative R. S. GRISCHKOWSKY AND J. LISTON differences already apparent. Results of con- tingency table analysis on mortality data are indicated by the Chi-Square/degrees of freedom figures provided in parentheses. In the test grouping all trials, mortality was significantly related to the trial number (186.53/33), treat- ment (396.02/44), time (greater mortality with increased time (213.13/99), and temperature (142.01/121). Considering trials 1, 2, and 3 each alone, mortality was associated with treatment, time, and temperature. For trial 4 alone, with all treatment groups combined, mortality was allied with treatment and time. Comparing LTC and HTC, the high- temperature group had higher mortalities in all trials grouped (135.23/11) and individually for trials 1-4. In the test comparing mortality and trial number for LTC and HTC (130.66/33), all treatments and trials support the observation that mortality was more rapid in the summer than in the winter. Specifically comparing the treatments of HTC and HT + UV, the mor- tality of the two groups for all trials together (4.22/11) and individually is not significantly different. This result confirms observations that UV generally did not decrease mortality. Com- paring the treatments HTC and HT + V. anguillarum. mortality was higher in the inoculated groups with all trials grouped (40.60/11) and with trials 1, 3, and 4 in- dividually. Regarding the treatments HTC and HT + TM-50, the addition of the antibiotic significant- ly decreased mortality in the two trials, 3 (6.92/1) and 4 (7.87/2), in which it was employed, ployed. DISCUSSION These data implicate V. anguillarum and V. alginolyticus as principal causes of heat-induced laboratory mortality, because they are most frequently isolated from heart blood and pericardial fluid of diseased oysters. Other organisms, e.g. Aeromonas. may also be in- volved. Mortality monitoring and computer analysis indicated an increased mortality rate among vibrio-exposed oysters. V. anguillanan and V. alginolyticus were both associated with moribund or dead oysters. We think they are facultative pathogens, since V. alginolyticus and V. anguillarum also occur in healthy oy- ters. Oysters held at high temp erature consistently died at a higher rate than oysters at low temperature. This finding sub- stantiates the reports of Lipovsky and Chew (1971, 1972, and 1973). These authors found 100% moilality in Pacific oysters at 20 C, with low mortalities at 10 C. and described V. parahaemolyticus as a "suspected marine moUuscan pathogenic bacteria." V. parahaemolyticus identification at that time was made by Dr. John Baross, on the basis of biochemical tests then available. New techniques for bacterial identification, including DNA homology, have indicated that some earlier identifications of V. parahaemolyticus may be in doubt, (Lipovsky and Chew, 1971; Anderson and Ordal, 1972; Vanderzant, 1973). Lipovsky and Chew later (1972) suggested these bacteria might more appropriately be called mesophilic vibrios. V. parahaemolyticus has been implicated as a pathogen of brown shrimp. Penaeus aztecus. in pond cultivation in the Gulf of Mexico (Van- derzant and Nickelson, 1970; Vandei-zant et al. 1971; and Vanderzant, 1973) and of blue crabs, Callinectes sapidiis. in Chesapeake Bay (Krantz et al. 1969). We isolated V. parahaemolyticus by direct plating at 25 C from one moribund oyster of the HT + V. anguillarum trough from trial 4, with confirmation by DNA homology. We used only direct plating techniques in this investigation. Perhaps the use of enrichment techniques (Fishbein and Wentz, 1973) might have significantly increased the recovery of V. parahaemolyticus. Lipovsky and Chew (1972 and 1973) found 18 C to be the apparently critical temperature for significant laboratory oyster mortality. Tem- perature, nutrient level, stress, and bacterial in- festation were indicated by the authors as decisive factors in laboratoiy oyster mortality. Exposure to elevated temperature for extended times increased bacterial numbers (total counts and mesophilic vibrio counts). This process may effectively stress oysters, enhance the virulence of vibrios in the laboratory and perhaps in the field under crowded summer conditions. Katkansky and Warner (1969, 1971) in Hum- boldt and Tamales Bays reported Crassostrea BACTERIAL PATHOGENS IN OYSTER MORTALITIES 89 gigas mortality was highest during June through August in 1968 and 1969. Scholz et al. (1971) substantiated the seasonal occurrence of Pacific oyster mortality in Case, Eld, and Tot- ten Inlets (Puget Sound) and indicated August through October, 1967, was the highest mor- tality period. They also estimated September mortality of commercial oyster stocks as the highest for the same year in Eld Inlet. Wedemeyer (1970) determined that stress caused by any number of environmental con- ditions can allow potential (facultative) fish pathogens to increase infectious processes. Perhaps a similar situation exists with oysters in the laboratory and in the environment when increased temperature may permit invasion of host tissue by vibrios. Detailed pathology of the oyster mortalities in the experimental trials is not known. It was obvious from examination of recently dead and moribund oysters that nearly all showed high enough counts of living bacteria in the heart blood and pericardial fluid to be observed easily in phase-contrast slide preparations. Healthy oysters frequently contained very low levels of bacteria in the heart blood, but never enough to be seen by direct microscopy. It might be postulated that a near-terminal event in the disease process is invasion of the blood by bacteria. Certainly the observation of large num- bers of bacteria (hundreds to thousands per field) in microscopic preparations of heart blood can be considered indicative of disease situation. This method provides a relatively rapid test to assay the disease potential in an oyster population. Addressing the treatment of bacteria-related oyster mortalities, this study has determined that oxytetracycline (Terramycin) in the form of TM-50 is effective in reducing heat-induced oyster mortality. Ultra-violet treatment of the water did not reduce heat-induced oyster mor- tality, although bacteria counts were decreased. Vasconcelos and Lee (1972), in determining the ability of Pacific oysters to purge themselves of microbial contaminants, found only coliform counts reduced by ultraviolet irradiation of se&water, whileVibrio/PseiuiomoTuts bacteria were not reduced in oysters by UV treatment. Sin- dermann and Rosenfield (1967) proposed the con- trol of mass invertebrate mortalities by main- taining production in artificial environments where disease could be controlled by UV treat- ment of filtered seawater, antibiotic treatment of water, maintainence of general sanitation, control of contaminants in phytoplankton cultures, and elimination of shellfish associates that act as in- termediate hosts of disease agents. Fryer et al (1971) considered manipulation of the en- vironment in ways which would favor the host and work to the disadvantage of the pathogen. TM-50 has been found effective for control of vibrio diseases by Umbreit and Ordal (1972) in goldfish, Carassius auratus and in salmonids by Anderson and Conroy (1970). It could be used in combination with other methods adequately to treat heat-induced bacterial-associated oyster mortality. The results presented in this paper should be expanded by related studies to identify agents causing mortalities under field conditions. ACKNOWLEDGEMENTS The authors express their gratitude to Mr. Earl Brenner for his donation of the oysters during 1972 and to Dr. Kenneth K. Chew for his continual guidance. Appreciation is ex- pressed to Dr. Erling J. Ordal for his con- firmation of vibrio isolates, using DNA homology' and change of Tm. Sincere thanks are extended to Dr. Frieda B. Taub and her associates for supplying Monochrysis lutheri throughout this investigation. We are indebted to Patricia Read for invaluable technical assistance in the bacteriological evaluation of field samples. Contribution No. 375, College of Fisheries, University of Washington. This study was supported in part by NSF Grant GH-40, under the Sea Grant Program. The Sea Grant Program is now in NOAA, U. S. Department of Commerce. The work reported here was part of a dis- sertation submitted by the senior author to the Graduate School, University of Washing- ton, in partial fuUfillment of the requirements for the degree of Doctor of Philosophy. 90 R. S. GRISCHKOWSKY AND J. LISTON LITERATURE CITED American Public Health Association. 1970. Recommended procedures for the examina- tion of seawater and shellfish, 4th ed. American Public Health Association, Washing- ton, D. C. 105 p. Anderson, J. I. W., and D. A. Conroy. 1970. Vibrio disease in marine fishes. In a Sym- posium of Diseases of Fishes and Shellfishes. S. F. Snieszko, Ed.. American Fisheries Society Special Publication No. 5, Washington D. C. p. 266-272. Anderson, Robert S., and Erling J. Ordal. 1972. Deoxyribonucleic acid relationships among marine vibrios. J. Bacteriol. 109: 696-706. Baross, J., and J. Liston. 1968. Isolation of Vib7-io parahaemolytieus from the Northwest Pacific. Nature 217: 1263-1264. Baross, J., and J. Liston. 1970. Occurrence of Vibrio parahaemolytieus and related hemo- lytic vibrios in marine environments of Washington State. Appl. Microbiol. 20: 179-186. Breese, W. P. 1971. Hot water and oysters. Proc. Nat. Shellfish. Assoc. 61: 7. Colwell, R. R., and J. Liston. 1962. The natural bacterial flora of certain marine invertebrates. J. Insect Pathol. 4: 23-33. Colwell, R. R., and A. K. Sparks. 1967. Properties of Pseudomoyias enalia, a marine bacterium pathogenic for the invertebrate Crassostrea gigas (Thunberg). Appl. Microbiol. 15: 980- 986. Colwell, R. R., T. E. Lovelace, J. Wang, T. Kaneko, T. Staley, P. K. Chen, and H. Tubiash. 