1967 mOCEEDINGS

NATIONAL SHELLFISHERIES ASSOCIATION t Volume 58

LIBRARY ratorv JUL 9 1968

WOODS HOLE. MASS.

I EDITORIAL COMMITTEE

ASSOCIATE EDITORS

Dr. Jay D. Andrews Dr. Daniel B. Quayle Virginia Institute of Mcirine Science Fisheries Research Board of Canada Gloucester Point, Virginia Nanaimo, B. C, Canada

Dr. Kenneth J. Boss Dr. Sammy M. Ray Museum of Comparative Zoology A. & M. College of Texas Harvard University Marine Laboratory Cambridge, Massachusetts Galveston, Texas

Dr. A. F. Chestnut Dr. Aaron Rosenfield Institute of Fisheries Research Bureau of Commercial Fisheries Carolina University of North Biological Laboratory Morehead City, North Carolina Oxford, Maryland

Dr. James E. Hanks Dr. Rudolf S. Scheltema Bureau of Commercial Fisheries Woods Hole Oceanographic Institution Biological Laboratory Woods Hole, Massachusetts Milford, Connecticut

Dr. Carl J. Sindermann Dr. G. Robert Lunz Bureau of Commercial Fisheries Bear Bluff Laboratories Wadmelaw Island Biological Laboratory Oxford, South Carolina Maryland

Dr. Albert K. Dr. J. C. Medcof Sparks Fisheries Research Board of Canada College of Fisheries of Biological Station University Washington St. Andrews, N. B., Canada Seattle, Washington

Dr. D. Winston Menzel Dr. Harry W. Wells Oceanographic Institute Department of Biological Sciences Florida State University University of Delaware Tallahassee, Florida Newark, Delaware

EDITOR

Arthur S. Merrill Bureau of Commercial Fisheries Biological Laboratory Oxford, Maryland PROCEEDINGS

OF THE

NATIONAL SHELLFISHERIES ASSOCIATION

OFFICIAL PUBLICATION OF THE NATIONAL SHELLFISHERIES

ASSOCIATION ; AN ANNUAL JOURNAL DEVOTED TO SHELLFISHERY BIOLOGY

VOLUME 58

Published for the National Shellfisheries Association by

Economy Printing Co., Inc., Easton, Maryland

June 1968

PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

Volume 58 — June 1968 CONTENTS

List of Abstracts by Author of Technical Papers Presented at the 1967 NSA Convention Abstracts: NSA Convention 1 Pacific Coast Section 10 H. E. Crowther BCF Role in Farming of the Sea 16 David A. Adams The States' Role in Management of Sea Resources 19 Jay D. Andrews Mortality Studies in Virginia. VII. Review of Epizootiology and Origin of Minchinia nelsoni „ 23 Herbert Hidu and James E. Hanks Vital Staining of Bivalve Mollusk Shells with Alizarin Sodium Monosulfonate 37 Ronald E. Westley Relation of Hydrography and gigas Setting in Dabob Bay, Washington 42 J. F. Caddy and R. A. Chandler Accumulation of Paralytic Poison by the Rough ( undatum L.) 46 John A. Couch and Aaron Rosenfield Epizootiology of Minchinia costalis and Minchinia nelsoni in Introduced into

Chincoteague Bay, Virginia - _ „ 51 Evan B. Haynes Relation of Fecundity and Egg Length to Carapace Length in the King Crab, Paralithodes camtschatica _ _..._ 60 John W. Ropes Hermaphroditism in the Surf , Spisula solidissima _ _ 63 Walter T. Pereyra Distribution of Juvenile Tanner Crabs (.Chionoecetes tanneri Rathbun), Life History Model, and Fisheries Management „ 66 Don Maurer and Kent S. Price, Jr. Holding and Spawning Delaware Bay Oysters {Crassostrea virginica) out of Season. 1. Laboratory Facilities for Retarding Spawning _ 71 Bruce A. Rogers, J. Stanley Cobb and Nelson Marshall Size Comparisons of Inshore and Offshore Larvae of the Lobster, Homarus americanus, off Southern New England _ _ _ _ 78 T. E. Lovelace, H. Tubiash and R. R. Colwell Quantitative and Qualitative Commensal Bacterial Flora of Crassostrea virginica in Chesapake Bay , 82 R. G. Westenhouse Developments in the Methodology for Glycogen Determination in Oysters _ 88 Wariboko Q. B. West and Kenneth K. Chew Application of the Bergman-Jefferts Tag on the Spot Shrimp, Pandalus platyceros Brandt 93 Elgin A. Dunnington, Jr. Survival Time of Oysters after Burial at Various Temperatures 101 Association Affairs _ 105

iii

LIST OF ABSTRACTS BY AUTHOR OF TECHNICAL PAPERS PRESENTED AT THE 1967 NSA CONVENTION

Victor J. Cabelli and W. Paul Heffernan Seasonal Factors Relevant to Fecal Coliform Levels In Mercenaria merceriaria 1 Walter J. Canzonier Present Status of Attempts to Tt-ansmit Minchinia nelsoni Under Controlled Conditions 1 A. Russell Ceurvels Development of a Saltwater Embayment for Molluscan Research 1 L. Eugene Cronin, Robert P. Biggs and Hayes T. Pfitzenmeyer Relations of Spoil Disposal to Shellfish Areas 2 Elgin A. Dunnington, Jr. Potomac Fisheries: Potential and Opportunity ..- 2 John L. Dupuy and Albert K. Sparks Gonyaidax washingtonensis, its Relationship to Mijtilus caUfornianiis and Crassostrea gigas as a Source of Paralytic Shellfish Toxin in Sequim Bay, Washington 2 A. Eble and A. Rosenfield The Enzyme Histochemistry of the Sporulation of Minchinia nelsoni in

Crassostrea virginica . . 3 John D. Fiske

The Massachusetts Estuarine Research Program _ 3 Adel Hamwi Pumping Rate of Mercenaria mercenaria as a Function of Salinity and Temperature 4 Dexter S. Haven and Kenneth W. Turgeon Influence of Small Quantities of Cornstarch and Dextrose on Glycogen Levels of Crassostrea virginica 4 Herbert Hidu Inshore Settlements of Crassostrea virginica in Delaware Bay 4 Victor S. Kennedy Temperature Mortality Studies on Mya arenaria 5 Warren S. Landers and Edwin W. Rhodes, Jr. Growth of Young , Mercenaria mercenaria, in Tanks of Running Sea Water 5 Clyde L. MacKenzie, Jr. Feeding Rates of , Asterias forbesi (Desor), at Controlled Water Temperatures, and During Different Seasons of the Year 6 R. W. Menzel

Hybridization in Species of Crassosf?-ea _ 6 John L. Myhre Some Cytochemical Observations on Miytchinia nelsoni Haskin, Stauber and Mackin, a Sporozoan Parasite of Crassostrea virginica 6 John L. Myhre and Harold H. Haskin Some Observations on the Development of Early Minchinia nelsoni infections in

Crassostrea virginica, and Some Aspects of the Host-Parasite Relationship . 7 Maria Panciera The Toxicity of Rhodamine-B to Eggs and Larvae of Crassostrea virginica 7 Maynard W. Presnell, Joseph M. Cummins and John J. Miescier Influence of Selected Environmental Factors on the Elimination of Bacteria by Crassotrea virginica 8 John W. Ropes

The Reproductive Cycle of Surf Clams from the New Jersey Coast _ 8 Carl N. Schuster, Jr. What's with a Salt Marsh? 9 Carl N. Schuster, Jr. and Benjamin H. Pringle Trace Metal Accumulation by the Oyster - - - _ 9 William N. Shaw Farming Oysters in Artificial Ponds — Its Problems and Possibilities 9 Webb Van Winkle The Effects of Additives on the Pumping Rate of Mercenaria mercenaria 9 Robert M. Yancey Surf Clam Research, 1966 - 10

ABSTRACTS NSA PACIFIC COAST SECTION

Muzammil Ahmed and Albert K. Sparks Chromosomes of Oysters, Clams and 10 C. Dale Becker and Gilbert B. Pauley A Parasite from the Ova of Crassostrea gigas 11 Terence H. Butler British Columbia Fisheries and Research Projects — Past and Present 11 David M. DesVoigne and Albert K. Sparks Wound Repair In Crassostrea gigas - - 11 John B. Glude The Effects of Oil from the Wrecked Tanker, Torrey Canyon, on Shellfisheries Resources of England and 11 C. Lynn Goodwin Subtidal Shellfisheries Development - - 12 R. B. Herrmann Seasonal Changes in the Condition of Crassostrea gigas and Possible Relations to Nutrient Sources _ - — - - - 12 Ethelwyn G. Hoffman Description of Laboratory-Reared Larvae oi Paralithodes platypus (Brandt) 13 Gilbert B. Pauley The Pathology of "Spongy" Disease in Freshwater Mussels - 13 Vincent A. Price and Kenneth K. Chew Post-Embryonic Development of Laboratory-Reared "Spot" Shrimp, Pandalus platyceros Brandt , - 13 Paul H. Reed Larval Rearing Studies of the Dungeness Crab, Cancer magister 13 C. S. Sayce and D. F. Tufts The Supplemental Feeding of Crassostrea gigas in the Laboratory - 14 Herb C. Tegelberg Some Effects of a Dense Razor Clam Set in Washington - 14 G. J. Vasconcelos, J. C. Hoff and T. H. Ericksen Bacterial Accumulation-Elimination Response of Pacific Oysters (Crassostrea gigas) and Manila Clams {Tapes philippinarum) Maintained under Commercial Wet 14 Storage Conditions - - - Michael E. Weatherby Studies on the Japanese Oyster Drill _ _ - 15

VI Proceedings of the National Shellfisheries Association Vohnne 58 — Jiaie 1968

ABSTRACTS OF TECHNICAL PAPERS PRESENTED

AT THE 1967 NSA CONVENTION

SEASONAL FACTORS RELEVANT TO disease agent of Crassosfrea virginica. Attempts to FECAL COLIFORM LEVELS IN infect have involved a variety of procedures. Among MERCENARIA MERCENARIA these are proximity to infected oysters or sus- pected alternate hosts, feeding with infected ma- Victor J. Cabelli and W. Paul Heffernan terial both forced and as a suspension in the Northeast Marine Health Sciences Laboratory aquarium water, inoculation into various sites such PHS, DHBW as muscle, heart, visceral mass and mantle cavity, Narragansett, Rhode Island and implantation of infected gill and mantle tissue. Failure to achieve infections has been a con- It is well known that during the winter in spicuous feature of all of these attempts. In spite latitudes where the water temperature falls below of failure in the primary objective, these experi- 10° C for considerable periods of time, very few ments have contributed to our knowledge of M. coliform organisms can be recovered from Mer- nelsoni. It has been established that: cenaria mercenaria even though they are growing M. nelsoni Plasmodia will persist in infected in heavily polluted waters. This could be due to a oysters kept in aquaria for at least 6 months more marked effect of low temperatures on up- though no multiplication was indicated. take as contrasted to elimination, resulting in a Natural infections can occur in running sea- gradual decrease of the organisms within the water systems under some conditions. . A second possibility is death of the organ- M. nelsoni Plasmodia can be transferred to a isms within the animal. In this report the effect of susceptible host and persist for as as 8 seasonal changes on the presence of fecal coliforms long days in the circulatory system. in from a polluted area is documented. The Both normal and infected tissue be trans- more marked inhibitory action by temperatures of may planted into a susceptible host, the Plasmodia 10° C and lower and turbidities greater than 10 ap- pearing to remain viable up to 2 months, Jackson turbidity units (JTU) on uptake as com- though infections do not spread to and develop in the host. pared to elimination of Escherichia coli by the There is some indication of a toxic factor asso- quahaug is demonstrated. Data on the effect of ciated with infected tissue. seasonal changes on the experimental uptake and elimination of E. coli by the animal are presented. Possible bases for these differences are discussed. DEVELOPMENT OF A SALTWATER EMBAYMENT FOR MOLLUSCAN RESEARCH PRESENT STATUS OF ATTEMPTS TO TRANSMIT MINCHINIA NELSONI UNDER A. Russell Ceurvels CONTROLLED CONDITIONS Division of Marine Fisheries Department of Natural Resources Walter J. Canzonier Boston, Massachusetts Oyster Research Laboratory The Massachusetts of Natural Re- N. J. Agricultural Experiment Station Department sources, Division of Marine Fisheries, has Rutgers University, New Jersey recently acquired an 8-acre saltwater pool and 8 acres of The value of a method for producing infections upland. The Division plans to develop this area of Minchinia nelsoni consistently under controlled for a marine laboratory and shellfish culture conditions in the laboratory has been appreciated station with emphasis on shellfish research. Larval since the early stages of investigation of this shellfish will be available in limited quantities to ABSTRACTS the cities and towns of Massachusetts for the re- GONYAULAX WASHINGTONENSIS, ITS habilitation of depleted shellfish areas. Particular RELATIONSHIP TO MYTILUS CALIFORNI- consideration will be given to the science of aqua- ANUS AND CRASSOSTREA GIGAS AS A culture as a sound economic use of the resources. SOURCE OF PARALYTIC SHELLFISH TOXIN IN SEQUIM BAY, WASHINGTON

John L. Dupuy ' and Albert K. Sparks RELATIONS OF SPOIL DISPOSAL TO SHELLFISH AREAS College of Fisheries University of Washingt07i L. Eugene Cronin, Robert B. Biggs Seattle, Washington and Hayes T. Pfitzenmeyer Several aspects of the relationship of Mytilus University of Maryland californian)is and Crassostrea gigas to the toxic Natural Resources Institute dinoflagellate. Gonyaulax washingtonensis, from Chesapeake Biological Laboratory Sequim Bay on the Strait of Juan de Fuca have Solomons, Maryland been studied using unialgal mass cultures for feed- ing experiments. Recent observations on the shallow-water dis- Five feeding experiments were conducted in posal of dredged fine-grain sediments from the vvhich mussels iM. californianus) received G. wash- upper Chesapeake Bay provide information use- ingtonensis at a concentration of 50 cells/ml. The ful in the selection of spoil disposal sites. Field feeding of these cells with a known amount of observations of the distribution of fine sediments toxin per cell to the has shown that when in water, the deposition of sediments on the bot- the standard extraction and mouse bio-assay is tom, and the short-term spread of those sediments used. 75 to 92 per cent of the toxin is found in the over an unexpectedly wide area was described. shellfish meats. This relatively vigorous uptake The possibile effects of such sediment on shellfish, of to.xin by the mussel is similar to the uptake and responses of some organisms to de- possibile under natural conditions. position was discussed, with special reference to In contrast, feeding and water pumping rate the upper Chesapeake Bay. Guidelines were sug- experiments with the oyster (C gigas) demonstra- gested for assuring adequate protection of shell- ted no measurable uptake of toxin after 3 months. fish beds and other important benthic communities. Two types of reaction have been observed to occur when the oyster was fed G. washingtonensis. The most common reaction was drastic curtail- FISHERIES: POTENTIAL POTOMAC ment of the volume of water filtered, with vigorous AND OPPORTUNITY clapping of the valves or complete cessation of Elgin A. Dunnington, Jr. pumping activity. The second reaction recorded was a 30 per cent reduction in pumping rates University of Maryland with large amounts of pseudofeces being produced. Natural Resources Institute No feces containing G. washingtonensis were ob- Chesapeake Biological Laboratory served. Concentrations of 20, 40, 80, and 120 Solomons, Maryland cells/ml curtailed or caused cessation of the During the four years since the implementation pumping activity. Culture medium, with cells re- of the Potomac River Compact of 1958 by the moved, added to filtered sea water did not ap- formation of the Potomac River Fisheries Commis- pear to affect the oyster's pumping rate. This sion, the commercial fisheries of this largest general physiological reaction would tend to lend tributary of Chesapeake Bay have been under support to the hypothesis that oysters under field joint Maryland and Virginia management. With conditions refuse to accept G. washingtonensis the assistance of both states, the new agency has initially but after acclimation partially accept this begun a progressive fisheries management pro- dinoflagellate as food. Results from sampling C. gram. Prior to this, disagreements between the gigas at Sequim Bay has shown a 2 week delay states concerning the utilization of Potomac River in its uptake of paralytic shellfish poison in com- fisheries had prevented effective management of parison to the . Furthermore, re- these valuable resources. The Potomac River sults from bio-assay of field samples have shown Fisheries Commission uses research and manage- that the levels of toxin in C. gigas are 3 to 4 times ment advice from Maryland and Virginia as a lower than in M. californianus. basis for planning and operations. This coopera- tive effort offers the promise of increased sea- I Present address: food production in an area having a great fishery Virginia Institute of Marine Science potential. Gloucester Point, Virginia. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION THE ENZYME HISTOCHEMISTRY OF THE Fisheries has been engaged in an extensive estu- SPORULATION OF MINCHINIA NELSONI IN arine research program. This program was pro- CRASS08TREA VIRGINICA < posed by the Marine Fisheries Advisory Commis- sion in December, 1960. Funds for the A. Eble and A. Rosenfield program were earmarked by the Legislature in 1962. The of the Department of Biological Sciences, Mercer objectives program as outlined in the Commission's County Community College, Trenton, New Jersey advisory report were; and 1. to provide comprehensive information U. S. Bureau of Commercial Fisheries about the current condition of the fish- Biological Laboratory, Oxford, Maryland eries in the inshore waters of the Com- monwealth, Five enzymes were surveyed for reactivity 2. to determine the factors responsible for throughout the sporulation process of Minchinia the decline of fishing industries in cer- nelsoni in Crajssostrea virginica: NAD diaphorase, tain areas, and non-specific esterase, acid and alkline phosphatase 3. to suggest methods of and malic dehydrogenase. Plasmodia and sporonts developing or improving local marine were generally reactive for all the enzymes fishery produc- tion. studied. There was usually a marked increase in With these objectives in the enzyme activity in the sporont stage relative to mind, Division is evaluating all of Massachusetts' the Plasmodium; this was probably associated with major estuaries. To date, 9 estuary studies have been an increase in metabolic activity as the sporula- completed. Two more are in tion process started. Early sporocysts manifested presently progress. The major study phases in each a strong activity for mitochondrial enzymes, NAD estuary consist of: 1) Re- view of the history of the marine diaphorase and malic dehydrogenase, but ap- fishery; 2) analysis of physical and chemical peared less reactive for the other enzymes. The characteristics; 3) inventory of the distribution and utilization of cytoplasm of the later sporocyst became quite shellfish, and 4) of the reactive for alkaline phosphatase, concentrations inventory distribution and utilization of finfish and crustacean of which could be seen clinging to the cytoplasm populations. In evaluating the shellfish resources of surrounding the mature spore walls. Acid phos- estuaries, primary concern has been to phatase present a similar picture. Mitochondrial given determining the volume and value of enzymes appeared highly reactive in the cytoplasm shellfish harvest and, of the whenever possible, to define harvests in terms of sporocyst especially in the immediate vicinity acre production. For instance, in the Pleasant of the spore, but sparsely distributed in the sporo- Bay estuary, a total of 11,255 bushels of plasm of the mature spore; usually 2-6 mito- quahaugs were harvested a chondria could be observed per spore. Concentra- during single year from about 640 acres of bottom. tions of non-specific esterase fell sharply as the Acreage production was valued at about 199 dollars sporocyst matured, although isolated granules per acre. In another study area, the value of the were observed in the cytoplasm of the sporocyst harvest was 371 dollars per acre. In contaminated as well as the sporoplasm of the mature spore. estu- aries, shellfish density studies have Oyster tissues infected with the sporulating pointed out the of extensive loss in resource utilization which stages Minchinia nelsoni (tubules of the diges- occurs because of tive gland) appeared to maintain a near-normal pollution. Biologists studying the Merrimack River estuary estimated that over level of enzyme activity; however, heavy concen- 300.000 dollars worth of soft-shell clams were not trations of the parasite all but replaced host being utilized because of tissue, leaving little glandular tissue available for annually pollution. In it was digestion. Quincy Bay, noted that with pollution abatement, the value of the soft-shell clam harvest could increase by about 35,000 dollars annually. Published estuarine reports are as THE MASSACHUSETTS serving ESTUARINE guideposts for the wisest management and utiliza- RESEARCH tion of PROGRAM our estuarine resources, as well as provid- ing direction for future estuarine research in John D. Fiske Massachusetts.

Department of Natural Resources Division of Marine Fisheries I This Boston, Massachusetts research was supported by Contract 14-17- 0003-111 with the U. S. Bureau of Since Commercial 1963, the Massachusetts Division of Marine Fisheries. ABSTRACTS

PUMPING RATE OF MERCENARIA tent, wet meat weight, shell volume, total volume, MERCENARIA AS A FUNCTION OF underwater weight, and air weight than controls. SALINITY AND TEMPERATURE Other supplements significantly increased gly- cogen content over the controls but had little effect Adel Hamwi on other growth parameters. Oyster Research Laboratory and Department of Zoology Rutgers University, New Jersey INSHORE SETTLEMENT OF Pumping rates of clams acclimated to tempera- CRASSOSTREA VIRGINICA tures ranging between 6 and 32= C and salinities IN DELAWARE BAY between 15 and 40 ppt were measured by a Herbert Hidu ' metered stream of sea water, colored by soluble non-toxic which dye, completely replaced the New Jersey Oyster Research Laboratory incurrent flow. Determinations were made at the Rutgers the State University acclimation and and also temperatures salinities, New Brunswick, New Jersey after sudden changes of salinity to higher or lower levels. Factors were defined which may be important The results indicated: in producing the consistently heavy oyster settle- ment on the Delaware tidal flats the (1) At a salinity of 25 ± 2 ppt: Bay along Pumping was completely inhibited at temperatures west coast of the Cape May Peninsula. A transect below 6= C and above 32° C. of 12 stations extending 4 5 mile offshore was for settlement over two seasons with A moderate and steady increase in the rate of measured water pumping occurred as temperature was raised from cement-board panels placed throughout the 7 to 12=' C. column. Oysters set heavily at the extreme inshore intertidal area and also 1/2 mile offshore at a No marked changes in the pumping rate were ob- the flats from served between 12° C and 18° C. slope area which separates deeper water. The settlement area coincided with Maximum pumping occurred at temperatures of slope larval concentrations. At the inshore 24 to 26° C. high oyster area, however, similar setting rates were asso- There was an abrupt decrease in pumping rate at ciated with a tenth of the larval concentra- temperatures above 26° C. only tion, indicating existence here of factors promot- (2) At temperature of 25 ± 1° C: ing stimulation of set. Pumping was completely inhibited below 15 ppt The offshore slope was the area of steepest and above 40 ppt. gradient in current velocity. The most inshore Maximum pumping rate occurred in the salinity water mass was characterized by relatively great range of 23 to 27 ppt. salinity and temperature increases at early flood The lower and upper salinity boundaries for 50 . per cent of the maximum pumping rate were larvae, when subjected to 18 ppt and 31 ppt respectively for nonacclimated Laboratory-reared similar salinity and temperature increases, were clams, 17 ppt and 34 ppt respectively for acclim- ated clams. stimulated to settle in response to the temperature, but not the salinity factor. A gregarious tendency in settlement, however, reduced the statistical significance of these findings. Most of the set INFLUENCE OF SMALL QUANTITIES OF was generally received on one of several exposed CORNSTARCH AND DEXTROSE ON cultch shells and often a heavy set was received GLYCOGEN LEVELS OF which was not related to experimental treatment. CRASSOSTREA VIRGINICA Further investigation of gregarious setting re- Dexter S. Haven and Kenneth W. Turgeon sponse revealed that individual cultch shells were not differentially preferable in oyster settlement. Virginia Institute of Marine Science However, the presence of 24-hour spat and 2month- Gloucester Point, Virginia old spat on cultch shells attracted set differentially Feeding experiments were carried out to deter- and stimulated settlement. mine the effect of cornstarch and dextrose on The offshore slope area may be important in the glycogen content and growth of Crassostrea vir- primary concentration of larvae. Inshore settle- ginica. Statistical analyses showed that starch- ment then probably is promoted by factors which fed oysters had significantly greater glycogen con- may draw in offshore larvae, such as easterly PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION winds and spring tides, and factors which stim- ard technique for rearing clam larvae. Unresolved, ulate settlement, such as a temperature increase however, is the most dependable way to rear post- and the presence of oyster populations which may set clams to a size at which they have gained con- promote a gregarious setting response. siderable immunity to predation by their principal enemy, the crab. Measures which will effectively protect recently-set clams in the field have not yet I Present address: University of Maryland, Na- been developed. An alternative approach to the tural Resources Institute, Chesapeake Biological problem is to rear the young clams to the proper Laboratory, Solomons, Maryland. size in the hatchery. The following account de- scribes our observations on the growth of young clams in sand in rectangular tanks 30 feet long and TEMPERATURE MORTALITY STUDIES 4 feet wide through which sea water flowed ON MYA A RENARIA continuously. Small clams grew faster than did larger clams. Victor S. Kennedy Growth increments of equal volumes of clams 18, 13, 10 and 8 mm long for periods of 30 to 117 days Hallowing Point Field Station produced the following growth ratios, respectively: Prince Frederick, Maryland 1.0, 1.9, 2.7 and 2.9. Recent growth estimates of the steam electric Clams from 10 to 18 mm long grew equally well industry reveal a 30 to possibly 256 times increase at planting densities of 8, 16 and 67 ml of clams foot of substrate. as num- in electricity requirements by the year 2010. This per square Expressed industry has at present the greatest non-consump- ber of clams per square foot planting density tive industrial demand for water as a heat trans- ranged from 8 clams 18 mm long to 428 clams 10 fer medium. The need for large volumes of cool- mm long. ing water is resulting in the location of many Clams from 3 to 18 mm long grew equally fast new installations on marine and estuarine en- at water flows from 15 to 56 gpm. At 9 gpm the vironments. There is a growing need for ecolog- growth rate of clams 8 to 18 mm long decreased ical assessment of heated water discharges in the by about 33 per cent and that of clams 2 to 5 mm aquatic environment, especially the effects on im- long, over 50 per cent. portant commercial and recreational species. When the tanks were fully occupied with shell- Studies of Mya arenaria, an important com- fish, growth of clams of all sizes was considerably mercial shellfish on the east coast have been better at all flow rates at the intake end of the initiated. These studies have used bio-assay techni- tank than at the discharge end; for example, ques to determine temperature mortality levels of clams approximately 12 mm long, at times, grew Mya that were held at a series of acclimation tem- almost twice as fast in the former location as in peratures ranging from 1° C to 30° C in 5-' incre- the latter. ments. Young-of-the-year clams and adults have Animal fouling in the tanks, especially by been used and the results indicate that, in gen- barnacles, mussels and tunicates, was severe. eral, Mya are not temperature resistant under Irradiating the water with ultraviolet light before summer conditions. Increasing acclimation tem- it entered the tanks controlled mussels but not peratures do not raise the lethal level to any barnacles or tunicates. Dense clusters of mussels extent. Young-of-the-year organisms are slightly which invariably concentrated near the intake of more resistant than adults. These results were dis- the untreated water tanks apparently reduced the cussed in relation to thermal loading. growth of clams throughout the tanks. Growth in these tanks was roughly half as much as in tanks receiving irradiated water. GROWTH OF YOUNG CLAMS, MERCENARIA Growth of clams in the tanks compared favor- MERCENARIA.IN TANKS OF ably with growth of transplanted clams in Mil- RUNNING SEA WATER ford Harbor, Connecticut, in Wickford Harbor, Rhode Island (Landers, unpublished data), and in S. Landers and Jr. Warren Edwin W. Rhodes, Home Pond, Gardiners Island, New York K

U. S. Bureau of Commercial Fisheries Biological Laboratory I Carrlker, Melbourne R. 1959. The role of physical Milford, Connecticut and biological factors in the culture of Crasso- The well-tested mass culture method for rearing strea and Mercenaria in a salt-water pond. Ecol. young oysters to metamorphosis is also the stand- Monogr. 29:219-266. ABSTRACTS

FEEDING RATES OF STARFISH, ASTERIAS SOME CYTOCHEMICAL OBSERVATIONS ON FORBESI (DESOR), AT CONTROLLED WATER MINCHINIA NELSONI. HASKIN, STAUBER, TEMPERATURES, AND DURING DIFFERENT AND MACKIN, A SPOROZOAN PARASITE OF SEASONS OF THE YEAR CRASSOSTREA VIRGINICA L. Clyde MacKenzie, Jr. John L. Myhre U. S. Bureau of Commercial Fisheries Oyster Research Laboratory Biological Laboratory N. J. Agricultural Experiment Station Milford, Connecticut Rutgers University, New Jersey Starfish were held for 28 days in trays of running sea water maintained at a series of con- The cytochemistry of Minchinia nelsoni was iCrassostrea stant temperatures. Ample numbers of oysters studied in infected oyster virginica) tissues. Nucleic acids were were provided as food. During this period, at 5.0°C, blood and in sectioned nucleal Azure each starfish ate an average of 2.3 oysters, at investigated with the Feulgen test. Three 10.0°C, 3 oysters; at 15.0°C, 4.2 oysters; at B differential stain, and enzymic digestion. were observed in M. 20.0°C, 5.0 oysters; at 22.5°C, 2.8 oysters, and at Feulgen-positive structures film-like 25.0°C, 1 oyster. Thus, the optimum temperature nelsoni: (1) A spherical, (Feulgen-posi- for feeding was 20.0 -C. Starfish lost weight when tive) structure containing evenly spaced, dense, on its surface. held with oysters at temperatures above 23.5 C. Feulgen-positive concentrations structure was found in at Observations of variations in seasonal feeding rates This nuclei, generally but often more were made from trays holding 20 starfish with the nuclear-cytoplasmic interface, oysters suspended in Milford Harbor. Starfish fed centrally located in large nuclei. The Azure B at low rates from mid-January to the end of March. stain and enzymic digestion confirmed the pres- ence of DNA. (2) A mass From then until late June or early July, feeding dense, Feulgen-positive of variable size (0.2 - and is often increased rapidly. After spawning, starfish fed at 4.0ju,) shape three- about one-third of the rate recorded in late June. observed in a single parasite. During a as few as 4 The period of low feeding, which appeared to be month period, September-November, cent of the associated with both high temperatures (above per cent and as many as 60 per were found to 22.5- C.) and spawning, lasted from July through parasites from individual oysters September. From late October, through December, have such structures. The Azure B stain and en- mass. (3) the rate of feeding increased again to about two- zymic digestion confirmed DNA in this of uncertain thirds of its level in late June and early July. The Intensely Feulgen-positive spheres were rate again became low by mid-January. location, but apparently in the c>^oplasm, infrequently found. The spheres, when present, were in great abundance in individual oysters. have been observed in blood HYBRIDIZATION IN SPECIES OF They only prepara- tions of infected oysters, never in tissue sections CRASSOSTREA i from these same oysters. Because of this labile R. W. Menzel characteristic, it has not been possible to confirm The structures are Florida State University DNA by enzymic digestion. to at the nuclear DNA con- Tallahassee, Florida thought originate centrations at the nuclear-cytoplasmic interface, have been made to Crasso- Attempts hybridize and may be the DNA of a newly-formed nucleus. strea angulata, C. cornmercialis, C. gigas, C. The presence of RNA was determined with the C. and C. in all iredalei, rhisophorae, virginica pos- Azure B differential stain and confirmed with sible combinations. Fertlization and cell cleavage enzymic digestion. The cytoplasm of M. nelsoni occurred in all reciprocal crosses except when C. varied from finely granular, rich in RNA, to fairly cornmercialis was one of the parents. The egg and clear, containing coarse RNA granules. sperm of this species were totally incompatible The nuclear-cytoplasmic interface was rich in with all the other The larvae of all the species. RNA, often masking the DNA found in this loca- crosses developed to the umbo stage and limited tion. The most commonly observed nucleus in success has been obtained in securing attachment - M. nelsoni was roughly spherical, about 1 2 ^ of the several hybrids. Meiotic chromosomes have in size with one prominent peripheral endosome been examined in all the species and mitosis in and a variable number of small, secondary periph- the species and hybrids. All species have a diploid eral endosomes. The secondary endosomes corre- number of 20. spond in size and location to the concentrations of DNA at the nuclear-cytoplasmic interface. The I Supported by NSF Grant GB-5034 primary endosome is Feulgen-negative and rich in PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

