The reproductive ecology of the calico scallop, Argopecten gibbus (Linnaeus), and mass mortality linked to a protistan

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The reproductive ecology of the calico scallop, Argopecten gibbus (Linnaeus), and mass mortality linked to a protistan

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Title:
The reproductive ecology of the calico scallop, Argopecten gibbus (Linnaeus), and mass mortality linked to a protistan
Creator:
Moyer, Michael Alan
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Tampa, Florida
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University of South Florida
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English
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x, 168 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Atlantic calico scallop -- Reproduction ( lcsh )
Dissertations, Academic -- Marine Science -- Doctoral -- USF ( FTS )

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General Note:
Includes vita. Thesis (Ph. D.)--University of South Florida, 1997. Includes bibliographical references (leaves 150-168).

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University of South Florida
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University of South Florida
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All applicable rights reserved by the source institution and holding location.
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023912501 ( ALEPH )
37682906 ( OCLC )
F51-00207 ( USFLDC DOI )
f51.207 ( USFLDC Handle )

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THE REPRODUCTIVE ECOLOGY OF THE CALICO SCALLOP, ARGOPECTEN G IBBUS (LINNAEUS), AND MASS MORTALITY LINKED TO A PROTIST AN by MICHAEL ALAN MOYER A dis se rtation s ubmitted in partial fulfillment of the requir e ment s for the degree of Doctor of Phi l o so phy D e partment of Marine Science Univer sity of South Florida April 1 997 Major Professor: Nor man J. Blake Ph.D.

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Graduate School University of South Florida Tampa Florida CERTIFICATE OF APPROVAL Ph. D. Dissertation This is to certify that the Ph.D. Dis s ertation of MICHAEL ALAN MOYER with a m ajor in Marine Science h a s been approved by the Examining Committee on January 16, 1997 as s atisfactory for the di s sertation requirement for the Doctor of Philosophy degree Examining Committee : Majo r Professor : Norman J Blake Ph D Member: Kendall L. Carder, Ph D Member: Sandra E Shumway Ph.D. Member: Joseph J. Torre s, Ph D Member : G a briel A. V a rgo Ph D

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Copyright by Mi c h ae l Alan Moyer 1 997 A ll r i g h ts rese r ved

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ACKNOWLEDGEMENTS I would like to thank my major professor Dr. Norman J. Blake for all that he ha s done to assist and support me throughout my graduate s tudie s. Mere words are not e nough to express my gra titud e for the help h e has provided me both profe ssio nally and personally. I would also like to th a nk th e members of my committee; Drs. Ken Carder Sandy Shumway, Jose Torre s, and Gage Vargo for their advice and comments. My wife Jill has put up with an awful lot over the course of thi s work and I thank her for her Jove, support, and patience. I want to recognize the s upport and guidance provided to me by my parent s, FrankS. Moyer and Carol K. Moyer, as well as my late grandmother, Helen L. Moyer. The love, support, and prayer s of the many friends who make up my extended family have a l so been invaluable Thi s research would no t hav e been poss ible without the help provided to me by the calico sca llop indu s try. My th anks to all of the captains a nd crews who assisted me with sample collection and to Southern Seafood Inc., and Lambert Seafood Inc for a llowing me access to their vessels I a m p ar ticularly grateful for the assistance provided to me by Don Stevens, Tommy Smith, AI Silchenstead, and Bill Lambert. I also extend my sincere appreciation to the members of Blake's Flakes who have assisted me in my re sea rch; B. Barber T Cuba, R. Erdman D Hesse lm an and R. Darden. Finally, I salute th e guidance, in s piration, and enthusiasm s h a red with me by the late Paul P. Yevich.

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TABLE OF CONTENTS LIST OF TABLES Ill LIST OF FIGURES v ABSTRACT VIII CHAPTER 1. INTRODUCTION CHAPTER 2. LITERATURE REVIEW 4 Th e Study Organism 4 The Fishery 8 Reproduction in Related Specie s I 0 The Effect of T e mperature and Food Abundance upon Reproduction 15 Hydrography of the Study Ar ea 19 Di seases in Bivalve s Link e d to Proti sta n s 23 C HA PTER 3 REPRODUCTIVE ECOLOGY OF ARGOPECTEN G IBB US 26 Introduction 26 Material s a nd Method s 28 Res ult s 33 Di sc u ss i o n 46 CHAPTER 4. TEMPERATURE AND THE REPRODUCTIVE CYCLE 56 Introduction 56 Materials and Method s 57 R es ult s 61 Di sc u ss ion 79

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CHAPTER 5. FOOD ABUNDANCE AND THE REPROD UC TIVE CYCLE 88 Introduction 88 Material s and Methods 90 Result s 92 Discu ss i on 108 CHAPTER 6 MORTALITY IN ARGOPECTEN GIBBUS LINKED TO A PROTIST AN OF THE GENUS MARTElL/A I 13 Introduction 113 Materials and Methods 114 Result s 114 Discussion 123 CHAPTER 7. DISCUSSION 134 Interaction of Temperature and Food Abundance 134 Natural Reproductive Cycle 145 Pathogenic Effects of Marteilia Upon th e Calico Scallop 1 46 Conclu s ion s 147 LIST OF REFERENCES 150 VITA End Page II

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LIST OF TABLES Table 1. Experimental trial dates and the treatment s used for each of the tria ls. 60 Table 2. Mean ( 1 sd) wet weights (g) of the gonad body component of Argopecten gibbus for all treatments grouped by experimental temp e rature 63 Table 3. Mean ( 1 s d ) wet weights (g) of the digestive diverticulum body component of Argopecte n gibbus for all treatments g rouped by experimental temperature. 64 Table 4 Mean ( 1 s d ) wet weights (g) of the mantle body component of Argopec t en gibbus for all treatments grouped by experimental temperature 65 Table 5. Mean ( I s d ) wet weights (g) of th e adductor muscle body component of Argopecten gibbus for all treatments grouped by experimental temperature. 66 Table 6. Mean ( 1 s d ) total body wet weights (g) of Argopecten g ibbus for all treatments g r o uped by experimental temperature 67 Table 7. Mean ( 1 sd) wet weights (g) of the go nad body component of Argop ecten gibbus for a ll treatment s grouped by experimental food level. 93 Table 8. Mean ( I sd) wet weight s (g) of the dige s tive diverticulum body component of Argopecten gibbus for all treatment s grouped by experimental food level. 94 Table 9. Mean ( 1 s d ) wet weights (g) of the mantle body component of Argopecten gibbus for all treatment s grouped by experimental food level. 95 lll

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Table 10. Table 11. T a ble 12. Table 13. Mean ( 1 s d) wet weights (g) of the adductor muscle body component of Argopect e n g ibbu s for all treatments grouped by experimental food level. Mean ( l sd) total body wet w e ight s (g) of Argopec ten g ibbus for all treatments grouped by experimental food l evel. Matric es of the mean gonad index s howing the interaction between temperature and food abundance for experimental trial s 2 to 5. Matrice s of the mean oocyte diameter showing the inte raction bet ween temperature and food abundance for experimental trial s 2 to 5. I V 96 97 136 137

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LIST OF FIGURES Figure 1 Location s of major commercial stocks of calico scallop s in the eastern United States and location s used for electrophoretic and mitochondrial DNA analy ses of variation between populations. 5 Figure 2. Area off the eastern coast of Florida in which sa mple s were collected for this s tudy. 30 Figure 3. Mean go nad indices including stan d ard deviation based upon dry tissue weights for field sample s collected from 1983 to 1991. 35 Figure 4. Mean dige s tive diverticulum indice s including standard deviation ba sed upon dry tissue weights for fie ld sample s collected from 1983 to 1991. 37 Figure 5. M ea n mantle indi ces including sta nd a rd deviation based upon dry tissue weights for field samp le s collected from 1983 to 1991 38 Figure 6. Mean adductor muscle indices including s tandard deviation based upon dry tissue weights for field sa mple s collected from 1983 to 1991. 39 Figure 7 Mean oocyte diameters including s tandard deviation for fie l d samples collected from 1983 to 1991. 41 Figure 8. Mean oocyte diameters for fie ld samples collected from 1983 to 1994 plotted agains t time of year. 43 Figure 9. Mean go nad indi ces includin g s tandard deviation based upon dry tiss ue weights for field samp le s collected from 1983 to 1994 plotted again st time of year. 45 Figure 10. Mean daily temperatures collected at the central thermograph s ite every two hours from October, 199 0 to October, 199 1 plotted against the time of year 49 v

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Figure I I. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Mean gonad indices including standard deviation based upon dry tissue wei ghts for experimental animals maintained at 15 C plotted against time of year from 1993 D and 1994 along with fie ld measurements from 1993 Oand 1994 + 70 Mean oocyte diameter including standard deviation for experimental animal s maintained at 1 5 C plotted agai nst time of year from 1993 D and 1 994 along with field measurements from 1993 Oand 1994 + 7 I Mean gonad indice s including standard deviation based upon dry tissue weights for experimental an im a l s maintained at 20 C plotted against time of year from I 993 and 1994 A a long w ith field measurements from 1 993 Oand I 994 + 73 Mean oocyte diameter including standard deviation for experimental animal s maintained at 20 C p l otted against time of year from 1 993 b. and 1994 A along w ith fie l d measurements from 1 993 Oand 1994 + 74 Mean gonad indices including standard deviation based upon dry ti ssue weights for experimental animals maintained at 25 C plotted against time of year from 1 993 0 and 1994 e a long with field measurements from 1 993 Oand 1 994 + 76 Mean oocyte diameter including standard deviation for experimenta l animals maintained at 25 C plotted against time of year from 1993 0 and 1994 e a long with field measurements from 1993 Oand 1994 + 78 Mean gonad indices including standard deviation based upon dry tissue weights for experimental animals maintained with no food plotted against time of year from 1993 D and 1994 along with fie ld measurements from 1 993 0 and 1 994 +. 100 Mean gonad indices including standard deviation based upon dry tissue weights for experimental animal s maintained wi th a low food ration plotted against time of year from 1993 1 994 A along with field m easurements from 1993 Oand 1994 + 101 Figure 1 9. Mean go nad indi ces including standard deviation based upon dry tissue wei ghts for experimental animal s maintained w i th a hi gh food ration plotted against time of year from 1993 0 and 1994 e a long with field measurement s from 1 993 0 and 1 994 + I 02 VI

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Figure 20. Mean oocyte diameter including standard deviation for experimental animals maintained with no food plotted against time of year from 1993 0 and 1994 along with field measurements from 1993 Oand 1994 104 Figure 21. Mean oocyte diameter including s tandard deviation for experimental animals maint a ined with a low food ration plotted against time of year from 1993 !J. and 1994 .._ along with field meas urements from 1993 0 and 1994 + 105 Figure 22. Mean oocyte diameter including standard deviation for experimental animals maintained with a hi g h food ration plotted against time of year from 1993 0 and 1994 e along with field meas urement s from 1993 0 Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. and 1994 + 106 Digesti ve diverticulum of a h eal thy calico scallop, Argopecten gibbus, collected off Cape Canaveral, Florida (scale bar i s 25 Jlm). 118 Digestive diverticulum of a calico sca llop, Argopecten gibbus, collected off Cape C anaveral, Florida exhibiting a heavy infection by a protozoan of the genus Marteilia. 119 Inte s tine of a healthy calico sca llop Argopecten gi bbus collected off Cape Canaveral, Florida (scale bar i s 50 Jlm). 121 Inte st ine of a calico scallop, Argopecten gibbus, collected off Cape Canaveral Florida exhibiting extensive occlusion of the lumen by mature s pore s of Mart eilia. sp. 122 Adductor muscle tissue of a healthy calico scallop, Argopecten gibbus, collected off Cape Canaveral, Florida (scale bar is 50 Jlm). 124 Adductor muscle ti ssue of a moribund calico scallop Argopecten gibbus, collected off Cape Canaveral, Florida which has been infected by Mart eilia sp. 125 Vll

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THE REPRODUCTIVE ECOLOGY OF THE CALICO SCALLOP, ARGOPECTEN GIBBUS (LINNAEUS), AND MASS MORTALITY LINKED TO A PROTISTAN by MICHAEL ALAN MOYER An Abstract Of a dissertation submitted in partial fulfillment of th e requirements for the degree of Doctor of Philosophy D e partm e nt of Marine Science Unive r si t y of South Florida Apri l 1997 Major Professo r : Norman J. Blake, Ph D V II I

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ABSTRACT The reproductive ecology of the calico scallop Argopect en gibbus (Linnaeus), was invest igated for a popul atio n located off Cape Canaveral, Florida. Monthly samples collected over an 8 yea r period indicate a semiannual reproductive cycle. The major spawn occurs in the spring between March and May, while a second, minor, spawn occurs in the late summer or fall, typically in July or between September and November. Not all individuals participa t e in the minor spawn an d thi s second spawn i s absent in some years. Spawning occurs when th e bottom water tempera ture s are> 20 C following a period of time a t lower tempera tur es Unlike the bay scallop, calico scallops as young as two months old undergo gametogenesi s a n d apparen tl y produce mature oocytes and spermatozoa; however, th e viability of these gametes remain s unknown. In laboratory experiments, gametogenic development occurred at all temperature and food level s during th e early s pring ; however, production of gametogenic material both in a bsolute terms and r elat ive to other body components was positively correlated with food level s and negati ve l y correlated with tempera ture. Spawning was observed in th e l aboratory in the lat e spring among some of the animal s held at 20 C. During the lat e summer gametogenic development was obser ved among th e animals maintained at 25 C, which was independe nt of the food leve l that was received. There was no evidence of gametogenic development among scallops held at !5 C in late summer and although gametogenic maturation was observed at high temperatures th e r e was no evidence of increa sed gametogen i c production in term s of either gonad weight or percent IX

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co mp ositio n i n these sca l lops. Oocy t e maturation was o b serve d a t all te mp e r a tur es an d f ood levels d u ring t he fall; however, pro d uct i on o f ga m etogenic ma t er i a l was n ot observed a m o n g a nim als h e l d a t I 5 C, a n d was p ositiv el y co rr e lat ed w i t h t e mp e r a tur e among the other tr eatme n ts. Gametogen i c p rod u ctio n durin g the fall was grea test w it h ris i ng t e mp e r a tures w h i l e ga m e t ogenic p r od u ct i o n d uring t he s p ring wa s g reate st with d ec lin i n g te mp erat ur e Mass mor t ality amo n g t he cal i co scallo p popu l atio n was obse r ved in early 1 989 a nd agai n i n 1991 Hist o l og i ca l examin ation o f scallo p s from t hese perio d s reveal ed t h a t the p r ox i mate ca use of t he observed mo r tal it ies was i n fec t io n by a prot i s t an of t he gen u s M a rt eilia. T here h ave b ee n n o r epo rts of thi s pathoge n inf ect in g sc all o p s p r i o r t o th e wor k i n this s t udy. Abs tr ac t A p proved:------------X M a j or P ro f ess or: N o rm a n J Bl a k e, Ph D Prof essor, D e par t m e n t of M a rin e S c i e n ce D a t e A p p r oved: /ttJ )b, /f/f/ v 7

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CHAPTER 1. INTRODUCTION The calico scallop Argopecten gibbus (Li nn aeus) s upport s one of the largest s hellfish fis heries in the state of Flo r ida. Annual l andings of as many as 40 million pounds of scallop meat have been reported for the sca llop beds located off Cape Canaveral, Florida (Blake and Moyer I 991). The calic o scallop fis h ery lo cated in Brevard county is th e l argest scallop fishery in the United State s and i s responsible for more than 1200 jobs in the l oca l economy ( R ockwood and Pompe, I 988). But de s pite its economic importance very litt l e re sea rch has been cond u cted upon the biology of the calico sc allop. The distribution and abundance of the calico scallop may vary dramatically from one season to the next (Allen and Costello, I 972; Peter s, I 978; Roe et al, I 971 ). The fluctuations in stock availability are related in part, to s pawning s ucce ss whic h in turn i s influenced by seve ral environmenta l factors; temperature i s thought to be the mos t important (Mi lle r et al, I 981 ). Th e in fluence of e n v ironment a l factors s uch as temperature and food avai l abi l ity upon r e production ha s been demon s trated for many bivalv e s pecies (Bayne, 1975 ; Giese, I 959; Giese and Pear se, 1974); alt h o u gh o nl y po s tulated for the calico scallop (Allen and Costello, I 972; Miller e t al, I 98 I). No work,

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however has been done to actually s how the relation s hip s b e tw ee n these environmental fac tor s and the variou s stages of the calico scallop' s reproductive cycle. The calico sca llop exhibits, in both habitat prefe r e nce and reproductive s tr a tegy many diffe rences from other member s of the genus Argopecten. The calico scallop is found in open marine waters on a hard s hell or sand bottom with the population reaching maximum den s ity at depth s of 35 to 65 met e r s (Allen and Costello, 1972). In contras t, other members of the genus Argopecten ( A circularis, A irradian s, A. nucleus, and A purpuratus) are all found in s h a llow water bay s sounds, and estuaries (Felix-Pico, 1991 ; Sastry, 1961 ; 1979; Seijo et al, 199 3; Wolff, 1988) All of the s pecie s of the genus Argo p ecten, except for A. gibbus, have their major spaw ning event in the fall while the major s pawning event in the calico scallop i s in the spring. In fact, in the northern hemi s phere members of this genus, except for the calico sca llop, exhibit an annual reproductive cycle with one spaw n in the fall while the calico scallop may have a s emiannual reproductive cycle in which there is a major s pawn in the s pring and a minor s pawn in the fall (Porter and Schwartz, 1976). Considerable research on th e influence of temperature, food availability and age upon reproduction ha s been conducted with other member s of the g enu s Argopecte n notably upon the bay sca llop, A. irradians. The reference s cited above indicate that the calico scallop employs a r e productive s tr a t egy that i s different in m a ny respects from the other members of the ge nus. Likewi se, the calico scallop i s found in a habitat that i s quite different from other spec i es of th e ge nu s and hence i s s ubject to different phy s ical conditions. While work on other spec i es of the genus Argopecten doe s point out areas 2

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that need to be explored w i t h th e calico scallop, it would not be prudent to attempt to extrapolate or apply the re s ult s of researc h con d uc ted upon other members of the genu s to the calico scallop wit h o ut conductin g s imilar studie s upon the speci es in question. Very little qu a litativ e an d no quantitative work has been done upon the reproductive biology of the calico scallop Since temporal patchine ss in population n umbers i s often r e lated to the reproductiv e cyc l e of an animal, proper management o f this f isher y resource necessitates a clear u nders tanding of the scallop 's reproductive cycle and the parameters w hi c h in flue nce the ti min g of repro ductive events. The o bjectiv e of this research was to examine the reproductive ecology of th e calico scallop A. gibbus, off Cape Canaveral FL. The p aramete r s m os t commonly linked to th e timin g of r eproductive events in scallops a r e t emperature and food abundance, l a bor a tory studies were conducted to examin e how eac h of th ese influence r e production in th e calico scallop. Vari a tion s in the timin g of reproduction within a popul a tion i s n o t the only factor which can cause fluctuation s in population level s. Popul a tion level s of this s hort lived species can fluctuate g reatly due to variations i n l arval transport. In addition mortality related to fishing pre ssure and di sease can a lso h ave an effect upon the population level. In both 1989 and 1991 massive mortalities were o b s erved in the calico scallop population off Cape Canaveral, Florida. Examination of scallop s collected during those events identified the proximal cause as the presence of a prot istan of th e gen u s Marte ilia. The results of th e study of these events and the impact that the parasite had upon the calico scallop both individually and as a population are a lso presented in thi s study. 3

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CHAPTER 2. LITERATURE REVIEW The Study Organism The calico s callop Argopec t e n gibbus occurs throughout the Gulf of Mexico and western North Atlantic Ocean, from th e northern s ide of the Antilles past so uthern Florida to Cape Hattera s, with small populations being found as far north as Delaware Bay ( Waller 1969) An a pparentl y s table population ha s also been observed in Bermuda ( W aller, 1973 ). V ariation a m ong three populations of calico s callop s collected from the Marquesas K eys in the Florida Ke ys, Cape Canaveral in Florid a and Cape Lookout North Carolina were compared us in g both morphometric and electrophoretic analysis and s howed no evidence of hi s tori cal divergence b e tween the population s (Krau s e 1994) This indicate s t h a t fre qu e nt migration between these populations which present s genetic isol a tion of the calico scallop s found in the three areas. Compari s on of mitochondri a l DNA variation betwe e n calico s callop populations collected from Appalachicola and Cape Canaveral, Florida was also con s ist e nt with the hypothesi s that th e re i s a share d common ge ne pool for the two popul a tion s (Blake a nd Graves, 1995) The calico scallop population s which h ave s upp orte d com mercial fis h eries and have been analyzed u s ing DNA a nd electrophoretic analy s i s are s h ow n in Figure I. The calico scallop i s generally 4

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CAPE LOOKOUT ATLANTIC OCEAN CAPE CANAVERAL GULF OF MEXICO Figure I MARQUESAS KEYS Locations of major commercial s tock s of calico scallops in the eastern Un i ted States and location s u s ed for electrophoretic and mitochondrial DNA analyse s of variation betw ee n population s. 5

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found in warm, open marine waters at depth s extending from 9 to 366 meters (Waller, 1969) although the maj or ity of sca llop s are located at depths between 35 and 65 meter s. The open shelf habitat preferred by the calico sca llop is markedly different from the shallow estuarine habitat in which other member s of the Argopecten genus are found and the calico sca llop appears to prefer a smooth bottom consisting of sand and hard shell (Drummond, 1969) The calico scallop s hell has between 19 and 21 ribs which are square in cross sec tion. Maximum s hell height (SH = straight line mea s urement from the umbo to the ventral edge of the s hell) i s ap proximately 64 mm. Both valve s are globose. The bottom valve is commonly whitish with very little color while the upper valve can be of many bright colors with a variegated or mottled appearance (Abbott, 1974). The pelagic larval period la sts for approximately 14 to 16 days with s pat set occurring at a s hell height of 0.25 mm (Costello et al, 1973) Growth is almost 2 mm I week or more for the fir s t 3 months re s ulting in sca llops of 10 mm shell height with an approximate age of 7 weeks and 25 mm 10 weeks (Allen, 1979; Allen and Costello 1972). The rapid growth phase la s ts for approximately 3 month s, at which time the calico scallop reache s a s i ze of approximately 28 mm SH (Allen, 1979) The growth rate then s teadily declines to le ss than 0.5 mm I week by the time the s callop reaches 35 mm SH at approximately 6 months of age (Blake and Moyer, 1991 ). Although the scallop may live as long a s 24 months and reach 64 mm SH, the normal life span i s 18 20 months with the scallop s attaining an average s hell height of 54 mm (Allen and Costello, 1972; Roe et al, 1971) 6

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The calico scallop i s a protandrous functional hermaphrodite (Allen and Costello, 1 972; Broom, 1 976). In laboratory st udie s male gametes are released first with discharge of eggs commencing 30 minutes to 1 hour after the sperm has been released and continuing for up to seve r a l hour s (Coste ll o et al 1 973). The delayed relea se of eggs helps reduce the chance of se lf-f ertilizatio n Miller et al ( 1979 ) found that scallop s as small as 20 mm SH contained eggs and spe rm and appeared ready to spawn. Singhas ( 1992) also reported finding scallops of< 30 mm SH in early to late developing stages with mature eggs with diameter s in excess of 40 IJm. The calico scallop appears to be the only scallop species in which reproductive maturity can be obtained in less than 3 months. The viability of any l arvae produced by these young scallop s has not, however, been determined. Most early re sea rch relied upon gonada l color changes to determine the reproductive cycle of the calico scallop (Allen and Costello, 1972; Allen, 1979; Costello e t al, 1973 ; Miller et al, 1979 ; Miller et al, 1 981; Roe et al, 1971 ). Initially spawning was thought to occur in the Cape Canavera l Florida scallop population between February a nd June with ris ing water temperatures possibly triggering spawning (Peters, 1978; Roe et al, 1971 ). Research on the hydrogr a phy of this region has shown, however, that th e bottom water temperature decrea ses during the s pring (Leming, 1979). Allen ( 1979) a nd Miller e t al ( 1981) have since postulated that the cold e r water triggers spawning in the s pring. Using spat tr aps, Miller et al ( 1981) determined that so me s pawning mu s t be occurring year-round, although the high est period of spawning ac tivity occurred between November and June In his doctoral dissert ation Kirby-Smith ( 1970 ) exami ned the North 7

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Carolina calico scallop population a nd con c lud e d that in that area spawning occurred only in the s pring All of thes e workers u sed color c h a n ges to determine the reproductive s t ate of th e scallops alt h o u g h it has been s hown th a t more accurate mean s of d e t e rminin g the r eproductive s tate of biv a l ves in c lud e the ratio of gonad ti ssue to total anima l ti ss ue (s h ell excluded) by w eig ht n ormal l y r e f e rr e d to as the go nad ind ex (Giese, 1959 ; Gies e a nd Pear se, 1 97 4 ) and hi s tological s taging an d oocyte diamet e r measureme nt s (Barbe r a nd B lake, 1991 ). The re has only been o n e s tud y t o date w hi c h has ut ilized thi s method t o examine the repr o duction of the calico sc allop. Porter and Schwartz ( 1976) utilized gon a d indice s and found th a t the mai n s pawn occurred between April and May but note d that a second spawn poss ibly took place in S e pt e mber. A later s tudy of the North Car o lin a population by Sin g h as ( 1 992) u s in g both gonad indice s and hi s tolo g ical anal ysis, a lso found that s pawnin g occu rred in the North Caro lin a population only once a year in the winter o r s prin g (November to April) No st udie s have used hi s t o lo g ical methods to s tud y r e production in the calico scallop in Florida waters. The Fishery D e nse b e d s of cal ico scal l ops were fir s t discove red off C ape Look o ut and Cape H atte r as, North Carolina i n 194 9 ( Che s tnut 1 95 1 ). Because of th e ir s m all s ize relative t o th e bay scallop A. irradians it was f e lt th a t h a nd s hu c king of the cal ico scallop would be economicall y unfeas ible Shrimp trawlers opera tin g in the Gulf of Mexic o r eported l arge 8

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populations of calico scallops in 1949-50 but active exploration did not begin until April of 1954 (Bullis and Ingle, 1959; Carpenter, 1967). Between 1955 and 1963 periodic production occurred in the Florida Middle Grounds and off Panama City and Appalachicola (Bullis and Ingle, 1959; Carpenter, 1967). The immense scallop beds located off of Cape Canaveral were first noted in May, 1960 by the Bureau of Commercial Fisheries with commercial concentrations located in depths between 29 and 51 meters (Bullis and Cummins, 1961 ). During the 1960's a hand shucking fishery was developed in North Carolina which by 1966 was able to harvest close to 2 million pounds of scallop meat annually (Cummins, 1971) but with the large beds located off Cape Canaveral and the arrival of automated shucking machines the North Carolina fishery rapidly decreased in size. Commercial fishing trials began off Cape Canaveral in October, 1967 and automatic shucking was introduced in 1969 (Cummins, 1971; Cummins and Rivers, 1970; Castagna and Duggan, 1971 ). Since that time the Cape Canaveral beds have been the primary source of calico scallops. The Cape Canaveral scallop beds extend for 200 miles along the east coast of Florida centered on Cape Canaveral. The largest concentrations of scallops, however, are located between Melbourne, Florida and New Smyrna Beach, Florida in depths of 27 to 64 m (Drummond, 1969). These beds have enabled the development of a strong calico scallop industry in Cape Canaveral. The Cape Canaveral scallop beds appear to be the only calico scallop population which is maintaining itself. The calico stocks in the Gulf of Mexico and off North Carolina fluctuate greatly in size. Irregular stocks in which the population fluctuates 9