1973. Vibrio parahaemolytieus - isolation, identification, classification, and ecology. J. Milk Food Technol. 36: 202-213. Dixon, W. J. Ed. 1968 BMD Biomedical Computer Programs. University of California Press, Berkeley. 600 p. Fishbein, Morris and Barry Wentz. 1973. Vibrio parahaemolytieus methodology for isolation from seafoods and epidemic speci- mens. J. Milk Food Technol. 36: 118-123. Fryer, J. L., J. S. Nelson, and R. L. Garrison. 1972. Vibriosis in fish. Progress in Fishery and Food Science. University of Washington - Publications in Fisheries - New Series 5: 129-133. Grischkowsky, Roger Saft. 1973. Studies of the nature of Pacific oyster (Crassostrea gigas, Thunberg) mortality. I. Implications of bac- terial pathogenicity and II. Pathogenicity testing of vibrios on chinook salmon (On- corhynehus tshaivytscha. Walbaum) and Pacific oysters. Ph. D. Dissertation, Univer- sity of Washington. 152 p. Kaneko, Tatsuo, and Rita R. Colwell. 1973. Ecology of Vibrio parahaemoh/tieus in Chesa- peake Bay. Bacteriol. 113: 24-32. Katkansky, S. C, and R. W. Warner. 1969. Oyster disease and mortality study yearly report for the period January 1, 1969 to December 31, 1969. California Dept. Fish and Game to U. S. Bur. Comm. Fish. (Mimeograph report). Katkansky, S. C, and R. W. Warner. 1971. Oyster disease and mortality study yearly report for the period January 1, 1970 to December 31, 1970. California Dept. of Fish and Game to LI. S. Dept. Commerce, National Marine Fish- eries Service (Mimeograph report). Kiehn, E. D., and R. E. Pacha. 1969. Characteri- zation and relatedness of marine vibrios patho- genic to fish: deoxyribonucleic acid homology and base composition. J. Bacteriol. 100: 1248- 1255. Krantz, G., R. R. Colwell, and T. E. Lovelace. 1969. Vibrio parahaemolytieus from the blue crab Callineetes sapidus in Chesapeake Bay. Science 164: 1286-1287. Lipovsky, Vance P., and Kenneth K. Chew. 1971. A preliminary report on Pacific oyster (Crass- ostrea gigas) mortality after transfer from a natural bed into 10 C and 20 C water in the laboratory. Proc. Nat. Shellfish. Assoc. 61: 9. Lipovsky, V. P., and K. K. Chew. 1972. Morta- lity of Pacific oysters (Crassostrea gigas): the influence of temperature and enriched seawater on oyster survival. Proc. Nat. Shell- fish. Assoc. 62: 72-82. Lipovsky, Vance P., and Kenneth K. Chew. 1973. Laboratory control of Pacific oyster mortality by manipulation of temperature and nutrient concentration. Proc. Nat. Shell- fish. Assoc. 63: 3. Liston, J., and J. Baross. 1973. Distribution of Vibrio parahaemolytieus in the natural envi- ronment. J. Milk & Food Technol. 36: 113-117. BACTERIAL PATHOGENS IN OYSTER MORTALITIES 91 Sakazaki, R. 1969. Halophilic vibrio infections. hi Food Borne Infections and Intoxications. Ed. H. Reiman, Academic Press, New York. P. 115-129. Sakazaki, Reichi. 1971. Present status of studies of Vibrio parahaemoiyticus in Japan. Proc. Symposium, U.S.F.D.A., Washington, D. C. Sakazaki, Reichi, Setsuo Iwanami, and Hideo Fukumi. 1963. Studies on the enteropatho- genic, facultatively halophilic bacteria. Vibrio parahaemoiyticus I. Morphological, cultural, and biochemical properties and its taxonomi- cal position. Jap. J. Med. Sci. Biol. 16: 161-188. Scholz, Albert J., Ronald E. Westly, and Marvin A. Tarr. 1971. Pacific oyster mass mortality studies. State of Wash. Dept. Fish. Seasonal Summary Report No. 3. May 1967 to April 1968. (Mimeograph report). Sindermann, Carl J., and Aaron Rosenfield. 1967. Principal diseases of commercially important marine bivalve mollusca and Crustacea. Fishery Bull. 66: 335-;385. Spotte, Stephen H. 1970. Fish and Invertebrate Culture, Water Management in Closed Systems. Wiley-Interscience, a Division of John Wiley & Sons, Inc., 'New York. 145 p. Taub, Frieda B. 1971. Algal culture as a source of feed. In Proceedings of the First Annual Workshop World Mariculture Society. Ed. James W. Avault, Jr. Louisiana State Uni- versity Division of Continuing Education, Baton Rouge, Louisiana. 179 p. Thomson, W. K., and D. A. Trenholm. 1971. The isolation of vibrio parahaemoiyticus and related halophilic bacteria from Cana- dian Atlantic shellfish. Canadian J. Micro- biol. 17: 545-.549. Tubiash, H. S. 1971. Soft-snell clam, Mya arenaria, a convenient laboratory animal for screening pathogens of bivalve moUusks. Appl. Microbiol. 22: 321-324. Tubiash, H. S., R. R. C«lwell, and R. Sakazaki. 1970. Marine vibrios associated with bacillary necrosis, a disease of larval and juvenile bivalve mollusks. J. Bacteriol. 103: 272-273. Umbreit, W. W., and E. J. Ordal. 1972. Infec- tion of goldfish with Vibrio anguillarum . Amer. Soc. Microbiol. News 38: 93-96. Vanderzant, C. 1973. Vibrio parahaem.olyticus: a problem in mariculture? J. Milk Food Technol. 36: 135-139. Vanderzant, C, and R. Nickelson. 1970. Isola- tion of Vibyio pnrahaemolyticuH from Gulf Coast shrimp. J. Milk Food Technol. 33: 161-162. Vanderzant, C, R. Nickelson, and P. W. Jud- kins. 1971. Microbial flora of pond-reared brown shrimp (Penaeus aztecus). Appl. Microbiol. 21: 916-921. Vasconcelos, G. J., and J. S. Lee. 1972. Microbial flora of Pacific oysters (Crassostrea gigas) subjected to ultraviolet irradiated seawater. Appl. Microbiol. 23: 11-16. Wedemeyer, Gary. 1970. The role of stress in disease resistance of fishes. In A Sym- posium of Diseases of Fishes and Shellfishes. S. F. Snieszko, Ed. American Fisheries Society Special Publication No. 5, Wash., D. C. p. 30-35. Proceedings of the National Shellfishenes Association Volume 6i - 197J^ TISSUE GRAFTS IN THE AMERICAN OYSTER, CRASSOSTREA VIRGINICA Walter J. Canzonier^ OYSTER RESEARCH LABORATORY NEW JERSEY AGRICULTURAL EXPERIMENT STATION RUTGERS - THE STATE UNIVERSITY NEW BRUNSWICK, NEW JERSEY ABSTRACT Tiss-ue implantation was one of several techniques used in a study of the trans- mission of the oyster disease agent Minchinia nelsoni. The fate of several tissue types, plus response to inert materials, was followed for up to 35 days by periodic sampling and histological examination. Both normal and infected gill and mantle tissue were capable of fusing unth the recipient and persisting as recognizable en- tities for at least the period of observation. The response of the recipient appeared to be influenced by the nature of the implanted material. Viable gill and mantle tissue and paraffin shims elicited a minimal host response. There was some leucocytic infiltration of the immediate area and the appearance of fusiform cells along exposed surfaces followed by re-establishment of epithelia. In the case of moribund tissues or implants of digestive gland, there was a more intense response in the form- of leucocytic infiltration followed by a rapid dissociation of the im- plant. In this case the fusiform cells were also found oriented around the implant. There was no evidence for transfer of M. nelsoni to the recipient oysters. INTRODUCTION With the exception of the rather extensive studies on pearl formation by implanted mantle tissue in pearl oysters, few studies on the fate of tissue transplants in bivalve molluscs have been recorded. Pearl formation is reviewed by Alver- des (1913) and Tsujii (1960). Drew and deMorgan (1910), Butcher (1930), Cashing (1957), Tripp (1961) and Chemin (1966) have described various aspects of host response to a variety of implanted tissues in gastropods and bivalves. Canzonier (1963) reported the results of initial attempts to implant tissues in Crassostrea virginica. However, the only detailed histological description of the fate of implants in bivalves is that of DesVoigne ' Present address: Institute di Biologia del Mare-CNR, Riva Sette Martiri. .30122 Venezia, Italia. and Sparks (1969) concerning the reaction of Crassostrea gigas to homologous mantle tissue. Recently, Cheng and Galloway (1970) have report- ed in detail the response of the freshwater snail Helisoma duryi normale to both allografts and xenografts from three species. Cheng and Rifkin (1970), in their review of cellular responses of marine molluscs to helminth parasites, have also summarized and discussed the reported instances of the observed responses elicited by a variety of materials that have in some way come in contact with the internal tissues of molluscs. As part of a series of experiments designed to transmit Minchinia nelsoni (Haskin et ai. 1966), a haplosporidan parasite causing extensive mor- talities of Crassostrea virginica, both infected and normal tissues were successfully transplanted in the American oyster. Though the primary ob- jective of transmitting infections of this pathogen 92 OYSTER TISSUE GRAFTS 93 in the recipient oysters was never realized, several aspects of host response, tissue repair, and implant incorporation are clearly demon- strated in some of the preserved material. In this series, sufficient material was available to illustrate some of the mechanisms of tissue repair and host -implant interaction which are the result of deliberate manipulations performed under relatively well-controlled conditions. Though there still remains some question as to the suitability of the design of such experiments, it was felt that the observations correspond suf- ficiently with those occurring under normal con- ditions to be of value in interpreting some aspects of molluscan tissue responses. MATERIALS AND METHODS Recipients were normal C. virginica (8-12 cm long) tonged in the Navesink River, New Jersey. They were maintained in a flow-through seawater system at Pierces Point, Cape May on the eastern shore of Delaware Bay. Temperatures ranged from 14 - 20 'C with salinities from 18- 24 %o . Normal donors were of the same origin as the recipients, maintained in the aquarium system until used. Infected donors were oysters from Delaware Bay or James River, Virginia, transplanted to the tidal flats of Cape May for infection studies. On some occasions, tissues