RNA. In larger nuclei the primary and secondary epithelium of the water tubes and enters the endosomes are generally more centrally located. epithelium of intraplical water spaces, passes The nucleoproteins were investigated with the through the ostia and infects the epithelium of the Alkaline Fast-Green procedure of Alfert and gill filaments. At the base of an infected demi- Geschwind for the detection of histone, and the branch, the parasite continues its surface growth Picric Acid-Eosin Y procedure for protamine. by infecting the base of adjacent demibranchs, the Histone was found in all confirmed DNA loca- palps, or the mantle epithelium. tions. The primary endosome and often the entire This epizoic stage lasted about one month after nucleus give a positive reaction for protamine. which some parasites penetrated to subepithelial The Periodic Acid Schiff (PAS) test for carbo- tissues. The time of penetration varied from oyster hydrates was consistently negative for the nucleus. to oyster and suggested the possibility of an The cytoplasm generally contains fine PAS-posi- "epithelial barrier". Epithelial sloughing is often tive granules. associated with advanced lesions, and some lesions have large accumulations of leucocytes outside the gills, indicating that widespread ciliary paralysis SOME OBSERVATIONS ON THE DEVELOP- may occur. Observable host response to the lesion is infiltration of the infected often MENT OF EARLY MINCHINIA NELSON! IN- leucocytic area, with of the tissue and of FECTIONS IN CRASSOSTREA VIRGINICA, AND swelling phagocytosis the SOME ASPECTS OF THE HOST-PARASITE parasite. RELATIONSHIP In some oysters many of the parasites have a sharply delineated outer membrane, often accom- John L. Myhre and Harold H. Haskin panied by a withdrawal of cytoplasm from the membrane. This condition is associated with an Oyster Research Laboratory, N. J. Agricultural increase in the size of the parasite nuclei and the Experiment Station and Department of Zoology, appearance of coarse cytoplasmic RNA granules. Rutgers University, New Jersey When phagocytized, M. nelsoni generally appears Data from 4 lots of oysters [Crassostrea virg- condensed with densely staining nuclei and cyto- inica) imported to the Cape Shore flats of plasm. Delaware Bay at various times during the sum- mer of 1966 indicate that one lot became infected with Minchinia nelsoni sometime during the first THE TOXICITY OF RHODAMINE-B TO EGGS 4 days of exposure (July 2-5). Another of the AND LARVAE OF CRASSOSTREA VIRGINICA 4 lots imported on July 6 indicates that few, if any, new infections occurred for more than a Maria Panciera month following this 4-day infection period. This U. S. Bureau of Commercial Fisheries chance occurrence provided the closest approach Biological Laboratory yet to a controlled infection experiment. Milford, Connecticut Individual oysters in the infected lot were com- pared as to number, location, size and shape of Rhodamine-b. a fluorescent dye, is being used the developing lesions; the appearance and rela- extensively to trace water movements in estuarine tive number of parasites in each lesion; and the environments. Since the larvae of various com- host response to each lesion. mercially important shellfish comprise an im- Minchina nelsoni infections originate from a portant segment of the biota of estuaries, it is small locus, possibly a single infective particle, in worthwhile to determine the effect of this dye the water tubes of the gills. Only one lesion was ob- on their survival and growth. served per oyster with the exception of one oys- Observations were made on the toxicity of the ter which had two separate lesions. This may dye to eggs and larvae of Crassostrea i-irginica. indicate that the oyster encounters few infective Six decimal dilutions of Rhodamine-b, ranging particles or that few of the total number of parti- from 100 ppm to 0.001 ppm, were tested in the cles establish an infection. laboratory. Eggs were exposed to the dye for 48 The fastest growth rate occurs in the earliest hours. Larval exposures were begun with 2-day-old detectable lesions. A generation time (doubling of individuals and maintained in the appropriate the population) of about 24 hours was calculated. concentration of dye until all animals died, or The parasite appears to be "epizoic" in these metamorphosis began. small early lesions, being restricted to the laby- At 100 ppm Rhodamine-b, no eggs developed. At rinth of folds and crevices formed by the finger- 10 ppm there was some development to the like projections of the epithelium of the water straight-hinge stage, but 27 per cent of the in- tubes. The parasite spreads up and down the dividuals were abnormal. Development at con- 8 ABSTRACTS

centrations of 1 ppm or lower was comparable to form bacteria at varying rates. The rate of coli- control cultures. form and fecal coliform elimination was much Larvae exposed to 100 ppm Rhodamine-b died greater when salinities exceeded 16 ppt. With within 2 days. At 10 ppm they survived as well as salinities below 7 ppt the rate and extent of coli- those in control cultures but their growth was form and fecal coliform elimination were greatly retarded 10-17 This retarding effect reduced. initially by fj,. on growth was temporary, occurring during the Results of salinity studies indicated that the first 2-3 days. Throughout the rest of the experi- time required to achieve satisfactory levels of oys- ment their rate of growth was equal to that of ter depuration was dependent, to a great extent, the controls. Survival and growth of oyster larvae on salinity. Development of a method(s) of sal- at concentrations of 1 ppm or lower was compar- inity control, nondeleterious to shellfish activity, able to that in the control cultures during the test could be important in a plant design that would period. permit more precise prediction of acceptable end- point depuration. Such a method! s) could also per- mit establishment and continuous operation of de- INFLUENCE OF SELECTED ENVIRONMENTAL puration plants in shellfish areas subject to re- FACTORS ON THE ELIMINATION OF curring periods of low salinity. BACTERIA BY CRASSOSTREA VIRGINICA Turbidity study: Data from this study demon- strated that the per cent reductions of Esrherwhia W. Presnell, M. Cummins Maynard Joseph coli after 24 hours of elimination were comparable and John J. Miescier in oysters exposed to sea water having average turbidities of 22.3 and 69.4 Jackson Gulf Coast Marine Health Scienres Laboratory 8.8, 19.3, Pnblir Health Service Turbidity Units (JTU). The difference in reduction of E. coli in to 69.4 JTU and 8.8 Dauphin Island. Alabama oysters exposed JTU turbidities was 0.002 per cent. Results would Studies were conducted to determine the influ- indicate that a highly effective filtration system ence of seawater flow rates, water temperature, will not be needed for oyster depuration plants on salinity, and turbidity on the rate of bacterial the Gulf Coast. elimination by the oyster, Crassostrea virginica. Seaivater flow rate study: Results indicated that with proper system design and physiologically THE REPRODUCTIVE CYCLE OF SURF CLAMS favorable conditions of water temperature and FROM THE NEW JERSEY COAST rates of coliform and fecal salinity, comparable John W. Ropes coliform elimination by the oyster could be ob- U. S. Bureau Commercial Fisheries tained with seawater flow rates ranging from of 0.5 to 5.0 liter/oyster/hr. Biological Laboratory Water temperature study: Oysters acclimated to Oxford, Maryland warmer waters (26.7-29.6^0 and transferred to During the past 3 1/2 years nearly 2,500 gonad

cooler waters ( 18.9-23.4'^ C) eliminated coliform tissues of Spisula solidissima (Dillwyn) from the bacteria at a lower rate throughout 48 hours of New Jersey fishery have been collected and pro- exposure than oysters retained in the warmer cessed for histological examination to determine waters. The rate and extent of fecal coliform the seasonal reproductive cycle. The adult clams, elimination were comparable at both of the above with a shell length of 120 mm or more, were caught temperature ranges. by commercial hydraulic dredges in the vicinity of Transferring oysters acclimated to cooler waters Barnegat Lightship, at water depths of 18 to 32 m. (16.3-22.0'^C) to warmer water (24.4-35.1 = 0) had Samples of 25 clams were taken monthly in the no apparent influence on the rate of bacterial winter and bimonthly during the spring, summer, elimination after the first 24 hours of exposure. and fall when active gametogenesis and spawning During this 24-hour interval, oysters exposed to occur. the warmer waters eliminated bacteria at a higher Five arbitrary categories were established to rate. describe stages of the reproductive cycle. The time Experiments conducted to date have employed and duration of spawning is basic information temperatures in excess of 16°C. Further studies necessary to understand the time of larval occur- are needed to determine the influence of lower rence in the water and the eventual settling of temperatures on bacterial elimination rates by the juveniles on the bottom. The categories are: early oyster. active gametogenesis, late active gametogenesis, Salinity studies: Oysters exposed to salinities of ripe, partially spent, and spent. 5.8 to 25.8 ppt eliminated coliform and fecal coli- A biannual gamete maturation sequence cul- PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

in the minating spent condition indicated that the 2.5 gpm flow rate and water temperature at 20 it clams had 2 spawning cycles each year. Spent 1°C; salinity varied, 31 ± 2 ppt. gonads were collected in July and August and in The most obvious effects were observed in the mid-October, 1962 and 1963, and in July and August oysters exposed to cadmium and lead. Cadmium- and early November, 1964. In 1965, a single matura- exposed specimens had very little shell growth, tion and occurred in spawning mid-September to lost pigmentation of the mantle edge and digestive late October. The delay in spawning and single diverticulae, and suffered high mortalities. Com- annual cycle was related to the colder environ- parable morphological data on the lead-exposed mental not temperatures observed in the 3 earlier oysters were not obtained, but significant mor- years of the study. talities also occurred in that experiment.

WHAT'S WITH A SALT MARSH'' FARMING OYSTERS IN ARTIFICIAL PONDS — ITS PROBLEMS AND POSSIBILITIES Carl N. Shuster, Jr. William N. Shaw Northeast Marine Health Sciences Laboratory PHS, DHEW U. S. Bureau of Commercial Fisheries Narragansett, Rhode Island Biological Laboratory Oxford, Maryland The role of tidal marshes in estuarine produc- Four 1/4-acre tivity, particularly their impact upon shellfisheries, artificial saltwater ponds were is not fully understood at this time. Nevertheless, put into operation in the fall of 1964 at Oxford, certain undeniable trends — such as losses of Maryland. Three oyster culture studies were ini- tiated in the shellfish growing areas due to encroaching pollu- ponds: production of seed oysters: tion, the search for improved methods of shellfish comparative growth of 7 strains of Chesapeake and culture, and the increase of research-generated in- Bay oysters, response of oysters to 4 types of formation — are sufficient incentives, from a man- bottom. In addition, a 2-year ecological study was made of the invertebrate agement viewpoint, to explore the possible signifi- succession and pond colonization. cance of tidal marshes and comparable ecological units to shellfisheries. About 10 spat per 30 shells were caught in the A tidal marsh is a well-defined natural com- first attempt to produce seed oysters in an artifi- munity and can be characterized by the kind and cial pond. The seed originated from the introduc- tion extent of its plant life. Attention is directed to- of 4 to 5 million straight-hinged larvae (48 hours ward such communities because they provide food old). In the comparative growth study, all for shellfish. The nature of a middle Atlantic tidal strains grew at about the same rate, but meat marsh is considered in some detail, as is the quality was lower than in similar groups suspended relevancy of certain of its aspects to shellfish pond in a natural pond. Condition of oysters (percent- culture. age of solids) declined on all 4 types of bottom from an apparent lack of food; thus the effect of bottom type was overshadowed by other factors. Future will TRACE METAL ACCUMULATION BY studies refine methods of producing THE OYSTER seed oysters, and develop methods of feeding pond- held oysters. We believe that artificial ponds have Carl N. Shuster, Jr. and Benjamin H. Pringle a commercial potential for culturing oysters which will be determined by further biological research. Northeast Marine Health Sciences Laboratory PHS, DHEW Narragansett, Rhode Island THE EFFECTS OF ADDITIVES ON THE In 10-week and 20-week studies, Crassostrea vir- PUMPING RATE OF ginica was subjected to nitrate salts of lead (0.025, MERCENARIA MERCENARIA 0.05, 0.1 and 0.2 ppm) and of chromium (0.05 and 0.1 ppm I, copper (0.025 and 0.5 ppm), zinc (0.1 and Webb Van Winkle i 0.2 ppm), and cadmium (0.1 and 0.2 ppm), respec- Oyster Research Laboratory tively. The 10-week study was conducted on sum- R)ttgers U^iiversity, Ncu- Jersey mer-harvested oysters: winter-harvested specimens were used in the 20-week study. About 200 oysters The effects of various additives on Mercenaria were placed in each of several 50-gallon tanks in mercenaria pumping rate were investigated. The a flow-through seawater system maintained at a additives tested included simple organic com- 10 ABSTRACTS pounds (glucose and glycine), hormones (acetyl- information on fishing areas, catch, effort, clam choline, adrenalin, and serotonin), and gonad lengths, and amount of small clams discarded at extracts (male and female Mei-cenaria and sea. The major fishing areas are off Point Pleasant Mytilus). Clams were maintained in sand in run- and Cape May, New Jersey, in depths of 24 to 110 ning sea water, and additives were administered feet. Point Pleasant and Cape May-Wildwood are directly into the incurrent siphon through a the principal ports. Average daily catch per boat catch pipette hooked up to a flowmeter and reservoir. was 328 bushels of clams and average per The simple organic compounds and adrenalin hour was 38 bushels. Boats fished an average of 9 and serotonin occasionally, but not consistently, hours a day. Average length of clams landed was stimulated pumping. Acetylcholine inhibited pump- 151 mm at Point Pleasant and 130 mm at Cape ing at 10'^ — 10"3 M. Male and female Mercen- May-Wildwood. The amount of clams discarded at aria gonad extracts stimulated pumping as well as sea was negligible. spawning. As in the oyster the active principle of Two research cruises were made in 1966. The the male gonad extract is associated with the spring cruise assessed surf clam density and distri- in to bottom and sperm and is not water soluble. bution relation type topography in the area from Montauk Point, Long Island, to Ocracoke Inlet, North Carolina. The fall cruise re- Present address: Department of Biology peated stations established in 1965 between Mon- College of William and Mary tauk Point, Long Island, and Cape Hatteras, North Williamsburg, Va. 23185 Carolina. Only minor changes were found in the distribution or density of surf clams since the 1965 cruise. SURF CLAM RESEARCH, 1966 A study to establish growth rates of young surf clams was completed in 1966. Marked and un- Robert M. Yancey marked young clams in Chincoteague Inlet, Vir- ginia, were observed from October, 1964, through U. S. Bureau of Comrnercxal Fisheries March. 1967. The mean length of 1-year-old clams Biological Laboratory is 45 mm: 2-year-old, 69 mm and 3-year-old, 91 mm. Oxford, Maryland Surf clams in the major fishing area off New In our continuing study of surf clams iSpisula Jersey usually spawn twice a year, in summer and solidissi^na), four major research areas were in- in fall, but spawned only once (late September) in vestigated in 1966: fishery statistics, population 1965. Analysis of water temperature records from densities and distribution, growth rates, and re- Barnegat Lightship indicated that the sum of production. daily temperatures above 0^ C between spawnings Port sampling, interviews with boat captains, may be more important to the spawning pattern and day trips aboard the fishing vessels provided than abrupt temperature changes.

NSA PACIFIC COAST SECTION

CHROMOSOMES OF OYSTERS, CLAMS numbers of some of the species examined are: AND MUSSELS 20; 20; Crassostrea gigas 20; Saxidomus giganteus and S. nuttallii 38 Muzammil Ahmed and Albert K. Sparks each; and Mytilus californianus and M. edulis 28 College of Fisheries each. University of Washington Work on the hybridization of the 2 clams, S. Seattle, Washington giganteus and iS. nuttallii, and the 2 mussels M. Several species of bivalves from the Northwest californianus and M. edulis, is in the preliminary Pacific coast of the United States have been stages. In the respective reciprocal crosses only a examined for their chromosome complements and few eggs cleaved. Results of the experiments and other aspects of chromosome cytology. The diploid morphology of the chromosomes are discussed. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 11

A PARASITE FROM THE OVA OF basic biology, started around 1917 and have con- CRASSOSTREA GIGAS tinued with interruptions until the present. Re- cent crab work deals with population dynamics of C. Dale Becker and Gilbert B. Pauley Queen Charlotte Island stocks. Larval stages of around and Biology Department shrimps were described 1930, growth Battelle-Northivest rates and hermaphroditic reproduction determined. included ex- Richland, Washington Since 1953 shrimp projects have ploratory fishing, further life history studies, and During a recent (1966) summer mortality in a study of the dynamics of an inshore population. Crassostrea gigas, 5 mature female animals were In 1964 an experiment to introduce Atlantic lob- collected in Humboldt Bay, California and ex- sters was started. amined histologically. An unidentified parasite was observed in the cytoplasm of the ova of 3 of these Several different of the oysters. stages WOUND REPAIR IN CRASSOSTREA GIGAS parasite were observed in the cytoplasm, but never in the nucleus. Single and multiple infections David M. DesVoigne and Albert K. Sparks were observed, with as many as 10 parasites in an Fisheries individual ovum. College of Some infected eggs were necrotic and accom- University of Washington an acute or chronic reac- panied by inflammatory An experiment was conducted to investigate tion. However, even in the most infected heavily histologically the wound healing process in Crasso- no evidence was seen of fibrous oyster, encapsula- strea gigas. Sterile wounds were inflicted in the tion which is observed in ex- frequently oysters visceral mass adjacent to the palps and the pro- to bodies or posed foreign pathogenic organisms. gression of healing was followed. Internally, the Neither an nor inflammatory response granula- leucocytes rapidly filled the wound channel, elon- tion tissue was observed the of the among eggs gated and formed a scar. At the surface, leucocytes uninfected female oysters. formed a band under lying the mantle epithelium The of this to summer relationship parasite and replaced damaged epithelium. Eventually mortalities in Humboldt is unknown — and Bay these leucocytes formed a pseudoepithelium which since the probably unimportant, parasites appear became indistinguishable from the adjacent normal to be confined to the ova. columnar epithelium.

BRITISH COLUMBIA CRUSTACEAN THE EFFECTS OF OIL FROM THE WRECKED FISHERIES AND RESEARCH TANKER, TORREY CANYO'N, ON SHELLFISH- PROJECTS — PAST AND PRESENT ERIES RESOURCES OF ENGLAND AND FRANCE Terence H. Butler John B. Glude Fisheries Research Board of Canada Biological Station, Nanaimo, B. C. Bureau of Commercial Fisheries U. S. Fish and Wildlife Service The most valuable fishery in British Columbia is Seattle, Washington for Dungeness crab (Cancer magister). From 1961 to 1965 annual catches averaged 3.7 million pounds, Crude oil released shortly after the tanker, with a landed value of 486 thousand dollars. Crabs Torrey Canyon, grounded on Seven Stones Reef are taken almost exclusively by traps, with a off Southwest England came ashore along the small amount caught incidental to fish trawling. coast of Cornwall, England and Brittany, France. The main fishery is near the Queen Charlotte Is- Measures to control the spread of oil included lands, and smaller areas are located around Van- spraying of 1/2 million gallons of detergents or couver Island and near the FYaser River mouth. solvent-emulsifiers at sea. In addition, over 2 Trawling yields most of the shrimp production million gallons of detergent were applied ashore consisting of 5 species of Pandalus. Another in England to remove the oil from sandy beaches species, the prawn (P. platyceros), is trapped. Im- and rocky headlands. Adverse biological effects portant shrimp grounds are found around Van- were increased by application of detergents which couver Island and near Vancouver. Annual catches, are much more toxic than oil. 1961 to 1965, averaged 1.5 million pounds with a In France, use of detergents was prohibited ex- landed value of 238 thousand dollars. cept on certain beaches important to tourism and Crab investigations, at first concerned with unimportant to commercial fisheries. 12 ABSTRACTS

Actual loss to commercial fisheries in both Eng- suitable here because of large rocks in the sub- land and France was small but this was largely strate. Therefore, modification of these dredges because of fortuitous circumstances of geography, or the development of new types will be necessary. currents and weather which prevented contamina- Information obtained in the study will be tion of estuaries where oysters are farmed. Similar utilized to establish long-range management pro- oil spills in other areas could have disasterous grams for subtidal clam resources in the Puget consequences for commercial shellfish resources. Sound area.

SUBTIDAL SHELLFISHERIES DEVELOPMENT SEASONAL CHANGES IN THE CONDITION OF CRASSOSTREA GIGAS AND POSSIBLE C. Lynn Goodwin RELATIONS TO NUTRIENT SOURCES

Washington Department of Fisheries R. B. Herrmann Brinnon, Washington Research Department Weyerhaeuser Company A survey of the subtidal hardshell clam popula- Longview, Washington tions of Puget Sound has recently been initiated by the Washington State Department of Fisheries. A continuing study of Pacific oysters in Grays Actual field work started in July of 1967; there- Harbor, Washington, in 1966 and 1967, involved fore this report will be largely confined to objec- sampling oysters from 1964 imported Japanese tives and methods. seed at two stations. Growth and condition of the The primary purpose of this program is to en- oysters from average weights, and total solids courage the private development of commercial and glycogen contents, are reported. Per cent total fisheries for subtidal clams. The study consists of solids of oysters from both stations was lowest in 3 phases: (1) To conduct with scuba divers under- winter and highest in summer. The weight of dried water surveys of hardshell clam populations in total solids followed a similar cycle except for specific areas of Puget Sound; (2) to develop, in pronounced drops which occurred after spawning. cooperation with industry, appropriate gear and Very little weight recovery of total solids occurred harvest methods for clam species present; (3) to during the subsequent fall and winter months. The develop management practices for subtidal popula- level of glycogen storage in the oysters was highest tions of commercial clams and . in late spring and dropped to a minimum in sum- Survey work completed thus far has been con- mer. fined to Kilisut Harbor near Port Townsend. Phytoplankton standing crops, and levels of Buoys are first placed around the perimeter of the particulate organic carbon, chlorophyll A, organic survey area and equally spaced transects are laid phosphate, and dissolved and particulate carbo- out. Divers take bottom samples of 2 sq ft each hydrate based on weekly sampling at both stations at equal intervals along each transect with a are also reported. These features are of interest modified 4-in venturi gold dredge. Sample ma- since they may be utilized as food by oysters or terial is caught in baskets of the appropriate mesh may reflect levels of food utilized by oysters. size, hauled aboard the boat and clams are identi- Phytoplankton standing crops were relatively low fied and grouped according to size. in midsummer and winter with maximum abun- Commercial quantities of geoducks (Panope dance during the spring and fall blooms. On gererosa), native little necks (Venerupis stam- the average samples from the more seaward inea), horse clams (Schizothaerus nuttalU), and station had fewer phytoplankton organisms. Num- butter clams (Saxido7nus giganteus) have ap- bers alone, however, were of limited value for com- peared in the samples. Wet weight of clams has paring plankton biomass from the two areas. varied from a few ounces to over 4 Ibs/sq ft in Organic phosphate and chlorophyll A follows the samples taken in Kilisut Harbor. phytoplankton cycle except in midwinter when and From the limited data it appears that subtidal chlorophyll A levels were lower organic clams are most abundant in areas of tidal cur- phosphate levels were higher. rents rather than the heads of bays. Subtidal clam Dissolved and particulate carbohydrate levels abundance populations are frequently composed of a single seemed correlated with phytoplankton levels size group, apparently the result of a "good set in late spring, summer, and early fall. Winter environmental year". Test diving in Hood Canal has shown com- seemed more influenced by other the car- mercial quantities of butter clams as deep as 60 ft conditions. Conclusions based on organic below tide level. bon data were limited because of the imprecision Dredging in Puget Sound has indicated that com- of the method. Relative levels of carbon, however, mercial dredges used on the East Coast are not as well as those for chlorophyll A, organic phos- PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 13 phate, and particulate and dissolved carbohydrate Ozette River in Washington. Seventy-five of these averaged higher at the station located further animals possessed large watery lesions on the foot, upbay. which have a resilient characteristic similar to a Of the five environmental features investigated, sponge; hence the name "spongy" disease. In some chlorophyll A levels correlated more significantly animals, the large spongy lesions were replaced with weight changes of oyster total solids. For by scarred wounds, in many instances bordered by the more seaward station, r ^ 0.62 (P<.01): for multiple small papillary lesions that also had a the upbay station, r = 0.57 (P<.01). A better re- spongy resilience. lation was obtained using the product of log tem- Histologically, the large watery lesions are perature and chlorophyll A levels with weight edematous areas in which some of the normal changes of total solids expressed as per cent (re- muscle tissue has been replaced by fibrous con- lative growth). For the seaward station, r =z 0.79 nective tissue. The epithelial covering over the (P<.001) and for the more upbay station, r = 0.75 afflicted area is disorganized, necrotic, lacking or (P<.001). reduced to a squamous lining. Those animals that possess scarred wounds and multiple small papil- lary lesions have a subacute inflammatory reac- DESCRIPTION OF LABORATORY-REARED tion with well-developed granulation tissue and LARVAE OF collagen deposits. This disease affected only the

PARALITHODES PLATYPUS (BRANDT) i foot of the mussels. Ethelwyn G. Hoffman U. S. Bureau of Commercial Fisheries POST-EMBRYONIC DEVELOPMENNT OF Biological Laboratory LABORATORY-REARED "SPOT" SHRIMP, Alike Bay, Alaska PANDALUS PLATYCEROS BRANDT Larvae of the blue king crab, Paralithodes Vincent A. Price and Kenneth K. Chew platypus (Brandt), were hatched and reared in the laboratory. All larval stages obtained developed in College of Fisheries, a manner similar to the development reported for University of Washington other lithodid anomurans. In culture, P. platypus "Spot" shrimp were reared successfully in the had 4 zoeal stages and a single glaucothoeal stage. laboratory from eggs stripped from females The feature which distinguishes all zoeal stages of caught in Dabob Bay, Washington. Eggs were this species from zoea of the other two North incubated in circulating bubbling sea water at Pacific species of Paralithodes {P. caintschatica 53°F. Hatching occurred after 41 days while eggs and P. breinpes) is the presence of 9 pairs of tel- on ovigerous females held at the same tempera- son processes (including a hair-like second process) ture took 22 days longer. Larvae were cultured in rather than 8 pairs. Glaucothoe of P. platypus have floating 800 ml beakers and were fed newly one more pair of spines in the branchial region of hatched brine shrimp nauplii. Six stages were the carapace than do those of P. camtschatica. recorded before the post-larval stage. The shape of Glaucothoe of P. platypus have 15 of pairs spines the telson was characteristic for each of the six on the dorsal surface of the carapace — not includ- stages but was not of diagnostic value for older ing the spines of the frontal area (rostral complex) stages. Larvae were also kept at 51°, 53°, and or the suborbital spines — whereas the glaucothoe 55°F. Survival was best at low temperatures in the of P. Camtschatica have 14 pairs of spines, and the early stages but larvae at higher temperatures glaucothoe of P. brevipes have 13 pairs. survived the longest. Larvae at 55^' F moulted 4.5 days ahead of larvae at 51 °F. Highest mortality I This in full in J. Fish. Bd. Bd. paper appears occurred during the moulting process. Can. 25(3):439-455, 1968.

THE PATHOLOGY OF "SPONGY" DISEASE LARVAL REARING STUDIES OF THE IN FRESHWATER MUSSELS DUNGENESS CRAB, CANCER MAGISTER ' Gilbert B. Pauley Paul H. Reed Biology Department Oregon Fish Commission Battelle-Northivest Newport, Oregon Richland, Washington The Dungeness crab is an important Oregon In March, 1967, 123 freshwater mussels (Mar- resource. Large fluctuations in annual landings garitana margaritifera) were collected from the prompted studies to determine the extent to which 14 ABSTRACTS selected environmental factors may limit crab It appears that supplemental feeding improves abundance. Studies were conducted during the oyster condition at all times of the year, but that larval development, a critical period of crab life the optimum feeding rate varies by season or as a history. result of changes in physical parameters. Preliminary work developed culture methods for rearing crabs through the larval period with good survival. Crab larvae were reared in 250 ml Erlen- SOME EFFECTS OF A DENSE RAZOR CLAM meyer flasks. The nauplius of the barnacle, SET IN WASHINGTON Balanus glandula, was found to be a suitable food organism. Relatively equal numbers of barnacle Herb C. Tegelberg nauplii were fed in each culture flask by using Washington State Department of Fisheries an automatic pipette. Good survival was main- Aberdeen, Washington tained until crab zoea metamorphosed into the cannibalistic megalopa. A dense set of razor clams, Siliqua patiila Dixon, Subsequent work established the effects of com- occurred on beaches north of Grays Harbor, binations of temperature and salinity on survival Washington in 1966. Patches of clams up to sev- and growth of crab zoea. Zoea were tolerant of eral acres in extent and 1 to 3 inches deep were greater ranges of temperature and salinity than noted for 2 to 3 weeks in June along 10 miles of are normally found in the ocean. Survival of beach. Set reached a record ma.ximum of 13,600 zoea exceeded 70 per cent within temperature and clams per ft- in a single sample for this unusually salinity ranges of 50 to 64'= F and 25 to 30 ppt early set. respectively. Development time was inversely re- The 1966 year class persisted at a high abun- lated to temperature. dance level for 7 months, but grew at a greatly re- duced rate. Growth of older clams was also ad- versely affected. There were indications that plank- I in the Commercial Fisheries Supported part by ton concentration was depressed north of Grays Act (P. L. 88-309) Research & Development Harbor during the period of dense set. Availability of clams was adversely affected be- tween September and December. Recovery of THE SUPPLEMENTAL FEEDING OF marked clams by standard digging methods aver- CRA8808TREA GIGAS IN THE LABORATORY aged 5 per cent per dig north of Grays Harbor compared to about 20 per cent per dig at 2 beaches C. S. and D. F. Tufts Sayce south of Grays Harbor. Washiyigton Department of Fisheries Ocean Park, Washington BACTERIAL ACCUMULATION-ELIMINATION Three laboratory experiments were conducted RESPONSE OF PACIFIC OYSTERS using cornstarch as a supplemental food for (CRASSOSTREA GIGAS) AND MANILA CLAMS Crassostrea gigas. Starch was fed at 17 mg/1, (TAPES PHILIPPINARUM) MAINTAINED 34 mg/1, and 68 mg/1. Water flow was main- UNDER COMMERCIAL WET STORAGE tained at 10 l/oyster/hr in a continuous flow sys- CONDITIONS tem. Temperatures and salinities were recorded G. J. Vasconcelos, J. C. Hoff and T. H. Ericksen but not controlled during each 32-day experiment. In Experiment 1, a noticeable increase in condi- Pacific Northwest Marine Health tion index occurred in oysters fed at 17 mg/1 over Sciences Laboratory nonfed laboratory control oysters, but greater in- Gig Harbor, Washington creases occurred in condition index of field-con- The accumulation and elimination of bacteria trol oysters over laboratory oysters. Results of by Pacific oysters and Manila clams held in com- 2 showed that supplemental feeding Experiment mercial wet storage was studied under varying at 17 and 34 increased condition of mg/1 greatly hydrographic and climatological conditions. A over nonfed and field-control oysters laboratory series of 3 experiments was conducted in 2 loca- oysters. In Experiment 3, oysters were fed at 17, tions in the southern Puget Sound area. In each 34, and 68 mg/1 with less conclusive results. Oys- experiment, samples of commercially harvested ters fed at 17 and 34 mg/1 did better than those shellstock, depurated shellstock and overlying fed at 68 mg/1, while the difference was highly water were collected at various periods through a significant between supplementally fed and nonfed complete tide cycle. Bacteriological examination oysters treated as groups. included the most probable number (MPN) of coli- PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 15

form and fecal coliform bacteria and 20-'C plate drill; (1) Egg cases and juveniles are more suscep- counts using MacLeod's seawater agar. Water tem- tible to toxicants and environmental changes than perature, salinity, turbidity and precipitation data adult drills or oysters; (2) male drills are attracted were also collected. Changes in coliform and fecal to water-born substance released by egg laying coliform levels in sea water correlated closely females, and (3) preliminary observations on with the tidal cycle. Shellstock collected from sex of the drills indicate changes in relative abun- various sections of the float showed similar rapid dance or distribution of male and females as the responses to changes in bacterial levels in the drills increase in size. If this last point is true, a water. Examination of depurated oysters or clams control method may be possible by interfering positioned at various locations in the float indi- with normal reproduction of the drill population. cated that these shellfish rapidly attained coliform Future research will continue to examine these and fecal coliform levels similar to those of the phenomena as basis for control methods. naturally harvested shellfish. This study is being Past research in chemical control of the drill has continued to include other types of commercial shown that, due to the physiological similarities of floats in the Puget Sound area. the adult drills and oysters, a complete chemical control may not be safe or economically feasible. Future studies in chemical control will test STUDIES ON THE JAPANESE OYSTER DRILL candidate chemicals; (1) against the more sus- ceptible egg cases and juvenile drills, (2) as caustic Michael E. Weatherby poisons that would eliminate all drills from barren oyster ground, (3) as barriers to prevent reinfesta- Washmgtoii Department of Fisheries tion of oyster beds, and (4) for their ability to sup- Brinnon, Washington press the breeding potential of the adult popula- During the last 20 years, much research has tion. been directed towards control of the Japanese oys- Past research in physical controls has shown ter drill, Ocinebra japonica. In this report past re- that barriers are not effective unless used in con- search and plans for further research is discussed. junction with improved chemical repellants. Im- Past research on biological control has dis- proved spawning site collectors and other means covered the following vulnerable points in the life to collect egg cases or drills warrant further in- cycle and behavioral patterns of the Japanese vestigations. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

BCF ROLE IN FARMING OF THE SEA' H. E. Crowther

U. S. BUREAU OF COMMERCIAL FISHERIES WASHINGTON, D. C.

There are many conflicting reports about the which 15 grams should be high-grade animal pro- abundance of fish and shellfish in the and tein. their potential as a source of food for the millions Only a portion of the potential annual world fish of people existing on inadequate diets through- catch would be composed of edible species of types out the world. Some uncertainty also exists in the now accepted by the American people. In fact, Dr. minds of many scientists about the role of aqua- Chapman breaks the potential catch down into culture in augmenting this food supply. three sizes of fish according to length: over 10 It is not difficult to find articles written by "ex- inches, 5 to 10 inches, and under 5 inches. Most perts" who say that food from the sea is a myth, species presently in demand in the United States, and that only through agricultural techniques can with the exception of mollusks and some crusta- we hope to supply the food needed by our expand- ceans, are longer than 10 inches and are relatively because the ing population. On the other hand, many marine expensive public wants particular scientists have demonstrated through their own flavor, texture, and appearance. The herring-like calculations that the oceans can supply the needed fishes which form a major part of the present catch are food. There is no doubt about my belief. I am con- world included in the 5 to 10-inch cate- vinced by the evidence presented that the oceans gory. The greatest volume of protein in the ocean, have been overlooked and neglected as a source of however, is in animals less than 5 inches long. for the all-important animal protein. These, most part, are not considered desir- At the Law of the Sea Institute meetings at the able as human food and therefore are inexpensive in the United States. Of course, shellfish whether University of Rhode Island in June, 1967, several mollusks or , fall within the speakers presented some exciting estimates of logically category of the more expensive species. possible annual yields of fishery resources from If wild fish and shellfish in the ocean can be the oceans. The present world marine catch is harvested in the amounts predicted, why bother approximately 46 million metric tons. Dr. M. B. with fish farming? The answer is that aquacul- Schaefer has estimated that the total annual ture could provide a steady and probably increased potential is 200 million metric tons. Drs. W. M. supply of the high-priced now in greatest Chapman and H. Kasahara estimate 2,000 million demand. My view is that industry has the metric tons, while Dr. W. R. Schmitt's estimate is potential for producing tremendous quantities of 4,000 million metric tons, or 100 times the nearly fish and shellfish in the relatively near future. I world catch. Which of these is present predictions also see the possibility of producing large crops of the most accurate is of little significance, accord- less expensive fish through marine fanning, but ing to Dr. Schaefer, because an annual production not within our lifetime. One of the reasons for this of 200 million metric tons, if fed directly to people, latter prediction is that we have far too little basic would provide about 15 grams of fish per day for information on the techniques which must be used. 6 billion people — twice the present world popula- Also, the cost of producing the less expensive fish the cost of tion. The total daily protein requirement of an would not permit us to compete with the wild For ancho- individual is estimated at about 80 grams, of harvesting species. example, vettas are now landed in Peru for about $15 per ton. Hake, from the new resource discovered off

I Remarks of Mr. H. E. Crowther, Director of the Washington and Oregon, are harvested for ap- Bureau of Commercial Fisheries, before a meet- proximately $18 per ton; and anchovies in Cali- ing of the 59th Joint Annual Convention of the fornia for about $20 per ton. It would be almost im- Oyster Institute of and the Na- possible for fish farming to compete with these tional Shellfisheries Association at Boston, Mass- costs in the United States. achusetts, on July 17, 1967. With this background, let us look at some aspects