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greatly from year to year wit h out a clear pattern are often found when recruitment i s s tron gly dependen t on the hydrographic conditions (Orensanz et al 199 1 a). Occasionally large populatio n s are present in these areas but the population s appear to b e unable to maintain themsel ves. It h as bee n s u ggested by Kirby-Smith ( 1 970) t h at th e popul ations located on the North Carolina s helf may be maintained by recruitm ent of l arvae tran sported b y the Gulf Stream from more souther l y popul atio n s, includin g th e mass ive beds located off Cape Can ave r al. A simi lar mechani s m may be responsible for maintaining the population s found in the Gulf of Mexico. Reproduction in Related Species Tn m arine invert ebrates the reproductive or gametogen i c cyc l e refer s to I ) the accumul ation of nutrient s for utili zatio n in gametogenes i s, 2) prolife r at ion of gonial cells and their diffe r e ntiati o n i nto gametes 3) th e accumu l ation of r ipe gametes, 4) spawnin g (the re lease of the gametes), and 5) a rest i ng or spe nt perio d during w h ich remainin g gamete s a r e reabsorbed or expel led (G iese and Pearse 1 974; G i ese, 1 959). The timi n g of th e reproduc tive cycle range s from continuous to biennial. In polar and temperate zon es most species exhibi t an an n ua l r eproduct i ve cyc l e a l tho u g h as th e l atitude dec r eases th e bre e ding period tends to expand (Giese 1 959) In s pecies w hi c h exten d over a wide lati tudi n a l range reproduction appears to be continuous near th e equator and seasonal away from th e equator (Pearse, 1 968; Giese and Pearse 1 974). Tropical s pecies may a lso 1 0

PAGE 25

exhibit an annual reproductive cycle but seem more likely to exhibit a semiannual cycle than specie s from temperate zones. Regulation of gametogenesis is accomplished through various endogenous and/or exogenous controls. Endogenou s control systems include the nervou s, endocrine, and n e uroendocrine systems while exogenous controls include temperature, photoperiod salinity and food abundance (Giese and Pearse 1974). Of the se, the most studie d has been temperature, although th e role of food abundance has received considerable attention in recent years. Because of the long his tory of commercial exploitation, the most s tudied scallop in North America is the sea sca llop Placopecten magellanicus. In the sea scallop, researchers have generally found an annual reproductive cycle with spawning during a two month period in the fall but with th e timing of the commencement of spawn in g varying from July through October (Barber et al, 1988 ; Giguere et al, 1994 ; MacDonald and Thompson, 1986 ; 1988 ; Robin so n eta!, 1981; Thompson 1977). R ece ntly, however there have been indi catio n s that in certain areas, notably the Mid-A tlantic Bight and Georges Bank, a second spaw n occurs in April or May (Dibacco, 1 991; Dibacco et al, 1995; Dupaul et al, 1989). An early summer spaw n (June ) by the sea scallop, di s tinct from the main fall spawning event, ha s also be e n reported by Naidu ( 1970) Studies on the Mid-Atlantic Bi g ht ha ve indi cate d th a t in terms of both the gametogen ic output and the temporal stability the spring spaw n is more important than the fall spaw n to the sea scallop population in that area (Sc hmitzer eta!, 1991; Kirkley and Dupaul 1991 ). A summ ary of th e reported spaw nin g periods for th e sea scallop s how s that spawn ing ha s II

PAGE 26

been reported for every month of the year except for March (Barber and Blake, 1991 ). Many researchers h ave sugges ted that the difference s in the timing of reproduction in the sea scallop are related to latitude. Based on the reported s tudies northern population tend to spawn only once a year and typically begin spawning in Augu s t or September while so uthern population s off of Georges Bank and farther south exhibit greater variability both in terms of when and how often they spaw n each year. Considerable work has also been conducted upon the bay scallop, Argopec te n i rradians. There are two s ub spec i es, A. i. irradians a nd A. i. co n centr icus, which have been s tudied exten sive l y Research on the reproductiv e cycle has s hown that both s ub s pecies of the bay sca llo p exhibit an a nnual reproductive cycle but the timing of spaw nin g varies tempor a lly both between the s ubspeci es a nd with latitude. In A. i. concentric us s p awni ng occurs during a two month period falling between July and November (G utsell, 1930 ; Sastry, 1961; 1963 ; 1966a ; 1966b ; 1970a ; 1970b; Barber and B l ake, 1981 ; 1983; Barber 1984) The spaw ning period commence s later as the latitude d ec rea ses throu g hout it s ran ge In A. i irradians s p aw ning commences earlier than in A i. concentricus with s pawnin g occurring between May and August. Again the trend i s for s pawning to b ot h commence and finish later in th e year as th e latitude decreases (Taylor a nd Capuzzo, 1983; Sastry, 1966b ; 1970a; Bricelj et al, 1987 a; Barber and Blake, 1991 ). There are three other member s of the genus Argopecten in which reproduction has b e en s tudied to so me extent. In the so uth ern hemisphere, work by Wolff ( 1988) on the Peruvi a n scallop A. purpuratus, indicated a reprodu c tive pattern more typical of tropical s pecie s. The Peruvian sca llop i s r epor t e d to exhibit a continuous reproductive pattern 12

PAGE 27

Even so, peaks in reproductive activity occurred between February and May (late summer and fall in the southern hemisphere). The Catarina scallop, A. circularis, is found on the west coast of Mexico in Baha California. The Catarina s callop has a semiannual reproductive cycle in Mexico with spawns in February to March and October to November (Felix-Pico, 1991) while in Panama only one annual spawn, in April, has been reported (Villalaz and Gomez, 1987 ). The final member of the genus Argopecten the Caribbean scallop, A. nucleus, is considered the closest living relative of A. gibb us (Waller, 1991 ) Studies in the Caribbean Sea off Venezuela indicate that the Caribbean scallop has a protracted spawning period extending from June to October (Seijo et al, 1993). With a life span of only 8 to I 0 months, this is in agreement with the continuous reproductive pattern associated with tropical species. Among scallops of the genu s Chlamys the specie s seem to be almost evenly divided between annual and semiannual reproductive cycles. In the northern hemisphere the Icelandic scallop C. islandi ca as well as a number of species from the Isle of Mann and Scotland (C. distorta C. septemradia ta C. striata, C. ti ge rina) the scallop exhibits a n annual reproductive cycle. All of these s pecies have a single spawning period occurring between June and September (Reddiah, 1962; Ansell, 1974 ; Sundet and Lee, 1984; Giguere, 1994). In the southern hemisphere the Australian doughboy scallop, C. asperrima, also spawns between June and September but being in the southern hemi sphere that corresponds to winter or sprin g (O'Connor and Heasman, 1996) It s hould be noted that two related Australian scallop species Mima c hlamys gloriosa and M. 13

PAGE 28

Leopardus also have a sing l e annual spawning event in the winter to sp ring (August October) and fall (May) r espective ly ( Robin sTroeger and Dredge, 1993). The black scallop, C. va ria the queen scallop, C. opercularis, the Chiloe scallop C. amandi, the Jicon sca llop, C.farreri, and the Huagui scallop, C. nobilis, among others all show evidence of a sem iannual reproductive cycle. Shafee and Lucas (1980) reported that in the black scallop a complete or major spawn ing event occurs in September or October, with a partial or minor s pawnin g even t taking place in May or June. Studies conducted upon the queen sca llop indicate that the major spawn take s place between June and August while a partial spawn may also occur in January (Soe modihardjo 1974; Taylor and Venn, 1979 ). The Jicon scallop from northern China has spaw ning peaks in May to June and September to October while the Huagui scallop found in southern China precedes it by about a month in both cases (April to May and August to September) (Lou, 1991 ). Finally the Chiloe scallop fro m Chile in the sout hern hemi sphe re spawns in l ate Novembe r to December and late February to April (Jaramillo et al, 1993). Two other import ant commercial s p ec ie s are the Japan ese common s callop Patinopecten yessoensis, and the European g re at sca llop P ecten maximus. The Japanese scallop spaw n s once a year in the spring between March and Jun e (Yamamoto, 1964; Maru 19 76; Ito, 1 99 1 ). The great scallop on the other hand presents a much more confus in g picture. In Ireland a nd the I sle of M a nn the scallop s spawn twice a year in April to May and Au gust to September (Gibson, 1956 ; Maso n, 1958 ; Ansell et al, 1991 ) In Scotland a nd Bay of Bres t France they spaw n only once in th e summer June to July and July to August, r espectively while in La Rochelle France the s ole annual spawning 14

PAGE 29

event takes place in the spring (April to May) (Comel y, 1974 ; Lubet et al, 1987 ; Lubet, 1 959; Ansell et al 1991 ) To summarize, the majority of scallop spec i es exhibit an annua l rep r oductive cycle and among those, spawning normally occurs in the summer or fall. The exceptio n s to this include severa l specie s from the sout h ern hemi sp her e as well as the Japanese s callop in which spawning occurs in the winter o r spri n g There are three species in which there seem to be latitudin a l difference s with regards t o ann ual or semiannua l reproductive patterns ( Plac o p ec t e n ma ge llani c us, Pecten maximus, and Argopect e n circu l aris) In addi t ion to the s e three and the cal ico scallop the b alance of the species reported to have a semiannual reproductive pattern are members of the genus Chlamys. Amon g those scallop s which s pawn more than o nce a year the major spaw ning event, when identified, normally occur s during the fa ll. The only other member of the Pectinidae family which reportedly s hares the calico scallops semiannua l reproductive cycle with the major spawning e vent in t h e s pring and a minor spawning event in the fall i s Pla c opect e n magellani c u s at the s outhern extent of it's distribution. There are s everal members of the genus Argopecten which exhibit a continuous reproductive pattern The Effect of Temperature and Food Abundance upon Reproduction Temperature and food abundance are cons id e red to be among the most important factors involv e d in controlling the reproductive cycle of marine invertebrates (Gie s e, 1959; Giese and Pearse 1974 ; Sas try, 1979) It has been show n that high temperatures 15

PAGE 30

and I or low food le vels r es ult in a decline of b o dy condition. While gonadal development may continue or eve n increase there will be a reduction in fec undity and I or a resorpti o n o f gametes (Bayne e t al, 1 975) In bivalve s, temperature i s the major tri gge r o f both sy nchronization of th e bre e din g cycle and progression from one s tage to the n ex t (Bayne, 1975 ) This may b e acco mplished eit h e r by the rat e of temperature change or by th e temperature increa s in g or decreasing to a n abso lut e value (Bayne, 1976). In some cases th e food abundance m ay ac tually be more important than t empera ture and may serve as the triggering s timulu s, allowing progre ss ion through the var i ous reproductive s t ages includ ing s pawnin g (Bay ne 1975 ; 1976 ; B ay ne a nd Newell 1983; Dis alvo et al, 1 984). Considerab le re searc h h as been conducted r e l at in g the effect of temperatur e and food ab und a n ce up o n r eproduc tion in sca llop s. There a re two a r eas of reproductive ph ys iolo gy in w hich temperatur e a nd food a bund a n ce can b e important. The fir s t area i s gam e tog e nesi s an d fecundit y a n d th e seco nd a r ea i s s p a wnin g a nd s pawnin g synchrony ( B a rber and B l ake, 1991 ). In the bay sc allop th e r e are two contr o l point s in the reproductive cycle which a r e controlle d by ei ther food a bund a n ce or temperatur e (Sas try 1966a; I 968; l 970b; Sas try and B l a k e, 1971 ). Sp ec ifically an a bundance of foo d appears to b e ne cessary for the sc allo p s to proceed f rom the primary germ cells (s t age I ) to devel opment of oogonia and s p e rmato gonia (stage IT) while th e t e mp era tur e mus t b e e l evated t o a threshold temp e r ature of approximate l y 1 5 Centi g r ade (C) in order for d e velopment to continu e to th e cytopla s mic g rowth phase (s t age Ill) a n d the temperature mu s t b e e levated to > 20 C 1 6

PAGE 31

for completion of maturation and spawning (Stage V) (Sastry, I 966a; 1970b; Blake, 1972; Blake and Sastry, I 979). In addition, spawning in A. i. concentricus appears to be triggered by a decrea se in temperature following a summer maximum (Barber and Blake 1983: Sastry 1963) Laboratory studies on the effect of food concentration and temperature upon the reproductive cycle of Argopecten ventricosus (=A. circularis) found no relationship between temperature and reproductive condition (Villalaz, I 994). However, he does indicate that temperature may be important factor for initiation of gametogenesis and s pawning. In addition the maximum gonad index was observed at low food concentrations and was attributed to energy tran sfe r from the dige s tive gland to the gonad while high concentrations seemed to favor growth over gametogenic development. In the sea scallop th ere is some disagreement as to the relative role of temperature and food abundance in controlling the reproductive cycle. Thompson (I 977) reported that gonad growth and differentiation begins only when temperature is low and food levels are high. In later s tudies MacDonald and Thompson ( 1985b) indicated that high temperature, in addition to high food levels was necess a ry for increased reproductive output. Barber et al ( 1988 ) also found that a low temperature and food level resulted in reduced reproductive activity in the sea scallop, although in their opinion the controlling f ac tor was the food level and not the temperature. Finally, Naidu (1970) suggested, with regards to the se miannual reproductive cycle he observed in Canadian waters that a temperature decrea se in the fall and a temperature increase in the spring may have played a role in the initiation of the two spawning events although he noted that spawning occurs 17

PAGE 32

during periods of physical di s turbance associated with high wind s and rough seas. Des ro s iers and Dube ( 1993 ) s howed that flowing seawa ter independent of temperature or food changes was an effective method for inducing spawning. Further work appears to be neces sary to differentiate between th e effects of temperature and food abundance upon the sea scallop. Other scallop species also s how evidence that temperature and food abundance influence reproduction. In the Japan ese sc allop P ec t en yessoe nsis gametogenes i s is accelerated by increased temperature and spa wning is triggered by a s udden temperature increa se above a critical minimum temperature (Ya mamoto, 1951; 1952) Yamamoto also found that the intensity of the reproductive cycle is related to the food level. Research on the Peruvian scallop, Argopec ten purpuratus, suggests that high temperature favors gametogenic maturation a nd spawning and that temperature is negatively correlated w ith the gonad ind ex (Wo lf f, 1 988). However, spawning has been induced in the Peruvian scallop u s ing only a n increa se in the food concentration ( Disalvo et al, 1984) so it may be that in this species the abundance of food is actually more imp ortant than temp e rature. It has been suggested that food r a ther than temperature i s the important parameter regulating the reproductive cycle in the Chiloe scall op, Chlamys amandi as well (Jarami llo et al, 1 993). The requirement of food level s sufficient to maintain the animal, as well as temperatures that do not stress the animal, in order to maximize reproductive output is int uitive. If there are insufficient re so urce s ga metogene s i s can not begin, and an increase in s tress re s ult s in a decrease in both reproductive activity and fecundity (Bayne et al, 18

PAGE 33

1983) Since both reproduction a nd growt h depend upon an energy level in excess of that required for routine metaboli sm, i t can be expected that changes in the food level will therefore have an effect upon both r ep r od uction and growth. Many s tudies have s hown that increa se d food level s do indeed re s ult in increased gro wth. Specifically, work on the bay sca llop (Kirby-Smith and Barber 1974) sea sc allop (MacDonald and Thomps on, 1985 a), ro ck scallop, Hinnit es multiru go sus (Le ighton, 1979 ) and P ecten maximus ( M ason, 1957 ) have s hown th a t growth and food levels are posi tively correlated N a tural food l eve l s are probably such that they are alway s a limiting factor to g row t h (Bayne and Newell, I 983 ). If ins ufficient food is available (or stored in the animal) game t oge ne s is will not occur. It is diffi c ult to separate the effect s of temperature and food level in the field since in many cases both factors are linked to the same phy s ical pro cesses. Increased temperature does re s ult in an incre ase in the m e t a bolic rate which will result in J ess energy being available for reproduction and a possible reduction in the growth rate (Ba rbe r and Blake, 1986; MacDon a ld a nd Thompson, 1985a ). Hydrography of the Study Area Calico scallops are most abundant ne a r coastal prominences s uch as Cape Canaveral FL, Cape Lookout NC, and Cape San Bias, FL (Blake and Moyer 1991 ; Brand, 1991 ). The largest calico sca llop b e d s are located off Cape Cana veral between 2 8 N an d 29 30' N at depth s ran g in g from approx imately 35 to 65 m ( Figure I ). 19

PAGE 34

Upwelling events are common in this a rea a nd have been extensively studied b y phy s ical ocea n og rapher s. Capes and s ho a l s induc e upwelling on the down s tream s ide ( north in this case) due to change s in s h e lf water vorticity ( Blanton et al, 1981 ). Cape Canaveral i s the only prominent cape so uth of the Carolina' s in the South Atlantic Bight ( SAB), a nd thi s has a s ignificant effect upon coastal upw e lling (Leming and Mooers, 1981 ) A number of studies have reported the intrusion of colder Florida Current w a ter along the sea floor of the continental s helf due to meanders of the Florida Current and frontal eddie s (A tkin so n 1985; Leming, 1979; McClain e t al, 1988 ; Oey e t al, 1987 ). These intru s ion s are stron gest during the summer in the SAB and are mo s t pronounced off east central Florida (Atki n so n 1985; Blanton e t al, 1981 ). This co lder water may intrude as much as 35 to 40 kilometers s horeward of the continental shelf break (Lee e t a l 1 982) and is greatest in the re g ion extending from 50 kilometer s north to 50 kilometers so uth of Cape Canaveral (Leming a nd Mooers, 1981) In this re g ion intrusion i s most pronounced during July and August when it intrudes to waters of 40 to 60 m depth ( Bl a nton et al 1981 ). This type of intru s i o n may leave s tranded bodie s of colder water on the shelf for up to 4 weeks ( McCl ain et al, 1988 ) and is po ssi bly encouraged by the pr e dominantly southea s t s ummertime winds ( Smith 1987). The onshore intru s ion of cold Florida Current water normall y begin s in l a te M arc h o r April and continues to move o n s hor e until July or August (Leming, 1979). Lee et a l ( 1984) s tated that the low es t temp erat ure s a re ge nerally found in May and July with t h e 18 C isotherm near or ins hor e of 1 8 m ete r s water depth. They also reported that the 15 C isoth e rm during their s tud y was ins h ore of 55 meters depth only in May a nd July 2 0

PAGE 35

Other processes may enable the intrusion of cold Florida Current water onto the conti nental shelf at other times during the year. Cyclonic cold core frontal eddies may occur in the spri ng (Ap ril ) where they may l ast up to 3 weeks as well as at other times during the year for periods ranging from 2 days to 2 weeks (Lee and Atkinson, 1983; Lee et al, 1982) Short term intrusions during the winter which allow shoreward flux of colder water may be caused by frontal eddy and shelf break upwelling (Oey 1986 ; McClain et a l 1988; Smith, 1987). Intrusions durin g the winter are often caused by water overriding the surface waters rather than transport along the sea floor as occurs in summertime intrusions (Oey et al, 1987). Larval retention in the presence of eddies has been cited as being respon sib le for local abundances in Patinop ecten yessoens is as well as Chlamys tehuelcha and C. patagonica (Yamamoto, 1964; Orensanz et al, 1991 b) The s upply of nutri ents onto the continental shelf in this area depends mainly upon the intru sio n of Florida Current water (Lee and Atkinson, 1983; Oey et al, 1987). Deeper Florida Current water at Jess than 20 C conta in s a significant amount of nutrients and is the most imp ortant source of nutrients in this region (Atkinso n 1985 ). The highest chlorop hyll concentratio n s are positively correlated with the most intense periods of upwelling (Yoder et al, 198 1 ). For this re ason, upwelling is the most important process controlling phytoplankton on the con tinental shelf of the southern region of the SAB (Yoder et al, 1983). The nutrients from th e upwelling of cold water lead s to a phytoplankton bloom and high zoop lankton abundance on the middle and outer shelf of the SAB l ast in g for up to 3 weeks (PaffenhOfer, 1985). Yoder et al ( 1983) found that production averaged 21

PAGE 36

almost 2 g Carbon m -2 d -1 during upwelling events which were present for more than 50 %of the time between November and April and re s ulted in phytoplankton production up to 5 times greater in the upwelled water than in the nutrient-poor overlying water. Value s as high as 6 g Carbon m -2 d-1 were observed in April a nd elevated production associated with upwelling events was also observed in August. Phytoplankton bloom s within upwelled waters on the outer s h elf (>40 m ) in the region off C ape Canaveral occur durin g all s eason s of the year and are accompanied durin g th e w a rmer months (March to October) by phytoplankton blooms which penetrate to the middle and inner shelf due to s ub s urface intru s ion s (Yoder, 1985; 1991; Lee et al, 1991 ). Primary production in the middle shelf waters off C ape C a naveral which are effected by larg e intrusions can ave r age as much as 1.9 g C arbo n m -2 d -1 for a t le ast 40 days during the summer (Yoder e t a l 19R5). Sinc e the arriva l of colder w a ter brings with it the nutrient s ne cessary for abundant food production, it i s probable that the correlation between concentration and l ocation of calico scallop bed s and where the intru s ion of nutrient-rich water is s tronge s t i s s i g nific a nt. Further work by Lee et al ( 1984 ) indicate s that the Florida Current impart s a northward momentum to the SAB shelf water with a net onshore flow in the southern part of the bight where the s helf be g ins to w id en just so uth of Cape Can a veral. The ga ined momentum is rele ase d by offshore momentum at 30 N. The area with a net onshore flow of nutrient-rich w a ter thu s correspond s to the same area that has traditionally contained th e mos t productive cal ico s callop beds. 22

PAGE 37

Diseases in Bivalves Linked to Protistans Mass mortalitie s in sca llops are a widespread phenomenon but can be the result of many causative agents including temperature extremes, s torm s, predator outbreaks, oxygen depletion, algal blooms, diseases, and other factors (Orensanz et al, 1991a). I suggest that the mass mortalitie s observed during the course of thi s study are due to a disease Although several diseases have been identified in bivalves other unknown disease s may also occur. Much of the lack of information i s s impl y due to the fact that few people have done any re sea rch in this area. Nonetheless, there are some protistan disea ses that have been identified and in some cases have received considerable attention. The most comprehensive summary of the Protozoa pathogens of bivalves i s found in Sindermann ( 1 990). Mos t of the work to date h as been on the Pro tozoa pathogens of oysters of which 6 primary p at ho gen ic species have been i dentified and s tudied while 3 additiona l spec ies have been identified in which the pathology i s le ss clear T h e most extensively s tudied i s MSX disease which i s caused by Haplosporidium nelsoni and h as re s ulted in m ass mortalitie s of the eastern oyster s ince 1957 (Haskin e t al, 1966; Haskin and Andrews, 1988). The r e l a ted oyster pathogen H. costale was a l so identified over 30 years ago and is res pon s ibl e for considerable mortalities in the eastern oyster (Wood and Andrew s, 196 2; Andrew s eta/, 196 2). The third Protozoa pathogen found in the eastern oyster is P erkinsus marinus which i s re s ponsible for "Dermo" disease. Thi s di sease was identified eve n earlier and was th e dominant patho ge n id e ntified in oysters linked to mort alities in the Gulf of 23

PAGE 38

Mexico and Chesapeake Bay pri o r to the di scovery of H. nels oni (Mackin et al 1950; Andrews, 1 988). A related s p ec ie s, P. karlssoni, was recently identified in the bay scallop and whi l e no tra ns mi ss i on o f th is para s it e to other species h as been observed they hav e prov e n to be very pers i ste nt in t h e bay scallop (McGiaddery e t al 1991 ; 1993). Other oysters are a l so pron e to protozoan p a thogens. The European flat oyster, Ostrea eduli s, i s the id e ntified ho s t for a t l east two pathogens resu l ting in mass mortaliti es. Bonamias i s i s the n ame given t o a disease caused by the pathogen Bonamia ostreae Thi s pathogen h as been cited as re s pon s ible for mas s mortaliti es which have quickl y sp read to mo s t of the oyste r b eds i n Europe (Comps et al 1 980, Brehelin e t a l 1982; Grizel et al, 1 988). Th e final major g roup of pathogen s link e d to mass mortalities in oyst e r s a re members of th e ge nu s M a rt e ili a. First to be identified was M. refring ens which ha s been lin ked t o Abe r dis ease in 0 ed uli s on th e Frenc h coas t s ince the l ate 1960' s (Comps, 1970 ; Fi g u e r as and Montes, 19 88). Two additional s pecies M sydn eyi and M. len gehi are pathog e n s of Saccostrea commercia lis from Au s tralia and S. c ucullata from t h e Pers i a n Gulf, respect i vely (Wo lf 1972; Comps, 1 976; Fi g u eras a nd Montes, 1 988). Several a dditi o nal protozoa n pat h ogens have been reported but do n o t appear to be res p o n s ibl e for the s tron g p at h o l ogy not e d in th e preceding examples. These pathogens in c lud e H exami ta nelsoni, Nemat opsis os tr e arum, Steinhausia ovicola and some a dditi o n al s p ecies of H aplospo ridiwn (S pra g u e a n d Orr 1 9 55 ; Sch li cht and Mackin 1968; Lege r and H olla n de, 191 7; Si n de r mann, 1990). Additional Proti s t patho ge n s hav e b ee n r epo rt e d in mu sse l s, cl a ms, and scallops. Reported patho ge n s of va r ying v irul e nce in mu sse l s in c lud e H tum efac ientis, M. maurini 24

PAGE 39

an d S mytilovum (Tay l or, 1966 ; Auffret and Poder, 1 985 ; Sprague, 1 965; Sindermann 1 990) In the clams pathogens include H yalokloss i a pelseneeri an d Pseudoklossia sp. (Leger, 1 897; Morad o eta!, 1984 ; Spark s 1 98 5). Members of the ge nu s P seudok l ossia have a l so be en reported as pathogens of the scallo p s Pecten m axi mu s an d Argopecten irradi ans a nd may re s ult in morta l ities (Karlsso n 1991 ; C aw th o rn et al, 199 1 ; McGladdery et a l 19 93) Coccidia can establish heavy kidn ey inf ections in th e bay scallop a nd m ay result in k i dney dysfunction or tiss ue destru ct i o n (Leibov it z e ta!, 1 984; Karl sso n 1991 ) Several bacterial di seases have appeared in sc allop s Both ch l amy d ia an d rickettsia have been identified in bay and sea scallops a nd m ay in some instances lead t o morta lit y (Ge tchell 1991 ; Karl sso n 1 991; Gulka eta! 1983). The ex tent to which di seases are responsible for mass mortalitie s as opposed to o th e r po ss ibl e causes of natural mortalit y are by and large unknown for bivalves in ge n e r a l and scallops i n particular. 25

