Use of a synthetic polypeptide to determine the sex and reproductive status of field-caught red grouper, Epinephelus morio

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Use of a synthetic polypeptide to determine the sex and reproductive status of field-caught red grouper, Epinephelus morio

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Title:
Use of a synthetic polypeptide to determine the sex and reproductive status of field-caught red grouper, Epinephelus morio
Creator:
Jarrell, Jennifer Lee
Place of Publication:
Tampa, Florida
Publisher:
University of South Florida
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Language:
English
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xii, 107 leaves : ill. (some col.) ; 29 cm.

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Subjects / Keywords:
Epinephelus morio ( lcsh )
Vitellogenin ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )

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General Note:
Thesis (M.S.)--University of South Florida, 2000. Includes bibliographical references (leaves 85-92).

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
027432404 ( ALEPH )
45442682 ( OCLC )
F51-00150 ( USFLDC DOI )
f51.150 ( USFLDC Handle )

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USE OF A SYNTHETIC POLYPEPTIDE TO DETERMINE THE SEX AND REPRODUCTIVE STATUS OF FIELD-CAUGHT RED GROUPER, EPINEPHELUS MORIO by JENNIFER LEE JARRELL A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department ofMarine Science University of South Florida May2000 Major Professor : Raymond R. Wilson, Jr., Ph.D.

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Graduate School Uni vers ity of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Master's Thesis This is to certify that the Master's Thesis of JENNIFER LEE JARRELL with a major in Marine Science has been approved by the Examining Committee on December 3 1999 as satisfactory for the thesis requirement for the Master of Science de gree Examining Comm ittee: }hi)fe ssor : Raymond R. wA?oii7 Jr. Ph .D.

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Dedication This thesis is dedicated to all those who never let me lost faith in myself, but especially to my parents who instilled in me the belief that I could do anything. I would also like to thank those who supported me with the gift of friendship throughout my graduate education I would do a great injustice without also mentioning Dr. James Porter who is greatly responsible for my success as a marine scientist. Without his guidance and encouragement while at the University of Georgia, it is doubtful that I would have pursued a career in marine biology.

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Acknowledgements My research project would never have been completed without the help and guidance of many different individuals Captain Ed Thompson, the crew, and "regulars" of the MN Friendly Fisherman were irreplaceable in helping me to acquire field samples and in teaching me to become a better fisherman The RN Bellows captain and crew were also patient and helpful in field sampling Sample processing could not have been completed without the guidance and expertise of the FMRI Aquatic Health Lab I would like to thank all my friends and co-workers for immense support for my entire stay in the Department ofMarine Science Specifically I'd like to thank the following people: Brian Donahue, Kim Donaldson, Erin Lipp, Dan Merriman, Chris Simonello, Fred Stengard, and Tracey Sutton I would also like to thank the Department of Marine Science the Old Salts Fishing Club, the Aylesworth Foundation the Parrotheads of Tampa Bay, and the Von Rosenstiel Family for supporting various portions of this research Lastly, I'd like to acknowledge my committee for support throughout my years at USF. Dr. Gary Mitchum never let me lose momentum Dr. Jose Torres allowed me to take part in a variety of unforgettable research experiences. Finally, Dr. Ray Wilson was a source of constant encouragement and knowledge and a great fishing partner.

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List of Tables List of Figures List of Symbols and Acronyms Abstract I. Introduction Red Grouper Fishery Regulations Landings Table of Contents Economic Importance Current Stock Status Fishing and groupers Red Grouper Biology Distribution Habitat Feeding Reproduction Hermaphroditism Gonad Morphology Sexual Maturity Spawning Egg and Juvenile Development II Research Development and Design Review of Vitellogenesis and Immunological Methods Vitellogenesis Immunological Studies Experimental Design III Methods Estradiol Stimulation of Red Grouper Synthetic Peptide Production Enzyme Linked Immunosorbant Assay Field Collection Histology lll lV Vl X 1 1 2 3 4 4 5 7 7 9 10 11 11 14 15 16 17 19 19 19 21 23 25 25 26 27 29 31

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BCAAssay 32 SDS-Polyacrylamide Gel Electrophoresis/Western Blotting 33 Optical Density 34 IV Results 35 Vitellogenin stimulation 35 Enzyme Linked Irnmunosorbant Assay 3 5 Ontogenetic Development/Seasonal Maturity 37 Regressed/Immature Female 37 Mature Ripening Female 38 Spent Female 41 Transitional 41 Ripening Mature Male 42 Post-Spawning Male 43 Epinephelus morio 53 Population Structure 57 Optical Density Index 59 V. Discussion 65 Vitellogenin Production and ODI Measurements 65 Population Structure 70 VI. Conclusions 80 References 8 5 Appendices 93 Appendix 1 One and Three Letter Symbols for the Amino Acids 94 Appendix 2 PAS/MY Staining Protocol 95 Appendix 3 Hemotoxylin and Eosin Staining Protocol 96 Appendix 4 BCA Protein Assay Protocol 97 Appendix 5. 15 Residue Sequence BLAST Search Results 98 Appendix 6 12 Residue Sequence BLAST Search Results 1 00 Appendix 7 Western Blot Results for high/low ODI for red grouper 102 Appendix 8. Western Blot of typical gag female 105 Appendix 9 Gag Histological Pictures 107 Appendix 10. Western blotting results for Estradiol-stimulation experiment 108 11

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List of Tables Table 1. Grouper species of commercial and recreational importance Table 2. Artificial peptide sequences Table 3. Monthly collection of E morio Table 4. Monthly collection of M microlepis 6 26 29 29 Table 5 Histological description of each developmental stage 39 Table 6 Results ofK.ruskall-Wallis between developmental stages for 270 kD band 62 Table 7 Results ofK.ruskall-Wallis between developmental stages for 187 kD band 63 lll

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List of Figures Figure 1. Map of sampling area 30 Figure 2 Absorbance(562) versus dilution series ofbleed 2 36 Figure 3 Absorbance(562) versus dilution series ofE2-stimulated fish 36 Figure 4. E. mario regressed mature female and typical immature female 43 Figure 5 E mario early ripening female and mid ripening female 44 Figure 6. E mario typical late ripening and ripe female 45 Figure 7 E. mario spent females 46 Figure 8 Ripening male E. moria 47 Figure 9. Late ripening male E. mario 48 Figure 10. Spent male E moria 49 Figure 11. Transitional and female portraying precocious sperm crypts, E. mario 50 Figure 12. Early transitional E. mario 51 Figure 13. Percentage of female red grouper in various developmental stage 54 Figure 14. Percentage of female red grouper in vitellogenesis by month 54 Figure 15. Percent of female E moria in various stages of vitellogenesis 55 Figure 16. Mean GSI values for female E mario for each month 55 Figure 17. Percentage of male red grouper in each developmental stage 56 Figure 18. Mean male red grouper GSI for each month 56 Figure 19. Length frequency distribution 57 iv

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Figure 20. Length frequency distribution for male E. morio 58 Figure 21. Length weight relationship for E. morio 58 Figure 22. ODI value for 270 kD band for each developmental stage 60 Figure 23. ODI values for 187 kD band for each developmental stage 61 Figure 24. Mean ODI +SE for each developmental class for MW 270 kD band 64 Figure 25. Mean ODI +SE for each developmental class for MW 187 kD band 64 Figure 26. Western blot oflow and high ODI values for 270kd band 102 Figure 27 Western blot oflow and high ODI values for 270kd band 103 Figure 28. Western blot of high/low ODI values for 270kd band 104 Figure 29. Western blot of gag grouper females 1 05 Figure 30. Gag Histology Pictures 106 Figure 31. Comparison of FIM high and low ODI 270kD value s .. 107 v

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List of Symbols and Acronyms A(562) absorbance at 562 nanometers AB atretic bodies ALPP Alkali Labile Protein Phosphorus ANOVA Analysis of Variance AP Alkaline Phosphatase BCA Bicinchoninic Acid BCIP 5Bromo-4-Chloro3Indolyl Phosphate BLAST Basic Local Alignment Search Tool oc degrees Celsius CA cortical alveoli CPUE catch per unit effort Cu Copper DO degenerating oocyte DTT Dithiothreitol E2 Estradiol EDTA Ethylenediaminetetraacetic Acid EEZ Exclusive Economic Zone ELISA Enzyme Linked Imrnunosorbant Assay EtOH Ethanol Vl

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FFWCC FMFC FGOC FER FLR FMR FIM FOM FR FS g GMFMC GVM HMW IgG kD kg KLH M mg MLR mm mM Florida Fish and Wildlife Conservation Commission Florida Marine Fisheries Commission (now FFWCC) Florida Governor's Ocean Committee female early ripening female late ripening female mid ripening female immature final stage of maturation female ripe female spent gram or gravity Gulf of Mexico Fishery Management Council germinal vesicle migration high molecular weight Immunoglobulin G kilodalton kilogram Keyhole Limpet Hemo cyanin molar milligram male late ripening millimeter millimolar Vll