16 FARMING THE SEA 17

of farming the sea by first drawing an interesting problems faced by shellfish growers today. parallel with the development of agriculture. At Oxford, extensive studies in shellfish culture The United States is the recognized world leader are also now undenvay. Techniques in catching seed in agriculture, a position which has brought much oysters from shells suspended from rafts are being prestige, and admiration from many countries. developed. In addition, off-bottom culture of oys- Agriculture achieved this status in the United ters in both natural ponds and in man-made ponds States through simultaneous development of four are being investigated. courses of action: (1) improvement of the environ- Also at Oxford, we hope eventually to extend ment, (2) plant and animal husbandry, (3) agri- our studies of disease to all marine animals, not cultural engineering, and (4) product develop- just shellfish. As sea fanning develops, problems ment. In addition, knowledge gained from research with diseases undoubtedly will become more and has development been effectively transmitted serious and it will be necessary to learn how to to the farmer and agricultural industry through cope with them. The Bureau of Sport Fisheries and extension activities, including demonstration and Wildlife has already made many important ad- training programs and nontechnical publications. vances in knowledge and control of diseases of In developing commercial fish farming we can freshwater fishes. gain much by considering the history and accom- At Galveston, Texas, our Bureau has made some plishments of American agriculture. In a sense, promising beginnings at shrimp culture. Through we have a blueprint to follow. Tlie important lesson the work being done in state and other labo- inherent in the history of agricultural develop- ratories as well as our own, we visualize eventually ment is that progress has followed sound and a thriving shrimp farming operation to provide adequately financed research. Much aquacultural select products for the gourmet trade. research is — following this blueprint but slowly. These are the programs of the present. Now let At present, aquatic husbandry and environmental us look at future possibilities. improvement are emphasized, but we recognize the need for additional studies of engineering and pro- SEA FARMING duct development. Such studies will lead eventually to the development of farms or factories to produce There has been much talk recently about farm- high-value, high-quality fish and shellfish to pro- ing the edges of the sea. This approach has many vide variety to our diet. attractive features, but we find the prevalent con- Now let me briefly review some studies of aqua- cept too narrow. Although there are intriguing culture being carried on by the Bureau of Com- possibilities for culture of marine animals and mercial Fisheries. plants under some form of private ownership, Scientists at our Boothbay Harbor, Maine, labor- there will always be resources to be harvested as atory are acquiring knowledge of the importance common property and we see some exciting and of suitable habitat to lobsters by using SCUBA completely untested avenues to be explored. These divers to observe lobster behavior and the rate of include i and we have broadened our definition of colonization on a man-made reef. The relation be- aquaculture to include such activities): tween the surf clam and its ocean environment a. Altering the Environment. — Why do is being studied at our Oxford, Maryland, labora- some relatively large areas of the ocean pro- tory. At other laboratories, pesticide and radio- duce 300 pounds or more per acre per year, biology studies are to our of the adding knowledge yet the average fishing yield of the world accumulation and effect of these pollutants on ocean is less than two pounds per acre? Can commercial species. yields in "desert" areas be improved by al- At Milford, Connecticut, we have had some success tering the environment? The answer is yes, in developing artificial culture methods for oysters but we do not understand the mechanisms and clams, some of which are being tested on a nor are we able to anticipate the costs at all commercial scale. Our new laboratory at Milford, well. when in full operation, will carry forward this work more rapidly. Already we are investigating b. Positive Approach to Waste Disposal. — the genetics of mollusks, with a view to develop- Domestic and many industrial wastes contain ing new strains and hybrids with more desirable the essential nutrients for plant growth and characteristics. increased production of animals. Yet our in- Also at Milford we have developed some methods discriminate disposal of wastes into the na- of protecting shellfish against predators in the tural environment brings out the harmful, natural environment. When perfected and ap- rather than the potentially beneficial effects. proved, these techniques, coupled with an assured Why not examine the feasibility of controlled source of seed from hatcheries or other protected waste disposal to enhance fishery produc- environments, can eliminate some of the major tion? This could be much cheaper than the 18 HAROLD E. CROWTHER

inevitable alternative, which is to remove neighboring states. Catfish production has these wastes entirely from the natural cycle. been rising sharply and the potential exists in a five-state area in the Middle South to in- Production Ocean Fishes. — c. Hatchery of crease our total domestic fish production at Some of the earliest to stabilize and attempts least fivefold. In fact, if we set costs of pro- marine were to improve fishery production duction aside for the moment, there is no rear numbers of in hatcheries large young reason why we could not supply much of the and release them into the natural environ- increasing world requirements for animal ment. Extensive effort in this direction was protein well into the future from privately and on e.xerted for many years many species, controlled waters in the United States. flounders, and lobsters. among them; cod. Private production offers attractive relief The died slowly, but its demise was concept from most of the baffling and virtually in- inevitable, because it no demon- produced soluable problems of production from public strable effect and was not conducted scienti- waters. Private production could well be the Our molluscan culture studies have fically. key to survival of some of our commercial a better understanding of what we provided fisheries. There is no good reason to question must know to successfully operate marine the feasibility of private commercial produc- hatcheries. We feel this idea should be tion in fresh waters, but techniques, costs, and believe that marine reexplored many and markets require careful examination. can be reared through the critical species These questions deserve vigorous inquiry. early stages when mortality is high, if Let me close by reiterating my feeling that scientific methods are followed carefully. We aquaculture of high-value fish and shellfish holds have no estimates of the cost of large-scale great promise for the United States fishing in culture, but we do know that the potential dustry. In view of the increasing demands on the rewards are Such techniques might great. estuarine areas for recreation, urbanization, and provide the only practical mechanism by industrialization; controlled farming of some which fluctuations in survival of abundant species may be the only way we can maintain or schooling fishes could be reduced or elim- increase production of the valuable species. There inated. This in turn could be the key to suc- is no question that our expanding United States cessful management of fisheries based on population will require more food; fishei-y pro- widely fluctuating resources like menhaden ducts can supply some of this food. Aquaculture or, for that matter, almost any estuarine can assist in this increased production and provide resource. Such developments could be funda- the kind and quality of fish and shellfish that mental to the success of a fish protein con- the public of the United States will be seeking. centrate industry at home or abroad. A For some species of fish and shellfish the re- massive effort is necessary before we can wards of research will come soon, since the states, evaluate the potential of hatchery techniques. industry and the Bureau of Commercial Fisheries d. Pond Fish Culture. — An attractive alterna- have already accumulated a substantial amount of tive to all the difficulties inherent in achiev- data on the production and management of clams ing rational management of ocean resources and oysters. For others, the payoff may not be would be to concentrate on scientific pond within our lifetime but the potential is so great fish culture in fresh waters and in selected that we owe it to future generations to begin the coastal areas. While we have been preoc- basic studies now. I hope budget conditions will cupied with the hypothetical potential of the soon be such that the Bureau of Commercial Fish- vast ocean, exciting developments have been eries can carry out its share of this needed re- going on almost unnoticed in Arkansas and search. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

THE STATES' ROLE IN MANAGEMENT OF SEA RESOURCES David A. Adams NORTH CAROLINA DEPARTMENT OF CONSERVATION AND DEVELOPMENT RALEIGH, NORTH CAROLINA

"From a conservation viewpoint, the fisheries more individuals, and in preventing gross over- are perhaps the most poorly managed of all our fishing, they cannot increase populations above national resources". Dr. Lionel Walford made the the level already provided by nature. True man- above statement more than 20 years ago; it's about agement must have this as its goal — to provide a time to take a hard look and see if he is right, and greater harvestable crop than would have been if so, to find out who's responsible and what can available otherwise — and we're a long way from be done about it. achieving that goal. We inherited our basic philosophy of fisheries Let's explore a few of the reasons why the administration from the English, who, since the States have not been able to do a decent job of Magna Charta, have considered wild animals to be managing these resources. First, the prime man- tool the common property of all citizens, held in trust agement that has been employed has been of the by the Crown in its sovereign capacity. Following regulation catch. If overfishing is a signi- the Revolution, the sovereign power was trans- ficant limiting factor, then catch restrictions are ferred to the various States, and remains there to- biologically valid. If they are imposed in such a manner as day. Jurisdiction over game and fisheries re- to effect an increased monetary re- turn unit of fisherman then sources, per se, has never been vested in the Fed- per effort, they are eral government. The Federal government must economically valid. If a particular catch restric- tion is its effect derive what authority it has over these resources really worthwhile, should be noticeable in from the commerce clause, from broad inter- the fishery after a reasonable period of time. Take a look at the pretations of the "general welfare of the United catch restrictions in State. States", from treaties, and from a liberal inter- your How do they measure up? A number of states have size pretation of its power to "make all needful rules limits on various of marine fish. These and regulations respecting the territory or other species regulations have ap- been property belonging to the United States." parently enacted because of the feeling that fish should have the to There appears to be little doubt, then, that the "every opportunity grow up and reproduce." Whether or not reduction of the States have the basic authority, and the obliga- juvenile is reflected in the size of tion, to manage the marine resources within their population or whether the size of the boundaries. We hear quite a bit (from the States) spawning population, is the size of the about the States' rights and authority: we hear spawning population limiting very little from the States about how well they succeeding generation, is rarely considered, and even more known. Yet laws have lived up to their obligations. rarely are enacted and officers are to enforce them. Of In all honesty, I think most of us will have to required course, the size of is admit that the States, in general, have done a legal many species adjusted downward until pretty miserable job. About the only management through political manuevering the restriction is techniques that have been practiced (with the essentially meaningless, or such as "unless exception of oyster rehabilitation work) have been qualifications obviously injured or dead" are aimed at limiting the harvest. While these may written into the law. What fish could have been effective in spreading the catch among not become "obviously injured" when landed? Many States have laws which restrict the size of certain units of gear (headrope of trawls, I Remarks of Dr. David A. Adams, Commissioner length of seines, number of dredges, etc.). These of the Division of Commercial and Sports Fish- restrictions may reduce the harvest, and may be eries, before a meeting of the 59th Joint Annual biologically justifiable, but they do it by reducing Convention of the Oyster Institute of North the efficiency of a commercial enterprise and plac- America and the National Shellfisheries Associa- ing it at an economic disadvantage in competition tion at Boston, Massachusetts, on July 17, 1967. with other industries.

19 20 DAVID A. ADAMS

My point is this. If there is some reasonable we must be willing and able to do something biological evidence that a population is being over- about it. then the harvest should be reduced. Fish- fished, Finding these limiting factors takes time, money, should be the method that ing regulated through and trained manpower. At the present time, State results in the reduction in harvest with the 2 greatest agencies are spending about $18 million per least effect on Limits on fishing efficiency. entry year, about a fourth of which is Federal aid, on into the fishery, seasons, catch quotas, selective research and development work, and have more (where are all to meth- gear possible), preferable than 600 persons employed in this area. All coastal ods which cut into profits by reducing fishing States except Florida, Virginia, Mississippi, and efficiency. North Carolina are now spending more for re- There are a few examples of effective manage- search and development than they are for law ment through catch restrictions. The Pacific halibut enforcement — a commendable reversal of past fishery has profited through imposition of a quota- history. Only Virginia does no research and de- by-area system; the Alaska salmon fishery was velopment within its marine fisheries agency (this probably saved by rather rigid restrictions backed function being carried out by Virginia Institute of up by biological monitoring i though the economics Marine Science), while Texas spends almost $6 of some of the gear restrictions may be argued): million per year on these activities. Intensity of the Hudson River shad run increased following effort, then, is not too bad — ranging from 0. 1 per restriction of Let me fishing mortality. emphasize, cent to 15-(- per cent of the value of each State's that the last of these however, only examples commercial catch — and is increasing each year. was a result of State the halibut is effort: fishery The time element is one which we can't do too international commission and Alaska governed by much about, since it is difficult to reconstruct the was then a territory administered the Federal by past, but one which will decrease in importance in government. future years as backlogs of information ac- There is a biological principle which holds that cumulate. the size of populations is limited by some single Once the necessary information is available, factor or group of factors working together. These utilizing it in a management program takes guts limiting factors change in time and space, may and. usually, money. There are few fisheries ad- and be different for different may entirely species. ministrators who don't have guts — they can't last That is to that the size of the east coast say long without them. Most States now employ pro- population of shad be limited the inac- may by fessional people in these positions, people who have of sufficient that the cessibility spawning waters; at least some research training, are in the business west coast sardine population may be limited by as a career, and who are somewhat resistant to the of anchovies; that the middle competition political pressure. It is not easy to face a group Atlantic coast menhaden population may be of irate fishermen who feel that a decision which on the limited by overfishing juvenile stocks; that injured them personally was a result of prejudice be limited southeastern oyster population may by or politics, even when such a decision is backed up insufficient cultch; and that the North Carolina by reasonably adequate data, and would benefit estuarine population of penaeid shrimp may be the State fishery as a whole. Those of us who are limitea by extreme winter temperature. However, unwilling to stand up to such groups had better the Hudson River example shows that shad in get out. river at limited that were one time apparently by Being willing to effect logical management pro- fishing pressure; anchovy competition apparently grams is one thing, being able may be another until was not significant the sardine population thing entirely. Let us look first at the biological was reduced through overfishing; even if the implications and then at the socio-political ones. In fishing pressure on juvenile menhaden is reduced, order for an agency to manage effectively a popul- destruction or deterioration of nursery areas may ation of animals, it must have jurisdiction over the prevent this fishery from ever reaching its former entire geographic range of the population. If level; adequate cultch may be available in middle Alaskan salmon are being taken in excessive num- Atlantic and New England waters, but oysters bers by foreign vessels on the high seas, there is cannot increase because of disease problems and little that the State of Alaska can do about it. If insufficient set; and winter temperatures may not Louisiana's estuarine fisheries are being damaged be nearly as limiting on Gulf shrimp as they may be in more temperate waters. All I am trying to say is that there is always something or a group 2 Does not include Alaska, Delaware. Massachu- of somethings that holds a population at a given setts, New Jersey, and Washington, from whom level. If we want to increase that population, we information was not received in time for in- must first find out what that factor is and then clusion. STATES' MANAGEMENT OF SEA RESOURCES 21

carried down the by pesticides Mississippi River, can get a bit excited at times, too). The same piece the State of Louisiana has little control over the of bottom cannot support an oyster fishery, a situation. even Things get worse when parts of haul seine fishery, be dragged by shrimp trawls, the same lie in estuary different states, as is the —and serve as a bathing beach and recreational area case in Long Island Sound. Delaware Bay, Chesa- some decision as to its best use must be made, peake Bay. Mississippi Sound, and the mouth of the and any such decision will cause repercussions Columbia River. All this is merely to illustrate from the supporters of the losing cause. The State's that, although the individual States have the re- responsibility to maintain a public fishery does sponsibility for managing estuarine and marine not necessarily conflict with its right to lease, or fisheries, geography prevents their doing a decent otherwise grant franchises for private manage- job even if they could otherwise do so. The alter- ment of oyster grounds, but the proper balance natives are interstate compacts or agreements of between support and encouragement of private some sort (how effective have these been?) or enterprise and maintenance of a public resource Federal control through either constitutional can be a pretty thorny problem. So amendment or some pretty far-fetched interpreta- far, I've painted a rather bleak picture of State tion of existing constitutional law ( how much management of marine fishery resources. chance does this have?). The picture is not entirely black; progress is being The socio-political factors are even more com- made and some promising courses of action are plex than the biological ones. Let us take for an open to us. Once the limitations on State manage- ment have been example a species which lives out its life entirely realized, we can begin to do some- about them. within a single body of water, within the juris- thing State diction of a single State, and one about which we A may not have jurisdiction over all the waters entered a have a considerable volume of biological in- by given species during its life- but it formation — oysters. You will remember that at time, can do a decent job of stewardship the beginning I pointed out that fisheries are a during the time that species is in its waters. It can public resource, held in trust by the State for the take steps to alter environmental conditions which are benefit of all its citizens. Yet there are among the detrimental to its total fisheries. It can correct citizens about as many points of view as there are legal inequities which are based on prejudice rather people. Regardless of how well founded in fact a than sound reasoning and fact. It can make certain that catch restrictions first resource program is. it must have the support of are of all necessary, and that a significant segment of the public or it is doomed. they are accomplished through the most Distasteful as it may be at times, those of us in efficient means biologically and the least disrup- tive fisheries administration are painfully aware that economically. But it can only do these things if individual people cast the ballots which elect the one important element, largely absent in past man- legislatures which appropriate the funds which agement, comes to the forefront. In the past, oyster- sustain our programs, which pass the laws which men have spoken for oystermen, shrimpers for we must enforce, and which, in many cases, can shrimpers, purse seiners for purse seiners, charter reshuffle agencies and personnel with an amazing boatmen for charter boatmen, beach fishermen for beach amount of dexterity. Getting back to oysters, the fishermen, ad injmitinn. As a result, much of the State has an obligation to maintain a public oys- State agencies' efforts have been concerned with ter fishery. To do so it should employ the most attempts to resolve disputes among these efficient management tools applicable to the waters various factions, disputes usually based on selfish concerned, and it must weigh the effect of its motives and short-sighted objectives. As long as management upon other, sometimes incompatible, fisheries administrators must serve as referees in- uses of the basic fisheries resource. In some stead of being able to devote their time to consid- waters, natural reproduction and growth may eration of the entire fisheries resources and plan for their sustain a fishery (with adequate protection) if present and future utilization, the resource cultch is available, and cultch may or may not be is going to continue to slide downhill as it is doing available; in others, seed must be brought in from now. As long as State legislatures are continually other waters, and the users of those waters may exposed to conflicts among many types of re- be so vociferous in their objections to the removal source users, criticism of their State resource of seed oysters that the State's efforts are effective- agency, and garbled assemblages of conflicting ly thwarted. Sometimes a local fishery must be facts that are confusing to say the least, they will sacrificed for the greater common good (as is the never see the need for a comprehensive program of case with some development projects), and the ire resource utihzation, nor will they see fit to fund ad- of the local is oystermen something to be heard. equately those agencies charged with this respon- Sometimes a proposed development project must sibility. The solution, then, is for each segment of be modified or eliminated in order to protect valu- coastal fishermen — clammers, oystermen, shrimp- able nearby oyster resources (and land developers ers, sport fishermen of all kinds — to see them- 22 DAVID A. ADAMS selves as a part of a greater whole, and to support which all segments are dependent continues to their State's efforts, through their legislature or decline. other governing body, to such a degree that their In summary, then, the individual States have the State will be able to employ and retain competent responsibility for managing their fisheries as a men in sufficient quantities and with sufficient public resource. They are prevented from doing an financial backing to initiate and implement sound, efficient job because of both biological and socio- comprehensive programs for the total fishery. This political reasons. The States are increasing their takes a little personal sacrifice, for decisions of efforts toward sound management (within the the agency will invariably benefit one segment above limitations and with the assistance of the more than another, or injure one segment more Federal government). The true value and intelli- than another, and personal sacrifice is not too gent management of coastal fisheries will be common a quality among modern man. Without realized only when competitive users are willing such an approach, however, we will continue to to compromise their differences and support a be faced with a continual stream of petty bicker- comprehensive resource program witliin their ing while the health of the basic resource upon various States. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

OYSTER MORTALITY STUDIES IN VIRGINIA. VII. REVIEW OF EPIZOOTIOLOGY AND ORIGIN OF MINCHINIA NELSONI '' Jay D. Andrews VIRGINIA INSTITUTE OF MARINE SCIENCE GLOUCESTER POINT, VIRGINIA 23062

ABSTRACT

Intensive epizootics in Crassostrea virginica caused by Minchinia nelsoni (MSX) show no signs of abating in lower Chesapeake Bay. Prevalences of the pathogen have commonly exceeded 50% in susceptible stocks during the first year of ex- posure; rnor-talities of 50 to 70% occurred during the first year and slightly lower losses in succeeding years. Disease activity increased in isolated lots of oysters from 1963 to 1966 during a drought period with high salinities. Native and planted oysters were extremely scarce in the lower Chesapeake Bay hence density of popul- ations appears not to be important for disease activity. Seasonal patterns of death rates have remained stable through eight years of observations but morbidity has occurred earlier in drought years. These patterns are influenced by time of import to areas of unexposed susceptible oysters of disease prevalence. ,. . Laboratory-bred progeny from susceptible (unexposed) and selected (by MSX) parents were held to marketable size in areas where MSX is intensively active with- out serious losses (10 to 20% annually). Early exposure appears to be important for subsequent survival of large oysters; hence, acquired resistance is postulated. M. nelsoni has not produced epizootics of oysters in the high-salinity environment of the Seaside of the Eastern Shore of Virginia; M. costalis (SSO) regularly causes a sharp mortality there in May-June each year. Since these congeneric syynpatric pathogens are very similar morphologically, it is postulated that their annual life cycles are similar, with June the period of sporulation. MSX appears to be highly virulent since death of the host usually occurs before sporulation which is rare. A hypothesis on the origin of a virulent strain ivhich arose in Delaware Bay in 1957 and appeared to spread to Chesapeake Bay in 1959 is based on seed oyster transplantations between the areas.

INTRODUCTION (Haskin, Canzonier and Myhre, 1965). Early pat- terns of prevalence and mortality were reported Nearly ten years of activity of the oyster by Haskin in 1959 (personal communication). The pathogen, Minchinia nelsoni, in the estuaries of the disease was found in Chesapeake in 1959 middle Atlantic coast have been observed. Mortal- Bay (Andrews and Wood. 1967) where it quickly be- ities first appeared in Delaware Bay in 1957 and came epizootic. In 1966, the organism was named the pathogen was first observed by Stauber in 1958 and described as a haplosporidian by Haskin, Stauber and Mackin (1966), and further linked with this group of parasites by Couch, and 1 Farley Contribution No. 263, Virginia Institute of Marine Rosenfield (1966) on the basis of rare spores. The Science. Research supported in part by U. S. Fish morphology and life cycle are incompletely known and Wildlife Service, Bureau of Commercial and sporulation is rare. Most information is based Fisheries, Contract 14-17-0007-330, Subproject on field studies and collections. cul- 3-6-R-l. Laboratory tures and infections have not been attained. The 2 Manuscript submitted for publication on July 26. organism seems to be highly virulent and exten- 1967. sive damage has been inflicted on the oyster in-

23 24 JAY D. ANDREWS

dustry of the middle Atlantic states, particularly losses are acknowledged (usually less than 10 per in Chesapeake and Delaware Bays. cent annually) but are not explainable. Deaths from the The epizootiology was described by Andrews fungus organism, Dermocystidium, were (1966) from data collected through 1963. Subsequ- e.xcluded for the most part by isolation of trays 1965). of live ently, three dry years have brought some changes (Andrews, Many samples oysters in the intensity and the seasonality of "MSX," as were examined each year from private and public in- to follow disease activities in the organism is commonly called. Additional beds various com- formation on the effects of age and history of mercial operations. These tests have aided greatly has in that data oysters is presented. The program of research confirming tray were representative been broadened from epizootiology of susceptible of oyster beds. Mortalities oysters to include a search for resistant strains are expressed as death rates per of oysters. A discussion of possible origins and life month (30 days) regardless of period between cycles is offered from the meagre clues provided examinations. In line graphs, the death rate is by field studies. Finally, some findings that may plotted at the end of the period of observation, permit oystermen to limit losses in afflicted areas hence refers to the period preceding the point. are presented. Death rates were obtained by dividing number of This paper presents evidence and discussion on deaths during the period by number alive at the three primary subjects: 1) confirmation, changes beginning of the period. Plotted death rates cannot and refinements of epizootiological concepts given be added to obtain cumulative mortalities. Total earlier; 2) evidence for innate and acquired im- cumulative mortalities for peaks are often given munities in oyster populations; 3) hypotheses on below the curves with end points chosen as illus- the origin and life cycle of the pathogen. The trated by vertical bars in Figure 1. Prevalences in dramatic appearance and persistent depredations all graphs are given as number of cases of MSX by M. nelsoni demand some attempt to rationalize per 25 live oysters. No data are given for Dermocy- its origin and history. stidium because it was not involved in these studies. METHODS In referring to the history of oysters, the terms early-and late-summer exposure (or infections) Disease activities have been monitored with are used because a sharp break occurred in pat- cohorts of oysters in plastic and wire mesh trays. terns of morbidity and mortality about 1 August Wild oysters transplanted from disease-free areas (Andrews, 1966). Early-summer exposure applies are referred to as "imports." The designation to all oysters imported to an endemic area from "progeny" is restricted to laboratory-bred groups November through July whereas late-summer in- spawned from parents chosen for their history of fections were initiated in August, September and exposure to MSX. Groups of 300 to 1000 oysters October. have been held on oyster grounds in legged trays. The term "selection" is used in the phenotypic Regular examinations and counts have provided sense of choice of breeding stock by the activity of accurate death rates and eliminated predation and MSX and has no implications of phylogenetic physical causes of deaths. Little attempt has been changes because genotypes are unknown. The made to control density of oysters. Each cohort terms infectiveness or infectiousness refer to the was held as a separate population until numbers ability of the pathogen to produce disease. declined to about 100 oysters. Samples of live oysters were taken sparingly and all gapers (dead RESULTS oysters with meats) were examined. Methods of handling trays and processing oysters have been MSX in Susceptible Im^mrts described previously (Andrews, 1966; Andrews and The patterns of prevalences and mortalities for Wood, 1967). the years 1960-63 were derived from exposure of Several stations have been established in Vir- susceptible James River oysters. Each year new ginia waters and often duplicate trays have been lots of the same oyster stocks were imported and maintained at one or more stations (Andrews and observed until depleted. Intensity of MSX activity Wood, 1967). "Off VIMS" refers to an oyster bed increased in the drought years of 1964 to 1966. one-quarter mile offshore of the laboratory. Data Timing of events was earlier and death rates were from VIMS pier trays have been avoided because higher but the basic patterns prevailed (Andrews, of Dermocystidium occurrence. Seasons of importa- 1966). The following data confirm earlier observa- tion and histories listed in graphs are important tions under conditions of higher infective pressure factors for various cohorts. Seasonal patterns of by MSX. morbidity and mortality were the chief parameters Mortality data from spring and late-summer sought. Conclusions are drawn only when differ- imports of susceptible oysters are compared for ences were large in magnitude. "Background" two-year periods in Figure 1. Spring imports are OYSTER MORTALITY IN VIRGINIA 25

Y 21 14 SEP 1964 -^ - -— Y 18 13 AUG 1963 —» ^0 4 14 26 JAY D. ANDREWS

the imported stocks were dead. In late-summer imports, infections became clinical well ahead of deaths although this did not occur in earlier years (Andrews, 1966). The same patterns occurred in 1966 with higher mortalities — particularly in spring imports (Fig. 2). Y28 and Y29 are duplicate trays except that the latter was located at AMOCO station three miles below Gloucester Point in the York River. It will be noted that the late-summer import (Y25), although showing patent infections in early winter, did not exhibit deaths until June of the year after import. Y35 had high prevalences in fall and winter and deaths began in winter and accelerated slowly through the spring of 1967 (not shown). By 1 June 1967, Y35 and a duplicate tray (Y34) had about 15 per cent mortality from MSX. Death rates increased steadily through cold winter and spring months without following the typical end-of-winter pattern. Both mortalities and pre- valences in 1966 appeared to be higher than in 1964 and 1965. MSX activity in 1966 is compared for several

1966

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC OYSTER MORTALITY IN VIRGINIA 27

were over 50 per cent in 1967. Note that — January in the same waters where imported susceptibles prevalences were not much in Potomac higher concurrently exhibited mortalities of 60 to 70 per oysters than in other but death groups rates were cent per year. Fortunately, some data have been very high and more peaked in August and Septem- accumulated to explain these contrasting experi- ber. These groups of oysters illustrate the im- ences with MSX. portance of timing and history of if imports Two groups of native trayed oysters exposed to severe losses are to be avoided, as the next lots the severe epizootics of 1965 and 1966 are shown further demonstrate. in Figure 4. Y27 contained Deep Rock oysters al- most marketable in size. It is clear MSX in Native Oysters and Progeny in that the 50 per Endemic Areas cent prevalence of MSX when imported to VIMS in March was greatly reduced in April and May, One important factor in the epizootiology of a and a light mortality of short duration indicates disease is the age of host organisms. It was noted recovery of these oysters from infections. early that young oysters had low death rates but Tray Y33 containing VIMS piling oysters (1964 it was assumed this was related to size and that year- class) showed no evidence of a typical summer MSX gradually selected populations more vigor- mortality and prevalence of MSX was low. It is ously as oysters became larger. This was the ex- important to note that little selection of the perience with the disease caused by Dermocysti- piling population occurred unless it happened at a dium. MSX infections in spat were observed in the very small size when "boxes" (attached valves of dead 1958 epizootic in Delaware Bay (Haskin, personal oysters) were detached. communication, 1960). easily A comparison of susceptible imports and labora- For most studies of MSX patterns, James River tory-bred progeny is given in Figure 5. Susceptible oysters 2 to 3 inches in length were imported. oysters are represented by trays of new imports in James River is the primary source of seed oysters 1965 and 1966. The progeny group PIO was bred in in Virginia although other seed areas have been the summer of 1964 and averaged three inches in developed since 1963. In lower Chesapeake Bay, the fall of 1966, hence represents MSX-exposed small seed oysters are subject to predation; hence progeny as yearlings in 1965, two-year olds in 1966, the use of large susceptible James River seed etc. Death rates and MSX prevalences reveal oysters is natural and practical. dramatic differences in susceptible and exposed In March 1965, Virginia opened, for the first lots. PIO is perhaps the most resistant of 13 time, public beds of the Piankatank and the Great progeny groups of the 1964 yearclass being moni- Wicomico rivers for seed oystering. in Beginning tored. It was bred from rare survivors of millions 1963, these beds had been planted with reef shells of bushels of oysters decimated by MSX in Mob- obtained by a suction dredge. Samples of seed jack Bay. Tray PIO has not shown any of the oysters in January and April 1965 showed the 1963 mortality peaks typical of epizootics caused yearclass to be 60 per cent infected with MSX and by MSX and prevalence has been low small 1964 20 cent consistently yearclass spat per infected on (June 1967). the same bed. Due to crowding, few oysters were over an inch in length. A group containing both yearclasses was moved to VIMS in November 1964. 1966 Incidence of MSX declined in spring and summer of 1965 without much mortality and two years of monitoring revealed that losses were - Y 27 lOMAR 1966 relatively » 13 low. These observations increased interest in the i Y 33 27 JUL 1966- use of small, young oysters for rehabilitation of oyster beds although attempts to breed disease- resistant oysters were begun in the summer of 1962. Other observations and reports indicated that native oysters in areas epizootic for MSX were producing some marketable oysters. The Nan- semond Ridge area of Hampton Roads produced a sizable crop of oysters between 1960 and 1965. Deep Rock bed off the mouth of the Piankatank JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC River was observed to have good survival and marketable oysters in the epizootic years of 1964 FIG. 4. MSX activity in native oysters from Deep to 1966. Additionally, a native spatfall in Septem- Rock (Y27) and VIMS pier (Y33) in 1966. Y33 oys- ber 1964 on at Gloucester Point has pilings grown ters were placed in trays in July but had been nearly to market size without appreciable losses exposed at VIMS from setting. 28 JAY D. ANDREWS

I I I 1 1 I I I I 1 I I I 1 1 J I I I u L^_ JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

FIG. 5. Susceptible oysters (Y23, 1965 imports and Y28, 1966 iinports) versus laboratory-bred progeny (PIO) off VIMS. Horseheads were older and slightly larger in both years but all selection by MSX shown on this graph. VIMS station.

More typical of the level of MSX in progeny are MSX. An example of a progeny group reared from groups P6 and P7 whose parents were the sur- unselected (susceptible) parents is P14 shown in vivors of several years of MSX selection at VIMS Figure 7. These oysters bred in February 1965 com- in trays (Fig. 6). TTiese groups show small from Horsehead parents are essentially mortalities at expected times of MSX-caused parable in size to 1964 progeny. Low prevalences deaths, although the peak was very late in 1965 and late moderate mortality were exhibited in in the as yearlings, and peaks are barely detectable in 1966. The two groups of progeny depicted 1966 as two-year olds. The winter loss of P7 is not upper graph of Figure 7 are from MSX-selected until typical of progeny, and factors other than MSX parents. Both were held in Ames' Pond were involved. Prevalences, which usually were March 1965, then moved to Horsehead Rock in the only one-third or one-fourth as high as in suscep- upper James River seed area where MSX is not of three tible oysters, peaked in May (typical) but losses prevalent. In April 1966 at a size nearly were not commensurate. Total losses in trays at inches, P13A was moved to VIMS for exposure to two and one-half years of age and market size MSX. Mortality and prevalences were surprisingly levels were about 35 per cent in each tray. low although typical in timing. Background Low prevalences and mortalities were not con- of mortality in disease-free low-salinity areas are fined to oysters reared from MSX-selected parents exhibited by P13, which was a control group held (survivors of epizootics). It is probable on the basis at Horsehead Rock. of population sizes alone that the native oysters P14, raised from unselected parents, appears to as which survived epizootics (in three separate river have as good a record of resistance to MSX systems) were derived mostly from upriver breed- progeny bred from selected parents. It was one of ing populations which had little or no selection by the few groups held at VIMS from time of setting.