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CHAPTER 3. REPRODUCTIVE ECOLOGY OF ARGOPECTEN GIBBUS Introduction Marine inve r tebr ates display a va riet y of different repr od uctive cycles which can be either continuous or rh ythmic. Di fferen t types of rhythmic reproductive p at tern s in c lude bienni a l a nnu a l semiannu a l monthl y or even week ly but in all cases r ep rodu c tion follows a distinct se rie s of s tep s which compose th e gametog e nic cycle Virtually all of the scallo p s p ec ie s st udi ed to d a te exhi bit some type of rhythmic r ep rodu c tiv e cycle. Thu s, after gametes are r e l eased, there i s a period of rel a tiv e reproductive in ac tivit y while th e a nim a l s acc umul a t e e ner gy r ese rv es a nd gonadal tiss ue i s rege n e rat e d Because o f thi s, most of th e animal s in a g iven popul a tion will s p aw n s ynchronou s l y b ase d upon the local environmental condition s (G i ese 1959 ; Giese and Pearse, 1 97 4 ; Sastry, 1 9 7 9; Barbe r an d Blake 1991) Most s c allop s pecies seem to have eit h er a n a nnual or sem ia nnu a l reprodu ctive cy cl e. In s pecies which ex t end over a w ide latitudinal range th e r eproductive cycle seem s t o become s horter (Pearse, 1 968; G ease and Pear se, 1 974) Thu s tr opica l spec ies a r e more lik e ly to ex hibit a se mi a nnu a l cyc l e while tem p era t e s p ecies m ore often exhibit an a nnu a l cycle. Exceptions t o thi s g u i d eli ne are, however plentiful. The sea scallo p h as an 26

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annual reproductive cycle in northern latitude s but often shows a semiannual cycle in southern latitudes (Dupaul et a l, 1989; Barber and Blake 1991 ). On the other hand the bay scallop which, while overlapping the di s tribution of the sea scallop in part, also inhabits lower latitudes ex hibit s an annual reproductive cycle throughout its range (Barber and Blake 1991 ). The timing of the annual spawn in the bay scallop does get progressively later with decrea s ing latitude (Barber and Blake 1983). Only limited work has been done on reproduction in the calico scallop. Work in North Carolina has indicated t h at in those waters the calico scallop only spawns once annually in the spring (Kirby-Smith, 1970 ; Singhas 1992 ) In Florida, researchers have also found a spawning event in the spr ing (Allen a nd Costello, 1972; Allen, 1979; Costello et al, 1973; Miller et al, 1979 ; Miller et al 1981; Peters, 1978; Roe et al, 1971 ). All of the work upon calico scallop reproduction in Florida has utilized a large population located off of Cape Canaveral. All of the Florida researchers noted above used color changes as indicator s of reproductive state which is not as accurate as body tissue indices or hi s tological analysis (Giese, 1959 ; Giese and Pe arse, 1974; Barber and Blake, 1991 ). Using body-component indices a seco nd s pawning period in September has been noted in the Cape Canaveral population of calico scallops (Porter and Schwartz, 1976). The purpose of this study i s a detailed analysis of the reproductive cycle of the Florida population of the calico scallop Both body-component indices and hi s tological analyses will be examined in order to prepare a detailed, nons ubjective description of the reproductive cycle. This quantitative information i s reported over a multi-year period in order to insure that t e mporally variable characteristics are noted and quantified. 27

PAGE 42

Materials and Methods All samples were collected either from research vessels or commercial vessels The commercial vessels were typically 'Gulf style' shrimp trawlers modified for sca lloping by installing shorter boom arms. The commercial vessels typically fish two modified shrimp or otter trawls one from each side while the research ves s els typically tow a single trawl from th e s tern (Blake and Moyer, 1 99 1 ). All scallop samples and field measurements of physical parameter s were collected on the eastern coast of Florida off Cape Canaveral in an area extending from 27 42' N to 29 20' N at depths which ranged from approximately 17 to 72 meters All of the 12 I field scallop samples used in this study were located within the polygon s hown in Figure 2 except for the 5 samples indicated. The majority of the samples were collected in depths ranging from 35 to 55 meters. When possible each samp l e consisted of 20 scallops for determination of body component indices and between 16 and 20 scallops for histological analysis. Occasionally only analysis of body-component in dices was conducted and occasionally only histological analysis was conducted. In addition there were times when it was not possible to obtain enough scallops to provide 1 6 to 20 sca llops for each of the two analyses. When more than one disti nct size cla ss was present, indicating s callops of differing ages, the sample was either s ubsampled based on size or, if additiona l scallops from th at loc ation and date were available, comp l ete samples were analyzed for each size class. 28

PAGE 43

Figure 2 Area off the eas tern coa s t of Florida in which samples were collected for thi s s tudy All s ample s were collected from within the polygon except for five sample s which are marked with a e on the figure Thermographs were deployed at three s ite s within the study area during 1990 and 1991 which have been marked with the letters N, C, and S

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80 30' ,-----, I I I '' I I ', I I I I I I I I I I I I I I I \ I \ \ \ I I I \ I \ \ I I I I I I I I I I I I I I I I I \ I I I \ I ' ' \ \ ' I I \ I I I I I ' \ \ I \ I \ I \ I I I \ I \ I \ I \ 30 ' ' \ ' ' \ I '. ... -___ I

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Upon return to t h e laboratory the shell height and length of each scallop was measured u sing vernier calipers acc urate to 0.1 mm. Shell height is a straight line measurement extending from the umbo to the ventral edge of the s hell, while shell length i s the maximum s traight lin e measurement from the posterior to the anterior edge of the scallop shell. Vi s ual observations were recorded with regard to all body components. The scallops utilized fo r body-component indice s were dissected into their component parts (go nad s, digestive diverticulum and kidneys, adductor muscle, and the remaining mantle and gills). Each component was placed into a tared aluminum weighing pan and the weight of th e tissue determined to 0.1 mg u sing a Mettler balance. Weights were calculated for each component both before and after the tissue had been dried at 60 C for 7 day s for comparison of wet and dry weights and indices. Mean bodycomponent weights and body-component ind exes for each sample, defined as Index = (weight of body-component I weight of whole a nimal [tissue only]) X 100 were calculated u sing both the wet and dry ti ssue weights (Giese, 1959; Sas try and Blake, 19 7 1 ; Barber and Blake, 1983). A total of 2,024 scallops collected in the field were analyzed for thi s portion of the s tudy Reproductive development was also monitored hi sto logically. After the physical measurements and v i s ual observations already noted were completed, scallops analyzed histologically were removed from their s hell s and placed in Helly' s fixative made with zinc chloride (Barszcz and Yevich, 1975) After 2-3 hours they were bi sected sagittally and returned to the fixative for a tota l fixation time of 20 hours Scallop halves were then trimmed of excess tis sue, placed into cassettes and rinsed for 1624 hours in a self31

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siphoning water bath. The tissues were processed through 6 changes of Tissue Dry dehydrant (2 hours each), 3 changes of UC-670 clearing agent (I hour each), and 2 changes of liquid paraplas t (MP 56 C) (I hour each) in an Autotechnicon -Duo (Technicon In s truments Corporation) The processed tissues were then embedded in Paraplast, and sectioned (5 f1m) using a Spencer 820 rotary microtome. The resulting slides were then stained with hematoxylin and eosin, using standard procedures (Luna, I 968; Yevich and Barszcz, 1977). Occasionally slides were stained with Cason's Trichrome Stain instead of hematoxylin and eosin as outlined in Chapter 6 (Cason, I 950). At least two slides were made from each scallop processed. For this portion of the study a total of 1 ,851 scallops collected in th e f i e ld were processed. The finished s lides were examined and photographed u sing a Zeiss Photomicroscope III, during which the gametogenic stage of each scallop was determined (Sastry, 1966 a; Blake, I 972). The gametogenic stage was also monitored by measuring 50 oocytes from each animal using an image analysis system (Boeckler VIA-170) attached to the Zeiss photomicroscope (Barber and Blake, 1981; 1983 ; Hesselman et al, 1989). Only oocytes of roughly s pherical s hape in which a nucleu s was observed were utilized. The outlines of th e 50 selected oocytes from each animal were traced using the image analysis system and the s iz e of the enclosed area was then automatically calculated and downloaded to the computer. The calculated areas have been converted to nominal diameter values (the diameter of a perfect circle with the same area) (Mann et al, 1994 ) The conversion to nominal diameters i s solely for ease of comparison to other studies and it should not be construed that these represent the actual diameter of the eggs. Most of 32

PAGE 47

the prev iou s hi s tolo gica l st ud ies of repr oductio n in bivalves ha ve reported the maximum diameter obse rved using the cri t eria for oocyte selecti o n previou s l y delineated (Barber and Bl ake, 1991 ). The mean value for eac h a n ima l ( 50 oocytes) w i thin a sample w as calc ulat e d a nd those re sult in g mean s were th e n used to give an overall me a n oocyte di a m e ter for eac h sa m p l e o r s ub sample. T empe rature measurements of the b ottom temperature durin g the s tudy were condu c ted in two mann e rs. Wh en possib l e Ex p e nd a ble Bathythermograph s (XBTs) were deployed during samp lin g trips or sepa r ate trips for that purpo se. The XBTs h ave a s ensitivi ty of 0.01 a centigrade an d measure the temperature every 0.6 m e ter s from the s u rface to t he bottom of the water column. In addit i on 6 thermograph s f r o m Ryan Instrum e nt s were deploy e d at three s it es (2 per si t e) in the scallop collection area. The l ocatio n s of these site s is i n dicat e d o n Figure 2 The thermograph s, which have a rang e of 5 to 30 C a nd a sen s itivit y of 0.1 o C were deployed from Septemb er, 1990 until October, 1 99 1 and rec orde d the bot tom tempera tu re on c e every two h o urs. After October, 1 991 the remaining thermographs were used for samplin g th e bottom temp e rature during sampl e collect i ons instead of XBTs. Results The digestive diverticulum, mantle and ad ductor m u scle dry weights are all highly corr e l ated with both shell height and s h ell length with value s for the Pe arso n Produ c t Moment Correlation of greater t han 0.72 in all cases (p < 0.001 ). This is not unexpected 33

PAGE 48

since a larger ani mal contains more tissue and is indicative of somatic growth. The wet weights for the above components s how an even higher level of correlation with shell height and length (0.83, 0 90, 0.8I, p < 0.00 I). The gonad dry weight, on the other hand which is more indicative of gametogenic growth does not show correlation values as high as those seen in the other body components While still s howing a s ignificant correlation of 0 .52 (p < 0 .00 I), the gonad weight shows variations in d ependen t of purely so m a tic growth. More useful for thi s stu dy are the body-component indice s data. The gonad index data show a cyclical pattern of increase prior to spawning and a decre ase after spawning. The value of th e mean gonad index for the samples collected from 1983 through the sp ring of 1991 v aries within a range of I .6 to 28 2 % For those years in which data are complete there i s a peak in the gonad index in the spring of each year ( Figure 3). The exact timing of this event varies from one year to the next but generally falls between Februar y and May indicating that the spring s pawn commences in March. In a ddition there are seco ndary peaks in evidence in some of the years, these second spawning event s are particularly noticeable in 1986 and I988. There is no e vidence of a seco nd peak in 1984 or 1 987 In other years either there are gaps in collection which preclude determination of the timing of spa wning events durin g parts of those years based upon the gonad index d ata ( 1985, 1990) or there are differences within the population indicating th a t only some of the sca llops may be participating in a second s p awn. When either additional sample s on a d ate or a s ubset of s callops within a sample 34

PAGE 49

1984 1985 1986 1987 1988 1989 1990 DATE re 3. Mean gonad indices including standard deviation based upon dry tissue weights for field samples collected from 198 to 1991. When multiple s amples had different values the sample with fewer indi victuals was designated 2. When tht were difference s within samples they were divided into 1, 18, and 1C in decreasing order of prevalence.

PAGE 50

had gonad index values significantly different from the majority of scallops within a sample those scallops have been identified separately in Figure 3. There is no evidence of a discernible pattern in the digestive diverticulum index for the same period (Figure 4). There are some small decreases in the digestive index occasionally appearing coincident with large decreases in the gonad index in both 1984 and 1986. There is, however, no significant correlation between the two parameters By and large there is no similarity between the two patterns. Although the values observed for the mean digestive index range from 5.7 to 19.0% the majority of values are concentrated between 10 and 15 percent and the standard deviation for a sample is often far greater than the change in the mean value for successive samples. The values for the mean mantle index varies greatly with values ranging from 17.9 to 50.0% (Figure 5). Like the digestive diverticulum, however there is no correlation between the mean mantle and gonad indexes. The changing values for the mantle index do not seem to follow a pattern from one year to the next. There is a highly significant negative correlation of -0.70 (p < 0.001) between the mean gonad index and the mean adductor muscle index. The values for the adductor muscle index range from 25.4 to 64.8 % (Figure 6). Decreases in the adductor muscle index correspond with increases in the gonad index and increases in the adductor muscle index correspond with decrease s in the gonad index. The correlation is not perfect as some of the peaks observed in the gonad index are not observed in the adductor muscle index. The increases in the adductor mu s cle index are also not quite as sharp as the decrease s in the gonad index The initial incre a se in the adductor muscle index 36

PAGE 51

30 25 ....-... ::R 0 >< w 0 20 z ::J .....J ::J fj. u 15 f-0::: w > 0 w 10 > f-U) w <..9 5 0 1984 1985 fj. 1986 1987 DATE 1988 ---1 1989 2 fj. 18 0 1C I 1990 re 4. Mean digestive diverticu l um indices inc lu ding standard deviation based upon dry tissue weights for fie ld sa mpl es collected from 1983 to 1 991. When multiple samp l es had different values the samp l e with fewer individua l s was designated 2. When th e r e were differences within samp l es they were divided into 1 lB, and lC in decreasing order< preva l ence.

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65 60 55 50 >< 45 w 0 I I z w 40 I ....J 1z 35 -< t:.. 30 25 20 15 1984 I I I t:.. I I I I I I I i I I I : I I 1985 1986 1987 DATE 1988 ------1 2 t:.. 18 0 1C I 1989 4 I I I 1990 e 5 Mean mantle indice s includin g stand a rd deviation b ased up o n dry tiss u e we ight s for f ield s ampl es colle cted f rom to 1 991. Wh e n multipl e sa mpl es h ad diff ere nt valu es th e sampl e with fewer indi vi du a l s was d esig nated 2. Wh en th e : w ere differences within samp l es th ey w e r e di v ided into 1 lB, a nd lC in d ec r eas in g order o f preval e n ce.

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75 70 __._ 1 + 2 A 18 o 1 C J 65 ..--.. 0 ...._, 60 >< w 0 z 55 w _J u 50 (/) :::> 0:.:: 45 0 1u 40 :::> 0 0 <{ 35 30 25 1984 1986 re 6. Mean add u ctor m u scle ind i ces in cl ud in g sta n dard deviatio n b ase d upon dry t i ssue weigh t s for fie ld samp l es co ll ectec from 1983 t o 1 99 1 Whe n mult i ple samp l es had different val u es t h e sam p l e wit h fewer i nd i v idual s was designa t ed 2 W h en t h ere were d i fferences wit h i n samp l es th ey wer e divi ded into 1, lB, and l C i n decreasing order of preva l e n ce.

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corresponds to the initial decrease in the gonad index which occurs during spawning. The loss of tissue mass in the form of eggs and sperm results in the remaining adductor muscle constituting a larger portion of the remaining tissue. If that were the only factor, however, a similar increase of lesser magnitude would be expected in the digestive diverticulum and mantle indexes. There is a negative correlation between the gonad and mantle indexes of -0.21 possibly indicating this decrease, but it is not significant, and the mantle index itself has a highly significant negative correlation of -0.43 (p < 0.001) with the adductor muscle index. The digestive diverticulum index has a nonsignificant correlation of 0.15 with the gonad index and -0.19 with the adductor muscle so it shows no evidence of a relationship between loss of gonadal material and a concomitant increase in the digestive diverticulum index. The adductor muscle index continues to increase after the gonad index has stopped decreasing indicating that factors other than a simple loss of gonadal material are involved. The histological data support the information obtained from the body-component data with regard to the number and timing of spawning events. Figure 7 shows the mean oocyte diameter for scallop samples collected from 1983 through April, 1991. The mean values obtained for each individual scallop were averaged to calculate an overall mean for each sample. When two distinctly different groups of scallops were present on a particular date the mean value for the two groups is reported with the group containing fewer individuals placed in the second group. Oocytes increased in size from 5 to as much as 40 11m in diameter as oogonia developed into mature oocytes. Spawning events can be seen as sharp decreases in the mean oocyte diameter as mature oocytes are 40

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45 40 35 ........... C/) c 0 30 ...... u E ...._..., 0:::: 25 w Iw +>-::2: 20 ........ <( 0 w 15 I->-u 0 0 10 5 0 Figure 7. 1984 1985 1986 1987 1988 1989 1990 DATE Mean oocyte diameters including standard deviation for field samples collected from 1983 to 1991. When there were differences within the samples they were divided into 1 and 2 in decreasing order of prevalence. The fitted line represents a bicubic spline smoothing of the means from group 1.

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released and compose a smaller proportion of the remaining oocytes. Spring spawning events can be seen in each year from 1984 through 1990 except for 1989. A lack of scallop samples during the first half of 1989 precludes identification of a spring spawning event in that year. A second spawn in the fall or summer of the year can be identified in each year from 1986 through 1988 with the possibility of a second spawn in both 1985 and 1990 as well. In 1984 there clearly was only one spawn. At least one spawn occurred in 1989 but the exact timing of that spawn can't be determined due to the low numbers of scallops available for sampling during that year (see Chapter 6). The exact timing of the spawns seen from the above data varies from year to year. As was noted with the gonad index the first spawn generally occurs between March and May while the second spawn, when present, occurs between July and November. The second spawn is more variable in the timing of its occurrence. This can be seen more clearly by looking at the distribution of the mean oocyte diameters for each individual scallop and sample collected from the field throughout the study (1983 to 1994) and plotted against the time of year (Figure 8). This graph also includes the mean oocyte diameter of all animals collected within each month with the standard deviation. Statistical examination using analysis of variance indicates that the changes in the mean oocyte diameter are highly significant (p
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45 40 l .. .. : I 3l. .. : 11 : : i (J) ... c: . 30 . E .._.. .. 0::: .. 25 .. w : p ,. : I-' !: w : 20 <( u,) 0 ... : w 15 I-r 0 0 0 10 5 0 j F : [ . .-----...: I' : : . I M A II l A: :i' :;: .&. : ... i M ' I ; . 1 : ": : :.1 . ; .... : I : j j MONTH :.i. I !. : ; .... A , : .i s J 1 .&. 2 J ... : ... . 0 N IIi I . 1 .. ' f D Figure 8 Mean oocyte diameters for field samples collected from 1983 to 1994 plotted against time of year. The small circles are the mean value s for the individual scallops within the samples. The means for the samples are represented by 1 and when there were differences within the samples 2 .A. in decreasing order of prevalence. The mean oocyte diameter including standard deviation for all field samples collected during the study by month e has also been plotted and includes a fitted line which represents a bicubic spline smoothing of these values

PAGE 58

Oocyte development commences during late December or January and continues on into March when virtually all of the scallops contain mature oocytes with a mean diameter of greater than 30 to 35 !Jm. Thi s graph indicates evidence of spawning as early as March but more often the scallops remain ripe until April or May when the major spawn of the year generally takes place By June the sca llops are usually spent or redeveloping oocytes. The seco nd s pawn of the year, when it occurs, can take place at any time between July and December. An early spring spawn s eems to re s ult in an early second spawn. The second spawn is most often see n in either July or between September a nd November. Accept for years in which a seco nd spawn was noted in July, scallops sa mpled in August contained only imm a ture oocytes and oogonia. When spawning was observed in July mature oocytes were observed as late as the first few days of August. A large peak is observed in the monthly means in the spring repre se nting the major spawn while a small peak can be noted in the fall which represents the fall spawn. Because the fall spawn is so variable from one year to the next it can not be clearly seen when the a nimal s collected in each month are averaged over an eleven year period. Figure 9 shows the mean gonad index values for all samples and subsamples collected between 1983 and 1994 plotted by time of year. In addition the mean gonad index for all animals collected in each month regardless of year has been plotted along with the standard deviation an d a fitted line. The s pring spawn can be clearly seen a lthough the precise timing i s var iable from year to year which re s ults in a flattening of the overall trend Upon spawning the gonad index drops rapidly over a short period of time due to the release of gametes. The mean oocyte diameter drops as well but by a 44

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............ 0 _. >< w 0 z 0 <( Vl z 0 <.9 Figure 9. 30 I I I 1 2 18 1C I I I 1:. ... 1:. 0 I I ----.____ MEAN I I I 25 t +: I I I I I : I I 1 .. I I I II I I, I I I I I I I I I I I II I ,. ... I I 20 1:. i I I 1:. I Ill A 1:. I 1:. I I I : I 0 I I I I I 1 1 ''II I I I I I I 1 1 l l j l I I I II 11 ,1111 I I I I I +.: 1111' I I If:. I I 1:. I I 15 I I I I I I I II I I ? I 4 1 111 i I I I I I I I 1111 I I I I I I I IIIII I I I I ... I I I I tt 'II' I 1:. 14. ... I I 10 I I It I I + I I I .. I I I I I I I I I i +t , I Jl I I I I I I I ,. l II I 1 111 AI II I I I' I II .. I I.:, t t.A .II 5": F 0 I I I .II I ... 1 -I : II I I I I I I JA. I I ... 't I I ' I ... I tt. 0 .. + j F M A M j j A s 0 N D MONTH Mean gonad indices including standard deviation ba s ed upon dry tis s ue weights for field samples collected from 1983 to 1994 plotted against time of year. When multiple s amples had different values the sample with fewer individuals was designated 2. When there were differences within samples they were divided into 1 lB, and lC in decreasing order of pre v alence. The mean gonad index including standard deviation for all scallops collected in the field throughout the study has also been plotted and includes a fitted line which represents a bicub i c spline smoothing of these v a lue s

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smaller amount as not all mature oocytes are typically released. The seco nd spawns are not as clearly defined due to increased temporal variability of the second spawn. Evidence of a seco nd spawn can be observed, however, in late July, and in the fall. Examination of the mean monthly gonad index values utili zing anlysis of variance to compare consecutive months indicates that the peaks obseved in both the spring (March and April) as well as the fall (October) are signif icant. The results of analysis of variance comparisons for consecutive months indicate that the changes oberved from December through August are highly sig nificant (p
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Thus the Florida calico scallop exhibits a semiann ual reproductive cycle with a major spawn in the spring between March and May and a sma ller spawn of le ss temporal stability in July or between September and November. This is notably different than the ann ual reproductive pattern found in th e North Carolina population (Kirby-Smith, 1970; Singhas, 1992). Singhas ( 1992) reported that the spr ing spawn in the North Carolina population occurred between November and April with spaw ning occurring over a protracted period January and April in 1990 and between November, 1990 and February, 1 99 1 th e following year. A failure of the North Carolina sca llop population in February, 1991 prevented additional sampling (Singhas, 1992). It is unknown whether this event is related to the mortality observed in the Florida population which is discussed in Chapter 6. In Florida the spring spawn occurs slightly later in the year, between M arch a nd May This is sim ilar to the latitudinal reproductive trend in timing of th e reproductive cycle noted in Argopecten irradi ans. In the bay scallop maximum gonada l development i s observed in June and July in Massachusetts, in August and September in North Carolina, and in l ate September and early October in Florida (Sastry 1966b; 1970a ; Barber and Blake, 1983; Barber, 1984). Whereas most of the Florida calico scallops spawn in the spring, only a portion of the population participates in the fall spaw n This may explain why earlier re searc her s often failed to note this minor spawning event. The histological evidence indicate s that some rip e individual s are present at a lmo st all times of the year. While spaw ning tends to be locally synch ron ous, individuals can spawn at just about any time during the year. This explains the year-round presence of some calico larvae in spat traps noted by Miller 47

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et al ( 1981 ). The mere presence of scallop spat is not sufficient in and of itself to predict when major spawning events have taken place The variability within samples which can be noted in conjunction with the second spawn in the graphs of both the oocyte diameter and the gonad index (Figures 8 and 9) indicate that not all of the scallops appear to participate in the seco nd spawn. This is particularly clear in the graph of mean oocyte diameter in which individual scallops s howing no evidence of maturation were always present with scallops maturing for the seco nd spawn In the spring, or main spawn, all of the scallops collected were undergoing gametogenic development. Early studies s ugge s ted that the spring spawni ng was possibly triggered by rising w a ter temperature s (Peters, 1978; Roe et al, 1971 ). Following the discovery of the pre se nce of cold-water intrusion s into this area in the spring (Leming, 1979) other researchers proposed that the colder waters triggered the spring s pawning (Allen, 1979 ; Miller et al, 1981 ). The bottom temperature a t the central thermograph location in the center of the scallop beds was recorded every two hours from September, 1990 until October, 1991 except for a 15 day gap in June, 1992 The daily averages for 1991 and the last three months of 1990 are p l otted in Figure 10. Although there will be variation from year to year this gives a pictur e of the temperature s typically experienced by the scallop population. Comparison of the data for gametogenic development expressed in Figures 3, 7, 8, and 9 with the representative temperature measurements from Figure 10 indicates that gametogenic development i s grea test in both the spring and fall when the water temperature is low Comparison of the data also indicates that s pawning occurs when the 48

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--u w a:: t-\0 w a.. :! w t-Figure 10. 27 24 21 181 15 . J F M A M J J A s 0 N D MONTH Mean daily temperatures coUected at the central thermograph site every two hours from Octobe r I 990 to October I 991 plotted agn.inst the time of year.

