Reproductive energy metabolism in the bay scallop, Argopecten irradians concentricus (Say)

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Reproductive energy metabolism in the bay scallop, Argopecten irradians concentricus (Say)

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Title:
Reproductive energy metabolism in the bay scallop, Argopecten irradians concentricus (Say)
Creator:
Barber, Bruce J.
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Tampa, Florida
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University of South Florida
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English
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x, 122 leaves : ill., map ; 29 cm

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Bay scallop ( lcsh )
Dissertations, Academic -- Marine science -- Doctoral -- USF ( FTS )

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Thesis (Ph. D.)--University of South Florida, 1984. Bibliography: leaves 109-119.

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University of South Florida
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University of South Florida
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030076238 ( ALEPH )
12735631 ( OCLC )
F51-00164 ( USFLDC DOI )
f51.164 ( USFLDC Handle )

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REPRODUCTIVE ENERGY METABOLISM IN THE BAY SCALLOP, ARGOPECTEN IRRADIANS CONCENTRICUS (SAY) by Bruce J. Barber A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida December, 1984 Major Professor: Norman J. Blake

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Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Ph. D Dissertation This is to certify that the Ph.D. Dissertation of Bruce J Barber with a major in Marine Science has been approved by the Examining Committee on September 14, 1984 as satisfactory for the dissertation requirement for the Ph. D degree. Examining Committee: Lawrence < Torres E S Van Vleet Member: G A Vargo/

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Bruce J. Barber 1984 All Rights Reserved

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ACKNOWLEDGEMENTS I would like to thank committee members J.M. Lawrence, J.J. Torres, E.S. Van Vleet, and G.A Vargo for valuable advice and assistance on various aspects of this research. I am also grateful to J.J. Torres, G.A. Vargo, and G.E. Rodrick for providing laboratory facilities and equipment. Numerous individuals assisted in the collection of animals for this study; I am especially grateful to Paul Behrens, Dave "Dunk" Martin, and Gregg "Does It Deeper" Brooks. I would also like to thank Florida Power Corporation (St. Petersburg, Fl.) for providing water temperature data. This undertaking would not have reached fruition without the aid of N.J. Blake, my faculty advisor. He has provided both financial and spiritual support throughout my tenure as a graduate student. His insights into all aspects of life make him a mentor in the true sense of the word. Last but not least, I want to thank my wife, Laurie, for sharing my aspirations, providing moral support, and making numerous sacrifices on my behalf. ii

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TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW Growth and Reproduction Biochemical Composition Substrate Catabolism 14C Incorporation The Study Organism CHAPTER 3 : GROWTH AND REPRODUCTION Introduction Materials and Methods Results Discussion CHAPTER 4 : BIOCHEMICAL COMPOSITION Introduction Materials and Methods Results Discussion CHAPTER 5 : SUBSTRATE CATABOLISM Introduction Materials and Methods Results Discussion CHAPTER 6: 14c INCORPORATION Introduction Materials and Methods Results Discussion CHAPTER 7: DISCUSSION LIST OF REFERENCES iv v viii 1 4 4 6 9 12 13 1 7 17 18 22 29 38 38 39 41 49 56 56 57 60 68 74 74 75 77 86 96 109 APPENDIX : CRITIQUE OF BIOCHEMICAL PROCEDURES 120 iii

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LIST OF TABLES Table 1. Mean ( sd) dry weights (g) of mantle, digestive gland, adductor muscle, and gonad body components of A. irradians concentricus 24 Table 2. Mean ( sd) indexes (%) of mantle, digestive gland, adductor muscle, and gonad body components of A. irradians concentricus 26 Table 3. Lipid, glycogen, and protein levels (%dry wt.) of pooled adductor muscle, mantle, digestive gland, and gonad body components of A. irradians concentricus 42 Table 4. Mean ( sd) rates of bay scallop (A. irradians concentricus) oxygen consumption, carbon dioxide production, and ammonia excretion at the environmental temperature and salinity of the various sampling dates; (n=6) 61 Table 5. CPM mgDW-1 ( sd) for bay scallop irradians concentricus) digestive gland, adductor muscle, and female gonad body components 1, 4, 7, and 10 days after ingesting radioactive cells 79 Table 6. CPM mgDW-1 ( sd) lipid, carbohydrate, and protein for bay scallop irradians concentricus) digestive gland (DG), adductor muscle (AM), and female gonad (FG) body components 1 and 4 days after ingesting radioactive cells 82 Table 7. Energy content (J) of bay scallop irrndians concentricus) digestive gland, adductor muscle, mantle, and gonad body components (based on biochemical composition data); reproductive effort as the ratio of gonad energy content to total energy content x 100 107 iv

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LIST OF FIGURES Figure 1. Map of Anclote Estuary, where bay scallops irradians concentricus) were collected 19 Figure 2. Drawing of bay scallop (A. irradians concentricus) with left valve removed showing shell height measurement and body components; A, anus; AD, adductor muscle; DG, digestive gland; F, foot; FG, female gonad; G, gills; K, kidney; LP, labial palps; M, mantle; MG, male gonad; PC, pericardium; (after Sastry, 1979) 21 Figure 3. Mean monthly shell height of irradians concentricus and mean monthly water temperature of Anclote Estuary; all points represent the average of all means obtained within that month 23 Figure 4. Mean oocyte diameter of A irradians concentricus for the years 1979-1981; vertical lines represent 1 s d about the mean 27 Figure 5 Mean monthly oocyte diameters of A. irradians from Massachusetts, North Carolina, and Florida populations; Massachusetts and North Carolina data from Sastry (1970); Florida data presented as a monthly average of all means obtained within that month 31 Figure 6 Protein, glycogen, and lipid contents of pooled A. irradians concentricus adductor muscle tissue 44 Figure 7 Protein, glycogen, and lipid contents of pooled irradians concentricus m antle tissue 46 Figure 8. Protein, glycogen, and lipid contents of pooled A. irradians concentricu s digestive gland tissue 47 v

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Figure 9. Protein, glycogen, and lipid contents of pooled irradians concentricus gonad tissue 48 Figure 10. Relationship between adductor muscle glycogen content and mean oocyte diameter in A. irradians concentricus (Y = + 796.6; r = -0.83) 50 Figure 11. Mean oxygen consumption rate of A. irradians concentricus as a function of environmental temperature ( sd); (R = 0.031T + 0.074; r = 0. 79) 63 Figure 12. Mean carbon dioxide production rate of A irradians concentricus as a function of environmental temperature (1 sd); (R = 0.104T1.842; r = 0.78) Figure 13. Mean ammonia excretion rate of A irradians concentricus as a function of environmental salinity ( sd); (R = 2 .486S + 0.172.532; r = 0 .61) Figure 14. Seasonal variation in mean O/NH3 ratio of A. irradians concentricus (1 sd); fitted line is a third order polynomial, circled point excluded Figure 15. Seasonal variation in mean respiratory quotient (RQ) of A irradians concentricus ( sd); fitted line is a third order polynomial Figure 16. Relative seasonal incorporation of 14C by gonad, digestive gland, and adductor muscle body components of A. irradians concentricus Figure 17. Relative seasonal incorporation of 14C by lipid, carbohydrate, and protein fractions of A. irradians concentricus digestive gland tissue Figure 18. R elative seasonal incorporation of 14C by lipid, carbohydrate, and protein fractions of A. irradians concentricus adductor muscle tissue Figure 19. Relative seasonal incorporation of 14C by lipid, carbohydrate, and protein fractions of A. irradians concentricus female gonad tissue vi 64 65 66 67 80 85 87 88

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Figure 20. Schematic diagram showing the energy storage cycle in relation to the exogenous and endogenous factors regulating reproduction in A irradians concentricus (after Sastry, 1975) 100 Figure 21. Schematic diagram summarizing the reproductive energy metabolism of irradians concentricus; circled numbers indicate the order of mechanism invocation 104 vii

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REPRODUCTIVE ENERGY METABOLISM IN THE BAY SCALLOPt ARGOPECTEN IRRADIANS CONCENTRICUS (SAY) by Bruce J. Barber An Abstract Of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida December, 1984 Major Professor: Norman J. Blake viii

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Reproductive energy metabolism, the manner in which the major lipid, carbohydrate, and protein substrates are used in relation to gamete production, was investigated for a Florida population of the bay scallop, Argopecten irradians concentricus (Say). Seasonal studies on growth and reproduction, biochemical composition, substrate catabolism, and 14c incorporation identified locations within scallop body components of specific energy substrates and revealed the timing of substrate utilization with respect to specific gametogenic events. A. irradian s concentricus exhibited a seasonal cycle of energy storage and utilization in response to the reproductive process. During the resting stage, somatic body components (digestive gland, adductor muscle, and mantle) increased in weight and protein content. Adductor muscle glycogen level steadily increased, and maximal digestive gland lipid level began to decrease, coincident with the initiation of oogenesis in June. Overall metabolism at this time was primarily supported by digestive gland lipid catabolism. Shortly after the onset of oocyte cytoplasmic growth in July, maximum somatic body component weights and protein contents were attained and adductor muscle glycogen level began to decrease. Adductor muscle glycogen was the major catabolic substrate during ix

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this period. Vitellogenesis occurred in September, as the gonad exhibited gains in weight and lipid and protein contents and oocytes increased in number and diameter. Adductor muscle glycogen utilization was followed by a steady decrease in adductor muscle weight and protein content. Metabolic energy during this phase was supplied primarily by adductor muscle protein catabolism. Spawning in early October was preceeded by maximum oocyte diameter. Digestive gland lipid provides the threshold level of nutrients required for the initiation of gametogenesis in A. irradians. Both digestive gland lipid and adductor muscle glycogen provided material for oocyte production. Adductor muscle protein is catabolized only after the other stores are depleted and supports reproduction indirectly. The utilization of adductor muscle protein may represent an adaptation to a latitudinally changing e nergetic situation that results in Florida scallops having smaller oocytes and a shorter life-span than more northern populations. This in turn may ultimately limit the southern distribution of A. irradians t o west central Florida. Abstract approved: Norman J. Bf?ke Professor of Marine Science Date of Approval X

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1 CHAPTER 1: INTRODUCTION Many marine bivalves exhibit cycles of energy storage and utilization that are closely linked to annual reproductive patterns (Giese, 1959, 1966, 1969; Bayne, 1976; Sastry, 1979). Although the specifics of these cycles are species and location dependent, it has generally been found that with an adequate food supply lipid, carbohydrate, and/or protein substrate is accumulated in one or more body components prior to gametogenesis and subsequently utilized during the energy demanding gametogenic process. The initiation of egg and sperm growth appears to require the accumulation of a threshold level of nutrient reserve (Gabbott and Bayne, 1973; Sastry, 1975) that serves to remove the gametogenic process from an uncertain food supply (Bayne, 1976). The study of reproductive energy metabolism is concerned with the ways in which organisms cope with the energetic demands posed by reproduction. It deals with the manner in which the major lipid, carbohydrate, and protein fuels are used by an organism for gamete production. It involves not only the biochemistry and physiology of the animal as a whole, but with the inter-relationships between the different body tissues, their energy requirements, and

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the metabolic transform a tion of the energy reserves as related to repro du ction. For marine bivalves, the relationships between recently ingested food, nutrient reserves, and gonad development are unclear. Observed inverse correlations 2 between digestive gland and gonad body component indexes s uggest that reserves may be transferred directly from the digestive gland t o developing ova (Sastry, 1968; Sastry and Blake 1 971; Vassallo, 1973). The depletion of glycogen reserves during vitellogenesis suggests that this substrate may be converted to lipid which is then incorporated into gonadal material (Gabbott, 1975, 1976). Gametogenesis could a lso take place indirectly at the expense of protein reserves if the supply and/or composition of incoming food i s not a dequate for supportin g maintenance metabolism during the reproductive period (Gabbott and Bayne, 1973). In spite of the importance of these types of relationships to the reproductive process, relatively little investigation of bivalve reproductive energy metabolism has been attempted. The aim of this research was to investigate the reproductive e nergy metabolism of the bay scallop, Argopecten irradians concentricus (Say). The project involved four separate but inter-related seasonal studies, with the overall goal of determining the cycle of energy storage and utilization that accompanies reproduction in the bay scallop. The accomplishment of this goal provided

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information complementary to current knowledge concerning the factors regulating gametogenesis in this species. 1) Growth and reproduction 3 Body component indexes and tissue weights identified periods of tissue formation (somatic and germinal) and degradation, thereby giving a general indication of energy storage location and the timing of utilization of these stores with respect to reproduction. Histological examination of gonads provided a quantitative means of assessing gamete development. 2) Biochemical composition Analysis of body component biochemical compositions identified the locations and amounts of specific substrates (lipid, carbohydrate, protein) within the animal and how they were stored and utilized in relation to reproductive events. 3) Substrate catabolism Physiological measurements resulting in O/NH3 and C02/02 (RQ) ratios were used to identify sources of metabolic energy over the course of the reproductive cycle. 4) 14c incorporation A radiotracer (14c) provided a means of directly monitoring energy substrate gain and loss (storage and utilization) within individual body components of the organism over its gametogenic cycle.

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CHAPTER 2 : LITERATURE REVIEW Growth and Reproduction Growth is an increase in weight that occurs when assimilated energy exceeds what is required for maintenance. Since molluscs have no localized nutrient 4 storage depots such as the glycogen-storing vertebrate liver, the subdermal and omental adipose tissue of mammals, or the fat bodies of lower vertebrates, they store energy rather generally in various body components (Giese, 1969). The body component index (Giese, 1959) represents the proportion of the whole animal that each component occupies at any one time. For bivalve somatic tissue, an increase in body component weight indicates energy substrate storage, while a decrease indicates utilization or transfer. For germinal tissue an increase indicates reproductive development and a decrease indicates spawning. Body component indexes have been used to monitor growth and thus determine the relationships between growth and reproduction in several species of marine bivalves. Sastry (1966a, 1970) found reciprocal relationships between digestive gland and gonad indexes of Argopecten irradians from Massachusetts and North Carolina populations, with the Massachusetts relationship being more

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pronounced; adductor muscle indexes decreased during the latter portion of the reproductive cycle (Sastry, 1979). In Tivela stultorum all somatic body component indexes (wet weight basis) remained unchanged during the breeding season while the gonad index increased 1967). Placopecten magellanicus adductor muscle and 5 digestive gland indexes decreased at the same time that the gonad index was increasing (Robinson et 1981). Seasonal changes in body component indexes should be viewed in conjunction with actual body component weights, as a change in index does not necessarily signify a change in actual tissue weight. The adductor muscles of Chlamys septemradiata (Ansell, 1974b) and C. opercularis (Taylor and Venn, 1979) showed clear seasonal cycles in tissue weight, with a decrease occurring over the period of gonad differentiation. The gonad of Pecten maximus increased in weight at the same time that somatic tissues decreased in weight (Comely, 1974). Digestive gland and adductor muscle weights decreased in conjunction with gonadal weight increases for Patinopecten yessoensis (Fuji and Hashizume, 1974) and Placopecten magellanicus (Robinson 1981). Cycles of growth and reproduction in marine bivalves are related in that gonad growth, which is an indication of an increase in both the size and number of gametes, is generally separated in time from somatic growth. Increase in gonad weight and index is usually accompanied by a

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concurrent decrease in the weights and indexes of one or more somatic body components, suggesting that the utilization of certain stored reserves is involved in the reproductive process. Biochemical Composition 6 Growth is a form of energy storage in that accumulation of the lipid, carbohydrate, and protein substrates in growing body tissues can be utilized at a later date to satisfy metabolic requirements. Triacylglycerols, sterol esters, and hydrocarbons frequently reach storage levels in lamellibranch d igestive gland and ovarian tissues (Lovern, 1964; Giese, 1966, 1969; Lawrence, 1976; Sargent, 1976). The most prominant carbohydrate stored in marine bivalves is glycogen, with considerable amounts accumulated in mantle, adductor muscle, digestive gland, and gonad tissues (Galtsoff, 1964; Giese 21_., 1967; Walne, 1970; Robinson 21_., 1981). Protein, although not deposited intracellularly like lipid and carbohydrate stores, can also serve as an e nergy source. Its utilization is associated with cellular destruction of non-mantle (Gabbott and Bayne, 1973) and adductor muscle (Adachi, 1979; Taylor and Venn, 1979) tissues. Many studies have investigated seasonal changes in whole animal biochemical composition. The earliest studies of this type have been reviewed by Korringa (1952),