1966

1964 PROGENY

1964 PROGENY ^ i I I i

I

f 5 S JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

FIG. 6. MSX activity in laboratory-bred progeny at VIMS (P6 and P~!). Average size in 1966 was 65 mm in June and 75 mm in November. OYSTER MORTALITY IN VIRGINIA 29

1966 have sustained this conclusion through 1966. In 0->PI3 —» i i 1962, prevalence data for gapers and live oysters PI3A —» were presented M. costalis 20 indicating (SSOl was much more active than M. nelsoni (MSX) in native oysters. Since there is a possibility that native oysters have developed some resistance to MSX, data from susceptible James River imports were chosen to demonstrate the inactivity of MSX on the Seaside. No attempt has been made to com- ir JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC pare the level of activity of SSO in the six years illustrated. James River oysters, imported to the Seaside in 1959, survived unexpectedly well for 12 to 14 months (Figs. 8 and 9). A sharp mortality, caused by SSO, occurred in May-Juno in 1960 but there was no evidence of epizootics from MSX in either year. Prevalences of MSX were low in live oysters. Fall samples are particularly significant because SSO is not clinically evident then. Four relatively isolated bays on the Seaside gave similar patterns JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC of little mortality and low MSX morbidity during a two-year period. High prevalences of SSO were FIG. 7. MSX activity in progeny from selected found in gapers during the May-June epizootics in as (PIS and P13A) and unselected (PlJf) parents. 1960 demonstrated previously for native oysters VIMS station except the control group P13 at (Andrews et ah, 1962). Horsehead, James River, an area free of MSX. Two imports in later years are depicted in Figure 10. The mortality patterns and low mor- bidity from MSX again demonstrate the inactivity of M. nelsoni on the Seaside. The graphs also show hence exposed intensively to MSX. that oysters go through their first SSO epizootic without losses and that this MSX Activity on the Seaside of Eastern Shore exposure apparently initiates infectons which result in Mortality patterns and prevalence data indicate an epizootic the next June The Island very little MSX activity in the bays of Seaside of May- period. Long oysters were as hence Virginia. Monitoring for MSX on the Seaside in imported spat were rather small and for a full-scale 1959 and 1960 resulted in the discovery of Seaside young SSO epizootic in 1963. Disease of oysters caused by Minchinia costalis Mortality and prevalence data are given for two (Andrews, Wood and Hoese, 1962). It was reported trays of native oysters in Hog Island Bay (Fig. in 1962 that oysters on the Seaside did not exhibit 11). Mortalities were low and erratic except for the epizootic patterns of MSX and subsequent data the deaths caused by M. costalis each June. Deaths

25 FEB 1959 HOG ISLAND BAY «feTl 15 MAY 1959 OUTLET BAY ^ 40 - a: 0-35-

^'•-f^V^7* ***>^^*y JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

FIG. 8. Susceptible Horsehead oysters on the Seaside of Eastern Shore Virginia (S3 and S7). The mortality peak in 1960 was caused by SSO. MSX was not active as indicated by absence of typical mortality peaks and scarcity of infected oysters. 30 JAY D. ANDREWS

S 12 16 JUN 1959 COBB IS BAY

S 13 16 JUN 1959 SWASH BAY

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

FIG. 9. Horsehead oysters in two additional Seaside bays reveal little MSX but a strong SSO kill in 1960 (S12 and S13).

in tray S49 for the period ending 30 September of devastation. My first description of the epi- 1966 are of unknown accidental cause and sliould zootiologj' of MSX was based on five years of field be disregarded. Prevalences in these groups, al- studies (1959-1963). The oyster industry in lower though low, suggest that MSX played a minor role Chesapeake Bay was destroyed in these first years in the 1966 deaths in S49. The summer death curve of MSX activity which were considered catas- of this tray is spread much wider than in typical trophic. Yet, the past three years (1964-66) have SSO epizootics. brought increasingly intensive activity during a MSX has not caused enough deaths in eight period of drought in the Bay. The range of MSX, years of monitoring tray oysters on the Seaside to or at least the area of damage, has been extended produce a recognizable peak in mortality curves. up the Bay and its tributaries in irregular pat- E.xposure of susceptible oysters indicates that the terns not associated with salinity alone. In 1964, environment is unsuitable in some way. The the James River seed area was invaded seriously regular occurrence of MSX on the Seaside sug- but not in later years. In 1965, the disease caused gests that its failure to become epizootic may not extensive damage in Pocomoke Sound, the lower be due to scarcity of infective materials although Maryland tributaries of the Bay, and in the upper nothing is known of dosage. In contrast, SSO is an Rappahannock River — all of which had been important cause of deaths but can be avoided for marginal areas for MSX in earlier years. These nearly two years by proper timing of imports. extensions of MSX activity were not maintained in 1966 and 1967. DISCUSSION Epizootiology The epizootics of oysters caused by Minchinia The studies of recent years have essentially con- nelsoni show no signs of abating after eight years firmed the patterns of morbidity and mortality re-

JAN FES MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC

FIG. 10. Imports monitored from 1962 through 1966 revealed typical SSO peaks but very little MSX activity (S37 and S^S). OYSTER MORTALITY IN VIRGINIA 31

549 NATIVES, HOG IS BAY —

JAN FEB MAR APR HAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAfl APR MAY JUN JUL AUG SEP OCT NOV DEO

FIG. 11. MSX activity in native oysters iS/il and SiO) is similar to that in susrepti- bles—without clear mortality peaks from MSX and with low prevalences.

ported for MSX earlier. A clearer understanding December 1966. The death rates accelerated slowly of the epizootiology has resulted from exposure of from about 1 per cent per month in the late fall to oysters of a greater variety as to age and history. 5 per cent per month in May without a distinctive Increasing intensity of the disease challenged pattern. These rates are very low for oysters 60 to susceptibles, resistant progeny and old survivors 80 per cent infected with MSX, and most diseased and provided new insights of timing of infections oysters followed the usual pattern of high mor- and sporogony. Sporulation, although still rare, talities in June and July of the second year. The has occurred more frequently in resistant progeny apparent occurrence of two separate types of in- than in susceptible oysters. Mostly the susceptible fections seems to resolve now into a matter of oysters were monitored in earlier years to obtain intensity of infectivity or dosage followed by tem- patterns of mortality and morbidity. The life cycle perature reduction. The end-of-winter mortality is slowly being pieced together without the bene- peak is absent in late-summer imports despite high fits of cultures and infections in the laboratory. prevalences. MSX has been active in Delaware Bay for ten Patterns of Morbidity years. Delaware and Chesapeake bays are the two Prevalence of MSX in live oysters has increased major areas damaged by MSX and massive collec- during the drought years of 1964-66. Susceptible tions of data are available for both. It would be Horsehead Bar oysters have been used for monitor- very useful to have careful comparisons of data ing prevalence each year. In earlier years, 50 per and of interpretations of epizootiology. cent prevalence was about maximum but in recent Patterns of Mortality years samples exceeding this level are common in Patterns of mortality have remained essentially all seasons. stable in timing but death rates have increased in Winter and spring imports acquired typical June recent years. Season of import is critical in sub- infections, evident in July, e.xcept that clinical in- sequent time and level of deaths. Winter and fections have occurred slightly earlier in drought spring imports begin dying from mid-July to late years — particularly in 1964 when deaths began August. Oysters imported in winter and spring ex- before mid-July. The minimum observed period hibited lower first-year deaths than those brought from import to deaths for populations has been six in during the early-summer period of infectivity weeks; hence MSX infectivity must have been (June and July). This was observed in earlier intense by 1 June in 1964. Imports in mid-June that years but not appreciated fully until the 1966 ex- year resulted in late infections and deaths be- perience with Potomac River oysters (Fig. 3). ginning 1 August. For many years the absence of deaths (and Late-summer imports have shown progressive patent infections) in late-summer (August through changes in timing with increases in infective pres- October) imports was an enigma. These groups sure as shown by susceptible controls. Previously, usually lived for eight to ten months with deaths infections were apparently acquired promptly beginning in June of the second year. In recent after import but were not clinically detectable years of incieased MSX activity, this pattern has until ]\Iay of the following year. Beginning with been broken. The change is more evident in pre- the first of lour drought years in 1963. a gradual valence data than in death rates but August 1966 change in the time of appearance of clinical infec- imports began dying from MSX in November and tions was observed. In 1963, oysters imported 14 32 JAY D. ANDREWS

September had no infections in late January 1964 features of MSX in lower Chesapeake Bay are the when first sampled. By early April 1964, infections persistent patterns of widespread infections with- were evident and prevalence reached 56 per cent out regard for number or proximity of other oys- in late May before deaths began. In 1964, mid- ters. Susceptible oysters held in trays with infected August imports already had 25 per cent clinical lots do not obtain infections earlier or in greater infections in late January 1965 and nearly 50 per numbers than others grown in relative isolation. cent in Februai^. It is probable that some infec- Most epidemiologists would probably regard this tions were clinical cases in the late fall of 1964 but as evidence of another host whether alternate or no sampling was done because patent infections one independent of oyster populations. The long were not expected. In 1965, August imports showed period of infectiousness of MSX (about 5 months) 25 per cent infections in December and prevalence and continuous infection pressure are factors of reached over 60 per cent in May 1966. Finally, in interest in this regard. The obvious dispersion of 1966 mid-August imports had begun to die in late micro-particles in tidal estuarine waters makes it October with 30 per cent infections and prevalence difficult to imagine direct transmission over climbed to 80 per cent in December. widely spaced areas where oysters are relatively In recent years intensive MSX activity has scarce. Yet the period of apparent infective pres- demonstrated that throughout the warm season sure matches the period of oyster deaths from If another host is it some susceptible oysters developed clinical infec- MSX rather closely. involved, tions in about two months after import. This must release infective elements for a long period motile and distributed implies that dosage of infective materials is vari- also. Large highly widely and scianid fishes able by seasons and years. Early summer (late organisms, such as blue crabs seem May to early July) appears to be consistently the which are attracted to oyster communities, hosts. The period of most intensive infectivity and the most likely as possible alternate many shortest incubation periods follow. Incidence of varied situations where oysters have become in- and MSX (percentage of oysters infected in a given fected tend to exclude fouling organisms period) is higher from early summer exposure bottom infauna if proximity is required. Oysters been than prevalence data indicate because deaths re- essentially free of oyster associates have move morbid oysters continuously. In late-summer imported and infected before fouling organisms infections, deaths are usually delayed for 8 to 10 could accumulate. Sandy, shelly and muddy bot- months, hence the highest prevalences are ob- toms show no apparent differential effect on in- served in May. Cool temperatures intervene and fection activities. delay the development of late-summer infections Innate vs Resistance hence no major change in mortality patterns Acquired of two of populations occurred even in the years of high MSX activity. Progeny types parental were monitored in our program to select oysters The conclusions from these many years of ob- resistant to M. nelsoni. Unexposed susceptible servations are that interaction of dosage, suscepti- stocks and survivors of MSX-ravaged populations bility of oysters (including effects of timing of im- were chosen for breeding. The survivors from epi- portation) and temperature produced the patterns zootic areas were estimated to comprise less than of infectivity and mortality described. These 10 per cent of the originial populations after five factors tend to obscure a pattern of continuous years of MSX activity. Evaluation of resistance in exposure at variable levels of infective particles. progeny tests was based on the phenotypic char- Apparently infections are established only during acters of mortality and prevalence rates. As con- the warm season from May through October when trols and as indicators of MSX activity, native oysters are dying most rapidly. This does not pre- Horsehead oysters grown for two to four years in clude exposure during the cold months of Novem- an area free of the pathogen were imported. In the ber to May at a low level of infective particles. years 1964 to 1967, MSX generated intensive in- of oysters and dosage of infective Susceptibility fective pressure in these susceptible controls. particles are only very crudely measured in field Progeny of both susceptible and selected parents studies by monitoring stocks of common history exhibited low levels of MSX activity and of mor- and origin at one station. I must assume steady At first this was attributed to their small virulence of MSX to conclude that dosage has in- tality. size and young age, but all progeny continued to creased during the drought years. Susceptibility exhibit resistance to MSX as they grew to market has probably not increased, although it appears to size at ages of two and three years. Progeny were be modified by time of import and age (size) of reared in trays off VIMS in the same environment oysters at first exposure. Evidence on the im- in which controls succumbed to MSX (Fig. 12). It portance of early exposure is presented below. is important to note that early selection of Contagiousness progeny did not occur. Spat were tested when Perhaps the most intriguing and dismaying moved from the pond to VIMS and MSX was OYSTER MORTALITY IN VIRGINIA 33

MSX PREVALENT AREAS MSX FREE AREAS Mobjack Boy, VIN4S, etc Upper James River Seed Area

VERY LOW NO MSX LOW LOW LOW HIGH I960 - 1967

1 1 1 ' '

MARKET 1^: MARKET „ MARKET _ FEW OYSTERS OYSTERS OYSTERS SURVIVORS 34 JAY D. ANDREWS

erations in MSX-prevalent areas may be en- out the year although this has not been demon- most dur- hanced more by acquired than innate resistance. strated. Since oysters are dying rapidly The immediate practical apphcations of early ing these five months of demonstrated infectivity, the source exposure to reduce losses from the disease caused this is indirect evidence that oysters are of and source by M. nelsoni make it imperative that this aspect of infections. The problems dosage of resistance be explored and exploited fully. If of infective particles are not easily solved in field effective in practice, it reduces the immediate need studies. for producing seed from genetically resistant SSO exhibits a long incubation period (6 to 9 strains of oysters under controlled conditions of months) during which neither clinical evidence of hatcheries and ponds. The effects of predation on the organism nor physiological evidence of reac- host be observed. studies spat and young oysters must be considered by oys- tion by the can MSX termen in Chesapeake Bay where most seed oys- have yielded a confused picture on period of in- ters are large when planted. Most seed oysters cubation to clinical cases. Individual oysters ex- in 5 weeks originate in low-salinity sanctuaries where disease posed in early summer may show MSX late is absent or scarce, hence sources of stocks ex- or 5 months, whereas oysters first exposed in in two posed to MSX must be considered. summer may show physiological effects weeks (under water weighing) but delayed ap- Comparison of Life Cycles of MSX and SSO pearance of clinical cases may extend to nine SSO and MSX appear to be poorly adapted months. Again dosage and susceptibility are prob- parasites of oysters. Both tend to kill their hosts ably involved in ways now obscure and produce before sporulation is completed. A carefully at- puzzling patterns of prevalences and mortalities. tuned parasite, such as Bucephalus, exhibits low Once patent, SSO infections develop rapidly, be- pathogenicity and does not upset the feeding come intensive and kill oysters promptly. Tissues in the ability of its host — at least until late of nearly all organs are filled with sporocysts and has had parasitic relationship when the parasite are quite disrupted, but usually epithelia are left ample time to complete its reproductive cycle. intact and not invaded. MSX can kill with equal Oysters show little reaction to Bucephalus al- rapidity but no clear relation between intensity of though the parasites castrate their hosts and rob infections and deaths is seen. Susceptible oysters them of nutritive materials. which die in the first summer often have light A comparison of the two Minchin'm parasites of infections with little disruption of tissues. Those in survive infections oysters reveals several important differences oysters which with persistent of the adaptation of life cycles. These factors of season- until late winter or early summer following of (one after infection) have inten- ality, exposure, incubation period, intensity year year usually infections and their implications about life cycles sive infections and reactions to match. Yet tissue will be discussed. SSO is much more regular in its disruption is not as serious as in .SSO cases. seasonal patterns of mortality and prevalence. In conclusion, it is postulated that the normal This sharp seasonality was a primary reason for life cycle of M>7ichinia species in oysters is an describing M. costalis as a separate organism in annual affair with mortalities and new infections 1962. However, without sporulation stages and occurring in early summer (June mostly in Vir- spores, which were common and regular in occur- ginia). SSO is moderately adapted to its host in rence, the description of SSO as a new species terms of seasonality, level of mortality and limita- would have been risky in 1962. Plasmodial stages tion of morbidity in oyster populations. This were not easily separated from those of M. nelsoni; pathogen is limited to high-salinity areas in its the characters used were recently described by environmental range. Its pathogenicity is high and Couch (1967). SSO disappears from oysters (clini- a large proportion of infected host individuals die cally) in July and does not reappear usually until before sporulation is completed. Since oyster meats March and April, although occasional infections are rapidly destroyed after death, only a small have been seen as early as December. Sporulation proportion of gapers produces mature spores. The occurs rapidly in May and June and oysters die or potential number of spores is very high. recover in close synchrony. In contrast, MSX MSX appears to be a closely related species occurs in oysters throughout the year and kills parasitizing oysters in more brackish waters. Ap- them continuously in patterns related to time and parently, some recent event of mutation or hybrid- intensity of exposure, susceptibility and tempera- ization has increased its virulence greatly. This ture. virulent strain is so pathogenic that the annual SSO appears to require exposure of oysters to cycle is interrupted very early in most oysters by an epizootic to initiate infections. MSX produces death of the host. Sporulation is rare but it has infections in newly-imported oysters for at least been observed in nearly all months of the year. the five continuous warm months and probably June appears to be the month when sporulation oysters are exposed to infective particles through- normally occurs. Timing of infections, deaths and OYSTER MORTALITY IN VIRGINIA 35

sporulation have all been disrupted by high patho- cases now on slides). Despite the rarity of spores genicity. The usual fate of such a destructive (fewer than 1 per 1,000 cases of MSX prior to parasite as MSX would be to decimate its host 1966), I believe the sequence of sporulation stages population, hence reducing its own occurrence. But and invariable occurrence of MSX Plasmodia are MSX continues to thrive with greatly reduced oys- strong evidence of correct identification as a hap- ter populations, and the source of infective parti- losporidian. The similarity of morphology and the cles and the mode of transmission remain a placing of both pathogens in the genus Minchinia mystery. reinforce the concept of closely related species. I Hypothesis on Origin of MSX agree with Couch (1967), who described concur- rent The origin of marine epizootics is always diffi- infections, that affinity of the two pathogens is close. cult to explain. However, it seems worthwhile to record the fragments of information available and It is worth noting some other similarities and to attempt an explanation of events. In Virginia differences between MSX and SSO. Both exhibit a we have positive evidence that MSX was not im- frequent failure to complete their cycle to a portant as a disease agent in the decade of the mature resting spore. However, SSO usually pro- 1950's prior to 1959. The preponderance of data duces sporulation stages whereas MSX rarely consists of mortality records of many groups of does. In our paper on the epizootiology of SSO oysters over many years. The patterns of oyster (Andrews et ah. 1962), we reported over half of deaths were clearly linked to Dermocystidium the gapers with spores but our criterion was to marinum. Losses were confined to the late warm report the most advanced stage seen. Many gapers season each year. A few hundred permanent slides had only immature spores and often few of these. of gapers confirm the absence of MSX and high Therefore, most SSO materials were being re- prevalence of D. marinum. leased to open waters as sporocysts without ma- A tenuous but important link in the evidence on ture spores. In some recent years, spores have origin is the finding of two pre-epizootic cases of been comparatively difficult to find in smears and MSX — one in 1953 among 49 fungus-killed oys- sectioned gapers during SSO epizootics although ters six years before MSX became active. One cysts of sporoblasts are common. A further simi- must conclude that M. nelsoni was endemic but larity is the occurrence in both pathogens of a quite inactive in the 1950's. If this assumption is stage without distinct nuclei but with numerous accepted, upon slender evidence, the question punctate chromatin granules (probably schizogo- arises as to how MSX became epizootic. A theory ny) preceding formation of sporoblasts. This stage of cyclic activity with fluctuations in susceptible is distinctive in morphology but it is never ob- host populations seems improbable with more than served unless sporogony occurs. Enlargement of a decade of known inactivity when oysters were Plasmodia to sporocysts begins with this stage. abundant followed by eight years of continuous Field experiments by carefully timed imports epizootics. indicate that periods of infectivity match those of In 1959, while monitoring for MSX, the haplo- oyster mortalities for both MSX and SSO. The sporidian parasite, Minchinia costalis, was dis- infective period for SSO appears to be less than covered and later named by Wood and Andrews two months, with June the most important month. (1962). Seaside organism (SSO), as this pathogen Although oysters are infected with SSO during a was called, has never been found on the western June mortality period, the pathogen is rarely shore of Chesapeake Bay although it appears to found before March of the following year. In con- be endemic on Eastern Shore, particularly on Sea- trast, MSX kills oysters around the year but chief- side. The pattern of a sharp peak of mortality in ly in the warm months, June through November, late May and June was a dominant reason for inclusively. Evidence has been presented that high separating SSO from MSX. In addition, sporula- levels of infection of MSX can be attained by ex- tion stages were observed progressing to typical posure in any one of these six months except haplosporidian spores. Considerable effort was Novmber. MSX, too, tends to exhibit "hidden" in- expended in convincing colleagues that two sepa- fections for periods up to nine months. rate organisms were involved, for the early Plas- The purpose of these discussions of similarities modia are remarkably similar. of MSX and SSO drawn from epizootiological In 1966, Couch et ah associated some rare Min- studies is to demonstrate the close relationship of chinia spores with MSX. These spores, found only the pathogens. It is ironic that in the early 1960's in epithelia of liver tubules whereas smaller my efforts were directed at proving differences in SSO spores occur in all tissues within epithelial the pathogens whereas now I am emphasizing con- walls, were observed first at VIMS in 1960. Occur- generic relationships. On Seaside, the pathogens rence of MSX spores has been extremely low in are living sympatrically but only SSO produces gapers and live oysters, but always typical MSX typical infection and mortality patterns there. Plasmodia have been present with spores (about 20 The next episode of our reconstruction of the 36 JAY D. ANDREWS origin of MSX is based upon well known events ACKNOWLEDGMENTS and their timing but presumes interbreeding of I am most grateful to Dr. J. L. Wood, Head of MSX and SSO for which there is no evidence. The the Department of Microbiology at VIMS, and his history of seed oyster planting from the Seaside of Microtechnique Section for preparation of slides Eastern Shore to Delaware Bay is well known. In of oyster tissues. We now have a collection of the first half of the decade of 1950-60, over half the 90,000 preserved oysters, most of which have been seed planted in Delaware Bay came from Seaside sectioned and stained on permanent slides. My ap- where SSO is prevalent. Although generally lim- preciation is also extended to Mr. Michael Frier- ited to environments nearly oceanic in salinities, man who has diagnosed most samples in recent SSO does live in Bayside creeks and lower Dela- years for occurrence of MSX. The samples of ware Bay (Haskin, personal communication). In James River oysters from the Seaside of Eastern view of the sudden appearance of a virulent strain Shore were obtained by Mr. Michael Castagna to of MSX in Delaware and Chesapeake bays only whom I am indebted for monitoring trays and beds two years apart, it seems quite logical to me that in that area. a new strain of Minchinia could have arisen in Delaware Bay from the proximity of native and Virginia oysters. No evidence that MSX was pres- ent in Delaware Bay prior to 1957 is available, but LITERATURE CITED if endemic in Chesapeake, transplanting would al- Andrew, J. D. 1964. Oyster mortality studies in most surely have scattered it along the coast with- Virginia. IV. MSX in James River public seed in its tolerance range. beds. Proc. Nat. Shellfish. Ass. 53( 1962) :65-84. If we assume that a new virulent strain arose, it Andrews, J. D. 1965. Infection experiments in na- could be to follow the expected epizootiologically ture with Dermocystidium niarinum in Chesa- patterns of a newly introduced disease. The pat- peake Bay. Chesapeake Sci: 6:60-67. terns of kill are very similar in both bays (Haskin Andrews, J. D. 1966. Oyster mortality studies in et ah, 1965). Mortalities began in late spring and Virginia. V. Epizootiology of MSX, a protistan early summer of 1957 and 1959 in Delaware Bay pathogen of oysters. Ecology, 47:19-31. and Chesapeake Bay, respectively. The following Andrews, J. D. and J. L. Wood. 1967. Oyster mor- summer severe mortalities occurred in all beds tality studies in Virginia. VI. Historj' and dis- within the tolerance of MSX. In both bays salinity tribution of Minchinia nelsoni, a pathogen of the initial large-scale infection occurred in late oysters, in Virginia. Chesapeake Sci. 8:1-13. summer (well documented for Chesapeake Bay) Andrews, J. D., J. L. Wood and H. D. Hoese. 1962. and patterns of infection and mortality typical Oyster mortality studies in Virginia: III. were established in both bays the second summer. Epizootiology of a disease caused by Haplo- In each bay a localized center of infection was sporidium rostale Wood and Andrews. J. In- observed from which the disease spread. In Dela- sect Pathol. 4:327-343. ware Bay this center consisted of imported Sea- Couch, J. A. 1967. Concurrent Haplosporidian in- side oysters but in Chesapeake Bay it occurred in fections of the oyster, Crnssostrea virginira native transplants no different from beds in sur- (Gmelin). J. Parasitol. 53(2) :248-253. rounding areas. It is hard to believe that this close Couch. J. A., C. A. Farley and A. Rosenfield. 1966. of events two could be accid- parallel years apart Sporulation of Minchinia nelsoni (Haplo- ental or caused natural or by changes cycles. sporida, Haplosporidiidae) in Crassostrea vir-

an of this of : 1529-1531, Perhaps important part hypothesis ginica ( Gmelin i. Science. 153(3743) a new virulent race of MSX lies in the environ- Haskin, H. H., W. J. Canzonier and J. L. Myhre. — for lack mental tolerances of the two pathogens 1965. The history of "MSX" on Delaware Bay often referred to of accurate knowledge of causes, oyster grounds, 1957-1965. Amer. Malacolog. as "salinity tolerance." It has been noted that SSO Union Bull. 32:20-21 (Abstract). whereas requires high salinity environments MSX Haskin, H. H., L. A. Stauber and J. G. Mackin. in salinities and does not flourishes best moderate 1966. Minchinia nelsoni n. sp. (Haplosporida, in the areas produce typical epizootics optimum Haplosporidiidae): Causative agent of the for The fluctuations in MSX at SSO. activity Cape Delaware Bay oyster epizootic. Science, 153 May (Haskin et al., 1965) are notable in com- (37421:1414-1416. parison with persistent activity in lower Chesa- Stauber, L. A. 1961. Immunity in invertebrates, peake Bay. I suspect lower Delaware Bay is mar- with special reference to the oyster. Proc. Nat. ginal in some years for both MSX and SSO al- Shellfish. Ass. 50(1959) :7-20. though the reasons are vague. One can only specu- Wood. J. L. and J. D. Andrews. 1962. Haplospori- late that this environment provided suitable con- dium costale (Sporozoa) associated with a ditions for mutation or hybridization which gave disease of Virginia oysters. Science 136(3517): rise to a virulent race. 710-711. Proi-ecdings of the National Shellfisheries Association VdlAone 58 — June 1968

VITAL STAINING OF BIVALVE MOLLUSK SHELLS WITH ALIZARIN SODIUM MONOSULFONATE Herbert Hidii ' and James E. Hauks U. S. BUREAU OF COMMERCIAL FISHERIES BIOLOGICAL LABORATORY MILFORD, CONNECTICUT

ABSTRACT

Alizarin sodium mo7u>sulfonate icas used for i^ita! staining of shells of larval and jKjst-metamorphir hard- and soft-shelled rlams, Mercenaria mercenaria and Mya arenaria. In post-metamorphic clams a week-long immersion in 5 to 20 ppvi alizarin resulted in peripheral and medial deposition of red shell material. Larval M. mer- cenaria, when subjected to 0.25 to 0.75 ppm alizarin for 48 hours, produced distinc- tive check ma7-ks on shells ichich may have value in marking. The long persis- tence (at least 18 months iti the present studyi of metabolically induced red shell coloration gives this method an advantage over more superficial marking techniques used in ecological research.

INTRODUCTION hard- and soft-shelled clams, Mercenaria mer- cenaria and Mya arenaria. Also, alizarin has pro- Shells of living mollusks have been marked for duced distinctive check marks on shells of larval later identification in various scientific studies by hard-shelled clams. painting (Carriker, 19551. tagging (Posgay, 1961), notching (Rounsefell, 19631, and application of fluorescent material (Tufts, 1967). Larvae of MARKING POST-LARVAL BIVALVES the soft pelecypods have been marked by staining In the initial trials, post-metamorphic M. mer- tissue with neutral red (Loosanoff and Davis, cenaria were successfully marked by holding them 1947). These methods have been successful for in a solution of alizarin in sea water. Clams 2 to their purpose to greater or lesser degree but all 3 mm in width across the shell Were produced in have disadvantages that limit their general appli- the hatchery by spawning and rearing techniques cability. Most require the handling of individuals, summarized by Loosanoff and Davis (1963). One which precludes marking the great number of hundred clams were placed in each of several glass animals which are often necessary to obtain mean- 1-liter beakers containing concentrations of aliz- ingful ecological data. A method such as painting arin of 0.25 to 20 ppm. Other beakers contained may produce ephemeral marks or may cause untreated animals as a control. All were reared mortality through injury, limiting its usefulness in for 7 days at 24° C in sea water that had been fil- studies requiring identification of marked in- tered and treated with ultra-violet light; the water dividuals after a time lapse. and the alizarin concentrations were renewed at been used Alizarin sodium monosulfonate has 2-day intervals. A mixture of live flagellates, in vital staining of vertebrate bone (Lillie, 1952). Isochrysis galbana, Dunaliella euchlora, Mono- Also, Turner ' used alizarin dye in marking the calcareous tubes of the marine polychaete Hy- droides dianthus. These that applications suggested 1 Present address: Univ. of Maryland. Natural Re- alizarin also be used as a vital stain for the might sources Institute, Solomons, Md. shells of bivalve mollusks. The experiments reported here demonstrate the 2 Turner, H. J., Woods Hole Oceanographic In- incorporation of alizarin red coloration in the de- stitution, Woods Hole, Mass. Personal Com- position of new shell material in post-metamorphic munication.

37 38 H. HIDU AND J. E. HANKS rhrysis lutheri, and ChloreUa 580, was fed every medial shell that was deposited when clams were day at an approximate rate of 0.01 ml of packed returned to natural conditions. cell volume per liter culture per day (Davis and Guillard, 1958). MARKING LARVAL BIVALVES Clams were active and feeding at all test con- Larval M. mercenuria, when exposed to alizarin, centrations of alizarin during the treatments. a distinctive check mark on the shell. Shell exteriors soon became stained uniformly produced The larvae, like the animals, were dark red. post-larval initially treated tor 7-day periods. Concentrations After treatment, clams were held in screened of alizarin below 0.10 ppm produced no ill effects cages in Milford Harbor for later observation. or distinctive marking; concentrations higher than Under outdoor summer conditions all clams grew 0.25 ppm stopped growth and produced high mor- rapidly and soon lost the general dark red colora- tality. If, however, larvae were subjected to 0.25 tion of alizarin. The red margins of the shells, to 0.75 ppm alizarin for 48 hours and then re- however, representing areas of growth during turned to untreated sea water, growth of larvae alizarin treatment were retained as red bands and had faded verj' little up to 18 months after treat- ment (Figures 1 and 2 show alizarin marks 6 FIG. 1 and 2: External and Internal shell surfaces months after treatment ). The red bands were most of alizarin-inarked juvenile M. mercenaria, 6 distinctive at the higher dosage rates of alizarin, months after marking. The stnaller clam at right between 5 and 20 ppm, and appeared to be excel- shows red band one alizarin lent marks. single after 7-day immersion (5 ppm) whereas the larger individual In an attempt to place more than one red band at left shows two bands that represent two sepa- on a single clam, the clams of the original experi- rate markin.g treatments; size bears no relation- ment were subjected to a second 7-day exposure of ship to treatment. Shell length parallel to hinge alizarin (8 ppm in this trial) after a month's post- line of largest individual — 6 mm. marking growth in the natural environment. The clams again acquired general dark red coloration. When returned to natural conditions the red color FIG. 3: Cross-section of shell perpendicular to line mercenaria re- again faded, but a second red band was formed hinge of juvenile M. which ceived alizarin treatment 6 months representing shell growth during exposure to the single before Red striation reveals site shell de- second alizarin dosage (Figs. 1 and 2). sacrifice. of position during term of alizarin iinmersion. Shell Results of trials with M. arenaria were equally length to hinge line — 11 mm. good. Juvenille M. arenaria were subjected to a parallel 7-day treatment with 5 ppm of alizarin under con- ditions described above and then held under na- FIG. 4: External shell surface of alizarin-marked tural conditions for observation. Six months after juvenile M. arenaria 6 months after marking. treatment these clams had added new shell and Visible is red band and general red discoloration had also retained red bands on the shells (Fig. 4). of the shell representing alizarin deposition with In addition to the persistent red bands, the general peripheral and medial shell growth during the red coloration of the shell had not faded to normal term of alizarin treatment. Shell length parallel to coloration, but has remained light pink (Fig. 4). hinge line — 2 cm. The pink shell, contrasting with the white color of shell later also a distinctive deposited produced FIG. 5: Cross-section perpendicular to hinge line effect. Similar results were obtained with marking of juvenile M. arenaria which received single the coot clam, Mulinia lateralis. alizarin treatment 6 months prior to sacrifice. Red Cross-sections were made of shells to determine striation reveals site of shell deposition during the site of alizarin deposition. Sections of M. mer- term of alizarin immersion. Shell length parallel cenaria shells marked with a single red band re- to hinge line — 2 cm. vealed a red striation running with the laminae and the thickness of extending through complete FIG. 6: Late stage larval M. mercenaria showing the shell 3). Sections of M. arenaria shells (Fig. check marks produced by ifS-hour immersion in showed that the red coloration was on deposited O.JiO ppm alizarin. These shell marks are distinct the entire inner surface of the shell and extended from a later demarcation line between the pro- to the outer surface in the area which was periph- dissoconch shell of the larva and the dissoconch eral at the time of treatment (Fig. 5). Colorless shell of the metamorphosed individual. Shell shell beneath the red to line — about 200 band represents additional length parallel hinge fx. VITAL STAINING OF BIVALVE SHELLS 39

FIG. 1 FIG. 2

FIG. 3 FIG. 4

;^

FIG. 5 FIG. 6 40 H. HIDU AND J. E. HANKS resumed and a very distinctive check mark re- the animal. After 18 months the marks had faded sulted (Fig. 6). Immersion in 0.30 to 0.40 ppm for very little. 48 hours produced a light check mark recognizable Certain difficulties might be expected in the field in all individuals. With increasing concentrations use of the alizarin technique. Marked animals re- check marks became more pi-onounced, until at leased in natural water undoubtedly will become 0.75 ppm larvae greatly indented the shell before dark or discolored, thus obscuring the mark on the resumed. for normal growth was Larvae subjected shell exterior. It may be necessary to hold the 48 hours to concentrations of alizarin above 0.75 captured animals in sea water away from a sub- ppm were unable to resume normal growth. strate for several days to bleach shells sufficiently M. mercenaria larvae of all sizes from 115 to marked individuals. jj. distinguish Alternatively, to 225 shells of live mollusks could be filed for straight-hinge veligers up jx pediveligers lightly were marked in this manner, although success was examination of subsurface shell for traces of red somewhat more difficult to achieve than in the marking. If the animals were to be sacrificed, the technique used for post-larval clams. Many cul- interior margins or complete cross-sections of tures of marked clam larvae were reared past shells could be examined. metamorphosis; the check marks could be readily M. mercenaria shells on occasion contain na- identified as long as the larval shell itself was tural purple striae which might be mistaken for in the Considerable work recognizable juveniles. an alizarin mark. Nearly all natural striations of remains in the value of this method testing general this type instead of running the complete thick- of but it could be used for marking, probably ness of the shell are confined to the medial third other as well. marking species of the shell. The natural striae are probably varia- tions of natural purple pigmentation of certain DISCUSSION areas of the inner translucent region (Shuster,

1957 », and seem to be associated with less than in shells of M. mer- The red bands post-larval optimum growth conditions in M. mercenaria. Such of cenaria and the bands and pink discoloration M. coloration has not been observed in M. arenaria. arenaria appear to result from the deposition of Trials are being continued to determine the gen- alizarin in the new shell added during treatment. eral applicability and limitations of the method in The deposition of red shell was first suggested by molluscan research. No doubt other species of the rapid fading of red coloration in all areas of pelecypods and also gastropods can be similarly the shell except in the area which was peripheral marked. during the exposure to alizarin. Later, cross-sec- tions of shells (Figs. 3 and 5) revealed sharp alizarin bands only on shell surfaces which were ACKNOWLEDGMENT medial and peripheral during time of treatment. thank Mr. Manton Jr., for An adequate food supply favoring shell deposi- We Botsford, photo- tion during alizarin treatment would appear to be graphs and Dr. Ravenna Ukeles for supplying the determining factor in the laying down of a algal foods for experimental animals. distinctive mark, although present experiments have not conclusively demonstrated this. Adequate LITERATURE CITED food may be particulary important in marking smaller juvenile animals because their growth Carriker, M. R. 1955. Critical review of biology rates may depend more on immediate food supply and control of oyster drills Urosalpinx and than on a stored food reserve (which might be Eupleura. U. S. Fish and Wildlife Service, Spec. more important for larger, more mature mollusks). Sci. Rept. Fish. No. 148, 150 p. The alizarin marking method will be useful in Davis, H. C. and R. L. Guillard. 1958. Relative ecological research requiring the marking of a value of ten genera of micro-organisms as number of shellfish with a durable large highly foods for oyster and clam larvae. U. S. Fish limitations on the number of in- mark. The only and Wildlife Service, Fish. Bull. 58:293-304. dividuals that can be marked are the ability to Lillie, R. D. 1952. Histopathologic Technic. The keep them under such conditions that adequate Blakiston Co., Philadelphia, 300 p. shell deposit is formed while the animals are being treated and the number of animals that can be Loosanoff, V. L. and H. C. Davis. 1947. Staining of procured either through collection of wild stock oyster larvae as a method for studies of their or through hatchery rearing. Marks appear to be movements and distribution. Science 106- stable and are expected to persist for the life of (2763): 597-598. VITAL STAINING OF BIVALVE SHELLS 41

Loosanoff, V. L. and H. C. Davis. 1963. Rearing of Rounsefell, G. A. 1963. Marking fish and inver- bivalve mollusks. Advanc. Mar. Biol. 1:1-136. tebrates. U. S. Fish and Wildlife Service, Fish. Leafl. 549. 12 Posgay, J. A. 1961. Tagging as a technique in p. population studies of the sea . In ICNAF Shuster, C. N. 1957. On the shell of bivalve North Atlantic Fish Marking Symposium. Int. mollusks. Proc. Nat. Shellfish. Ass. 47:34-42. Comm. for the N. W. Atlantic Fish. Spec. Publ. Tufts, N. R. 1967. Topical labeling of shellfish. No. 4, p. 268-271. Proc. Nat. Shellfish. Ass. 57:73-76. Proceedings of the National Shellfisheries Association Volimie 58 — June 1968

RELATION OF HYDROGRAPHY AND CRASSOSTREA GIGAS SETTING IN DABOB BAY, WASHINGTON

Rot/a/d E. Westley DEPARTMENT OF FISHERIES STATE SHELLFISH LABORATORY BRINNON, WASHINGTON

ABSTRACT

Studies have been carried out since 1952 on the reh(tionshii> of the depth of the surface layer of irarm icater to the success of oyster setting in Dabob Bay. During this time it has been absented that depth of the xvater layer is just as important as numbers of larvae per sample in determining the intensity of oyster setting. Over the years information collected enables a close esti7nate of the probable number of spat per shell that will result from given numbers of oyster larvae in quantitative plankton saynples.