of in- fected gapers (moribund oysters) were used in lieu of living materials. Various procedures for exposing soft parts had been developed and tested but the most successful involved forcing the hinge ligament until it part- ed and propping the valves in an open position with a wooden shim at the anterior end. Oysters maintained in this condition were flushed frequently with a stream of water to remove ac- cumulated feces and mucus. Other procedures such as the grinding of windows, though suc- cessful, caused difficulty in interpretation of tissue response because of local trauma by con- tact of the soft parts with the rough periphery of the window. There was also a tendency for the exposed surface to secrete new periostracum with subsequent shell formation, an undesirable com- plication. Tissues were removed from the mantle or gill, cut into approximately 3x5 mm pieces and placed in filtered seawater. The implants were pushed beneath the lateral surface of the visceral mass in proximity to the overlying mantle. In a few cases, the tissues were inserted between the bases of the demibranchs or in the free areas of the mantle. Earlier data have indicated that deep penetration of the visceral mass resulted in ex- cessive rejection of implants. The instrument used to insert the implant was a blunt lancet-like probe. It was constructed by flattening the end of a No. 16 gauge soft iron wire and sharpening the flattened portion to resemble an arrow head. In a series of 10 experiments conducted over a 4 year period, 207 oysters received implants of in- fected tissue and 159 received normal tissue. A previous series of 3 experiments using infected tissue from gapers had not been successful in achieving fusion of implants to recipients, though they provided useful material for the evaluation of response mechanisms, as well as leads for the development of suitable procedures. Parallel implants of paraffin and agar slivers were performed to evaluate response to this type of injury. Samples of oysters were removed at intervals of 1-35 days after implantation, opened, examined grossly, and fixed in either Zenker's Acetic (5%) Fixative or in Davidson's Fixative (modification of Shaw & Battle, 1957) and prepared for histological examination according to routine procedures. Although a certain percentage of the oysters were dead or moribund at the time of sampling, only active oysters (pumping and healthy in gross appearance) were considered representative of normal responses. Mortality Oysters implanted with normal living tissues sustained a 21% overall mortality for the series of 10 experiments. Overall mortality for those receiving infected tissues was essentially the same, 23%. There was no overt time pattern for the mortalities. They appeared to be more closely correlated with oyster condition and en- vironmental parameters than with the implant procedure per se. Of the several factors noted as influencing survival, the most significant were water temperature (optimum 14 - 16 C), tur- bidity (it was occasionally necessary to use sand filtered water) and maintenance of adequate cir- culation in the aquaria. Mortality in the in- 94 W. J. CANZONIER «C' 1 FIG. 1. Gill implant, 3 days post-transplantation (P-T). Notice minimal infiltration and initiation of fusion. 10 X obj. dividual experiments ranged from 0 - 60%, reflect- ing variations in these factors. Rejection of Implants Mechanical Rejection. Rejection of some im- plants occurred within the first 24 hours in all experiments. This appeared to be primarily mechanical in nature; involving a squeezing out of the implanted tissue accompanied by an oozing of aggregated leucocytes. This response was associated with the site of implantation, oc- curring most frequently where there was a minimum of firm tissue surrounding the lesion and a maximal potential for compression of the tissues by mechanical or hydraulic forces. Im- plants in the dorsal portion of the visceral mass anterior to the adductor muscle experienced almost 100% rejection. In other sites the percent *. •*^« ^ ' SfaaT^i-^A ^i!^»)t»^^,-.' '.':-jr. ■ ~ •i r< j,'/;*j»7^-'^\ FIG. 2. Gill implant as in Fig. 1, showing area of fusion. 25 X obj. /-at .vj •;^' 3 FIG. 3. Gill implant, 25 days P-T. Notice integrity of epithelial lining and juncture of implant with recipient. J^O X obj . loss of at least 1 piece of tissue per oyster ranged from 0 - 50%. Chronic Rejection. There was no evidence in the material studied of a chronic response and subsequent rejection or resorbtion of an implant that had become successfully established. However, it should be pointed out that these ex- periments were not primarily designed to study such phenomena and were of rather short duration (max. 35 days). Recipient Response Infiltration. Leucocytic infiltration in the case of living implants and inert materials was usually minimal, though always present to a limited degree (Figs. 1-4). However, when gaper FIG. 4. Mantle implant, 25 days P-T. Leucocytk cuffing of blood vessel occurs frequently in nor- mal oysters at this location and is probably not associated irith implant. 20 X obj. OYSTER TISSUE GRAFTS 95 ^BS'-^i'^Ni^:^*- Imp •wa;- * , ;^m'^z> .1 r» *ht V « 4 Imp ^ l'^^ N%« .^6- FIG. 5. Moribund mantle tissue inplant three days P-T. Note complete infiltratioti of iniphuit by re- cipient leucocytes. iOA'obj. F'IG. 6. Same section as in Fig. 5 showing nature of response to moribund tissues. Note fusiform cells peripheral to the implant. 40 X obj. tissues were implanted, mobilization of leucocytes, beginning in the first 24 hours, was often very intense and eventually resulted in the complete infiltration of the implant (Figs. 5-6). This resulted in rapid loss of implant integrity and after 3 days cell boundaries were no longer recognizable and nuclear staining was diffuse. Tlie Plasmodia of M. nelsoni degenerated in these situations, becoming eosinophilic, agranular and losing nuclear detail within 2-3 days. Tissue Repair. Within 1 day, the injured sur- faces showed evidence of repair in the form of fibrocyte-like cells aligned on all exposed tissue surfaces not in direct contact with subepithelial portions of the implant or adjacent tissues of the recipient. These cells increased in number to several layers deep (Figs. 7-8). These cells were fusifonn, 20-30 ;um in length and 2.5 ^im maximum diameter. The nuclei were elongate, 3.5-5.5 X 1.5-2.5 pxr\ and densely stained with hematoxylin. After 3 days, there was often ^'f^!^^ FIG. 7. Tissue repair on outer surface of visceral mass, 10 days P-T. lOX obj. FIG. 8. Tissue repair in deep connective tissue of viscernl mass. 15 days P-T. Note densit fusiform cells. JfOX obj. 8 // (f layer if 96 W. J. CANZONIER evidence of an accumulation of fine granular yellow material (refractile to both hematoxylin and eosine in and around these cells). Eventually the cavity or exposed outer surface became covered by small epithelial cells. The origin of these cells is difficult to ascertain from the material at hand. The fusiform cells were possibly the descendents of leucocytes that had migrated to the area. It appears that the epithelial cells may migrate or grow outward from the intact epithelium that surrounds the lesion (Fig. 7). In the case of implants, the epithelial cells of the grafted tissue appeared to contribute to the re-establishment of the epithelial layer. Indeed, in the case of the lining of cavities surrounding implanted mantle, the cells were more characteristic of the graft epithelium than the cells of the epithelium that had been penetrated (Fig. 4). Even in the case of gill implants, it was often impossible to determine where the epithelium of the graft terminated and that of the recipient began (Figs. 3 and 9). Fusion of Implants Unsuccessful. In the case of gaper tissue or pieces of digestive gland we never observed fusion of implant to recipient. These tissues always disint^rated and an abcess filled with leucocytes and cellular debris was all that remained after 10-15 days (Fig. 5). Successful. Living gill and mantle tissues, both normal and infected with M. nelsoni, fused with the host tissues and remained viable and iden- tifiable for up to 35 days. After the initial period of mechanical rejection, during which as many as 50% of the oysters lost at least 1 implant, the retained tissues showed evidence of successful fusion in at least 85% of the oysters surviving to the time of sampling. Survival of the implant was apparently not significantly influenced by the presence of M. nelsoni Plasmodia, since success with infected tissue was equal to that with nor- mal tissues, provided the implant was living and otherwise healthy. Gill tissue retained its integrity for the entire period of observation, even when completely con- tained within a closed abcess without access to external surfaces (Figs. 9, 10 and 11). The only major change observed was a swelling and delamination of the chitinous rods (Figs. 10, 11 and 13). Mantle tissue, being composed of only 2 major cell types, Leidig cells and epithelium, assumed the appearance of an extension of the tissues at the site of the implant. However, as mentioned above, the nature of the epithelium that lines the .ChR ^ •'. •'*NiA% FIG. 9. Gill implant in free poHion of mantle, 25 days P-T. The cavity is lined with a low epithelium and is relatively free of leucocytes and debris. 10 X obj. FIG. 10. Gill implant in connective tissue of visceral mass. 35 days P-T. Note there is no indication of incapsulation and only a slight increase in the density of the tissues surrounding the implant. 10 X obj. OYSTER TISSUE GRAFTS 97 •- K 'iff <«• ' FIG. 11. Zone of fusion of gill implant 35 days. P-T. Other than swelling of chitinous rods there is little change in the composition of tissues. 25 X obj. FIG. 12. Zone of juntion of implanted mantle shown in Fig. J^, 25 days P-T. It is not pos.