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temperature is normally increasing from values below 20 to 21 o C Miller et al ( 1981) suggested that temperatures below 22. 5 C were necessary for spawning The data presented here suggest that low temp e ratures indicated are perhaps more important for reproductive development than specifically for spawning. In this study, spawning has often been observed when temperatures were greater than 22.5 C. It is not possible from the data to determine if spawning, which happens over a relatively short time scale, is correlated with either temperature increase or decrease since significant changes in the water temperature with periodicities of I to 2 weeks are superimposed upon the longer periodicity trends and detailed temperature records were not available for most of the s tudy. The exact timing of spawning events is not generally known since sampling dates were generally a month apart except for research cruises. The cold-water intru s ion onto the shelf in July and August may be crucial for the second spawning event in the Florida calico scallop population, and cold-water intrusion onto the scallop grounds is most pronounced during this portion of the year (Leming and Mooers, 1981 ). Temperatures lower than 20 C in this region typically originate in the s ub s urface nutrient-rich Florida Current waters (Atkinson, 1985 ; Lee and Atkinson, 1983 ; Oey et al, 1987). Thi s upwellin g of water onto the shelf has been cited as the most important process controlling phytoplankton abundance on the continental shelf in this region (Yoder et al, 1983) In part because the Gulf Stream is farther off shore in North Carolina than the Florida Current i s off Cape Canaveral, less upwelling occurs in the shelf waters off North Carolina and nutrients are less often advected into shelf waters than in northern Florida (Atkinson et al 1982 ; Blanton et al, 1981 ). The decrease in 50

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bottom temperature observed in Florida during August does not occur in North Carolina (Atkinson, et al, 1983) The Gulf Stream is not as tightly coupled to the shelf break in the Carolina Cape as the Florida Current is off Cape Canaveral and hence chlorophyll concentrations are highest in the winter in North Carolina and highest in the summer off northern Florida (McClain et al, 1988) The presence of colder waters in the late summer months and resulting increases in phytoplankton production in the waters off Cape Canaveral may be part of the reason the Florida scallop population can spawn twice a year while the North Carolina population only spawns once a year in the winter to spring months. Gametogenic development in the fall generally occurred, however as the water temperature was rising following the influx of cold water and the highe s t prolonged bottom temperatures are observed between September and November. This does not mean that the colder waters or increased food supply resulting from those waters is not necessary for the scallops to proceed with the gametogenic cycle for the second spawn. The temperatures experienced by the Florida calico scallop population during reproductive development are si milar to the temperatures reported for the North Carolina bay scallop population (Sa s try, 1966a; 1970a). They are approximately go C colder than the temperature s for the Florida bay scallop population (Barber, 1984) This reinforces the observation that the calico sca llop in Florida inhabits an environment far different from the Florida bay sca llop. Temperature i s not the only factor important in influencing the reproductive cycle. Without the availability of s ufficient energy the scallop population is not able to undergo 51

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the costly process of reproduction. In the bay scallop the reproductive cycle is dependent upon both the temperature and the food supply as exogenous factors (Sastry, 1963; 1966a; 1968). These exogenous factors may result in changes in the stage of neurosecretion which are believed to serve as endogenous factors in regulation of the gametogenic cycle (Blake, 1972; Sastry, 1975; Blake and Sastry, 1979; Sastry, 1979; Barber and Blake, 1991 ) Studies on the reproductive energetics of the bay scallop have often considered the question of energy transfer from various body-components to the gonad. Negative correlations between the digestive diverticulum index and the gonad index have been noted in bay scallop populations from Massachusetts and North Carolina (Sastry, 1970a). A negative correlation between the gonad index and adductor index was also found in the North Carolina population (Sastry, 1979) Barber ( 1984) found no correlation between the gonad and digestive diverticulum indexes but a negative correlation between the gonad index and both adductor index and dry weight. In Massa chusetts bay scallops reproductive energy is being supplied by the digestive diverticulum (Sastry and Blake 1971). The adductor muscle in addition to the digestive diverticulum may provide reproductive energy to the bay scallop in North Carolina (Sastry, 1979). Barber (1984) concluded that the adductor muscle had an increasingly important role as an energy reservoir for reproduction in the bay scallop in Florida while the digestive diverticulum did not serve as an energy reservoir for this population. 52

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The current study on calico scallops, on the other hand, found a positive correlation, not statistically significant between the gonad index and the digestive diverticulum index A highly significant negative correlation was observed between the gonad index and adductor index but there was no correlation with either dry or wet weights. The adductor muscle remains relatively constant during reproductive development and then increases rapidly following spawning. This indicates that, under normal condition s sufficient energy is being consumed to meet the energetic costs of gametogenic growth and there is no need for the other tissues or organs to serve as an energy reservoir for reproduction It may also signify that during the period of rapid gametogenic growth there is little or no energy left over for somatic growth. After spawning, the energy that had been utilized for reproduction may be redirected towards somatic growth. Because of the me s h size used to collect the scallops in this study only a limited number of small scallops< 30 mm were normally captured. These small scallops were not routinely fully analyzed but were monitored. Periodically small scallops were collected which exhibited evidence of being in advanced stages of gametogenic maturation and samples of the s e scallops were analyzed. Histological analysis shows that scallops as small as 19 mm, presumably Jess than 2 months old, contained mature oocytes with a mean diameter of 36.5 7.3 f..im. Although it is unknown whether or not these scallops would be a ble to produce viable offspring it confirms the visual observations made by Miller et al ( 1979) that these young scallops can undergo gametogenesis. This was also observed by Singha s ( 1992) in calico scallops collected in North Carolina. The 53

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ability of calico scallops of< 3 months age to undergo gametogenesis has not been reported in other members of this genus. Attempts to induce 3 month old bay scallops to undergo gametogenic development were completely unsuccessful despite the application of various temperature and food regimes (Blake, 1972). Singhas ( 1992) reported mean oocyte diameters as high as 54 11m for ripe scallops collected in North Carolina. This value is considerably higher than the values observed for the calico scallop in Florida presented in this study in which ripe scallops had a mean oocyte diameter of no more than 40.5 jlm. There are two possible explanations for this difference. A trend of decreasing maximum mean oocyte diameters with decreasing latitude has been observed in the bay scallop with maximum values of 90 11m in Massachusetts, 70 11m in North Carolina, and 38 11m in Florida (Sastry 1970a; Barber, 1984). However in the bay scallop, the same researchers found that the maximum gonad index also decreased with latitude. Singhas (1992) found a maximum mean gonad index of 21.1 % which is less than the 25.8 % observed in this study. A second explanation is differences in the way the mean oocyte diameter was calculated in the two studies. Singhas (1992) measured the 3 largest oocytes found in each of 5 follicles while the first 50 oocytes which contained a nucleus were used in this study. The result is that a larger number of small oocytes are included in the means reported for this study. The maximum oocyte diameter observed by Singhas (1992) in calico scallops from North Carolina was 60.20 11m which is very similar to the maximum oocyte diameter of 61.85 11m found in field samples collected for this study from the waters off of Cape Canaveral. Oocytes of 50 to 60 11m were observed in ripe scallops 54

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analyzed for this study but the pre s ence of additional smaller oocytes lowered the overall mean oocyte diameter found in the s e s c a llops. Unlike the bay scallop no decrease in the maximum gonad index or oocyte diameter with decreasing latitude was observed in the calico scallop. 55

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CHAPTER 4. TEMPERATURE AND THE REPRODUCTIVE CYCLE Introduction In bivalve s the reproductive cycle i s genetically controlled but responds to changes in environmental factors (Sastry 1979). The two exogenous factors most often involved in the regulation of reproduction in scallops are temperature and food abundance Both are often cited as playing a role in the timing and extent of gametogenesis, fecundity, and spawni ng (Barber an d Blake, 1991 ) External factors often are expressed through endogenous factors such as neuro se cretion level s changing in respon se to changes in the external environment (Blake, 1972; Blake and Sastry, 1979). Temperat ure i s most often cited as influencing reproduction in scallops (Sastry, 1979). Thi s influ e nce can include the need for sp ecific minimum or maximum temp e rature s in order for s pecific stages of the reproductive cycle to commence or increased gametogenic output or maturation with changing temperature levels. It may also represent the pre se nce of high nutrient s leading to an increase in the food supply or differences in metabolic requirements. Gonadal differentiation in conjunction with minimal temperature s has been observed in Placopecten ma gellanicus, Patinopecten yessoensis and Chlamys varia among others ( Thompson 1977 ; Maru, 1976 ; Burnell, 56

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1983) In order for gametogenesi s to proceed a threshold temperature must often be attained and increased reproductive activity is typically associated with increasing temperature (MacDonald and Thompson, 1985b; Yamamoto, 1951; 1952; Wolff, 1988; Sastry 1966a; 1970a) In tho se spec ies present over a wide range of latitudes different populations may require different minimum threshold temperatures in order for continuation of the gametogenic cycle (Barber and Blake, 1983 ; 1991; Barber, 1984) The purpose of thi s stu dy i s to examine the role of temperature as an exogenous regulator of reproduction in the calico s callop The work to date on the calico scallop has been descriptive in nature and has not examined the regulatory mechanisms involved. Examination of the response of the gametogenic cycle to variations in the controlled temperature will allow determination of the role which environmental temperatures may play in the timing of reproductive proce sses in the Florida population of the calico sca llop. Materials and Methods Laboratory studies on the relationship between temperature and the reproductive cycle of the calico scallop were conducted in 1993 and 1994. Approximately 1500 scallops were collected on each of 5 occasions during this time period from commercial scallop trawlers and transported live b ack to the laboratory. Subsamples of 20 scallops each were analyzed for body-component indices and his tological condition respectively as 57

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described previously in Chapter 3. The remaining scallops were placed in experimental treatment s as described below. Twelve 115-liter glass tanks were constructed with dimensions designed to allow the placement of 3 tanks in each of 4 commercial chest freezers Sufficient space was allowed for a water jacket with at least 5 centimeters of space on all four sides and the bottom of each tank A pump in the water jacket insured that the water was in constant motion. A temperature controller was attached to each freezer with the temperature probe placed in the middle tank of the three tanks in each freezer. The controller allowed power to be switched between the freezer and water heaters as necessary in order to maintain the desired experimental temperatures of 15, 20, or 25 C. This system maintained the temperature within the tanks to within 0 1 o C of the desired temperature Within each freezer the three tanks received 300, 100 or 0 ml, respectively, of phytoplankton I scallop I day as a food source. This corresponds to 9 x 1 0 7 3 x 1 0 7 or 0 cells/scallop/day, respectively. These food levels correspond to high or abundant low, and no or starved food levels in subsequent discussion. The food was introduced into the system continuously using a peristaltic pump in which seawater was pumped out of the tanks at the same rate at which the concentrated phytoplankton s olution was pumped into the tanks The results of the varying food level s upon reproduction are detailed in Chapter 5. The seawater used in each tank was collected from the dock next to the laboratory and pumped through both s and and diatomaceous-earth filters to a 500-liter storage tank. The salinity of the seawater used in all experiment s was raised from the normal Bay 58

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salinity, which ranged from 20 to 30 ppt during the experiments, to 35 ppt using sea salts. Once the sa linity of the s eawater had been adjusted the water was sterilized by adding I ml of bleach to every 2 liters of seawater. After 24 hours, sufficient sodium thiosulfate was added to the seawater to neutralize the bleach. The seawater was not utilized for an additional 24 hours after the introduction of the sodi um thiosulfate at which point neither bleach nor sodium thiosulfate wa s present at detectable l evels Each tank contained a gravel filtration and aeration system. Water was fi lt ered through the gravel by suction created within tubes underlying th e gravel bed by air stones l ocated in tubes at each of the four corners. An additional plastic rack was placed in each tank in order to increa s e the area on which the scallops co uld be placed in order to guard against overcrowding Lids were placed on each tank to reduce evaporation and transfer of water between the tanks. The temperature within the tanks was recorded every 2 minutes and the salinity checked and adjusted if necessary twice each day. A total of 1 200 scallops were placed in the experimental tanks for each of the 5 trials (I 00 scallops I tank). The dates for each trial and experimental conditions are listed in Table 1. Twenty scallops were removed from each experimental tank at two week intervals from th e start of the experiment until either there were no longer any scallops l eft in a particular tank or 8 weeks had passed. The 20 scallops removed on sampling days from each tank ( or remaining scallops if l ess than 20) were divided into equal subsamples Animals from the first subsample from each tank were ana lyzed for body component we i ghts and indices while scallops from the second subsample were prepared histologically. The methods for these analyses is described in Chapter 3. 59

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Table 1. Trial# 2 3 4 5 Experimental trial dates and the treatments used for each of the trial s. Dates Temperature Food le vel #of Tanks 14 Feb 93 to 12 Apr 93 15 C 0 rnl/scallop /day 2 15 C 100 ml/scallop/day 2 15 C 300 rnl/ sca llop/da y 2 25 C 0 ml/scallop / d ay 2 25 C 100 ml/scallop/day 2 25 C 300 ml/scallop/day 2 16 Jul 93 to 13 Sep 93 15 C 0 ml/scallop/day 2 15 C 100 ml/scallop/day 2 15 C 300 mllscallop/day 2 20 C 0 ml/scallop/day 1 20 C 1 00 rnl/sca llop / day 1 20 C 3 00 rnl/scallop / day 1 25 C 0 ml /sca llop / day 1 25 C 100 ml /sca llop/day 1 25 C 300 ml/scallop/day 1 08 Feb 94 t o 05 Apr 94 15 C 0 ml/scallop/day 2 15 C 100 ml/sca llop / da y 2 15 C 300 ml/scallop/day 2 20 C 0 ml/ sca llop/day 20 C 100 mllscallop/day 1 20 C 300 ml/scallop/day 1 25 C 0 ml/scallop/day 1 25 C 100 ml/scal1op/day 1 25 C 300 ml /scal1op/ day 1 04 May 94 to 29 Jun 94 15 C 0 ml /scallop/day 2 15 C 100 ml /sca llop / day 2 15 C 300 m1/scallop/day 2 20 C 0 ml/scallop/day 20 C 100 ml/scal1op/day 20 C 300 ml/ s callop/day 25 C 0 ml/scallop/day 25 C 100 mllscallop/day I 25 C 3 00 m1/scallop/day 1 15 S ep 94 to I 1 Nov 94 15 C 0 ml/scallop/day 2 15 C 100 m l/scallop/ day 2 5 C 300 mll sca ll o p / d ay 2 20 C 0 ml/ s ca llop /day 1 20 C 100 m 1 /sca llop / day 1 20 C 300 ml/scal1op/day I 25 C 0 mllscallop/day 2 5 C I 00 ml/scallop/day 25 C 300 ml/scallop/day 60

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Trial number 1 consisted of two sets of three tanks at 25 C and two sets of three tanks at 15 C. High mortality in all of the tanks maintained at 25 C necessitated an adjustment in the later four trials. For a ll subsequent trials two sets of three tanks were maintained at 15 C, one set of three tanks at 20 C and one set of three tanks at 25 C. Trial number three was conducted at the same time of year as trial number one in order to examine what happens when the scallops are maintained at 20 C during that portion of the year as well as comparing the two different years during which sampling took place Results Over the course of the five trials body-component analysis was performed upon 2,017 calico scallops and histologic a l analysis on an additional 2, 037 scallops. An a lysis of the results obtained from the two sets of tanks maintained at 15 C showed no significant differences. The sca llops from the tanks maintained under identical experimental conditions was therefore pooled before further analysis. For this portion of the study scallops from the three different food levels at each temperature level were also combined prior to analysis. The mean shell height of the animals at the start of the five trials ranged from 44.9 to 49.4 mm with standard deviations of 1.9 to 3.4 mm. This indicate s that in all cases the sca llop s u sed were more than 6 months old at the start of each trial (Blake and Moyer 1991 ). The mean weights of eac h of the body components as well as the total body weight of the animal declined in all ca s es except those noted below This was true for 61

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both wet and dry weights. This was expected since one-third of the anima ls were receiving no food (0 cells/scallop/day) and only one-third were receiving abundant food (9 x 1 0 7 cells/scallop/day). The mean wet weight and standard deviation for each treatment for the gonad, digestive diverticulum, mantle, adductor muscle and total body are shown in Tables 2 3, 4 5, and 6 re s pectively. In all of the components except for the gonad the amount of weight l oss for each body component as well as the total body increased with increasing temperature. The mean adductor mu s cle wet weight declined at all temperatures, in all trials as measured from the start of each trial to the l as t sample collected at each temperature. This loss ranged from -13 to -69 % of the initial weight. An initial increase in the adductor muscle wet weight at I 5 C during the first two weeks of both trial three (February-April) and trial four (May-June) was negated by the end of each trial. The mean mantle weight declined in every case except for those scallops maintained at 15 C in trial five (SeptemberNovember) in which there was basically no change over the course of the trial. In trial three at 15 C and in trial four at both 15 and 20 C initial increases in the first 2 to 4 weeks were negated by the conclusion of that trial at 8 weeks. The amount of change in the mantle weight ranged from+ 1 to -36 % of the initial wet weight. The digestive dive r ticulum weight also dropped at all temperatures and trials except for trials one and three (both February April) when sca llop s were maintained at 15 C. In trial one the se scallops exhibited a I 4 % increase over the course of the 62

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Table 2. Mean ( 1 sd) wet weights (g) of the gonad body component of Argopecten gibbus for all treatments grouped by experimenta l temperature. Date and Treatment Gonad Date and Treatment Gonad ---------------------------------------------------------------------------------------------18 Feb 93 Field 0.457 .116 05 Apr94 15C 0.814 322 01 Mar 93 15 C 0.664 202 20 C 0.702 284 25 C 0.511 .138 04 May 94 Field 0.648 .153 15 Mar 93 15C 0.676 .303 18 May 94 !So c 0.995 .266 25 C 0.421 .235 20C 0.807 .217 29 Mar 93 l5C 0.593 .235 25C 0 729 .191 12 Apr 93 15 C 0.562 .249 01 Jun 94 l5C 0.900 231 14 Apr 93 Field 1.124 .114 20C 0 718 .210 16Jul93 Field 0.767 .209 25 C 0.577 .160 02 Aug 93 15 C 0.867 .222 15 Jun 94 15 C 0.718 .188 20 C 0.910 .243 20 C 0.657 271 25C 0.759 .217 25 C 0.428 .068 16 Aug 93 15 C 0 741 .203 29 Jun 94 15 C 0.728 .161 20C 0 773 .202 20C 0.599 .122 25 C 0.610 .112 15 Sep 94 Field 0.456 .148 30 Aug 93 15 C 0.720 .214 30 Sep 94 15C 0.495 .161 20 C 0.628 .222 20C 0.404 .11 0 13 Sep 93 15 C 0.660 248 25 C 0.396 .138 20 C 0.881 .000 14 Oct 94 l5 C 0.434 169 08 Feb 94 Fie l d 0.578 .159 20 C 0.365 .160 22 Feb 94 15 C 0.942 278 25C 0.421 .168 20C 0.785 .238 28 Oct 94 l5C 0.379 .099 25C 0. 725 236 20C 0.487 292 08 Mar 94 15 C 0.806 .294 25C 0.521 241 20 C 0.661 .203 11 Nov 94 l5 C 0.460 168 25 C 0 .6 79 .190 20 o c 0.492 203 22 Mar 94 l5C 0.787 .257 25C 0 553 .40 1 20 C 0.698 .355 23 N ov 94 Field 0.412 .087 25C 0.676 .324 63

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Table 3 Mean ( I s d) wet weights (g) of the digestive diverticulum body component of Argo pe c ten g ibbu s for all treatments grouped by experimenta l temperature. Date and Treatment Dig Diverticulum Date and Treatment Dig Divertic u lum ---------------------------------------------------------------------------------------------18 Feb 93 Fie ld 0 623 .129 05 Apr94 15 C 0.798 .210 01 Mar 93 15 C 0.746 0.207 20 C 0.670 .147 25 C 0.555 .084 04 May 94 Field 0 843 1 1 3 15 Mar 93 15 C 0.709 .202 18 May 94 15 C 0.910. 154 25 C 0.542 119 20 C 0.752 .136 29 Mar 93 15 C 0.645 0 1 86 25 C 0.722 .131 1 2 Apr 93 15 C 0.709 0.226 01 Ju n 94 15 C 0.814 .155 1 4 Ap r 93 Field 1.066 0 1 19 20 C 0.699 .1 09 16 J u l 93 Fie l d 1.022 0.220 25 C 0 625 .1 04 02 Aug 93 1 5 C 0.974 .178 15Jun94 1 5 C 0 749 .140 20 C 0.924 0.192 20 C 0 672 132 25 C 0 868 0.173 25 C 0.617 .137 16 Aug 93 l 5 C 0.904 .154 29 Ju n 94 15 C 0.784 140 20 C 0.865 0 .161 20 C 0 710 146 25 C 0.819 0 127 15 Sep 94 Field 0 .911 1 1 9 30 Aug 93 15 C 0 952 .197 30 Sep 94 15 C 0.858 .147 20 C 0.873 0. 1 67 20 C 0 804 .12 1 13 Sep 93 15 C 0.889 0.19 1 25 C 0 716 .119 20 o c 0.886 000 14 Oct 94 15 C 0.823 .149 08 Feb 94 Field 0 798 0.104 20 C 0.698 .11 2 22 Feb 94 1 5 o c 0 823 .146 25 C 0 684 111 20 C 0.728 .139 28 Oc t 94 l5 C 0.796 .134 25 C 0.634 099 20 C 0.704 .201 08 Mar 94 1 5 C 0.819 0 .195 25 C 0.679 .160 20 C 0 644 .129 1 1 Nov 94 1 5 o c 0.868 .195 25 C 0.607 .134 20 C 0.680 148 22 Mar 94 15 C 0.765 193 2SOC 0 673 .090 20 C 0.685 0.140 23 Nov 94 Fiel d 0.713 .100 25 C 0.709 0. 163 64

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Table 4 Mean ( 1 sd) wet weights (g) of the mantle body component of Argopecten gibbus for all treatments grouped by experimental temperature. Date and Treatment Mant le Date and Treatment Mantle ------------------------------------------------------------------------------------------------------18 Feb 83 Fie l d 4.169 803 05 Apr 94 15 C 3 770 .545 01 Mar 93 l5 C 4.195 781 20C 3.442 .478 25C 3 .2 68 649 04May 94 Field 3.925 .696 15 Mar 93 15C 4.202 .967 18 May 94 l5 C 4.356 .656 25C 2.666 588 20 C 4 274 .742 29 Mar 93 15C 3.887 .803 25 C 3.604 .562 12 Apr 93 15 C 3 944 .70 8 01 Jun 94 l5C 4.254 .612 14 Apr 93 Field 4.2 22 .61 0 20C 3.618 .427 16 Jul 93 Field 4 855 .696 25 C 3 .2 38 .528 02 Aug 93 15C 5.031 .834 15Jun94 15 C 3.976 631 20C 4.512 .749 20 C 3.543 .472 25 C 3.993 593 2SOC 3.057 .441 16 Aug 93 15 C 4 734 .737 29 Jun 94 l5 C 3 828 .566 20 C 4 533 783 20 C 3.481 .438 25C 4 .2 09 502 15 Sep 94 Field 4 153 .533 30 Aug 93 l5 C 4.814 0.923 30 Sep 84 15 C 4.168 .450 20C 4 387 .894 20C 3 930 .498 13 Sep 93 l5 C 4.496 .755 25 C 3.597 .487 20 C 4.435 000 14 Oct 94 l5 C 4.120 .432 08 Feb 94 Field 4 146 .4 8 4 20C 3.736 545 22 Feb 94 l5 C 4 026 .50 I 25 C 3.479 .490 20C 3.760 567 28 Oct 94 15C 3 992 .555 25C 3.342 .453 20C 3.582 531 08 Mar 94 l5 C 3.926 .551 25 C 3 520 575 20C 3 519 561 11 Nov 94 l5 C 4.194 .553 25 C 3.289 .468 20C 3.714 .545 22 Mar 94 15 C 3 679 .5 7 1 25 C 3.264 .312 20C 3.410 .482 23 Nov 94 Field 4 20 l .484 25C 3 .28 6 .483 65

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Table 5. Mean ( 1 sd) wet weights (g) of the adductor muscle body component of Argopecten gibbus for all t rea t ments grouped by experime n tal tempera t ure. Date and Treatment Adductor Muscle Date and Treatment Adductor M u scle --------------------------------------------------------------------------------------------------18 Feb 93 Field 2.630 .780 05 Apr 94 l5 C 1 .916 .539 01 Mar 93 l 5 C 2.428 .544 20 C 1.526 .416 25 C 1.474 .404 04 May 94 Field 2.128 .388 1 5 Mar 93 1 5 C 2.197 0 756 1 8 May 94 l5C 2 297 .432 25C 0.810 .343 20C 2 .103 505 29 Mar 93 l5 C 1.823 .578 25C I. 646 .400 12 Apr 93 15 C 1.821 .495 01 Jun 94 15 C 1.915 .445 14 Apr 93 Field 2 .2 85 565 20 C 1.513 272 1 6 Jul 93 Field 2.715 .641 25C 1.133 391 02 Aug 93 15 C 2.820 0.823 15 Jun 94 1 5 C 1.620 .399 20 C 2.446 .539 20 C 1.185 329 25 C 2.10 1 .616 25 C 0 955 340 16 Aug 93 15 C 2.582 0.7 1 1 29 Jun 94 15C 1.325 .354 20 C 2.30 I 0.639 20 C 1.140 .354 25 C 1.828 .424 15 Sep 94 Field 3.698 .583 30 Aug 93 15 C 2.472 .743 30 Sep 94 l 5 C 3.685 593 25C 2.029 .624 20 C 3.425 .578 13 Sep 93 15C 2.0 1 3 .626 25 C 3.1 90 .502 20 C 1 .289 0 000 14 Oct 94 15 C 3 575 .435 08 Feb 94 Fie l d 2 556 0.483 20C 3.286 .409 22 Feb 94 l 5 C 2.542 .38 5 25 C 2.855 428 20C 2.475 .455 28 Oct 94 l5 C 3.328 .5 1 6 25C 2.170 .41 1 20C 2.826 563 08 Mar 94 1 5 C 2 337 .498 25 C 2.46 7 .56 1 20 C 2.030 0.600 11 Nov 94 1 5C 3.233 .484 25 C 1 770 .409 20C 2.727 .529 22 Mar 94 15 C 2.11 0 0.502 25 C 1.918 .028 20 C 1 .703 0 384 23 Nov 94 F i eld 3.462 .398 2SOC 1 5 1 2 0.345 66

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Table 6. Mean ( 1 sd) total body wet weights (g) of Argopecten g i bbus for all treatments grouped by experimental temperature Date a n d Treatment Total Body Date and Treatment Tota l Body ---------------------------------------------------------------------------------------18 Feb 93 Fie l d 7 878 .683 05 Apr 94 15 C 7 298 .247 01 Mar 93 15 C 8.032 .557 20 C 6.339 .024 25 C 5 808 1.126 04 May 94 Field 7.544 1.124 15 Mar 93 15 C 7 784 .035 18 May 94 15 C 8 558 324 25 C 4.438 .125 20 C 7 936 .407 29 Mar 93 15 C 6.947 .558 25 C 6.701 .099 12 Apr 93 15 C 7.035 .491 01 Jun 94 15 C 7.882 1.149 14 Apr 93 Fie ld 8.697 .261 20 C 6.548 .736 16 Jul 93 Field 9.3 59 494 25 C 5 573 .973 02 Aug 93 15 C 9 692 .836 15 Jun 94 15 C 7 063 .144 20 C 8 791 1 373 20 C 6.058 .917 25 C 7 722 .257 25 C 5 057 .879 16 Aug 93 15C 8 960 .465 29 Jun 94 15 C 6.665 .993 20 C 8.472 .375 20 C 5 930 895 25 C 7.466 .907 15 Sep 94 Field 9.220 1.257 30 Aug 93 l5 C 8.959 .694 30 Sep 94 15 C 9.205 1.086 20 C 7.916 .574 20 C 8.562 .085 13Sep93 15 C 8.058 .478 25 C 7 899 .990 20 C 7.491 .000 14 Oct 94 15 C 8.953 .989 08 Feb 94 Field 8.078 .867 20 C 8.084 .964 22 Feb 94 15 C 8 333 .066 25 C 7.439 .872 20 C 7 7 48 1.1 77 28 Oct 94 15 C 8.497 033 25 C 6.872 0.913 20 C 7.599 .208 08 Mar 94 l5 C 7.888 1.249 25 C 7.187 .150 20 C 6.855 1 .291 11 Nov 94 l5 C 8 755 .127 2SOC 6.346 .988 20 C 7.613 1.048 22 Mar 94 l5 C 7.340 .255 25 C 6.409 .410 20 C 6.496 .058 23 Nov 94 Field 8.788 909 25 C 6 184 .083 67

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experiment while in trial three there was no change in the mean wet weight. Changes in the digestive diverticulum wet weight from the initial weight ranged from+ 14 to -27 %. The total body wet weight also decreased at all temperatures in all trials from start to conclusion of each trial. The total wet weight of scallops maintained at 15 C either increased or did not change (trial five) during the first two weeks but declined later in each trial. Some rebounding in the total weight occurred between weeks six and eight during some treatments (the same rebound was observed in digestive diverticulum and mantle tissue) but overall there was a net loss of tissue weight with values at the conclusion of each trial with a mean tissue loss of 5 to 44 % of the initial total body wet weight. The situation in the gonad is not as straight forward as it is in the other tissues The net change in the gonad weight during the trials for the various treatments ranged from +41 to -34% of the initial gonad wet weight. In trials one and three increases in the gonad weight were obs erved at all of the temperatures examined except for no change at 25 C in trial one. The amount of the gonadal tissue gain increased with decreasing temperature. In trials two (July-September) and four (May-June) there was generally a decrease in the gonad wet weight. In trial four initial increases at all temperatures were later reversed resulting in no net change in the gonad wet weight at 15 C and net losses at 20 and 25 C. In trial two increases in gonad wet weight at 15 and 20 C in the first two weeks were reversed in the remaining weeks and there was an overall decline in the gonad tissue weight at all three temperature s. The amount of gonadal tissue loss increased with increasing temperature. The gonad wet weight trends for trial five 68

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(September November) a re very different from what was observed in the other trials and body components. There was no change in the gonad wet weight at 15 C a slight increase was observed at 20 C and a greater increase was seen at 25 C. Unlike the other tissues and trials for trial five the rate of gonad tissue gain increased with increasing temperature. The gonad index based upon dry weights for scallops maintained at 15 C is shown in Figure 11. Between February and April there is an increase from initial values of 5% and 7 % to concluding values of 8% and 13% for scallops from 1993 and 1994 respectively Between May and July the gonad index again showed an increase from 8% to 13 % Scallops from July to September and September to November at 15 C exhibited no change in the gonad index which remained at approximate l y 6 % and 3 % respectively. Increases observed in the experimental animals during the early spring, in 1994, and in the fall, were greater than those observed in the field during the same period while scallops in the field in the early spring of 1993 exhibited increases in the gonad index greater than those obs erved in the experimental animals The mean nominal oocyte diameters for the scallops kept at 15 C are depicted in Figure 12 From February to Apri I 1993 oocyte diameters remained constant at a large size of approximately 32 !l m. In the same period of 1994 the initial oocyte diameter was smaller than the year before at a pproximately 23 11m and the oocytes of the experimental animal s reached 32 11m in 6 weeks and remained at that size. From May to the beginning of July the oocyte diameter of experimental animals steadily increased in size from initial values of 22 11m to 32 11m in average size. For scallops from July to September oocyte 69

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-....) 0 .......... 0 ......... >< w 0 z 0 <( z 0 (!) 24 l 20 16 12 8 4 0 Field 1993 + Field 1994 I D 15C 1993 15C 1994 I It I I I + ____ ,_ __ . A S J Figure 11. M J J F M A MONTH 0 N D Mean gonad indices including standard deviation based upon dry tis sue weights for experimental animals mainta i ned at 15 C plotted against time of yea r from 1993 0 and 1994 along with field measurements from 1993 Oand 1994 +

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38 ...-.. C/) 32 c 0 ,_ E ..__... 0::: 26 w 1-w <( 20 0 w !;:: 0 14 0 0 8 Figure 12. J + F M A M J J MONTH A s 0 0 Field 1993 + Field 1994 D 15C 1993 15C 1994 t N D Mean oocyte diameter including standard deviation for experimental animals maintained at 15 C plotted against time of year from 1993 0 and 1994 along with field measurements from 1993 O and 1994 +.