INTRODUCTION moved out of the bay by the action of northerly winds. Since the first importation of the Japanese Continued observations on spawning and setting oyster (Crassostrea gigas) into Washington State, of the Pacific oysters in this area have provided there has been interest in the possibility of local additional information on other aspects of oyster reproduction of this species. While the main source setting in northern Hood Canal. The purpose of of seed oysters has continued to be from Japan, this report is to summarize this information, to periods of seed shortage and the complete un- present a better understanding of the occurrence availability of oyster seed from Japan during of oyster setting in Dabob and Quilcene bays, and World War II have focused increasing attention on to provide a better basis for interpreting the local areas where reproduction of this oyster (now summer bulletins issued on spawning and setting called the ) occurs. One such area, of the Pacific oyster in Hood Canal. first recognized in 1936, is the Dabob Bay, Quilcene area of northern Hood Canal. Bay DISCUSSION Schaefer (1938) observed that successful setting occurred in Dabob and Quilcene bays with some Dabob Bay is a large, deep inlet located on the regularity, and setting intensity was adequate for western side of Hood Canal (Fig. 1). The bay is commercial utilization. Chapman and Esveldt about 10 miles long and 1 1/2 miles wide. Quilcene (1943) made further observations of Pacific oyster Bay is a branch of Dabob Bay opening about half- setting in northern Hood Canel, and found setting way down the west side of Dabob Bay at Whitney intensity to be highest in Dabob Bay. Subse- Point. During summer, waters of this area stratify quently, Glude and Lindsay (unpublished manu- thermally (Westley, 1956). script) worked extensively on Pacific oyster re- As part of the prediction service to the oyster production and developed methods for prediction industry, an effort is made to forecast both time of time of setting. and intensity of spatfall. Prediction of setting Disappearance of oyster larvae from the water intensity is quite important to the commercial prior to setting was occasionally observed and oystermen since it is generally believed that a posed a perplexing problem in the successful minimum of 10 spat per adult Pacific oyster shell prediction of the occurrence of oyster setting. is necessary to make cultching (the placement of Westley (1956) found that the waters of the bay strings of Pacific oyster shell in the water to were thermally stratified and that the surface collect oyster spat) economically feasible. Since warm water layer which contained the larvae was forecasting setting intensity is difficult at best,

42 HYDROGRAPHY AND OYSTER SETTING 43

FIG. 1 Map of Dabob Bay, Quilcene Bay area.

these forecasts have been restricted to prediction may be occurring in pockets (Westley, 1956). In of no setting (0 to 2 spat per shell), light setting developing the information for Figure 2 every (3 to 10 spat per shell), and commercial setting effort was made to present the average condition (over 10 spat per shell). of the surface layer immediately prior to setting, The procedure used in the past to forecast in- but particularly in years of a limited surface layer, tensity of oyster setting was to collect quantitative this information is not as representative of the plankton samples, determine the number of ad- entire bay as would be desirable. vanced larvae per unit of water, and relate this to Examination of the data in Figure 2 and Table 1 subsequent spatfall (Lindsay, Westley and Sayce, indicates a relation between the depth of the warm 1958). In carrying out this procedure, it was water layer and the intensity of spatfall, and observed that rather serious discrepancies some- shows that the deeper the layer, the greater the times occurred in northern Hood Canal between number of spat produced by the existing oyster the predicted spatfall, based on the numbers of larva population. These data indicate that depth larvae in the water, and the actual set. of the layer is at least as important as number of Evaluation of the available data indicated an larvae in determining intensity of spatfall. That apparent relationship between the depth of the such a relationship exists seems logical since in the warm water layer and the intensity of the oyster case of thermally stratified waters, depth of the spatfall. As previously mentioned, removal of the surface layer is another dimension of the oyster surface warm water layer had been found to be larva's environment, because the larvae cannot partially responsible for the set failure in Dabob survive in the cold waters below. The data clearly Bay. Thus extensive observations were made each demonstrate that any time the 17°C water is less year on the temperature and depth of this surface than 5 ft deep, there is almost no possibility of warm water layer. successful commercial setting. The data also in- Figure 2 presents vertical temperature observa- dicate that any time the depth of the 17°C water tions in Dabob Bay each year from 1952 thru 1965, is between 5 and 10 ft, the chances of successful immediately prior to oyster setting. Table 1 pre- commercial setting are greatly reduced unless sents the average number of advanced Pacific numbers of advanced larvae per''20 gal sample are oyster larvae per 20 gal sample, and resultant set 50 or more. in numbers of spat per shell. It should be pointed Table 1 shows that when the layer is over 10 ft out that the data presented on vertical tempera- in depth, good commercial setting can result from ture distribution is far from perfect. The depth of relatively low numbers of advanced larvae per 20 the surface warm layer is not always uniform gal sample. over the entire bay, and at times the warm water The depth of the warm water layer early in the 44 ROLAND E. WESTLEY

8

16

24

16 17 18 19 20 WATER TEMPERATURE °C HYDROGRAPHY AND OYSTER SETTING 45

TABLE 1. Relationship of depth of water layer, num- bers of larvae, and intensity of Pacific oyster spatfall in Dabob Bay.

No. advanced No. spat per Depth of Pacific larvae adult Pacific Year 17°C water per 20 gal* oyster shell** 1952 Proceedings of the National Shellfisheries Association Volume 58 — June 1968

ACCUMULATION OF PARALYTIC SHELLFISH POISON BY THE ROUGH WHELK (BUCCINUM UNDATUM L.)

J. F. Caddy and R. A. Chandler FISHERIES RESEARCH BOARD OF CANADA BIOLOGICAL STATION, ST. ANDREWS, N.B., CANADA

ABSTRACT

Whelks fed for several weeks on molluscan tissue containing high concentrations of paralytic shellfish poison (P.S.P.) accumulated toxin in the digestive gland. This phenomenon probably accounts for the high concentrations of P.S.P. recorded in from Quebec and is believed responsible for several cases of paralytic shell- fish poisoning reported from the province following human consumption of lohelks. No toxin was found in extracts of the muscular parts containing the hypo- branchial and salivary glands, intrinsic sites of poisonous secretions. Starvation of ivhelks, or feeding on a non-toxic diet, resulted In rapid elimination of poison. The digestive gland/body weight ratio was found to be a sensitive in- dicator of starvation in Buccinum.

INTRODUCTION known to feed upon shellfish, in addition to its normal scavenging habit. Twelve mild cases of poisoning were reported by An experiment was conducted to determine the a fishery officer from the vicinity of Godbout, accumulation of P.S.P. whelks fed on toxic Quebec (Fig, 1), during the summer of 1966, which by shellfish. Parallel with whelks fed on were suspected to be due to the consumption of experiments non-toxic diets acted as controls. whelks, Buccinum undatum L. (Table 1). The ex- Other sources of planation offered locally was that beach seepage possible poison in neogastropods from a sawdust mill had contaminated the whelk must be considered here: a toxin, acrylylcholine, beds. However, the symptoms shown by the victims was reported from B. undatum (Whittaker, 1960), This closely resembled those of paralytic shellfish has some neuromuscular blocking action and poisoning (P.S.P.) which often follows consump- is secreted by the hypobranchial gland. Other tion of toxic shellfish (Medcof, Morin, Nadeau and closely related gastropods have a toxin similar in Lachance '). action to P.S.P. and located in the salivary gland 2). In Shellfish poison is accumulated by bivalves as a (Fig. testing for paralytic shellfish poison- the action of these toxins ex- result of feeding upon the dinoflagellate, Gonyau- ing, possible was cluded lax (Prakash, 1963). The poison stored in the by sampling the digestive gland separately from these other digestive gland of the bivalve has been shown to "toxic" organs. be biochemically identical with that extracted Bioassay technique for P.S.P. is based upon the from the flagellates (Schantz, Lynch, Vayvada, effect of extracts of poisonous shellfish when in- Matsumoto and Rapoport, 1966), The suggestion jected into white mice. The lower limit of sen- was made that whelk toxicity might have resulted sitivity of the bioassay technique occurs at 44 ng from a diet of toxic bivalves, since B. undatum is of poison/100 g of tissue. Any scores below this value will be referred to as negative. All extracts made during the course of the experiment were 1 Medcof J. C, N. Morin, A. Nadeau and A. prepared from raw Buccinum tissue. Lachance. MS, 1966. Survey of incidence and risks of paralytic shellfish poisoning in the METHODS province of Quebec. Fish. Res. Bd. Can., MS Rept. (Biol.), No. 886, 22 p. Whelks used in the experiment were captured

46 SHELLFISH POISON IN BUCCINUM 47

TABLE 1. P.S.P. scores recorded by Nadeau and Lachance for various parts of rough whelks ex- pressed in micrograms per 100 g of raw flesh (after Medcof et al. ' ).

Location 48 J. F. CADDY AND R. A. CHANDLER

Hypobranchial Gland

Digestive Gland

Ovary

-Salivary Gland

FIG. 2. Point of bisection of the whelk used to separate the digestive gland from the muscular tissues (containing the salivary and hypobranchial glands).

TABLE 2. Toxicity values.

Scallop livers Date Sample SHELLFISH POISON IN BUCCINUM 49

mately 3 g, so that at least 20 whelks were needed -Fed - Liver -h" Starved to provide each sample of digestive gland. Ex- tracts of the muscular organs (including salivary and hypobranchial glands) were also prepared and tested for toxicity.

RESULTS

Preliminary tests of whelk livers for toxicity values 44 gave below fxg of poison which is the lower limit of sensitivity of the assay method. After feeding one week on the toxic material, the whelks in tank C showed a measurable score for P.S.P.. although whelks from tank A remained negative. Subsequent to the first week, all samples from the test tanks showed positive readings, ex- ° for Fed Herring 40 cept one occasion (tank C. third week). An- imals in the test tanks showed wide fluctuations in toxicity. No close correlation was found between toxicity readings for the two test tanks except that both showed maximum scores at the end of the feeding period. A maximum value for toxicity of 79 /ig of poison was reached for tank C in the sixth week of the experiment; on the same date, tank A yielded a value for P.S.P. of 110 ^g (Table 2, Fig. 3). Possibly even higher levels might have been attained if the numbers of animals had been sufficient to continue the experiment. Control tanks B and D gave negative readings for toxicity on all occasions tested. An interesting side feature of these data was provided by the marked fluctuations noted in the ratio of the weight of the upper part of the body (including the digestive gland) to the rest of the body (liver/body ratio). This can be attributed to 7 14 21 28 35 42 49 the changing volume of the digestive gland (or Number of Days liver) under different conditions of feeding. Whelks in both tanks showed similar fluctuations. FIG. 3. Fluctuaiio7is in liver toxicity and liver/body A fall in the value of the ratio ratio for ivhelks fed a diet of toxic scallop livers average liver/body from 0.25 to 0.17 in the first week of the over a six-week period. (Points lying on the ab- experi- ment may be assumed to reflect the initial reluc- scissa correspond to toxicity values of 44 ng of tance of the animals to change over from a diet of poison, the lower limit of sensitii-ity of the assay herring. the third week, ratio had technique.) By liver/body returned to its initial value (Fig. 3). Confirmation of the interrelationship between starvation and a reduction in the liver/body ratio detached from the sides of the tank and mixed was provided at the end of the experiment (Second before the was netted out. thoroughly sample phase. Table 2). At the end of the sixth week the Shells were broken with a ham- by tapping gently toxic diet was terminated, tank A were fed herring and the soft extracted. The mer, parts carefully and tank C starved. Tank A showed a pronounced whelks were bisected above the mantle just cavity rise in liver/body ratio, while tank C showed a in order to separate that part of the contain- body corresponding fall. The toxicity of both samples the and ing salivary hypobranchial glands (pos- fell away sharply. sible intrinsic sources of toxin) from that contain- All tests for toxicity carried out on extracts of the (main site of P.S.P. ac- ing digestive gland the rest of the body gave negative results. cumulation in other ). When 60 g of the digestive gland fraction ("whelk liver") had ac- DISCUSSION cumulated, both tissue fractions were weighed to 0.1 g. Individual whelk livers weighed approxi- The results reported here are preliminary; 50 J. F. CADDY AND R. A. CHANDLER nevertheless they do establish certain points of interesting light on selectivity of feeding in whelks. current interest: Other species of neogastropods have been shown 1. The experiment succeeded in demonstrating to feed selectively on one prey species until it is no that toxin uptake may take place when whelks are longer available: a period of starvation then pre- fed a diet of toxic bivalves. There was a progres- cedes a changeover to another species of prey sive though irregular rise in toxicity of the diges- (Fischer-Piette, 1935). This phenomenon may also tive gland during the course of the experiment. explain the drop in liver/body ratio observed at 2. No positive toxicity scores were shown by the beginning of the period of toxic diet. that fraction of the body tissues containing most The rapid elimination of toxin suggests a of the muscular organs and the salivary and simple method of processing toxic whelks for hypobranchial glands. This confirms the results of human food, namely, maintaining them in running Medcof et al. ' who found low toxicity scores for sea water for several weeks prior to consumption. muscular tissues of toxic whelks collected in One incidental feature of the toxicity readings Quebec. for the scallop livers was the progressive loss of The irregular rise in toxicity of the digestive toxicity when this material was maintained at gland throughout the experiment may be due to —13°C. Further studies will be necessary to one or both of the following factors: establish the extent of this phenomenon, but it is 1. The long intervals between feeding periods. clear that the procedure of holding samples of 2. The small size of the assay samples. At least material for toxicity analysis in a frozen state 100 g of tissue or a number of small samples must be approached with some caution. should have been used on each occasion. However, this would have necessitated a large-scale experi- ACKNOWLEDGMENTS ment which was impossible under the circum- stances. Our thanks go to Mr. Hazen Boyd of the St. Andrews Fish who Certain reservations must be made in applying Inspection Laboratory prepared our conclusions to the natural environment: the extracts, and Dr. A. D. Tennant of the Public Health Division, of Na- 1. The highest level of toxin attained by whelks Engineering Department tional Health and Welfare, Ottawa, who performed in the course of the experiment was 110 ^g. This the is considerably below the levels of toxicity en- bioassays.

i countered in nature ( Medcof et al. recorded scores as as for in LITERATURE CITED high 1,600 jxg whelk digestive glands the Godbout, Quebec, area). Bourne, N. 1965. Paralytic shellfish poison in sea 2. Scallop livers were not highly favoured as a (Placopecten niageUanicus Gmelin). diet by whelks. There was always a proportion of J. Fish. Res. Bd. Can. 22(5) :1137-1149. the food left by the beginning of the next feeding Dakin, J. 1912. XX Buccinum. L.M.B.C. Memoirs, period. A number of empty shells found in the London. Published Williams and test tanks at the end of the experiment suggests by Norgate, 115 that some cannibalism occurred, although whether p. E. Histoire d'une mouliere. this was the cause of mortality was not established. Fischer-Piette, 1935. One curious feature noted was the presence to- Bull. Biol. Fr. Belg. 69:152-177. wards the end of the experiment of several whelks Prakash, A. 1963. Source of paralytic shellfish in both test and control tanks which had aband- toxin in the Bay of Fundy. J. Fish. Res. Bd. oned their shells although still crawling actively. Can. 20(4):983-9"96. The rapid fall in toxicity following both starva- Schantz, E. J., J. M. Lynch, G. Vayvada, K. Mat- tion and feeding on a non-toxic diet is particularly sumoto and H. Rapoport. 1966. The purifica- interesting, and suggests that prolonged storage tion and characterisation of the poison pro- of toxin as found in Placopecten (Bourne, 1965) duced by Gonyaulax catenella in axenic cul- may be absent in Buccinum. If this is true, whelks ture. Biochemistry, 5:1191-1195, showing high toxicity scores in nature must have Whittaker, V, P. 1960. Pharmacologically active been feeding predominantly on toxic shellfish to choline esters in marine gastropods. Ann. N. maintain their levels of toxin. This throws an Y. Acad. Sci. 90(31:695-705. Proceedings of the National SheUfisheries Association Volume 5S — June 1968

EPIZOOTIOLOGY OF MINCHINIA COSTALIS AND MINCHINIA NELSONI IN OYSTERS INTRODUCED INTO CHINCOTEAGUE BAY, VIRGINIA

John A. Couch and Aaron Rosenfield U. S. BUREAU OF COMMERCIAL FISHERIES BIOLOGICAL LABORATORY OXFORD, MARYLAND

ABSTRACT

The introduction of Crassostrea virginica into Chincoteague Bay, at Franklin City, Virginia, ivas studied in relation to haplosporidan epizootics from September, 1963 to September, 1966. The introduced oysters (1962 year class) were from Harris Creek, eastern shore of Chesapeake Bay, an area where Minchinia nelsoni and M. costalis (Haplosporida: Haplosporidiidae) have not been found. Eight introductions of about 1200 oysters each were made in September, December, March, and June during 2 successive years. Generally, mortality followed a similar course among oysters in all introductions. Oysters in the two quarterly introductions in September died during the following August-September, after 11-12 months of exposure, in numbers that indicated an epizootic. Other quai-terly pairs of introductions in December, March, and June of both years also experienced similarly significant mortalities in the same August- September period, after 3-9 months exposure. Mortality was not significant during the wi7iters following any of the introductions. The second mortality peak for all introductions came in May-June of the second summer of exposure. A relatively lower death rate jiersisted after the May-June epizootic until the end of the study. Total periods of exposure to Chincoteague Bay waters ranged from 16 months to 21f months and cumulative mortalities, to September 1966, ranged from 65.5 per cent to 76.5 per cent. Among oysters introduced during the first year of the study, Minchinia nelsoni was first observed in April of the first spring; its prevalence declined during May and June, then increased in July, August, and September preceding and concurrent with the first mortality peak. Among oysters introduced during the second year of the study, M. nelsoni ivas first seen in late July. Prevalence of the parasite in- creased during August and September, concurrent with the first mortality peak ex- perienced by these oysters. The prevalence of M. nelsoni during the first and second winter of exposure was high in oysters examined, but accompanied by significant mortality. Prevalence of M. nelsoni in early May of the second spring dropped slight- ly but overlapped the first incidence of M. costalis. Prevalence of M. costalis reached its peak (concurrent with heavy spore pro- duction) in May-June of the second year of exposure, during the second mortality peak, when M. nelsoni icas also common. Concurrent plasmodial and spore infections of the two sjjecies were found during May and June. After early July, M. costalis was not found in oysters from any of the introductions. After the Jime mortality of the second year prevalence of M. nelsoni was low until the end of the study. The epizootiology of these parasites in Chincoteague Bay is compared with long- term studies by others in lower Chesapeake Bay, and Delaware Bay.

51 52 J. A. COUCH AND A. ROSENFIELD

INTRODUCTION Bay was an enzootic-epizootic area for Minchinia nelsoni and M. costalis. Our objectives were: 1) After the of the discovery oyster haplosporidan to determine annual and seasonal parasite pre- termed "MSX" and later named Minchinia nelsoni valences in susceptible oysters; 2) to determine Stauber, and Mackin, 1966), another (Haskin, the relationships of season of introduction and Minchinia costalis (Wood and And- haplosporidan, length of exposure to detection of infections and was described from of Seaside rews, 1962), oysters mortalities: 3) to determine cumulative mortality Wood, and Hoese Virginia. Subsequently, Andrews, in introduced populations after varying periods of (1962) the of M. costalis reported epizootiology exposure. This paper reports the results of this and M. nelsoni for sea-side areas of Virginia, and 3-year epizootiological study of M. nelsoni and M. Andrews (1966, 1967) further described the effects costalis and compares the epizootiology in Chin- M. nelsoni on of lower Chesa- of oyster populations coteague Bay with that in areas studied by others. peake Bay, Virginia. Haskin, Canzonier, and Myhre (1965) described briefly the epizootics caused by METHODS M. nelsoni in Delaware Bay. General these in- epizootiological findings by Beginning in September. 1963, experimental were; vestigators groups of oysters were introduced at 8 quarterly-

) of 1 Two haplosporidan parasites oysters intervals, over a period of 2 years, into Chin- of the eastern United exist in coastal waters coteague Bay. Virginia, and held there for vary- States. ing periods of time (Table 1). The first 4 introduc- 2) These parasites are found in high pre- tions (September. 1963 — June, 1964) were dupli- valences in certain oyster populations and cated chronologically by the last 4 (September, may cause severe oyster mortalities. 1964 — June, 1965). A study year was from 1 3) Mortality patterns associated with these September to 31 August. The oysters (1961 year parasites are usually repetitious yearly and, set) were obtained from Harris Creek, a portion to some degree, predictable. of Chesapeake Bay, where Minchinia nelsoni and 4) A possible way to re-establish oyster popu- M. costalis have not yet been found. A sample of lations in epizootic areas is to seek innate 50 oysters from each introduction was examined resistance in some percentage of the sur- microscopically before exposure in Chincoteague vivors and develop a resistant stock from the Bay. No haplosporidans were found in any of the progeny of these survivors (Haskin, personal 8 quarterly base samples, nor in any of the trayed communication, 1967). Andrews (1966) ex- control oysters kept in Harris Creek and ex- pressed this concept as "raising herd im- amined periodically during the second year of munity on a genetic basis." introductions. Mortalities of these control oysters Considerable work has been done on the history were extremely low. (Haskin. 1962: Haskin et ah. 1965; Mackin, 1961; Each introduction consisted of approximately Andrews, 19671, morphology (Haskin ct ah, 1966; 1200 oysters distributed among 4 wire trays—about Couch. Farley, and Rosenfield. 1966; Couch. 1967), 300 to 400 per tray. These trays were suspended portions of the life cycle (Myhre. 1966; Myhre, about 1 foot above the bottom to reduce siltation personal communication, 1967; Farley, 1967), and and to prevent predation. Silt accumulations and histochemistry (Eble, 1966) of these parasites. fouling organisms were removed at each sampling Previous studies (Wood and Andrews, 1962; period. One tray of each introduction was used Haskin et ah, 1966) suggested that Chincoteague throughout the study for mortality observations

TABLE 1. Periods of exposure and cumulative mortalities of trayed Harris Creek oysters maintained in Chincoteague Bay.

Date Date Exposure Cumulative Introductions introduced terminated time (months) mortality (%) 1 EPIZOOTIOLOGY OF MINCHINIA IN OYSTERS 53

only, and was not sampled. Twice each month the observation trays were raised, and the dead oys- ters counted and removed. Total monthly (cumula- tive) mortalities of each introduction were deter- mined. Dead oysters (gapers with remaining tissues) found in the observation trays were fixed, sectioned, and e.xamined for haplosporidans. The remaining 3 trays in each introduction con- tained about 900 oysters. Each month 25 oysters of each introduction were taken randomly from among the 3 trays. Oysters were fixed in David- son's fixative, sectioned and stained with Harris' hematoxylin, eosin, and Ziehl's fuchsin (Farley, 1965). Single sections of 7 ^ from the area of the gills, palps, and stomach of each oyster were examined for M. costalis and M. nelsoni. Percent- age of infection of monthly samples (n=:25 oysters o J per introduction) is expressed either as initial in- Introduilion. Introductiont cidence (first level of infections for each introduc- tion) or prevalence levels of infection). (monthly FIG. 1. Mortality curves for 8 introductions of oys- Oysters of samples were ac- monthly diagnosed ters in Clmicoteague Bay, Virginia. Solid line re- to the method of (1952) for cording Ray the pres- 1 presents introductions through Jf. Broken line re- ence of the fungal pathogen, Dennonjsfidium presents introductions 5 through 8. Vertical scale marinum (Mackin, Owen, and Collier, 1950), a is in per cent and mortality points are plotted as species whose generic designation has recently percentage dead each month of original number of been to changed Lahyrinthomyxa (Mackin and oysters in each introduction. Ray, 1966). No fungal infections were found in samples of oysters from this study.

and 7, and 4 and MORTALITY OF OYSTERS 8, respectively, all introductions experienced initial peak mortalities during the following 1). The mortality patterns in introduced oyster September (Fig. Susceptible oysters introduced in June populations are described first, followed by the (introductions 4 and 8) had mortalities in incidences and prevalences of Minchlnia costalis September (3-month exposure) com- to mortalities and M. nelsoni as probable causes of mortalities. parable in oysters introduced more than 9 The quarterly introductions were numbered months earlier (introductions 1 and 5). Therefore, as far as the first consecutively from 1 through 8. Curves of percent- period of mortality is concerned, of age monthly mortality for each introduction are length exposure was not as signifi- cant as of presented in Figure 1. seasonality introduction. in Oysters introduced in September 1963 and 1964 Oysters all introductions had to be exposed to did not die in significant numbers until the sum- Chincoteague Bay for at least 12 months (i.e. Fig. 1, introductions 4 and 8) mer and early fall (curves 1, 5 — lowermost before they suffered the curves in Fig. 1). Oysters introduced in December, peak mortality of May and June. Note that during the first and June of March, and June also experienced a similar period May exposure, mortal- ity was low in of first mortality (Fig. 1, curves 2,6; 3,7; 4,8). The relatively most introductions. Cumulative first real peak of mortality occurred in September. mortality for each introduction of During the winter after the initial summer- oysters is given in Table 1. All groups of oysters introduction through-October mortality period, mortality was (except 3) experienced over 70 per cent very low. A slight mortality occurred in March mortality. It is pertinent that the magnitude in the of cumulative was not second-year introductions (Fig. 1, curves 5, mortality necessarily pro- to the duration of 8) ; portional 6, this preceded by 1 month the major mortality exposure. All introduc- tions period for these introductions (broken line). The experienced approximately similar cumula- tive period of high death rate (30-38 per cent) occurred mortalities after two summers and one win- in ter of (e. May and June. In 3 of the 4 first-year introduc- exposure g., compare introductions 1, 5 tions with 4, 8 in 1). (Fig. 1; curves 1, 3, 4 — solid line) the May- Fig. June mortality peaks approximately equaled their preceding September-October peaks. INITIAL INCIDENCES AND MONTHLY Even though introductions 1 and 5 were exposed PREVALENCES OF MINCHINIA NELSONI 4, 7 and 9 months before introductions 2 and 6, 3 Initial incidence designates the first appearance 54 J. A. COUCH AND A. ROSENFIELD of Minchinia infections. Prevalence represents the level of infection (percentage of sample infected) in a given introduction at any given time after initial incidence. Figure 2 shows initial incidences and monthly prevalences for Minchinia nelsoni (curves 1-8) and M. costalis (histograms) in living oysters. In the first year of introductions, M. nelsoni was first observed in March and April in exposed introduc- tions (Fig. 2, introductions 1, 2, 3). These infec- tions consisted of Plasmodia largely restricted to the gill and mantle tissues. Initial infections were high in these introductions during these two months. The fourth introduction was made in June.

SON EPIZOOTIOLOGY OF MINCHINIA IN OYSTERS 55 active predators and scavengers (i.e. small crabs and fish I quickly consumed the meats of dying and dead oysters in the trays. Table 2 shows pre- valences of the two species of Minchinia in dead oysters recoverable during the two heavier mor- tality periods (those figures are combined data from all 8 introductions!. Plasmodia! infections of M. nelsoni were heavy in most of the oysters that died during September and October. There was much evidence of tissue replacement and damage caused by the Plasmodia, particularly in weak and dying oysters. No M. costaUs infections were found during the September-October mortality.

TABLE 2. Prevalences of two species of Minchinia m dead oysters during the tivo peak mortality periods.

M. nelsoni Sept. Oct. May June 56 J. A. COUCH AND A. ROSENFIELD

Depending upon the season of introduction, oys- postulate [Koch's], that of regular association, has ters can become infected within 1 month and ap- been satisfied indirectly," is applicable to both M. parently mortalities can occur within 3 months nelsoni and M. costalis in Chincoteague Bay. (Figs. 1, 2). It is pertinent to the present discussion to con- RELATION OF INFECTION TO sider another parasite of the oyster that was once REPRODUCTIVE CAPACITY OF OYSTERS indicted as a pathogen, but was later found to have little effect on the host. Prytherch (1940i Little has been reported on the influence of described Nematopsis ostrearum, a gregarine par- haplosporidans on their host's physiology or de- asite (Eugregarinina: Porosporidae), and thought velopment. One potentially detrimental effect a it was a serious pathogen of oysters. Spores of this parasite may have upon a population is to reduce parasite are found in tissues of the oyster, which that population's reproductive capacity by directly acts as a vector to transmit the parasite to its de- or indirectly impeding gametogenesis. Some in- finitive hosts, mud crabs, when the crabs consume direct evidence of this effect is presented in Table infected oyster tissues. The stages (spores) of the 3 for oysters infected with Mtiichinia nelsoni and gregarine do not multiply in the oyster. Sprague M. costalis. These data were gathered from living introductions 7 (1955, p. 102) stated: "Any effect of the parasite oysters sampled from and 8 in on the host must, therefore, be caused by these April through September, 1966 (main period of original invaders; there can be no effect (as in gametogenesis) after at least 11 months of ex- most Sporozoa and in pathogenic bacteria) at- posure. All of these oysters were adults. Before tributable to multiplication of the parasites within this period (April-September) oysters in these in- the oyster." Therefore, intensity of infection by troductions had high prevalences of M. nelsoni, and spores of Nematop.sts is directly proportionate to during the period both M. nelsoni and M. costalis the number of infective stages contacting the oys- were present in living oysters in each group (Fig. ter, and not to proliferation, or metabolic increase, 2). or activity of the parasite within oyster tissues. The essential points to be inferred are: 1) Unin- The passive spore held within oyster tissues is fected oysters (52.3 per cent of total) were most perhaps less pathogenic than a proliferating para- successful in producing gametes (fully developed site that is actively metabolizing and increasing sperm or eggs); 2) infected oysters were least in large numbers. Minchinia nelsoni and M. costalis successful in producing gametes (only 7 per cent), appear to have the necessary qualifications for and 3) comparisons between infected and unin- pathogens. Each species apparently increases vege- fected oysters showed that a slightly higher num- tativel.v within the oyster; distinct life-history ber of infected oysters were without gonadal stages are reproduced within the host. Host tissue development. is replaced drastically by the parasites. Preva- It is intriguing to speculate about the history of lences of the parasites were substantial in living, the gravid, uninfected oysters because the groups dying, and dead oysters in epizootic areas during from which the above oysters were obtained had mortality periods. Thus, Andrews' (1966, p. 30) a previous high prevalence of M. nelsoni and an statement. "By reason of a very extensive ex- initial incidence of M. costalis. Did these oysters perience with the distribution of MSX and asso- recover from infections in time (before April or ciated mortalities, it is probable that the first May) to begin active gametogenesis? Was there

TABLE 3. Relationship of Minchinia infections and host gonad conditions (1966).