^ble to de- termine the boundary between implant and recipient. W X obj. site of the implant more closely resembles the higher columnar cells of the free mantle border than the epithelium covering the central visceral mass (Fig. 12). In the case of tissues infected with M. nelsoni, the parasite appeared normal in a few implants after 25 days. In no case was there evidence for transfer of the infection to the recipient oysters (Fig. 14). In a few oysters the parasites appeared to mingle with the tissues of the recipient but it was not possible to determine whether this was the result of active migration, or if these sites merely represented an area of fusion where it was impossible to distinguish reliably between the tissues of the recipient and implant. Occasionally, other organisms were associated with the lesions created by the disintegration of tissues in the area of unsuccessful implants. In three cases, the flagellate Hexamita inflata in- fested the debris-filled ulcers. In two cases, a fungal mycelium (Mackin, 1962) had become established in the zone between implant and host tissue. L ^ vV _ . >« t FIG. 13. Gill implant 35 days P-T, shounng the swelling ufchituwus )od.s. ^it X obj. FIG. 14. Gill tissue infected with Minchinia nelsoni, 25 days P-T. W X obj . *^>-s*j^.-, W. J. CANZONIER DISCUSSION Reports of tissue transplantation in molluscs range from successful fusion of the implant with the recipient to rejection or encapsulation. In the case of implants of mantle tissue to form pearl sacs in Pterin martensii. there is apparently con- sistent success using techniques perfected over many years by the Japanese pearl industry (Tsujii, 1960). The site of implantation, the physiological state of the implanted tissue and its origin are all important factors determining both graft acceptance and pearl formation. Successful fusion of implant to host has been reported by DesVoigne and Sparks (1969) for C. gigas. These authors state that rejection of mantle tissue im- plants was common, "but considerably less than 50%." They do not state exactly the frequency of successful implant fusion, mentioning only "one specimen, after 280 hr," showing incomplete fusion with the host and "an implant which had fused completely with the host tissue in the palp region was observed at 448 hr." The latter case closely resembles the histological picture of suc- cessful fusion in C. virginica. They specifically note that in this case the "fusiform cells typical of healing were not pi-esent." We have observed that such fusiform cells are generally confined to the repair of exposed surfaces hence their absence in an established implant is not surprising. DesVoigne and Sparks also indicate some of the more critical factors that affect implant ac- ceptance and fusion, including the size and site of incision in the host, the importance of mechanical factors and nature of the implanted tissue. In other bivalves, successful fusion has been reported in the case of implanted autologous gill tissues in the scallop Gibbus borealvi (Butcher, 1930). Partial success in transplantation of man- tle tissue in another scallop, Pectcn iiTodMns. has been reported by Cushing (19.S7). This report (ab- stract — no details) implies that the site of im- plantation or operative procedure may influence survival of the graft. Apparently sites deep in the visceral mass are not suitable for maintenance of transplanted tissues. The failure of Drew and deMorgan (1910) to achieve successful grafting of gill tissue in Pecten maximus is possibly due to a combination of fac- tors. The adductor muscle, chosen by them as a site for implantation, is an environment not mechanically compatible with the much softer gill tissues. We are also in agreement with Feng (1967) in his criticism of their technique which involved forcing the implant through the bore of a hypodermic needle, probably causing excessive traumatization. The report by Tripp (1961) of the successful transplantation of foot tissues in Australorbis glabmtus illustrates the degree of success possible when certain selected tissues are transplanted to a suitable site (in this case cephalopedal sinus) with a minimum of tauma to implant and recipient. The failure of Cheng and Galloway (1970) to have implants of digestive gland persist in H. duryi normale. using procedures similar to those of Ti'ipp, could possibly be due to an excessive release of lytic metabolites by the cells of the transplant. Early experiments using digestive gland tissue in C. virginica always resulted in disintegration of the implants and large areas of necrosis in the recipient oysters. Drew and deMorgan (1910) reported similar results when they used digestive gland implants in scallops. The fact that the intensity of response varied when different donor species were used by Clieng and Galloway (1970), indicates that there was some degree of compatibility for tissues originating from the same species, the failure to survive and fuse with the recipient being related to some factor common to all the tissues used. The accumulated observations of this series of experiments with C. virginica, in addition to results of similar investigations with other species, indicate some factors of primary im- portance in successful tissue transplantation in molluscs. The type and physiological state of the implanted tissue must be considered. Tissues, such as digestive gland, with a potential for producing large quantities of extracellular en- zymes, are likely to create an unsuitable en- vironment at the site of implantation. Likewise, excessive injury to, or disruption of such tissues in the recipient is not desirable. Provided there have been no irreversible changes due to toxic or other factors, it is also possible to transplant tissues infected with at least 1 quite virulent parasite, (M. nelsoni). Though this procedure did not prove to be a successful means of trans- mitting the disease, it might be further in- vestigated as a means of studying the fate of this OYSTER TISSUE GRAFTS 99 and other parasites in hosts of varying suscep- tibility. The response of the recipient to implanted material depends on the nature and subsequent fate of the implant. If the implant is viable, ac- ceptable to the recipient, and becomes established with sufficient contact with the recipient to maintain essential metabolic functions, there is likely to be minimal response (e.g., leucocytic in- filtration or encapsulation), at least for the duration of the experiments reported. In the case of inert material (paraffin), no excessive infiltration into the area was noted but the normal responses to an injured surface pro- ceed to produce an "encapsulation" of the insert. In these cases there seems to be some displacement of the connective tissue surrounding the implant. We would agree with Cheng and Rifkin (1970) that some of the flattened cells that form the "encapsulation" are merely flattened Leydig cells. However, it is evident that distinct fusiform cells ("fibroblastic" cells of Cheng) are also involved in such encapsulation since they can be traced to outer portions of the lesion where the compressive factor is not present. It is not possible to determine the origin of these cells from the material described but there is an in- dication that they are the descendents of leucocytes that have migrated to the area in response to the injury. Some degree of leucocytic infiltration, commencing soon after injury, always precedes the appearance of fusiform cells. Infiltration by hemocytes has been noted as a response to both mechanical injury in C. y/'/n.s' (DesVoigne and Sparks, 1968, 1969) and tissue destruction by ionizing radiation in the same species (Mix and Sparks, 1971). The latter authors indicate that some of these cells at first form aggregates or cell "nests" on the surface and that subsequent migration and transformation of these cells re-establish the integrity of the tissue surface. Armstrong et al. (1971), reporting on wound healing in the abalone, Haliotis cracherodii noted a sequence of leucocytic in- filtration, appearance of "fibroblast cells" and re- establishment of epithelium by migration or ex- tension of cells from intact areas. The overall histological picture was slightly different from that observed in C. virginica because of the rather muscular nature of the abalone tissues. Similar responses were noted for the en- capsulation of the cestode Tylocephalum in C. virginica (Rifkin and Cheng, 1968), though we have not observed the fibrous elements ap- parently characteristic of capsule formation. A somewhat different response resulted when the tissues were not viable. There was a marked influx of leucocytes into the area surrounding the implant and then into the implant itself. Even gross examination of a stained section through such a site will reveal an area of intense leucocytic aggregation ("inflammation"). Fusiform ("fibroblastic") cells appear within one day in the area adjacent to the implanted material. This sequence of events appears to be identical to the response to non-viable implants in Pecten described by Drew and deMorgan (1910). Tsujii (1960) also implied a similar response in the case of "death of the graft" or "inflamed pearl sac." We have occasionally observed a corresponding phenomenon in cases of infection with the larval trematode Bucephalus cucnhis in oysters. Normally sporocysts of this parasite, even though displacing and disrupting large areas of host tissue, elicit no overt response. However, when moribund or parasitized, the sporocysts elicit an intense influx of leucocytes into the surrounding tissues (unpublished observations). It seems that this type of response is primarily dependent on a chemical stimulus from the site of the lesion or invading foreign materials and less dependent on mere physical disruption of the tissues. In the case of successful