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Galtsoff (1964), G iese (1969), and Walne (1970). More recent studies include those of Donax vittatus (Ansell, 1972), Abra alba (Ansell, 1974a), Nucula sulcata (Ansell, 1974c), Tellina tenuis (Ansell and Trevallion, 1967), a nd Mytilus edulis (Dare and Edwards 1975 ; Pieters et 1979). The general finding was that carbohydrate reached maximum levels early i n the gametogenic cycle and decreased toward the time of spawning 7 Studies of seasonal v a riations in the biochemical composition of individual body components have been less numerous but have provided considerably more i nformation regarding the r e lati o n s h i p s between nutrient allocation a nd reproduction i n marine bivalves. In Mercenaria mercenaria carbohydrate levels in t h e foot, mantle, and siphons decreased with a corresponding increase in gonad protein and lipid levels during gametogenesis ( A nsell et 1964). Tivela stultorum gonad and digestive gland carbohydrate levels a nd digestive gland lipid leve l reached maxima prior to peak reproductive activity a n d declined as gonad lipid a nd protein levels increased duri n g the latter stages of gametogenesis . 1967). Reid (1969) found that a n increase in the gonad lipid level of Tresus capax during gametogenesis was accompanied b y decreases i n go n a d glycogen and digestive gland lipid levels. G lycogen and protein in the m antle were utilized by Mytilus edulis during gametogenesis

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8 (De Zwaan and Zandee, 1972; Gabbott and Bayne, 1 973; Bayne, 1976; Zurburg 2.l, 1979); digestive gland glycogen apparently replaced mantle glycogen reserves after spawning (Thompson 2.l, 1974). Mori (1975) found that d igestive gland lipid and adductor muscle glycogen levels decreased prior to gonad development in Patinopecten yessoensis. In Chlamys septemradiata (Ansell, 1974b), Pecten maximus (Comely, 1974), and Chlamys opercularis (Taylor and Venn, 1979), adductor muscle glycogen and protein contents declined while gonad lipid content increased. Adachi (1979) found that the adductor muscle protein level in Tapes philippinarum decreased by two-thirds during the period of gonad growth. Pollero et 2.l (1979) noted a reciprocal relationship between gonad and other tissue lipid contents in Chlamys tehuelcha. Digestive gland lipid and carbohydrate and adductor muscle carbohydrate contents in Placopecten magellanicus were depleted as gametes matured and gonadal lipid content reached a maximum (Robinson et 2.l 1981). What is apparent from these studies is that marine bivalves have seasonal biochemical cycles of energy storage and utilization that are closely related to reproductive cycles. Nutrient reserves are accumulated in various body tissues during periods of somatic growth when food is abundant. Carbohydrate (mainly glycogen) appears to be of primary importance, but lipid and protein reserves a lso

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play important roles. Reserves from one or more body components may subsequently be catabolized to support maintenance metabolism when food is scarce and/or the production of gametes, depending upon the timing of reproduction in relation to seasonal food supplies. Substrate Catabolism 9 Seasonal body component weights, indexes, and biochemical compositions, while providing quantitative measurements of when and where particular energy reserves are being stored and utilized, give no indication of relative rates of substrate accumulation and depletion. Rates of oxygen consumption, carbon dioxide production, and ammonia excretion, however, when converted to O/NH3 and C02/02 molar ratios, do provide indexes of the catabolic balance between protein, carbohydrate, and lipid substrates within an animal (Richardson, 1929; Corner and Cowey, 1968; Mann, 1978). The ratio of moles oxygen consumed to moles ammonianitrogen excreted provides an index of the relative amount of protein to non-protein (lipid, carbohydrate) catabolism occurring at a particular time. If the amino acids resulting from protein catabolism are deaminated and totally excreted as ammonia while the carbon skeletons are fully oxidized to carbon dioxide and water, the theoretically minimum O/NH3 ratio is 9.33 (Bayne, 1973a). Anything higher than this means that relatively

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more non-protein (lipid and carbohydrate) substrate is being catabolized. The O/NH3 ratio has been used sparingly in the study of marine bivalve physiology. For M ytilus edulis, O/N H 3 ratios were found to be greater than 10 300 just prior to gametogenesis but 70-110 over the rest of the year (Bayne and Th ompson, 1970; Bayne, 1973a, 1973b; Gabbott and Bayne, 1973). Also, the O /NH3 ratios of Donax vittatus and M. edulis were lower for starved a nimal s than fed animals (Ansell and Sivadas, 1 9 73; Bayne, 1973b) Additional information on the relative contribution of t h e variou s s ubstrates to energy metabolism is provided b y the respiratory quotient (RQ), defined as the ratio of the moles of carbon dioxide produced to the moles of oxygen consumed (Richardson, 1929). When carbohydrate is oxidized, all of the oxygen utilized form s carbon dioxide, so for every mole of oxygen consumed, o n e mole of carbon dioxide results, givin g an RQ of 1 0 When substrates other than carbohydrate are catabolized, som e of t h e o xygen form s water, and the RQ is less than 1.0 about 0 79 for protein and 0 71 for lipid. A value greater than 1 0 indicates a carbohydrate t o lipid conver s ion (Gabbott, 1975; Mori 1968, 1975) and a value less than 0 6 s uggest s t h e conversion of lipid to carboh ydrate (Mori, 1968). Due to the difficulty of accuratel y measuring carbon dioxide in seawater, few RQ values have been obtained for

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marine bivalves. 11 Bruce (1926) found monthly RQ values for M edulis to vary from a low of 0 25 after spawning to a high of 1.31 coincident with the accumulation of carbohydrate prior to gametogenesis. The RQ of Crassostrea virginica varied between 0.51 and 1.44 over a year, but showed no definite seasonal trend (Galtsoff, 1964). Mori (1968) investigated the RQ of various tissues of C. gigas a n d found that gill tissue RQ remained close to 1 0 all year, digestive gland tissue RQ was 1 5 prior to spawning and dropped to 0 7 during spawning and less than 0 6 after spawning, and pallial margin tissue RQ was between 1.0 and 2 0 during the early stages of sexual maturation. For Patinopecten yessoensis digestive gland tissue, RQ fell from 1 3 to 0.7 during the period of gonad development (Mori, 1975). The physiological indexes O/NH3 and RQ complement the biochemical compo sition work by indicating relative levels of substrate catabolism. It appears that both indexes are highest prior to and during the initial stages of gametogenesis, indicating a predominantly carbohydrate based catabolism. Both indexes tend to decline as gametes mature and spawning aproaches, indicating increased lipid and protein catabolism. Minimum indexes occur after spawning in conjunction with recovery and the start of another cycle.

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12 Incorporation Seasonal changes in body component weights and biochemical compositions and physiological indexes provide only circumstantial data with respect to energy metabolism. Monitoring the distribution of a radiotracer within the body of an animal over time, however, can provide direct information on the allocation of tracer material relative to a particular function. The specific radiotracer, its molecular configuration, its method of introduction, and its method of detection can be tailored to best attack a particular problem. Radiotracers have been used for the examination of nutrient utilization and distribution in relation to gamete growth in only a few marine bivalve species. Allen (1962, 1970) fed 32p (orthophosphate) in both dissolved and particulate form to several species, including Venus striatula and Mytilus edulis, and found the digestive gland to be the main site of assimilation, with concentrations falling with time as it was distributed to other tissues; V. striatula with maturing gametes showed a slightly higher amount of 32p in the gonad relative to those with either immature or fully developed gametes. A transfer of injected 14c leucine from the digestive gland to the gonad in Argopecten irradians was associated with oocyte development (Sastry and Blake, 1971). Vassallo (1973) cannula-fed 14c lipid (Chlorella extract) to Chlamys hericia and found

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that lipid activity in the gonad increased as digestive gland lipid activity decreased. Thompson (1972) fed 14c labeled algal cells to edulis during the 13 time of nutrient storage and found that over a four-day period, loss of activity in the digestive gland was matched by a gain in mantle and adductor muscle tissues. When M edulis undergoing gametogenesis was fed 14c labeled food, extraction of the lipid fraction from spawned ova revealed that 24% of the total radioactivity was in the lipid fraction, with 62% of that being in the form of triglyceride (Bayne, 1975). Results of studies employing radiotracer methodology have directly reinforced some of the relationships between energy metabolism and reproduction that previously could only be surmised. Nutrients from ingested food are first assimilated in the digestive gland. They are then transferred to storage locations during periods of somatic growth where they accumulate until reproduction begins. During gametogenesis these reserves, along. with some portion of recently ingested nutrients from the digestive gland are mobilized and incorporated int o gonadal tissue. The Study Organism The bay scallop, Argopecten irradians (Lamarck) is an epifaunal filter feeder that inhabits shallow, protected estuaries from Cape Cod to the northern Gulf of Mexico (Gutsell, 1930). Three living taxa of this superspecies

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are recognized along this range: irradians irradians (Lamarck) from Massachusetts to New Jersey, A. irradians concentricus (Say) from New Jersey to South Carolina and western F l orida to eastern Texas, and A. irradians amplicostatus (Dall) from central Texas to Mexico (Clarke, 1965; Waller, 1969). The life history of irradians has been reviewed 14 by Risser (1901), Belding (1910), and Gutsell (1930). Its reproductive cycle is characterized by functional hermaphroditism, a high fecundity, and a high degree of synchrony within a population. Spawning commences in June and July in Massachusetts (Sastry, 1966b, 1970) and Rhode Island (Risser, 1901), late July in North Carolina (Gutsell, 1930; Kirby-Smith, 1970), and early August in northern Florida (Sastry, 1961). Larvae are planktonic and reach late veliger stage (184 12 days after fertilization; settlement by prodissoconchs (190 pm) occurs 15 days after fertilization (Sastry, 1965). Spat will attach to various substrates, but viable scallop populations are most often associated with seagrass beds (Gutsell, 1930; Dreyer and Castle, 1941; Marshall, 1947). Growth i s rapid, with scallops from Rhode Island reaching 55 mm in shell height by age 6-7 months and 85 mm at age 18-20 months (Risser, 1901). In North Carolina, the maximum mean shell height of 65-70 mm is attained by age 18 months (Gutsell, 1930; Kirby-Smith, 1970), and a maximum mea n shell height of 65 mm is attained by Florida scallops

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15 by age 12-14 months (Sastry, 1961). Although a few scallops may live 24 months and spawn twice, the vast majority spawn only once at age 12 months and live less than 20 months (Risser, 1901; Belding, 1910; Gutsell, 1930; Marshall, 1960). The factors regulating reproduction are better understood for the bay scallop than any other marine bivalve. The duration of the resting stage and the minimum age at which gonad growth begins are most likely genetically fixed for a population (Blake, 1972; Sastry, 1975). However, once minimum age, temperature, and food supply criteria are met, oocyte growth is initiated in conjunction with t h e transfer of nutrients from the digestive gland (stored during the resting stage) to the gonad (Sastry, 1963, 1966a, 1968; Sastry and Blake, 1971). Once the gonad has accumulated a certain amount of gametogenic material, oocyte development to maturation is independent of food supply, provided temperatures remain above the required minimum (Sastry, 1975). After spawning, a decrease in water temperature and available food seems to limit the duration of the spawning period (Sastry, 1968). A feedback control mechanism linking a neurosecretory cycle with the various gametogenic stages appears to regulate the transfer of reserves to gonads and the normal growth of oocytes to maturation (Blake, 1972; Blake and Sastry, 1979; Sastry, 1975, 1979). Over its geographical range, the reproductive cycl e of

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A. irradians varies both as the result of genetic divergence and as the phenotypic response of a singl e genotype. Successive events in the reproductive cycle of 16 Massachusetts and North Carolina scallop populations occur at different times of the year and at different environmental temperatures (Sastry, 1970, 1979). Scallops from Massachusetts transplanted to North Carolina failed to initiate gametogenesis, s uggesting that variation in t em perature requirements for gonad growth is genetically different for the two populations (Sastry, 1966b). However, such differences in temperature requirements do not exist between North Carolina and north Florida populations, indicating that s ligh t differences in the reproductive cycles of these populations are non-genetic adaptions to environmental differences (Sastry, 1961, 1979).

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CHAPTER 3: GROWTH AND REPRODUCTION Introduction Reproductive success i s closely linked to the environment and ultimately determines the geographical range of a species. As a result, variations in the 17 reproductive cycles of marine invertebrate species often accompany change in latitude (Giese, 1959; Vernberg, 1962; Giese and Pearse, 1974; Sastry, 1975). These variations are the result of intraspecific physiological differences caused by genetic divergence, phenotypic adaptation, or a combination of both (Sastry, 1979). It was originally postulated that marine bivalves have a species-specific temperature at which breeding begins and that this temperature i s c onstant over the entire geographic range (Orton, 1920; Nelson, 1928). Subsequent investigation revealed exceptions to this general rule (Loosanoff and Nomejko, 1951; Korringa, 1957), suggesting that factors other than temperature se are involved in the regulation of reproduction. For the bay scallop, A. irradians, food and temperature criteria have to be met before gonad growth is initiated (Sastry, 1963, 1966a, 1968). Thus, scallops from North Carolina initiate gametogenesis later in the year and at a higher temperature

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18 than scallops from Massachusetts (Sastry, 1966b, 1970). Little is known about the life history of the bay scallop in Florida. The present study defines the seasonal cycle of growth and reproduction of the bay scallop in Florida at the extreme southern limit of its distribution. Particular attention is given to the relationships between body componen t weight fluctuations and gametogenesis, thereby allowing a more complete analysis of the latitudinal variation in scallop reproductive energy metabolism over its entire geographic range. Materials and Methods For this and all subsequent sections, scallops were hand collected by divers from the Anclote Estuary near Tarpon Springs, Florida (Figure 1), a n d returned to the laboratory in aerated containers. The collection of scallops was dictated by their size and availability. In May, first-year scallops were about six months old a n d large enough to initially discern in the seagrass. Shortly after spawning in October, mortality of the population prevented further collection. For this portion of the study, 20-30 scallops were collected between May and November, in the years 1979-1981 In 1980 and 1981 bottom water temperature at the collection site was continuously recorded with a Ryan J Thermograph, and monthly averages were calculated. Mean scallop shell height, the dorso-ventral

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.... 1 km ,.:.. .::.'17 .... ..._lb .._o .. e> 82 50' Figure 1 Map of Anclote Estuary, where bay scallops irradians concentricus) were collected 19

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20 measurement along a straight line tangential to the curvature of either valve (Figure 2), of 20-30 individuals was measured to the nearest 0.1 mm with vernier calipers. Mean body component dry weights and mean body component indexes (Giese, 1959), defined as I= (dry wt of body component/ dry wt of entire animal) X 100, were calculated. Ten scallops were divided into gonad, digestive gland, adductor muscle, and mantle (remaining tissue) body components (Figure 2), with each component being placed into a tared, aluminum weighing pan and dried to a constant weight at 60 C. Reproductive development was monitored histologically. Six to 10 scallops were removed from their shell, bisected sagitally, and fixed for 16-20 h in Helly's fixative made with zinc chloride (Barszcz and Yevich, 1975). Scallop halves were trimmed, placed in cassettes, and rinsed for 16-24 h in a self-siphoning water bath. Cassettes were processed through six changes of S-29 dehydrant, three changes of UC-670 clearing agent, and two changes of liquid Paraplast in an Autotechnicon-Duo (Technicon Instruments Corp.). Gonad sections were embedded in Paraplast, sectioned (6-8 pm) using a Spencer 820 rotary microtome, and stained with hematoxylin and eosin (Luna, 1968). Mean oocyte diameter and gametogenic stage were determined by measuring the maximum diameter of 50 oocytes from each scallop, using a compound microscope with an ocular micrometer (Sastry, 1966a; Blake, 1972). Observations on