Months EPIZOOTIOLOGY OF MINCHINIA IN OYSTERS 57

a genetically controlled resistance mechanism in a (7 — 10 months of exposure) (Haskin et al., 1965). certain percentage of the oysters of this group These losses are higher than experienced by any- which was activated by physiological changes at single introduction in Chincoteague Bay during a the onset of gametogenesis? Had these oysters comparable time period. escaped initial infection completely? The first two questions cannot be answered with presently COMPARISON OF DISEASE IN existing information. The answer to the last ques- CHINCOTEAGUE AND LOWER tion is probably "no", because prevalences of M. CHESAPEAKE BAYS nclsoni in the source introduction had been so high in the preceding winter as to insure that some Andrews (1967) pointed out that after 8 years of these oysters had been infected at one time. of high prevalences of Minchinia nelsoni in lower Chesapeake Bay, there seemed to be no sign that COMPARISON OF DISEASE IN this parasite was diminishing in either prevalence CHINCOTEAGUE AND DELAWARE BAYS or virulence. The same conclusion apparently held for both Minchinia nelso^ii and M. costalis in The patterns of mortality in introduced oysters Chincoteague Bay as late as 1966 (present study). in Delaware Bay (Haskin et al., 1965) attributed As in Delaware and Chincoteague Bays, patterns to Minchinia nelsoni are, as in Chincoteague Bay, of oyster mortality attributed to M. nelsoni in influenced largely by season of introduction rather lower Chesapeake Baj- depended more on season than duration of exposure. Spring and early-sum- of introduction than on duration of exposure (see mer introductions have first mortalities in the Andrews, 1966, Figs. 3, 7. 8). Oysters introduced in late summer and early fall of the same year. Late- early spring had first epizootic mortalities in winter mortalities, reported for Delaware Bay, September and October, second mortality in late were not found for oysters in the present study. winter (February), and heaviest mortality in June Late-summer and fall introductions had first mor- through October of the second year of exposure. talities in the following late-winter and June re- This sequence was similar to that of mortalities in spectively. This timing differs from the September comparable introductions in Chincoteague Bay introductions (1, 5) of the present study in that (Fig. 1) with the following exceptions: 1) Certain introductions 1 and 5 had first epizootic mortalities seasonal mortalities (i.e. September) in Chesa- in September, 12 months after initial exposure. The peake Bay were slightly higher than comparable sharp June mortality peak reported by Haskin mortalities in Chincoteague Bay; 2) the late-winter et al. (1965) for fall introductions (8 to 10 months mortality in Chesapeake Bay did not occur in of exposure) was not found in Chincoteague Bay Chincoteague Bay (at least not to a recognizable for chronologically identical introductions. The degree, and 3) the second summer mortalities June mortalities were attributed to M. nelsoni in (Andrews, 1966. Fig. 7) were spread over a longer Delaware Bay. whereas most dead oysters from period (June-September) than those in Chinco- the June mortalities (12-22 months exposure) were teague Bay (May-July). heavily infected with M. costalis in Chincoteague Slight differences in times of initial incidences Bay. Minchinia costalis occurs rarely in Delaware of M. nelsoni have been found for lower Chesa- Bay (Haskin et al., 1966) and therefore is prob- peake and Chincoteague Bays. Initial infections in ably not a significant cause of mortality there. lower Chesapeake Bay usually occurred in May Generally the high prevalences of M. nelsoni (Andrews, 1966), whereas in the present study the preceding and associated with mortalities in majority of initial infections came in late July and Chincoteague Bay were similar to those in Dela- August (Fig. 2), except for the possible atypical ware Bay. Haskin et al. (1965) and Haskin (per- aborted infections in introductions 1, 2, and 3. sonal communication, 1967) also reported that re- A 2-year prevalence pattern for M. nelsoni in cently certain high prevalences of M. nelsoni in epizootic areas of lower Chesapeake Bay was pres- Delaware Bay were not paralleled by heavy mor- ented by Andrews (1966, Fig. 8). These data can be talities. High winter prevalences of M. nelsoni in compared with Figure 2 in the present study in Chincoteague Bay also were not associated with Chincoteague Bay. Andrews presented an ex- mortality. The reasons for failure of mortality to trapolated curve of prevalences based on data col- coincide with epizootic prevalences of M. nelsoni lected over a 5-year period, whereas in Figure 2 in the two areas at certain times are not under- of the present study actual monthly prevalences stood presently, and are possibly not indentical. (percentage infected of 25 oysters per month) are Seasonal mortality peaks in Delaware Bay are given for 8 introductions. The actual seasonal apparently higher than comparable peaks in prevalences determined in the present study do not Chincoteague Bay, at Franklin City, Virginia. differ radically from Andrews' curve of prevalence Spring introductions in Delaware Bay lose from for lower Chesapeake Bay. Minor differences were: 65 to 85 per cent of the oysters by November 1) The few high (abortive ?) prevalences of M. 58 J. A. COUCH AND A. ROSENFIELD

nelsoni in Chincoteague Bay in Marcli and April cology and host-parasite relationships are better were not found by Andrews in newly introduced understood, very little can be said definitely oysters in lower Chesapeake Bay; 2) winter preva- about resistance of any kind in the oyster. lences of M. nelsoni in Chincoteague Bay seemed, on the average, to be higher than in lower Chesa- ACKNOWLEDGMENTS peake Bay, and 3) spring and early summer preva- lences of the second year were not as high in We thank Dr. Jay D. Andrews, Virginia Institute Chincoteague Bay as they were in lower Chesa- of Marine Science, who suggested certain techni- peake Bay. The significance of these similarities ques used in this study: Dr. Carl Sindermann who and differences may be open to some questions; encouraged us and offered valuable suggestions; these studies are not entirely comparable because Mr. George Ward, who was responsible for gather- of differences in location and years of study, ages ing samples and data, and Mr. Thomas Carver, of oysters introduced, and origin and susceptibility who participated in the early implementation of of stocks and perhaps experimental methods. the study. Cumulative mortalities in the two areas for com- parable periods of exposure were closely similar LITERATURE CITED (Andrews. 1966, Table 5; present study. Table It. Andrews, J. D. 1966. studies Minchinia costalis played no known role in Oyster mortality in Virginia. V. of a lower Chesapeake Bay mortalities, but was found Epizootiology MSX, protistan pathogen of oysters. 47:19-31. to be a significant contributor to deaths of oysters Ecology, Andrews, J. D. 1967. studies in on Seaside Virginia (Andrews et ah, 1962). The Oyster mortality VI. and distribution of mortalities and prevalences attributed to M. Virginia. History Minchinia nelsoni, a of in costalis for Seaside V^irginia were essentially pathogen oysters, Virginia. Sci. 8:1-13. duplicated during the present study in Chinco- Chesapeake Andrews. J. D., J. L. Wood and H. D. Hoese. 1962. teague Bay. a similar and nearby locale. Oyster mortality studies in Virginia. III. of a disease caused PRACTICAL IMPLICATIONS OF THE Epizootiology by Haplo- cost ale. Wood and Andrews. J. STUDY sporidium Insect PRESENT Path. 4:327-343. Couch, J. A.. C. A. and A. Apparently it would be possible to manipulate Farley Rosenfield. 1966. oysters introduced in Chincoteague Bay to avoid Sporulation of Minchinia nelsoni (Haplo- high mortality from haplosporidans. If frayed sporida, Haplosporidiidae) in Crassostrea vii-- oysters were to be placed there for relatively short ginica (Gmelin). Science, 153:1529-1531. J. A. periods to improve their flavor or for fattening, it Couch. 1967. Concurrent haplosporidian in- would be necessary to introduce them in the fall fections of the oyster, Crassostrea virginica and harvest them before the following June to ( Gmelin 1. J. Parasitol. 53:248-253. avoid the September mortality. If losses of from Eble, A. F. 1966. Some observations on the season- 10 to 30 per cent could be tolerated, oysters should al distributions of selected enzymes in the American as revealed histo- be introduced in the fall ( September), maintained oyster by enzyme through one winter and subsequent summer, and chemistry. Proc. Nat. Shellfish. Ass. 56:37-42. harvested the second winter without exposing them Farley, C. A. 1965. Acid-fast staining of hap- in to the second high mortality period in June ( Fig. losporidian spores relation to oyster patho- 1). Exposure of oysters for two summers and a logy. J. Invert. Path. 7:144-147. winter results in cumulative losses of approxim- Farley, C. A. 1967. A proposed life cycle of ately 70 per cent (Table 1; — also see Shaw, 1966). Minchinia nelsoni (Haplosporida, Haplosporidi- Evidence for stocks of oysters resistant to idae) in i'ie American oyster, Crassostrea vir- Minchinia nelsoni (developed from naturally se- gmica. J. Protozool. 14:616-625. lected, innately resistant parents) has been re- Galtsofl, P. S. 1964. The American oyster Crasso- ported by Raskin (personal communication, 1967). strea vuginica Gmelin. U. S. Fish and Wildlife The present study was not planned to seek or Service, Fish. Bull. 64:1-480. demonstrate resistance in newly introduced oys- Haskin, H. H. 1962. The current research program ters. The only suggestion of possible resistance in of the Oyster Research Laboratory. In: Proc. these oysters was highly indirect and of uncertain First Nat. Coastal Shallow Water Res. Conf. significance. The fact that a certain percentage (19(;ii. Nat. Sci. Found, and Off. Nav. Res. of each introduction did not die (after 2 years of 207,^09. exposure) suggests that some oysters may be less Haskin, H. li.. W. J. Canzonier and J. L. Myhre. susceptible to the lethal effects of the parasites 1965 The history of "MSX" on Delaware Bay than others. No observation on the possibility of oytier grounds, 1957-1965. Amer. Malacolog. acquired immunity was made. Until the microe- Union Bull. No. 32:20-21 (Abstract). EPIZOOTIOLOGY OF MINCHINIA IN OYSTERS 59

Haskin, H. H., L. A. Stauber and J. Mackin, 1966. phology of Nematojisis ostreariim sp. nov., a Minrhinla nelsoni n. sp. (Haplosporida, Haplo- gregarine parasite of the mud crab and oys-

sporidiidae) : causative agent of the Delaware ter. J. Morphol. 66:39-65. Bay oyster epizootic. Science, 153:1414-1416. Ray, S. M. 1952. A culture technique for the diag- Mackin, J .G. 1961. Status of researches on oyster nosis of infection with Dennocystidium mar- diseases in North America. Proc. Gulf Carib. inum Mackin, Owen, and Collier in oysters. Inst. 13:98-109. Science, 116:360-361. Mackin, J. G.. H. M. Owen and A. Collier. 1950. Shaw, W. N. 1966. The growth and mortality of Preliminary note on the occurrence of a new seed oysters, Crassostrea virginica, from protistan parasite, Dermorystidium marinum Broad Creek, Chesapeake Bay, Maryland, in n. sp. in Crassostrea virginica (Gmelin). high- and low-salinity waters. Proc. Nat. Shell- Science, 111:328-329. fish Ass. 56:59-63. Mackin, J. G. and Sammy M. Ray. 1966. The Sprague, V. 1955. Neinatopsis ostrearum and N. ta.xonomic relationships of Dermoci/stidium prytherchi (Eugregarinina: Porosporidae) marinum. Mackin, Owen, and Collier. J. Invert. with special reference to the host-parasite Pathol. 8:544-545. relations. J. Parasitol. 41:89-104. Myhre, J. L. 1966. Some histochemical observations Wood, J. L. and J. D. Andrews. 1962. Haplospori- on "MSX". Proc. Nat. Shellfish. Ass. 56:5-6 (Ab- dium costale (Sporozoa) associated with a stract). disease of Virginia oysters. Science, 136:710- Prytherch, H. F. 1940. The life cycle and mor- 711. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

RELATION OF FECUNDITY AND EGG LENGTH TO CARAPACE LENGTH IN THE KING CRAB, PARALITHODES CAMTSCHATICA Evan B. Hayties U. S. BUREAU OF COMMERCIAL FISHERIES BIOLOGICAL LABORATORY AUKE BAY, ALASKA

ABSTRACT

The fecundity of female king crabs, collected in Kachemak Bay, Alaska, in July, the 1967, increased with carapace length, but egg length did not. For ci'dbs of than same size, the mean number of eggs carried was significantly greater for crabs in a similar study in Kacheynak Bay in July, 1960.

INTRODUCTION In this report I describe the relation of fecundity and egg length to the carapace length of female S. Bureau of Commercial Fisheries The U. king crabs collected July 14-25, 1967, in Kachemak Auke Alaska, has Biological Laboratory, Bay, Bay. These data are compared with the findings of the of started a long-term study reproduction of Bright et al. (1960) to determine any changes Paralith- the commercially important king crab, in these relations in 1960 and 1967. odes camtschatica (Tilesius), in Kachemak Bay off Cook Inlet, Alaska. Female king crabs spawn SAMPLE COLLECTION AND TREATMENT in early spring and carry the developing embryos on their pleopods for about one year. It is ex- Crabs were taken in pots similar to those used pected that during this time fecundity (the num- in the commercial king crab fishery. The pots ber of eggs or developing embryos) will decrease. were set 30 to 40 m deep and were usually checked Therefore, fecundity is being determined periodical- every other day. Carapace lengths (posterior These ly during the year to measure this decrease. margin of the right eye socket to the mid-point of data will be used to derive equations for estimat- the posterior margin of the carapace) of ovigerous ing the average fecundity of the population at crabs were measured to the nearest 0.1 mm. To each sample date and to predict the probable num- ensure representation of all sizes of ovigerous ber of larvae that would result. crabs, 90 were selected on the basis of their cara- Although many authors have reported on the pace length over a range of 98 to 175 mm. The number of eggs carried by the king crab, only egg masses, with the pleopods attached, were re- Sato (1958), Bright, Durham, and Knudsen (1960) moved from the females by excising the pleopods and Rodin ' determined the number of eggs over at their base and placed in plastic bags. the entire size range of ovigerous crabs. Sato The number of eggs in each mass was deter- based his studies on king crabs from the North mined in the laboratory by sampling. Each egg Pacific Ocean off Nemuro, Japan; Rodin used mass was weighed (after blotting) to an accuracy king crabs from the Bering Sea; and Bright et al. of ztO.Olg, and a sample of eggs (about 5 per cent did their research on king crabs collected in July of the total weight of each mass) was removed, in Kachemak Bay — the same area and time of weighed, and counted. The count-weight ratio was year that I sampled. used to estimate the total number of eggs in the mass. For counting, the eggs in each sample were separated from the hairs of the pleopods by soak- solution of sea water I Rodin, V. E. 1966. Soviet works of 1965 on the ing the sample in a saturated and distribution of Kamchatka crab supply conditions and sodium hydroxide for about 24 hours, ParaUthodes camtschatica (Tilesius) in the south- then rinsed several times in 10 per cent formalin. indicated a eastern part of the Bering Sea. (unpublished Repeated counting of several samples manuscript) counting error of 0.08 per cent.

60 FECUNDITY OF THE KING CFIAB 61 400

• • 350 -

300 -

Y= -247,400+ 3,3!8.8X CO Q 250 z <

z> gzoo

CO o 150 UJ

100

• • 50

_L _L 100 110 120 130 140 150 160 170 180

CARAPACE LENGTH OF CRAB (MM )

FIG. 1. Estimates of fecundity of 90 king crabs collected in Kachemak Bay in July, 1967 and a computed linear regression of fecundity on carapace length.

RELATION OF SIZE NUMBER AND OF EGGS The cause of the wide range in is un- TO CARAPACE fecundity LENGTH known, but it is possible that an exceptionally low number of eggs results when a female mates with In general, fecundity increased as carapace a less than fully capable male. Powell and Nick- length increased. There was a wide range in the erson (1965) found that, although a male number of eggs carried by crabs of any given king crab can mate successfully with at least five carapace length, and this range increased with females, it may not be capable of all carapace length (Fig. 1). The greatest range was fertilizing the eggs of subsequent females. This in crabs whose carapace lengths were between 150 inability may result in many unfertilized eggs, which do not ad- and 170 mm. Females in this size group had 25,030 here to the pleopod hairs. This has an to 390,000 eggs. To describe the relation of possibility important bearing on the king crab be- fecundity to carapace length, I computed a linear fishery, cause only males are harvested. regression of fecundity (Y) on carapace length (X): To compare my estimates of numbers of eggs with those of Bright et al. (1960). who Y presented = -247,400 -f 3,318.8X their data as the mean number of eggs for each 62 EVAN B. HAYNES

100 no 120 130 140 150 160 170

CARAPACE LENGTH OF CRAB (MM )

FIG. 2. Relation of mean length of 50 eggs to carapace length for 31 female king crabs collected in Kacheinak Bay in July, 1967.

10-mm size class of crab. I computed a linear re- 0.97 mm — almost the same as the mean of 0.98 gression of the number of eggs (Y) on carapace mm calculated from measurements of 480 eggs length (X) for their data. Analysis of covariance by Bright et al. (1960). was used to compare the regression coefficients and adjusted means of the two groups of data. LITERATURE CITED At the 1-per cent level the regression coefficients were not different (F 5.336; significantly ^ Bright, D. B., F. E. Durham and J. W. Knudsen. 94 but the mean number of d.f.), adjusted eggs 1960. King crab investigations of Cook Inlet, in was — about my study significantly greater by Alaska. Dep. Biol., Allan Hancock Found., 11,000 (F= 751.6; 95 d.f.) — than that cal- eggs Univ. S. Calif., Los Angeles, 180 p. culated from the data of Bright et al. (1960). Powell, G. C. and R. B. Nickerson. 1965. Reproduc- The lengths of 50 unpreserved eggs from each tion of crabs, Paralithodes camtschatica of 31 crabs were measured (with an ocular micro- king (Tilesius). J. Fish. Res. Bd. Can. 23:101-111. meter) to determine if the size of the eggs in- creased with the size of the crab (Fig. 2). The Sato, S. 1958. Studies on larval development and mean egg lengths varied among the crabs but fishery biology of king crab Paralithodes could not be related to carapace length. The mean camtschatica (Tilesius). Bull. Hokkaido Reg. length of 1,550 eggs measured in my study was Fish. Res. Lab. 17; 1-102. Proceedings of the National Shellfisheries Association Volume 58 — June 1963

HERMAPHRODITISM IN THE SURF CLAM, SPISULA SOLIDISSIMA

John ir. Ropes U. S. BUREAU OF COMMERCIAL FISHERIES BIOLOGICAL LABORATORY OXFORD, MARYLAND

ABSTRACT

A single her>naphro4itic surf clam, Spisula solidissima (Dillwyn), was found among 2,500 clams collected to determine the seasonal reproductive cycle in the mid- Atlantic bight. Standard histological techniques were used to prepare stained sections of all gonads for detailed microscopic examination. The hermaphroditic specimen contained clearly distinguishable testicular and ovarian alveoli with developing germ cells of both sexes. The rarity of hermaphroditism in the surf clam, is discussed in relation to this condition in other pelecypods.

Hermaphroditism has not been reported for the hermaphroditic clam. The tubelike alveoli are the surf clam, Spisula solidissima (Dillwyn), a closely packed in the cross-sectioned gonad; in- pelecypod that normally is dioecious (Belding, teralveolar cells are sparse between the basement 1910), The simultaneous production of male and membranes of connective tissue. As is typical of female germ cells within an individual is common active gametogenesis, primordial germ cells form in many gastropods, but is relatively rare in a layer of several cells in one area of the gonad pelecypods (Coe, 1943, 1944; Galtsoff, 1961). (Figs. IB & IC). Spermatogonia, and primary A hermaphroditic surf clam was collected on and secondary spermatocytes occur respectively June 19, 1965, in an experimental jet dredge from the basement membrane toward the lumina (Merrill and Webster, 1964) towed at a site 34 of alveoli, and spermatids and sperm are in the miles (ca. 63 km) east of False Cape, North centers. In comparison with the ripe phase, how- Carolina (75° 10' W. Long., 36° 38' N. Lat.). ever, the alveoli contain few germ cells, and Water depths ranged from 90 to 105 feet (ca. 27 especially sperm. Typical of the ripe developmental to 32 m). This drag was one of many made during phase, sperm form a dense mass in the lumina of a study underway since 1962 to determine the alveoli in another area of the gonad (Fig. ID). seasonal reproductive cycle of surf clams. Eleven Primordial germ cells lie nearest the basement clams averaging 142 mm in shell length were membrane, and are less numerous than sperm. taken in the 5minute tow. The gonads were pre- The ovarian alveoli contain early developing served in Bouin's fixative, and standard histolog- ovocytes, typical of active gametogenesis, although ical used to a techniques were prepare 7 ^-thick sec- few ripe ovocytes are present in the lumina tions for microscopic examination. (Figs. IB, IC, & ID). Early ovocytes protrude The hermaphroditic condition was not detectable from the basement membrane, advanced ovocytes in the freshly opened clam. Color differences be- are attached by stalks, and ripe ovocytes lie free tween gonads of male and female surf clams are within the lumina and contain amphinucleoU not obvious, but in the slide preparation a differ- (Allen, 1953). ence in stain intensity is apparent between the Variability in development throughout a gonad male and female alveoli (Fig. lA). The darkly is common; it was observed by Coe (1932) in the stained portions of the gonad contain male germ California oyster, Ostrea lurida, a species that cells: the lightly stained portions contain female undergoes a sex reversal and also has true her- germ cells. The specimen is an example of maphroditic individuals in its populations. Coe bilateral hermaphroditism (Coe and Turner, 1938). (1943) hypothesized that in ambisexual oysters the Both active gametogenic and ripe developmental sex-differentiating mechanism apparently fails to phases are present in the testicular alveoli of the function normally. A tendency for sperm to ma-

63 64 JOHN W. ROPES

*;i>sy^5^«' ^^ft-^.i d o 'oc -ff, 'HA I '•*"!* /* ^^;.-V^

^- " *i^ •5*5^;'?^ '''&^

FIG. 1 Sections of the gonad of a hermaphroditic surf clam, Spisula solidissima. (A) a macroscopic view of the section showing three areas of testicular alveoli stained -more intensely than the adjacent oimrian alveoli. A millimeter scale is included in this photograph and arrows point to ynicroscopic en- largements (lOOX) of areas containing testicular alveoli. (B & C) Two areas of testicular alveoli con- taining mostly jrrimordial germ cells and primary and secondary spermatocytes. (D) Another area of testictdar alveoli containing dense masses of m,ature sperm,. Ovarian alveoli are adjacent to the testicular alveoli in all of the enlargements and contain developing ovocytes.

ture in advance of ova in one area of the surf clam normal gonads. The single hermaphroditic surf gonad (Fig. ID), but at the same rate as ova in clam in a sample of nearly 2,500 must be an other alveoU (Figs. IB & IC) is perhaps a devia- anomaly and would, then, be termed an "accidental tion from the normal developmental process. functional ambisexual" by the classification of Coe (1943). Typically dioecious pelecypods produce few hermaphrodites. Coe and Turner (1938) found only LITERATURE CITED three hermaphroditic soft-shell clams, Mya are- naria, in about a thousand obtained near New Allen, R. D. 1953. Fertilization and artificial Haven, Connecticut. Shaw (1965) found no her- activation in the egg of the surf-clam, Spisula maphrodites in 800 soft-shell clams from Chesa- solidissima. Biol. Bull. 105:213-239. peake Bay, Maryland, and Ropes and Stickney Belding, D. L. 1910. Growth and habits of the sea (1965) found no hermaphrodites in 1,400 Mya clam (Mactra solidissima). In: Rep. Comm. collected from nine areas along the New England Fish. Game 1909, Commonwealth Mass., Public coast. Loosanoff (1936) reported only two her- Doc. 25:26-41. in hundred Merce- maphrodites several quahogs, Coe, W. R. 1932. Development of the gonads and naria mercenaria. The discovery of first one and the sequence of the sexual phases in the Cali- then another hermaphroditic sea scallop, Placopec- fornia oyster (Ostrea lurida). Bull. Scripps ten magellanicus, by Merrill and Burch (1960) Inst. Oceanogr., Univ. Calif., Tech. Sen was interrupted by an inspection of about 3,000 3:119-144. HERMAPHRODITISM IN THE SURF CLAM 65

Coe, W. R. 1943. Sexual differentiation in mollusks. Merrill, A. S. and J. R. Webster. 1964. Progress in I. Pelecypods. Quart. Rev. Biol. 18:154-164 surf clam biological research. In: Sindermann, Coe, W. R. 1944. Se.xual differentiation in mollusks. C. J. (ed.). The Bureau of Commercial Fish- II. Gastropods, amphineurans, scaphopods, and eries Biological Laboratory, Oxford, Mary- cephalopoda. Quart. Rev. Biol. 19:85-97. land: Programs and Perspectives. U. S. Fish Coe, W. R. and H. J. Turner, Jr. 1938. Development Wildlife Service, Circ. 200, p. 38-47. of the gonads and gametes in the soft-shell Ropes, J. W. and A. P. Stickney. 1965. Reproductive clam <.Mya arenaria). J. Morphol. 62:91-111. cycle of Mya arenaria in New England. Biol. Galtsoff, P. S. 1961. Physiology of reproduction in Bull. 128:315-327. molluscs. Amer. Zool. 1:273-289. Shaw, W. N. 1965. Seasonal gonadal cycle of the Loosanoff, V. 1936. Sexual phases in the quahog. male soft-shell clam, Mya arenaria, in Mary- Science, 83:287-288. land. U. S. Fish Wildlife Service, Spec. Sci. Merrill, A. S. and J. B. Burch. 1960. Hermaph- Rep., Fish. No. 508, 5 p. roditism in the sea scallop, Placopecten magel- lanicus (Gmelin). Biol. Bull. 119:197-201. Proceedings of the National Shell fisheries Association Volume 58 — June 1968

DISTRIBUTION OF JUVENILE TANNER CRABS (CHIONOECETES TANNERl RATHBUN), LIFE HISTORY MODEL, AND FISHERIES MANAGEMENT'

Walter T. Pereyra U. S. BUREAU OF COMMERCIAL FISHERIES EXPLORATORY FISHING AND GEAR RESEARCH BASE SEATTLE, WASHINGTON

ABSTRACT

A large population of Tanner crabs was found at depths from 250 to 1,050 fm southwest of the Columbia River mouth. Analysis of trawl-catch data revealed that juvenile crabs ivere distributed deeper than the adult population. The adult popula- tion teas not stationary. Females icere most abundant at depths of 350 to 375 fm throughout the year. In contrast, the male population was centered at shallower depths during the spring and summer, shifting deeper during the ivinter breeding season. Adult females bearing eggs u-ere found in catches made at all seasons and egg development proceeded on a yearly cycle, beginning in the winter. The biolo- the observed gical and environmental factors responsible for life history pattern are briefly discussed.

INTRODUCTION used on all cruises. Stations from 50 to 475 fm were sampled four times a year with a modified Bur- During a cooperative investigation by the 94-ft (400-mesh) Eastern fish trawl having a li-in eau of Commercial Fisheries and Atomic Energy mesh liner in the cod end (Greenwood, 1958). fish and Commission of the deep-water demersal Stations at depths greater than 475 fm were invertebrate populations off the northern Oregon sampled intermittently with a 43-ft flat shrimp coast, a sizeable population of a large majid crab trawl (Schaefers and Johnson, 1957) or a 70-ft Chionoecetes tanneri Rathbun, was found at depths semi-balloon shrimp trawl (Greenwood, 19591. The from 250 to 1,050 fm. Because this crab is found shrimp trawls were constructed of li-in mesh of its in relatively deep water, little was known throughout. One-hour drags were usually made to distribution, abundance, or life history prior at depths from 100 to 475 fm, but drags were this study. In two papers resulting from this study limited to a half-hour at 50 and 75 fm because of I on the distribu- (Pereyra, 1966 and 1967) reported the large quantities of fish taken in shallow water. crabs tion, abundance, and reproduction of adult The trawling time varied between 0.6 and 1.5 hr and their commercial potential. With the presenta- for hauls made at stations deeper than 475 fm. tion of data on distribution and size of juvenile Trawling time was considered as that time elaps- 2 it is now to crabs in this paper, possible develop ing from the moment the desired amount of cable of a plausible life-history model for this species was out until retrieval of the cable was begun. All the en- deep-water crab and to elucidate major drags were made during daylight at an average to it. vironmental factors related vessel speed (relative to land) of about 2.6 knots. Throughout all drags, the gear was maintained at METHODS as constant a depth as possible. Despite the diversity of fauna taken, all catches The study was made between 1961 and 1966 off the northern coast on a trackline running Oregon ) This work was supported in part by A.E.C. agree- southwest from the mouth of the Columbia River. ment No. AT(49-7)-1971-Mod. No. 1. Stations were established at 25-fm depth intervals 2 not under- from 50 to 500 fm and at 50-fm depth intervals Juvenile crabs are those which have from 550 to 1,150 fm. A similar station pattern was gone their puberty molt.

66 DISTRIBUTION OF TANNER CRABS 67

were thoroughly enumerated. Tanner crabs in All immature crabs found in the interval from these catches were counted bj' sex and, when time 325 to 475 fm were less than 50 mm in carapace permitted, weighed with hand scales. The sex of width. All subadult crabs (i.e., immature crabs a crab may be determined by the shape of its ab- greater than 50 mm) were taken at depths greater domen regardless of the size of the crab or degree than 475 fm. The complete absence of the imma- of maturity. A representative sample of Tanner ture crabs larger than 50 mm in the interval from crabs the was taken, and carapace width of each 325 to 475 fm suggests that, as development pro- crab was measured to the nearest millimeter using gresses, the smaller crabs are either lost, owing Vernier calipers. Carapace width was considered to predation or disease, or as a consequence of as the greatest measurement across the branchial some type of "territorialism," move horizontally region of the carapace but did not include the off the trackline or vertically down into the 500-to lateral spines. 650-fm zone.

In this discussion, the calendar year is divided As is shown in Figure 1, the size structure of the into four 3-month periods as follows; winter populations of immature male and female crabs (January through March), spring (April through over the two depth intervals 500 to 650 fm and June I, summer iJuly through September I, and fall 750 to 850 fm was not homogeneous. Larger im- (October through December). This division is mature crabs were relatively more abundant in consistent with seasonal hydrographic phenom- the 500-to 650-fm interval than from the deeper ena, which show a lag effect; i.e., the greatest interval (750 to 850 fm). Assuming that all crabs amount of incident energy reaches the ocean sur- at depths greater than 650 fm do not eventually face in the trackline area during June and July, die, it seems reasonable to envision an immigra- but the highest surface temperatures occur during tion of the young crabs into the 500-to 650-fm zone August and September (Tully, 1964). as growth proceeds.