implants, "in- flammation", infiltration by leucocytes, is usually minimal. Variation in the intensity and extent of initial response appears to be common to other bivalves and dependent on several fac- tors including site of injury, nature of the foreign material introduced and the species involved (Mikhailova and Prazdnikov, 1961; Pauley and Sparks 1966, 1967; Pauley and Heaton 1969; Prazdnikov and Mikhailova, 1965). One aspect of response to tissue implants has scarcely been touched upon: the maintenance of the implant over extended periods and the sub- sequent long-term response of the recipient. In the series reported here, 35 days was the maximum period of observation. The persistence of the pearl sac in the Japanese culture 100 W. J. CANZONIER procedure is perhaps the best evidence available for long term compatibility. In that particular case there is some indication of a change in the cellular composition of the implant (Tsujii, 1960). This change is probably due to a shift in prominence of some particular cell type. Mantle implants in C. virginica appeared to be composed of only one major cell type, perhaps the one most favored by the environment at the site of im- plantation. The deterioration of the chitinous rods in gill implants might indicate a reduction of the cells responsible for the maintenance of these components. It is evident that interpretation of histocom- patibility and recipient response to tissue grafts in bivalve molluscs must take into account several factors that influence the potential for successful fusion and maintenance of the implant. Some of these factors are suggested in the various studies cited. However, these factors are still poorly understood while other aspects of preliminary host response, tissue repair and im- plant fusion mechanics remain unexplored areas for investigation. ACKNOWLEDGEMENTS I would like to express my deepest appreciation for the inspiration, encouragement and con- structive suggestions and criticism graciously provided, during the many years of my in- volvement in these studies, by a most un- derstanding and able teacher, the late Dr. Leslie A. Stauber. I am also indebted to Dr. H. H. Haskin for providing both the physical facilities and the essential guidance for the effective execution of the research reported. I would like to thank Dr. S. Y. Feng for his criticism and advice, and Theresa M. Menko for technical assistance in the preparation of the manuscript. This research was supported by a grant (Con- tract No. 14-17-007-887) from the U.S. Bureau of Commercial Fisheries (NMFS) to Dr. H. H. Haskin. LITERATURE CITED Alverdes, F. 1913. Ueber Perlen und Perlbildung. Z. Wiss. Zool. 105:598-633. Armstrong, D. A., J. L. Armstrong, S. M. Krassner and G. B. Pauley. 1971. Experimental wound repair in the black abalone, Haliotis cracherodii J. Invertebr. Pathol. 17:216-227. Butcher, E. 0. 1930. The formation, regeneration, and transplantation of eyes in Pecten (Gibbus borealis). Biol. Bull. 59:154-164. Canzonier, W. J. 1963. Histological observations on the response of oysters to tissue implants. Natl. Shellfish. Assoc. Ann. Conv. (Mimeo abstract). Cheng, T C. and P. C. Galloway. 1970. Trans- plantation immunity in mollusks. The histoin- compatibility of Helisoma duryi normale with allografts and xenografts. J. Invertebr. Pathol. 15:177-192. Cheng, T C. and E. Rifkin. 1970. Cellular re- actions in marine molluscs in response to helminth parasitism. In S. F. Sniesko (ed.), A Symposium on Diseases of Fishes and Shell- fishes. Am. Fish. Soc, Spec. Publ. No. 5, p. 443-496. Chernin, E. 1966. Transplantation of larval Schistosoma mansoni from infected to unin- fected snails. J. Parasitol. 52:473-482. Gushing, J. E. 1957. Tissue transplantation in Pecten irradians. Biol. Bull. 113:327. (Abstract). DesVoigne, D. M. and A. K. Sparks. 1968. The process of wound healing in the Pacific oyster, Crassostrea gigas. J. Invertebr. Pathol. 12:53-65. DesVoigne, D. M. and A. K. Sparks. 1%9. The reaction of the Pacific oyster, Crnssostrea gigas. to honiologous tissue implants. J. Invertebr. Pathol. 14:293-300. Drew, G. H. and W. deMorgan. 1910. The origin and formation of fibrous tissue produced as a reaction to injury in Pecten maximus, as a type of Lamellibranchiata. Q. J. Microsc. Sci. 55:595-620. Feng, S. Y. 1967. Responses of molluscs to foreign bodies with special reference to the oyster. Fed. Proc. 26:1685-1692. Haskin, H. H., L. A. Stauber and J. G. Mackin. 1966. Minchinia nelsoni n.sp. (Haplosporida, Haplosporidiidae): Causative agent of the Delaware Bay oyster epizootic. Science, 153:1414-1416. Mackin, J. G. 1962. Oyster disease caused by Dermocystidium marinum and other micro- organisms in Louisiana. Publ. Inst. Mar. Sci., Univ. Tex. 7:132-229. OYSTER TISSUE GRAFTS 101 Mikhailova, I. G. and E. V. Prazdnikov. 1961. Two questions on the morphological reactivity of mantle tissues in Mytiltis edulis L. Tr. Murni. Morsk. Biol. Inst. 3:125-130 (transl.) Referat. Zhur. Biol. 1962, 16Zh:118 (Abstract). Mix, M. C. and A. K. Sparks. 1971. Repair of digestive tubule tissue of the Pacific oyster, Crassostrea gigas, damaged by ionizing radiation. J. Invertebr. Pathol. 17:172-177. Pauley, G. B. and A. K. Sparks. 1966. The acute inflammatory reaction of two different tissues of the Pacific oyster, Crassostrea gigas. J. Fish. Res. Board Can. 23:1913-1921. Pauley, G. B. and A. K. Sparks. 1967. Observa- tions on experimental wound repair in the adductor muscle and the Leydig cells of the oyster Crassostrea gigas. J. Invertebr. Pathol. 9:298-309. Pauley, G. B. and L. H. Heaton. 1969. Experimental wound repair in the freshwater mussel Anodonta oregonensis. J. Invertebr. Pathol. 13:241-249. Prazdnikov, E. V. and I. G. Mikhailova. 1965. Morphological reactivity of mussel mantle tissues at some stages of ontogeny: information on the problem of embryonic immunity. Tr. Murm. Morsk. Biol. Inst. 5:194-225. (transl.) Referat. Zh. Otd. Vypusk. Obshch. Vop. Patol. Onkol. 1965, 5:8 (Abstract). Rifkin, E., T. C. Cheng and H. R. Hohl. 1969. An electron microscope study of the constituents of encapsulating cysts in Crassostrea virginica parasitized by the cestode Tylocephalum sp. J. Invertebr. Pathol. 14:211-226. Shaw, B. L. and H. I. Battle. 1957. The gross and microscopic anatomy of the digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J. Zool. 35:325-347. Tripp, M. R. 1961. The fate of foreign materials experimentally introduced into the snail Australorhis glabmtiis. J. Parasitol. 47:745-751. Tsujii, T. 1960. Studies on the mechanism of shell and pearl formation in molluscs. J. Fac. Fish. Prefect. Univ. Mie, 5:1-70. Proceedings of the National Shellfisheries Association Volume 6U - 197i THERMAL TOLERANCE OF OYSTER LARVAE, CRASSOSTREA VIRGINICA GMELIN, AS RELATED TO POWER PLANT OPERATION Herbert Hidu'' Willem H. Roosenburg Klaus G. Drobeck Andrew J. McErlean Joseph A. Mihursky CHESAPEAKE BIOLOGICAL LABORATORY UNIVERSITY OF MARYLAND SOLOMONS, MARYLAND ABSTRACT The upper thermal tolerance of Chesapeake oyster larvae (fertilized eggs, ciliated gastrulae and 2-day veliger larvae) was determined for 10-second to 16- hour exposures. A biphasic temperature tolerance curve was noted with time of exposure, the inflection point coming at 2 hours. Consideritig all life stages tested, at 2 hours significant mortality (LD ,„ ) came at just over 30 C, unth slight increases in effect noted up to 16 hours of exposure. Tolerance levels in- creased with decreasing exposures less than 2 hours, until at 10 seconds significant reduction came at approximately W C. Tlie relevance of these find- ing.^ to power plant design and. operation on estuanes is discussed. INTRODUCTION The increasing use of estuarine waters by electric power generating stations for condenser cooling has necessitated a more detailed un- derstanding of the tolerances of shellfish larvae and other entrained organisms to high tem- peratures. Mihursky and Kennedy (1967) state that the doubling time for electricity needs in the United States is now 6 to 10 years. From 1960 to 2010, for example, there is anticipated a 30-to 250-fold increase in electricity demand. Thus, more and larger coal-fired or less ef- ficient atomic electric plants will be constructed in the near future. Since most choice sites in fresh water have been used, and fresh water volumes, in many cases, are inadequate for the ' Present Address • University of Maine, Ira C. Darling Cen- ter. Walpile, Maine 0.1573. large capacity plants planned, this expansion may take place in estuarine and marine waters. Vast quantities of cooling water, up to 2.7 million gallons per minute in larger plants, may be drawn from the environment, heated at condensers to engineering optima and released back to the environment. Entrained species may be subjected to high temperatures for periods of one minute to several hours depending on the design of effluent canals and provision for cooling by mixing with "tempering water". ("Tempering water" is an engineering in- novation which adds ambient temperature water to the cooling water just after condenser passage to allow conformance with certain state and federal regulations which only stipulate maximum temperatures at the point of release to the environment.) The temperature tolerance of estuarine 102 THERMAL TOLERANCE OF OYSTER LARVAE 103 FIG. I. Experimental thrnnal gradient block choiring: A-teftt tube environment with 11 tubes on the thertnal axis: B-syringes to allow simultaneous injection of experimental animals on the 11 tube axis; C-hot and cold water sources and block circulation airangement; D-block temperature sensors and re- cording apparatus. molluscan larvae has drawn considerable research interest. Loosanoff, et al. (1951) found that if the fertilized eggs of the hard-shelled clam, Mercenaria mercenaria, were placed at 33 C, development to abnormal veliger larvae resulted. However, if