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LP F K MG FG G 21 DG PC AD M SHELL H E IGHT A Figure 2. Drawing of bay scallop irradians concentricus) with left valve removed showing shell height measurement and body components; A, anus; AD, adductor muscle; DG, digestive gland; F, foot; FG, female gonad; G, gills; K, kidney; LP, labial palps; M mantle; MG, male gonad; PC, pericardium; (after Sastry, 1979)

PAGE 34

22 spermatocyte development were also made. Results Mean shell height increased from an early May value of 39.9 mm to over 60. 0 mm by August (Figure 3). However, from the latter part of August into November, scallop shell heights remained relatively constant, averaging between 60.0 and 65.0 mm. The period of rapid somatic growth indicated by the initial increase in mean shell height was also seen as an increase in the mean dry weights of the mantle, digestive gland, and adductor muscle body components (Table 1). From May to August mean mantle dry weight increased from 0.26 to 0 .89 g, mean digestive gland dry weight increased from 0.17 to 0.61 g, and mean adductor muscle dry weight increased considerably, going from 0.41 to 2.08 g. From early August onward, the mantle and digestive gland mean dry weights remained relatively constant, around 0.76 and 0.57 g, respectively. However, after early August the adductor muscle exhibited a steady and rapid decrease in mean dry weight, reaching a minimum of 0 .68 g by early November, less than one third its August maximum. In contrast to somatic growth, gametogenic growth, as reflected by mean gonad dry weights of less than 0.1 g, was relatively low in May and June. Increase in mean gonad dry weight was more apparent between July and September, with a maximum value of 0.36 g being reached in early October

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E E '-' I w I w I 00 z < w 0 70 0 '-' w 60 < 50 w 40 w 30 w < 20 z 1 0 < w J F M A M J J A s 0 N MONTH Figure 3. Mean monthly shell height of A. irradians concentricus and mean monthly water temperature of Anclote Estuary; all points represent the average of all means obtained within that month 23 0

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24 Table 1 Mean ( 1 sd) dry weights (g) of mantle, digestive gland, adductor muscle, and gonad body components of A. irradians concentricus Date Hantle Dig. Gland Ad. Muscle Gonad 9 May 81 0 .26 .08 0 .17 .08 0.41 .22 0 05 03 17 May 79 0 .38 .08 0.18 .04 0 .60 .21 0 .04 .01 31 May 79 0 .40 .07 0.21 .04 0 .64 .19 0 06 .01 14 June 79 0.55 .06 0.39 .05 1.17 .17 0 09 .03 19 June 81 0 .39 .05 0.23 03 0 .66 .09 0 05 .01 28 June 79 0.66 .08 0.49 .07 1.49 .19 0.11 .04 6 July 80 0.49 .06 0.33 .06 0.92 .22 0.09 .04 11 July 79 0 66 .06 0.49 .06 1.33 .21 0 .20 .05 16 July 81 0 .53 .13 0.34 .08 0.90 .29 0 06 .01 26 July 79 0 .68 .15 0 45 .13 1.46 .43 0 .15 .08 9 Aug 79 0 .89 .11 0 .61 .1 0 2 08 .38 0 .20 05 14 Aug 80 0 .81 .12 0 .58 06 1. 76 .48 0 .23 .04 24 Aug 79 0 .82 .13 0 .50 .06 1.61 .32 0 .16 03 28 Aug 81 0.80 .14 0.60 .11 1.69 53 0 .24 .07 7 Sept 79 0.81 .15 0.60 .16 1.40 .33 0 .24 .11 11 Sept 80 0.78 .10 0.60 08 1.57 32 0.32 .07 22 Sept 81 0 .80 .11 0 .62 .13 1.53 .31 0.32 .06 26 Sept 79 0 71 .05 0.52 06 1.07 23 0 .27 .07 8 Oct 81 0 82 .12 0 .62 .05 1.18 .21 0 30 .03 10 Oct 79 0.66 .13 0.55 .10 0 .92 .33 0.36 .10 17 Oct 79 0.64 .12 0 .55 .12 0.69 .16 0.25 .07 31 Oct 79 0 .67 .09 0.57 .04 o. 73 .16 0.26 .04 4 Nov 81 0.66 .03 0.55 .07 0.68 .11 0 .28 .09

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25 (Table 1). Mean gonad dry weight decreased after early October, indicating that spawning had commenced. The rapid decrease in mean adductor muscle dry weight that occurred in conjunction with the increase in mean gonad dry weight is reflected more clearly by the body component indexes (Table 2). For the period of somatic growth, May through early August, all body components maintained a relatively constant proportion of the whole animal. The mean adductor muscle index remained about SO%, the mean mantle index about 27%, the mean digestive gland index about 17%, and the mean gonad index about 5%. However, from late August through early October, during the period of greatest gametogenic growth, the mean adductor muscle index fell from 54.6 to 35.9% while the mean gonad index increased from 5.2 to 14. 5%. Mean mantle and digestive gland indexes during the reproductive period remained essentially constant, about 27 and 21%, respectively. Mean oocyte diameters (Figure 4), reinforced the information obtained from the mean gonad dry weights and indexes. During May and June, little ovarian development took place as most follicles were completely undifferentiated or contained primar y germ cells or oogonia. Overall mean cell diameters were less than 15 with large standard deviations reflecting the variation between animals. By July oogonia were developing into oocytes and undergoing cytoplasmic growth, as mean oocyte

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26 Table 2 Mean ( 1 sd) i ndexes (%) of mantle, digestive gland, adductor muscle, and gonad body components of irradians concentricus Date Mantle Dig. Gland Ad. Muscle Gonad 9 May 81 30.9 .6 19.2 .6 44.7 .5 5.1 .0 17 May 79 32.2 .4 15. 3 .3 48.5 .9 3.9 .6 31 May 79 31.0 .9 16.6 8 47. 5 .2 4.8 0 14 June 79 25.0 .6 17.8 .0 52. 8 .5 4.3 2 19 June 81 29. 4 .3 17.0 .4 49. 8 .4 3.8 .4 28 June 79 23.8 .4 18.0 9 54.0 2 4.2 3 6 July 80 27. 1 .4 17.9 3 49.9 .9 6.3 4 11 July 79 24. 8 .2 18.5 .8 49.4 .5 7.3 .3 16 July 81 29. 1 .6 18. 6 7 48.9 2 3 5 6 26 July 79 25. 1 .2 16.6 .8 52.9 .8 5 4 .3 9 Aug 79 23. 8 .2 16. 2 .2 54. 6 .1 5 4 .0 14 Aug 80 24. 2 .8 17. 4 .8 51.6 .6 6.8 .5 24 Aug 79 26.7 2 16.4 .2 51.8 .1 5.2 .8 28 Aug 81 24.6 .8 18. 5 .2 49 6 .9 7.3 .2 7 Sept 79 27.0 8 19. 8 .6 45. 6 .2 7.7 4 11 Sept 80 23. 9 .2 18.4 .2 47 7 0 10. 0 6 22 Sept 81 24. 7 1 19.0 .1 46.4 .8 9 9 7 26 Sept 79 27.8 6 20.4 .8 41.3 8 10.4 .0 8 Oct 81 27. 9 .5 21.5 .1 40.3 .6 9.6 .8 10 Oct 79 26. 9 .4 22.7 .7 35. 9 3 14.5 7 17 Oct 79 30. 3 .0 25. 9 .8 32. 0 .6 11.9 .8 31 Oct 79 30. 1 .3 25. 7 .5 32. 5 .0 11.7 5 4 Nov 81 30.7 .2 25.3 .2 31.4 .1 12.7 .1

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,..... E :::1. 0: w 1w <( 0 w I->(.) 0 0 z <( w 40 30 20 10 j EARL v ooGENESIS I CYTOPLASMIC GROWTH lvtTELLOGENEstsl SPAWNING MAY JUNE JULY AUG SEPT OCT NOV MONTH Figure 4. Mean oocyte diameter of A. irradians concentricus for the years 1979-1981;-vertical lines represent 1 sd about the mean 27

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28 diameter exceeded 20 Oocytes continued to grow, with vitellogenesis, as evidenced by vitelline membranes, occurring in September. A maximum mean oocyte diameter of 38 was attained in late September. Spawning commenced in early October as evidenced by a sharp decrease in mean oocyte diameter, as mature oocytes became proportionally less numerous. Differentiation of the testicular portion of the gonad was much more rapid than in the ovarian portion. Spermatogonia and spermatocytes were found in May and June, but from July through September spermatozoa predominated. Thus, mature sperm were evident much earlier in the year than mature eggs. A decrease in spermatozoa density after 10 Oct indicated spawning and coincided with the decrease in m ean oocyte d iameter. The period of rapid somatic growth from May thro ugh August was correlated with an increase in mean monthly wate r temperature from around 26 C to over 30 C (Figure 3). The cytoplasmic growth stage of gametogenesis was initiated in July when water temperature exceeded 28 C. From July through September, during the reproductive period, mean water temperature averaged 30 C. Spawning occurred in October when mean water temperature dropped 5 C, to 25 C. Average water temperature continued to decrease to a January minimum of 12 C, then began to increase.

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29 Discussion The annual cycle of growth and reproduction in the Florida bay scallop is consistent on a year to year basis. Growth is minimal i n winter, but increases in April and May as mean monthly water temperature increases. Somatic growth, seen as increased mean shell height, and increased mean mantle, adductor muscle, and digestive gland dry weights, continues through early August when maximum size and weight were attained. The cytoplasmic growth phase of oogenesis is initiated in July and continues through September when maximum oocyte diameters are found. During the period of reproductive development, a reciprocal relationship between adductor muscle and gonad dry weights and indexes indicates the utilization of energy reserves for gamete synthesis. Spawning commences in early October in conjunction with a decrease in water temperature. After spawning the population as a whole experiences increased mortality, resulting in few scallops living through a second winter. Specific information now exists on the reproductive cycle of irradians from three locations along its entire geographica l range of Cape Cod, Massachusetts to Tampa Bay, Florida (Gutsell, 1930). Several reproductive latitudinal trends that wer e noted between Woods Hole, Massachusetts (latitude 42 N) and Beaufort, North Carolina (latitude 35 N) populations (Sastry, 1966b, 1970) are continued to the Tarpon Springs, Florida population

PAGE 42

(latitude 28 N), even though it is geographically separated from the others by the Florida peninsula. 30 The most obvious of these trends is the timing of the reproductive cycle which is linked to water temperature and food availability criteria at each of the locations. With decreasing latitude, the temperature required for the initiation of the gametogenic cycl e increases, and as a result, reproductive cycles occur later in the year (Figure 5). In Massachusetts, gametogenesis begins in April when water temperature is about 10 C, and gametes reach maturity in June and July when water temperature approaches 23 C (Sastry, 1966b, 1970). In North Carolina, gonad growth begins later (June), with water temperature exceeding 20 C, and culminates in August and September when water temperature is 26-28 C (Sastry, 1966a, 1970; Kirby-Smith, 1970). In Florida, cytoplasmic growth begins in July with water temperature around 28 C, with maximal mean gonad dry weight, gonad index, and oocyte diameter being observed in late September and early October when water temperature is near 30 C. If temperature itself controls reproduction in bivalves as suggested by Nelson (1928), southern populations would spawn earlier in the year than more northern populations, as critical threshold temperatures would be reached sooner. To the contrary, each population of A. irradians initiates gametogenesis at a temperature which increases with decreasing latitude. A

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,.... E ::L a: w w <( 0 w >-(.) 0 0 z <( w 3 1 ---MASSACHUSETTS 100 NORTH CAROLINA --FLORIDA 90 80 70 I 60 I ---, I \ I \ 50 I \ I \ 40 / \ / ........... \ / ...... ..... \ 30 / ...... / \ ...... ....... /' / \ ...... 20 / / / / \ y ...... '/ / 10 / \ .......... \ \ F M A M J J A s 0 N D MONTH Figure 5 Mean monthly oocyte diameters of A. irradians from Massachusetts, North Carolina, and Florida populations; Massachusetts and North Carolina data from Sastry (1970); Florida data presented as a monthly average of all means obtained within that month

PAGE 44

32 more likely explanation for this, which has been demonstrated in the laboratory, is that scallop oocyte growth is dependent upon food supply and a certain minimum temperature (Sastry, 1963, 1966a, 1968). Reproduction, being an energetically expensive process, is dependent on an adequate food supply that is present in ample quantities at different temperatures and times of the year at the different latitudes. Endogenous factors such as neurosecretion appear to be the regulatory mechanism between the exogenous factors of food and temperature and the completion of the reproductive process (Blake, 1972; Blake and Sastry, 1979; Sastry, 1979). Th e ability of an organism to reproduce is dependent upon an adequate supply of energy to meet maintenance, as well as reproductive requirements, during its gametogenic cycle. Whether adequate energy is available depends on the availability and quality of food and the metabolic rate of the animal, which to a certain extent is influenced by environmental temperature. Although far from conclusive, primary productivity studies in the areas of concern reveal a decrease in potential scallop food supply with decreasing latitude, as shown by a southern New England value of 308 gC m-2 yr-1 1976), North Carolina values up to 153 gC m-2 yr-1 (Thayer, 1971), and an Anclote value of 93 gC m-2 yr-1 (Johansson and Hopkins, 1972). In addition, the metabolic rate of A irradians may increase with decreasing

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33 latitude (and increasing mean temperature), with individuals from Massachusetts having a respiration rate of 0.07 ml 02 g-1 h-1 at 20 C (Van Dam, 1954) and individuals from North Carolina having a considerably higher rate of 0.11 ml 02 g-1 h-1 at the same temperature (Kirby-Smith, 1970). The Florida bay scallop is adapted to even greater average temperatures and may have an even greater overall respiration. (This apparent lack of latitudinal adaptation is discussed in Chapter 5.) It is reasonable to suggest that with decreasing latitude the bay scallop has a smaller food supply and possibly a greater metabolic rate, resulting in less energy being availabl e for reproduction. A latitudinal shift in the source of reproductive energ y for A. irradians over its range which is a result of the balance between food suppl y and metabolic rate at the three locations, tends to support this contention. A negative correlation exists between the digestive gland and gonad indexes over the reproductive periods of the Massachusetts and North Carolina scallop populations; however, only the Massachusetts correlation is statisticall y significant (Sastry, 1970). This indicates that at least in Massachusetts scallops, reproductive energy is being supplied from nutrients in the digestive gland. In fact, a transfer of 14C leucine from the digestive gland to the gonad of irradians during oocyte development has been demonstrated (Sastry and Blake,

PAGE 46

34 1971). In North Carolina, in addition to the decrease in digestive gland index, a 10% decrease in adductor muscle index occurs during the reproductive period (Sastry, 1979), suggesting that this body component also plays a part in providing reproductive energy at this latitude. In Florida, no correlation exists between digestive gland and gonad indexes, but a 20% decrease in adductor muscle index as well as a two-thirds reduction in dry weight is found over the period of gametogenesis. Thus, because of an increased reduction in index associated with gametogenesis, the adductor muscle appears to increase in importance as a reproductive energy reservoir with decreasing latitude. The size of the bivalve digestive gland and its importance as a storage organ vary as the result of seasonally changing food supplies and reproductive states 1974). Therefore, the extent to which the digestive gland operates as a nutrient storage organ in bay scallops might be expected to decrease with decreasing latitude as available food supplies generally decrease. In Massachusetts, scallop reproduction is supported directly through short-term storage of the digestive gland as a result of abundant food and low overall metabolic rate. In North Carolina, with ambient phytoplankton levels being less and metabolic rates being greater, the storage function of the digestive gland becomes less important and scallops have to draw on reserves stored in the adductor muscle. With the Florida