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MR MRG MS mV MW NaOH NaN3 NBT run NaCl NMFS ODI PBS PBST PMSF ppt SAFMC SDS-PAGE SL SPR TL TR Vt male ripe male ripening male spent millivolts molecular weight Sodium Hydroxide Sodium Nitro Blue Tetrazolium nanometer or nautical mile Sodium Chloride National Marine Fisheries Service Optical Density Index Phosphate Buffered Saline Phosphate Buffered Saline with 1% Tween-20 Phenylmethylsulfonyl Fluoride parts per thousand South Atlantic Fishery Management Council Sodium Laurel Sulfate -Polyacrylamide Gel Electrophoresis standard length spawning potential ratio total length transitional vitellogenin Vlll

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j.lg J.!l 2X micorgram microliter two times the concentration IX

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USE OF A SYNTHETIC POLYPEPTIDE TO DETERMINE THE SEX AND REPRODUCTIVE STATUS OF FIELD-CAUGHT RED GROUPER EPINEPHELUS MORIO by JENNIFER LEE JARRELL An Abstract Of a thesis s ubmitted in partial fulfillment of the requirements for the degree of Master of Science Department of Marine Science University of South Florida May 2000 Major Professor: Raymond R. Wilson, Jr. Ph.D X

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Due to the relationship established in the literature between plasma estradiol levels serum vitellogenin, and gender the use of vitellogenin (Vt) measurements to determine the gender of fish is logical and straightforward. To possibly exploit this relationship in a future study of sex reversal rates of red grouper in situ the present study examined the gonadal histology changes prior to, and immediately following, the spawning season of red grouper, Epinephelus morio, in relation to the variations in plasma Vt determined by immunogenetic probing i.e. antibody bindin g to a Vt synthetic peptide Synthetic peptides were composed from conserved vitellogenin sequences from Oncorhyncus mykiss and Fundulus heteroclitus, and anN-terminal sequence from gag Mycteroperca microlepis. Peptide synthesis, cojugation to KLH and antibody production was done by Genosys Biotechnologies. Gonadal histology was employed as ground truth for the sexual state of the study fish. Blood and gonad specimens of red grouper were collected on recreational and research vessels from February until August of 1998 Gag samples were also taken during the same period Ninety-five red grouper were sampled, with 75 histological females 19 males and 1 early transitional fish. All gag were females. Blood samples from fish were expelled into anticoagulant and held on ice until centrifuged and plasma was taken off and frozen at 80C. Samples were later analyzed by Western blotting for reactivity with synthetic peptide antibodies. Female red grouper were histologically determined to be either regressed/immature ( 49) early ripening (6) mid ripening ( 4) late ripening (7) ripe (2), or spent (4). Male red grouper were ripening (4) late ripening (5), ripe (6) or spent (4). One fish was an early transitional. 25 of the gag samples were determined to be regressed/immature and 3 were early ripening females Xl

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Blood samples were taken from 92 red grouper and 28 gag. Western blot s showed all gag samples to be virtually identical in Vt content but variation existed among red grouper samples Two protein bands appeared to react most intensel y with the s ynthetic probe in red grouper blots The molecular weights of these bands w e re 187 kD and 270 kD. The 187 kD band was stimulated in estradiol-treated red grouper with definite higher levels produced in test versus control fish However no significant differences s een among any of the developmental stages with respect to the 187 kD band nor between histological male and females. The 270 kD band s howed a significant peak early / mid ripening female fish. Oocytes were discernible in male red grouper indicating a lack of hormonal control" in sex-reversed male fish This lack of hormonal control in protogynous grouper could explain the occurrence of vitellogenin levels comparable to that found in female fish. The present study is in agreement with previous studies that have noted the occurrence of vitellogenin in male fish This indicates that vitellogenin is not a female specific protein and should not be referred to as such Abstract Maj Professor : Raymond R Wi ,.Jr. Ph. D As ocate Professor, Department of Marine Science Date Approved : _____ ___!,_,--!'------Xll

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I. Introduction Red grouper, Epinephelus morio, is assigned to the teleost family Serranidae that includes both synchronous and sequential hermaphrodites. Sequential protogynous hermaphrodites, fish that begin adult life as female and become male later in their lifecycle, are prevalent in the genera Epinephelus and Mycteroperca (Bannerot 1984; Sadovy and Shapiro, 1987) For species such as red grouper that have a relatively discrete distributional range, the ability to change sex allows greater reproductive potential in isolated populations (Smith, 1975). Even though Epinephelus morio and Mycteroperca microlepis comprise the majority of the commercial grouper catch in the Gulf of Mexico little is known of their reproductive biology and the consequences that conservation measures and fishing pressure might have on the population structure Red grouper fishery Red grouper, Epinephelus morio, is the most important commercial grouper species in the Gulf of Mexico, making up ai1 average of 69% of the grouper catch for 1986-1989 (Schirripa, et al., 1999; Rivas 1970 ; Moe, 1969; Jarvis, 1935: 3). The fishery is regulated by the state of Florida and the federal government. The Florida Marine Fisheries Commission (FMFC) has jurisdiction up to 9 miles off Florida's coast. The federal government takes over within the US Exclusive Economic Zone (EEZ) that

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extends out to 200 miles offshore. In 1976 the Magnuson Fishery and Conservation Act excluded foreign fishing within 200 miles of the U.S. shoreline and also created regional councils that are responsible for managing their own fisheries throughout the United States The Gulf of Mexico Fishery Management Council (GMFMC) regulates the EEZ of Florida's west coast and therefore the grouper fishery. The range of red grouper also extends into the jurisdiction of the South Atlantic Fishery Management Council (SAFMC), but the bulk of the fishery is located from Tampa southward (Schirripa et al. 1999) Regulations In 1985 Florida set a size limit of 18 inches total length for grouper. This was followed in 1986 by a daily aggregate bag limit of 5 grouper per recreational fisherman and prohibition of the use of longline gear in state waters. In 1990, FMFC set the si z e limit to 20 inches total length for red black gag and yellowfin grouper, and the GMFMC followed the 20 inch size limit and 5 grouper bag limit in the same year. Currently the GMFMC commercial quota for shallow water groupers is a gutted weight of9.8 million pounds annually (Schirripa et al. 1999) The most recent changes in grouper regulations will occur with the NMFS approval of Amendment 9 to the GMFMC's Reef Fish Fisheries Management Plan (FMP). The GMFMC approved an increase in size limit to 24 total length (TL) for black, and gag grouper and a complete closure of the commercial fishery for red, black and gag grouper during the peak spawning period. 2

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The recreational size limit will increase immediately to 22" and increase an inch a year until reaching 24". Landings The commercial harvest of grouper in the Gulf was not reported by species until 1986 Prior to 1986, all species were combined into a category called "unclassified grouper" (Schirripa et al., 1999). This poses a problem for determining the historical significance of the species and the changes occurring in size and weight of individuals in the Gulf population. After species were designated, statistics for the commercial catch illustrate the importance of the red grouper. Red grouper make up greater than half of the total commercial grouper harvest in the Gulf of Mexico (Goodyear and Schirripa 1991 and 1993). The preferred methods of capture for the Gulfs fishery of red grouper is hand and power lines or bottom longlines. With the introduction of longline gear in the 80 s the total poundage of grouper landed increased from a low of about 5 million pounds in 1970 to a high of 12.5 million pounds in 1982 (Goodyear and Schirripa, 1993). However since then commercial landings have shown a slow decline to approximately 55% of the 1982 high and estimates of the recreational harvest for 1997 are the lowest since 1981. Average size of the catch composition also appears to be declining (Schirripa et al., 1999). 3

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Economic Importance Data from the National Marine Fisheries Service Fisheries Statistics Division shows that the Gulf is second only to the Pacific Coast and Alaska in total landings for all fish species (incl u ding shellfish) in 1996 and 1997. Grouper rank fourth in the U.S fishery when comparing total pounds landed to total dollars made, only swordfish bigeye tuna and bluefin tuna have a larger value per pound (excluding shellfish) (Fisheries of the United States 1997) According to a recent report to Florida's governor by the Governor s Ocean Committee (FGOC) in 1997 the commercial fishing industry landed 121 million pounds of seafood including species that are provided to the United States almost exclusively by F lorida The Florida Sea Grant College Program estimates that the commercial industry associated with Florida seafood provides the state with $900 million annually and $600 million in commercial seafood related wages. The recreational sector also contributes vastly to Florida s economy The Tampa/St. Petersburg area is especially important to the grouper fishery as the greatest part of the recreational and commercial harvest come from Tampa southward and roughly half of the harvest from the Tampa/St. Pete area alone. Since grouper landings were separated into species in 1986, red grouper have made up about 2/3 of the total grouper catch (Schirripa et al. 1999; Goodyear and Schirripa, 1993) Current stock status Currently of the 18 grouper species that are important both recreationally and 4