PAGE 86

diameters remained relatively constant at approximately 1211m although the standard deviation for these animals was rather large. For scallops maintained at 15 C between September and November the oocyte diameter increased from 6 to 14!lm but again there was a high variability among the experimental animals in this trial at this temperature. The increases in oocyte diameter in experimental animals at this temperature were identical to those observed in the field during the early spring of 1993 and larger than was observed in the field during the early spr ing and fall of 1994. The values for the dry gonad index of the four trials during which scallops were maintained at 20 Care shown in Figure 13. Between February and April the gonad index of the experimental animals s teadily increased from 7 to 14 %. Between May and the end of June the gonad index the experimental animals also increased from 8 to 12 % but appeared to level off between week 6 and week 8. Scallops maintained in the laboratory at this temperature between July and September exhibited a slight increase in the gonad index from 5 to 7 %. Finally, sca llop s maintained in the lab from September to November remained at the initial gonad index of approximately 2% for the first 4 weeks before increasing to mean values of roughly 6 %at the end of that trial. The increase in the gonad index was greater in the early spri ng and fall of 1994 for the experimental animals than was observed in the field during that time. The increase in the early spring of 1994 in the experimental animals at this temperature was comparable to the increases observed in the field at this time during 1993. Nominal oocyte diameters for animals held at 20 Care presented in Figure 14. The oocyte diameter between February and April followed the same pattern as the 72

PAGE 87

-..) UJ ......... 0 ........... >< w 20 16 0 12 z 0
PAGE 88

-...) ....-... (/) c 0 ...... u E ........... 0::: w I-w <( 0 w I->-() 0 0 38 32 26 20 14 8 ? + A 0 Field 1993 + Field 1994 A 20C 1993 A 20C 1994 t J F M A M J J A s 0 N D MONTH Figure 14. Mean oocyte diameter including standard deviation for experimental animals maintained at 20 C plotted against time of year from 1993 .6. and 1994 ,._ along with field measurements from 1993 0 and 1994 +.

PAGE 89

animals kept at 15 C. The animals increased within 6 weeks from an initial value of 23 to 32 11m and remained at that level. Between May and the end of June the oocyte diameter increased from 22 to 32 11m during the first 4 weeks and then remained at a large size with a slight decrease in the oocyte diameter at the end of June. Scallops increased slightly from an initial value of 12 11m in the middle of July to approximately 16 11m at the end of August. There was only one scallop available from the 20 C treatment during the sampling at 8 weeks for this trial. The oocyte diameters for animals at 20 C from September to November increased from 6 to 21 11m during the first 6 weeks and then remained constant. The standard deviation is quite large for some of these means. Again s callops in the field in the early spring and fall of 1994 did not exhibit increases in oocyte diameter comparable to those observed in the experimental animals but were similar in the early spring to field observations from 1993. Difficulty in maintaining s callops at 25 C resulted in a reduction of the number of scallops available for sampling throughout the five experimental trials. With the exception of the last trial high mortality normally resulted in the termination of this treatment after 4 to 6 week s a s opposed to 8 weeks In addition the number of individuals available for s ampling late in each trial was smaller than de s ired at this temperature. The values for the dry gonad index of the animals maintained at 25 Care displayed in Figure 15. Between February a nd late March the gonad index increased from approximately 6 to 13 % with slightly lower value s obs erved in 1993 relative to 1994. The gonad index increa s ed during May f rom 8 to 12 % and then decreased to 9 % by mid June. There wa s a s light increa s e in the gonad index from 5 % in mid July to 7 % 75

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24 20 16 f-........... "#. ......... >< w 0 12 -...) z f-0\ 0 <{ z 0 <.9 8 f4 0 J Figure 15. I I I I I I I I I I 0 Field 1993 Field 1994 o 25C 1993 25C 1994 t ... 4 : 4 0 l t ( t 4 .. .. .. ( 4 i + t : .. ,, .. ......... .. : : F M A M J J A s 0 N D MONTH Mean gonad indices including standard deviation based upon dry tissue weights for experimental animals maintained at 25 C plotted against time of year from 1993 0 and 1994 e along with field measurements from 1993 Oand 1994 +.

PAGE 91

in mid August. The gonad index remained relatively constant from mid September to mid October at 2 % with perhaps a slight increa s e and then increased steadily to approximately 10% with a very large standard deviation by the middle of November Increases in the gonad index in the early spring of both years were similar to each other and the field observations for this time period in 1993. The experimental animals in the fall again exhibited larger increase s in the gonad index than were observed in the field. Figure 16 depicts the nominal mean oocyte diameters for those scallops which were maintained at an experimental temperature of 2SO C. As with the other temperatures between February and the end of March the oocyte diameter in 1993 remained at a high value of approximately 32 11m. In 1994 the increase from the initial 23 11m was more rapid than at the other two temperatures with the scallops reaching 32 11m in size in less than 4 weeks and continuing to slowly increase to approximately 34 11m by 6 weeks at which time sampling had to be s u s pended at thi s temperature. During May the oocyte diameter increased very rapidly in the from 22 to 32 11m during the first 2 weeks remained at that level through the 4 week sample and then decreased to 26 11m with a large standard deviation by the 6 week sample in the middle of June. Again no further scallops at this temperature were available for sampling at the 8 week point of this trial The oocyte diameter increased from 1211m in the middle of July to 20 11m in the middle of August. Large standard deviations are apparent in the lab samples and sampling at this temperature was suspended aft e r 4 weeks due to a lack of available animals. There was a steady increa s e in the oocyte diameter from 6 lliTI in the middle of September to 28 11m by the end of October after which it rem a ined steady until the middle of November. This 77

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-....) 00 ____ ,_ __ 38 32 0 ...... (.) E 26 w 1w <{ 20 0 w (.) 14 0 0 8 t tt t ? r I + <> Field 1993 + Field 1994 o 25C 1993 25C 1994 4 + J Figure 16. F M A M J J A s 0 N D MONTH Mean oocyte diameter including standard deviation for experimental animals maintained at 25 C plotted against time of year from 1993 0 and 1994 e along with field measurements from 1993 Oand 1994 +.

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was the only trial in which sufficient s callops maintained at 25C survived long enough to enable collection of a sample at 8 weeks. The same comparisons between experimental and field animals observed in the gonad index at this temperature were present in the oocyte diameter data as well. Increases similar in the early spring to those found in the field in 1993 and larger increases in experimental animals in the fall. Discussion The data on body-component weights and indices in the field studies reported in Chapter 3 indicated that sufficient food was generally present for gametogenesis to take place without catabolizing tissue from other body components This was not the case in laboratory conditions when suboptimal diets in terms of either quantity or quality were supplied In this study one-third of the animals in the temperature experiment received no food and an additional one-third of the animals received a ration that may have been suboptimal at higher temperatures. Increases in the temperature result in an increase in the energy required for routine metabolism in marine mollusks (Bayne, 1975; Bayne and Newell, 1983). The increase in the rate of weight loss with increasing temperature in this study with the exception of gonadal tissue corresponds to the increased energy requirements of the scallops which would be expected due to increases in the metabolic rate associated with higher temperatures. The fact that the gonadal tissue did increase in weight in many of the treatments in this study despite decreases in other tissue indicates that unlike the 79

PAGE 94

results seen for scallops in the field, scallop maintained in the lab under at least some conditions were catabolizing non-gonadal tissue in order to support gametogenesis. In the bay sca llop, energy transfer from the digestive diverticulum and adductor muscle has been shown for the northern subspecies (Sastry, 1970a ; Sastry and Blake, 1971; Sastry, 1979). In the southern subspecies located in Florida, no relationship was found between the digestive diverticulum and the gonad, but the adductor muscle serves as an increasingly important energy reservoir with a two-thirds reduction in the adductor muscle weight noted during gametogenesis (Barber, 1984; Barber and Blake, 1981; 1985 ; 1986). Large decreases in the adductor muscle weight were also observed in many of the treatments used in these experiments and it may be that the calico scallop was utilizing energy s tored within the adductor muscle to fuel the gametogenic process when sufficient resources were not present. Another possibility is that the diet provided in the lab, which consisted of a single s pecies of phytoplankton (Tetraselmis suecica) may not have provided all of the nutrients required by the calico sca llop for gametogenesis resulting in the scallops catabolizing other ti ss ues to replace whatever was missing from their diet. The maximum oocyte diameter obtained in the laboratory among ripe scallops was usually approximately 5 le ss than had been observed in scallops at the same stage of gonadal development in the f ield (Chapter 3) Again this could be due to deficiencies within the experimental diet although the values observed within the lab did fall within the range observed for scallops in si milar reproductive states in the field. 80

PAGE 95

In the early spring of 1993 sca llops began the trial with very mature oocyte s, and these oocytes remained at a high s tate of reproductive development at both 15 and 25 C. In 1994 the oocytes qui ckly matured at all temp e ratur e but faster at 25 C than at the other two temperatures. In all cases during both years the gonad i nd ex increased throughout this period of the year, as much as doubling or more over the 8 week period. There are a few difference s b etwee n the different temperature s u sed. With increasin g temperature there is a slight inc rease in the gonad index but this i s probably an artifact of the increa se d Joss of non-gonadal tissue at higher temperatures. The gonadal weight increase i s ac tually largest in the a nimals maintained at 15 C. Not all of the tissue lost is the re s ult of the tran sfe rence of energy to meet the demand s of gametogenesis. A portion i s due to increa se d standard metabolism rate s at higher temperatures. The maximum oocyte diameter observed at this time of year was the same at all treatments but may have been a tt a ined s lightly fa s ter at higher temperature s in the early spring of 1994 .. In the period from May through June late spring, there are also some differences observed between the three temperatures u se d within this experiment. In all cases the oocytes present incre ase d in s ize to mature oocytes within the 30 to 40 micron ran ge. The rate at which the oocytes increased in diameter from the initial value of 22 11m to mature oocytes increa se d as the temperature was rai se d At the low temperature, oocytes continued to mature throu g hout the trial while sca llops at higher temperatures not only matured but began to decrease after reaching peak values consistent with mature oocytes. This decrea se after reaching maximal values was greatest at 25 C. The gonad index data also reflect a peak at 25 C, a plateau at 20 C a nd continuation of the increa se in the 81

PAGE 96

gonad index at 15 C. This is a s trong indication that some spawning occurred among the scallops maintained at 25 C and po ss ibly among those maintained at 20 C although the latter is questionable. Decreases in oocyte diameter and gonad index are classic indicators of spawning in scallops and are commonly cited as indicators of spawning in various scallop species (Barber and Blake, 1991). Some additional explanations for the decrease in oocyte diameter and gonad index would include vacuolization and lysis or resorption of oocytes and preferential catabolism of gonadal tissue or storage of energy in other body components. The former has be e n observed in bay scallops that were exposed to temperatures below a thre s hold required for further gametogenic development for prolonged periods of time (Sa s try 1966a ; 1968 ) Histological examination of scallops in thi s s tudy showed no evidence of oocyte lys i s or vacuoli z ation of the cytoplasm in any of the anim a ls S i nghas ( 1992) reported observations of atresis among unspent eggs at thi s time of the year for calico scallops from North Carolina but no evidence of atretic eggs was observed in any of the scallops examined in Florida at this or any other time of the year. All of the body components during this trial decreased in weight during the course of the experiment. That eliminate s the possibility of decrea s e s in the gonad index due to increases in storage of energy reserve s in non gonadal tis s ue but does support the idea that gonadal material wa s being catabolized in order to support other metabolic functions at the elevated temperature. If that the occurred there should have been histological evidence. This evidence was lacking and it would not account for the decreasing oocyte diameters. 82

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There are several reasons for the large standard deviations associated with scallops collected late in this treatment at higher temperatures. Only 19 scallops were available among the three tanks maintained at 25 C instead of the 60 scaJ!ops that would normaJ!y constitute the pooled sample for that temperature. Among those 19 scallops visual observations of the gonadal condition divided them into two different states (ripe and spent). This was reflected in slight differences in the gonad index and lar ge differences in the mean oocyte diameter where ripe scallops had a mean oocyte diameter of 31 to 32 J.lm whi le spent sca llops had an average oocyte diameter of 9 J.lm. Although no spawning was observed in any of the tanks during experimentation it is possible that limited s pawning in the scallops maintained at 25 C may have occurred and went unnoticed. The high mortalitie s experienced among the experimental animals being maintained at that temperature necessitated water changes on a regular basis. The small number of individuals present at that point in the tanks maintained at 25 C would also have increased the difficulty of detecting any spawning at that time. Scallops collected in the fie ld in July of 1993 were in a partially spent or spent condition, s pawning having begun in the field several weeks earlier. This is reflected in the s mall oocyte diameter s and reduced gonad index. The scal l ops maintained at 15 C remained in this state throughout the 8 week trial. There was no evidence of oocyte development or increases in the gonada l index. The actua l bottom water temperature during this period of the year is normally less than 20 C (Fig ur e 1 0) There were some mature oocytes present but the overall reproductive condition of the scallops did not change. With increasing temperature there were indications of gametogenic activity. 83

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Increased maturation of oocytes and an elevation in the gonad index was noted at both 20 C and at 25 C among so me but not all of the scallops exposed to higher temperatures with increases in the oocyte diameter particularly pronounced among some of the sca llops maintained at 25 C This is in agreement with the field data discu ss ed in Chapter 3 where spawns occurring l a ter in the year had a le ss distinct signal than the spring spawn in part due to incomplete participation in gametogenic maturation among the population Increa sed gametogenic maturation at higher temperatures i s most clearly seen in the final trial which was conducted from September to November when the bottom water temperature in the field is typically greater than 23 C. Again there was little evidence of development at 15 C. Minimal oocyte development was observed at this temperature and no change was observed in either the gonad index or the gonad weight. Increases in gonad weight, gonad index a nd oocyte diameter were observed at 20 and 25 C. In this tri a l changes in the gonad weight, gonad index and oocyte diameter were all positively correlated with temperature Increases in the gonad weight were also observed during the s pring gametogenic cycl e but during that time period the rate of change in the gonad weight was negatively correlated with increasing temperature. With regards to the effect of temperature alone upon the gametogenic cycle in the calico sca llop the spring and fall cycles are quite different. Development in the early spring is largely uncoupled from the environmental temperature with maturation occurring at all temperatures. Greater incre ases in the amount of gonadal material is observed at lower temperatures which indicates that while gonadal development proceeds at higher temper a tures, lower temperatures may result in an increase in the quantity of 84

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eggs and sperm developed by the scallops. Elevated temperatures may have slightly increased the rate of oocyte maturation and do appear to play an important role in spawning during the spring. The initiation of sp awning has often been linked to temperature. Both temperature increases and decreases have resulted in the initiation of spawning in various scallop species. Spawning associated with increasing temperature has been observed in Amusium balloti (Heald and Caputi, 1981 ), Chlamys opercularis (Ursin, 1956), Patinopect e n yessoensis (Wakui and Obara, 1967), Pecten alba (Sause et al 1987), and P. novaezelandiae (Bull, 1976). Decreasing temperature was linked to initiation of spawning in Hinnites multirugosus (Jacobsen, 1977) Other species which spawn more than once a year such as Pecten maximus and Placopecten magellanicus have been observed spawning in conjunction with both increasing and decreasing temperatures (Stanley, 1967; Naidu, 1970). Subspecies may also have differing responses to temperature with regards to the initiation of spawning. The northern subspecies of the bay scallop Argopecten irradians irradians spawns in conjunction with an increasing water temperature (Sa stry, 1970a; Taylor and Capuzzo, 1983 ; Bricelj et al, 1987a) while the southern subspecies A. i. concentricus s pawns when the water temperature is decreasing (Barber and Blake, 1981: 1983) Miller et al ( 1981) working with the calico scallop suggested that spawning occurred when the temperature dropped below 22.5 C while earlier work by Peters ( 1978) and Roe et al ( 1971) indicated that spawning was possibly triggered by rising temperature. This work supports the latter viewpoint that spawning is triggered by rising 85

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temperature in the calico scallop. The experimental evidence of spawning found in the trial conducted from May through June wa s only observed at higher temperatures and was most pronounced at the highe s t experimental temperature examined. The experimental results presented here as well as the temperature and reproductive cycle information from the field discussed in Chapter 3 all show a correlation of spawning with increasing temperature. In the late summer and fall the gametogenic cycle does appear to be partially coupled to the environmental temperature Maturation of oocytes commenced at high temperature s and did not occur at low temperatures Whether or not development would have occurred after a period of exposure to elevated temperatures or whether low temperatures prior to the start of the trial in the environment were necessary in order for the elevated temperatures to initiate maturation was not determined. It is possible that a regulatory process simi l ar to the switching mechanism put forth for the bay scallop i s a l so present in the calico sc allop with environmental temperature and or food conditions initiating or delaying growth of the oocytes (Sastry, 1968; 1970b ; Sastry and Blake 1971;Bl ake, 1972 ;BlakeandSastry, 1979 ;BarberandBlake, 1991) Furtherwork would be nece ss ary to determine if these switches are indeed present in the calico sca llop. Miller et al (1981) indicated that temperatures below 15 C and greater than 27 C may be fata l to the calico scallop. Based on the current study the lower l ethal limit is below 15 C. Calico sca llop s do quite well at 15 C for extended periods of time and have been maintained in the laboratory in temperatures as low as 10 C for s horter durations without any observed mortality. The upper l etha l limit on the other hand, is 86

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probably somewhat less than 27 C. Temperatures in the field rarely exceed 27 C and then only for short periods of time (Figure I 0). Temperatures above 25 C are also present for no more than 3 to 4 weeks at a time and high mortality was always observed during experiments conducted at this temperature after 4 to 6 weeks. The idea of lethal limits is somewhat deceptive anyway since the duration of exposure is often not of sufficient duration for long term detrimental effects to manifest themselves. Long term exposure may prove lethal at temperatures which an animal can tolerate for short periods of time. If the animal is unable to survive long enough to reproduce at a particular temperature that temperature may be outside of the lethal limits for the species regardless of any ability to tolerate that temperature for short periods of time. 87

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CHAPT E R 5. FOOD ABUNDANCE AND THE REPRODUCTIVE CYCLE Introduction The seco nd factor considered an important exoge n ous regulator of reproduction in scallops is food abundance. The r elat i ons hi p between food supp ly and gametogenesis has been examin ed in several species of scallops. Food ab und ance can be important in both th e reg ul atio n of developmental stages and the initiation of spaw nin g events I n P ecten m .a:xim. us high food levels are re quired for oocyte maturity in the late sprin g ( Lubet et al, 1987) w hil e i n Placop ecten magellanicus high foo d leve l s in conjunction with low tempe r at ur es a r e n ecessary for gametoge n ic deve l opm ent (Thompson, 1 977). In Chlamys va ria gametogenes i s begin s when maximum ava il able food i s present ( Burn ell, 1983) and spaw ning h as been associated with abu ndan t foo d i n both C. amandi (Jaramillo et al, 1 993) a n d C. opercularis ( Broom and Mason, 1 978). On the other hand spaw ning h as a l so been linked to a low food supply in C. ope r c ulari s ( T aylor and Venn 1979). In t h e ge nu s Argopecten co n s ider a bl e work has b een done o n t h e relatio ns hip between food a bundance a nd game togenic deve l opment i n the bay sca llop. Studie s in the 88

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bay scallop indicated that different populations of the same species could vary greatly. Populations in Florida and North Carolina showed a correlation between gonad growth and food levels (Sastry 1961; 1963; 1966a; 1968) while gonadal development did not coincide with enhanced food levels for a population from Massachusetts (Sastry, 1970a). In this case the interaction between temperature and food levels is cited as an explanation for the differences between populations. In the Peruvian scallop A. purpuratus increases in gametogenesis and fecundity were linked to increased food and temperature levels resulting from El Nino events (Dlanes et al, 1985; Wolff, 1987 ; 1988) Villalaz (1994) studied the role of food abundance and temperature in reproduction of A. ventricosus and concluded that gametogenesis occurred during low phytoplankton densities with the scallops exhibiting somatic growth during high phytoplankton den s ities There have been no s tudies to date looking at the role food levels may have in regulation of the reproductive cycle of the calico sca llop. It has been proposed that the introduction of colder, nutrient rich waters onto the s helf may be linked to spawning of the calico scallop (Allen 1979; Miller e t al, 1981) Without examination of the role food level plays in the reproductive cycle it is not pos s ible to determine whether low temperatures or enhanced food level s associated with intrusion of water onto the shelf are linked to the regulation of reproduction in the calico scallop. The purpose of this study is to clarify this s ituation by exa mining the effect different food level s may have upon the reproductive cycle of the calico sca llop. 89

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Materials and Methods The design of the experiments presented in this s tudy has already been presented in Chapter 4. The same animals used in the temperature study were used for examination of the relationship between food abundance and reproduction. The methodology involved in body-component and histological ana lysi s was described in Chapter 3. Scallops were fed a monogenic culture of the flagellate Tetraselmis suicica. Tetras e lmis has been found to be an excellent food s ource for laboratory experiments on bival ves in general (Bayne and Thompson, 1970; Walne, 1970 ; Thompson et al, 1974) as well as scallop s spec ifically (Wikfors e t al, 1995 ; 1996; Di xon et al, 1996). Previou s work in the laboratory on the bay sc allop had also utilized Tetra selmis as the food source (Barber and Blake, 1985 ). The Tetraselmis was cultures in a series of 2 liter (L), 20 L, and 110 L containers using f/2 enrichment media and standard methodology ( Guill ard, 1983). Cultures were allowed to attain a concentration of 3 x 1 0 5 cells/m l prior to u se. The large batch culture s u sed in the se experiments had a m aximum concentration of approximately 4 x 105 cells/mi. This was probably due to light limitation despite the insertion of lights into the se culture tanks. Cultures maintained in two liter flasks routinel y attain concentrations of 2 x 1 0 6 cells/mi. The Tetraselmis u s ed t o feed the experimental animals was dispensed at a concentration of 3 x 105 cell s /ml on a continuous basis using peristaltic pumps. Th e twelve tanks were divided into 4 sets of 3 tanks each. In each set the 3 tanks received 90

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either 0, 100, or 300 cell s /ml/scallop/day hereafter referred to as no food, low ration and high ration respectively. The initial density of scallops in each tank was 100 animals per tank The amount of algae flowing to each tank was adjusted as necessary to maintain the proper amount of food for each scallop in that tank. Every two weeks, for a maximum of 8 weeks, 20 animals were removed from each tank for reproductive analysis as previously described (Chapters 3 and 4) The food level s were chosen to represent poor, limited and abundant food levels. High food levels provided 9 x 1 0 7 cells/scallop/day while low food levels correspond to 3 x 1 0 7 cells/scallop/day. This compares well with Villalaz ( 1994) who utilized a high food level of 6.6 x 1 0 7 cells/scallop/day and a low food level of 1 6 x 1 0 7 cells/scallop/day in his study of reproduction in Argopecten ventricosus That study utilized a mixture of Isochrysis galbana and Chaetoceros calcitrans as a food source. Great care was taken to insure that the tanks remained as clear as possible of additional food sources or consumers. All epibionts, predominantly barnacles, were removed from the scallops prior to placement in the experimental tanks. All water used within the tanks and culture operation was carefully monitored and sterilized to prevent the introduction of other organisms into the system. The treatments used for each of the five trials and dates of tho s e trials has already been presented in Table l. For this study the sample from all of the sca llops at a given food level for the three experimental temperatures ( 15, 20, 25 C) were combined prior to analysis. 91

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Results The same 4 054 scallops u se d for the temperature study are analyzed in this s tudy As expected tissue lo ss was greatest among the scallops receiving no food. In all cases, for all body components, tissue lo ss was negatively correlated with food level and tissue gain was positively correlated with food leve l except for those cases when no differences were observed between two or more of the food levels The mean wet weights and standard deviations for the four body components (gonad, digestive diverticulum mantle adductor muscle) and total body weight ( s hell excluded) are presented in Tables 7 8, 9, 10, and 11, respectively At all three levels of food, the gonad s howed increases in weight in at least some cases. With no food, the gonad weight often increa se d initially but then returned to at or s lightly below the initial levels by the end of a particular trial. The gonad weight decreased in all treatment s during the trial conducted from July to September. In the other four trial s the gonad weight increased or held constant in the case of low rations and increased in the case of high rations. The dige st ive diverticulum weight decr ease d during all trials in scallops receiving no food, and in all but trial one (February to April) for sca llops receiving low rations. There were fluctuations during experiments indicating that early weight losses in the digestive diverticulum were later reversed to some degree in scallops maintained in low ration an d no food conditions. The overall loss in this component was at times small enough to be stat i s tically insignificant in sca llop s with a low ration. In scallops with a 92