DISTRIBUTION OF JUVENILE CRABS LIFE HISTORY MODEL OF THE TANNER CRAB Juvenile crabs were taken from 325 to 1.050 fm in depth with greatest availability occurring be- In this discussion I will draw extensively from tween 350 and 800 fm (Table 1). No seasonal recently published data from this study on the availability patterns could be detected as sampling seasonal reproductive cycle and patterns of bathy- at depths greater than 475 fm was not conducted metric distribution of adult crabs (Pereyra, 1966). during the fall and winter periods. This information together with the distributional

TABLE 1. Numbers of juvenile Tanner crabs per 1-hour drag by depth, spring 1961 — spring 1966. 68 WALTER T. PEREYRA

data on juvenile crabs presented in the preceding — IMMATURE FEMALES 500- eSOtOthoms section will be used to the life N = 95 X = 804 develop history — - tothomi ^Q- 750 650 model. Where data are lacking, such as for the N < 68 X ; 68 8 larval period, the pattern will be inferred from the life cycles of other species of Tanner crabs. Throughout this treatment the reader is referred to Figure 2. Pereyra (1966) reported that the population structure of the adult male and female Tanner crabs (C. tanneri) changes quite dramatically with depth. Females are fairly stationary, with the ap- IMMATURE MALES parent abundance of the population centered be- — 500-650 toihomi N>86 X>758 tween 350 and 375 fm. In contrast, the males are — 750-850foIh657 quite migratory and exhibit a seasonal movement pattern that repeats itself for all years studied. In the spring and summer the population of males is centered at 275 to 300 fm, but as winter approaches the entire male group shifts downward and oc- 60 70 80 90 cupies the same depth interval as the females. Corapoce Width (mm ) Following a short period of aggregation with the FIG. 1. Percentage size composition of juvenile female group, the males return to the shallower Tanner crabs taken in two depth intervals, spring environment, separating the sexes once again. 1961t. In shifting from 275 to 350 fm, the male mode,

Sea Surface DISTRIBUTION OF TANNER CRABS 69

i.e., the center of male availability, is displaced during the January to March period, water tem- about 2i nautical miles down the continental slope. peratures are isothermal above approximately II spread out over the 6-month period while this 55 fm (Tully, 1964). The only major discontinuity transition is occurring, the shift would average would be the permanent halocline, which is 40 about 80 ft per day along the slope. Thus, one can to 85 fm thick, situated 40 to 55 fm beneath the see that on the average this displacement is quite surface. This halocline should not be an impenetr- gradual. able barrier to active vertical movement. As the male population performs the seasonal Although the length of time that the larvae are shift downward, maturation of eggs, which are in the surface layers is not known, it is assumed carried by the females, progresses. The process of that they remain there well into the summer, maturation, which commences with the ovulation growing and progressing through various molts of new eggs in late winter at the time of intermix- in the zoeal and megalopal stages. With the attain- ture of the male and female populations, also fol- ment of some advanced larval stage, they would lows a yearly cycle. Hatching (i.e., emergence of end their planktonic phase, settle to the bottom, the zoea) occurs during the winter, about one and take up a benthic existence. year after the eggs are ovulated. Since it has been assumed that the planktonic The phenomenon of downward shift of the larvae would be in the surface layers throughout male population into depths of maximum avail- the summer, it is logical to assume that they would ability of female crabs concurrent with the hatch- be subjected to the surface currents prevailing ing of the fully developed eggs and the subsequent during that period. In the trackline area the north- ovulation of new eggs is not a fortuitous circums- westerly wind pattern, which dominates the off- tance. On the contrary, the sequence of events and shore environment throughout the summer, causes their annual regularity strongly suggest that the surface waters to be directed offshore (Barnes downward movement of adult males is a pre- and Paquette, 1957). Such a pattern would also requisite to breeding and thus can be called a distribute the developing larvae farther offshore, breeding migration. Because of the slow rate at so that at the time of their descent to the bottom which this migration takes place, I strongly sus- in late summer, the small crabs would be distri- pect that considerable random feeding movement buted at depths greater than the adult breeding is associated with it. population from whence they came some months The abrupt change in the type of eggs carried before. by the female crabs during the winter is good This conjecture as to the distribution of the evidence that ovulation takes place during this juvenile crabs on the bottom is substantiated by period of population overlap. At the beginning of distributional data presented in the preceding the period, in January, 99.5 per cent of the females section. The fact that not all juveniles were found carry dark, fully-developed eggs, but at the end of at greater depths and that some were taken at the the period, in March, only 20 per cent still carry deeper fringes of the adult group indicates that dark eggs. The remainder are newly berried crabs. this dispersion pattern is more widespread than Although the time of the year when the eggs postulated. The variable nature of the offshore are hatched is now fairly well known, the fate of currents, as suggested by Barnes and Paquette the emerging zoea can only be conjectured. As- (1957), may be partially responsible for the wide suming that the zoea of this species react similarly dispersal of post-larval crabs. to those of Chionoecetes opilio, which were found From the above it appears that recruitment to by Matsuura (1934, in Katoh, Yamanaka. Ochi and the adult population occurs from the depths. Ogata, 1956) to lead a planktonic life near the sea Differences in the size of immature crabs at surface, we might expect to find the newly hatched depths greater than those at which the majority zoea of C. tanneri also situated in the upper water of adult crabs are found also support such a re- strata. Assuming that the upward movement of cruitment pattern. the emerged zoea to the surface is fairly rapid (by The above life history model for the Tanner surface is meant the upper 25 fm), they should be crabs appears to be consistent with the observed present in this zone from January to March or into facts. The weakest aspect concerns the fate of the summer, depending upon the rate of develop- the zoea from the time they hatch or are released ment. by the female until they settle to the ocean floor. The hydrographic structure of the northeastern Lack of data makes discussion of this feature Pacific Ocean during the winter should permit strictly conjectural. such a vertical movement to take place readily. Whereas a strong thermocline exists in the upper MANAGEMENT IMPLICATIONS zone during the summer, this feature begins to dis- OF THE MODEL sipate with the onset of cooling processes during the fall and winter. At the time of zoea emergence If harvesting of the Tanner crab resource should 70 WALTER T. PEREYRA become economically feasible, information on the Greenwood, M. R. 1958. Bottom trawling explora- seasonal patterns of distribution of the various tions off southeastern Alaska, 1956-1957. U. S. segments of the population as depicted in this Fish and Wildlife Service, Com. Fish. Rev. model could be used to manage the fishery to per- 20(12) -.9-21. mit maximum utilization at the minimum bio- Greenwood. M. R. 1959. Shrimp exploration in cen- logical cost. The proposed management scheme tral Alaskan waters by M/V John N. Cobb, would involve both time (season) and sex con- July — August 1958. U. S. Fish, and Wildlife siderations. Service, Com. Fish. Rev. 21(7):1-13. During the spring and summer the adult male Katoh. G., I. Yamanaka, A. Ochi and T. Ogata. and female populations are fairly well segregated, 1956. General aspects on trawl fisheries in the with the males in shallower water than the Japan Sea (In Japanese, English summary). females. By fishing only from April to September Bull. Jap. Sea Reg. Fish. Res. Lab. (Niigata, when the sexes are separated and keeping only Japan) 4:1-331. Transl. avail., p 293-305, Transl. male crabs, several beneficial results would be Program, U. S. Fish and Wildlife Service, Bur. im- achieved. First, recruitment should not be Com. Fish., Wash., D. C. be reduced paired. The stock of males would by Matsuura, Y. 1934. On the ecology of Chionoecetes. but the be- the fishery, suspected polygamous (In Japanese). Dobutsugaku Zasshi, 46(511): havior of the males by multiple matings would 411-420 (Not seen). maintain the female stock in a state of high fer- Pereyra, W. T. 1966. The bathymetric and seasonal Also, the on male crabs would not be tility. fishing distribution, and reproduction of adult Tanner to the because they occupy damaging juveniles crabs, Chionoecetes tanneri Rathbun (Brach- distinct areas. Second, males would be taken when yura: Majidae), off the northern Oregon coast. are concentrated over the shallow portion of they Deep-Sea Res. 13(6) :1185-1205. their depth range (275 to 300 fmt, and at a time Pereyra, W. T. 1967. Tanner crab — an untapped of the year when the weather is most favorable Pacific resource. Nat. Fish. 48(4):16-A. for offshore fishing. These factors would facilitate Schaefers, E. A. and H. C. Johnson. 1957. Shrimp their capture and reduce costs, thereby making the explorations off the Washington coast, fall fishery more attractive economically. 1955 and spring 1956. U. S. Fish and Wildlife Com. Fish. Rev. 19(l):9-25. LITERATURE CITED Service, Tully, J. P. 1964. Oceanographic regions and assess- in the seasonal Barnes, C. A. and R. Paquette. 1957. Circulation ment of temperature structure near the Washington coast. Proc. 8th Pac. Sci. zone of the North Pacific Ocean. J. Fish. Res. Congr., Oceanogr. 3:583-608. Bd. Can. 21(5) :941-970. Proceedings of the National Shellfisheries Association Volume 58 — June 196S

HOLDING AND SPAWNING DELAWARE BAY OYSTERS (CRASSOSTREA VIRGINICA) OUT OF SEASON L LABORATORY FACILITIES FOR RETARDING SPAWNING. ' '

Don Maurer and Kent S. Price, Jr. UNIVERSITY OF DELAWARE MARINE LABORATORIES LEWES, DELAWARE

ABSTRACT

Laboratory facilities and techniques for retarding the natural spawning of Crassostrea virginica from Delaware Bay are described. Oysters were held in the laboratory from 11 May 1967 until September and October 1967 and January 1968 at which times spawning was successftilly stimulated. This work demonstrates that oysters do not resorb even when held for periods up to 8 months in the laboratory if proper temperatures and quantities of ivater are provided the brood stocks. The importance of proper conditioning is stressed.

INTRODUCTION (1963) were the first to publish results of oyster conditioning and spawning experiments from Del- This study was undertaken to develop laboratory aware Bay. Haskin (unpublished reports) made techniques for holding, conditioning, and spawn- reference to an unpublished report by Dupuy who ing the American oyster, Crassostrea virginica had conducted spawning experiments on Delaware (Gmelin), from the Delaware Bay region. Our Bay oysters in 1960 and reported unpublished work main objective is to expand the spawning season on conditioning experiments by Hidu and Taylor. in order to produce oyster progeny on a year In other reports Haskin (unpublished report'') round basis for research and hatchery require- offered new experimental data on conditioning ments. Success in artificial spawning of oysters and rearing native oysters carried out by Hidu and from waters of the southeastern United States has Mulhern. Spawning experiments treated in Has- been limited (Andrews, Haskin, Hidu, Ritchie, and kin's reports were concerned primarily with in- Shaw, personal communications). The purpose of season spawning. this paper is to outline techniques and to describe The basic factor involved in laboratory facilities for holding oyster brood stock controlling spawn- ing in oysters is temperature. That is, one can (from Delaware Bay) for 8 months and subsequ- retard spawning with low and ad- ently spawning them. temperatures vance spawning with high temperatures (Loosan- The important publication on rearing of bivalve off and Davis, 1963). Crassostrea normal- mollusks by Loosanoff and Davis (1963) has virginica ly spawns in Delaware sometime in mid- served as a model for comparable studies through- Bay sum.mer when the water has risen to out the United States. Their equipment, techniques, temperature around 26 C. and experimental design have been followed or We were faced with having to hold modified by many workers. Loosanoff and Davis large quantities of brood stock at relatively low

' This research was supported in part by the 3 Haskin, H. H. Disease-Resistant Oyster Program, Bureau of Commercial Fisheries, U. S. Fish and October-December 1964 Quarterly Report to U. S. Wildlife Service, under contract 14-17-0007-507, Bureau of Commercial Fisheries. Subproject 3-49-R-l. ^ Haskin, H. H. Disease-Resistant Oyster Program, 2 Contribution No. 49, University of Delaware July-September 1966 Quarterly Report to U. S. Marine Laboratories. Bureau of Commercial Fisheries.

71 72 D. MAURER AND K. S. PRICE, JR.

temperatures in order to retard spawning and yet changers are connected to an oil-fired furnace provide these oysters with enough food to promote (150,000 gross B.T.U. s/hr) which also heats the proper gonadal condition. We therefore placed con- laboratory. Fresh water heated in the boiler of siderable emphasis on our brood stock holding the furnace is circulated through the interior of room, the seawater supply, and refrigeration sys- the heat exchangers while the sea water flows tem. over the exterior surfaces of the exchangers. The Our results indicate that it is possible to retard water temperature is controlled by means of a natural spawning in Delaware oysters for at least thermostat sensing the sea water as the water 6 months and still cause them to spawn in a passes from the heat exchanger box to the holding relatively normal manner in the laboratory. How- tanks. ever, spawning responses of oysters in our ex- In each tank are refrigeration coils constructed periments suggest the need for an additional of 1-in copper tubing that has been coated with than period of conditioning at higher temperatures 3 layers of epoxy paint. Each coil is approximately our holding temperatures. 46 ft long and is suspended 6 inches above the bottom of the tank at the upper edges of the

METHODS AND PROCEDURES plastic-coated trays in which the oysters are kept ; i.e., the oyster trays are below the coils. The coils Facilities are connected to a 3-ton refrigeration compressor The University of Delaware Shellfisheries Lab- which is located outside of the Shellfisheries Broadkill oratory is located on the bank of the Laboratory. Water temperature in each tank is within 3 miles River, adjacent to Roosevelt Inlet, individually controlled by means of a solenoid of the mouth of Delaware Bay and near the town valve in the freon line leading from the compressor this build- of Lewes, Delaware (Fig. 1). Within to the coil. The solenoid valve is activated by a This room is ing is the brood stock holding room. thermostat sensing the seawater temperature in rectangular (8 ft by 10 ft) with fully insulated the appropriate tank. A Dunham-Bush air cooler are two 100 walls 8 feet high. In the room gallon regulates the air temperature within the brood plywood tanks (4 ft X 4 ft X 1 ft) which are in- room. on sides and bottom with 4-in thick slabs of sulated Several important safety features have been styrofoam. built into the system. One is the insulated walls feet under A Teel submersible pump located 2 of the room and tanks. In the event of a complete the surface of the Broadkill River con- supplied power failure, water temperatures in the holding sea to each tank tinually running water through tanks would rise very slowly. Another feature is 1-in black well Sea water is drained plastic pipe. the safety thermostat controlling the seawater the means of an overflow stand- from tanks by pump. In the event of a refrigeration compressor in the tanks pipe which maintains the water depth failure or malfunction of the seawater heating 12 inches. A thermostat at approximately safety system, the safety thermostat would turn off the submersible off should the tem- shuts the pump pump preventing warm sea water from entering stock tanks rise above the perature in the brood the brood stock holding tanks and the water in level. Water in the tanks is prescribed holding these tanks would equilibrate with that of the reduce strati- continuously aerated to temperature brood stock holding room air temperature. Air should fication and to provide sufficient oxygen temperature would of course be maintained at a be shut off. the seawater supply relatively low level — about 15 °C. These safety When the water in the Broadkill River is features are intended to prevent a sudden heat the hold- warmer than the temperature desired in shock (exposure to water of 28 to 30 °C) which ing tanks, the refrigeration system is activated. might prematurely stimulate brood stock to spawn. However, when Broadkill River water is cooler than the temperatures desired in the holding Holding and retarding brood stock tanks, the heating system is activated. In order On 11 May 1967, 784 adult oysters were removed to produce 20 °C and 15 °C water when River from the field and divided equally between the two temperatures are below 15 °C, the water is first brood stock holding tanks. Oyster stocks were heated to 20 °C. Half of this water is diverted segregated in plastic-coated wire baskets (Marl- to each of the two holding tanks and the water boro Wire Goods Co., Marlboro, Mass.) which in one tank is cooled to 15 °C by the refrigeration have inner dimensions of 19 3/4 in X 17 1/4 in X system. Although this is inefficient, it was the 6 in. Four baskets containing oysters were placed only course of action open to us with our available on the floor of each tank. These oysters were held equipment. in the tanks until 6 September 1967 after which River water is heated by passing it through an time small groups of oysters were periodically re- epoxy-coated steel box containing two karbide moved for spawning experiments until 15 January, heat exchangers (Union Carbide). The heat ex- 1968. Daily temperature ranges in each tank were HOLDING AND SPAWNING DELAWARE OYSTERS 73

5/. Jones R.

Murderkill R.c^^

Mispillion R.

Broadkill R.

Lewes -Rehoboth Canal

MILES

FIG. 1. hocations of original sources of brood stock taken from Delaware Bay and its tributaries in relation to the University of Delaware Shellfisheries Laboratory. The numerical part of the brood stock code name indicates either the year the parents of these laboratory-reared stocks were initially acquired from these locations (1 — Bowers 62 and Bowers 63, 2 — Red Buoy 63, 3 — Clamshell 64, k — Cape May eif, 5 —Broadkill 6Ji) or the year when brood stocks were acquired from these locations for in- clusion in these spawning experiments (5 — Broadkill 67, 6 — Mispillion 67). Rehoboth 66 is labora- tory-reared stock (frovi mixed Delaware Bay parents) which was placed in the field for 1 year at site 7 before inclusion in these spawning experiments. 74 D. MAURER AND K. S. PRICE, JR.

determined by means of a maximum-minimum Spawning techniques thermometer. Oysters in each tank were provided 6 September — 9 October 1967: In general, with approximately 3 gpm of Broadl. These were Bowers 1962, IS October — 3 November 1967: Groups of 2 to 8 Bowers 1963, Red Buoy 1963, Clamshell Delaware oysters were removed from the brood tank 1964, Cape May 1964, Broadkill 1964, Rehoboth (18.0 °C) and emersed in heated, unfiltered, 1966, Mispillion 1967, and Broadkill 1967. These running sea water in 12 in X 12 in X 5 in plastic stocks were selected on the basis of several trays at temperatures of 28 to 30.0 °C. After 1 1/2 criteria: 1) Field mortality studies, 2) presumed hours the water was turned off for about 30 resistance or susceptibility to MSX infection. (The minutes to allow for closer examination of oyster first 5 stocks are considered presumed resistant pumping action. This procedure was repeated stock because they were collected from areas of several times a day and on consecutive days until high incidence of MSX infection. The last 3 are spawning occurred. Between each day's experi- considered presumed susceptible stock because ments, the oysters were held in 12 in X 16 in X they were collected from areas of low incidence of 12 in fiberglass aquaria. During these interim MSX infection), 3) laboratory-reared progeny periods, the water generally ranged from 23 to (Bowers 1962, Bowers 1963, Clamshell Delaware 26.0 ^C with occasional lows of 19.0 °C. Water 1964, Broadkill 1964, and Rehoboth 1966 were was changed at least once daily and aerated con- reared and set in the laboratory). tinuously. In some experiments, the thermal treat- Temperature exposures of oysters held for ment was combined with a chemical stimulus spawning varied somewhat due to minor incon- (sperm or egg suspension). After the 90-min sistencies in temperature control systems and to thermal period, the water was stopped and the modifications applied by the investigators. Oys- sperm or egg suspension was placed in the plastic ters in both tanks experienced an average daily trays. Sometimes several suspension increments range in temperature of about 4 °C which was were added before the warm water was again due to lack of refinement in temperature control. turned on. In a few cases, an oyster, induced to The following temperature-exposure schedule was spawn by thermal stimulus, was placed in a set up in a rather arbitrary manner: 1) Experi- shallow glass tray with oysters which had not yet ments initiated on 6 September 1967 — the hold- spawned. The spawning oyster supplied natural ing tank was set and held at an average temp- sperm which is preferable to stripped sperm in erature of 16.1 °C for 118-147 days prior to experi- such experiments. In the one case where spawn- ments. 2) Experiments initiated on 18 October ing did not occur, the experiment was terminated 1967 — holding tank readjusted by investigators after 7 days. and held at an average temperature of 18.0 "C for 2 January — 19 January 1968: Groups of 7 to 10 8-15 days. The oysters had previously been held oysters were removed from the brood tanks in for 151 days at an average temperature of 16.1 ^C. pairs (a stock from the 19.4 -C holding tank was 3) Experiments initiated on 2 January 1968 — matched with the same stock from the 15.0 °C these oysters were placed in the holding tank on 11 holding tank) and emersed in heated, unfiltered, May 1967. The temperature of the tank was set running sea water in plastic trays. The water and maintained at an average temperature of 11.6 temperature was raised gradually from about 25.0 °C for 152 days after which time the tank tem- to 30.0 °C. After 90 minutes, the water was stopped perature was increased by the investigators to an for about 30 minutes. This procedure was repeated average of 15.0 ^C for the next 47 days. The oys- several times a day and on consecutive days until ters were then divided into two groups. One spawning occurred. Between each day's experi- group was kept at 15.0 ^C for an additional 42-49 ments the oysters were held in the plastic trays. days while the other group was exposed to an During these interim periods the water generally average temperature of 19.4 °C for 36-49 days. ranged from 19 to 23.0 "^C. Water was changed at The temperature history of each oyster stock is least once daily and aerated continuously. If the presented in the results section of this paper. oysters had not spawned, a chemical stimulus was HOLDING AND SPAWNING DELAWARE OYSTERS 75 introduced on the fourth day of testing. In these in 7 of the 16 trials; females produced viable eggs. cases, heated water was turned off periodically Fertilized eggs were produced by 4 of the 7 and sperm or egg suspensions were placed in the stocks tested. Several batches of larvae were trays of standing water. Sometimes several gamete carried to setting. Spawning occurred from 1 hour suspension increments were added before tlie and 15 minutes to 128 hours and 30 minutes after warm water was again turned on. On the fifth initial stimulation, with an average elapsed time day of testing, the experiments were terminated. of 18 hours and 42 minutes. Oysters held at relatively low temperatures RESULTS (15.0 °C) did not seem to spawn as readily as those held at higher temperatures (19.4 °C) during Apparently the brood stock holding tanks were January experiments (Table 3). Successful spawn- a reasonably healthy environment since only 46 ing in both sexes producing viable fertilized eggs oysters died during the total laboratory holding occurred in 5 of 7 groups held at 19.4 °C, while period of 248 days. This represents a mortality of males only spawned in the sixth group (Table 3). 6 per cent for the 8 months. Larvae produced by the three stocks represented During September and early October at least by these groups were successfully reared to setting. one sex spawned in each of the 17 experiments in- In all cases where spawning was successful, no volving Delaware Bay oysters (Table 1). Spawn- sperm or egg stimulus was necessary. Spawning ing occurred in both sexes in 12 of the 17 trials. occurred from 1 hour and 30 minutes to 77 hours Fertilized eggs were produced by all 9 stocks and 45 minutes with an average elapsed time of 41 tested. The fertilized eggs were viable and several hours and 36 minutes. Oysters held at 15.0 ^C batches of larvae were carried to setting. Spawn- were spawned successfully in only 3 of 6 trials with ing occurred within 50 minutes of initial stimu- both sexes spawning in just 2 trials (Table 3). lation (thermal only in this case) in the most rapid Spawning occurred within 76 hours and 30 minutes response but did not occur until 120 hours and 25 of initial stimulation in the most rapid response minutes after the initial stimulation (both thermal but did not occur until 96 hours and 33 minutes and gamete stimuli were applied within the first after the initial stimulation in one experiment. hour) in one experiment. The average delay be- The average delay between initial stimulation and tween initial stimulation and spawning was 32 spawning was 83 hours and 24 minutes. hours and 48 minutes. The spawning response times given for Septem- Table 2 indicates that spawning occurred in one ber, October, and January experiments are pro- sex in 15 of 16 experiments in late October and vided to give some measure of spawning success. early November. Both males and females spawned These times should not be used as a direct com-

TABLE 1. Spawning success in Delaware Bay oysters (Crassostrea virginica) held in the laboratory from 11 May 1967 until 6 September through 9 October 19S7. 76 D. MAURER AND K. S. PRICE, JR.

TABLE 2. Spawning success in Delaware Bay oysters (Crassostrea virginica) held in the laboratory from 11 May 1967 until 18 October through 3 November 1967. These oysters were held initially at an average temperature of 16.1 °C for 151 days before being exposed to 18.0 °C water for periods of 8 to 15 days. HOLDING AND SPAWNING DELAWARE OYSTERS 77

TABLE 3. Spanning success in Delaware Bay oysters (Crassostrea virginica) held in the laboratory from 11 May 1967 iintil 2 January through 19 January 1968. These oysters were held initially at an av- erage temperature of 11.6 °C for 152 days before being exposed to water of 15.0 °C for ift days. After that oysters were exposed to water of 15.0 °C or 19.4 °C as indicated. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

SIZE COMPARISONS OF INSHORE AND OFFSHORE LARVAE OF THE LOBSTER, HOMARUS AMERICANUS, OFF SOUTHERN NEW ENGLAND

Bruce A. Rogers, J. Stanley Cobb and Nelson Marshall GRADUATE SCHOOL OF OCEANOGRAPHY UNIVERSITY OF RHODE ISLAND KINGSTON, RHODE ISLAND

ABSTRACT

Comparisons are presented of lobster larvae taken toward the edge of the con- tinental shelf and larvae taken close inshore in southern New England. In the first stage the larvae from offshore, presumably spaicned in that area, were significantly larger than those from, inshore, presumably spawned near the coast. This con- sistant contrast in size was not found in the later stages.

The American lobster, Homarus americanus, taken over the shelf south of this line, first-stage occurs from the edge of the continental shelf well specimens were most abundant at those stations up into the estuaries. There is considerable inter- farther from shore while fourth-stage larvae est in the degree of reproductive isolation between were most abundant closer to shore, suggesting inshore and offshore populations. Tagging studies an onshore drift of the larvae with time. and both morphometric and biochemical analyses Figure 2 shows the characters measui-ed. Mea- of the juveniles and adults are being applied by surements were taken at a magnification of 100 others in attempts to identify sub-populations. An using a dissecting microscope with an ocular exploratory effort to distinguish between larvae micrometer. Counts were made on four meristic collected offshore and inshore off southern New characteristics, i.e., spines on the two claws and England is reported herein. the rostrum, and setae on the telson, but the re- The larvae measured were collected in the sults were inconclusive and are not reported. summers of 1965 and 1966 by the University of Con- A comparison between the means for the in- necticut Marine Laboratory and the Graduate shore and offshore larvae was made for each School of Oceanography of the University of character measured for each of the four larval Rhode Island. All specimens were originally pre- stages, using "Student's" T test. The measure- served in formalin; the majority were later trans- ments for inshore and offshore samples for each ferred to 70 per cent alcohol. The locations of the character within each stage were tested for stations and areas from which larvae were col- equality of variance using an F test, and the ap- lected are shown in Figure 1. propriate calculation of the T statistic, contingent Observations on surface circulation (Bumpus on the result of this test, was made. The hypothe- and Lauzier, 1965; D. F. Bumpus, personal com- sis tested for each measurement was that inshore munication; G. S. Cooke, personal communication) mean equals offshore mean; the alternate hypoth- indicate gyres and thus some tendancy toward con- esis is that inshore mean does not equal off- tainment in Rhode Island Sound and Block Island shore mean. Table 1 summarizes the results of tests Sound whereas from the edge of the Continental of this hypothesis for each of the 12 characters Shelf there is a net drift toward these inshore considered on each of the four stages. waters which may average as high as four miles In the first stage, 7 of the 12 means were signifi- per day during the larval period. Accordingly we cantly different between inshore and offshore have considered the larvae from stations in the larvae. Of these total length, carapace length and sounds and bays and in the coastal gyres as in- telson length are considered to be the most reliable shore, while those from over the continental shelf because they are sharply defined. Since these well south of a line from Montauk Point to (iay characteristics are a function of overall size, it ap- Head are considered offshore. Among the larvae pears from the data that in the first stage, off-

78 COMPARISON OF LOBSTER LARVAE 79

FIG. 1. Stations from which the larvae -measured were taken.

TABLE 1. Means of morphometric measurements (in mm) on larvae of the lobster, Homarus ameri- canus, collected off southern New England. Where the means of inshore and offshore groups differ significantly for a particular character, the level of significance( a J is shown in a third column.

Measurement 80 B. A. ROGERS, J, S. COBB AND N. MARSHALL

THIRD STAGE LOBSTEB FOURTH STAGE LOBSTER

FIG. 2. Larval stages of the lobster, Homarus americanus (after Hadley, 1906). Numbers indicate characters 'measured. For the first, second and third stages the end of the segment just anterior to the telson was measured at the base of the spines sirice the true end of the segmeyit was difficult to discern. — 1 — Total length 7 Eye diameter 2 — Carapace length 8 Telson length 3 — Length of right claw 9 Width of telson If Length of left claw 10 Length of longest telson spine 5 — Width of right claw 11 Length of first abdominal segment 6 — Width of left claw 12 Length of fifth abdominal segment COMPARISON OF LOBSTER LARVAE 81

shore larvae are larger than those taken inshore. A. Lund of the University of Connecticut Marine This consistent contrast in size was not found in Laboratory for the loan of their collections, to the later stages though some of the comparisons John Ross Wilcox and John R. P. French, III for suggest that meaningful patterns might unfold if assisting in these studies, and to Professor Harlan more data were available. We assume that the C. Lampe for advice relative to the analyses pres- larvae taken offshore are the progeny of offshore ented. spawners and larvae from inshore are from in- shore spawners. In fact for the first stage, show- LITERATURE CITED ing the differences reported, we are quite con- fident of this, but we can only speculate as to Bumpus, Dean F. and Lewis M. Lauzier. 1965. Sur- face whether differences noted are of genetic origin circulation on the continental shelf off or reflect such influences as the size of the brood eastern North America between Newfound- lobsters, the size of the eggs, or other environ- land and Florida. Serial Atlas Marine Environ- mental factors. ment, Folio 7, Amer. Geogr. Soc, 40 p. Hadley, Philip B. 1906. Regardng the rate of ACKNOWLEDGMENTS growth of the American lobster. Special Paper 24, 36th Annu. Rep. Comm. Inland Fish., The authors are indebted to Professor William Rhode Island., p. 153-236. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

QUANTITATIVE AND QUALITATIVE COMMENSAL BACTERIAL FLORA OF CRASSOSTREA VIRGINICA IN CHESAPEAKE BAY

T, E. Lovelace, H. Tubiash and R. R. Colwell DEPARTMENT OF BIOLOGY, GEORGETOWN UNIVERSITY, WASHINGTON, D. C, AND U. S. BUREAU OF COMMERCIAL FISHERIES BIOLOGICAL LABORATORY, OXFORD, MARYLAND

ABSTRACT

The natural commensal flora of the oyster has been under stitdy since July, 1966. Two areas of Chesapeake Bay were selected for investigation: Marumsco Bar, where severe mortalities occur annually, and Eastern Bay, a productive coinmercial oyster harvesting area. Water, mud, oyster mantle fluid ayid oyster gill tissue were examined for quantitative and qualitative estimates of total viable aei-obic hetero- trophic bacteria. In general, the two areas of Chesapeake Bay which were sampled are very similar in pH, dissoli^ed oxygen, seasonal temperature range and salinity. Little quantitative variation ivas obser-i^ed between samples ivithin each area and be- tween areas. The ivater counts ran at approximately 4 x lO'/ml., the mud 10*-10^/ml, oyster gill tissue 10^-10^/gm and the mantle fluid 5 x 10*/ml. Quantitatively, there was also little or no seasonal or environmental variation. From, counts made on basal salts medium and on distilled water medium, salt-requiring bacteria appear to be a significant part of the bacterial flora of both areas. From the generic dis- tribution of the bacterial types for the tivo areas tested, it ivas found that 81 per cent of the Marumsco Bar ivater, mud and animal samjiles consisted of Vibrio, Pseudomonas and Achromobacter spp., whereas the Eastern Bay representation of

these genera was 1^6 per cent. Cytophaga/Flavobacterium spp. was the do^ninant group in the Eastern Bay samples. The Marumsco Bar area harbors significantly greater munbers of Vibrio spp. Overall the Eastern Bay area represents a "balanced" microbial population.

INTRODUCTION and Willapa Bay, Washington, included a some- what greater incidence of Pseudomonas spp. (Col- The natural commensal flora of marine in- well and Sparks, 1967). vertebrate animals has not been studied to any Several investigators have induced pathological great extent. There is some published work on conditions in 1960; and bacteria isolated from shellfish but the focus has oysters (Tripp, Pauley Sparks, 1965), or succeeded in producing bacterial been chiefly on the public health aspects of the infection 1966). Pseudomonas a animals and their environment. In 1959 the natural (Feng, enalia, bacterial pathogen for C. gigas, was recently flora of Crassostrea gigas was investigated (Col- described Colwell and (1967). well and Listen. 1960). The research effort was by Sparks Since have undertaken of directed primarily to obtain information which July, 1966, we a study the natural commensal flora of Crassostrea vir- would aid shellfisheries technologists in establish- in areas ing handling methods for prevention of product ginica Chesapeake Bay. Two were selected for our severe spoilage. It was subsequently established that the investigation: Marumsco Bar, where mortalities occur natural flora of C. gigas is composed of organisms annually, and Eastern Bay, a representing the genera Pseudoinonas, Achromo- productive commercial oyster-harvesting area. The bacter, Flavobacterium and Vibrio (Colwell and work has been undertaken to obtain information Liston, 1960). Dead and dying oysters in popula- concerning the microbial ecology of C. inrginica tions of C. gigas grown in Hood Canal, Oyster Bay in its natural habitat and possible involvement of

82 COMMENSAL BACTERIAL FLORA IN THE OYSTER 83

TABLE 1. Etivironmental parameters — Marumsco Bar and Eastern Bay. 84 T. E. LOVELACE, H. TUBIASH AND R. R. COLWELL

TABLE 3. Comparison of shipboard and laboratory total viable counts. Figures from replicate counts. COMMENSAL BACTERIAL FLORA IN THE OYSTER 85

TABLE 5. Generic distribution of bacterial types in samples tested.*

Taxonomic Group Marumsco Bay Eastern Bay Vibrio spp. 46% Pseudornonas spp. Achroinohacter spp. Corynebacterium spp. Cytophaga/Flavobacterium spp. Micrococcus /Bacillus spp. Enterics** Other*** 86 T. E. LOVELACE, H. TUBIASH AND R. R. COLWELL

TABLE 7. Generic distribution of bacterial types in the mud, ivatcr and animal samples* COMMENSAL BACTERIAL FLORA IN THE OYSTER 87

ACKNOWLEDGMENT Colwell, R. R. and J. Liston. 1961b. Ta.xonomic re- lationships among the pseudomonads. J. Bac- This work was performed cooperatively by teriol. 82:1-14. Georgetown University and the Bureau of Com- R. R. and A. K. 1967. mercial Fisheries Biological Laboratory, Oxford, Colwell, Sparks. Properties of Pseudomonas a marine bacterium Md. Georgetown University's contribution was sup- enalia, for the invertebrate Crassostrea ported in part by BCF contracts 14-17-003-129 and pathogenic 14-17-003-149. gigas (Thunberg). Appl. Microbiol. 15:980-986. Feng, S. Y. 1966. Experimental bacterial infections LITERATURE CITED in the oyster, Crassostrea virginica (Gmelin). J. Invert. Pathol. 8:505-511. Colwell, R. R. 1964. A study of features used in L. R. the diagnosis of Pseudomonas aeruginosa. J. Moffett, M. and R. Colwell. 1968. Adansonian Gen. Microbiol. 37:181-194. analysis of the Rhizobiaceae. J. Gen. Microbiol. Colwell, R. R. 1968. Collecting the data. Tests and 51:245-266. testing methods. In: Manual on Nnmeriral Pauley, G. B. and A. K. Sparks. 1965. Preliminary . Amer. Soc. Microbiol. Publication. observations on the acute inflammatory reac- Ch. 2 (In Press). tion in the Pacific oyster, Crassostrea gigas Colwell, R. R. and J. Liston. 1960. Microbiology (Thunberg). .J. Invert. Pathol. 7:248-256. of shellfish. Bacteriological study of the na- Quigley, M. M. and R. R. Colwell. 1968. Properties tural flora of Pacific oysters (Crassostrea of bacteria isolated from deep-sea sediments. gigas). Appl. Microbiol. 8:104-109. J. Bacterid. 95:211-220. Colwell, R. R. and J. Liston. 1961a. A bacteriolog- R. 1960. of of in- ical study of the natural flora of Pacific oys- Tripp, M. Mechanisms removal ters (Crassostrea gigas) when transplanted to jected microorganisms from the American oys- various areas in Washington. Proc. Nat. Shell- ter, Crassostrea virginica (Gmelin). Biol. Bull. fish. Ass. 50:181-188. 119:273-282. Proceedings of the National Shellfisheries Association

Volume 58 •— Jmie 1968

DEVELOPMENTS IN THE METHODOLOGY FOR GLYCOGEN DETERMINATION IN OYSTERS R. G. Westenhouse RESEARCH DEPARTMENT WEYERHAEUSER COMPANY LONGVIEW, WASHINGTON

ABSTRACT

Glycogen separation and assay time -were substantially reduced by using the phenol tnethod for colorimetric analysis. Na2S0i addition prior to alcohol precipita- tion eliminates heating or overnight standing. Tentative results on longer NaOH digestion show higher glycogen recovery. Sa77i- ples were compared by using 1/2 hr and 1 1/2 hr tissue-digestion periods. Tissue frozen one week at — 7°C gave low glycogen recovery using the 1 1/2 hr digestion period comparison.

INTRODUCTION pattern of alkaline tissue digestion, alcohol pre- cipitation and end-product analysis. This procedure In the general assessment of oyster condition a requires a relatively long analysis time. Literature common method of analysis is the measure of oys- examination showed that assay time can be re- ter solids. Shaw, Tubiash and Barker (1967), duced by colorimetric analysis of the separated Galtsoff (1964), Engle (1950) and others have pro- glycogen. posed and used various methods for total solids A in metho- determination. corollary requirement changing dology was data relativity. With carbohydrates, Shaw et al. (1967) state that "the most precise different methods measure different chemical measure of quality is determination of glycogen species. Oyster glycogen assays of prior years content in oyster tissue." In an assessment of our must be related to newer techniques. objectives for an oyster study, it was concluded Montgomery (1957), Kemp, Andrienne and Van Heijningen that glycogen provided a better index of physio- (1954), and van der Vies (1954) have used colori- logical condition. Total solids analysis provides a metric assay of the Lo more rapid means of determining condition rela- separated glycogen. Eraser, and (1966) used both the colorimetric and tive to commercial value. With use of the glycogen Dyer methods. In the assay, the physiological state of the oysters can enzymic assay selecting phenol be related to the overall condition as determined method of Dubois, Gilles, Hamilton, Rebers and Smith (1956) it had visually or with total solids. For example, periods already been shown (Mont- of lowest glycogen storage occur at spawning when gomery, 1957, Eraser et al., 1966) that the phenol the presence of sex products detracts from market method was directly related to alkaline prepared acceptability. Relationships of glycogen and en- glycogen from cod muscle and guinea pig liver vironmental factors have been shown by Herrmann and muscle. Montgomery has compared phenol (1965') and Herrmann and Westenhouse (1966M. glycogen with concanavalin glycogen (Cifonelli Until recent years, glycogen assay (Calderwood and Smith, 1955). Eraser et al. (1966) used the and Armstrong 1941) had followed the classical glucose oxidase and phenol comparisons with good agreement on KOH prepared glycogen.