fertilized eggs which had been raised to normal veligers at 24 were placed at 33, then normal rapid veliger grow^th ensued. The authors stated that this observation coincided with the view of Pelseneer (1901), that young cleavage stages of molluscan eggs exhibit a narrower temperature tolerance than the later life history stages. Davis and Calabrese (1964) investigated the interaction of temperature and salinity on development of fer- tilized eggs and growth and survival of veliger larvae of M. mercenaria and oysters, Crassostrea virginica. With clam larvae, they found that development, growth, and survival were not affected generally between 17 and 30. However, at non-optimal salinities, temperature tolerances were much reduced. Oysters showed a similar pattern, with fertilized eggs not tolerating 32.5. The fully devebped veligers sur- vived and grew optimally at 32.5 but not below 22.5 C. Stickney (1964) investigated the tem- perature tolerance of New England and Chesapeake Bay stocks of the soft-shelled clam, 104 H. HIDU, W. H. ROOSENBURG, K. G. DROBECK, A. J. MCERLEAN AND J. A. MIHURSKY Mya arenaria. He found that optimal survival of fertilized eggs to veligers occurred at 15 and 18 C but was reduced drastically at 22.5 and 28 C. Similarly, veliger lai-vae grew well at 18 and 22, but all died at 28.4 C. Ambien't conditioning temperatures for adults was between 2 and 13 C. Stickney suggested that there were dif- ferences in temperature tolerances between the New England and Chesapeake stocks, possibly genetically controlled, although his experimental results appeared not to be decisive. The role of temperature of acclimation of adult spawning stock of bivalve mollusks in the temperature tolerance of their pelagic embryos and larvae appears to be nonrelevant since gonad production and spawning are largely temperature dependent. Numerous studies have demonstrated that for a given species and race, spavming and thus larval occurrence takes place wdthin a rather narrow temperature range, probably no greater than ±5 0. [See for example, Loosanoff (1937, 1942) and Pfitzenmeyer (1962).] Our experiments were designed to determine the short-term (10 seconds to 16 hours) upper thermal tolerance of oyster embryos and 2-day veliger larvae. The experimental design was dictated, in part, by a consideration of engineering specifications for 2 plants newly constructed in the Chesapeake Bay, in close proximity to oyster producing areas (Morgan- town on the Potomac and Calvert Cliffs Nuclear on the Bay proper). Both of these plants employ tempering water and are required to meet the 1967 Maryland and Federal tem- perature regulations, which specify seasonal dif- ferentials and effluent maxima. In contrast to older designs, such as the Chalk Point plant on the Patuxent Estuary, total transit times for cooling water at condenser temperature is measured in minutes rather than hours. These experiments attempt to simulate thermal change which will be experienced by entrained larvae in the two types of plant design. Appreciation is expressed to Drs. L. Eugene Cronin, T. S. Y. Koo and Mr. Elgin A. Dunnington for helpful advice throughout the project. Summer students. Miss Judith L. Baab, Patricia E. Albert and Sandra Wrenn aided in experimental work. MATERIALS AND METHODS Six experiments determined the short-term temperature tolerance of oyster larvae as follows: I'br these experiments, a multivariate tem- perature block (Keller, et ai. 1968) was used. The aluminum block (Fig. 1) is 4" x 24" x 15", witl, 8 rows of 11 one-inch diameter holes into which test tubes were placed. On the left side of the block, cold water was circulated; the right side was heated by circulating warm Experiment " lemperature Kange L Duration of Exposure 2-l6 hours Larval Stage 1 17-37 G fertilized eggs 2 27-37 C 1- 8 hours fertilized eggs 3 26-44 G 10-sec, -1 hour 6-hour ciliated gastrulae 4* 29-49 C 10-sec, -1 hoiir fertilized eggs 5* 29-50 G lO-sec, -1 hour 6-hour ciliated gastrulae 6* 29-49 G 10-sec, -1 hour 2-day veliger larvae * utilized the same brood of larvae THERMAL TOLERANCE OF OYSTER LARVAE 105 water. A linear temperature gradient was developed along the 11-tube axis which was regulated by controlling the temperatures of the circulating waters. Eight rows of tubes per- mitted replication of treatment and allowed in- vestigation of the effect of an additional factor, in this case, time of exposure. Adult Chesapeake Bay oysters were first con- ditioned and spawned in the shellfish hatchery at Solomons by the techniques described by Hidu, et ai, (1969). Conditioning temperatures ranged between 20 and 25 C for several weeks. Spawning stimuli included 1 to 4 hours of fluc- tuating temperatures ranging from 25 to 30 C. Naturally -spawned eggs then were brought back to 24 C in stock cultures with egg densities at approximately 60/ml . Salinities for all ex- periments ranged between 10 and 15 %» . Stock cultures of fertilized eggs were then transported 25 miles to the Hallowing Point Field Station where the temperature block had been brought to equilibrium at the desired tem- perature range. Test tubes in the block con- tained 30 ml of salt water collected at Solomons and filtered to 10 ^. Larvae were then simultaneously introduced into a full row of 11 test tubes from 5 cc syringes mounted in a rack (Fig. 1). This resulted in densities of about 300 larvae per test tube or approximately 10/ml . The larvae were left in the test en- vironments for the allotted time, after which they were simultaneously removed and poured into large test tubes containing about 300 ml of Solomons sea water at 24 C. Larvae were in- cubated for 48 hours at 24 C to enable the eggs or gastrulae adequate time to develop to nor- mal veligers, and in the case of the veligers, to decompose and leave only the empty shell, if they suffered mortality during the temperature exposure. At 48 hours, larvae were poured into a disposable beaker containing an adequate amount of formalin, so that they were con- centrated on the bottom of the beaker. The volume of water in the beaker was reduced to about 30 ml by careful siphoning, after which all the larvae were washed into vials and preserved with buffered formalin for counting. Counts of larvae were made by first decant- ing the vial and placing the entire sample on a Sedgwick-Rafter cell. In the case of ex- periments using the fertilized eggs and gastrulae, counts were made of resulting normal 2-day veliger larvae. Normal is defined as a larva between 70 and 80 n, measured parallel to the hingeline, and with a straight-line hinge. A marginally stressful treatment will produce all types and degrees of abnormalities (Loosanoff and Davis, 1963). In the case of the veliger experiment, mortality was measured by counting live and dead larvae. A dead larva, af- ter 48 hours, only left an empty shell. Within each experiment, two replicate runs (of 11 en- vironments) were made at a specific time of ex- posure. To determine whether embryos suffered mor- tality due to exposure to the block and associated manipulations, separate control lar- vae were incubated in the 300 ml test tubes for 48 hours at 24 C. There were no significant dif- ferences in survival between these and those held in the block at known optimal tem- peratures in the first experiment, so the prac- tice was discontinued. Further, high percentage retrieval rates of the innoculated larvae at op- timal temperatures in all remaining ex- periments attested to the suitability of the block environment for the experiments. Temperature in the block was monitored by spot checking (stem thermometer) and con- tinuously by thermistor probes (YSI — Telethermometer Model 47) accurate to .2 and .5 C, respectively. Because of stem lag and delay in cycling time, temperature values for the short-term experiments (10 sec and 1 min) had to be corrected. This was done in a separate study in which temperatures were simultaneously determined by telethermometer and stem thermometer. Regression analysis was used to compare the apparent (YSI) and actual (stem thermometer) values. These were highly correlated (r = .994) and yielded the following equation: actual temperature ( C) = .794 ap- parent temperature + 5.7. This equation was used to correct temperature data for short-term runs. Analysis of actual and apparent tem- peratures for times greater than 1 minute showed no appreciable differences and the YSI values were used directly. Analysis of Data From a given experiment, the relative per- 106 H. HIDU, W. H. ROOSENBLIRG, K. G. DROBECK. A. J. MCERLEAN AND J. A. MIHURSKY centage survival for each pair of vials in a replicate was computed using maximum sur- vival pair in the entire experiment as the base (Fig. 2). This technique has been used by Davis (1958, 1964) and others. These data were plotted as a response surface and lines of 80, 60, 40, 20 and 0 percent survival interpolated by in- spection. There is some danger in slightly overestimating percentage effects by the procedure, however, the proximity of survival contour lines indicates critical areas of thermal effect. Data were further analyzed by calculating LDIO, 50, and 90% values for all experiments combined LD50 values for fertilized eggs, ciliated gastrulae and veliger larvae were con- sidered separately. This was accomplished by probit analysis (Finney, 1962). To validly use the probit analysis, it was necessary to eliminate natural (background) or nontreatment mortality from being confounded with that due to experimental treatment (See Davis and Calabrese, 1964; Tables 4-6). This was done as follows: Each time-group (10 sec, 1 min, etc.) within an experiment was considered a homogeneous treatment group and background mortality was estimated from inspection of the raw data for that particular group. In most cases, a marked change in percentage mortality occurred as higher