PAGE 47

35 population having even less available food and possibly an even greater metabolic rate, the storage function of the digestive gland is negligible (i.e., all ingested food is immediately assimilated and distributed to other body components). The extra metabolic demand of gametogenesis appears to be met almost entirely by the long term storage of reserves accumulated in the adductor muscle prior to gametogenesis. Thus, with decreasing latitude, incoming food appears to satisfy a smaller portion of the energy requirement, necessitating a larger contribution from the adductor muscle. Another trend that is apparent when viewing reproduction in the bay scallop over its geographic range is likely related to the reproductive energetics at the three latitudes. With decreasing latitude, maximal mean gonad indexes and oocyte diameters become smaller. In Massachusetts scallops, maximal mean gonad indexes approach 20% and in North Carolina 17% is the maximum (Sastry, 1970). In Florida, the maximum is even less, reaching only 14%. Maximum mean oocyte diameters also follow this trend (Figure 5), reaching 90 pm in Massachusetts scallops, about 70 in North Carolina scallops (Sastry, 1970), but only reaching 38 in the Florida bay scallop. If less energy is available for reproduction after maintenance metabolism is met as latitude decreases, less gametogenic material might result, and less energy could be available to individual larvae. The differences in maximal egg

PAGE 48

diameters at the three locations result in North Carolina and Massachusetts ova potentially having 6 and 13 times, respectively, the amount of energy contained in Florida ova, assuming similar biochemical compositions. Larval survival is related to egg size, as shown by the study of Kraeuter al. (1982), in which smaller A. irradians eggs were found to have a significantly poorer survival rate than larger ones. This finding may hold true within a scallop population regardless of mean egg diameter, but whether larval survival in the Florida population is generally reduced because of its relatively 36 small mean oocyte diameter is unknown. Obviously survival is great enough to maintain a population at this location with little presumed recruitment from other areas. Scallop life span varies latitudinally and may be related to the cost of reproduction within the variou s populations. In Massachusetts, scallops commonly live to the age of 24 months (Belding, 1910), and in North Carolina, the majority of scallops live to be 20 months old (Gutsell, 1930). In Florida, scallop mortality noticeably increases after spawning commences at the age of 12-14 months. Calow (1979) notes that there is a negative correlation between the amount of energy invested in reproduction (as a proportion of total energy expenditure) and the subsequent survival of the parent. This may be related to reduced rates of turnover and repair in somatic tissue as a result of the diversion of energy to the gonad

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37 (Calow, 1981). On the basis of adductor muscle reserve 'utilization, the cost of reproduction for irradians increases with decreasing latitude, possibly accounting for this decreased longevity. Chapter 7.) (This is discussed further in If the range of a species is dictated by its ability to reproduce successfully over a spectrum of environments, then reproductive success can be measured in terms of reproductive energetics. The way that irradians meets the cost of reproduction over its latitudinal range differs as the result of varying environmental factors and is reflected by differing amounts of gametogenic material produced and differing life spans. The bay scallop in Florida appears to be operating at its energetic limit, being able to allocate just enough energy to reproduction to insure populational viability. This may act to largely limit the southern distribution of A irradians to west central Florida.

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38 CHAPTER 4: BIOCHEMICAL COMPOSITION Introduction Seasonal cycles of energy storage and utilization in marine bivalves are generally attributed to reproductive activity (Giese, 1959, 1966, 1969; Bayne, 1976; Gabbott, 1976; Sastry, 1979). In general, energy is stored prior to gametogenesis when food is abundant in the form of lipid, protein, and carbohydrate substrates, and subsequently is utilized in the production of gametes when metabolic demand is high (Gabbott, 1975; Bayne, 1976). The specifics of which substrates are important, where in the animal they are stored, and how the timing of their utilization is related to reproduction vary between species as well as between populations of the same species (Giese, 1969; Bayne, 1976; Sastry, 1979). Even though a large literature exists on seasonal changes in tissue weights and biochemical in marine bivalves (e.g., Galtsoff, 1964; Giese, 1969; Walne, 1970; Sastry, 1979), few studies have related energy substrate storage and utilization to specific reproductive events. Gabbott (1975) observed a synchrony between glycogen depletion and oogenesis in female Mytilus edulis. Adachi (1979) found that the adductor muscle

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protein level in Tapes philippinarum decreased by 'two-thirds during the period of gonad development, and Pollero et (1979) noted a reciprocal relationship between gonad and remaining tissue lipid contents in Chlamys tehuelcha. 39 A irradians requires digestive gland and adductor muscle reserves to support reproduction, with the relative amounts contributed from each varying latitudinally (Sastry, 1966, 1968; Chapter 3). This section investigates the energy storage cycle of irradians concentricus in Florida by defining the amounts, locations, and timing of utilization of specific nutrient pools within this species over its reproductive cycle. Materials and Methods Thirty scallops were collected at approximate biweekly intervals between May and October, 1979 from the Anclote Estuary. Analyses of body component dry weights (n=lO) and reproductive stage (n=6-10) were performed as described in Chapter 3. Average lipid, glycogen, protein, and ash levels (% dry weight) were determined for adductor muscle, digestive gland, gonad, and mantle (remaining tissue) body components. The body components of ten scallops were frozen and lyophilized in a Labconco 8 freeze dryer. Like tissues were pooled, ground into a powder using a mortar and pestle, and further dessicated under a vacuum over

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silica gel. Average biochemical contents were calculated by multiplying average levels by mean component weights (Giese, 1967). Total lipid was determined using the gravimetric method of Barnes and Blackstock (1973). A 0.2-0.5 g 40 portion of dried, ground tissue was homogenized with 80 ml of a 2 : 1 (v/v) chloroform-methanol mixture i n a Waring blender. The homogenate was filtered through a Whatman No. 1 filter, and the blender and residue were rinsed with solvent, bringing the final volume to 100 ml. The filtrate was purified by shaking in a separatory funnel with 20 ml 0 .9% aqueous sodium chloride. This mixture was allowed to separate overnight at 4 C, with the lower, lipid-containing phase bei ng drained into a tared beaker. Beaker solvent contents were evaporated before final weighing of the lipi d extract. The determination of glycogen was made using the phenol-sulfuric acid method of (1956). A portion of dried, lipid extracted tissue (containing 10-90 pg glycogen) was placed in a test tube with 2 ml distilled water, 1 m1 5% phenol in water, and 5 ml concentrated sulfuric acid. After cooling for 30 min, the optical density of the orange-yellow solution was read at 490 nm on a Beckman DU-2 spectrophotometer. A calibration curve was constructed using oyster glycogen as a standard. The Folin phenol method of Lowry al. ( 1951) was used to measure protein level. A 5 .0-10. 0 mg portion of

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41 dried, ground tissue was mixed with 10 ml 0.1 N NaOH in a test tube and left overnight at room temperature. A 0 5 ml aliquot of this solution was placed in another test tube with 5.0 ml "Reagent C". Tube contents were mixed and allowed to stand 30 min at room temperature, after which 0.5 ml "Reagent E (1 N Folin-Ciocalteau's reagent) was added with immediate mixing. After 2 h the optical density of the blue solution was read at 750 nm on a Beckman DU-2 spectrophotometer, with bovine serum albumin serving as the standard. For ash determination, a 0.5 g portion of dried, ground tissue was placed in a tared crucible and combusted in a muffle furnace at 550 C for 6 8 h. Results Lipid, g lycogen, protein, and ash levels (% dry weight) of the four body components are given in Table 3 Adductor muscle lipid level was consistentl y low, ranging from 3 7 to 5.6%. Ash level increased slightly from a minimum of 5.8% on 14 June to a maximum of 12.8% on 31 October. Adductor muscle glycogen showed a definite seasonal trend, increasing from 11 .5% on 17 May to 28.4% on 11 July and then decreasing steadily to a minimu m value of 3.4% on 3 1 October. Protein was the major adductor m uscle component, ranging from 40.0 to 55.1% The mantle showed no seasonal trend in biochemical composition Glycogen was the least important in terms of

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42 Table 3 Lipid, glycogen, and protein levels (%dry wt ) of pooled adductor muscle, mantle, digestive gland and gonad body components of A. irradians concentricus -----------------------------------------------------------------------Adductor Muscle Mantle ------------------------------------------------------Date Lipid Glyc. Prot. Ash Lipid Glyc. Prot. Ash ---------------------------------------------------------------17 May 4 2 11.5 54.3 7.8 6 4 3 5 33. 8 19.4 31 May 3.7 13.7 51.4 6 2 5.7 3 1 33. 8 19.7 14 June 4.3 22. 3 51.1 5 8 6 4 3 6 33.0 18. 7 28 June 4.5 25.8 40.0 7.4 6 7 4 8 33.3 20.2 11 July 4.2 28. 4 48. 2 6.4 6 7 4 1 39.4 18.6 26 July 3 8 20. 6 45. 5 6.9 6 2 3 2 37.8 20. 6 9 Aug 4 8 16.0 44.7 8.7 6 3 3 4 35. 3 19.9 24 Aug 4 8 14. 0 51.3 8 3 6.3 2.6 39. 6 21.7 7 Sept 3 9 12.0 54.1 8.2 5.6 3.1 38.7 20.6 26 Sept 5 1 9 7 47. 9 7.2 6.6 3.5 32. 6 18. 0 10 Oct 5 6 5 8 53. 7 8 4 6 9 2 3 33. 5 20. 2 17 Oct 4.3 6 5 53. 9 9.9 6.1 3.5 34.6 20.4 3 1 Oct 5.5 3 4 55. 1 12.8 6 6 2.4 32. 0 22.5 -----------------------------------------------------------------------Digestive Gland Gonad -----------------------------------------------------Date Lipid Glyc Prot. Ash Lipid Glyc Prot. Ash ----------------------------------------------------------------------17 May 14. 0 10. 6 30 1 11.9 8 2 4.7 38 1 18.2 31 May 9 7 8 7 30. 9 13.9 6 7 4 6 35. 2 18. 1 14 June 20. 0 10.7 29. 7 12.9 7 6 4.9 40. 0 24. 1 28 June 24. 0 12. 3 27.0 11.0 9 2 5.7 34 0 19. 7 11 July 14. 6 11.6 36.6 14.5 9 7 6.1 40. 3 18. 0 26 July 14. 5 12.8 26. 7 14. 8 9 9 4 7 34.7 23. 6 9 Aug 17. 6 12.1 26. 9 1 3 6 8 8 4.8 35.2 19. 4 24 Aug 8 4 10.7 26.8 1 8 8 8 0 3 8 33.7 24. 8 7 Sept 6 8 1 5 3 29. 1 15. 9 8 9 5 0 41.1 16.9 26 Sep t 9 3 14. 0 29.7 12. 8 11.8 5 7 38. 8 13.1 10 Oct 7 7 11. 0 30. 7 1 5.7 11.3 5 4 39. 6 15. 0 17 Oct 7 3 15.2 31.5 15.2 9.6 6 0 41.7 17.1 31 Oct 6 7 12. 2 30.3 16. 1 9.5 4.1 40. 7 17.9

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quantity, ranging from 2.3 to 4.8%. Mantle lipid maintained a consistent level between 5 0 and 7 0%. Ash was the second most plentiful component and ranged from 18 0 to 22. 5%. Protein was the major component and maintained a consistent level between 32 0 and 39. 6%. 43 Protein was also the major component in the digestive gland, comprising 26 7 to 36 .6% of the total. Digestive gland lipid reached a maximum of 24% on 28 June but decreased to 8.4% on 24 August, remaining below 10.0% for the rest of the study period. G lycogen and ash levels maintained relatively consistent levels, ranging from 8.7 to 15.3% and 11.0 to 1 8 8%, respectively. Gonad lipid level was 6.7% on 3 1 May and increased to 11 .8% on 26 September. Glycogen was consistently low, ranging from 3 8 to 6.1%. Protein was the major component, comprising between 33. 7 a nd 41.7% of the total. Gonad ash level was relatively high and ranged from 13 1 to 24 .8%. Biochemical conte nts (level X mean dry weight) represented the actu a l amounts of each substrate available at any time and served to better define the pattern of growth, energy storage and utilization, and reproductive development and spawning indicated by the mean dry weights. The adductor muscle exhibited a large seasonal variation in glycogen and protein contents (Figure 6). Adductor muscl e glycogen content increased from 69.0 m g on 17 May to a maximum of 384 mg on June 28, a nd then decreased steadily to a 31 October minimum of 24.8 mg. The

PAGE 56

44 MONTH Figure 6. Protein, glycogen, and lipid contents of pooled A. irradians concentricus adductor muscle tissue

PAGE 57

45 bulk of adductor muscle growth between 17 May and 9 August was accounted for by an increase in protein content, which increased from 326 mg to 992 mg. After 9 August protein content declined to a 17 October value of 372 mg. Adductor muscle lipid content remained low and showed little change during the study period. The mantle tissue was composed primarily of protein (Figure 7). An increase in protein content from 128 mg on 17 May to 325 mg on 24 August accounted for mantle growth during this period. After 7 September, protein content declined to 214 mg on 31 October. Mantle lipid and glycogen contents remained low and showed no seasonal variation. Digestive g land lipid content increased rapidly from 25.2 mg on 17 May to a maximum of 11 8 mg on 28 June but was depleted to initial levels by 24 August (Figure 8). Digestive gland protein increased from 54.2 mg on 17 May to a maximum of 179 mg on 11 July and remained essentially stable thereafter. Digestive gland glycogen content increased from 19.1 mg on 17 May to 60.3 mg on 28 June, fluctuating little for the duration of the study Gonad biochemical content (Figure 9) reflected the pattern of gonad growth and gametogenesis determined in the previous chapter. Between 17 May and 24 August, lipid content increased from 3.3 mg to 12.8 mg, and protein content rose from 15.2 mg to 53.9 mg. After August, protein and lipid was accumulated in the gonad at a greater

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46 ,.... 0) 350 e LIPID E GLYCOGEN ......, PROTEIN 1-300 z w 1-250 z 0 200 () ....J 150 <( () 100 ::;! w :c 50 () 0 m w (.!) 1z ::::> 0.. ::::> <( w ..., (f) MONTH Figure 7. Protein, glycogen, and lipid contents of pooled A. irradians concentricus mantle tissue

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,..... 200 0) E t-z 150 w t-z 0 () ....J <2:: () 100 50 :c () 0 (D e LIPID GLYCOGEN A PROTEIN w z ;:) J > ...J ;:) J MONTH 1-0.. w C/) 1-(.) 0 47 > 0 z Figure a Protein, glycogen, and lipid contents of pooled irradians concentricus digestive gland tissue

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,..... 0') E ......, r125 z w r-100 z 0 (.) 75 ...J <2:: (.) 50 w I 25 (.) 0 en LIPID GLYCOGEN PROTEIN w z :::::> -, > ...J :::::> -, ._ a. w C/) MONTH ._ (.) 0 > 0 z 48 Figure 9. Protein, glycogen, and lipid contents of pooled irradians concentricus gonad tissue

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49 rate. By 10 October protein content reached a maximum of 143 mg, and lipid content attained a 40. 7 mg maximum. After 10 October gonad protein content fell to 104 mg, and average lipid content fell to 24.0 mg. Gonad glycogen content remained consistently low over the course of the study. The three apparent energy stores (digestive gland lipid and adductor muscle glycogen and protein) were utilized differently in relation to the gametogenic cycle. Digestive gland lipid content reached storage levels early in the gametogenic cycle, and its utilization was associated with the beginning of the synchronous growth phase. Adductor muscle glycogen content also was accumulated early in oogenesis, and its utilization was significantly correlated to the increase in mean oocyte diameter during cytoplasmic growth and vitellogenesis (r 0 .83, P < 0.025). This relationship is shown in Figure 10. Adductor muscle protein content reached a maximum during late cytoplasmic growth and was utilized during the later gametogeni c stages and spawning as the lipid and glycogen reserves were depleted. The protein and lipid nutrients stored in developing gametes in the gonad were released upon spawning, leaving the scallop virtually devoid of energy reserves. Discussion Energy reserves in A. irradians concentricus are

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"" 0> E 400 '-" tz w 300 tz 0 (.) 200 z w (!) 0 100 (.) >-_J (!) 10 20 30 40 MEAN OOCYTE DIAMETER (1Jm) Figure 10. Relationship between adductor muscle glycogen content and mean oocyte diameter in A. irradians concentricus (Y = -20.37X + 796.6; r = -0.83) 50