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commercially in the South Atlantic and Gulf of Mexico only one species, Mycteroperca phenax (scamp), is recognized as not overfished This status is recognized only in the South Atlantic region as scamp status is unknown in the Gulf. The most recent stock assessments published by NMFS determined red grouper to be overfished in the Gulf, and found gag grouper approaching an overfished state (Schirripa et al., 1999; Schirripa and Legault, 1997). Black grouper is of unknown status in the Gulf and overfished in the South Atlantic region according to NMFS. See Table 1 for a listing of important grouper species in Gulf and South Atlantic regions their management agencies and current stock status. In 1997, the National Marine Fisheries Service also designated Florida for the ninth year as top state for saltwater fishing activity (FGOC, 1998). With predictions for numbers of tourist anglers to rise to nearly 6 million in the next l 0 years, Florida's fisheries face ever-increasing pressure and require more detailed knowledge in order to manage them for the future. Fishing and groupers Huntsman and Schaaf ( 1994) stated . fishery managers have immediate need for information about the interaction of protogyny and fishing In protogynous groupers, the rate of change is crucial to know because if the population is heavily fished during spawning times, the females may not be able to convert to functional males quickly enough to allow successful reproduction of the population. The long life spans large size at maturity, slow growth, catchability (i.e. aggregations), and sexual characteristics of these economically important fish are believed to render them much more susceptible to 5

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Tab l e 1. Groupe r species of corrunerica l and r ecrea t iona l importance i n the Gulf and South Atlantic and their status according to 1998 NMFS Report to Congress-Status of U.S. Fisheries and NMFS most recent stock assessments Species Management Unit Status Epinephelus adscensionis (rock hind) GMFMC / SAFMC UK/UK Epinephelus cruentatus (graysby) SAFMC UK Epinephelus drummondhayi (speckled h i nd) GMFMC / SAFMC UK/OF Epinephelus jlavolimbatus (yellowedge) GMFMC / SAFMC UK/UK Epinephelusfulvus (coney) SA FMC UK Epinephelus gutta/us (red hind) GMFMC/SAFMC UK/UK Epinephelus itajara Uewfish) GMFMC / SAFMC OF/OF Epinephelus morio (red) GMFMC / SAFMC OF/OF Epinephelus mystacinus (misty) GMFMC / SAFMC UK/UK Epinephelus nigritus (Warsaw) GMFMC / SAFMC UK/OF Epinephelus niveatus (sno1ry} GMFMC / SAFMC UK/OF Epinephe!us stria/us (nassau) GMFMC/SAFMC OF/OF Mycteroperca bonaci (black) ** GMFMC/SAFMC UK/OF Mycteroperca interstitia/is (yellowmouth) GMFMC/SAFMC UK/UK Mycteroper c a mic rolepis (gag) ** GMFMC / SAFMC AOFIOF Mycteroperca phenax (scamp) GMFMC / SAFMC UKINOF Myc teroperca tigris (tiger) SAFMC UK Mycteroperca venenosa (yellowfm) GMFMC / SAFMC UK/UK a closed fishery ** regulauons extst that protect spawnmg aggregati Ons o r season UK= unknown s tatus OF= overfished status AOF= approaching overfished status NOF = not overfi shed overfishing than gonochoristic species (Coleman et al., 1999; Huntsman and Schaaf, 1 994; Bannerot, 1984). Sadovy (1990) reviewed grouper management needs for assessed stocks in the Western Central At l antic and found sharp declines in average size and weight lowered l andings and catch per unit effort (CPUE) and loss / r eductions in spawning stock size at aggregations for most stocks To better unders t and the management of these species, Sadovy (1990) calls for increased information in several areas. T h e reproductive biology of grouper is one area stressed as lacking in information. Specifica ll y, i nformat i on on the number and location of spawning aggregations, the relationship between fecundity and body size, sex ratio, and the size at sex change if it occurs (Sadovy 1990). Coleman et al. (1999) also noted the need for increased understanding ofthe reprod ucti ve modes of 6

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reef fish such as grouper. The authors specifically call attention to the fallacies of spawning potential ratio (SPR a ratio of the female reproductive potential of an unfished vs a fished population) in estimating management measures for hermaphroditic stocks especially when so little is known concerning how male decline affects reproductive potential (Coleman et al., 1999). In order to determine the rate of sex reversal and the interactions that are responsible, a field study of grouper must be undertaken. Before this can be successful a method to accurately identify the sex of individual fish and the initial sex ratio of the study population should be developed. Red grouper are not sexually dimorphic, therefore frequent sacrifice ofthe fish is necessary for study. However LeBail and Breton (1981) and other authors have developed assays for other teleosts requiring only a small amount of blood from the animal in order to determine its gender. These non-invasive technique s should be further studied and qualified for utility in management applications. The present study was undertaken to develop a non-inva s ive technique to better understand red grouper biology and the rates and interaction s involved in s ex reversal. Red Grouper Biology Distribution The center of abundance for red grouper is the west F l orida shelf and the Gulf of Mexico (Moe, 1969). Red grouper also comprise the most important fisheries resource on the continental shelf of the Yucatan on Campeche Bank Mexico (Arreguin-Sanchez et 7

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1., 1993). E. morio, predominantly a continental species, is reported to be abundant in the Colombian Caribbean and off ofNE Venezuela (Jory and Iversen 1989). The northernmost extent of red grouper is documented as 50 nautical miles (nm) east of Cape Fear, North Carolina and the southernmost extent to 107 nm NNW of Cayenne French Guiana (Rivas, 1970). Rivas dismissed reports of more northern ranges ofthe species as due to "stragglers." However, Smith ( 1971) described a distribution from Massachusetts to Brazil including the Antilles and Bermuda. Jory and Iversen (1989) suggested that the more northern records for red grouper described in the literature are the likely result of larval transport, as in other grouper species (Thompson and Munro, 1978) Moe (1969) described the offshore movement of E. morio Young grouper tagged in shallow environments remained there for several years, and with increasing age began to appear in commercial catches offshore. Grouper larger than SL and older than 5 years left the near shore environment for deeper water Jory and Iversen (1989) found these older fish move to water at least 36m deep and stay near selected reefs for extended periods oftime. Moe (1969) pointed out that this migration coincided with sexual maturity. This observation is supported by Bullock and Smith (1991) who found small red grouper at shallow water (3-18 meter depth) reefs off Southwest Florida Brule and Deniel (1993) reported that the inshore population of Campeche Bank Mexico were composed of juvenile females while offshore collections were comprised of juvenile and adult females transitionals, and adult males. Moe (1969) documented one individual as moving 45 miles in 466 days and a group of 22 tagged fish moving 18 miles in 50 days. Commercial fishermen have reported seasonal movement in adult populations as well as aggregations in the water off 8

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Lower Matecumbe in the Florida Keys (Bullock and Smith, 1991 ). In the northern Gulf, red grouper appeared to be much more abundant during the warm season (May-October) and catch records indicated greater landings in the southern Gulf during the cool months (November-April) (Rivas, 1970). Habitat Red grouper are benthic, sublittoral inhabitants preferring rocky reef bottoms between 3 and 130 meters. The fish occupy crevices, ledges, and caverns in the limestone (Moe, 1969). However, in contrast to other groupers, specimens have been taken extensively on low relief hard bottom on the west Florida shelf (Bullock and Smith, 1991 ). Red grouper prefer to stay near the substrate and avoid mid water. E. morio can be found sharing habitat with other less common groupers such as snowy grouper (Epinephelus niveatus), Warsaw grouper (E. nigritus), rock hind (E adscensionis) Kitty Mitchell (E. drummondhayi), red hind (E. guttatus), yellowfin grouper (Mycteroperca venenosa), scamp (M phenax), gag (M microlepis), yellow-mouth grouper (M. interstitia/is), and black grouper (M bonaci) (Rivas 1970). Smith (1971) pointed out that grouper in general seem to have a "compelling need for cover." This leads to the co occurrence of species on habitat favored by grouper. Juveniles (less than 200 mm SL) are rarely found in collections from the West Florida shelf. They are sometimes found in seagrass beds, passes and rock formations, but are not found in numbers comparable to M microlepis (Koenig and Coleman, 1998; Bullock and Smith, 1991; Smith, 1971; Moe, 1969). Small red grouper have been 9

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reported to wedge themselves into crevices (Moe 1969). This behavior could explain the paucity of samples using traditional collecting gear. According to Moe (1969) the center of inshore abundance for juveniles appears to be the Florida Keys, while offshore they are found in low density over hard bottom throughout the Gulf at 36 meters Houde ( 1982) reported Epinepheline spp larvae in the eastern Gulf of Mexico from 1 0200m but samples were more commonly collected at s tations between the 10 and 50m isobaths F e eding Species of the subfamily Epinephelinae (includes grouper coral trout, rock cod etc ) are the most common top level predator on coral reefs (Thresher 1984) Grouper capture prey by opening the mouth and dilating the pharynx This action enables fish to inhale" prey (Smith 1971 ). Red grouper have been described as unspecialized carnivores that feed on small fishes of many species crabs lobsters s hrimp s, octopuse s squids and other crustaceans Although crustacean s dominated stomach cont e nts larger grouper consumed a higher ratio of fish as size increased (Moe, 1969) Jory and Iversen (1989) reported Keys' populations to feed on lutjanids sparids other fishes shrimps stomatopods, and spiny lobsters. In samples collected from the Campeche Bank Mexico juvenile red grouper prey was dominated by reptant crustaceans the diet not significantly changing with increasing si z e (Brule 1993). Adults occupy the top nich es in the complex reef food webs along with other generalized, opportunistic predators such as other groupers sharks, snappers (Lutjanidae), king mackerels (Scombridae), and jacks 10