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Table 7. Mean ( 1 sd) wet weig ht s (g) of the gonad body component of Argopecten gibbus for all treatments gro uped by experimental food l evel. Date and Treatment Gonad Date and Treatment Gonad -------------------------------------------------------------------------------------------------------18 Feb 93 Field 0.457 .116 22 Mar 94 None 0.584 .196 01 Mar 93 None 0.531 1 72 Low 0.718 .289 Low 0 597 .144 High 0.921 .292 High 0.792 .203 05 Apr 94 None 0.635 .206 15 Mar 93 None 0.461 .112 Low 0.742 .241 Low 0.618 .344 High 0.954 .374 High 0.851 .258 04 May 94 Field 0.648 .153 29 Mar 93 None 0.447 173 1 8 May 94 None 0.848 .309 Low 0.575 .224 Low 0.876 .227 High 0.756 202 High 0 921 .248 12 Apr 93 None 0.395 .116 01 Jun 94 None 0.689 .249 Low 0.488 .135 Low 0.782 .178 High 0.802 .255 High 0.849 .287 14 Apr 93 Field 1.124 .114 1 5 Jun 94 None 0.632 .170 16 Jul 93 Field 0.767 .209 Low 0 662 .247 02 Aug 93 None 0.829 .247 High 0 722 239 Low 0.856 .190 29 Jun 94 None 0.635 .128 High 0.880 .249 Low 0.710 .147 16 Aug 93 None 0.739 .213 High 0.754 .179 Low 0.791 .200 15 S e p 94 Field 0.456 .148 High 0.698 .183 30 Sept 94 None 0.473 .166 30 Aug 93 None 0.637 .169 Low 0.399 .12 1 Low 0.664 .239 High 0.470 .153 High 0.775 .2 10 14 Oct 94 None 0 413 .192 13 Sep 93 None 0.593 .136 Low 0.372 .1 05 Low 0.725 0.368 High 0.455 185 High 0.690 .216 28 Oct 94 Non e 0 393 .161 08 Feb 94 Field 0.578 .159 L ow 0.423 159 22 Feb 94 None 0.861 .278 High 0.500 .275 Low 0 873 .253 II Nov 94 None 0.428 .170 High 0.811 .29 2 Low 0 .44 3 .174 08 Mar 94 None 0.638 193 High 0.573 .192 Low 0.715 .271 23 Nov 94 Field 0.412 .087 High 0.857 .259 93

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Table 8. Mean ( 1 sd) wet weights (g) of the digestive diverticulum body component of Argopecten gibbus for all treatments grouped by experimenta l food level. Date and Treatment Dig. Diverticulum Date and Treatment Dig. Diverticulum -----------------------------------------------------------------------------------------------------18 Feb 93 Field 0.623 .129 22 Mar94 None 0.602 .092 01 Mar 93 None 0.618 .133 Low 0.717 .143 Low 0 621 .120 High 0.867 .176 High 0.925 .196 05 Apr 94 None 0.622 .134 15 Mar 93 None 0 565 .121 Low 0.754 .116 Low 0.648 .1 86 High 0 897 .225 High 0.860 .170 04 May 94 Field 0.843 .113 29 Mar 93 None 0.537 122 18 May 94 None 0.757 .150 Low 0.596 .162 Low 0.815 .134 High 0.801 .159 High 0 .900 .186 12 Apr 93 None 0.522 1 09 01 Jun 94 None 0.678 .120 Low 0.668 .137 Low 0.747 .138 High 0 935 .187 High 0.789 .182 14 Apr 93 Field 1 066 .119 15 Jun 94 None 0 656 .119 16 Jul 93 Field 1 .022 .220 Low 0.714 .139 02 Aug 93 None 0.939 .18 3 High 0.767 .151 Low 0.940 .206 29 Jun 94 None 0.700 .123 High 0.935 .168 Low 0.762 .119 16 Aug 93 None 0.873 .177 High 0 832 .152 Low 0.901 .143 15 Sep 94 Field 0.911 .119 High 0 882 .145 30 Sept 94 None 0 794 .118 30 Aug 93 None 0.872 .160 Low 0.778 .128 Low 0.864 .165 High 0.854 .176 High 1.047 .191 14 Oct 94 None 0.713 .123 13Sep93 None 0 819 .150 Low 0.732 .123 Low 0 781 .145 High 0.827 .166 High 1.038 .160 28 Oct 94 None 0.680 .144 08 Feb 94 Field 0.798 .1 04 Low 0.712 .125 22 Feb 94 None 0.752 .119 High 0 853 180 Low 0.733 .139 11 Nov 94 None 0.671 .122 High 0.772 .195 Low 0.818 .161 08 Mar 94 None 0.657 .1 09 High 1.00 1 .167 Low 0.663 167 23 Nov 94 Field 0.713 .100 High 0.851 .216 94

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Table 9. Mean ( 1 sd) wet weights (g) of the mantle body component of Argopecten gibbus for all treatments grouped by experimental food level. Date and Treatment Mantle Date and Treatment Mantle -------------------------------------------------------------------------------------------------18 Feb 93 Field 4.169 .803 22 Mar 94 None 3.340 .520 01 Mar 93 None 3.942 .941 Low 3.548 .577 Low 3.703 .643 High 3.750 .498 High 4 560 .722 05 Apr 94 None 3.625 .551 15 Mar 93 None 3 905 .785 Low 3.673 .452 Low 3.607 .098 High 3.727 .621 High 4.623 .008 04 May 94 Fie l d 3.925 .696 29 Mar 93 None 3.834 .611 18 May 94 None 3 992 .727 Low 3.593 .009 Low 4.244 .71 1 High 4.233 .626 High 4.206 .727 12 Apr 93 None 3.682 .645 OJ Jun 94 None 3.736 .724 Low 3.706 .653 Low 3 813 .724 High 4.442 .568 High 3.973 .642 14 Apr 93 Field 4.222 .610 15 Jun 94 None 3.706 .652 16Jul93 Field 4.855 696 Low 3.728 .556 02 Aug 93 None 4.562 .942 High 3.846 .7 1 5 Low 4.788 907 29 Jun 94 None 3.612 .513 H igh 4.674 755 Low 3.814 .403 16 Aug 93 None 4.685 .849 High 3.848 .676 Low 4.701 702 15 Sep 94 Fie l d 4.153 .533 High 4.52 1 .689 30 Sept 94 None 3.959 .435 30 Aug 93 None 4.982 .794 Low 3.981 .536 Low 4.360 1 .027 High 3.957 599 High 4.867 .825 14 Oct 94 None 3.780 594 13 Sep 93 None 4.448 .613 Low 3.813 .463 Low 4.169 0.922 High 3.997 56 1 High 4.787 .641 28 Oct 94 None 3.819 .690 08 Feb 94 Fie l d 4.146 .484 Low 3.721 .524 22 Feb 94 None 3 858 .579 High 3.8 1 3 561 Low 3 778 .592 I I Nov 94 None 3.983 .531 High 3 729 .563 Low 3.977 .609 08 Mar 94 None 3.6 1 8 .606 High 4.120 .698 Low 3.576 .654 23 Nov 94 F i e l d 4.20 I .484 High 3.826 .503 95

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Table 10 Mean ( I s d) wet weights (g) of the adductor muscle body component of Argope c t en g ibbus for all treatment s grouped b y ex perim e ntal food level. Date and Treatment Adductor Mu sc le Date and Treatment Adductor Mu s cle ------------------------------------------------------------------------------------18 Feb 93 Field 2.630 .7 8 0 22 Mar 94 None 1.938 .480 01 Mar 93 None 2.297 532 Low 1.8 89 .5 48 Low 1.985 .638 High 1.948 .499 High 2.594 .574 05 Apr 94 None I .882 .581 15 Mar 93 None 2 093 .693 Low 1.873 .485 Low 1 .667 .916 Hi g h 1.672 522 High 2.403 .764 04May 94 Field 2 128 .388 29 Mar93 None 1 .852 0.527 18 May 94 None 2.010 .5 10 Low 1.5 97 .680 Low 2.143 .486 High 2.020 .450 High 2.104 549 12 Apr 93 None I .642 .475 01 Jun 94 None 1 540 .489 Low I .797 .534 Low 1.606 .472 High 2.023 0.417 High 1.712 .562 1 4 Apr 93 Field 2.285 565 15 Jun 94 None 1 .4 1 4 .434 16 Jul 93 Fie ld 2.715 .641 L ow 1.370 .470 02 Aug 93 None 2.610 .863 High 1.498 .434 Low 2.595 .675 29 Jun 94 None 1.310 .381 High 2.498 .757 Low 1.268 256 16 Aug 93 No n e 2.392 .64 2 High 1.296 .416 Low 2.537 .778 15 Sep 94 Fi e ld 3.698 .583 High 2.386 .695 30 Sept 94 None 3.424 662 30 Aug 93 None 2.497 .688 Low 3.485 .57 5 Low 2.240 .716 High 3.580 564 High 2.389 .796 14 Oct 94 None 3.2 1 8 561 13 Sep 93 None 1 .9 20 .513 L ow 3.291 .409 Low 1.760 .5 8 7 High 3.459 .548 High 2.260 .694 28 Oct 94 None 3.086 .670 08 Feb 94 Fie l d 2. 556 .483 Low 2.893 651 22 Feb 94 None 2.553 .427 Hi g h 3.059 599 Low 2.356 .444 11 Nov 94 None 3.017 513 Hi g h 2.388 .41 7 Low 3 071 .719 08 Mar 94 None 2. 045 .588 Hi g h 3.034 .533 Low 2 103 .523 23 Nov 94 Field 3.462 .398 High 2.228 555 96

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Tab l e 11. Mean( 1 sd) total body wet weights (g) of Argopecten gibbus for all treatments grouped by experimental food level. Date and Treatment Tota l Body Date and Trea t ment Total Body -------------------------------------------------------------------------------------------18 Feb 93 Field 7 878 .683 22 Mar 94 None 6.463 1 .042 01 Mar 93 None 7.387 667 Low 6.872 I 322 Low 6 905 .389 High 7.486 1.198 High 8 870 .522 05 Apr 94 None 6.764 .314 15 Mar 93 None 7.025 .64I Low 7.042 .972 Low 6.540 .348 High 7.250 .413 High 8.737 .058 04 May 94 Field 7.544 .124 29 Mar 93 None 6 670 .27 1 18 May 94 None 7.607 1.481 Low 6 .361 .906 Low 8.078 .393 High 7.811 .030 High 8.131 .578 12 Apr 93 None 6.241 .249 01 Jun 94 None 6.644 1.401 Low 6 660 1.278 Low 6 948 1.370 High 8.203 .209 High 7 .3 23 .395 14 Apr 93 Field 8.697 l.26I 15Jun94 None 6.407 1.197 16 Jul 93 Field 9.359 .494 Low 6.474 .151 02 Aug 93 None 8.940 1.984 High 6.832 .356 Low 9 .178 .752 29 Jun 94 None 6.257 .966 High 8.987 .627 Low 6.553 700 I6 Aug 93 None 8.689 .560 High 6.729 1 209 Low 8 930 .475 15 Sep 94 Field 9.220 1.257 High 8.487 .345 30 Sept 94 None 8.650 .993 30 Aug 93 None 8.988 .362 Low 8.642 .211 Low 8.128.813 High 8 .861 .343 High 9.078 .737 14 Oct 94 None 8.125 .230 13Sep93 None 7.779 .225 Low 8 209 .893 Low 7.435 520 High 8 738 I. 205 High 8.776 .407 28 Oct 94 None 7.978 .290 08 Feb 94 Fie ld 8.078 867 Low 7 749 .184 22 Feb 94 None 8.024 I61 High 8 225 208 Low 7.740 .248 II Nov 94 None 8.099 .06I High 7.700 .22 5 Low 8.309 I.389 08 Mar 94 None 6.959 .3 1 6 High 8 727 .333 Low 7.057 .415 23 Nov 94 Fie ld 8.788 .909 H i gh 7.762 255 97

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high ration the digestive diverticulum weight increased in trials conducted in February to April and September to November. In trials conducted between May and September early decrea ses were later restored resulting in no net change in the digestive diverticulum weight. Mantle weights decreased in a ll sam ples maintained in low rations and no food. The degree of tissue loss in these samples was highest in trials conducted between February and April, an d July to September. The overall loss was very small in other cases. At high ration s, the mantle weight had no overall change except for the two trials conducted between February and April. In 1993 there was a slight increase during thi s period while a distinct decrease was observed in 1994. The latter is probably a more accurate picture si nce scallops were not maintained at 20 C in the first trial. The last few sa mple s obtained during trial I consisted primarily of scallops maintained at 15 C due to high mortalities in the scallops maintained at 25 C. The adductor muscle is the one body component in which the weight never increa se d in any of the trials or food levels. There was an overall decline in the adductor muscle weight even at h ig h ration s At lower rations and no ration s the decline in adductor muscle weight observed was progressively greater. The adductor muscle weight did not change for the first several weeks of trials from February to April in scallops receiving high rations nor the first couple of weeks during trial 4 (May and June) for all three food levels. The overall decrease in the adductor muscle weight ranged from 16 to 40%. 98

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Due to the decrea se in the adductor mu sc le weight, which t ypica lly compri ses 30 to 50% of the tissue mass, the total body weight declined in all but one instance. The exception is for sc allop s r ece iving high ra tio n s during trial 1 (February to April, 1993). The overall decrease for sca llop s receiving a high ration was small during samples conducted after July, and large for trials commencing in February (1994) and May. The mean gonad ind ex with sta ndard deviation s for all five trials for scallops receiving no food, low ration, and high ration are s hown in Figur e 17, Figure 18, and Figure 19, re spec tively. The gonad index increa se d in all cases during the trials conducted between February and April (Trials 1 and 3). Both initial and final level s were greater in 1994 than in 199 3 and the rate of increase was also greater in the former. There was no difference in the increase between scallops receiving no food and those receiving a low ration. In scallop s which received a high ration the gonad index increased at double the rate observed in the other two rations. During May through June (Trial 2) the go nad index again increased during the course of the trial at all three food levels. The growth rate was le ss for sca llops receiving a high ration than had been observed earlier in the yea r (Trial 3 ) There i s a positive correlation between food level and the increa ses in the gonad ind ex. From July to September only very small increases in the gonad index are observed. It appears that the gona d index in sca llops receiving a low r at ion increased more than the other two treatments. This is due in part to differen ces within the scallops at low r a tion as some exhibited increases and others expressed a decrease in the gonad 99

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2 4 20 16 .--.. ';:?. 0 ...._.. >< w 0 12 z 0 <{ z 0 8 (.') 4 0 Field 1 99 3 Fie l d 1994 0 No Food 1993 No Food 1994 t i? t + I t t J F M A M J J A S 0 N D MONTH Figure 1 7 Mea n gonad in d ices inclu d i n g standard deviation based u pon dry tiss u e weigh t s for experimental anima l s maintained with n o food plotted against time of year from 1993 0 a n d 1994 a l ong with field measuremer from 1 993 0 and 1994 +

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24 20 16 r-..-.... ;:R 0 .._ >< w 0 12 z 1-0 <{ z 0 8 (!) r4 1-0 J Fig u re 18. I I I I I I I I I I I I I 0 Field 1993 Field 1994 /::,. Low Rat i on 1993 Low Ration 1994 t J t t J i j j + t t L I I I I I I F M A M J J A s 0 N D MONTH Mean gonad i n dices i ncl u ding sta n dard deviation b ased upon dry t i ss u e weights for expe r iment al anima l s main t a i ne d wi t h a low food ration plotted agains t time of year from 1993 fl. and 1994 along with field measureme nts from 1 993 0 and 1 994 +

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24 20 16 I---:::R. 0 X w f.-0 12 z 0 <( z 0 8 (.9 4 I-0 J Fi gure 1 9 I I I I I I _l I I I I I I I I I I 0 Field 1993 Field 1994 0 High Ration 1993 High Ration 1994 f I I) ) ) t t <;> ) t t ? + I t + I I I I I F M A M J J A s 0 N D MONTH Mean gonad indi ces including s t a nd ard deviation based upon dry ti ssue weigh t s for experimental an imal s maintained with a high food r at ion plotted against t ime of year from 1 993 0 a nd 1 994 e along with field measurements from 1993 Oa nd 1 994 +

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index or remained at initial levels. The slight increase see n in the other two treatments may be an artifact of the decreasing adductor muscle weights. In the final trial conducted from September to November there is no clear evidence of overall increa ses in the mean gonad index among the scallops which were given either low rations or no food. There were small increases exhibited within individual animals within those examined at these treatments An increase in the gonad index is clearly present in the mean gonad index for scallops in high ration conditions. Again not all of the animals exhibited this increase which can be seen in the large standard deviation associated with these samples. The large standard deviations are due in part to the pooling of scallops maintained at different temperatures. Mean nominal oocyte diameter s for the trials for all scallops maintained with no food, low ration and high ration, ar e depicted in Figure 20, Figure 21, and Figure 22. There are difference s in the mean oocyte diam e ters during the period from February to April between 1993 and 1994 but no differences were seen within those two trials among scallops maintained in the presence of differing rations of food. In 1993 the scallops had an initial mean oocyte diameter of 32 j..lm and maintained that size throughout the study. In 1994 the initial oocyte diameter averaged 23 IJm. During the first 6 weeks of the trial the oocyte diameter s teadily increased at all three treatment s to a value of 32 j..lm. The oocyte diameter remained at 32 IJm for the balance of this trial a t all rations From May through June the oocyte diameter increa se d from 22 IJm to 32 IJm at all three treatments. This increa se took 8 weeks under low ration and no food conditions and was attained in 6 weeks by sca llop s maintained in a high ration environment. A slight 103

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38 ...-... 32 e (.) E .......... 0:: 26 w 1w <( 20 0 w (.) 14 0 0 8 t I f + 0 Field 1993 + Field 1994 o No Food 1993 No Food 1994 + J Figure 20. F M A M J J A s 0 N D MONTH Mean oocyte diameter including standard deviation for experimental animals maintained with no food plotted against time of year from 1993 D and 1994 along with field measurements from 1993 Oand 1994 +.

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38 C/) 32 c 0 ...... 2 E .......... 0:::: 26 w w l 0 :2: Vl <{ 20 0 w G 14 0 0 8 Figure 21. J I I I + ... . . . . . F M A M t J J MONTH 0 Field 1993 + Field 1994 t:.. Low Ration 1993 Low Ration 1994 l + A S 0 N D Mean oocyte diameter including standard deviation for experimental animals maintained with a low food ration plotted against time of year from 1993 A and 1994 _. along with field measurements from 1993 0 and 1994 +.

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0 0\ .......... C/) c 0 '-() E ......... 0::: w Iw :2 <( 0 w I->-(.) 0 0 38 32 26 20 14 8 + 0 Field 1993 + Field 1994 o High Ration 1993 High Ration 1994 ... t J F M A M J J A s 0 N D MONTH Figure 22. Mean oocyte diameter including standard deviation for experimental animals maintained with a high food ration plotted against time of year from 1993 0 and 1994 e along with field measurements from 1993 0 and 1994 +.

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decrease is observed in the mean oocyte diameter s of sc allop receiving high rations between weeks 6 and 8. The l a rge standard deviation for scallops receiving a low ration reflects that some of the scallop s maint ained under this ration had mean oocyte diameters that were approximately 32 J..lm and others had mean oocyte diameters that were much smaller. From July to September (Trial 2) the mean oocyte diameter remained relatively constant at 12 to 14 J..lm in all treatments. The large s tandard deviations reflect that differences were present on an individual basis possib l y due to varying responses among scallops maintained at the three temperature levels. From September to November increa ses in the mean oocyte diameter were observed under all three level s of ration. The baseline value of 6 J..lm for thi s trial had a s tandard deviation of le ss than I micron. The experimental values during this trial exhibited the large s t variations seen during thi s study Scallops that received no food and those receiving high rations rapidly increa sed to mean diameter s of 20 J..lm. The decrease in oocyte diameter between week 6 and week 8 in sca llops receiving no food is largely due to the absence of any sca llop s maintained at 25 C during the latter sampling period which consisted of equal number s of scallops maint a ined at 15 and 20 C The scallops in a low ration environment also exhibited a n increa se in oocyte diameter but the increase was not as great as was observed in the other two treatments The l arge standard deviations again reflect both temperature difference s and the fact that some scallops within each treatment had minimal or no increase in the mean oocyte diameter while others had very large increases. 107

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The gonad indices of animals observed in the field in the early spring of 1993 increased at the same rate as that which was observed in the lab in 1993 in scallops receiving a high ration and in 1994 at both high and low rations. There was no change in the mean gonad index in the field during the fall unlike the laboratory where increases were observed in scallops held at low and high rations. The mean oocyte diameter observed in the field during the early spring of 1993 had the same trend as was observed in the lab in 1993 and 1994 at all ration levels. In the fall no change was observed in the mean oocyte diameter in scallops collected from the field while large increases were observed at all ration levels in the laboratory. Discussion The increases in tissue weight observed in all of the body components, except for the adductor muscle, indicate that the high ration was sufficient for normal metabolic processes. At the higher temperatures, this ration may not be sufficient for growth, but examination of the scallops maintained at 15 C with a high ration indicated that increases or maintenance of both the adductor muscle and total body weight occurred during at least portions of all trials. Since the weight of tissue is related to the size of the animal small variations are to be expected. At higher temperatures, increased metabolic costs may preclude net growth of the animals at the rations used. As expected, increases in the ration resulted in either increases or no change in the tissue weight in all components. 108

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When exposed to rations higher than those utilized in this study scallops began to produce pseudofeces The production of pseudofeces is one of the processes used by scallops to control the amount of food ingested in response to increasing suspended particulate loads (Bricelj and Shumway, 1991 ). The production of pseudofeces often results in inhibited growth and abnormal development in bivalves (Winter, 1978). Maximum growth rates in other bivalves have been found at moderate algal concentrations and are diminished at high food levels (Malouf and Bricelj 1989). This was also observed in queen scallops fed Tetraselmis suecica with the scallops exhibiting maximum growth at cell densities of 1 ,000 to 3,300 cells/ml and diminished growth at 13,000 cells/ml (Richardson et al, 1984). This indicates that an increase in the ration used in this study would most likely have resulted in diminished assimilation by the scallops It would be preferab l e to utilize a mixture of phytoplankton and diatoms in future studies rather than a monogenic culture. A large number of both benthic and pelagic, diatoms and phytoplankton, are consumed by the filter feeding scallops (Davis and Marshall, 1961; Vernet, 1977; Shumway et al, 1987, Brice1j and Shumway 1991). Different species provide for a variety of nutritional requirements within the scallop. The maximum oocyte diameter found in scallops maintained in the lab was approximately 5 11m lower than was observed in scallops in the same reproductive state which had been collected in the field. This may be due to an inability of the phytoplankton species used as a food source, T. suecica, to provide for all of the nutritional needs of the scallop The optimal ration for any species must be based upon empirical observations and varies 109

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greatly with the nutritional value and culture conditions of the species composing the diet (Enright et al, 1986; Coutteau et al, 1994). Ideally studies would utilize the species composing the natural diet of the animal. Cons iderable work ha s been done on the relationship between food supply and gametogenesis in the bay s callop. Sastry (1966a; 1968; l970a) found a decrease in the gonad index, and resorption of oogonia and primary oocytes at all temperatures during the initial s tages of gametogene s is when scallops received no food and contained no reserves. When these scallop s had minimal reserves gametes were developed and released at temperatures of 25 and 30 C while gonad indexes decrea sed and oocyte s were resorbed in animals maintained with no food at 15 and 20 C. The conclusion was that a minimal food reserve was nece s sary for the initiation of gametogene s i s in addition to temperature requirement s This is not the cas e in the current study with the calico s callop. Maturation of oocytes, and increase in gonad index occurred in scallops receiving no food which were collected in a resting state during September. Maturation of oocyte s and increases in the gonad index are enhanc e d b y increased ration during this time period but the gametogenic cycle proceeds regardless of the food supply. The amount of food s upplied to the scallops had no effect upon maturation of oocyte s in term s of either the values obtained at all other times of the year. In the late spring the rate of increa s e in the oocyte diameter was greater for scallops which received high rations. The slight decrease observed in the oocyte diameters of s callops maintained under high ration condition s between the 6th and 8th weeks of the trial may represent the 110

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spawning noted in Chapter 4 among some of the individuals. There is no evidence of spawning in the gonad weights or indices during the late spring trial at any of the rations. There is a significant relationship between the amount of food and both the gonad index and weight in all cases except for the late summer (July to September). Statistical examination using analysis of variance indicates that increases in the food supply received result in highly significant (p
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no effect upon the ga meto ge nic cycle although temper a ture does have an effect (Chapter 4) Incr eased food le ve l s may also accelerate the rate at which the sc allops develop mature oocytes. Int eract i o n s of t em perature and food availability are also important and are di sc u sse d in Chapter 7. 112

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CHAPTER 6. MORTALITY IN ARGOPECTEN GIBBUS LINKED TO A PROTISTAN OF THE GENUS MARTElL/A Introduction I n December of 1 988 fishermen began to r eport findin g in c r eased numbers of dead a nd dyin g scallop s. By early January 1 989 th e r e was evidence of widespread mortality. By the end of January 1 989 th e popu l at i o n had decrea se d to the point that no scallop s could b e fo und by either commerci a l or r esearc h trawler s. By the summer of 1989 th e population h ad r e bound ed s ufficiently for r egula r monthly sampling of the popul a tion t o resume. Population l eve l s became l arge eno u g h fo r commer cial fis hin g t o r esume by t h e beg inni ng of 199 0. There wa s no evidence of any further problem s until J anuary, 1 991 w h e n mortality was agai n observed throu g hout the calico scallop popu l ation. By th e end of F e bruar y, 1991 the scallop population h ad once again been reduced to minimal level s an d commercia l fis hin g was s u s pended. Commercial fis hin g of calico scallops off Cape Canavera l d i d not r es ume until December of 1 992. T h e causes of th ese mass mortalities could n ot be directly ass ociated with phy s ical environmental factor s. Seaso n a l pattern s of temp e rature, salinity a nd u pwe llin g even t s o ve r the Cape Canavera l scallop grou nd s hav e been m e a s ur e d s ince 1 983 and no m ajor 113

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deviation s h ave been observed which co uld contribute to these mass mortalitie s. In an attemp t to a sc ertain a biological cause of the s e ma ss mortalities a hi sto pathological analysi s of calico scallop s collected in the field from 1983 throu g h 1994 wa s made. Materials and Methods S c allop s were collected from the Cape Canaveral fishing gro und s as de s cribed in Chapter 3. Since 1983 s ample s were ob t ai n e d when po ssi bl e o n a monthly ba s is. These r o utin e collection s were augmen t ed occas i o n ally b oth by intensive samplin g on specific resear c h c rui ses, and by ad dition a l collect i o n s made in re s pon se to ab normal events s uch as increased mortalities. Scall op sample s were processed for hi s t o lo gy as ha s a l so been previo u s l y described in Chapter 3. In a ddition to s t a inin g with H e matoxylin and Eos in some of the resulting s lid es were sta in e d w ith Cason's Trichrom e stain for con n ective tissue (Caso n 1 9 50 ). The fini s h e d slides we r e exam ine d a nd ph otog r ap h e d using a Zeiss Photomi c r oscope ill. Results A total of 54 scallo p s collected ove r a 1 month period December 1988 to J an u ary, 1 989 were examin e d hi s tolo gica ll y durin g the first period of incr ease d mortality. In 50 o f these a n ima l s a protozoa n par asi t e t entatively identified as b e l o n ging to the ge nus M arteilia was found in t h e digest i ve and b asop hili c ep ith e lial cells of th e tubu l es of t h e 114