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accumulated during the somatic growth period (prior to August) in the form of lipid in the digestive gland and glycogen and protein in the adductor muscle. During the gametogenic period (July-September), these reserves are depleted as developing gametes in the gonad accumulate lipid and protein. Decrease in digestive gland lipid 51 corresponds with the initiation of gametogenesis in July. Adductor muscle glycogen is utilized over the entire reproductive period (July-September). Adductor muscle protein is depleted during the later gametogenic stages and spawning (August-October). Spawning (the release of gametes) in October is reflected by a sharp decrease in gonad weight, protein and lipid contents, and oocyte diameter. After spawning, nutrient reserves are at a minimum and scallop mortality increases. Energy storage cycles similar to this are described for the pectinids Chlamys septernradiata (Ansell, 1974b), Pecten maximus (Comely, 1974), Patinopecten yessoensis (Mori, 1975), opercularis (Taylor and Venn, 1979), and Placopecten magellanicus 1981). In addition to its function in digestion, the molluscan digestive gland is responsible for nutrient storage and the transfer of assimilated food to the body tissues (Owen, 1966; Van Weel, 1974). gland lipid is an important energy reserve with respect to bivalve reproduction. In yessoensis (Mori, 1975) and

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magellanicus 1981), decreases in "digestive gland lipid contents occur in conjunction with 52 gametogenesis and an increase in gonad lipid levels. Lipid is accumulated in the digestive gland of A. irradians concentricus prior to gametogenesis, and its utilization is associated with the initiation of oogenic activity. Carbon is transferred between scallop digestive gland and gonad tissues in response to gametogenesis (Sastry and Blake, 1971; Vassallo, 1973), suggesting a mechanism whereby fatty acids (and other biomolecules) are transferred directly from the digestive gland to the gonad to be incorporated in developing ova. Rapid fluctuation in digestive gland biochemical composition, as seen in this study, may be the result of a changing food supply. Seasonal fatty acid changes in Chlamys techuelcha are related to phytoplankton fatty acid composition (Pollero 1979). However, other studies indicate that bivalve fatty acid compositions are species oriented rather than diet dependent, indicating active fatty acid metabolism (Watanabe and Ackman, 1974; 1979). In the Anclote Estuary, total primary productivity is fairly constant over the period of study, but species composition is variable (Johansson, 1975), possibly affecting bay scallop digestive gland biochemical composition. septemradiata (Ansell, 1974b), maximus (Comely, 1974), yessoensis (Mori, 1975),

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opercularis (Taylor and Venn, 1979), magellanicus 1981), the utilization 53 of glycogen and protein reserves occurs over the winter in association with gametogenesis. However, since adductor muscle weight loss during this period is greater than can be accounted for solely in terms of gonad development, part of these reserves undoubtedly support maintenance metabolism as well, as the result of seasonally minimum energy intake. The general synchrony between glycogen utilization and gametogenesis in marine bivalves suggests that stored glycogen is converted to fatty acids which are incorporated into developing ova (Gabbott, 1975, 1976). The role of protein as an energy reserve is less well defined. In A. irradians concentricus, adductor muscle glycogen and protein reserves may have different roles. Glycogen appears to be utilized primarily for oogenesis since its depletion is significantly correlated to increase in oocyte diameter during cytoplasmic growth and vitellogenesis. Adductor muscle protein utilization occurs after glycogen depletion and takes the form of cellular destruction rather than a decrease in actual protein level. Most likely it is used to meet maintenance requirements during the later stages of oogenesis and spawning at a time when food is becoming less abundant. Therefore, adductor muscle glycogen is utilized initially and probably supports gametogenesis more directly than adductor muscle

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54 protein. The significant inverse relationship between digestive gland and gonad indexes of irradians noted by Sastry (1966a, 1968, 1970, 1975) is not observed for the bay scallop in Florida. Instead, a large decline in adductor muscle index occurs over the period of gonad growth, indicating that in this population the adductor muscle is the major energy storage site. The relative importance of different body components as energy storage sites, being related to the reproductive cycle, is highly adaptive and is the result of genetic divergence or non-genetic adaptation to environmental differences over the geographical range of irradians. The seasonal cycle of energy reserve storage and utilization in A. irradians and its relationship to the reproductive cycle reflect the complex interactions existing between food supply, temperature, growth, and gametogenesis (Sastry, 1963, 1966a, 1968, 1970, 1975; Sastry and Blake, 1971; Blake and Sastry, 1979). Growth, which is exhibited mainly as an increase in protein content, and nutrient storage in the form of an increase in digestive gland lipid and adductor muscle glycogen and protein occur in the spring at a time of increasing water temperature and food availability. Digestive gland lipid provides the threshold levels of stored energy required for initiation of oocyte growth and is utilized over the early stages of gametogenesis, possibly being transferred

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55 directly to the gonad. Adductor muscle glycogen is negatively correlated to oocyte diameter over the length of the the gametogenic cycle, and is utilized primarily for oogenesis, possibly being converted to oocyte fatty acids. Adductor muscle protein is utilized during the later gametogenic stages, primarily providing maintenance energy as food suppl y decreases. Spawning occurs in the autumn, triggered by a decrease in water temperature, leaving scallops virtually devoid of energy reserves and possibly contributing to the increased mortality seen at this time.

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56 CHAPTER 5: SUBSTRATE CATABOLISM Introduction Physiological rates vary seasonally as the result of internally mediated metabolic changes and/or in response to environmental factors. Although seasonal trends in the oxygen consumption rates (Bayne, 1976; De Vooys, 1976; Widdows, 1978) and ammonia excretion rates (Bayne, 1973a, 1973b; Widdows, 1978; Mann, 1979a, 1979b) of marine bivalves have been correlated to changes in annual reproductive cycles, the fact that these physiological processes do not always respond similarly to seasonal environmental changes precludes their general use as indicators of the balance in catabolism between the different nutrient reserves in the tissues (Bayne, 1976; Bayne and Scullard, 1977; Mann, 1978). Rates of oxygen consumption, ammonia excretion, and carbon dioxide production, when converted into O/NH3 and C02/02 (RQ) molar ratios, provide indexes of the catabolic balance between lipid, carbohydrate, and protein substrates within an animal (Richardson, 1929; Bayne and Scullard, 1977; Mann, 1978). If the amino acids resulting from protein catabolism are deaminated and totally excreted as ammonia while the carbon skeletons are

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57 fully oxidized to carbon dioxide and water, the "theoretically minimum O/NH 3 ratio (indicative of exclusive protein catabolism) is 9.3, with higher values suggesting greater non-protein (carbohydrate and lipid) catabolism (Bayne, 1973a). When carbohydrate is oxidized, all of the oxygen utilized forms carbon dioxide, resulting in a respiratiory quotient (RQ) of 1.0. When protein and lipid are catabolized, some of the oxygen forms water, resulting in respective RQ values of 0.79 and 0.71 (Richardson, 1929). A n RQ greater than 1.0 indicates a carbohydrate to lipid conversion (Mori, 1968, 1975; Gabbott, 1975). Bivalve RQ and O/NH3 values are highest at the onset of gametogenesis and lowest after spawning, indicating a shift in catabolism from carbohydrate to protein (Bruce, 1926; Mori, 1968, 1975; Bayne and Thompson, 1970; Gabbott and Bayne, 1973). In this study, seasonal O/NH3 and RQ physiological indexes were obtained in order to establish patterns of reproduction related substrate catabolism in the bay scallop, Argopecten irradians concentricus. This provided a means of identifying periods of catabolism of specific substrates, reinforcing the information obtained in the previous two chapters. Materials and Methods Scallops were hand collected by divers from the

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58 Anclote Estuary (Tarpon Springs, Florida) at 2-3 week intervals between May and September, 1982. Since seasonal cycles of growth and reproduction in A. irradians are synchronous on a year to year basis (see Chapter 3), additional collections were made in October and November, 1983 to provide a more complete data set. Bottom water temperature was obtained at the time of collection with a hand-held thermometer (.5 C). Scallops were returned to the laboratory along with extra seawater for maintenance, physiological rate measurements, and salinity and total alkalinity determinations. Fouling organisms were removed, and animals were placed in aquaria containing water obtained at the collection site maintained at environmental temperature (.0 C). Salinity was determined conductimetrically with an Autosal 8400 and total alkalinity was measured by titration with HCl, standardized with 1.00N NaOH. Determination of physiological rates began the morning after collection and was completed within 48 h Six scallops were placed in separate pyrex (closed) respiration chambers of about 1.8 1 volume filled with air saturated, filtered (1 pm) seawater obtained at the time of collection. Environmental temperature was maintained by a recirculating water bath (Forma Scientific 2067) connected to water jackets surrounding the chambers. Water within each chamber was magnetically stirred throughout the 2-4. 5 h (depending on scallop size) experimental period.

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59 Bacterial respiration was negligible over this length of time. At the end of the experimental period, the exact chamber water volume was measured and scallop tissue dry weight (DW) was obtained by drying to a constant weight at 60 C Oxygen uptake was measured by a polarographic electrode al., 1953) (calibrated at air saturated (100%) and nitrogen purged (0%) oxygen levels) which was i nserted into the chamber t hrough a sealed portal and connected to an amplifier and chart recorder. The amount of oxygen consumed was calculated as the difference between initial and final % saturation based on the oxygen concentration of the air saturated seawater, knowing its temperature and salinity. The rate of oxygen consumption was calculated in terms of ml oxygen consumed gDw-1 h-1. Carbon dioxide resulting from scallop respiration \vas recorded as the increase in chamber water [H+] as measured by a pH electrode inserted into the chamber through a sealed opening and connected to an Orion 901 Ionalyzer. Initial and final total carbon dioxide concentrations were calculated based on the change in pH, knowing total alkalinity and the apparent dissociation constants of carbonic and boric acids (Riley and Chester, 1971). Carbon dioxide production rate was calculated as ml carbon dioxide produced gDw-1 h-1. The amount of ammonia excreted was determined by

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subtracting final from initial chamber water ammonia concentrations, as determined with an Orion ammonia electrode (calibrated with NH4Cl) in conjunction with an Orion 901 Ionalyzer. Ammonia excretion rate was calculated in terms of pg ammania-N excreted gDw-1 h-1. 60 Mean rates of oxygen consumption, carbon dioxide production, and ammonia excretion were calculated from the replicate rates obtained for each sampling date. Relationships between physiological rates and environmental temperature and salinity (as both separate and combined independent variables) were investigated using regression analysis. Mean O/NH3 and C02/02 (RQ) physiological indexes for each sampling date were obtained from the replicate ratios. Seasonal trends in the indexes were fitted to polynomial regression equations. Results Mean rates of scallop oxygen consumption, carbon dioxide production, and ammonia excretion, along with the environmental temperatures and salinities occurring on the various sampling dates are given in Table 4 Mean oxygen consumption rate showed no obvious seasonal trend, with a minimum of 0 .72 ml gDW-1 h-1 occurring on 9 November and a maximum rate of 1.13 ml gDW-1 h-1 occurring on 15 June. For most of the study, mean oxygen consumption was between 0 9 and 1 0 ml gnw-1 h-1.

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Table 4 Mean ( 1 sd) rates of bay scallop irradians concentricus) oxygen consumption, carbon dioxide production, and ammonia excretion at the environmental temperature and salinity of the various sampling dates; (n=6) ------------------------------------------------------------------------------Date Temperature Salinity Oxygen Carbon Dioxide Ammonia Production Excretion (oC) (o/oo) (ml gJJ.V h -1) (ml gp.v1 h -1 ) (}lgN gp.v1 h-1) ------------------------------------------------------------------------------19 May 82 25.7 26.72 0.80 .10 0.51 .08 87 3 June 82 29.0 34.25 1.10 .18 1.00 2 7 75 15 June 82 31.7 34 .29 1.13 25 1.14 29 94 29 June 82 31.4 30.21 0.93 .10 1.19 24 88 18 July 82 29.5 31.59 1.04 0.15 1. 55 30 128 30 July 82 30.0 32. 3 1 0 .91 .05 1.61 .30 72 1 15 Aug 82 31.3 31.24 0.99 .17 1.56 .41 100 2 1 3 Sept 82 29.3 25.62 0.96 .08 1.30 0.27 140 24 Sept 82 26. 2 17.01 0.89 .13 1.13 26 130 11 Oct 83 26. 5 18.53 0.92 .05 0.66 .15 136 9 Nov 83 21.5 22.33 o. 72 .18 0.45 .07 92 2 Q'\ ......

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The mean rate of carbon dioxide production did vary seasonally increasing steadily from 0 51 ml gDw-1 h-1 on 19 May to a maximum of 1 61 ml gDw1 h 1 62 on 30 July and then consistently decreasing to a mi n imum of 0.45 ml gDW-1 h-1 on 9 November. Mean ammonia N excretion rate generally increased over the study period, from between 75 (3 June) and 87 (19 May) pg gDW1 h-1 to 1 36 ( 11 October) and 140 (3 September) pg gDW1 h 1 then falling to 92 p g gDw1 h-1 on 9 November. All three scallop physiological rates were significantly dependent up o n the combined effects of environmental temperature and salinity (P < 0.025, multipl e regression). Analysis of the environmental factors as separate independent variables revealed that both oxygen consumption (Figure 11) and carbon dioxide production (Figure 12) rates increased with increasing temperature (P < 0.005). Ammonia excretion rate increased with decreasing salinity (P < 0 .05) (Figure 13). Scallop O/NH3 ratio increased from 1 1 6 on 19 May to a maximum of 22 1 on 29 June, then decreased to a minimum of 8 9 on 24 September, and recovered slightly to 11 4 on 9 November (Figure 14 ) Scallop RQ increased gradually from 0 7 on 19 May to a maximum of 1.6 on 30 July and then steadily decreased to a 9 November minimum of 0 6 (Figure 15).