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(Carangidae). Members of the top trophic level compete for resources (Bannerot, 1984; Arreguin-Sanchez and Valero 1993). Due to this competition when a top level predator is reduced by overfishing or poor recruitment, the biomass of other predators is increased (Arreguin-Sanchez and Valero, 1993) Reproduction Red grouper have been classified as protogynous hermaphrodites (Moe 1969 ; Sadovy and Shapiro 1987; Johnson 1995). This mode of reproduction allows female fish to convert" to males at some point in their lifecycle. Smith (1975) suggested protogynous and protandrous life cycles would allow the most success in reproduction among species that can be reduced to few individuals On intermittent hardbottom habitat within the Gulf of Mexico the removal of large male red grouper from a population could render a social group (if it exists) devoid of reproduction, if females were unable to change sex. Hermaphroditism. Ross ( 1990) defined hermaphroditism a s the presence of both s ex functions at some time during the life of an individual. The Serranidae is repres e nted by both synchronous (having male and female function concurrently) and sequential (gonads develop first as one sex and then the other) hermaphroditism. Among S e rranus species synchronous hermphroditism is common (Smith, 1975). Epin e phelus mario and 11

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Mycteroperca microlepis both exhibit sequential protogynous hermaphroditism. In the wrasses (Labridae ) parrotfishes (Scaridae ), and large groupers, protogyny is the dominant reproductive strategy in species studied (Warner, 1984) In this reproductive strategy juveniles mature into females, and males result when a female changes sex due to some cue (Shapiro, 1987) Two different types of protogynous modes exist. Monandry, the first of these modes, occurs in Epinephelus morio and other grouper species. Monandric populations are composed of only secondary male fish (those that were first females). In diandric populations males are both primary (develop directly into males from juveniles) and secondary. Diandric protogyny is common among the wrasses (Labridae ). Ghiselin developed several models attempting to explain the existence of hermaphrodites ( 1969 197 4 ) One of those models, the size advantage model, was proposed to explain sequential hermaphroditism The size advantage model maintained that if it was more advantageous to an individual s reproduction (i e. they can increase the number of eggs produced or gametes fertilized) to be a large male and a small female then protogyny would be the evolved mode of reproduction. The reverse would be true for protandry (Ghiselin, 1974). The individual fish would produce more offspring as a hermaphrodite than if it remained male or female (Warner, 1984) The mating system of the species would affect the reproductive potential of each sex. For example, in a species that has a large male dominating spawning fertility as a male would rise dramatically at some large size, thus protogyny would be favored. Small males would be selected against due to inexperience and preference among females for large males in the population (Warner, 1975). If group spawning with no dominance and random 12

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pairing of individuals occurred, then small males and large females would be expected and protandry would be advantageous (Warner, 1984 & 1975) Warner (1984) suggested that the interplay of clear water in a tropical reef environment sedentary habits, and planktonic larvae allow for greater dominance by large males and thus the evolution of protogyny in the reef environment. Ross (1990) and Shapiro (1984) attributed sex change to social circumstances that either induced or inhibited reversal in an individual Shapiro (1984) discussed behavioral induction as a social control and concluded the size-advantage model proposed b y Ghiselin might be applied to behavioral induction by "paraphrasing" him: . .if an animal in one circumstance can reproduce more effectively as one sex, and under a different circumstance can reproduce more effectively as the other sex then it becomes advantageous for an individual to switch gender as it changes circumstances The independent variable becomes circumstance, meaning the number of co-resident males and females, the pattern of interaction between those fish, and the historical, sociodemographic events producing these conditions, and size/age drops from the formulation entirely Ross (1990) and Warner (1988) stated similar conclusions and supported size-advantage as a "general theory" under which each case of hermaphroditism could be understood individually. Ross (1990) defined sex change suppression (inhibition) as occurring when sex change is prevented through interactions in a social group (i.e. dominance) Sex c hange induction describes reversal that must actually be stimulated. This stim ulation can occur through behavioral cues or through sizeor sex-ratio induction Sex-ratio induction is caused by a change in the sex rat io of the resident population. Size-ratio induction stim ulates sex change by functional use of a "threshold ratio." When the ratio 13

PAGE 30

of the number of smaller fish to larger fish is altered (i e. by recruitment or mortality), sex reversal occurs. Gonad morphology. Gonads in Epinephelines are paired, posterior to and inferior to the swim bladder, and joined to form a common oviduct (Thresher, 1984). In general, grouper show little sexual dimorphism and sex must be determined by inspection of ripe gonads (Charnov, 1982). A complete mixture of male and female gonadal tissue occurs in the Epinephelines (Smith, 1975). Small crypts of testicular cells are mixed in the germinal epithelium of the lamellae (longitudinal folds of germinal epithelia) with ovarian tissue These crypts lie dormant within the lamellae of female fish (Shapiro, 1979; Thresher 1984 ). Sex reversal is apparent when crypts begin to invade the gonad and oocytes begin to degenerate (Shapiro, 1979; Thresher, 1984) Once reversal is complete spermatozoa pass into sperm sinuses located i n the center of the remnant lamellae, and into a central sinus on the dorsal surface of the gonad, then pass into the sperm duct developed between the oviduct and urinary duct (Moe, 1969). Most protogynous gonads are distinctive because the lamellar form of the ovary is retained in the testicular tissue The gonad also retains the hollow structure of the ovary. Atretic bodies and some first and second stage oocytes can also be found as remnants in sex reversed fish (Shapiro, 1979 ; Sadovy and Shapiro, 1987). Shapiro and Sadovy (1987) described the evidence for protogyny and surveyed the literature to identify species that had been labeled protogynous following the proper criteria Features suggesting protogyny as identified by Shapiro and Sadovy 14

PAGE 31

(1987) include: membrane lined cavity (remnant ovarian lumen) in testes; transitional fish; atretic bodies of oocytes in testes; sperm sinuses in gonad wall ; and production of transitional fish through social manipulations. The work ofMoe (1969) allowed definitive evidence for a protogynous mode in E. morio (Shapiro, 1987). Moe found all of the above criteria, but did not attempt to manipulate a population to produce transitional fish. However, protogynous Coryphopterus (gobiid) species examined to date have testes without ovarian structure (Cole and Robertson 1988; Cole and Shapiro,1990 and 1992). Species within the Serranidae have also been found to lose the remnant lumen structure to the ovary InAnthias squamipinnis males about halfhad no remaining membrane-lined cavity (Shapiro, 1981) In fish such as Coryphopterus species and A. squamipinnis without remaining ovarian structure, the best criteria to u se is artificial induction of sex reversal (Cole and Shapiro, 1990). Sexual Maturity. Moe (1969) found hi s tological evidence of sexual maturity in E. morio at 425500mm SL and 4-6 years of age with peak sexual maturity between 8-12 years. Sexual transition was reported to occur between the ages of 5 and 10 and Jory and Iversen (1989) found males reproductively significant at ;::: 1 0 years of age. Moe ( 1969) found males between the ages of7 and 10. Johnson (1995) found males between the ages of 4 and 10 (375-400mm TL), with largest size class being 725-750 mm TL and 6-8 years old. Transitionals were found in age classes 4 to 7 (460-680 mm TL) and females were most common from 4-6 years old (500-525 mm TL) (Johnson, 1995). Johnson (1995) pointed 15

PAGE 32

out that most of the fish caught were in the 4-6 year age group. The author suggested few fish are reaching the older size classes and relatively small females are converting to male fish. This would effectively reduce the mean size of fish in the Gulf population and concomitantly reduce the number of female and male fish that are reaching their full reproductive potential. Spawning. Eastern Gulf of Mexico E. morio populations spawn from January to May with a peak in April and May (Coleman, et al., 1996; Johnson, 1995; Moe 1969) The abundance ofEpinepheline spp. larvae found in the eastern Gulf coincides with this spawning peak (Houde, 1982). Campeche Bank populations in the western Gulf spawn earlier with the season stretching from September to March, and a peak is seen in January and March (Brule and Deniel 1993) Brule and Deniel (1993) possibly attribute this spawning season difference to temperature variation between the northern and southern portions of the Gulf during late fall/spring Although Moe (1969) did not find evidence ofmulitple spawning by individual fish, a 1993 study by Brule and Deniel found mature individuals with both post-ovulatory follicles and vitellogenic oocytes in stages II, III, and IV in January March They also found a high percentage of male fish with ripe testes from January-April. The authors suggested females may spawn more than once throughout the reproductive season in the Campeche Bank population Koenig (1993) also concluded from histological evidence that E. morio are batch spawners and release eggs over a protracted spawning season. 16