test temperatures were encountered. This inflection, from about 10% mortality to values of 30-50% or greater, could be determined by inspection and was used to estimate background mortality. Background mortality was estimated by averaging individual percentage mortalities below the inflection point. The number of in- dividual values used to estimate background mortality ranged from 2 to 7. The computed average mortality was substituted in Abbott's formula (1925) and each individual raw score corrected. Probits (Finney, 1962) of these per- centages were then taken. In most cases, the raw data were considerably smoothed by this practice and probit lines could be easily fitted to the data points. Probit lines were fitted by inspection to data points and LDIO, 50 and 90 points estimated from the line by determining the antilog of temperature for a particular probit. No attempt was made to calculate con- fidence intervals for a particular LD value. Past experience with such calculation has produced unrealistically small intervals ( + .01 C) that are not justified by the experimen- tal procedures employed. A more realistic estimate of confidence interval is probably on the order of + .5 C. However, in short-term experiments (4 hrs) there was variation between comparable life history stages run at different times. Although some slight, between-run variation existed, all short-term experiments yielded high LD points. RESULTS Data are expressed first as percentage sur- vivals in all e.xperiments (Fig. 2). Further, trends and relationships are clarified in plot- ting LDIO, 50 and 90 values with exposure time derived by combining data from all experiments (Fig. 3). The LD50 values obtained with the three life stages (fertilized eggs, ciliated gastrulae, and 2-day veliger larvae) are plotted in Fig. 4. Oyster larvae exhibited a direct relationship of mortality to duration of exposure to elevated temjierature and the relationship appears to be biphasic. For exposure times of 10 sec to 2 hrs (Fig. 3), all data points were fitted by a line having considerable slope. Thus, in short duration exposures, LDSO's, decreased rapidly up to 2 hrs exposure. From 2 to 16 hrs, the slope changed and LD50 values were close to 33 C. This second phase of tolerance was more in keeping with published data on the upper tolerances of bivalve mollusca. Exposure to temperatures below ambient for 1 to 16 hours was also detrimental to larval survival (Fig. 3). LDIO values came at ap- proximately 20 C and showed little trend with time of exposure. The stage of larval development appeared to be a significant factor in thermal tolerance as illustrated by plotted LD50 values obtained for fertilized eggs, ciliated gastrulae and 2-day veliger larvae (Fig. 4). Fertilized eggs were least tolerant with LD values falling ap- proximately 3 C below those obtained for ciliated gastrulae. Veliger larvae were the most tolerant with LD values occurring 8 to 12 degrees above those obtained for fertilized eggs. THERMAL TOLERANCE OF OYSTER LARVAE 107 % SURVIVAL OF FERTILIZED E6GS o SURVIVAL OF FERTILIZED EGGS b •. ■ .„__ , ■■ ■ "^ — _ ~~~---^ -^ -. ~~-— 60 ~~-— ^- % SURVIVAL OF CILIATED GASTRULAE % SURVIVAL OF 2-DaV VELIGER LARVAE EXPOSURE TIME (log scole) EXPOSURE TIME (loi FIG. 2. Calculated perce^itage survivals for all experiments setting the maximum sunnval part- udthin a treatment at lOCM. Percentage survival contours were extrapolated visually between data points. 108 H. HIDU, W. H. ROOSENBURG, K. G. DROBECK, A. J. MCERLEAN AND J. A. MIHURSKY DISCUSSION These experiments indicated that the early cleavage stages, the fert;ilized egg and ciliated gastrula, are considerably more temperature sensitive than later stages of development. This adds weight to the contention of other workers who have noted a similar effect with a variety of species. Pelseneer (1901), noted that the early cleavage stages of molluscan eggs are limited to a narrower temperature range than more ad- vanced stages. Loosanoff and Davis (1963) con- firmed these observations through extensive trials with oysters and the hard-shell clam, Mcrcenaria mercenaria. Brett (1960) found that fertilized embryos of salmon are also the most thermally sensitive life stage even though this is a short-lived stage. The sensitive fertilized egg and gastrula stage in oysters exists for 6 to 8 hours and the more tolerant veliger for two weeks. However, the argument that thermal regulations could discard consideration of the early stages because their short duration would make power plant predation insignificant is not valid. It should be realized that any subsequent stages evolve from the early stages and loss may have great later effect on recruitment. 10 2 0 3 0 LOG TIME SECONDS FIG. 3. Upper and loiver temperature tolerance combining all data for all oyster larval stages expressed as LD,,,. .„, and ,„ values. 50 40 2 30 20 CUIATEO GASrpUL A 10 SEC. 1.0 2.0 30 LOG TIME SECONDS 4.0 FIG. 4. Upper temperature tolerance of oyster fertilized eggs, 6-hour ciliated gastrula and 2- day veliger larvae expressed as LI)-„ valves. The time of exposure, as stated, greatly af- fects the temperature tolerance of larvae and this may have considerable bearing on future power plant design. For example, the Chalk Point Power Plant on the Patuxent estuary (Fig. 5), in its original design, took in cooling water at ambient temperatures, passed it over condensers where it was heated 6.5 C, after which it flowed back to the estuary in a long effluent canal. The excursion in the effluent canal to final discharge in the river took 2.7 hours with an approximate temperature loss of 0.5 C in transit. These experimental results in- dicate that during critical summer tem- peratures, effluent canals may become long killing chambers for entrained forms. At river temperatures of 24 C, which is optimal for oyster larvae, the effluent temperature of 30.5 C, no doubt, had detrimental effects on early oyster larval stages during 2.7 hours of transit. A more recent development (Morgantown Power Plant on the Potomac and recently. Chalk Point), one designed to allow a plant to conform to discharge regulations and yet obtain higher temperatures within the condensers, is the innovation of using "tempering water" in the system just after the condensers (Fig. 6). THERMAL TOLERANCE OF OYSTER LARVAE 109 CHALK POINT RIVER Intake ■ 2 7 Hour CONDENSERS EFFLUENT CANAL -y/- TEMPERATURE CHANGES /+6 5°C RIVER 2 C ^-.. FIG. 5. Schematic diagram of cooling water transit time and teynperatnre elevation at the Chalk Power Plant (in its origirml design) on the Patuxent estuary on Maryland Chesapeake Bay. MORGANTOWN 15 Minutes RIVER "*''"^ CONDENSERS EFFLUENT CANAL BOTTOM WATER TEMPERATURE CHANGES + 5.1°C /%. Tempering to 32 2°C RIVER BOTTOM WATER FIG. 6. Schematic diagram of cooling water transit time and temperature at the Morgantown Power Plant on the Potomac estuary on Chesapeake Bay. no H. HIDU, W. H. ROOSENBURG. K. G. DROBECK, A. J. MCERLEAN AND J. A. MIHURSKY Here, entrained forms will be exposed to very high temperatures within the condensers for about 1 minute and then cooled immediately to meet effluent temperature regulations. The ex- cursion in the effluent canal, before release again to the estuary, is expected to be less than 3 minutes. A shortening of exposure time, from hours to minutes, increases the temperature tolerance of oyster larvae by as much as 5-7 C. Even though the water temperatures may, by themselves, provide an adequate margin of safety, there could be other factors which may heighten temperature sensitivity of species. To establish a basis for proper regulations which will protect entrained forms will require the study of many aspects of the problem. These laboratory experiments should be augmented by depletion rate studies at specific power plant sites, effects of power plant biocides, the in- fluence of metals, turbulence from pumps, and rapid pressure changes. Laboratory experiments should test the most sensitive stages of various entrained forms, from planktonic primary and secondary producers to egg and larval stages of marine fishes. The wide differences between thermal tolerances of Mya larvae obtained from northern New England (Stickney, 1964), and those shown in this study in the Chesapeake area, as well as those of Loosanoff and Davis (1963) point out that studies and regulations should pertain to a specific geographical area. LITERATURE CITED Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Ent. 18:265-267. Brett, J. R. 1960. In: Thermal requirements of fish - three decades of study, 1940-1970. In: Biological Problems in Water Pollution, 27id Seminar, 1959. Robert A. Taft Sanitary En- gineering Center, Tech. Rept. W60-3, 110-117. Davis, H. C. 1958. Survival and growth of clam and oyster larvae at different salinities. Biol. Bull. Woods Hole 114:296-307 , and A. Calabrese. 1964. Combined ef- fects of temperature and salinity on develop- ment of eggs and growth of larvae of M. mercenaria and C. virginica. Fish. Bull. 63(3) 643-655. Finney, D. J. 1962. Probit analysis. Cambridge U. Press, Great Britain. 388 p., Illus. Hidu, H., K. G. Drobeck, E. A. Dunnington, Jr., W. H. Roosenburg, and R. L. Beckett. 1969. Oyster hatcheries for the Chesapeake Bay re- gion. N.R.I. Spec. Rept. No. 2, Contrib. No. 382, Natural Resources Inst., Univ. of Md., Solomons, Md. 18 p. Keller, E. C, Jr., C. S. Nagle, Jr., H. E. Keller, and D. C. Maxwell. 1968. The effects of saline-thermal-bacterial interactions on popu- lations of primary producers. Proc. Penn. Acad. Sci. 41:97-106. Loosanoff, V. L. 1937. Seasonal gonadal changes of adult clams, Venns mercenaria (L.). Biol. Bull, Woods Hole, 72:406-416. 1942. Seasonal gonadal changes in the adult oysters, Ostrea virginica, of Long Island Sound. Biol. Bull., Woods Hole, 82: 195-206. ., and H. C. Davis. 