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digestive diverticulum. A hi s topathological examination of mor e than 1200 scallops collected from 1983 until December, 1988 revealed only a handful of prior occurrences of this parasite. Two infected animals each were found in February and April of 1987 as well as in January of 1988. A relatively high 9 of 1 6 animals ana l yzed in March of 1988 were infected but no unu s ual mortality in the population was noted. One additional infected animal was collected in May of 1988 and 5 infected animals out of 30 examined were found in November, 19 88 jus t prior to the onset of the first noted mortalities. In many of the above cases the extent of the parasitic infection was not extensive within the infected animal. From January of 1989 until July 1989 scallop populations were s o low that no live scallops were found in the Cape Canaveral fish ing area. In July 1989 when it was again po ss ible to obtain samp les there was no evidence of the protozoan in any of the animal s examined. Sampling continued from that point at approximately monthly interval s but no evidence of the protozoan parasite was detected until February, 1990. In a total of 48 scallop s anal yzed from February and March, 1990 13 were found to be infected with Marteili a While no evidence of mortality was observed in the population in general fis hermen did occasionally report seei ng a greater incidence of "clappers" (recent l y dead scallop s) than usual at various point s throughout 1990 No additional infected scallop s we r e di scove red until Septemb e r 1 990, when extensive sampling during a research cruise turned up 3 infected animal s out of 120 animals on which histological examinations were conducted. 115

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The first evidence of the large scale return of the parasite was indications of increased scallop mortality reported by the fishermen at the end of 1990 and beginning of 1991. Samples of 20 scallops collected in late December, 1990, and January, 1991, contained 6 and 5 infected animals, respectively, but examination of 46 scallops obtained from three locations in February, coincident with increased mortality, again revealed the widespread presence of the protozoan Infection levels of 86%, 27% and 69% were detected for scallops collected from the northern, central and southern areas, respectively, of the fishing grounds. Overall, 60% of the scallops collected during February, 1991 exhibited evidence of the protozoan parasite. As was the case in 1989 within a few weeks it was no longer possible to obtain additional samples due to the virtual elimination of the population and closure until late 1992 of the fishery for economic reasons associated with the mortality. Samples obtained since that time revealed only one other instance where the protozoan was present. In 6 of 25 animals examined in June, 1991, the protozoan was present although the infection was not as extensive in most of those animals as had been previously observed. It should be noted that population levels in the summer of 1991 were still extremely low and very few animals were available for analysis for the balance of 1991 and most of 1992. Since that point, no evidence of the Marteilia infection has been observed either in the field samples or the more than 1900 scallops from laboratory experiments examined histologically through 1994. In all, more than 1800 scallops, collected between 1983 and 1992, were examined histologically for evidence of infection by this protozoan parasite. Of these the parasite 116

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was observed in a total of 131 scallops. In three instances, March 1988, January 1989, and February 1991, more than 50% of the animal s examined were infected with the parasite although mass mortalitie s were only a ss ociated with the latter two of these periods. Outside of the period s immediately preceding or following these instances, and February to March, 1990, only s cattered cases of one or two infected animals in a sample were typically observed. In both 1989 and 1991 almo s t 100 % of the natural population died within 4 weeks of the appearance of the pathogen. The digestive diverticulum of a healthy calico scallop and one which has been infected with the protozoan are s hown in Figures 23 and 24, re s pectively. On the bas is of light micro scopy the para s ite obs erved in the calico scallop appear s to meet all of the descriptors that have been established for members of the genus Marteilia (Grizel et al, 1974; Perkin s 1976; Perkins and Wolf, 1976; Figueras and Montes, 1988), and result s presented here s hall remain consi s tent with the terminology that they have employed in describing this parasite. The lumens of the tubules in the dige s tive diverticulum of infected s callops are filled with sporangiosori each containing approximately 8 presporangia or s porangia. Th e s porangiosori are easily discerned with either Hematoxylin and Eosin s taining or Cason's Trichrome stain. The Cas on's stain s hows the sporangia containing mature spores appearing pink agains t a blue background leading to rapid identification of animal s containing the pathogen. The sporangia in turn appear to contain 3 or 4 spore primordia. The sporangios ori examined in histological section s have a mean length (the longest axi s ) of 17 11m (range: 14 to 2211m; N=25). Plasmodia, the 117

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Figure 23. Digestive diverticulum of a healthy calico sca llop Ar&opecten gibbus, collected off Cape Canaveral, Florida (scale bar is 25 J..lm). 118

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Figure 24. Digestive diverticulum of a calico scallop, Argopecten gibb us, collected off Cape Canaveral, Florida exhibiting a heavy infection by a protozoan of the genus Marteilia. The pathogen has completely tubule epithelial cells but there is no evidence of invasion into surrounding tissue or host hemocyte response (scale bar is 25 !Jm). 119

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stage of the sporangiosori prior to the development of presporangia, were not observed in these samples. The percentage of the digestive diverticulum exhibiting evidence of the pathogen varies, but it appears to be progressive. The most extensive infection appears to be found in those animals at or near death (adductor muscle shrunken in size, shells gaping and slow to respond to tactile s timulation, mantle slightly withdrawn from the shell edge) at the time of collection. At that point virtually 100% of the tubules are infected. In those scallops exhibiting extensive infection, mature spores are also observed in the lumen of the intestine of the animal. The intestinal lumen of a healthy and infected calico s callop are shown in Figures 25 and 26, respectively. The spores, which range in size from 3.5 to 4.3 diameter, appear to be in the process of being excreted rather than invading surrounding tissue It is not known whether this is an attempt by the scallop to clear the pathogen, or if it is instead normal excretion of undigested spores as part of the life cycle of Marteilia No evidence could be found of the pathogen invading the surrounding epithelial cells or connective tissue. There was also no sign of hemocyte infiltration in response to the parasite. The parasite spores were occasionally seen mixed with food items in the gut indicating that ingestion may lead to the spread of the pathogen although the spores were not plentiful among the gut contents. The pathogen was observed only in the tubules of the digestive diverticulum or as spores within the gut or being excreted through the intestine. 120

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Figure 25. Inte st ine of a healthy calico sca llop Argopecten g ibbus, collected off Cape Canaveral Florida (sca l e bar is 50 Jlffi). 121

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Figure 26 Intestine of a calico sca llop Argopecten gibbus, collected off Cape Canaveral Florida exhibiting extensive occlusion of the lumen by mature spores of Marte ilia. sp. There is no evidence of penetration into the intestinal wall nor of a host hemocyte response by the scallop (scale bar is 50 11m). 122

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The only other pathology observed was in those scallops in which large portions of the digestive diverticulum were infected by the pathogen. These animals exhibit evidence of catabolizing body tissue This is particularly clear in the adductor muscle where extensive atrophy of the muscle bundles is evident. Figures 27 and 28 s how the adductor muscle of healthy and heavily infected calico scallops, respectively. Scallops typically derive some of their energy requirements from utilization of adductor muscle tissue when unable to extract sufficient energy from food intake There was no evidence that food levels were abnormal during either of the two epizootic episodes. The apparent inability of these heavily infected scallops to extract sufficient energy from available food levels to maintain routine metabolic energy costs may be due to the presence of the pathogen. The large numbers of spores located throughout the digestive tubules may have prevented the processing of ingested food as gut contents indicate feeding was occurnng. Discussion The protozoan parasite pre s ent in the calico scallops is an ascetosporan of the genus Marteilia This determination was based upon a comparison with the published descriptions of the genus Mart e ilia and it s constituent species (Perkins, 1976 ; Perkins and Wolf, 1976; Comps, 1976; Comps e t al, 1982, Comps, 1985) Aspects leading to this conclusion include the formation of approximately 8 sporangia within the sporangiosori, the formation of 3 to 4 spore primordia within the sporangia, the presence of refringent 123

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Figure 27. Adductor mu s cle tiss ue of a healthy calico s cal l op Argo p ec t en. g ibbu s, collected off Cape Canaveral Florida (s cale bar i s 50 11m) 124

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Figure 28. Adductor mu s cle tissue of a moribund ca lico sca llop, Argopecten gibbus, collected off Cape Canaveral, Florida which has been infected by Marteilia sp. The mu sc le bundles exhibit exten s ive atrophy although the parasite was found predominate l y in the digestive diverticulum (scale bar is 50 11m). 125

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bodie s, the s ize of the respective stages, the location of the parasite within the tubules of the di ges tive di ve rticulum the la c k of h emocy te in f iltration, and th e epizootiological patte rn observed. The f a ilure t o id e ntif y th e p rese nce of pl as modi a which norm a lly prec e de developm e nt of presporangial and s por e s tag es i s somewhat puzzling. There are currently five species identified in th e genus M a rt eilia (Figueras and M ontes 1988). These sp ecies have been identifi e d as pathogens primarily in oysters and mu sse l s from Europe and Australia (Comps, 1970; Wolf, 1972; Alderman 1979) The ascetosporan M arteilia refringens was th e fir s t s pe c ie s of Mart e ilia identified and s tudied (Comps, 1970 ). M refringens ha s b e en cited as the cause of mass mortalities in the edible oyster Ostrea edulis population of the Bretagne region of France s ince the early 1970' s (Bonami e t al, 1971; Grizel et al, I 974 ; Camps et al, I 975; Grizel, 198 3). The di sease syndrome was initi a lly term e d Ab er disease in referen ce to the estuaries in Bre tagne whe r e mort a liti es were first recor de d a nd was l a ter named di ges tive gland disease in reference to the m a in infection s it e in the ed ible oyster (Figueras and Monte s, 1988 ). A tentati ve life cycle for M r efr in gens ha s been proposed by Grizel et al. (1974) a nd later re v ised b y variou s authors ( L a uckner 1983; Figueras and Montes, 1988) As s tated in Figuera s and Mont es ( 1 988) primary infect i o n s are thou g ht to occur b y plasmodia in e pith e lia of the g ut the g ill s, or b o th Sporangia mature in the lumina of the digestive diverticul a and are discharged via the g ut. How the edible oyster become s infe c ted and by what stage it becomes infe c ted ha s n o t yet been determined. 1 26

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Experimental attempts to transmit the disease to healthy edible oysters in the laboratory have failed, a l though field experiments have been successful (Bal ouet, 1979) Aber disea se results in a sever e pathologica l response in edible oysters. Infected oysters become progressively emaciated, and the digestive gland becomes brown to pale yellow in color. When glycogen reserves have been depleted the mantle becomes translucent and shell growth ceases. The viscera l mass also loses its pigmentation and may appear shrunken and s limy in heavily infected individuals (Figueras and Montes, 1988). In the ed ible oyster incidences of M. refrin gens infections as high as I 00% have been reported for some of the estuaries in the Bretagne region of France (Bonami et al. 1971 ). Mortalitie s usually commence in May, peak in June through August, and diminish in the fall. Subclinical infections may persist throughout the winter and the surviving young plasmodia then reinitiate n ew clinical infections the following May (Balouet, 1979). Factors influencing the timing of disease transmission are unknown. The potential spacial extent of disease transmi ssio n also is unknown. While it has been shown that tran sp lanted oysters infect ed with Marteilia have transmitted the disease to indigenous stocks ( Balouet 1979), Alderman ( 1979) has reported that M. refrin gens was able to become establi she d in one site in Spain and infect indigenous oysters but was unable to cross 2 km of open water to infect an adjacent s ite. This despite the fact that the estuary is wholly marine, has little fresh water input and an adequate tidal exchange. Clearly there are factors involved which are not yet understood. 127

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Three other species, M y tilus edulis, Cardium edule, and Crassostrea gigas, have been identified as possible ho sts for Marteilia refringens (Camps et al, 1975; Gutierrez 1977; Cahour 1979). The percentage of the populations exhibiting infection is quite low (2.0-1 0.0%) for the se alternate spe cies and the protozoan appears to have minimal effect upon them. The second species of Marteilia discovered was M. sydneyi. This species has also been associated with massive mortalities in its hosts the Sydney rock oyster Crassostrea commercia/is, and C. echinata in Australia (Wolf, 1972; Perkins and Wolf, 1976). No other species hav e been reported as po ss ible hosts for this species. M. sydneyi appears to be especially virulent, with an incubation period of less than 60 days from early infection to death of the ho st (Figueras and Montes 1988) This is in contrast to M. refringens in which the edible oyster host does not exhibit mortality until perhaps one year or more after the initial infection. M. sydneyi has been linked with the loss of as much as 80% of the Sydney rock oy st er during intense epizootic episodes in Queensland and New South Wales (Wolf, 1972; 1979). Ultrastructural studies of this s pecies has revealed that it typically contains 16 sporonts in the sporangiosorus (Perkins and Wolf, 1976) instead of the 8 sporonts reported for the other four species of Marteilia. M. maurini has been identified as a parasite in both Mytilus galloprovincialis (Camps et al, 1982) and M. edulis (Auffret and Poder, 1985). In M. edulis infection rates as high as 70% were observed. The infection rate forM. ga lloprovincialis is lower (35%) but it appears to cause an inflammatory response, ma ss ive mucus secretion and 128

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infiltration of hemocyte s into the dige s tive diverticulum respon ses which have not been reported in any of the other Marte ilia ho s t s (Figueras et al, 1991 ). The remaining two species of Marte ilia are each associated with only one ho s t species to date. The parasite M. lengehi i s found in the epithelium of the s tomach in the digestive diverticulum of Crassostrea cucullata (Comps, 1976). The most recently discovered species isM. ch rist enseni a para s ite found in the epithelium of the digestive diverticulum of Scrohicularia piperata (Comps, 1985). Delineation of Marteilia to th e s p ec ie s level require s analysis of the ultrastructure utilizing electron microscopy The tran sie nt nature of Marteilia in the calico scallop population, coupled with the extreme virulence of the pathogen which resulted in the decimation of the natural population in le ss than a month, thwarted efforts to obtain the necessary samples for ultra s tructure analysis of this parasite. Some comparisons are how ever, poss ible It i s possible to eliminate M. sydneyi because 16 sporonts are not observed in the s poran g io sorus. In thi s st udy a s poran g iosorus mean length of 17 11m ( ran ge: 14 to 22 11m; N=25) was observed. Thi s is smaller than the mean sporangiosorus len g th reported forM. refringens of21 11m (range: 16 to 27 J.lm; N=25) ( Perkins and Wolf, 1976) but not by a great deal. Since differ e nt fixatives were u se d the difference could be due to differin g amounts of s hrinkage The size of the mature spores i s another comparison that can be m ade The lit erat ure reports spo re sizes for the 5 specie s of Marteilia as follows: M. refringens 2.6 J.lm, M. sydneyi 2.7 J.lm, M. maurini 2 to 3 J.lm, M. lengehi 5 to 6 J.lm, and M. christenseni 3.5 to 4.5 11m when mea s ured fro m hi s tol ogica l sect ion s (Perkins a nd Wolf, 1 976; Comps 129

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et al, 1982; Camps, 1976; Camps, 1985). The comparable value from this research is 3.5 to 4.3 11m. Based on thi s alone, M. christenseni would be the likely choice but the use of different fixatives in preparing the histo logical sec tion s make s it impossible to rely too heavily upon s uch simi laritie s It s hould be noted that Perkins ( 1976) reported a range of 3.5 to 4.5 11m for living, unfixed spo r es of M. refring e ns. Another possibility is that rather than extending the range and su itable hosts of a previously identified sp ecies this may indeed be a previously unreported s pecie s. Although the precise species remains undetermined the sudden appearance of Marteilia in North American waters i s the proximate cause for the epizootic disease which resulted in mas s mortalitie s of the calico scallop in 1989 and 1991. It is possible that this para s it e only becom es a factor when the animals are already experiencing stress due to other factors. The fact that there is no evidence of thi s s tr ess doe s not eliminate the possibility of syne r g isti c effects from a variety of factor s. Balouet ( 1979 ) has s u gges t e d that Aber disease as with other s hellfish diseases, may b e not so much a microbial di sease as one arising from unfavorable physicochemical factors in the seawater. What these factors may be i s entirely unknown. Th e pattern exhibited in the cas e of this e pizootic dis e ase follows the classic pattern of a marine s hellfish population exposed to a pathogen with which it has had no previous ex perienc e and to which it was s usceptible (Sindermann 1990) Althou g h initial indication s are that a s maller percentage of the population w as involved in 1991 as compared to 1 989 it is unclear at thi s point if a ny increased r es i s t a nce h as or will dev e lop among the survivors or their offspring. 1 3 0

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The rapid rebounding of the calico scallop population in 6 months as evidenced in 1989 may seem quite remarkable to those unfamiliar with the life hi s tory of this animal. The calico scallop has a total life span of only 18-2 4 months, reproductive maturity can be reached in as little as 71 days (Miller et al, 1979 ) and spawning occurs both in the spring and in the fall as noted earlier in C h apter 3. Scallops have a very high fecundity with each scallop producing 500,000 o r more gametes. A small number of scallops mos t likely survi ved the presence of t h e para si te and it takes the successfu l spawning of a only a few scallop s to generate million s of offspring. This in turn enables a very rapid increase in the size of the population in a very s hort period of time. The Marteilia parasite appears to cause the death of the scallop by preventing it from obtaining s ufficient nutrition from the water column possibly by interfering with the normal process of digestion in the tubules or by interfering with the biochemical transfer of stored nutrients. The presence of food within the gut indicates that the infected scallops were i ndeed still feed ing but that the inge sted food was not being digested perhaps due to disruption of normal phy s iological function of digestive epi t h elial cells. There i s no evidence of inflammatory or ot her immunolog ic al responses by the scallop to the parasite. Death is too rapid however, especially in the heavily infected scallops, to be solely the result of s tarvation. What other factors may be involved are currently unknown. The s ignificant population reductions caused by M. r efri ngens during epi zootic episodes of Aber disease in 0. edu lis had extens ive economic repercussions ( Grizel 1983; 1985). The economic impact arose not only from the mortalities but a lso the loss 131

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of oyster tissue weight. A comparison of the total wet weights of healthy and diseased oysters s howed th a t diseased oysters were lighter than healthy oysters by 25 30% for 18 month old oysters and 2035% in two year old oysters ( Morel and Tige, 1974 ; Figueras and Montes, 1988). The ma ss mortalitie s reported herein have had a similar drastic economic effect upon the calico scallop industry in Cape Canaveral (Blake and Moyer, 1991). Since the 1991 appearance of the parasite almost all of the producers have been forced out of busines s and the scallo p fishing fleet which numbered upwards of 100 vessels each making 5 to 7 trips a week has been disper sed to other fisheries. The future viability of the calico scallop as a commercial species i s unknown. It will however, take years for production level s to r et urn to the level s of the early 1980's even if there is no recurrence of the Mart e ilia parasite in the waters off Cape Canaveral. It is also unknown whether or not the calico sca llop will develop increa se d resistance to this pathogen or whether chronic infection s in the calico scallop will result in reduction of the size of the adductor muscle similar to the ti ss ue loss that has been observed in the edible oyster. Singha s (1992) report ed that the calico s callop population in North Carolina failed in the spring of 1991 with few life scallops present for collection in February of that year. This is the same time that the seco nd outbreak of Marteilia was observed in the population locat ed off Cape Canaveral. Unfortunately, Singhas apparently did not prepare histological sectio ns of the dige s tive diverticulum in the scallops used in her study and it is not known whether or not the mortality observed in North Carolina is due to Marteilia. Evidence of encyst ed organisms of Platyhelminth morphology were 132

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observed in the connective tissue along the gut wall of the intestinal loop which run s through the gonad, g ut wall deterioration and increa sed numbers of macrophages (Singhas, 1992). The encysted organisms were probably nematodes. This is the first reported incidence of Marteilia in North American waters as well as the first reported incidence of a member of the family Pectinidae serving as a host for this genus of ascetosporans. The so urce of thi s protozoan and extent of its range is not known. The histopathological evidence suggests that Marteilia was not present in calico scallops off Cape Canaveral prior to I 987, approximately 22 months before the first mass mortalities in the calico sca llop population were noted This s uggests that it may be a newly arrived species but the mode of its transportation is unknown. Shellfish relocation i s not utilized in t he calico scal lop industry nor among any other shellfish aquaculture industries in the affected areas so that vector can be disregarded. There is considerable freighter traffic from all over the world using port s on Florida's east coast including Cape Canaveral. It is conceivable that bilge waters from some of these freighters could have transported the protozoan into the area. While this is an easy speculation to make, it is virtually impossible to either prove or disprove after the fact. It i s also unknown if this parasite has infected other molluscan species of the western Atlantic or what other species from this area may serve as ho sts. 133

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CHAPTER 7. DISCUSSION Interaction of Temperature and Food Abundance Looking at the effects that temperature and food abundance have upon reproduction is usefu l but rarely i s n a ture so easi l y categorized. It ha s already been noted that cold water, rich in nutrients intrudes onto the scallop beds in the spring and l ate summer as a result of frontal eddy a n d she l f break upwelling as well as meanders of the Florida Current (Leming, 1 979; B l anton et al, 1981; Lee et al, 1982; Oey, 1986). This col d water is the mos t important so urce of nutrient s in t hi s region and the intru s ion of these waters large l y controls th e phytoplankton concentration s in this reg ion of the continenta l s h elf (Yoder et al, 1 983; Atkinson 1 985; McCl ain et al, 1988 ) Thus changes in temperature and food abundance are l i n ked alt h ough there will be a delay of up to two weeks between when nutrient rich waters mov e on to the shelf a n d response of th e phytop l ank ton popu l ation. The interre l ationships between temperature, food abundance, and gametogenes i s in the bay scallop have been exami ned for populations from North Carolina and Massachusett s (Sastry 1 966a ; 1968 ; 1970a ; Sastry and Blake, 1971). In North Carol in a scallop s at 20 C wh i c h received food proceeded w it h gametogenesis, while fed scallops 134

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at 15 C did not. When scallops were not fed, gametogenesis did not proceed at either temperature. It was concluded that an adequate food supply and increasing temperatures were necessary in order for gametogenesis to be initiated and proceed to the cytopla s mic growth phase. Later work indicated that once gametogenesis had been triggered and s callops had entered the cytoplasmic growth phase (stage ill) lowering the temperatures below earlier levels did not prevent furth e r development (Blake 1972; Blake and Sastry, 1979). After the scallops had entered stage ill of the gametogenic cycle food abundance increased in importance ( Blake, 1972; Blake and Sastry, 1979). The interrelationship s between temperature and food abundance also provide intere s ting information in this s tudy. The mean values for gonad index and oocyte diameter for each trial s eparated by both experim e ntal temperature and ration are presented in Table 12 and Table 13. Values u sed were as close to the end of the experiment as pos s ible High mortality in scallops maintained at 25 C did not always enable the collection of s amples from those treatments late in each trial. There were no differences observed in the oocyte diameter measurements between trial 1 and 3 for scallop s maintained in identical treatments at either 15 or 25 C. Trials I and 3 were conducted from February to April in successive years, 1993 and 1994 respectively, but trial I did not include s callop s maintained at 20 C. The results from trial 1 are not depicted in the above mentioned tables but did not conflict with any of the trends obs ervable from trial 3 or the other trials. For scallops from the early s pring increa se s in the gonad index are observed with increasing food ration at temperatures at or below 20 C. The gonad index also increased 135

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Table 12. Matrice s of the mean gonad index show in g the interaction between temperature and food abundance fo r experimental tr i als 2 to 5. Significant (* p<0. 05) and highly significant(* p
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Table 1 3. Mat rices of the mean oocyte diameter showing the interaction between temperature and food abundance for tria l s 2 to 5 S i gnificant (* p<0.05) and h igh l y significant(** p
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with temperature when scal lop s were sta rved and when the y received a low ration through 20 C. Maximum go nad index values occurred either with high ration at temperatures :'!> 20 C or with no food at 25 C. At higher rations, the gonad index was reduced at 25 C but remained s imilar to that observed under low rations at 15 C and both low rations and no food at 20 C. Change s in the gonad weight follow trend s s imilar to those observed in the gonad index but there are some differences. The gonad weight increa sed with decreasing temperature and increa s in g ration. Therefore the larg est amount of gametogenic material present at the end of the trial was in sca llops which received a high ration at !5 C, which a lmo s t doubled in weight, followed by scallo p s held at 20 C with high rations. An exception to these general trends was observed in sca llops maintained at 25 C. At this temperature sca llop s which received no food were found to have go nad weights larger than was observed in the scallops which received low ration s and equal to the gonad weights attained by the sca llops which received high rations. Although they had lower gonad indexes, the sca llop s held at 15 and 20 C with low rations had increase s in the gonad weight which equaled or exceeded those observed in scallops maintained a t 25 C at any of the food level s. A possible explanation for the reduction in gametogenic material observed at 25 C and low food rations compared to sca llops recei v ing no food is the increased metabolism which is associated with incre ased temperature. Clearly the calico scallop is ab le to catabolize other tissues fo r game t ogenes i s The amount of energy available for utilization by the scallop must be partitioned among respiration the energy costs 138

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associated with feeding production of waste material and growth (so matic a nd ga metogenic ) There h ave been no determinations of Q10 in th e calico scallo p but s tudies of both s ubspecies of bay sca llop h ave fo und Q10 values between 2 a nd 3 over similar temperatures (Kirby, 1970 ; Bricelj e t a l 1987b ; Bricelj and Shumway 1991 ). Thus it i s re aso nable to expect that the energy utilized for re s piration by the calico scallo p is much hi g her at higher temperature s than at low e r temp e ratures. The energy costs associated with feeding, and excretion associated with feedi n g, may have bee n g re a ter th a n th e e n e r gy which w a s being gai ned at hi g her t e mperature s. The t e mperatures normally experienced by the s callop durin g thi s time of th e year a re < 21 o C and often < 18 C ( Fi g ur e 9) due to upwelling of colder, nutri e nt ric h waters which also provides for an abundant food s upply Thus the scallop i s able to maximize produ c tion of gametogenic m ater i a l during thi s time of th e year. The r e is no difference in the s i ze of the oocytes with attainment of maximum sizes of 31-35 !J m in all treatment s but scallop s at hi g he r temperatures rea c h that m aximum faster th a n coun terpa rt s held a t lower temperature s. In the l a te spring, increases in th e rat ion r es ult in lar ge r investments in gameto ge ne s i s as indicat e d by both the go nad weight and index among scallo p s held at 15 C The level of oocytes m a tur a ti o n obse rv ed w as the sa m e at all lev e ls of r atio n indicating that food abundanc e was n o t n ecessary for continued development a t th a t point. At 20 C th e r e is evi d e n ce in bo th the go n a d ind ex a nd oocyte diameters t h at some spawnin g occu rred At 20 Cit appears from the oocy te diameters th at the extent of s pawnin g was po s itiv e l y corr e lated with th e ration th e scallop s r eceived. While the 1 39