PAGE 33

No evidence has been found indicating red grouper form spawning aggregations similar to the Nassau grouper, E. striatus (Colin, 1992 Smith 1972 Thresher 1984 ' ' Tucker et al. 1993), the red hind, E. guttatus (Shapiro et al. 1993), the gag, Mycte r op e rca microlepis (Gilmore and Jones, 1992 ; Coleman et al. 1996) the scamp M phenax (Gilmore and Jones 1992; Coleman et al., 1996) the tige r grouper, M tigris (Sadovy et al. 1994) or other grouper species. Red grouper are thought by some a uthors to spawn in small polygamous groups with no depth dependent spawning sites ( Coleman et al. 1996) Brule and Deniel (1993) found ripe females on various locations of Campeche Bank throughout the spawning season, supporting current thought on small a ggregation spawning by E. morio (1993) Egg/ Juvenile development. Moe (1969) estimated fecundity in 495-667 mm SL f e males at a mean of 1 469 000 Epin e phelus morio eggs are almost spherical and about 0 .95 mm in diameter They are unpigmented with a single oil droplet. Laboratory reared eggs hatched at 30-38 hours post-fertilization ( Colin et al. 1993) Newly hatched larva have a large yolk sac no fins, unpigmented eyes and a single oil droplet (Thresher 1984) Colin et al. (1993) found a salinity necessary for positive buoyancy for newly hatched eggs and 32ppt at late stage ( > 20 hour s post-fertilization). Spawning along inshore areas of the Gulf would be limited for red grouper as many places have salinitie s lower than 32ppt. Eggs are negatively buoyant at 25 ppt and undergo limited development (Colin et al., 1993). Larvae develop elongate dorsal and pelvic spines Colin et al. (1993) speculated that spine development deters predation Pelagic juveniles 17

PAGE 34

transform to benthic juveniles at about 20mrn SL (35-50 day s post hatch ) ( Colin et al,. 1993) Moe (1969) describes a 20mrn SL specimen taken at 36 6 m in the Gulf with reduced spine length and dark pigmentation, indicating the transformation from pelagic to benthic 18

PAGE 35

II. Research Development and Design Review of Vitellogenesis and Immunological Methods For the species of protogynous fishes that have been studied, the rate of sex reversal has been found to be relatively fast. In Serranus species when a male is removed from the group, a large dominant female becomes a male in about two weeks (Shapiro, 1984) Little is known concerning the rate of change in the economically important groupers To address the need for this information and the questions posed by Huntsman and Schaaf ( 1994) and Sadovy ( 1990) this study utilized a plasma protein thought to vary sea s onally and sexuall y in fishes including grouper. The protein utilized was vitellogenin which is produced during the process of vitellogenesis in female fish. Vitellogenesis The growth and survival of embryos that develop outside of the female depend on reserves of energy stored as yolk within the oocyte. Yolk is not synthesized in situ" but derived from the breakdown of a precursor protein vitellogenin. Vitellogenin (Vt) i s a female specific protein produced by the liver in response to stimulation by estradiol. Vt is released into the blood stream until taken up by growing oocytes where conversion to storage forms takes place (Ho, 1987). The inclusion of Vt into oocytes (through the 19

PAGE 36

blood capillaries of the theca then through channels between the follicle cells and finally by pinocytosis) and the subsequent sequestering of nutrient reserves (i e yolk) is responsible for the increase in size of ovaries seen during the reproductive period of many fishes (Wahli et al., 1981; Wiegad 1982) This sequestering ofyolk materials is known as exogenous vitellogenesis Once in the oocyte, vitellogenin is proteolytically cleaved into the yolk proteins lipovitellin and phosvitin (W ahli et al. 1981 ) De Vlaming et al. (1980) describe vitellogenin in Carassius auratus as the macromolecular precursor for oocyte yolk proteins. Some yolk proteins are also produced within and by the oocyte during endogenous vitellogenesis (Van Bohemen et al., 1982) Hepatically synthesized Vt in fishes is a dimer with a molecular weight varying from 350 kD (180 kD on SDS-PAGE) (Plectropomus leopardus) to 550 kD (Oncorhynchus mykiss) (Matsubara and Sawano 1992 ; Takemura and Teruya, 1997) The dimer is incorporated into the oocyte where it is broken down into phosvitin, lipovitellin, and possibly a third protein (Van Bohemen, 1982). DeVlaming et al. (1980) found that the resulting yolk proteins consist of fractions varying from 19-110 kD for lipotvitellin and from 7.6-14 5 kD for phosvitin when analy z ed on SDS-PAGE and Sephadex-gel filtrat ion respectively Vitellogenin was found to consist of three polypeptides ranging from 140-147 kD. In goldfish, these multiple polypeptides of vitellogenin are processed into the multiple yolk proteins within the oocyte (De Vlaming et al. 1980) The production of vitellogenin has been shown in numerous studies to be related to circulating levels of estradiol (E2). Mananos et al. ( 1997) found changes in plasma Vt of Di c entrarchus labrax to correlate with E2, oocyte classification and spawning period. 20

PAGE 37

High levels ofE2 and Vt were found throughout the spawning period (Mananos et al. '1997). In the rainbow trout (Oncorhynchus m ykiss) a direct relationship was found between increasing and vitellogenin in female fish (Whitehead et al., 1978 ; Whitehead et al., 1983) and Van Bohemen and Lambert found a similar relationship inS. gairdneri (1981). Steroid-induced synthesis ofVt has also been accomplished by the injection of estrogenic steroids in both male and female catfish, Heteropneustes fossi/is (Sundararaj and Nath, 1981 ) In the serranid Epinephelus akaara vitellogenin was induced by injection of a low dose of Estradiol-17f3 (Ng et al., 1985). An injection of this estrogen also stimulates vitellogenin production in immature females and male goldfish (De Vlamming et al., 1980) A recent study of E. morio found relationships between estradiol levels in male and female fish that varied seasonally (Johnson, 1995 ; Johnson et al. 1998) Females had high circulating levels ofE2 during the breeding season with an increase to a seasonal maximum. Males and immature females had low constant levels throughout the year. Johnson (1995) proposed there might be a potential u se of this hormonal variation to conduct field studies concerning sex reversal, without sacrifice of resident fish. Immunological Studies Assays for Atlantic salmon (Salmo salar) and brown trout (Salmo lruttafario) have been developed using the sex specific prote in vitellogenin (Le Bail and Breton, 1981) Le Bail and Breton (1981) demonstrated the rapidity and simplicity of immunoagglutination detection (i.e. recognition) of female fish as they undergo 21

PAGE 38

vitellogenesis when there is no sexual dimorphism Other methods have been used for the detection ofVt in Morone spp (Tao et al. 1993 ; Specker et al, 1995; Heppell et al. 1999) and other marine and freshwater teleosts (Craik and Harvey 1984). Sexing of Pacific halibut (Hippoglossus stenolepis) has also been accomplished through the use of an antiserum against vitellogenin (Matsubara and Sawano, 1992) Vt was detected in Hippoglossus hippoglossus mature females and changes in electrophoretic patterns were found according to developmental stage of fish (Norberg 1987). Denslow et al. (1997) developed monoclonal antibodies to detect Vt in a range of teleost species. Folmar et al. (1995) found that theN-terminal sequence from the vitellogenins of six different teleost species (striped bass, mummichog pinfish, brown bullhead, medaka yellow perch) and sturgeon was relatively conserved. Utilization of this conserved area allowed Heppell et al. (1995) to develop an assay that they proposed could be used as a universal assay for environmental estrogens, as well as vitellogenin assays for sex recognition in striped bass, Marone saxatilis, gag Mycteroperca microlepis and other species (Heppell et al. 1995 ; Heppell and Sullivan 1999; and Heppell et al., 1999). Denslow et al. (1997) used vitellogenin from several different species to determine cross reactivity of varying epitopes The authors found cross-reactivity with different bands suggesting that the epitopes chosen appear many times in Vt. The studies done by Heppell et al. (1995) and Denslow et al. (1997) demonstrate that the development of a universal immunogenetic probe for Vitellogenin is an achievable goal. 22

PAGE 39

Experimental Design Due to the relationship Johnson et al. (1998) and others described between sex, estradiol levels and vitellogenin, the use of vitellogenin to determine the gender of fish would be logical and straightforward. A sequence comprised of a relatively conserved region ofVt could be used to probe for vitellogenin in many species offish (Heppell et al., 1995) The entire nucleotide sequence for Fundulus heteroclitus, and partial data for Oncorhyncus mykiss and Acipenser transmontanus are readily accessible in GenBank. In addition, partial sequence data is available from several teleost species, including gag. The development of antibodies that would cross-react with many species, might then allow field assays to be developed. If such a test were developed it could have wide ranging applications both in fisheries management and aquaculture. For example, the development of a quick method for field use would allow researchers to tag a study population of grouper and manipulate the population in such a way that information concerning rates of sex reversal in situ could be obtained (Wilson, unpublished). Such manipulations would result in data concerning reversal rates and causes of initiation that would allow a more precise management of the species. Techniques that cause the least harm to the individual fish should always be of utmost interest to research scientists, especially when studying a population under duress from commercial and recreational fisheries. A "universal" assay (useful across many different species of fishes) could lead to more proper management of many hermaphroditic species as well as allow fish culturists and researchers to quickly and easily determine the reproductive status of stocks. 23