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,... I 1 5 ,... I Q 1 0 0') C\1 0 0 5 E TEMPERATURE (C) Figure 11. Mea n oxygen consumption rate irradians concentricus as a function of environmental temperature ( 1 sd); (R = 0.031T + 0.07 4 ; r = 0 .79) 63

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,..-I ,..-I 64 2.0 ..c 1 5 0 0> C\J 0 () 0 5 E 20 25 30 TEMPERATURE (C) Figure 12. Mean carbon dioxide production rate of A. irradians concentricus as a function of environmental temperature ( sd); (R = 0.104T1.842; r = 0.78) 35

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or-I ..c T'"" I Q Ol z I ('I) J: z Ol :::J. 65 150 t t 100 t 50 ____ __ ____ _u 15 20 25 SALINITY {%o) 30 Figure 13. Mean a mmonia excretion rate of A irr adians concentricus as a function o f enviro nmental salinity ( 1 sd); ( R = 2 .486S + 172.532; r = 0 6 1 ) 3 5

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0 < oc I z 0 66 25 20 1 5 10 0 t 5 r > w z u 0 < w 0 z 00 MONTH Figure 1 4 Seasonal variation in mean O/N H 3 ratio i rradians concentricus ( sd); fitted line is a third order polynomial, circled point excluded

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1z w 1--0 1.5 ::J 0 >a: 0 1-<( a: a.. C/) w a: 1.0 0.5 67 t z :::> a. (.) 0 :::> :::> <( w 0 z ""') ""') (/) MONTH Figure 15. Seasonal variation in mean respiratory quotient (RQ) of A. irradians concentricus ( sd); fitted -rine is a third order polynomial

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68 Discussion An attempt was made in this study to determine the catabolic shifts that occur with respect to reproduction in this animal in nature. Since the physiological rates were determined at environmental temperatures and salinities within 48 h of collection, the metabolic rates measured were probably closest to "routine" rates rather than "standard" (starved) or "active" (swimming or feeding) rates (Thompson and Bayne, 1972). Bivalve physiological rates such as those investigated in this study are dependent upon a number of factors including body size, salinity, temperature, and reproductive state, as well as activity level and acclimation history (Bayne and Scullard, 1977; Shumway, 1982). In this study, bay scallop oxygen consumption, carbon dioxide production, and ammonia excretion rates are dependent upon seasonal variations in temperature and salinity. However, to attribute changes in these physiological rates only to environmental factors would be an over-simplification of a complex relationship. Bay scallop reproductive development (as well as oxygen consumption rate and carbon dioxide production rate) parallels average water temperature at Anclote (Chapter 3). Reproductive development also covaries with environmental salinity (as well as ammonia excretion) in that the lower salinities occur later in the year when gametes are maturing and the adductor muscle is undergoing a large

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decrease in weight and protein content (Chapter 4). Both of these factors in themselves could contribute to the observed variations in physiological rates. On the basis of this study it is impossible to separate environmental factors from reproductive events as determinants of bay scallop physiological rates. Oxygen consumption rates (V02) have now been measured for three widely separated bay scallop populations. At 20 C, V02 in a Massachusetts population is 0.070 ml gWW-1 h-1 (Van Dam, 1954), and 0.11 ml gWw-1 h-1 in a North Carolina 69 population (Kirby-Smith, 1970). According to the regression equation found in this study relating oxygen consumption to temperature, Florida bay scallops have a V02 of 0.76 ml gWw-1 h-1 at 20 C. However, no information as to the acclimation history and reproductive state of animals in the Van Dam (1954) study is given. Also, a water temperature of 20 C occurs in Florida in March and November when only small, resting stage and large, post-spawn scallops are found (Chapter 3). In Massachusetts, 20 C is close to the yearly maximum and occurs when gametes are fully developed. In North Carolina 20 C is the temperature at which oocyte growth is initiated (Sastry, 1970; Kirby-Smith, 1970). Thus, because of differences in acclimation and reproductive states in study animals, there is little that can be concluded from these studies regarding the ability of A. irradians to

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compensate vo2 to temperature over its geographical range. Mea n O/NH3 and RQ values indicate that the source of metabolic energy within A. irradians changes over 70 the course of a reproductive cycle. Resting stage animals exhibit increasing indexes from May to June, indicative of a shift from lipid to carbohydrate as the primary substrate supporting metabolism. Over the gametogenic period maximum O/NH3 and RQ values signify a predominantly carbohydrate based metabolism, with carbohydrate being catabolized in late June (O/NH3 > 22, RQ = 1.0) and possibly converted to lipid from July through September (RQ > 1.0). After oocyte development is complete and spawning has commenced (October-November), minimum O/NH3 and RQ values signify that metabolism at this time is almost exclusively supported by protein catabolism. Although the two physiological indexes indicate the same general trends (i.e., a shift in catabolic substrate from lipid to carbohydrate to protein over a seasonal reproductive cycle), there are slight differences between them. Exclusive carbohydrate catabolism is never indicated by the O/NH3 ratio, coming closest in late June when RQ values around 1.0 indicate carbohydrate catabolism. In this sense the O/NH3 ratio is more an index of relative protein to non-protein catabolism rather than the more absolute index that RQ appears to be. During gametogenesis from July-September the RQ exceeds 1.0 (indicating that

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71 carbohydrate is being converted to lipid) while a declining O/NH3 ratio indicates a greater contribution from non-carbohydrate substrates. After spawning, however, both indexes indicate that protein is the primary catabolic substrate. The seasonal cycle of mean O/NH3 ratios found in this study indicate that the source of metabolic energy depends to a great extent on reproductive state. A similar marked seasonal shift from use of carbohydrate to protein to meet reproductive energy demands has been found for Mytilus edulis in that O/NH3 ratios greater than 300 found in the summer in conjunction with the accumulation of carbohydrate reserves drop to 70-110 for the rest of the year (Bayne and Thompson, 1970; Bayne, 1973a, 1973b; Gabbott and Bayne, 1973). Although the trends are similar, the O/NH3 ratios themselves are considerably higher for M. edulis than for A. irradians. This could be the result of a greater anaerobic capacity and need for glycolytic energy reserves in the intertidal M. edulis and the overall greater ammonia excretion rate of the subtidal irradians. The seasonal cycle of mean RQ values exhibited by A irradians also reflects changes in reproductive energy metabolism similar to those found for other species. Monthly RQ values for M edulis increase from a low of 0.25 after spawning to a high of 1.31 coincident with the early stages of gametogenesis (Bruce, 1926). Digestive

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gland and pallial ma rgin tissues in Crassostrea gigas (Mori, 1968) and Patinopecten yessoensis (Mori, 1975) have RQ values from 1.3 to 1.5 during the period of gonad development that decline to 0.7 before spawning and less than 0.6 after spawning. 72 The seasonal cycle of physiological indexes found in this study reinforce the previously defined cycles of body component weights, indexes (Chapter 3), and biochemical compositions (Chapter 4) and establish a pattern of reproductive energy metabolism for irradians concentricus. The metabolism of resting stage animals is lipid based as indicated by the indexes. Maximum digestive gland lipid level also occurs at this time, and its utilization is associated with the initiation of gametogenesis. Early in the gametogenic cycle, maximum physiological indexes are obtained, signifying that metabolism is carbohydrate based. This corresponds with maximum adductor muscle glycogen content which is utilized as gametogenesis proceeds, perhaps being incorporated directly into the lipid of developing oocytes. As gametogenesis is completed and the gonad attains maximum weight and lipid and protein contents, physiological indexes decrease. A decrease in adductor muscle weight and protein content over this period coincides with this shift to a protein based metabolism. Minimum indexes occur as spawning commences. Thus, O/NH3 and RQ physiological indexes accurately indicate the utilization of specific

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nutrient reserves that accompanies the various stages of reproduction in irradians concentricus. 73

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74 CHAPTER 6: 14c INCORPORATION Introduction Reproduction in marine bivalve molluscs is an energetically expensive process in which gamete production is often in competition with somatic growth for limited resources. Nucleic acids are required in the male for sperm production, and lipid and protein are mobilized in the female for the synthesis of eggs (Giese, 1959). The utilization of energy reserves often accompanies the gametogenic process (Bayne, 1976; Sastry, 1979). The seasonal changes in body component weights, biochemical compositions, and physiological indexes (O/NH3, RQ) observed in Chapters 3, 4, and 5 provide only indirec t information with respect to the ways in which the major lipid, carbohydrate, and protein fuels are used by irradians concentricus in relation to gamete production. Seasonally monitoring the distribution of a radiotracer within the body of an animal, however, can provide direct information regarding the storage and utilization of specific nutrient pools in response to gamete development. The use of radiotracers to investigate bivalve reproductive energy metabolism has been limited. The most

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75 comprehensive study involved the seasonal distribution of 14c and 32p in acid soluble, lipid, and protein fractions of several body components of Mytilus edulis (Thompson, 1972). The translocation of radiolabel from digestive gland to gonad components in conjunction with reproductive development was demonstrated for the scallops Argopecten irradians (Sastry and Blake, 1971) and Chlamys hericia (Vassallo, 1973). Allen (1962, 1970) studied the incorporation and release of 32p (orthophosphate) into tissues of several bivalve species. In the present study, incorporation of 14c into lipid, carbohydrate, and protein fractions of gonad, digestive gland, and adductor muscle body components of the bay scallop, Argopecten irradians concentricus, was monitored seasonally to characterize the intra-organ biochemical transformations associated with reproduction. Materials and Methods Thirty scallops were collected from the Anclote Estuary, Tarpon Springs, Florida, at monthly intervals from June to November, 1983. Upon return to the laboratory, fouling organisms were removed and scallops were placed in an aquarium containing water obtained at the time of collection, adjusted to environmental temperature ( C), which ranged from 21.5 to 31.7 C. Six scallops were dissected for determination of mean body component wet weight (WW) and dry weight (DW).

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76 The next day 24 scallops were placed into a separate feeding tank containing 20 1 water. 1.5 1 Tetraselmis culture (1.5 x 106 cells mi-l), inoculated 24 h previously with 140 pCi sodium (14c) bicarbonate (Amersham Corp.) and centrifuged and resuspended to remove unincorporated 14c, was dosed with a peristaltic pump into the feeding tank over a 6 h period. After feeding, the scallops were returned to the holding tank where they were maintained with non-radioactive Tetraselmis at the rate of 100 ml animal-1 day-1 for the duration of the experiment. Six scallops were sacrificed on each of 1, 4, 7, and 10 days after ingestion of the radiolabel, and 0.20 g (WW) portions of the female gonad, digestive gland, and adductor muscle body components were each separated into lipid, carbohydrate, and protein fractions using a scheme similar to that described by Holland and Gabbott (1971). Each tissue p iece was homogenized with a tissue grinder i n 5 ml 2:1 chloroform:methanol in a test tube. 1 m l 0.9% NaCl was added to each test tube, followed by thorough mixing. After separating overnight in a refrigerator, the lower phase (lipid fraction) was removed with a Pasteur pipette and transferred to a tared scintillation vial. After evaporating to dryness, vials were reweighed to obtain m g (DW) lipid. After lipid phase removal, 1 ml 20% TCA was added to the solution left in each of the test tubes to make a 5% TCA solution. The tubes were then heated in a

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77 boiling water bath for 30 min, cooled, and centrifuged. 'The supernatant (carbohydrate fraction) was poured (along with a distilled water rinse) into a scintillation vial and evaporated to dryness. The precipitate (protein fraction) was rinsed with distilled water into a tared scintillation vial, evaporated to dryness, and reweighed to obtain mg (DW) protein. Mg (DW) carbohydrate was derived from the difference between calculated total dry tissue weight and the sum of lipid and protein weights. Each of the biochemical fractions was solubilized in the scintillation vial with 1 ml NCS tissue solubilizer (Amersham Corp.); 10 ml OCS organic counting scintillant (Amersham Corp.) was then added to each vial. The radioactivity in each vial was counted in an Isocap 300 liquid scintillation counter (Nuclear-Chicago Corp.). Using appropriate corrections for quenching, results were expressed as counts per minute (CPM) mgDW-1 for each of the body components fractions. Total CPM for a particular body component was obtained by adding its respective lipid, carbohydrate, and protein fraction counts. Results Bay scallop carbon turnover was rapid enough that biochemical transformations (conversion, transfer, utilization, etc.) generally occurred within four days after ingestion of the radiolabel. After Day 4 (i.e. on Days 7 and 10), losses of 14C from all body components

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78 were generally found (Table 5). Therefore, results were expressed as the rate of change (slope) of CPM mgDw-1 between Day 1 and Day 4 on the various sampling dates. A positive slope indicated a net gain in radiolabel (relatively slow carbon turnover), and a negative slope indicated a net loss o f radiolabel (relatively fast carbon turnover). For plotting purposes a log transformation was performed on the slopes. Body component CPM mgDW-1 are given in Table 5. The digestive gland and gonad had considerably higher Day 1 counts than the adductor muscle in all months except November when adductor muscle CPM were almost double those of the digestive gland. In all months except September and October, gonad counts were higher than digestive gland counts on Day 1, probably accountable for by the fact that scallop intestine is intertwined throughout the female go nad and was impossible to exclude completely. Figure 16 illustrates the dynamics of 14c incorporation by the three body components over the reproductive cycle. In June and July, l4c losses between Day l and Day 4 in the gonad and digestive gland were offset by gain s in the adductor muscle. Turnover started to increase in August as oogenesi s commenced. By November, after spawning had occurred, 14c was being lost from all components very rapidly. The digestive gland exhibited significant 14c loss (P < 0.05, Duncan's New Multiple Range Test) between Day 1

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Table 5 CPM mgDw-1 ( sd) for bay scallop irradians concentricus) digestive gland, adductor muscle, and female gonad body components 1, 4, 7, and 10 days after ingesting radioactive cells 79 -----------------------------------------------------------------------Month Day 1 Day 4 Day 7 Day 10 -----------------------------------------------------------------------Digestive Gland June 1293 1 90 928 263 423 187 353 1 3 1 July 457 44 349 105 195 16 95 26 Aug 678 177 210 75 103 22 70 42 Sept 1197 431 399 109 196 47 133 27 Oct 1444 407 534 54 253 60 231 55 Nov 1797 9 41 527 155 299 80 221 55 Adductor Muscle June 143 80 500 83 220 88 204 88 July 52 22 187 75 99 20 62 35 Aug 163 40 120 26 121 53 106 31 Sept 337 172 147 42 247 49 144 45 Oct 419 49 379 52 229 77 251 52 Nov 2940 1 223 840 482 282 565 263 Female Gonad June 2220 654 2099 637 1327 710 699 234 July 1337 270 1282 319 679 293 442 129 Aug 929 270 978 16 427 99 313 87 Sept 1158 522 1469 426 602 97 455 128 Oct 1219 530 1884 394 1150 230 638 187 Nov 4682 3025 2220 1301 302 -----------------------------------------------------------------------

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-,.... Cl -,.... I Cl E a.. (..) I C'? .q-Cl 1 -,.... I Cl E a.. (.) "---./ -3 C) e GONAD 8 DIG. GLAND AD. MUSCLE 80 () z 0 < z J J 00 MONTH Figure 16. Relative seasonal incorporation of 14c by gonad, digestive gland, and adductor muscle body components of A. irradians concentricus

PAGE 93

and Day 4 for every month sampled, as assimilated carbon was transferred to other body components. This loss was relatively minor in June and July, but increased steadily from July through November. 81 Adductor muscle gains in 14c in June and July were significant (P < 0 .05, Duncan's New Multiple Range Test) and inversely correlated to digestive gland and gonad losses. Between July and August, as the gonad s lope went from negative to positive, the adductor muscle slope went from positive to negative. The slope remained negative from September through November, with losses being significant (P < 0.05, Duncan's New Multiple Range Test) in September and November. The gonad showed a loss of 14c between Day 1 and Day 4 in June and July, as reflected by negative slopes By August, a slight gain was found, which increased considerably in September and was significant (P<0.05, Duncan's New Multiple Range Test) in October when a maximum gain was seen. In November, a loss in gonad 14C occurred between Day 1 and Day 4 CPM mgDw1 substrate (lipid, carbohydrate, protein) in the three body components are given in Table 6 For the digestive gland, the carbohydrate fraction contained the greatest CPM mgDW-1 on both Day 1 and Day 4 in June and July, with the lipid fraction containing the second highest level of radioactivity. This trend was reversed from August through November, as the lipid

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Table 6 CPM mgDW -1 ( sd) lipid, carbohydrate, and protein for bay scallop irradians concentricus) digestive gland (DG), adductor muscle (AM), and female gonad (FG) body components 1 and 4 days after ingesting radioactive cells ----------------------------------------------------------------------------Month Day 1 Day 4 -------------------------------------------------------------------Lipid Carbo. Protein Lipid Carbo. Protein ---------------------------------------------------------------------------June DG 1002 907 3544 775 225 186 106 5004 750 AM 296 140 33 15 96 39 226 139 1459 608 194 82 FG 4376 2076 821 1762 395 2536 255 3515 1341 July DG 1071 714 2186 80 54 688 303 1829 715 176 83 AM 182 96 438 284 12 7 194 63 650 342 59 32 FG 1760 910 1928 949 546 299 1488 642 1432 502 833 Aug. DG 2913 734 1020 319 142 34 570 132 422 143 73 41 AM 196 44 1198 395 26 11 126 51 894 16 3 35 14 FG 1928 508 1142 384 554 201 2546 661 772 210 486 Sept. DG 4230 714 1247 431 612 298 1003 277 380 98 261 79 AM 279 117 1881 85 38 139 44 769 28 48 20 FG 2450 1260 595 1198 616 3749 783 426 909 00 N

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Lipid Oct. DG 4291 AH 571 211 FG 2840 Nov. DG 6204 AM 566 203 FG 7559 1982 Table 6 (cont'd) Day 1 Day 4 Carbo. Protein Lipid Carbo 2006 569 410 154 1021 87 869 125 9043 76 22 468 121 8568 1678 1972 5507 1080 209 2271 317 168 1634 526 556 201 32104 339 243 415 192 9878 17574 2768 2881 2675 187 Protein 315 61 137 40 1924 424 282 74 424 884 CX> w