1 Herrmann, R. B., Interim Report, Weyerhaeuser Company, 1965. METHOD DEVELOPMENT

2 Herrmann, R. B. and R. G. Westenhouse, Interim Newer colorimetric glycogen assay methods are Report, Weyerhaeuser Company, 1966. conducted in acid systems. Our previous method

88 METHODOLOGY FOR GLYCOGEN DETERMINATION 89

analysis. This will minimize the effects of non- glycogen carbohydrate contamination. Either method eliminates the alkaline conversion pro- ducts error. Dubois et al. (1956), Handa (1966) and Mont- gomery (1957) used 1 or 2 cc samples with an experimentally determined weight of added phenol. UJ It is often desirable to use larger sample volumes. u A curve for concentration z phenol was constructed < using 4 cc samples, 1 cc of the phenol solution and 10 cc concentrated 1 CD H2SO4. Figure shows 1 cc cr of 15 per cent (w/v) phenol is adequate for these O analysis conditions. 00 m In changing the quantitative method of determin- < ing separated glycogen, results were evaluated on an acidified and pH 8.5-9.0 neutralized extract. The CU2O method was always applied to pH 8.5-9.0 extract. Table 1 indicates small differences due to pH of the glycogen extract prior to analysis, as well as the basic differences in alkaline or acid assay method used. The small differences could be due to contaminating low molecular weight carbo- 50 100 ISO 250 200 300 hydrates or non-glycogen reducing groups. Separa- tion PHENOL, MILLIGRAMS procedures include sufficient washing to minimize the problem. Blanks carried through the entire procedure have consistently shown no filter FIG. 1 Absorbance 29 change for fig glycogen with paper contamination. the quantity of added phenol. Paired sample results by the CuiO method have shown some divergence. Although data are not presented, the paired sample results from phenol have shown better utilized Cu.O assay in an alkaline system. John- assay much agreement. This son and Fusaro (1965) reporting on the alkaline evidence supports that shown by Johnson and Fusaro is conversion of glucose, refer to the following (1965) and probably caused from general reactions of aldoses in heated alkaline localized heating during neutralization of the systems: glycogen hydrolyzate. Aldoses ^IntermediateX—>Enediol Overall analysis time was reduced approxi- ' > Ketose mately 50 per cent by eliminating the hydrolysis and use of the more With these potential alkaline conversion pro- step rapid phenol analytical ducts as a reference, the acid phenol method of procedure. Dubois et al. (1956) was selected as the best ap- There are many references to the anthrone ( Viles proach to assay time reduction. The method of and Silverman, 1949) method of carbohydrate Kemp et al. (1954), while being specific for determination. In a simultaneous comparison of hexoses, was rejected because it lacked sensitivity this method with the phenol and CuzO assays the and required a heating period for color develop- following data were developed (Table 2). Note the ment. Phenol assay allow ample dilution prior to anthrone method shows a larger test range. It is

TABLE 1. Method comparison per cent glycogen, wet weight. 90 R. G. WESTENHOUSE

TABLE 2. Tr-i method comparison — glycogen analysis.

CuaO Anthrone Phenol per cent wet per cent wet per cent wet 5 Sample Average* 1.50 1.42 1.41 Difference* .00* —.08 —.09 Range :0.02 :0.21 :0.02

* CU2O method used for reference.

** Each sample run in duplicate.

the author's experience that the anthrone method data in liver and muscle. Enzymic breakdown of is not as sensitive or reproducible as the other glycogen to monose and oligoglucose resulted in methods. In addition, the prepared reagent is not lowered values. stable, causing problems in daily data comparisons. An examination of our raw data has shown a Under proper conditions ( Westenhouse, 1964), how- cyclical variation in the analytical agreement be- ever, anthrone can be suitable for carbohydrate tween paired samples. These paired sample values and urea-formaldehyde analysis. (duplicate samples) tend to diverge in the winter- Van Handel (1965) reported a more rapid spring and converge in the summer-fall periods. glycogen precipitation technique that yielded im- To approach a resolution of this cyclic phenome- proved recovery after glycogen precipitation and non, duplicate samples of tissue were digested in 30 separation. The basic change is the addition of per cent NaOH for the normal 1/2 and 11/2 hr saturated Na2S04 solution to the KOH digest prior periods. These preliminary results, shown in Table to alcohol precipitation. After standing 15-30 min 4, indicate a higher percentage glycogen in sam- the precipitated glycogen can be centrifuged or ples digested for the longest period. This has oc- filtered. Comparative data by our older procedure curred during the seasonal period when analytical of overnight standing and the more rapid NazSOj- agreement was poorest by the previous data alcohol method are shown in Table 3. The phenol analysis. assay was used for these precipitation com- In a related experiment the same fresh homo- parisons. genized tissue was frozen for seven days at —7°C. Use of the NajSOj modification allows an almost Analysis of this frozen tissue, shown in Table 4, continuous procedure, eliminating the long wait- gave low recoveries. One tentative criteria for ing periods between analytical phases. glycogen assay may be the use of fresh tissue rather than frozen samples. This would apply only DISCUSSION if the refrigerated temperatures cannot be main- tained at dry ice values. A cursory assay of the Use of the developed glycogen methodology has low molecular weight carbohydrates washed from fresh tissue been centered on routine oyster glycogen assay. glycogen precipitates gave values of Through the time reductions afforded, additional approximately 0.5 per cent as glycogen. Glycogen- studies have focused on the digestion phase of wash solutions from frozen tissue have not yet glycogen analysis. been examined. Johnson and Fusaro (1966, 1967) have shown It cannot be concluded that less easily extractible the necessity for careful interpretation of glycogen glycogen forms are present in oyster tissue. In- creasing the digestion period may be necessary for quantitative recovery, particularly during the winter-spring assay periods. Continued testing TABLE 3. Glycogen precipitation — per cent glyco- should clarify these points. gen wet. While performing a routine standard check with new reagent glycogen, the unit Alcohol Precipitation Alcohol — NazSOi absorbency per of glycogen had increased over the Overnight wait 15-30 Minute wait original glycogen reagent. A thorough review of the data 2.99 3.16 calculations and standard preparation has shown 3.14 3.22 incorrect glycogen values were obtained. Sub- 2.71 2.72 sequent evaluation of the glycogen used in prepar- 2.98 3.01 ing the original standards has shown it was parti- 4.07 4.04 ally degraded. New glucose and glycogen stand-

I METHODOLOGY FOR GLYCOGEN DETERMINATION 91

TABLE 4. Tissue digestion — storage, per cent glycogen, ivet.

Month 92 R. G. WESTENHOUSE

Westenhouse, R. G. 1964. Spectrophotometric de- water to the larger sample, giving a total volume termination of urea-formaldehyde with anth- of approximately 50 cc. After mixing, stir in 115 rone. Anal. Chem. 36:1875-1876. ml ethanol to each .sample. Let stand 15-30 min. Decant the supernatant liquid through a What- man No. 54 filter paper. Wash the precipitated glycogen 4 times with 75 ml portions of 66 per cent ethanol. Decant with each wash step. Transfer the precipitate to the filter Wash twice APPENDIX paper. more with small volumes of 66 per cent ethanol.

Solution of Glycogen OYSTER GLYCOGEN DETERMINATION Attach a small piece of tubing, 1-2 in long, to each filter funnel. Close a pinch clamp on each Reagents, Special Equipment tubing extension. Fill the filter paper containing the glycogen with 75-90 ml water. Let 30 per cent NaOH boiling stand for 2 hr. the and allow Na2SO<. saturated solution Open pinch clamp the solubilized to drain into a 250 ml Ethanol, absolute glycogen volumetric flask. Repeat addition of hot water HoSOj, concentrated after closing the pinch clamp. One hour standing Glycogen, highest purity available is normally sufficient. Drain filtrate to the 250 ml Phenol, 15 per cent. Dissolve 37.5 g reagent volumetric flask. Wash the filter paper with small grade phenol in 250 ml water. portions of boiling water until the total filtrate Bauscli and Lomb Spectrophotometer with 1 in collected is about 240 ml. The at this cuvet washings stage should be clear and colorless. Method: If further analysis is to be delayed, add 2 cc concentrated H-SOi to each flask and store In Homogenization, Sampling, Digestion refrigerator. For immediate analysis, cool the Remove the tissue from 20 and let them oysters flasks to room temperature and dilute to 250 ml. drain 10 min. Add the drained oysters to a blender using maximum speed for 5 min. Glycogen Determination Weigh to the nearest mg, a 5 and 10 g sample Pipet 5 ml of the well-mixed glycogen filtrate to of homogenized tissue. Use a 10 ml plastic syringe another 250 ml volumetric flask. Dilute to mark for these transfers and weighings. Discharge the and mix well. weighted tissue into 400 ml beal^ers containing 10 Pipet 4.0 cc of this dilute glycogen to a 1 in B. and 20 ml 30 per cent NaOH (2 cc NaOH for each and L. cuvet. Pipet 4 ml distilled water for a gram of sample). When removing the samples for reagent blank. Add 1.0 cc 15 per cent phenol weighing, continuous agitation at very low speeds solution and mix well. Add 10.0 cc concentrated gives better duplication. H2SO4 mix and let stand for 10 min. Measure at Cover the beakers with a watch and 485 the blank for reference. glass place fji using reagent on a preheated steam bath. When solution tem- Calculation: peratures reach 80'^C let the tissue digest for 30 min. Average tissue digestion temperatures are from standard curve) (0.3125) <|U,g glycogen normally 88-93' C for most of the 30 min digestion (wet weight sample, grams) period. Occasional stirring gives a more homo- geneous mixture. ^ per cent glycogen, wet

Precipitation, Washing A standard curve was prepared from oven dried Remove the samples from the steam bath. Add glycogen. The calculation is based on glycogen 5 cc saturated Na2S04 to each hot sample, mixing values of 0.5 to 1.5 per cent glycogen, on a wet well. Add 30 cc water to the smaller sample, 15 cc basis. Proceedings of the National Shellfisheries Association Volume 58 — June 1968

APPLICATION OF THE BERGMAN-JEFFERTS TAG ON THE SPOT SHRIMP, PANDALUS PLATYCEROS BRANDT'^ ^ Warihoko Q. B. West and Kenneth K. Chew COLLEGE OF FISHERIES UNIVERSITY OF WASHINGTON SEATTLE, WASHINGTON

ABSTRACT

The Bergman-Jefferts ferromagnetic loire tag was used in two 50-day experiments on captive spot shrimp in saltwater aquaria. The tag was inserted into the ab- dominal m.usculature at the first somite. Tagged, untagged and wounded shrimp were used in this study. The wounded shrimp were processed in the same manner as the tagged ones, including puncture by the needle, and differing only in the lack of tags. A high incidence of mortality was generally observed among the test animals, but no marked difference was found between mortalities of tagged, untagged and wotmded shrimp. A mortality of shrimp occurred when the pH dropped below 6.6. The feeding habit and general activity of s/irunp were considerably affected by low pH and newly shed shrimp appeared to be more adversely affected. The percentage of shrimp that molted at least once ivas relatively high, 61.5 per cent in Experiment 1 and 72.5 per cent in Experiment 2, but only 2.5 per cent molted twice in both experiments and most of these were in the small size group (<20 mm carapace length). No difference in molting was observed between the tagged, un- tagged and wounded shrimp. No abnormalities that could be directly attributed to the implantations of the wire tags in the shrimp musculature were noted. Hence, the wire tag has great promise in identifying shrimp and other crustaceans for migration, growth and population studies.

INTRODUCTION tion growth, fishing pressures and mortality. Tagging studies previously conducted on crust- Tagging and marking^ methods have long been aceans have met with many difficulties, since used in population and behavior studies of animals. their exoskeleton is shed when molting occurs. Be- In fisheries research, several fish-tagging and cause of the loss of tags when these animals molt, marking techniques have been developed and when properly designed and executed, such mark-recap- ture experiments provide valuable information on 3 Present address: Fishery Resources and Exploita- fish population movements, measures of popula- tion Division, Food and Agriculture Organization of the United Nations Via delle Terme di Cara-

1 Contribution No. 284, College of Fisheries, Uni- calla, Rome, Italy. versity of Washington. 4 The term tagging is used to denote the applica- 2 The work reported here was part of a thesis sub- tion of a mechanical tag or other identification mitted by the senior author to the Graduate device. Marking refers to the use of mutilation, School, University of Washington, in partial ful- staining or other means of identification not re- fillment of the requirements for the Master of quiring the attachment or insertion of mechanics! Science degree. devices.

93 94 W. Q. B. WEST AND K. K. CHEW tagging techniques involving the use of external test animals were collected to observe changes in tags are generally limited to short term experi- hydrogen ion concentration (pH) and salinities ments on larger specimens. to which the shrimp were subjected in being trans- Because of their small size, shrimp are not suit- ferred from their natural environment to the able for tagging studies. In fact, previous tagging aquarium. Water temperatures were also taken experiments on peneid shrimp indicate that tags at the depths where the shrimp were collected. Re- increase the mortality rate (Lindner and Ander- sults of the hydrographic sampling prior to the son, 1956); thus, the interpretation of the results two experiments conducted are as follows: may be open to inaccuracies. Other techniques used, such as marking with biological stains and Experiment 1 dyes, also present problems. In addition to impair- 11/10/66 in field — pH 7.5, ing the attractiveness of shrimp as food and salinity 31.8 ppt and water rendering them more readily visible to predators, temperature 50.0" F at 35 fm. many stains and dyes often fade or are lethal 11/11/66 in — 7.2, within a short time after application (Klima, 1965) aquarium pH 29.6 and water and fast stains, such as Trypan Blue and Trypan salinity ppt 51.5°F Red, become increasingly toxic with age (Dawson, temperature 1957). 2 A possible solution to the problem of successful Experiment tagging of shrimp was recently developed by Jef- 1/13/67 in field — pH 7.8, ferts, Bergman and Fiscus (1963) for Pacific sal- salinity 30.1 ppt and water mon. This new tagging device permits the use of a temperature 48.0'- F at 38 fm. ferromagnetic wire tag, commonly referred to as 1/14/67 in aquarium — pH 7.2, the Bergman-Jefferts tag, which can be implanted salinity 29.8 ppt and water into the musculature beneath the exoskeleton of temperature 51.0'- F shrimp to preclude loss during molting. The mag- netized tag can be readily recovered by detection The pH of the aquarium was determined daily of its magnetic field either by the simple coil and salinity once each week throughout the ex- detector developed by Jefferts, or an electrometer. periments. Following the work of Jefferts et al. (1963), the A 25-watt incandescent lamp, placed over each wire tag was employed on lobsters by Squires test aquarium was turned on mechanically each (Woodland, 1966), and Tutmark, West and Chew morning (6:30 am) and off every evening (6:30 (1967) used it successfully on brachyuran crabs. pm). It required about 35 min for the lamps to The encouraging outcome of the latter preliminary reach maximum illumination in the morning and experiment made it desirable to conduct the pres- to be completely extinguished in the evening. ent study on the feasibility of using the Bergman- Thus, by means of this mechanical device de- Jefferts tag on spot shrimp and to determine dif- scribed by Chew'', the amount of light entering ferential mortality caused by the tag, it any. the test aquaria was controlled to simulate sun- rise and sunset conditions. MATERIALS AND METHODS Test Animals Aquarium The spot shrimp used for this study were all This study was performed at the University of collected off Broad Spit of Dabob Bay, Washing- Washington College of Fisheries' saltwater aquar- ton, at depths varying between 30 and 40 fm. The ium using four similar aquaria in each of two ex- test animals for Experiment 1 were collected on periments conducted on different dates. The inside November 9 and 10, 1966, with a Gulf Coast dimensions of each aquarium was 137 cm long, 61 shrimp trawl, and those for Experiment 2, on cm wide and 67 cm high. January 13, 1967, with the same gear. The water level in each test aquarium was main- Prior to the actual tagging, shrimp were con- tained at 56 cm throughout the study. Sea water ditioned to the salt water of the aquarium for from Puget Sound was recirculated through the about two weeks. During this acclimatizing period, aquaria at the rate of 5 1/min. The inflow and the shrimp were all measured from the base of outflow of water were near opposite ends of each the eyestalk to the posterior mid-dorsal edge of aquarium, about 128 cm apart. Throughout the study, the water temperature was maintained at 51.5°F (± 0.5°F). This tempera- " Chew, K. K. 1958. A study of the food preference Ocirtebra (—Tritonalia) ture is very close to the temperatures where the of the Japanese drill, Uni- test animals were collected, as shown below. japonica Dunker. Master of Science Thesis, Water samples were taken in the field when the versity of Washington. APPLICATION OF TAGS ON SHRIMP 95

the cephalothoracic exoskeleton (carapace length the mid-dorsal portion in order to avoid damaging in mm). the centrally located dorsal abdominal artery of The shrimp ranged in carapace length from the shrimp. 14 to 40 divided into mm mm, and were three size The minute tags enter the injector in the form of groups small ( < 20 mm), medium (21 30 of a continuous stainless steel wire. The first ab- mm) and (31 - 40 mm). In both large experiments dominal segment of each shrimp is pressed firmly most fell within the shrimp medium size classifica- into the plastic mold which positions the segment tion. on a hypodermic needle. This action triggers the During the entire period of the study, the test injector which cuts one tag and places it in the animals were fed flatfish every other day. Minced shrimp. A push-rod inside the hypodermic needle mussels iMytilus spp.), dead shrimp and other fish forces the tag through the needle, which retracts were used as food during the acclimatization ahead of the push-rod to avoid withdrawing the tag period, but this practice was discontinued because back into the needle. The implanting action of the of lack of constant supply. The flatfish, either needle takes only 1/4 sec. The actual sequence in fresh or frozen, was filleted and cut into small operation is detailed in Figure 1. One pieces. piece per shrimp was dropped into Immediately after tagging, all specimens were each tank or compartment. Food supplied in this examined visually for tag placement. Shrimp with fashion is eaten and allows for readily even dis- misplaced tags were rejected and replaced. If the tribution. tag was properly positioned, or if no tag was Tagging Method visible, the shrimp was then passed over a horse- The coded wire tags were fabricated from cold- shoe magnet to magnetize the tag (if present) and worked type 302 stainless steel and measured 1 mm then through the sensing unit called the Jefferts in length by 1/4 mm in diameter. They were im- electronic detector. If the detector was not planted with the Technical Research Company actuated, then the individual was either rejected Wire Tag Injector, Model 3, into the musculature or retagged. The detector includes two connected of the first abdominal somite, to the left side of parts — an audio unit and a sensing head. It

FIG. 1. Operation of Wire Tag Injector. 1) The coil of color-coded wire passes through the friction feed- rollers on the motor. 2) The wire enters the hole in the cutter bar. 3) The cutter bar solenoid pulls the cutter bar to the a left, shearing off tag of wire as long as the thickness of the bar. If) The hole in the cutter bar (carrying the tag) centers behind the hypodermic needle. 5) The rear solenoid drives a wire plunger through the hole in the cutter bar, pushing the tag through the hypodermic needle into the specimen. 6) The plunger retracts, leaving the tag embedded. 7) A spring pulls the cutter bar back to receive another tag, and the cycle is repeated. 96 W. Q. B. WEST AND K. K. CHEW

senses a magnetic tag in a shrimp passed through its sensing head and emits an audible signal. The tags are coded with longitudinal epoxy color stripes. These will allow a large number of unduplicated tags since as many as 6 colors may be applied to the wire simultaneously from a selection of 12 distinct hues. This yields at least S* different combinations if two stripes and two colors are reserved to denote the starting point and the direction of reading (Bergman et al.^).

Experimental Design Two experiments, designated as Experiments 1 Days and 2, were conducted. Each experiment was FIG. 2. Cumulative mortalities of shrimp in Ex- carried out over a period of 50 days — Experi- periment 1 with corresponding pH fluctuations. ment 1, from December 1, 1966, to January 20, TWz=Tagged and loounded shriynp. TU:=Tagged 1967, and Experiment 2, from February 2 to March and untagged shriinp. 24, 1967. In each experiment three categories of test animals were used. These categories were design- ated as tagged, untagged, and wounded with the were consequently left in the tanks. An indication latter two groups serving as controls. The wounded of the number of molting individuals was obtained shrimp were processed in the same manner as the by counting the cephalothoracic exoskeletons tagged ones. They were punctured by the needle (carapace) found in each tank. When bodies were but the tag was not inserted. found, shrimp which had died from the effects of In two tanks (Tanks 2 and 4i a group of tagged experimental procedures or other causes, were (Ti and untagged (U) shrimp were held to- distinquished from those eaten by their fellows gether in a one-to-one ratio. A second set of during or immediately after ecdysis, by the pres- tagged and wounded (W) individuals were placed ence or absence of a firm exoskeleton. together in two other tanks (Tanks 1 and 3). These were also in a one-to-one ratio. Thus, there was RESULTS AND DISCUSSION a duplicate set of tagged and untagged shrimp Mortality (TUl and TU2) and one of tagged and wounded The percentage of that died during the shrimp (TWl and TW2), all in the same ratio. All shrimp entire study was very high, ranging from 42.0 per four tanks contained equal numbers of shrimp cent in Tank 3 with TW2 shrimp in Experiment 2 (50). The shrimp in each experimental group were to 90.0 per cent in Tank 1 with TWl shrimp in selected to reflect the average size composition of Experiment 1. The cumulative mortalities of test the population. animals in Experiments 1 and 2 are shown in It was anticipated that molting shrimp might Figures 2 and 3 respectively, while Figure 4 shows be eaten by the rest. To partially combat this pos- sible problem, a plastic screen enclosure was in- stalled in each test tank to protect newly molted shrimp. This, in addition to the constant feeding, helped control the post molting cannibalism that occurred among the shrimp. Test tanks were checked at least twice daily for the presence of dead or molting animals and periodic counts were made of shrimp remaining in each experimental tank. Abdominal and cephalo- thoracic e.xoskeletons were removed whenever found. Shedding shrimp were observed to eat the branchial cast and associated structures, and these

5 Bergman, P. K., K. B. Jefferts, H. F. Fiscus and R. C. Hager, 1966. A preliminary evaluation of FIG. 3. Cumulative mortalities of shrimji in Ex- an implanted, coded wire fish tag. Washington periment 2 with corresponding pH fluctuations. State Department of Fisheries, Olympia, Wash- TW^Tagged and wounded shriinp. TU^Tagged ington, (unpublished manuscript) and untagged shrimp. APPLICATION OF TAGS ON SHRIMP 97

— Tank # Total tire study may be associated with several un- known factors caused the of 100 n possibly by deficiency the recirculating saltwater aquarium system where ra n the study was conducted. From the data presented in Figures 2 and 3, there is an obvious relation- 75- ship between mortality and pH; the mortality rate increases as the pH decreases. Until pH falls to 6.6 there seems to be little adverse effect on the 50- shrimp. It was also observed that at low pH, the test animals were relatively inactive. Usually, when- ^ ever food was introduced, the shrimp would 25- actively compete for it, but this did not occur LMS LMS LMS LMS I 234 during periods of low pH, especially below 6.6 when their seemed to be Experiment I feeding ability greatly affected if not completely inhibited. This peculiar 100 L= Large •^ behavior may be due to the fact that at a low pH M= Medium ^^ the ability of these animals to extract oxygen S= Small from the environment is seriously affected (Pruthi, r^ 75- ^ 1927; Tarzwell, 1957). It was noted that newly molted shrimp were found to be more adversely affected during the period in which the pH was less than 6.6. In most 50 ^ instance, over 80 per cent of all deaths during low pH occurred among the "soft-shell". Of course, one must not discount the fact that these "soft- 25- shell" have also been subjected to M. shrimp may other unknown conditions affecting their survival LMS LMS LMS LMS I 234 and well-being. Experiment 2 The differential mortality data are presented in Tables 1 and 2. To test differences in FIG. 4. Percentage mortality of shrimp in Experi possible and the ments 1 and 2. mortality due to treatment, size, replicate, interactions (treatment x size and treatment x replicate), a fully crossed, fixed model 3-way the percentage mortality within each size group in analysis of variance was conducted on the trans- each tanli. There appears to be no definite correla- formed data, using the ArcsinV Percentage trans- tion between size and mortality as shown in Figure formation (Snedecor, 1962). 4. Since the interactions (treatment-size and treat- The high incidence of mortality during the en- ment-replicate) were not significant at the 25 per

size TABLE 1. Mortality in Experiment 1 between tagged, wounded and untagged shrimp of various groups, held within four aquaria for 50 days.

Tank 98 W. Q. B. WEST AND K. K. CHEW

size TABLE 2. Mortality in Experiment 2 between tagged, wounded and untagged shrimp in various groups, held within four aquaria for 50 days.

Tank APPLICATION OF TAGS ON SHRIMP 99

molted at least once in each experiment did not each of the three treatment groups (tagged, survive the molt, and only 3 out of the 10 that wounded and untagged shrimp) are given in molted a second time survived. Tables 4 and 5. A fully crossed, fixed model three- Perhaps owing to a deficiency of some un- way analysis of variance was again used to test known component in the environment, the exo- the following hypotheses: skeleton of molted shrimp in this study did not 1) H,: no difference in the molting be- attain firmness even weeks after exuviation. Tarz- tween the three treatment groups; well (1957) asserts that some of the essential min- 2 1 H2; no size effect; erals in sea water becomes unavailable at low pH 3) H,: no replicate effect;

levels. This could retard the rate of calcification of 4 I H4: no treatment-size interaction; the new shell after molting. As mentioned before, 5) Hs: no treatment-replicate interaction. the experimental shrimp have frequently been ob- The data used for the analysis of variance test served to eat their discarded the tr exoskeletons immedi- were ansformed da ta of Tables 4 and 5, using ately after molting, a habit which may be nature's the Arcsin V Percentage transformation (Snedecor, device for furnishing the large supply of lime salts 1962). needed for rapid hardening of the new shell. Before the F statistics were computed for the The numbers and percentages of shrimp that variations due to the main effects (treatment, size molted at least once during the 50-day period in and replicate), the hypotheses for the interactions

TABLE 4. Percentage of shrimp of various size groups within different treatment groups (tagged, wounded and untagged), held within four aquaria that molted at least once in 50 days in Experiment 1. Tank 100 W. Q. B. WEST AND K. K. CHEW

TABLE 6. Analysis of variance to test differences in molting be- tween tagged, wounded and untagged shrimp of various size groups.

Source of Proreedinijs vf the National Shellfisheries Association Volume 58 — June 196S

SURVIVAL TIME OF OYSTERS AFTER BURIAL AT VARIOUS TEMPERATURES

Elgin A. Dtwuhigton, Jr. UNIVERSITY OF MARYLAND NATURAL RESOURCES INSTITUTE CHESAPEAKE BIOLOGICAL LABORATORY SOLOMONS. MARYLAND

ABSTRACT

Experimental burials of oysters were made 3 inches deep in containers of soil held in running sea water at five temperature ranges from less than 5°C to over 25°C. Smci'U'oZ time varied from 2 days in summer to 5 weeks in winter, showing a direct relationship to temperature.

INTRODUCTION 2 inches below the soil-water interface — mostly anaerobic, Accidental burial of oysters can be caused by 3 inches below the soil-water interface — en- storms; siltation due to high run-off or channel tirely anaerobic. dredging; oyster harvesting and planting activi- This gradient is similar to conditions observed in ties: smothering by vegetation or other organ- natural bottom having the same type of soil. isms; and probably b}' unknown agencies. Occa- In trial shallow burials, oysters buried 1/2 inch sionally, oysters being held in trays on or near the or less deep could usually clear their bills of sedi- bottom have been buried. Both natural and man- ment if the water was warm enough for active caused burial are widespread phenomena (Galt- pumping. Thus the 3-inch depth employed in this soff, 1964). experiment indicated survival under conditions Oysters were buried experimentally to study that did not permit recovery from buriaJ. how burial affects them. One result of these Initially, burial periods which would furnish the studies is an indication of survival time and rate desired information on survival could only be of decomposition at several temperature ranges. guessed. The work of Lund (1957a, b, c) was sug- gestive, and Wilson 2 experimented with burials METHODS but these authors dealt primarily with aspects of survival other than temperature. After the first Oysters from the lower Patuxent River were two series of burials were completed, it was pos- used and all were held in running sea water until sible to plan exhumations so that they were made dredge and handling damage to the shells was during periods of moribundity. Later experiments repaired. In each series, 8 oysters were buried 3 thus show progressively increasing decomposition inches deep in mixed sand and mud in each of 4 and deaths. polyethylene trays. Control oysters were placed on top of the soil and the trays were immersed in a large running seawater aquarium. ' Contribution No. 356, Natural Resources In- Observations of the soil in the trays and in glass stitute, University of Maryland. tanks showed the following conditions as indicated by the presence of reduced sulfur compounds: 2 Wilson, W. B. 1950. The effects of dredging on 1 inch below the soil-water interface — still oysters in Copano Bay, Texas. In Annual Report aerobic, — of the Marine Laboratory of the Texas Game, li inches below the soil-water interface Fish and Oyster Commission for 1948-1949, p. transitional, becoming anaerobic. 1-50.

101 102 ELGIN A. DUNNINGTON, JR.

oooe oooe 24-18° ooe© Falling ooe© ooe* SURVIVAL TIME OF OYSTERS AFTER oeG© BURIAL AT VARIOUS TEMPERATURES eeo© «©©• oo oo ^'O o© Over C'O ©© 25° oe ©© o© ©© O Live Oyster o© ©© e Clucker o© ©© © «© ©© Gaper • Box o o ©© o o ©© 15-20° o o ©© Rising o o o© o o ©© o o ©© oo ©© O G ©© o o o o o e © o o o o © » 10-15° o o o o © o © Rising o o o o © © © o o o o © © © o o o o © o o o o © o o o o o e e Less than o o e © o o e © o o e © o o e © o o © © o o © n o ©

J I L _L J L 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 DAYS OF BURIAL

burial. FIG. 1. Condition of oysters at exhumation after various periods of Each symbol represents a single oyster.

RESULTS AND DISCUSSION dead and later exhumations showed increasing decomposition. Figure 1 shows the results of burial at 5 dif- At 15-20^C, most oysters survived for a week, ferent temperature ranges. At mid-winter tempera- but all were dead after 2 weeks. When this ex- tures of less than 5-C, oysters lived for over 5 periment was conducted critical moribundity peri- weeks and decomposition was prolonged to over ods were still not established. 10 weeks. Pumping would not normally occur During mid-summer temperatures of over 25°C, when the water is this cold and experimental one buried oyster died in 2 days, 50 per cent were shell opening rarely happens at these tempera- dead in 4 days, and after a week only one was tures. alive. In the 10-15°C series, the first dead oyster was In the fall with ambient temperatures declin- found 3 weeks after burial. One week later, 7 were ing from 24-18° C, a similar pattern was found, SURVIVAL TIME OF BURIED OYSTERS 103

with the difference that oysters died a little more of toxic materials and bacteria from the mud slowly. when a buried oyster gapes may also be a signifi- Although there are marked differences in sur- cant factor in survival time and it probably vival times between the 10-15°C series and the hastens decomposition. 15-20 -C series, it appears that within these ranges (below 20'C) once a certain threshold has been ACKNOWLEGMENT reached, death follows in about one week. At higher temperatures, this critical point is reached I am indebted to Mrs. G. E. Drobeck for assis- in less than a week. tance with processing and some critical observa- All of the oysters were buried with the left side tions. down. When they were exhumed and examined it was observed that the left side of the visceral LITERATURE CITED mass decomposed before the right side and the posterior region decomposed before the anterior Galtsoff, P. S. 1964. The American oyster, Crass- region. The exposure to the mud which occurred ostrea virginica Gmelin. U. S. Fish & Wildlife when they gaped and remained open seems to Service, Fish. Bull. 64:1-480. have hastened decomposition. Lund, E. J. 1957a. A quantitative study of clear- In several of the oysters that were exhumed ance of a turbid medium and feeding by the alive, mud on the gills indicated that the valves oyster. Publ. Inst. Mar. Sci. Texas, 4:296-312. were opened temporarily before they finally gaped Lund, E. J. 1957b. Self-silting, survival of the and stayed open. oyster in a closed system, and reducing A direct relationship between temperature and tendencies of the environment of the oyster. survival time is shown by these experimental Publ. Inst. Mar. Sci. Texas, 4:313-319. burials. Earlier death at higher temperatures may- Lund, E. J. 1957c. Self-silting by the oyster and its be due principally to a higher metabolic rate and significance for sedimentation geology. Publ. more rapid consumption of reserves. The intrusion Inst. Mar. Sci. Texas, 4:320-327.

ASSOCIATION AFFAIRS

ANNUAL CONVENTION

The National Shellfisheries Association 1967 erica presented plaques to Dr. L. Eugene Cronin Convention was lield jointly with the Oyster In- and Dr. Leslie A. Stauber in recognition of their stitute of North America and the Oyster Growers contributions to the industry through scientific and Dealers Association of North America, Inc. research. Both the Bureau of Commercial Fish- from July 16 to 19 at Boston, Massachusetts. eries Biological Laboratory at Oxford, Md. and Officers and Executive Committee members the Exploratory Fishing and Gear Research elected for the term 1967-1968 were: Laboratory at Gloucester, Mass. received plaques President Harold H. Haskin for sea clam biological studies and gear research Vice President _ Albert K. Sparks respectively. Secretary-Treasurer Fred W. Sieling The Pacific Coast Section of the NSA met joint- Members-at-large _ Sammy Ray ly with the Pacific Coast Oyster Growers Associa- Roy Drinnan tion on August 23-25, 1967 at Olympia, Washing- James E. Hanks ton. Officers of the Section elected for the term Editor, NSA Proceedings 1967-1968 were: Arthur S. Merrill Chairman Kenneth K. Chew John W. Ropes of the Bureau of Commercial Vice-Chairman C. Dale Snow Fisheries Biological Laboratory at Oxford, Md. Secretary-Treasurer John C. Hoff was appointed Custodian of back issues of the The Pacific Coast Section has augmented its NSA Proceedings. membership by the addition of a number of A program of honors ceremonies took place, in crustacean research scientists. which Dr. David L. Belding was elected to Honor- Regular membership to the NSA is $6.00, library ary Membership in the National Shellfisheries subscription is $6.00, and patrons contribute Association. The Oyster Institute of North Am- $100.00 or more.

105

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H lABD +

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