1963. Rearing of bi- valve mollusks. In: F. S. Russell (ed). Advances in Marine Biologv', Academic Press, London. 1:1-136. W. S. Miller, and P. B. Smith. 1951. Growth and setting of larvae of Venus mer- cenaria in relation to temperature. J. Mar. Res. 10:59-81. Mihursky, J. A., and V. S. Kennedy. 1967. Water temperature criteria to protect aquatic life. Symposium on Water Quality Criteria. Sept., 1966, E. L. Cooper (ed), Amer. Fish- eries Soc. Spec. Publ. 4:20-32. Pelseneer, P. 1901. Sur le degre d'eurythermie de certaines larvaes marines. Bull. Acad. Belg. CI. Sci. 279-292. Pfitzenmeyer, H. T. 1962. Periods of spawning and setting of the soft-shelled clam, Mya arenarki, at Solomons, Maryland. Chesapeake Sci. 3(2):114-120. Stickney, A. P. 1964. Salinity, temperature and food requirements of soft-shell clam larvae in laboratory culture. Ecology. 45 (2):283-291. Proceedings of the National Shellfisheries Association Volume BJt - 197^ A PROPOSED METHOD OF WASTE MANAGEMENT IN CLOSED-CYCLE MARICULTURE SYSTEMS THROUGH FOAM-FRACTIONATION AND CHLORINATION ' Ramesh C. Dwivedy AGRICULTURAL ENGINEERING DEPARTMENT UNIVERSITY OF DELAWARE NEWARK, DELAWARE ABSTRACT A scheme of waste management independent of bacterial filters in closed cycle mariculture systems was presented. It recommended foamfractionation for organic removal to prevent build-iip of high ammonia levels in the system. Breakpoint chlonnation was recommended for the removal of remaining low levels of ammonia. Dechlorinatioyi was achieved through carbon filtration. An algal production system can be easily coupled in the system if mollusks are to be cultured. The non-bacterial filters remove contaminant more rapidly and completely and are not limited by disadvantages associated with the bacterial filters, such as excessive space requirements and build up of high nitrate in the system. INTRODUCTION Biological filters have generally been con- sidered a satisfactory method of maintaining water quality in closed system mariculture. However, they have some serious drawbacks. One is excessive space requirements due to overdesigning for emergency situations. The second is the possibility of the bacterial population being destroyed and the need for subsequent time interval to re-establish the bac- teria. Third, biological filters convert ammonia to nitrate which builds up in the water. High nitrate concentrations in the water may be un- favorable to organisms and also may promote undesirable algal growrth. Any incidence of high mortality of marine life will tend to overload a biological filter, Published as Miscellaneous Publication No. 680 with the approval of the Director of the Delaware Agricultural Ex- periment Station. Publication No. 6' in the Department of Agricultural Engineering. resulting in poor quality and further loss of marine life. Decaying animal tissues have two adverse effects on closed, system mariculture: the reduction of oxygen and the production of toxic ammonia. An alternative to larger biological filters is to extract the tissues direct- ly through the use of a foam-fractionating device, commonly known as a protein -skimmer, to prevent the build up of ammonia. Previous work (Dwivedy, 1973) established that the foam-fractionation process can be em- ployed effectively to remove dissolved and suspended organics from the water and thus to prevent build up of ammonia. However, foam- fractionation of water does not remove am- monia from the system. An alternative to biological filtration to remove ammonia from the system would be to chlorinate the water. It is well documented that ammonia is removed from the water upon addition of a sufficient quantity of chlorine (Baummer et al. 1969; Grif- fin, 1944; Harvill et al, 1942; Streeter, 1943; Tchobanoglous, 1970). Ill 112 R. C. DWIVEDY The purpose of this study was to work out a scheme of waste management through the use of non-bacterial filters in closed cycle mariculture systems. CHARACTERISTICS OF WASTE IN A MARICULTURE SYSTEM Organics are the primary components of waste in a mariculture system. The organics give rise to toxic ammonia and bacteria and also their decomposition produces substances that lower the pH of the water. Ammonia is also directly excreted by some marine organisms. Oxygen depletion is another serious problem if organics are allowed to decompose in the system. To illustrate the characteristics of waste in a typical mariculture system, various sources and types of waste that may be ex- pected in an oyster culture system are: decom- posed meat from dead oysters, feed residue (dead or live algae) and excretion products. Typical values for the constituents of oyster meat as computed for 100 gms of meat are: 9.8 g of protein, 5.6 g of carbohydrate, 2.1 g of fat and 80.5 g of water (Galtsoff, 1964). Parsons, et al. (1961) reported the chemical ratios of several species of algal cells. The values for Dunaliella salina, for example, are 1.43 protein/carbon, 0.8 carbohydrate/carbon and 0.15 fat/carbon. These values are ratios of components to carbon present in the cells. Although data on the chemical composition of oyster fecal material are not available, it is reasonable to assume that it contains large proportions of proteins and other nitrogenous compounds. NON-BACTERIAL FILTERS Foam-Practioyiation Process for Waste Removal The foregoing explanation of the charac- teristics of waste indicates that one would ex- pect large amounts of proteins and other nitrogenous compounds in the system. It is well known that proteins and some other nitrogenous compounds are excellent foam- producing agents (Gaudin, 1957). The presence of fat in the water provides high viscosity and high surface activity. Large amounts of heterogenous ions, naturally present in salt water, aid in foam formation. The author has evaluated the foam-fractionation process in detail for contaminant removal in a closed-cycle mariculture system (Dwivedy, 1973). It was found that the process removed organic matter, bacteria, dust particles, algae and some weak acids. The removal of organic matter resulted in the prevention of ammonia build up in the system (Fig. 1). The foam-fractionation process helped maintain the pH of the system, since probably some weak acids were being removed with the foam. Since ammonia build-up from decay of organic matter is controlled by the use of a foam-fractionation unit, the removal of the remainder of ammonia by chlorination is a possibility. Capacity of tanka * 40 gal. Rata of flou through. f oan-f ractlonatlon** 3 gpm Amount of oyster meat la each tank ■ 80 gram w Control tank I ^^'' (without any / r*'^"'^ filtering device) /I / Experiaental tank \.' (with a foam-fractiona- \ tlon unit) 7 14 21 28 35 Length of Experljnent, Days FIG. 1. Control on ammonia through foam- fractionation. (Redraum. from Dimvedy, 1973). WASTE MANAGEMENT IN M.ARICULTURE SYSTEMS 113 Theory of Ammonia Removal ivith Chlorine Ammonia could be removed from water chemically by adding chlorine to form monochloramine and dichloramine as in- termediate products and nitrogen gas and hydrochloric acid as end products. According to Sawyer and McCarty (1967), when elemental chlorine is dissolved in water, the following equilibrium equation takes place: CI, + H,0 = HOCl + hVcI" (1) Hypochlorous acid (HOCl) reacts with am- monia to form chloramines. This is a stepwise reaction : NH ;, + HOCl = NH , CI + H , 0 (2) NH . CI + HOCl = NHCl , + H , 0 (3) NHCl , + HOCl = NQ :, "+ H , 6 (4) In the presence of excess ammonia (NHs), nitrogen trichloride (NCli) reacts with am- monia to form nitrogen gas and hydrochloric acid as end products; as shovra by the followdng equation: NQ 3 + NH 3 = N , + 3 HCl (5) According to Griffin (1944) the process can be summarized and the equations be written in terms of NH , and CI o , as presented below: 2NH ., + 2 CI ,, = 2NH , CI + 2HC1 (6) NH ,, CI + CI 2 = NHCl , + HCl (7) NH , CI + NHCl = N,+ 3 HCl (8) 2NH3 + 3C1 ,= N,, + HCl (9) From equation (9) the theoretical amount of chlorine required per mg/1 of ammonia is about 6.3 mg/1. In practice, however, Griffin (1944) and Baummer et al. (1969) found that a slight excess of 10 mg/1 of chlorine was re- quired for each mg/1 of ammonia. Breakpoint Chlorination for Ammonia Removal When chlorine is added to water containing ammonia, the oxidation of ammonia and reduc- tion of chlorine take place provided the molar ratio of chlorine to ammonia is greater than 1.0. A substantially complete oxidation-reduction process occurs at about 2:1 and, if allowed suf- ficient time, this reaction leads to the disap- pearance from water of all the ammonia and oxidizing chlorine. This effect is called the breakpoint phenomenon (Saw^yer and McCarty, 1967). ,. 0 O 0.5 1.0 1,5 2.0 Mi>les of chlorine added per nole of anmon: FIG. 2. An idealized schematic diagram of breakpoint chlorination (Redrawn from Sawyer and McCarty 1967). Fig. 2 shows that the molar ratios of chlorine to ammonia are less than 1.0 between points 1 and 2 and chlorine is all in the form of chloramine in this region. Beyond point 2, a dechlorinating action apparently takes place un- til complete oxidation-reduction occurs at the breakpoint. Thus at breakpoint, theoretically all ammonia and all chlorine are removed from the water. However, Griffin (1944), pointed out that this is not the case in practice (Fig. 3). Although am- monia is completely removed, some residual chlorine is left in the water beyond the break- point. Therefore, some dechlorinating device Chlorine Applied, P. P.M. FIG. 3.. Ammonia removal through breakpoint chlorination (Redrawn from Otiffiru 19W- 114 R. C. DWIVEDY must be used to reduce chlorine to a safe level before chlorine-treated water can be introduced into the culture tanks. A definite time interval is required for the reaction between chlorine and ammonia to reach completion. Harvill ct ai. (1942) found this time to be 15-20 minutes at pH 7.7 at 85 F. Baummer rt