PAGE 40

Based on previous studies by Johnson (1995) and Johnson et al. (1998) which established a relationship between circulating plasma E2 and gender for red grouper plus the data presented by authors such as Mananos et al.,(1997), Ng et al., (1985), Whitehead et al., (1983), Van Bohemen and Lambert (1981) and DeVlamming et al. (1980) which correlate vitellogenesis with hormonal levels throughout the year in various species there appears to exist a quantitative relationship between reproductive condition and plasma levels of circulating Vt. This study examines the gonadal histology changes prior to and immediately following the spawning season of red grouper in relation to the variations in plasma protein Vt as detected by antibodies made to a specific artificial peptide. Gonadal histology was employed as "ground truth" for the sexual state of the study fish Due to the expense of radio-immunoassay and the possibility for false positives with polyclonal antibodies made to native Vt, antibodies made to a synthesized internal sequence of Vt were used. Specifically, this research addressed the following questions : 1 Are antibodies made to artificial peptides derived from known Vt sequences sensitive and accurate enough to detect seasonal variations in serum vitellogenin levels in red grouper ? 2 Is there a correlation between reproductive state as determined from gonadal histology and the level of serum vitellogenin detected among field caught male female and transitional fish ? 3 Can the assay be used for different grouper species ? 4. Does the assay hold much promise for the development of easy-to-u se field detection kits? 24

PAGE 41

III. Methods Estradiol stimulation of red grouper In order to conduct a dose-response experiment using estradiol to stimulate vitellogenin in red grouper fish were caught by hook and line in May 1998 and four were maintained at the University of South Florida until July of 1998. This experiment was designed to test whether E2 levels and Vt production are actually linked in red grouper as in other fish species. Vitellogenin produced from estradiol stimulation would be used to test the specificity of antibodies made to a synthetic peptide. All fish were allowed to acclimate in tanks for about one month In July of 1998, the fish were weighed and measured, and a dose was then calculated for each fish that amounted to 5mg Estradiol17P / kg bodyweight. The injection solution was 0 8 % NaCl (phosphate buffered saline), 1% EtOH, and amount of 17P Estradiol corresponding to 5mg/kg bodyweight (Sigma E-8875) Control fish received injections of 0 2ml of phosphate buffered saline with 1% EtOH Each fish received three injections in the week immediately preceding sacrifice. Upon sacrifice, blood was collected from the cardiac arteries with a syringe and expelled into a centrifuge tube containing anticoagulant (Phosphate buffered saline (pH 7.4) with 70 mM sodium citrate, 20 mM EDTA, and 05% PMSFChan, S.L. et al. 1991) at a concentration of .5mlbuffer / 1ml blood The samples were then iced and were immediately spun at 3000g 25

PAGE 42

for 1 0 minutes at 4 oc to separate plasma and red blood cells. The plasma samples from all four red grouper were analyzed by for protein content by 7.5% SDS-P AGE and subsequent western blotting to determine specificity of antibodies generated from the synthetic peptide Synthetic peptide production In order to compose an artificial vitellogenin sequence to be synthesized, coding DNA sequences were obtained from GenBank and from a then unpublished N-terminal region of purified vitellogenin from gag, Mycteroperca microlepis, (Heppell and Sullivan, UNC, personal communication; now published Heppell and Sullivan 1999). Two artificial peptides were made of 12 and 15 residues respectively (Table 2) Through alignment on PC-Gene of vitellogenin sequences from several different fish species a relatively conserved region was found. Species compared were Oncorhynchus mykiss and Fundulus heteroclitus. Table 2. Artt tcta peptt e sequences "fi I d enve om mtcro ep t s tss, an e d d fr M I 0 k d F h t e roclitu s. N-tennina l sequence from M microlepis YQVNFAPEFATGKTY Internal conserved sequence from 0. mykiss and F heteroc/itu s CGKADGEIRQEY *See Append t x I for Ammo Actd Codes A BLAST (Basic Local Alignment Search Tool) search was performed using the GENBANK program to check for other known protein sequences with a similar peptide sequence The only ones found that agreed with the above chosen sequences were part of the yolk precursor proteins thought to be formed from vitellogenin following sequestration into the developing eggs (see Appendix 5 and 6 for the BLAST results). 26

PAGE 43

Genosys Biotechnologies produced artificial pep tides of both sequences conjugated to a larger carrier protein keyhole limpet hemocyanin (KLH). KLH is derived from the mollusc, Megathura crenulata KLH represents the most different carrier for injection in a rabbit and will illicit the greatest immune response Once these peptides had been synthesized and conjugated they were injected together into two rabbits The resulting sera would therefore have both peptides repre s ent e d in the antibodies from each rabbit. Preimmune sera and sera from bleeds 1 through 4 were received, aliquoted and immediately frozen at 20C. ELISA analysis was carried out to determine which bleeds had the highest titer of antibody. Enzyme Linked Immunosorbant Assay Enzyme Linked Immunosorbant Assay (ELISA) was used to detect and determine levels of antibodies present in the bleeds. In this assa y, the antigen (synthetic peptide) was bound to a microtiter plate The antiserum that contained the anti-peptide antibody was then added to each well causing it to bind to the antigen Next, a secondary antibody was added that was an antibody to the animal that created the antibodies (e g. goa t anti rabbit IgG) This secondary antibody had an enzyme conjugated to it ; in this instance alkaline phosphatase The enzyme then reacted with a substrate to produce a color reaction the intensity ofwhich was used to determine the amount of primary antibody present, which in turn was stochiametric with the bound antigen In this particular case, a solution of the synthetic peptide sequence wa s made at 1 0 J.lg/ mL in sodium carbonate buffer (50mM, pH 9.6 with 02 % NaN ) Aliquots of the 27

PAGE 44

peptide or sodium carbonate buffer were then placed into each well of the micro titer plate and were incubated for 3 hours at room temperature. This antigen was then removed by washing with PBST buffer (Phosphate-buffered Saline with 1% Tween-20 ) The remaining sites were blocked with PBST with 5% nonfat-dry milk for one hour at room temperature. The plates were then washed again with PBST. A 1 : 50 dilution of the sera was prepared and serial dilutions made from 1:100 to 1:102 400. The dilutions were iced vortexed and added to the wells. The plates were then incubated overnight at 4 C After incubation the plates were washed with PBST to remove unbound antibodies For alkaline phosphatase detection, goat anti-rabbit IgG whole molecule/AP conjugate was used (Boeringher-Manneheim #1814206) at a dilution of 1:3000 in PBS-T. The secondary antibody was pipetted into each well and allowed to bind for 3 hours at room temperature The plates were then washed with buffer P-nitrophenol phosphate (Sigma-Fast) was added and allowed to react until a yellow color had developed 2M NaOH was added to stop the reaction and absorbance of each well was read at 405nm The procedure allowed determination of the proper dilution factor to use to detect the antibody as well as which bleed had the highest antibody titer. The above procedure was also done using estradiol-stimulated grouper sera (from the dose experiment) as antigen as well as the unconjugated synthetic peptide to determine the sensitivity and proper dilution when using native vitellogenin 28

PAGE 45

Field Co ll ection Red grouper and gag were sam pled b y hook and line February-August 1998 (Table 3 and 4) Sample s were taken from recreational and research vessels in the Gulf ofMexico (Figure 1). All s pecimens were weighed and tot a l length w as measured as in Johnson ( 1995). Table 3. Monthly Collection of E morio (JanAu g I998) Month Total Fema l e Male Transit iona l Feb ru ary 7 7 0 0 March I9 I8 I 0 April 2 I 16 4 I May I8 IO 8 0 June 4 3 I 0 Jul y II 9 2 0 August I5 I 2 3 0 T o tal 95 75 19 I Tabl e 4. Monthly Collection of M micro/ep is (Jan-Augl998) Month Total Fema l e Male Transitio n al February 5 5 0 0 March 5 5 0 0 April 7 7 0 0 May 3 3 0 0 Ju n e 4 4 0 0 July 5 5 0 0 August 2 2 0 0 Total 31 28 0 0 Gonads we r e remo ve d and immediately i ced and 2 ml blood samples were drawn from car diac arteries u s in g a syringe. The blood was immediat ely placed into sterile centrifuge t ubes containin g 1 ml of anticoagulant and was kept on ic e for fewer than 24 hour s until centrifugation a t 3000g for 10 min at 4 C. The pl as ma wa s the n taken off with a c hilled g lass pipette and fro zen at -80 C until analysis by SDS-PAGE and Western blotting 29

PAGE 46

w 0 + ' . . 4ft:. I I I I I 2 8 $00 ' I ' ' ' 'Ill .. ' '... w.ro.pa-. .....a 20 ... to ) eo I'. \ , c 1 c ,... '4750'> \_ I j -64500 I ' . \ \ \ \ \ }, \ _l-.. . . 8lSOO 0 Simple so t
PAGE 47

Histology Gonads were processed following Johnson (1995). Gonads were fixed in 10% formalin in 0 1M sodium phosphate buffer (pH= 7.4) for at least a week The samples were then rinsed in hourly tap water changes and transferred to 70% ethanol (EtOH) The gonad samples were grossed by cutting a cross section of about 3 mm at the junction of the two lobes If the gonad was too large a slice was taken from the center of the larger lobe Grossed samples were put back into 70% EtOH until dehydration in 95% EtOH for 2 hours. A series of infiltrations followed dehydration beginning with 50/50 solution of activated JB-4 (Polysciences Inc.) and 95% ethanol. After placing the samples into the 50% JB-4 solution, they were then refrigerated for at least 2 days on a shaker. The same process followed with 100% .TB-4 and two changes of fresh JB -4. The specimens were then embedded in plastic resin and faced with a LKB 2218 HistoRange microtome After facing, 3.5 !J.m sections were taken of each sample floated in a water bath placed on an acid-cleaned slide and dried on a slide warmer Three slides were made from each fish The sections were affixed to the slides by baking at 60C for at least 2 hours and stained with periodic acid Schiff-metanil yellow(P AS/MY) and hematoxylin (Quintero-Hunter et al., 1991), hematoxylin and eosin (H & E) or left blank for reference. Procedures for the stains can be found in Appendices 4 and 5 (Florida Marine Research Institute, Aquatic Health Lab, St. Petersburg, FL; personal communication). Samples were stained with H & E in order to allow comparisons between this study and those in the literature 31