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fraction contained the most CPM mgDW-1 and the carbohydrate fraction the second highest. The digestive gland protein fraction always exhibited the least 14c incorporation. Adductor muscle carbohydrate contained the most CPM mgDW-1 for both Day 1 and Day 4 in all months except June, when Day 1 scallops had more CPM mgDW-1 in lipid 84 and protein fractions than carbohydrate. The lipid fraction had the second highest CPM mgDW-1 at all times except for the Day 1 June sample and the November Day 4 sample in which protein was higher than lipid. Otherwise, the protein fraction had the lowest level of activity within the adductor muscle. For the gonad, CPM were highest in the lipid fraction for all months except July and November on Day 1 and June on Day 4. The carbohydrate fraction contained the most CPM mgDW-1 when the lipid fraction did not, and contained the second most when the lipid fraction contained the most. The protein fraction generally contained the least radiolabel except in September (Day 4) and in October (Day 1 and Day 4) when it exceeded the carbohydrate fraction. Digestive gland lipid activity was quite rapidly lost over the whole study period, with the rate of loss increasing after the July sample (Figure 17). Radioactivity increased in digestive gland carbohydrate in June, but decreased between Day 1 and Day 4 in succeeding months. Protein was the least dynamic of the digestive

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T-0 T-I 0> E a. () I C"') r1" 1 T-I 0 0> E -1 -2 () 85 e LIPID CARBOHYDRATE PROTEIN ---_\6_ ---------------------/ z 0 0 w 0 z ..., (/) MONTH Figure 17. Relative incorporation of 14c by lipid, carbohydrate, and protein fractions of irradians concentricus digestive gland tissue

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gland substrates, showing a slight slope increase in July but minor decreases in all other months. 86 For the adductor muscle (Figure 18), the lipid fraction had a small gain in radiolabel in July, but losses in all other months. Adductor muscle carbohydrate 14c incorporation showed a seasonal pattern in which decreasing gains were found in June and July and increasing losses occurred from August through November. Adductor muscle protein was the least variable, with positive slopes in all months but September, when a slight loss was noted. For the gonad (Figure 19), the lipid fraction showed a seasonal trend, with radiolabel loss between Day 1 and Day 4 greatest in June and less in July. In August, September, and October an increase in gain in radiocarbon lipid was followed by a drastic loss in November. The carbohydrate fraction showed a gain in radioactivity only in June, with losses occurring in the other months. For the protein fraction, a gain was seen in July, but losses were found in all other months. Discussion This study provides a complete account of the intra-organ metabolic transformations occurring in the bay scallop in response to reproductive energetic demands. Labeled food carbon is incorporated into digestive gland, adductor muscle and female gonad body component lipid, carbohydrate and protein skeletons at rates that vary over

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87 -or0 -e LIPID or- CARBOHYDRATE I & PROTEIN 0) 4 E ::E a. (.) I -('I) 1 0 -or-I 0) E ::E a. (.) -1 .....___, -3 (!) 0 ....J :::> -, MONTH Figure 18. Relative seasonal incorporation of 14c by lipid, carbohydrate, and protein fractions of A. irradians concentricus adductor muscle tissue--

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,r-.... T""" 0 T""" I Ol E a. 0 I .q-0 T""" I Ol E a. ('t) -1 -2 0 88 e LIPID 8 CARBOHYDRATE PROTEIN _J z ...J :::> n. () 0 :::> :::> <( w 0 z ...., ...., C/) MONTH Figure 19. Relative seasonal incorporation of 14c by lipid, carbohydrate, and protein fractions of irradians concentricus female gonad tissue

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the course of the reproductive cycle. Based on the study of Mathers (1972), in which radiolabeled amoebocytes were seen in the hemocoele within 6 h of 14c ingestion, 24 h provides ample time for the establishment of scallop "baseline" 14C activities. Differences in CPM mgDW-1 between Day 1 and Day 4 indicate the relative 89 rate of turnover of a particular carbon compound within a body component, with a slower turnover (positive slope) indicating anabolism and a faster turnover (negative slope) indicatin g catabolism. The fact that most of the radiolabel in all fractions was gone after Day 4 suggests that carbon turnover is higher in this species than in M edulis which was found to retain 14c for longer periods (Thompson, 1972). Over the six-month study period, body component carbon incorporation is divided into growth (energy storage) and reproductive (energy utilization) periods. June and July represent a period of nutrient storage in that radiocarbon losses from the digestive gland lipid and protein fractions are relatively small and are equaled by gains resulting from carbohydrate and protein anabolism in the adductor muscle. Apparent losses from gonad lipid and protein fractions at this time are probably due to unassimilated 14C in the intestine which had been excreted by Day 4. In August oogenesis is first evidenced by a gain in gonad lipid radiolabel in conjunction with an increased rate of 14c loss from digestive gland lipid and protein

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90 fractions. Overall, the adductor muscle loses radiocarbon for the first time during this period, with most of it in the carbohydrate fraction. More active oogenesis is indicated in September and October as radiolabeled lipid accumulates at an increased rate in the gonad. Digestive gland lipid and adductor muscle carbohydrate fractions both continue to lose radiolabel at increasing rates. The timing and magnitude of radiocarbon losses from these two body component fractions suggest that they are involved in the oogenic process. After spawning occurs in November, carbon is rapidly lost from all body component fractions, indicating that the physiological condition of these animals is poor. The importance of the digestive gland as the site of bivalve carbon assimilation storage and transfer (Owen, 1966; Van Weel, 1974) is seen in this study. The digestive gland has initially high activity that is lost as carbon is transferred to other body components. During the resting stage, digestive gland loss is relatively slow, with most of this apparently being incorporated into adductor muscle nutrient pools. As oogenesis takes place, the rate of loss of 14c from the digestive gland increases, possibly indicating a carbon transfer between these two body components. Initiation of gametogenesis in this species is associated with the depletion of digestive gland nutrient reserves (Sastry, 1966a, 1968), and radiocarbon is transferred from scallop digestive gland to gonad in

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91 association with game togenesis (Sastry and Blake, 1971; Vassallo, 1973). The digestive gland irradians concentricus functions as a short-term storage organ in that assimilated carbon from recently ingested food can be transferred to storage sites (growth centers) or rapidly turned over to meet i ncreased energetic demands associated with reproduction. In the digestive gland, lipid is the most important in terms of radiolabel content and metabolic participation, since it is the fraction most rapidly lost throughout the study. Lipid is a valuable energy substrate due to its high energy yield per unit weight (Giese, 1966). Digestive gland lipid stores decrease in conjunction with gametogenesis in the scallops Patinopecten yessoensis (Mori, 1975) and Placopecten magellanicus (Robinson 1981). The transfer of radiolabeled lipid from the digestive gland to the gonad of hericia suggests a mechanism for oocyte yolk synthesis whereby lipid is broken down into fatty acids and g lycerol in the digestive gland and transferred to the gonad where triglycerides and are synthesized in eggs (Vassallo, 1973). The existence of increased plasma lipid levels during oogenesis magellanicus (Thompson, 1977) and Crassostrea gigas (Allen and Conley, 1982) supports this proposed mechanism. In scallops, the adductor muscle is an important site of energy storage, the utilization of which is associated

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92 with reproductive development in Chlamys septemradiata (Ansell, 1974b), Pecten maximus (Comely, 1974), Chlamys opercularis (Taylor and Venn, 1979), and P. magellanicus (Robinson et al., 1981). --In this study energy storage is represented by a gain in adductor muscle radiocarbon in June and July which appeared to be supplied from the digestive gland. Once oogenesis is initiated, however, radiocarbon is initially lost from the adductor muscle. Adductor muscle reserves continue to be catabolized throughout the reproductive period, providing energy for reproduction. In A. irradians concentricus the adductor muscle is a long-term storage organ in the sense that nutrients stored prior to gametogenesis are used months later to support oocyte synthesis. Carbohydrate is the most metabolically active fraction in the adductor muscle. During the growth phase it is most rapidly synthesized and during reproduction it is most rapidly utilized. The importance of carbohydrate in bivalve energy metabolism is linked to its more efficient conversion of energy to ATP and its availability to anaerobic metabolism. A loss of adductor muscle carbohydrate reserves occurs over the reproductive period of the scallop species septemradiata (Ansell, 1974b), maximus (Comely, 1974), yessoensis (Mori, 1975), opercularis (Taylor and Venn, 1979), and P. magellanicus (Robinson 1981). This commonly observed synchrony between glycogen utilization

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93 and oogenesis suggests that carbohydrate reserves are converted to lipid in developing ova (Gabbott, 1975, 1976). The rate of incorporation of 14c into scallop gonad tissue is indicative of reproductive stage. During the non-reproductive period, radiocarbon is lost from the gonad between Day 1 and Day 4. During gametogenesis, the female gonad gains radiolabel at a rate that increases as oogenesis continues. The synthesis of gametogenic material is supported by reserves in the adductor muscle and recently assimilated carbon in the digestive gland. However, there is not a 1:1 correlation between 14C gains and losses over the reproductive period, most likely due to the metabolic "cost" of manufacturing oogenic material. The proportion of adductor muscle and digestive gland reserves that actually supports reproduction directly is not known, but it is evident that recently ingested food alone is not sufficient for supporting both maintenance and reproductive metabolisms. After spawning, gonad 14c is rapidly lost, as remaining oocytes are resorbed. The female gonad lipid fraction essentially parallels the oocyte growth curve for this population of irradians. Gonad lipid activity decreases during the resting stage but increases as oogenesis proceeds and fatty yolk accumulates in developing ova. Gonad lipid levels increase in conjunction with seasonal reproductive cycles for a number of scallop species, including septemradiata (Ansell, 1974b), maximus (Comely,

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94 1974), opercularis (Taylor and Venn, 1979), Chlamys tehuelcha (Pollero 1979), and P. magellanicus (Robinson et al., 1981). --These observations concerning the anabolism and catabolism of energy substrates over the reproductive cycle of A. irradians concentricus are comparable to those found edulis by Thompson (1972). Summer is a growth period in which carbohydrate, lipid, and protein reserves are deposited in mantle, digestive gland, and adductor muscle tissues and carbon turnover in the digestive gland is rapid. In the autumn, carbon turnover slows as temperature decreases. Carbohydrate and lipid reserves in the mantle and digestive gland are utilized over the winter to support the synthesis of nucleic acids, lipids, and proteins in developing gametes in the gonad. By early spring, gametes are mature and spawning commences. In contrast to irradians, gametogenesis in edulis occurs during the winter when animals are under nutritive and temperature stress (Bayne, 1976). Energy reserves stored the previous sum mer support maintenance and reproduction. M. edulis has a much greater metabolic reliance on carbohydrate than A. irradians, as indicated by relatively high 0/N ratios (Gabbott and Bayne, 1973) and the ability to accumulate large amounts of glycogen in mantle tissues (De Zwaan and Zandee, 1972). This may be the result of edulis being adapted to an intertidal habitat, undergoing periods of anaerobiosis.

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95 This research reinforces the results of the previous sections on seasonal variation in bay scallop body component weights and indexes (Chapter 3), biochemical compositions (Chapter 4), and substrate catabolism (Chapter 5) and provides a model for reproductive energy metabolism in this species. A period of growth and energy storage in which the adductor muscle increases in size and glycogen level occurs in the spring when food is abundant, prior to the initiation of gametogenesis. Metabolism is supported primarily by digestive gland lipid which is declining in level at this time. Early in the gametogenic cycle maximum O/NH3 and RQ ratios indicate a shift to carbohydrate as the primary respiratory substrate, as the adductor muscle reaches maximal weight and its glycogen reserves begin to be utilized. As these glycogen stores are depleted in conjunction with gamete development, the adductor muscle decreases in weight and protein content. Gametes are mature by the end of the summer, as evidenced by maximum gonad weight and oocyte diameter. Metabolism becomes primarily protein based as the adductor muscle continues to be catabolized. After spawning commences in the fall when food is less plentiful, energy reserves are depleted and scallop physiological condition is poor, as indicated by rapid carbon turnover. This cycle of energy storage and utilization is a reflection of the complex interactions that exist between the exogenous and endogenous factors controlling reproduction in irradians.

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CHAPTER 7 : DISCUSSION The results of this investigati o n describing the reproductive energy metabolism of A. irradians 96 concentricus complement previou s knowledge concerning the regulation of gonad development in this a nimal. In addition to understanding the influe nces of food a nd temperature on gonad g rowth (Sastry, 1963, 1966, 1968; Sastry and Blake, 1971) and how t h e overall process is coordinated by neuroendocrine feedback (Blake, 1972 ; Blake and Sastry, 1979), information now exists on how energy is cycled i nternally within the bay scallop, with the ultimate goal of maximizing reproductive success. The vegetative (non-reproductive) period for Florida scallops is between December and May. Growth prior to March is slow as the result of minimal water temperature and food suppl y In March, as water temperature and primary productivity i ncrease, scallops begin a period of rapid somatic growth. Digestive gland, adductor muscl e and m antle body com p onents increase in weight a nd protein content. With an abundance of food, energy stores are accumulated as lipid in the digestive gland and glycogen and protein in the adductor muscle. Based on unsuccessful attempts to induce gonad growth in three-month-old

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97 scallops, the length of the vegetative stage appears to be genetically fixed, perhaps because of energetic constraints (Blake, 1972; Sastry, 1975). Once the minimal age criterion is satisfied, a change in neurosecretory cycle stage (perhaps invoked by increased temperature) occurs (Blake, 1972). Scallops then become responsive to environmental stimuli for either initiation or delay of gonad growth (Sastry, 1975). If minimum threshold daylength, food, and temperature criteria are met, oocyte growth is initiated in conjunction with the transfer of nutrients from the digestive gland to the gonad (Sastry, 1963, 1966a, 1968; Sastry and Blake, 1971). In Florida, oogenesis is initiated between May and July, near the end of the energy storage period. A transfer of digestive gland lipid to the gonad may accompany this phase of reproduction. After a certain amount of gametogenic material is accumulated, oocyte development to maturation is independent of food supply, providing minimal temperatures are maintained (Sastry, 1968, 1975). This transition is marked by another change in neurosecretory cycle stage (Blake, 1972) and effectively removes the gametogenic process from an uncertain environmental food supply. In Florida scallops this strategy works because of the energy reserves which are utilized in conjunction with cytoplasmic growth beginning in July. Digestive gland lipid is catabolized initially, followed by adductor muscle glycogen

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98 utilization, and finally adductor muscle protein breakdown. vitellogenesis and oocyte maturation occur in September, as indicated by maximum mean oocyte diameter and maximum gonad lipid and protein contents. Spawning of mature oocytes is triggered by a combination of exogenous (temperature, daylength, ectocrine) and neurosecretory factors (Sastry, 1975, 1979; Blake and Sastry, 1979). Duration of the spawning period is limited by a decrease in water temperature and available food and a return of the neurosecretory cycle stage to that coinciding with the resting stage (Blake, 1972; Sastry, 1975). Florida scallops commence spawning in October in conjunction with decreasing water temperature. Spawning continues into the winter, but increased senescence after October limits the duration of effective spawning. The reproductive cycle of A. irradians can thus be described as a genetically controlled response to the environment. Although ultimately under genetic control, the various gametogenic stages are influenced by the interaction of various exogenous and endogenous factors. The reproductive energy cycle described in this study is an integral part of the reproductive process, acting as a buffer between the regulating factors and the actual production of gametes. The energy cycle itself is undoubtedly controlled by neuroendocrine and environmental factors but is critical to reproductive success in that it provides the energy and material required for gamete

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99 synthesis. A schematic diagram summarizing the regulation of bay scallop reproduction is provided in Figure 20. Even though this investigation reveals which substrates are important energy stores in A. irradians concentricus, where i n the animal they are stored, and the timing of their utilization with respect to gametogenic events, little evidence is available to indicate the fate of these reserves. The presence of an ample food supply and relatively constant respiration rate over the gametogenic period provides evidence that these reserves are indeed utilized by irradians concentricus for reproduction. Information exists that not only supports this contention, but suggests that three mechanisms are important in the reproductive energy metabolism of marine bivalves in general. The first mechanism involves the direct transfer of either stored or recently ingested fatty acids from the digestive gland to developing ova in the gonad. Digestive gland lipid reaches high levels in some bivalve species prior to gametogenesis and decreases as oocytes mature (Giese et al. 1967; Pollero et al. 1979; Robinson ---1981). Radioactive biomolecules move from digestive gland to gonad body components in conjunction with reproductive activity (Sastry and Blake, 1971; Vassallo, 1973). The existence of increased lipid plasma levels during the period of oogenesis supports the feasibility of lipid transport between body components via