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histological s tud y indicate s that some s pawning h a d occurred among scallops maintained a t 20 C a t all r a tion le ve l s an d th a t th e change observed is signif icant a m o n g scallops r e ceivin g low or high food ration s, th e go na d index va lues and go nad weights r e flect ev id e nce of spawning only among the scallo p s re ce iving no food or low rati o n s an d these changes are not significant. In th e sca ll ops held at 20 C which received hig h r at i o n s th e mean go nad index and weight continued t o inc r ease throu gho u t the study a l tho u g h they did a ppear to be levelin g off at th e e nd of thi s trial. If one assu me s that th ese scallops were ab le to attain a hig her go n ad ind ex i n itia ll y accom panied by a grea ter gon ad weig h t it i s possible that thi s decrease wou ld not h ave been n o ticed. Onl y a po rtion of the sca ll o p s examined in any of the treatment s s how a ny s igns of havin g s p awned. There is also evidence of s p aw nin g among some of the animals m a int a ined at 25 C. Ev id ence of s pawnin g is partic ul a rly s tron g in anim a l s receiving a low rat i on with a s i g nific a nt decrease in th e gonad ind ex (p<0 05) a n d oocyte diameter (p
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s callops continue to produce and store gametogenic material but exhibit no evidence of releasing tho s e gametes. This directly contradicts the hypothesis set forth by Miller et al ( 1981) that s pawning occurred in conjunction with water temperatures dropping below 22.5 C. The field studie s presented herein also indicate that Miller's hypothesis may be incorrect. Changes in the mean oocyte diameter and gonad index during the summer trial indicate that limited gametogenic development commenced at temperatures 20 C. At 15 C gametogenic development did not appear to be present at any ration level despite the significant increase in the gonad index observed at that temperature with low food ration. The apparent increase in the gonad index was not reflected in the gonad weights for this treatment and appears to be a result of accelerated l oss of other body tissues. This indicates that in order for s callops to undergo a second spawning event they may first require the e l evation of the temperature to some l evel for a period of time before gametogene s i s can proceed. The northern bay scallop does not proceed from development of oogonia (stage II) to cytoplasmic growth (stage III) until the temperature i s raised to 15 C for some period of time from lower temperature s ( Blake, 1972; Blake and Sastry, 1979). The lower temperatures prior to this are important in initiation of the gametogenic cycle in the bay scallop as anima l s which were not exposed to low temperature s at some point do not initiate gametogenesis (Sas try 1968; Blake 1972 ; Blake and Sas try 1979). It is possible that the s callops mu s t spend a period of time at low temperatures such are often ob served in the field during August before they are able to proceed with gametogenesis at higher temperature s It would be interesting to examine 141

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whether scallop s at thi s time of yea r showed evidence of gametogenic deve lopment at l o wer temperatures after ex po s ure to hig h t e mp e rature s for a brie f period of time. The oocyte diameter data from th e s ummer trial i ndicate s that so m e d eve l opment may a l so have taken place at temp e rature s $ 20 C particularly a mon g scallop s held at 25 C or those held at 20 C a nd provided a high ration. While some d eve lopm e nt did occur at higher temperature s the exte nt of any production would appear to be quite small. The gonad weight values show very li ttle evi d e nc e of gametogenic pro duction in a n y of the treatmen ts durin g th e summe r tri al. In tho se sca ll o p s maintained without a ny food ther e was a decrease in the gonad weight fro m initial valu es at all t emperat ur es. Gametogenic de ve l opment in the fa ll appears to exhibit so me distinct differences from development in the s pring. The re was evidence of ooc y te development in a ll trea tm e nts which incre ase d in co n j unction with hi g her temper a tur es. At 20 C oocyte development was positive l y co rr e l a t e d with r at i o n while at 25 C oocyte development was nega tiv e l y corr e l ate d with r at i o n Scallop s maint a ined a t 20 C with hi g h rati on e qu alled or ex c eede d the d evelopme nt ex hibit ed in any of th e ration treatments o f t he s callops maintained a t 25 C. The go nad ind ex and weight ge nera lly in c r ease d with t empera tur e and th e go n ad w e i ght a l so incr e as e d with r ation a t temperatures $ 2 0 o C durin g the fall trial. At 25 C the go n ad ind ex and weight valu es ex hibit n o clear tr e nd s with rat i on T hi s may again b e related t o in creased metabolic d emand s a t this temper a ture. The in crease in go nad index and weight w ith i n crea s in g t empe r at ur e i s the oppos it e o f w h at was observed in the earl y s prin g 142

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Scallops maintained at 15 C exhibited limited oocyte maturation but there was little evidence of gametogenic production based upon changes in tissue weights and indices at any of the ration s This is very different from the period of gametogenic production in the early spring when there was a strong correlation between ration and gametogenic production at thi s temperature. The oocyte diameter at the start of the early s pring trial was already 23 11m a s opposed to the 6 11m observed in the fall. Scallops in the early spring may have already been "turned on" by temperatures in the winter although the temperatures during that time of the year rarely exceed 23 C. Perhaps the elevated temperature requirement considered earlier had not been met during the fall trial but there is no evidence to s ugge s t thi s The bottom water temperature measured at the time of collection of the scallop s utili z ed in the fall trial w a s 22. 5 C. It is not known how lon g the scallops had heen exp o sed to this temperature prior to collection. This would indicate that if a s pecific temp e rature is required in the calico scallop in order to initiate the next step in gam e togenesis it is either> 22.SO C or this elevated temperature mu s t be pre sent for a time period long e r than the unknown amount of time in this cas e If the former were the case ther e s hould have been no development in any of the scallop s maintained at 20o C in the laboratory either since great care was taken to insure that once collected scallops were not exposed to temperatures greater than 20 C prior to plac e ment in the experimental tanks. This indicates that if a trigger temperature is pre s ent it s hould be no greater than 20 C. Gametogenic production in the fall is po s itively COITelated with temperature. Thi s i s the opposite of what was ob s erved in the spring. It would appear that for scallops in 143

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the fall a period of time at an elevated temperature is necessary in order for gametogenic production to proceed Since the s callop s us ed in the early spring trials were in a more a dvanced reproductive s tate at the s tart of the trials than those used in the fall it is pos sible that a period of time at an elevated temperature is also necessary in the spring but had occurred prior to sampling. Most, but not all, of the s callop s at high temperatures in the fall had entered into gametogenic development by the end of the trial. The gonad index during this fall period was only about half the value which wa s attained by ripe scallops in the spring. The initial value was lower but thi s may reflect the field observation that the fall spawn is smaller than the s pring s pawn in terms of the number of individuals involved and quantity of gametes released. Temperature s of 25 C for long periods of time result in negative impacts upon the calico scallop. Short term exposure to elevated temperatures may be beneficial but the scallops have diff i culty surviving prolonged, continuous exposure to this temperature for longer than 4 weeks. Observations of temperatures in the field indicate that value s in excess of 23 to 24 Care not normally present on the scallop grounds for longer than 3 to 4 weeks and are mo s t prevalent in the fall between September and December (Figure 9). Inability to with s tand high temperature s for prolonged periods may explain why the calico scallop is not often found in the nears hore F l orida waters inhabited by the Florida bay scallop. The mean monthly water temperature in the near-shore environment inhabited by the bay s callop in Florida exceeds 25 C from May through September and approaches 30 C during the s ummer months ( Barber I 984 ) 144

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Natural Reproductive Cycle The Florida calico scallop population spawns twice a year. The major spawn occurs in the spring between March and May. The exact timing of this spawn varies from year to year. Gametogenic development normally does not commence prior to January. All scallops are in a ripe condition by the end of March and contain large numbers of mature oocytes. The wate r temperatures on the scallop beds normally begin to increa s e during April and probably play a role in the initiation of s pawning. Once spawning has begun in a few individuals the presence of gametes within the water column apparently provides a chemical stimulu s that triggers spawning responses in mature individuals (Barber and Blake, 1991) The calico scallop may also spawn in the late summer or fall, generally in July or between September and November. The fall spawn is not as large as the spring spawn in terms of the number of individuals involved or the amount of gametogenic material which is released. In some years a second spawn is not observed probably due to improper environmental conditions Temperatures typically are minimal in late July and August with the intrusion of nutrient rich water onto the scallop beds which results in an increase in the available food supply. By September, temperatures begin increasing to maximal values for the year. These high temperatures are present for variable lengths of time into November. Thus spawning in the fall may also be associated with rising temperatures. 145

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Pathogenic Effects of Marteilia Upon the Calico Scallop The presence of the pathogen identified as a protistan of the genus Marteilia had not been identified in North American waters prior to identification in conjunction with the mortalities noted in the calico sca llop population in thi s s tudy Examination of pre se rved specimens indicate that this p aras ite had be e n pre se nt for almost two years prior to the fir s t evidence of high mortalities. Prevalence prior to the o bservation of mortality was extremely low and sporadic. Pathogens of this genus have been associated with mortality in other species in Europe and Australia but had not been documented in any other members of the Pectinidae family. Mortality within calico scallop s infected with Marteilia appears to occur very quickly. Populations were reduced to levels which precluded sampling within several months of the first mortalities noted in the area. Infection with the pathogen seems to spread very quickly within the ca lico sca llop population increasing from approximately 10% to almost 100 % of the population within le ss than a month. It is possib le albeit unlikely, that this pathogen i s a normal constituent in this area and species. The rapid and wide s pread mortality observed wou ld make the calico scallop an unsuitable host for the pathogen over time. The calico sca llop has a life span of less than two years and no evidence of this pathogen ha s been found in the histological examination of the almost I 000 anima l s collected during the four years preceding the first occurrence nor the more than 2,000 animal s collected in the four years since the last occurrence. Examination ha s failed to produce any alternative hosts for this pathogen 146

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among the mollusks in the area. Thus if this pathogen is normally present, the incidence within t h e scallop population must be extremel y l ow. If that is the case, the observed mortalities may be linked to changes in some aspect of the environment which allows for proliferation of the Marteilia within the calico scallop population. What those changes may be, i f they are indeed present, is not known. Conclusions I There are two spaw nin g periods in the calico scallop. The major spawn occurs in the sprin g, normally between March and May. A minor spawn occurs in the late summer or fall, typically in either July or between September and November. 2. Most of th e scallop s are in volved in the major spawn but only a portion of the population is involved in the minor spawn. In some year s the minor spawn is not observed. When the spring spawn occurs early in the year, the second spawn, if it occurs, is also early. 3. Spawning in the field occurs during periods when the bottom water temperature i s > 20 C following a period of time at lower temperatures. 4. Unlike the bay scallop the calico scallop i s able to undergo gametogenesis when less than three months o ld Two month o l d calico scallops can be fully ripe and contain mature oocytes and spermatozoa. It is unknown whether scallops of this s ize are able to produce viab l e game tes or larvae. 147

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5. In the bay sc allop both the maximum mean go nad index a nd oocyte diameter s in ripe sca llop s decline with d ec rea s ing latitude Comparison of th e max imum mean gonad index and oocyte diameters for th e calico sc allop in Florida with r e ported va lu es from North Carolin a indic a te s no differences betwe e n these populations. The l atit udinal decr eases observed in the bay sca llop s may not, therefore, be present in the calico scallop. 6. The reported leth a l limits for the calico scallo p need to be adjusted since the p r esent st ud y indicate s that the l ower l e th a l limit i s < 15 C and the upper l ethal lim it is< 27 C. Hi g h l eve l s of mortality are observed in calic o scallops m a intained at 25 C for l o n ge r th a n 3 to 4 weeks and scallops a ppe ar t o be able to wit h stand temperature s as l ow as a t l east 11 o C for ex te nd e d pe riod s of t ime. 7 In the sprin g incr ease d game to gen ic prod uction as d ete rmine d by go nad weights and indic es i s po s itively corr elated with rati o n and nega tiv ely corre l ated with temp erature Th e lar ge s t incr e a ses were obse r ve d in scallops maintained at 15 C w ith a hig h rati on The s ta ge of ga m etogenic deve lo pment atta in e d was identical a t a ll temperatur e a nd food l e v e ls. 8. Spawning was observed in the l a b in the l a t e sprin g at temperatures 20 C. There was n o evidence of s pawning amo n g anim a l s maintained at 15 C Spawning at this time may be pos iti ve l y corre l a t e d with ration. Not all of the indi v idu a l s at hig h temperatures in the ex p e rimental trial ex hibi ted evide nce of spaw nin g 148

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9 In the late summer some gametogenic development was observed among animals maintained at 25 C and possibly at 20 C. Thi s development was independent of the ration received by the scallops. No gametogenic development was observed in scallops held at I SO C This indicates an increase in temperature may be necessary in order for gametogenic development to proceed at this time of the year. There was little or no increase observed in the quantity of gametogenic material or gametogenic production during thi s period. 10. In the fall, so me maturation of oocytes wa s observed in all treatments, but gametogenic production was only observed in s callops maintained at 25 C or in scallops held at 20 C with increased ration There was no evidence of gametogenic production in scallops held at 15 C. 11. Gametogenic production during the fall was greatest with rising temperatures while gametogenic production during the spring was greatest with declining temperature. 12. The proximate cause of the massive mortalities observed in the calico scallop on the east coast of Florida during 1989 and 1991 was infection by a protistan of the genus Marteilia. This genus ha s not been reported in North American waters or as a parasite among scallops prior to the work conducted in this study. 149

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LIST OF REFERENCES Abbott, R. T. 1974. American Seashells. Van Nostrand Reinhold Company, New York, 663pp. Alderman, D. J. 1 979. Epizootiology of Marteilia refringens in Europe. Mar. Fish. Rev. 41: 67-69. Allen, D. M. 1979. Biological aspects of the calico scallop, Argopecten gibbus, determined by spat monitoring. The Nautilus 94(4): 107-119. Allen, D. M. and T. J Co s tello. 1972. The calico s callop, Argopecten gibbus. NOAA Technical report NMFS SSRF-656, 19pp. Andrews, J. D. 1988. Epizootiology of the disease caused by the oy s ter pathogen Perkinsus marin.us and its effects on the oy s ter industry. pp. 47-63. In: Disea s e Processes in Marine Bivalve Molluscs. W. S. Fisher (ed .) American Fisheries Society Bethesda Maryland, Special Publication 18. Andrews, J.D., J. L. Wood, and H D. Ho ese. 1 962. Oyster mort a lit y studies in Virginia. ill. Epizootiology of a disease caused by Haplosporidium costale Wood and Andrews. J. In sect Pathol. 4: 327-343. Ansell, A. D. 1974. Seas ona l changes i n biochemical composition of the bivalve Chlamys septemradiata from the Clyde Sea area. Mar. Bioi. 25: 85 -99. Ansell, A. D., J. C. Dao, and J Mason. 1991. Three Europea n scallops: Pecten maximus, Chlamys (Aequipecten) opercularis and C. (Chlamys) varia. pp. 715-751. In: Scallops: Biology, Ecology, and Aquaculture. S. E. Shumway ( ed.). Elsevier, New York. Atkinson, L. P. 1985. Hydrography and nutri ents of the southeastern U.S continental shelf. pp. 77-91. In: Oceanography of the Southeastern U.S. Continental Shelf, Coastal and Estuarine Science, vol. 2, L.P. Atkinson, D.W. Menzel, and K.A. Bush (eds.). AGU, Washington, D.C. 150

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Atkinson, L. P., T. N. Lee J. 0. Blanton and W. S. Chandler. 1983. Climatology of southeastern United States shelf waters. J. Geophys. Res 88: 4705-4718. Atkinson, L. P., L. J. Pietrafesa, and E. E Hofmann. 1982. An evaluation of nutrient sources to Onslow Bay, North Carolina. J. Mar. Res. 40: 679 -699 Auffret, M. and M. Poder. 1985. Recherches sur Marteilia maurini, parasite de Mytilus edulis sur les cotes de bretagne nord Rev. Trav. Inst. Peches marit. 47: I 05-109. Balouet G. 1979. Marteilia refringens: Consideration s of the life cycle and development of Aber disease in Ostrea edulis. Mar. Fish. Rev. 41: 64-66. Barber, B J. 1984. Reproductive energy metabolism in the bay scallop, Argopecten irradians concentricus (Say). Ph D. Thesis, University of South Florida, Tampa, 122 pp. Barber, B. J. and N.J. Blake. 1981. Energy storage and utilization in relation to gametogenesis in Argopecte n irradians concentricus (Say). J. Exp. Mar. Bioi. Ecol., 52: 121-134. Barber, B. J. and N.J. Blake. 1983. Growth and reproduction of the bay scallop, Argopec ten irradians (Lamarck) at it's southern distributional limit. J. Exp. Mar. Bioi. Ecol., 66: 247-256. Barber, B. J. and N.J. Blake. 1985. Intra -o rgan biochemical transformations associated with oogenesis in the bay scallop, Argopecten irradians concentricus (Say), as indicated by C 14 incorpor ation. Biological Bulletin 168(2): 39-49. Barber, B. J. and N.J. Blake. 1986. Reproductive effort and cost in the bay scal lop Argopecten irradians concentricus. Int. J. of Invert. Rep. and Dev. 10 : 51-57. Barber, B. J. and N.J. Blake. 1991. Reproductive physiology. pp. 377 -428 In: Scallops: Biology Ecology and Aquaculture. S. E. Shumway (ed.). Elsevier, New York. Barber, B. J., R. Getchell S. Shumway and D. Schick. 1988 Reduced fecundity in a deep-water population of the giant scallop Placopecten magellanicus in the Gulf of Maine, USA. Mar. Ecol. Prog. Ser. 42: 207-212. Barszcz, C. A. and P. P. Yevich. 1975. The use ofHelly's fixative for marine invertebrate pathology. Comp. Pathol. Bull. 7:4. 151

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Bayne, B. L. 1975. Reproduction in bivalve molluscs under environmental stress. pp. 259-277. In: Physiological ecology of estuarine organisms. F.J. Vemherg(ed.). University of South Carolina Press, Columbia, SC. Bayne, B. L. 1976. Aspects of reproduction in bivalve molluscs. pp. 432448. In: Estuarine Processes, Vol. I. M.L. Wiley (ed.). Academic Press, New York. Bayne, B L., P. A. Gabbott and J. Widdows. 1975. Some effects of stress in the adult on the egs and larvae of Mytilus edulis L. J. Mar. Bioi. Ass. U K. 55 : 675 -689. Bayne, B. L. and R. C. Newell. 1983. Physiological energetics of marine molluscs. pp. 407-515. In: The Mollusca, Vol. 4, Physiology, part J. K.M. Wilbur ( c d.). Academic Press, New York. Bayne, B. L., P. N. Salkeld and C. M. Worrall. 1983. Reproductive effort and value in different populations of the marine mussel, M y tilus edulis L. Oecologia 59: 1826. Bayne, B. L. and R. J. Thompson. 1970. Some physiological con sequences of keepin g Mytilus edulis in the laboratory. Helgolander Wiss Meeresunters 20: 526 552 Blake, N.J. 1972. Environmental regulation of neurosec reti o n and reproductive acti vity in the bay scallop, Aequipecten irradians Lamarck. Ph.D. disse rt a tion University of Rhode Island Kings ton, Rhode I s land, 161 pp. Blake, N.J. and M.A. Moyer. 1991. The calico scallop, Argopecten gihh uJ, fis hery of Cape Canaveral, Florida. pp. 899-911 In: Scallops: Biology, Ecology, and Aquaculture. S. E. Shumway ( ed.). Elsevier, New York Blake, N.J. and A. N. Sastry. 1979 N e urosecretory r e gulation o f oogenesis in the bay scallop, Argopecten irradian s irradians (Lamarck:). pp. 1 81-190. In: Cyclic Phenomena in Marine P lants an d A n imals. E T aylor and R G Hartnoll Pergamon Press, Oxford and New Y o rk Blake. S. G. and J E. Graves. 1995. Mitochon drial DNA varia tion in the bay %Callop, Argopect e n irradians ( La m arc k, 18 !9), an d the At lantic calico Argopect e n g ibb us (Li n naeus. 175 8 ) Journal Researclh t4{n; 79-85 B lanton, J. L. L. P Atkinson. L. J P ietrafesa, and T. N, Lee, 198L The intrn$ Ul01f11 of Gulf Stre a m w a ter across the c o ntinental shel f due to upwelling. Deep Sea Research 28(4) : 393--40.5, 1 52

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Bonami, J R. H. Griz el C. Vago, and J. L. Duth o it. 1971. Recherches sur un e maladie epizootique de l'hultre pl ate Ostrea edu lis Linne. Rev. Trav Inst. P ec hes marit. 3 5: 415-418 Brand, A. R 1991. Sc a llop Ecology: Dis tribution s and behavior. pp. 517-584. In : Scallop s: Biology Ecolo gy, and Aquacu ltur e. S. E. Shumway (ed.). E lsevi e r New York. Brehelin, M., J. R. Bonami, F C. Cousserans, and C. P. Vivares. 1982. Patholo g ie ani male-Exisrence d e formes plasmodiales vraies chez B onamia ost r eae p aras ite de l'hultre plate Ostr eae edulis. C. R. Hebd. Seance s Acad. S ci., Ser. ill 295: 45-48. Bricelj, V M ., J. Epp, and R E. M a l o u f. 1987a. Intraspecific variation in reproductive and s omatic growth cycles of bay s callops Argopecten irradians. M ar. Ecol. Prog. Ser. 36: 123-1 37. Bricelj, V. M., J. Epp and R. E. M a louf. 1987b. Comparative physiology of yo un g a nd o ld cohorts of b a y scallop Argopecten i rradi ans irr a dians ( L amarck) : Mortalit y, g rowth and oxygen consumption. J Exp. Mar. Bioi. Ecol. 1 12: 73-91. Bricelj, V. M. and S. Shumway 1991. Phy s iolo gy : Energy acquisition a nd utili z ation. pp. 305-346 In : Scallops: Bio lo gy, Ecology, a nd Aquaculture S E. Shumway (ed.). E l sev ier New York. Broom, M J. 1 976. Synopsis of biological d ata o n sca llo ps (Chlamys ( A equ ip ec t en) oper cularis ( Linn e), Argopecten irradians ( L ama r c k ), Argopecten gi bbus (L inna e us). FAO Fi s h. Synop. 114 44p p. Broom, M J a nd J. M aso n. 1 978. Growth an d spaw nin g in the p e ctinid Chlamys opercu l aris in relation to temper a tur e and ph y toplankton con cen trati o n. Marine Biology 47: 277 -285. Bull M. F. 1 976. A spec t s of the bi o l ogy of th e N ew Zealand scall op, P ec t e n novaezelandiae Reeve 1 853, in th e M a rlborou g h Sounds. Ph. D. Thes i s Victoria U niver s ity, W e llin gto n, New Zea l and. 175pp. Bullis H. R., Jr. a nd R. Cummins, Jr. 1961. An interi m report of th e Cape Canaveral scall o p bed. Commerc i a l Fi s h eries R ev iew 23(10 ): 1 -8 Bulli s, H R. Jr. a nd R. M In g l e. 1 959. A new f i s h e r y fo r scallops in western Florida. Proc. Gul f Carib b. Fi s h In st., II th Annu a l Session: 7578. 153

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Burnell, G. M. 1983. Growth and reproduction of the scallop, Chlamys varia (L.), on the west coast of Ireland. Ph.D. Thesis National University of Ireland, Galway, Ireland. 295 pp. Cahour, A. 1979. Mart eilia refringens and Crass os trea gigas. Mar. Fish. Rev. 41: 1920. Carpenter, J. S. 1967. History of scallop and clam explorations in the Gulf of Mexico. Commercial Fisheries Review 29: 47-53 Cason, J. E. 1950. A rapid one-step Mallory-Heidenhain stain for connective tis s ue Stain Technology 25: 225-226. Castagna, M.A. and W. P. Duggan 1971. Rearing the bay scallop, Aequipecten irradians. Proceeding s of the National Shellfisheries Association 61: 80-85. Cawthorn, R J ., R. J. MacMillan, and S. E. McGladdery. 1991. Epidemic of P seudok lossia sp. (Apicomplexa) in bay scallops Argop ecten irradians 14th Regional Fish Health Workshop, Nov 6-8, 1991 Halifax (Abst ract only). Chestnut, A. F. 1951. The oyster and other mollusks in North Carolina. pp. 141-190. In: Survey of Marine Fi s herie s of North Carolina. H. F. Taylor (ed.). The Univers ity of North Carolina Press, Chapel Hill, NC. Comely, C. A. 1974. Seasonal variations in the fle s h weights and biochemical content of the scallop Pecten maximus (L.) in the Clyde Sea area. J. Cons. Int. Explor. Mer 35:281-295. Camps, M 1970. Observation s sur les causes d'une mortalite anormale des hultres plates dans Ie bassin de M aren nes. Rev. Trav. In st. Peches marit. 34: 317-326. Camps, M 1976. Marteil ia lengehi n sp., para s ite de l'hultre Crassostrea cucullata Born. Revue des Trava ux de I Institut des Peches m a ritime s 40(2): 347-349. Camps, M. 1985. Etude morphologique de Mart e ilia christenseni sp. n. parasite du Iavignon Scrobiculari a piperata P. (Mollusque Pelecypode ). Rev. Trav. In st. Peches marit. 47: 99-104. Camps, M., H. Grizel G. Tige, and J. L. Duthoit. 1975. Parasite s nouveaux de Ia glande digestive des mollu s que s marins Mytilus edu lis L. et Cardium edule L. Comptes Rendus Hebdomadaire s des Seances de I'Academie des Science s, Serie D, Sciences N aturelles 281 : I 79-181. 154

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Yoder, J. A. 1985. Env ironm e nt al control of phytoplankton production on the southeastern U. S. continental s h elf. pp. 93-103. In: Oceanography of the Southeastern United States Continental Shelf, Coastal Estuarine Science, vol. 2. L. P. Atkin son eta/ (eds.). AGU, Was h ington D. C. Yoder, J. A. 1991. Role of Gulf Stream frontal eddies in forming phytop lankton patches on the southeastern shelf. Limnol. Oceano gr. 26: 1 103-1 1 10. Yoder, J. A., L. P. Atkinson, S. S. Bi shop, J.D. Blanton, T. N. Lee, and L. J. Pietrafesa. 1985. Phytoplankton dynamics within Gulf Stream intru sio n s on the southeastern United States continental shelf. Cont. Shelf Res. 4 : 611-635. Yoder, J. A., L. P. Atkinson, S. S. Bi shop, E E. Hofmann, and T. N. Lee. 1983. Effect of upwellin g on phytoplankton productivity of the outer southeastern United States continental shelf. Continental Shelf Researc h I: 385-404. Yoder, J. A., L. P. Atkinson, T. N Lee, H. H. Kim, and C. R. McClain. 1981. Role of Gulf Stream frontal eddies in forming phytoplankton patches on th e outer southeastern shelf. L imnol. Oceanogr. 26(6): II 03-1110. 16 8

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VITA Michael Alan Moyer i s originally from Nebraska He obtained his Bachelor of Art s with Honors from Cartha ge C ollege in Keno s ha, Wi scons in where he majored in Biolo g y and minored in Chemistry. While there he was active as a Stud e nt Government Senator Student Activities Board Vice-Pr eside nt and member of the Campu s Life student-facu lty committee. He was a lso a member of Honor s Fraternities in Biology Chemi s try and Theater. While pur s uin g hi s d octor ate Mr. Moyer received Fellowships and As s istant s hip s from th e University as well as Scho l arships a nd R esearc h Awards from the Sanibe l-Capti va Shell C lub Old Salt Fi sh in g C lub and Florida Power Squadron. He was selected for membership in the Omicron Delta Kappa Leadership Honor Society and twice by Outstanding Young M e n of America. He served as Chairm an of the USF St Peter s burg Lecture Serie s, Blood Dri ve Chairman, Student Go ve rnm e nt Senator and Parliamentarian He has al s o pre se nted pap e r s a t na t ional and intern a tion a l m ee tin gs


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