PAGE 48

Gonads were staged for maturity following Johnson (1995), Moe (1969), and Wallace and Selman (.1981). Oocyte growth and spermatogenesis are described by varying stages. These "stages" allow the characterization of the cellular and molecular aspects of gonad development. The gonads can be referred to classes" that describe the varying degree of maturity between fish. "Classes" refer to the seasonal and ontogenetic development of individuals (Johnson, 1995) After samples were assigned to respective classes, the development offish was compared to variations in Western blotting patterns. BCA Protein Assay All plasma samples were analyzed for total protein concentration by BCA (Bicinchoninic Acid) Protein Assay (Pierce BCA Protein Assay Kit #23225). See Appendix 4 for protocol. The BCA Assay depends on the conversion of Cu2+ to Cu + in alkaline conditions through the reaction of the peptide bonds of proteins with copper. Cu + is then detected by its reaction with BCA that results in an intense purple color. The absorbance maximum is 562 nm and the production of Cu + is a direct result of protein concentration and incubation time. Therefore the unknown sample concentrations can be estimated by comparison with known standards (BSA) (Walker, 1994). Samples were thawed and 5 Jll of each was aliquoted into replicate 1 ml cuvettes with 45 J.!l of 0.9% NaCl. One ml of working reagent was added to each tube and mixed well The tubes were then incubated for 2 hours at room temperature. The absorbance at 562 nm was read versus a water reference. The average A(562) reading for blanks was subtracted from the standards and samples A standard curve was plotted using the corrected 32

PAGE 49

readings for the standards versus the protein concentration in f.lg/ml. The curve was used to determine the protein concentration of each sample. The concentrations allowed for more precise comparisons between total plasma protein in field samples. SDS-polyacrylamide gel electrophoresis/Western Blotting Plasma samples remained frozen at -87 C until SDS-PAGE analysis. The s amples were loaded at a concentration of 2000 f.lg protein diluted 1: 1 with 2X sample treatment buffer (0.5M Tris-Cl, pH 6.8, 10% SDS, Glycerol Dithiothreitol (DTT), and Bromophenol Blue), separated by 7.5% SDS-PAGE on a Hoefer SE 400 Series vertical electrophoresis unit. The gel was then either stained with Coomassie BlueR250 or blotted onto Nitrocellulose with a Bio-Rad Trans-Blot Transfer Cell at 10 m V until complete transfer ofprestained standard (Bio-Rad Broad Range Prestained SDS # 161-0318) at 18 hours. Detection After blotting, nitrocellulose membranes were blocked overnight at 4C in PBS-T with 5% blocking reagent (in this case non-fat dry milk) The membrane was washed with as large volume as possible of PBST for two quick rinses once for 15 minutes and two times for 5 minutes. The membrane was then incubated for one hour at room temperature with the primary antibody (623-2) diluted at 1:3000 with PBS-T. The membrane was washed as above and incubated for one hour with the secondary antibody (goat anti-rabbit IgG / AP conjugate) diluted 1:2500 with PBS-T. The membrane was 33

PAGE 50

washed once for 15 minutes and four times for 5 minutes in fresh PBST Detection was completed following the protocol ofBio-Rad BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt/p-nitro blue tetrazolium chloride) Color Development Solution (Catalog #s: 170-6532 and 6539). One ml of stock solutions ofNBT and BCIP were added to 100 ml ofTris Buffer The membrane was then immersed in the solution and color allowed to develop for 10 minutes. The reaction was stopped by immersion in ddl water for 1 0 minutes The membranes were dried and stored for analysis Optical Density To allow comparisons of detected protein between month of collection and sex, blots were scanned in and read for optical density on a Bio Rad GS-700 Densitometer and analyzed with Multianalyst Software V. 1.0 The two bands that were detected at the greatest intensity were read for optical density inside rectangular boxes of equal area across all blots All blots were scanned at equal resolutions and pixel depth, and each band was analyzed inside a box of area 42.56 mm. A local background was figured for each box by the software and an adjusted volume for each box was found This adjusted volume was then compared across all samples in relation to the 2000 J..Lg protein concentration that was loaded for all samples. An Optical Density Index (ODI) was found for each sample: ODI = (Adjusted Volume for sample /20001-Jg plasma protein) X 100 KruskalWallis AN OVA was performed to check for correlations between stage of development and ODI values. 34

PAGE 51

IV Results Vitellogenin stimulation The fish which were injected with estradiol while maintained at USF, produced a protein that was apparent in stimulated fish but not control fish The SDS PAGE gel was Western blotted per methods discussed previously The resulting blot s hows the stimulated band is "recognized" by the synthetic probe (Figure 31, Appendix) The detected band was determined to be -187kD molecular weight. This band was asswned to be vitellogenin due to its occurrence in fish after E2 stimulation and its subsequent recognition by the synthetic probe Only purification and amino acid sequencing could produce a higher confidence in this asswnption. Enzyme Linked Immunosorbant Assay Results from the ELISA indicated that bleed two had the highest antibody titer. The serial dilution resulted in a working dilution of 1 :3000 for the primary antibody when used in western blotting detection protocols. The antibodies appeared to recognize both the synthetic peptide and native vitellogenin (i e the probe stuck to plasma from stimulated fish) (Figures 2 & 3). The pre-immune sera was also run in a dilution se ries to 35

PAGE 52

a = II) 1.2 .,-------------------, 0 ...,. 0 8 @ 0 6 = ,.Q 0.4 a.. 0 0 2 a = II) 0 ...,. @ y = ,.Q a.. 0 Col) ,.Q 1 2 4 8 16 32 64 128 256 512 1024 dilution of primary antibody (bleed 2) Figure 2. Absorbance versus dilution se ri es of bleed 2 s era with synthetic peptide (@[lO ug/ml]) as antigen 0 5 0.4 0.3 0.2 0.1 0 q, ,rv {vq, dilution of antigen Figure 3 Abso r bance ver s u s dilution se ries of sera from E2 s timulat ed fi s h (as antigen ) with primary antibody (bleed 2 s era ) at a dilution of I :3200. 36

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account for non-specific binding. The average absorbance value for the pre-immune sera was @ 405nm across all dilutions Ontogenetic Development / Seasonal Maturity Red Grouper Epinephelus morio samples totaled 95 from February of 1998 until August of 1998. Ofthese 95 samples, 19 were histologically determined to be male, 75 female and 1 transitional (See Table 3). A total of31 Mycteroperca microlepis were also collected within the same time period. Of those 31, 28 were histologically examined and all were determined to be female (Table 4). Gonads were classified following Smith (1965), Moe (1969), Brule and Deniel (1993 & 1999), and Johnson (1995). Descriptions of each gonadal class found and observations for individual fish follow. Although gonads were not preserved immediately upon extraction, postmortem lysis did not obscure the detail necessary to positively classify all samples taken Descriptions of various developmental stages were compiled from Smith (1965), Moe (1969) and Wallace and Selman (1981 & 1989). Table 5 describes gonadal development as used in this study Regressed/Immature Female Females in this developmental stage can be described in one of two ways, as an immature fish or a resting mature fish. Immature fish had an ovary that was small in diameter and lamellae enveloped closely by the muscular tunica. These descriptions corresponded to Moe's ( 1969) Classes 1 and 2 The periphery of the lamellae is 37

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composed of numerous primary oogonia with oocytes in stages 1 and 2 in the center of the lamellae. Atretic bodies should not be found, but yolk globules might be found Yolk globules indicate vitellogenesis might have occurred but evidence of prior spawning should not be apparent. Stage 1 oocytes (primary oocytes) have a nucleus with a single nucleolus and chromatin threads within the nucleus. Stage 2 oocytes are distinctive with a nucleus with several nucleoli around the periphery (perinucleolar stage). A fish that has undergone vitellogenesis and completed a spawn is considered a mature resting female. These fish are most often collected outside of the usual spawning season. The ovary is larger in diameter than in immature fish and the tunica is expanded because of enlargement of the ovary during spawning. Oocytes in stages 1, 2 and even 3 are present, but stage 2 oocytes numerically dominate the ovary. Atretic bodies are common, and some stage 3 oocytes may be entering the yolk vesicle stage. Stage 3 oocytes or cortical alveoli (CA) stage oocytes are beginning to acquire PAS+ yolk vesicles (CA). In late stage 3, a thin zona radiata or chorion begins to develop. A total of 49 red grouper were found to be regressed/immature females (Figure 4) and the majority were collected to either side of the April/May spawning peak The majority of gag (N=25) were determined to be in the immature or regressed developmental stage (Figure 30, Appendix). Mature Ripening Female An ovary in active vitellogenesis is defined as a mature ripening female and corresponds to Moe's (1969) Class 3. Fish in this stage are preparing for spawning. 38