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100 ENERGY STORAGE 40 -E ::::1. -a: w Iw -() 0 0 30 VEGETATIVE J F M A FOOD CRITICAL NEUROSECRETION EARLY OOGENES I S M J J A s SPAWNING 0 N D Figure 20. Schematic diagram showing the energy storage cycle i n relation to the exogenous and endogenous factors regulating reproduction in irradians concentricus (after Sastry, 1975)

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101 the hemolymph (Thompson, 1977; Allen and Conley, 1982). The second mechanism involves the conversion of carbohydrate to lipid in a manner similar to the glucose-fatty acid cycle in vertebrates (Gabbott, 1975, 1976). A general loss of glycogen reserves from somatic storage areas (which are not replaced until after spawning) often accompanies gonad lipid accumulation (De Zwaan and Zandee, 1972; Gabbott and Bayne, 1973; Zurburg 1979). High physiological indexes during the early stages of gametogenesis indicate the predominance of carbohydrate catabolism and the conversion of carbohydrate to lipid (see Gabbott, 1975; Mori, 1968, 1975). Th e existence of a functional Kreb's cycl e in bivalves is gener ally accepted, as evidenced by the presence of the carboxylic acid intermediates and e n zy mes involved in the cycle and the results of studies on the movement of 14C from glucose through the cycle to 14co2 (Gabbott, 1976). An increase in glucose-6-phosphate dehydrogenase activity associated with sexual development (Mori, 1967) suggests operation of the pentose phosphate pathway which would provide NADPH for fatty acid synthesis. In addition, Bayne et (1982) have documented a decline in the volume of adipogranular (glycogen storing) cells i n the mantle of M edulis during gametogenesis. The considerable advantage to the eggs and larvae of storing lipid (condensed energy source, buoyancy) outweighs the small cost (< 7%) of converting glycogen to lipid (Gabbott,

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102 1976). The third involves the breakdown of protein to indirectly support reproduction. Protein is laid down as new somatic tissue during growth periods, and utilized over the gametogenic period, most commonly in species that undergo oogenesis over the winter when food becomes scarce, and only after other reserves have been depleted (Bayne, 1976; Adachi, 1979). Protein breakdown thus appears to be a response to the combined stresses of gametogenesis and starvation which together can deplete lipid and glycogen reserves. Since there is no evidence that these protein reserves are directly utilized for gamete production, they most likely support maintenance metabolism, thus indirectly supporting gametogenesis. This is reinforced by the low physiological index values obtained during late gametogenesis and spawning periods (Mori, 1968, 1975). Based on the results of this study, all of these proposed mechanisms could be operating in bay scallop reproductive energy metabolism. Digestive gland lipid level decreases prior to the increase in gonad lipid level associated with oocyte growth. Radiocarbon losses in the digestive gland lipid fraction increase in conjunction with gains in the gonad lipid fraction. Thus, it is possible that fatty acids are directly transferred from the digestive gland to the gonad and incorporated into the lipid reserves of developing oocytes. Adductor muscle glycogen utilization is correlated with oocyte diameter

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over the period of cytoplasmic growth. In addition, 14c losses in the adductor muscle carbohydrate fraction increase concurrently with gains in the gonad lipid fraction. RQ values greater than 1 0 suggest the conversion of carbohydrate to lipid over the gametogenic period. It is likely, therefore, that adductor muscle 103 glycogen i s converted to fatty acids that are accumulated in the lipid yolk of oocytes. Adductor muscle protein utilization took the form of a decrease in content rather than a decrease in level and occurred in the late stages of gametogenesis after the other reserves had been depleted. O/NH3 ratios approached 9 0 at this time, indicating exclusive protein catabolism. Thus, it is reasonable that adductor muscle protein supports maintenance metabolism at a time when scallops are under maximum metabolic stress. The mechanisms thought to be important in the reproductive energy metabolism of irradians concentricus are diagramatically summarized in Figure 21. Reproduction in marine bivalves is energetically expensive, often involving a major portion of the annual energy budget (see Bayne, 1976). There is often a greater than 1 to 1 ratio between calories expended to produce gametes and calories contained in the gametes themselves. This is the "cost" of reproduction (Calo\v, 1979) and is undoubtedly the result of metabolic expenditures associated with the synthesis of gametogenic material, whether from direct transfer or conversion mechanisms.

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104 G) Figure 21. Schematic diagram summarizing the reproductive energy metabolism of irradians concentricus; circled numbers indicate the order of mechanism invocation

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105 Reproductive costs are usually measured as reproductive effort, or the proportion of total energy currently invested in reproduction (Hirshfield and Tinkle, 1975; Pianka, 1976). Number of gametes produced per parent (1), biomass of gametes produced per parent (2), biomass of gametes produced per biomass of parent (3), and energy invested in reproduction as a proportion of energy taken in (4) are all used to estimate reproductive effort (Calow, 1979). In terms of the physiological basis of reproductive cost, method (1) is unsatisfactory since gamete size is not considered and the cost of producing a large number of small gametes may be less than the cost of producing a small number of large gametes. Method (2) is unsatisfactory because it ignores parent size and its relative ability to stand the metabolic demands associated with gametogenesis. Method (3) is more satisfactory but is based on the assumption that metabolic economy is directly related to size of the parent. This assumption is probably incorrect since most physiological processes are not linearly related to body size. Method (4) expresses reproductive output in terms of nutrient input, and thus is the best measure of the cost of reproduction (Calow, 1979). Reproductive effort in irradians concentricus can be examined quantitatively after converting seasonal body component biochemical contents (Chapter 4) to energy equivalents using the conversions of 17.16 J mg-1 for carbohydrate, 39.55 J mg-1 for lipid, and 23.64 J

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mg-1 for protein (derived from Brody, 1945). values are given in Table 7 These 106 The ratio of gamete production to total production is generally higher for semelparous species than iteroparou s species, and in iteroparou s species, the ratio increases with successive breeding seasons (Bayne, 1976; Browne and Russell-Hunter 1978). This ratio has been estimated at 8% in one-year-old edulis (Bayne, 1976) and 3% in one yessoensi s (Fuji and Hashizume, 1974). Shafee and Lucas (1980) found gamete production to range from 4 to 10% of total production in Chlamys varia. Several semelparou s freshwater s pecies wer e found to average 29.9% (Brown e a n d Russell-Hunter, 1978). For A irradians concentricus, this ratio reached a maximum of 15.6% in early October in conjunction with maximum oocyte diameter and gonad weight (Table 7). Thus, by this method, the bay scallop is seen to invest considerably more energy in gamete production than other one-year-old iteroparous marine species, but not semelparous freshwater species. The cost of reproduction can also be estimated for A irradians concent r icus, if it is assumed that maximum body size is a reflection of energy taken in. For the bay scallop, somatic energy loss between early August and early October was 22710 J, and overall gonad energy gain was 4713 J (10 Oct value 17 May value) (Table 7), for a 4 8 to 1 cost ratio. Since respiration rate over this period was not related to reproduction (C hapter 5) a n d

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Table 7 Energy content (J) of bay scallop irradians concentricus) digestive gland, adductor muscle, mantle, and gonad body components (based on biochemical composition data); reproductive effort as the ratio of gonad energy content to total energy content x 100 ---------------------------------------------------------------------------------------------Date Dig. Gland Ad. Muscle Mantle Gonad Total Repro. Effort ---------------------------------------------------------------------------------------------17 May 2675 9942 4288 625 17530 3 .56 31 May 2775 10280 4380 754 18189 4.15 14 June 6663 20923 6092 1296 34974 3.71 28 June 9009 23681 7476 1525 41691 3.66 11 July 8238 24227 8392 2888 43745 6 .60 26 July 6576 23223 8151 1983 39930 4.97 9 Aug 9401 31848 10326 2529 54104 4 .67 24 Aug 5800 26598 10126 1892 44416 4.26 7 Sept 7496 23073 9784 3392 43745 7.75 26 Sept 6838 16155 7767 4029 34789 11.6 10 Oct 6858 14697 7309 5338 34203 15.6 17 Oct 7155 10788 7305 3825 29073 13.2 31 Oct 695 1 11738 7230 3675 29594 12.4 ..... 0 -....J

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108 primary productivity in the Anclote Estuary does not begin to decrease until October (Johansson, 1975), most of this energy utilization apparently went toward reproduction. The utilization of somatic energy reserves for reproduction (such as that exhibited by irradians concentricus) is not accounted for in current measures of the cost of reproduction (Pianka, 1976), but certainly warrants consideration. Reproduction is the dominant physiological process in the bay scallop. Gross seasonal variations in biochemical content, substrate catabolism, and 14c incorporation occur in this species as the result of the energetic transformations that accompany gametogenesis. The high metabolic cost of producing gametes in irradians may contribute to its relatively short life span. Unlike other bivalve species most of the energy used to meet this cost is provided by reserves in the adductor muscle. Unlike more northern populations, the bay scallop in Florida is operating at its energetic limit, and is able to provide just enough energy to reproduction to maintain a viable population. These differences are the result of a combination of genotypic and phenotypic environmental adaptational factors.

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109 LIST OF REFERENCES Adachi, K 1979. Seasonal changes of the protein level in the adductor muscle of the clam, Tapes philippinarum (Adams and Reeve) with reference to the reproductive seasons. Comp. Biochem. Physiol. 64A: 85-89. Allen, J.A. 1962. Preliminary experiments on the feeding and excretion of bivalves using Phaeodactylum labelled with 32p J M B 1 U K 42 . ar. io Assoc. : 609-623. Allen, J.A. 1970. Experiments on the uptake of radioactive phosphorus by bivalves and its subsequent distribution within the body. Comp. Biochem. Physiol. 36: 131-141. Allen, W.V. and H. Conley. 1982. Transport of lipids in the blood of the Pacific oyster, Crassostrea gigas (Thunberg). Comp. Biochem. Physiol. 71B: 201-207. Ansell, A.D. 1972. Distribution, growth and seasonal changes in biochemical composition for the bivalve Donax vittatus (da Costa) from Kames Bay, Millport. J. Exp. Mar. Biol. Ecol. 10: 137-150. Ansell, A.D. 1974a. Seasonal changes in biochemical composition of the bivalve Abra alba from the Clyde Sea Area. Mar. Biol. 25: 13-20. Ansell, A.D. 1974b. Seasonal changes in biochemical composition of the bivalve Chlamys septemradiata from the Clyde Sea Area. Mar. Biol. 25: 85-99. Ansell, A.D. 1974c. Seasonal changes i n biochemical composition of the bivalve Nucula sulcata from the Clyde Sea Area. Mar. Biol. 25: 101-108. Ansell, A.D. and P. Sivadas. 1973. Some effects of temperature and starvation on the bivalve Donax vittatus (da Costa) in experimental laboratory populations. J. Exp. Mar. Biol. Ecol. 13: 229-262. Ansell, A.D. and A. Trevallion. 1967. Studies on Tellina tenuis Da Costa I Seasonal growth and biochemical

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110 cycle. J. Exp. Mar. Biol. Ecol. 1: 220-235. Ansell, A.D., F.A. Loosmore and K.F. Lander. 1964. Studies on the hard-shell clam, Venus mercenaria, in British waters. II. Seasonal cycle in condition and biochemical composition. J. Appl. Ecol. 1: 83-95. Barnes, H. and J. Blackstock. 1973. Estimation of lipids in marine animals and tissues: detailed investigation of the sulphophosphovanillin method for "total" lipids. J. Exp. Mar. Biol. Ecol. 12: 103-118. Barszcz, C.A. and P.P. Yevich. 1975. The use of Helly's fixative for marine invertebrate histopathology. Comp. Pathol. Bull. 7: 4. Bayne, B.L. 1973a. Physiological changes in Mytilus edulis L. induced by temperature and nutritive stress. J. Mar. Biol. Assoc. U K 53: 39-58. Bayne, B.L. 1973b. Aspects of the metabolism of Mytilus edulis during starvation. Neth. J. Sea Res. 7: 399-410. Bayne, B.L. 1975. Reproduction in bivalve molluscs under environmental stress. In, Physiological ecology estuarine organisms, edited by F.J. Vernberg, University of South Carolina Press, Columbia, pp. 259-277. Bayne, B.L. 1976. Aspects of reproduction in bivalve molluscs. In, Estuarine processes, vol. 1, edited by M. Wiley, Academic Press, New York, pp. 432-448. Bayne, B.L. and C. Scullard. 1977. Rates of nitrogen excretion by species of Mytilus (Bivalvia: Mollusca). J. Mar. Biol. Assoc. U.K. 57: 355-369. Bayne, B.L. and R.J. Thompson. 1970. Some physiological consequences of keeping Mytilus edulis in the laboratory. Helgolander. Wiss. Meeres. 20: 526-552 Bayne, B.L., A. Bubel, P.A. Gabbott, D.R. Livingstone, D.M. Lowe and M.N. Moore. 1982. Glycogen utilization and gametogenesis in Mytilus edulis L Mar. Biol. Lett. 3: 89-105. Belding, D. 1910. A report upon the scallop fishery of Massachusetts. The Commonwealth of Massachusetts, 150 pp. Blake, N.J. 1972. Environmental regulation of neurosecretion and reproductive activity in the bay

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Prosser, C.L. 1973. (Editor) Comparative animal physiology. W.B. Saunders Company, Philadelphia, 966 pp. Vernberg, W .B. and F.J. Vernberg. 1972. Environmental physiology i marine animals. Springer-Verlag, New York, 346 pp Zar, J H 1974. Biostatistical analysis. Prentice-Hall, Incorporated, Englewood Cliffs, New Jersey, 620 pp. 119

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120 APPENDIX

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121 CRITIQUE OF BIOCHEMICAL PROCEDURES Pooling like tissues prevented the measurement of variability between individuals but did provide an average for the population (Giese, 1967), which was the goal of this study. Tissue lyophilization circumvented protein associated with heat drying and allowed the tissues to be finely pulverized, which aided the analyses (Giese, 1967). Lipid was extracted with the polar solvents chloroform and methanol, which removed structural (phospholipids) as well as storage (triglycerides) lipids (Giese, 1967). Blanks were eliminated by redistilling solvents prior to extraction. Even though the weight of extracted lipid (20-120 mg) was small in relation to beaker weight, it was well within the limit of the balance used (Mettler, 0.1 mg) The procedure was found to be accurate (near 100% recovery of pure lard) and precise ( 3%). The reagents used in the phenol-sulfuric acid method for sugars . 1956) are stable and produce a permanent color with a definite absorption peak, so control of conditions was not critical. The method is sensitive (pg range), precise (0. 01 0 .02 absorbance units) and accurate ( 2%) . 1956). The use of

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122 oyster glycogen as a standard allowed a quantification of the primary bivalve storage carbohydrate. The method used for protein analysis (Lowry et 1951) is also highly sensitive, measuring as little as 0.2 of protein with reasonable precision ( 10%) and little interference from free amino acids. However, color is not strictly proportional to protein concentration a nd the amount of color varies with different proteins (Lowry 1951). Therefore, it is assumed that standards and unknowns have similar protein compositions. The use of bovine albumin as a standard in this study may have resulted in reduced accuracy. Total lipid, carbohydrate, and protein tissue levels always fell below 100% This was partly due to the presence of salts and other inorganic materials. Insoluble polysaccharides and proteins and non-protein nitrogen compounds probably accounted for the remainder. In spite of shortcomings, the analytical procedures employed i n this study are among the best available for the analysis of marine invertebrate tissue. The methods used were kept constant throughout the study, and an attempt was made to minimize all potential sources of error so that meaningful results were obtained.


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