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Table 5. Histologial description of each developmental stage used to describe female male and transitional Epinephelus moria Gonad Development Regressed Female Early Ripening Female Mid Ripening Female Late Ripening Female Ripe Female Spent Female Transitional Ripening Male Late Ripening Male Ripe Male Spent Male Histological Description SI II oocytes dominate ovary SI II, early III oocytes present with early Sill Beginning to dominate ovary Stage III IV oocytes dominate ovary Stage IV oocyte s dominate ovary GYM evident hydration apparent POF present, S I II oocytes present with S IV degenerating Testicular tissue beginning to proliferate with greater than one stage of sperm development Spermatogonia, primary and secondary spermatocytes and spermatids, some ripe sperm Same as ripening with intralobular sinuses Beginning to develop and ripe sperm in ducts Spermatids and spermatozoa dominate, intralobular Sinuses are packed with sperm few spermatocytes Old crypts some sperm s till in duct s, new crypts of Spermatogonia and primary spermatocytes dev eloping 39

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Oocytes in stages 1, 2, 3 and 4 are present. Stage 3 dominate early, and then stage 4 dominate until spawning occurs Atretic bodies are not apparent due to oocyte development. Stage 4 oocytes have a thick zona radiata, yolk vesicles and globules. Late stage 4 oocytes are indicated by germinal vesicle migration (GVM) that indicates a final push towards hydration and spawning Stage 5 oocytes, also called Final Stage of Maturation (FOM) oocytes, are distinct as they are the maximum size of development ,and the nucleus or germinal vesicle disappears and the yolk fuses as the egg becomes "hydrated". Mature ripening females include females described as early, mid, late ripening, and ripe. Early ripening females are those with oocytes in the early Stage 3 phase beginning to dominate the ovary. Mid ripening females have late stage 3 and stage 4 oocytes dominating the ovary. Late ripening females have a majority of late stage 4 oocytes. Ripe females have oocytes exhibiting germinal vesicle migration (GVM) that indicates the onset ofFOM. Takahashi et al. (1991) found no fish fully in FOM and attributed this to the short time period for this process. Goetz (1983) describes GVM as following or in some cases occurring during vitellogenesis. The nucleus (or germinal vesical) migrates to the oocyte edge and begins to breakdown. The oocytes also become more transparent during this time due to coalescence ofyolk and lipid (Goetz 1983). In this study no females were sampled that had ovaries fully in FOM. Therefore fish that exhibited GVM were assumed to be within a few hours/days of spawning and classified as ripe A total of 6 female red grouper were early ripening, 4 were mid-ripening, 7 late ripening, and 2 ripe (Figures 5 & 6). Early ripening females were collected in March mid-ripening in March/ April, late-ripening in March through May and ripe fish in 40

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April/May. For gag only 3 fish were found to be active females and all were classified as early ripening (Figure 30 Appendix) Spent Female An ovary that has just completed spawning and is developing into either a mature resting female or a transitional fish is considered spent. (Moe 1969). The diameter is reduced from that of ripening/ripe fish. The gonad structure is disrupted and oocytes in stages 3, 4, and 5 (if present) are degenerating. Yolk globules are scattered and atretic bodies are forming throughout the gonad. Post ovulatory follicles (POFs) are present and numerous. POFs are being resorbed into the gonad. Four fish were determined to be spent females (Figure 7) and were collected in May / June. Transitional This class is defined by the development of an ovary into an early testis. Crypts of spermatogonia are formed in the germinal epithelium on the periphery of the lamella e. Spermatocyte development is also occurring in the transitional gonad. Active proliferation of primary spermatocytes is the primary characteristic (Moe, 1969 ; Class 5). However, Moe (1969) also called attention to the fact that obvious crypts of spermatocytes and precocious spermatids were found in juvenile and mature resting females. He credited this male tissue as due to a lack of firm hormonal "control and 41

PAGE 58

stated that they may represent a slow transition that will intensify when the proper hormonal stimulation occurrs. One transitional fish was collected in April (Figure 11 & 12). Ripening Mature Male A mature male dominated by the later stages of sperm development is defined as a ripening male (Moe, 1969 ; Class 8). Secondary spermatocytes and spermatids are in most crypts and spermatozoa are beginning to collect in the dorsal sinuses. Late ripening males were distinct as the testes had sperm ducts filled with tailed spermatozoa Four fish were sampled as ripening male fish (Figure 8). Months of collection were March through May. Late ripening males numbered 5 (Figure 9) and were samp led in April/May. Ripe Male A fully developed testis is characteristic of ripe males (Moe's ( 1969) Class 9) Crypts contain spermatids or tailed sperm and the dorsal collecting sinuses are packed with spermatozoa Spermatogonia and spermatocytes are rare. Intralobular sinuses are developing and sperm crypts breakdown. These intralobular sinuses are thought to empty into the dorsal sin uses at spawning. Six ripe males were collected (Figure 9). The first ripe male was collected in April, four were collected in May / June, and one was taken in July. 42

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Post-spawning Male A testis dominated by stromal tissue and developing crypts of spermatogonia is characteristic of a spent male (Moe s (1969) Class 1 0). The old crypts are empty but a few spermatozoa remain in the intralobular and dorsal sinuses. Four spent males were collected at the end of the spawning peak in July / Augu s t (Figure 10). 43

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B. Figure 4. E morio 4A. Regressed mature female with atretic bod ies ( AB) p r e s ent in the periphery of the lamellae indicating prior spawning. Note presence of S I (arrow) and S2 (perinucleolar stage oocytes) 48. A typical immature female ovary with Sl and S2 oocyte s Note the tight envelopment of the tuni c a 44

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Figure 5. E morio 5A. Early ripening female with late early stage 3 oocytes (arrows) that are beginn ing to enlarge and were used in this study to denote early ripening fish. 58. Mid-ripening female tha t has stage 3 oocytes in varying stages dominating the ovary. Cortical alveoli development is seen in early mid 3 (see I), while in late stage 3 (see 2) the chorion thickens and yolk globules begin to appear 45

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Figure 6 E morio 6A Typical late ripening female with late S3 (III) oocytes dominating the ovary with numerous early S3 still present. 68. Ripe female with late S4 (IV) oocytes dominating the ovary were considered ripe females for the purposes of this study Late S4 oocytes lose the integrit y of the nucleus yolk vesicles coalesce 6C. Ripe female late S4 oocyte showing GYM GYM happens quickl y and is believed to indicate eminent spawning 46

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Figure 7 E mor i o spent females collected at the end ofthe spawning season 7A. Spent female with numerous POF (Post ovulatory follicles) present and degenerating oocyte s (DO) Later spent female than in 78. 78. Note the presence of Sill and SJV oocytes along with degenerating oocyte s and POF s (arrows) 47

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Figure 8 8A. Ripening male E morio. Note all stages of sperm development exist and few oocytes remain in testes. 88. Same as A. with greater number of remnant oocytes present. In both fish the crypts are still well formed. 48

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Figure 9. 9A Late ripening male E morio Intralobular sinuses are well-developed with spennatozoa present but ducts in gonad wall are not packed with ripe spenn (not shown) 98. Male with numerous S2 oocytes remaining. This fish was classified as a ripe male due to the predominance of ripe spenn and the development of duct work in the gonad wall that was packed with spenn This male accounted for the peak of vitellogenin density in ripe male fish 49

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Figure 10 Spent male E morio lOA. Disordered structure of male gonad is typical of all spent fish in this study Although sperm still exists in sperm ducts (SO) the teste shows no signs of spermatic act iv ity lOB An example of a spent male with sparse ripe sperm in ducts and an atretic body (AB) present indicating prior female activity 50

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B. Figure 11. Transitional and female portraying precocious sperm crypts in E morio. 11 A Example of a precocious regressed female fish S I and SII oocytes present along with scattered sperm crypts (arrows). 11 B Early transitional red grouper Note pockets of sperm deve l oping along with SII and early stage III oocytes. 51

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B. Figure 12. 12A. Early transitional E morio with greater than one stage of development apparent in sperm Crypt. 128. Early transitional fish with evidence of degeneration (DG) of later stage oocytes. 52

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Epinephelus morio The developmental classes for the 75 female red grouper are plotted against month of collection (Figure 13) Percentage of spent, vitellogenic, and regressed fish per month are also plotted (Figure 14). It is evident that the spawning season for red grouper is from March to May with a peak in vitellogenic fish occuring in May The peak is further illustrated when stage of vitellogenesis is compared monthly (Figure 15) The majority of late ripening/ripe fish were found in May This also coincides with the peak in GSI seen in April/May (Figure 16). Spent females begin to appear in May and the majority are found in June (Figures 13 & 14). A drop in GSI also indicates the end of the spawning season (Figure 16). February, July and August samples were wholly composed of immature or mature resting stage fish. The GSI for these developmental stages shows little variation across all months (Figure 16) All male fish sampled were reproductively active. Mature ripening males were further divided into ripening and late ripening fish. In the plot of stage of development versus month (Figure 17), a May-June developmental peak in evident. Spent males began to appear in June and were 100% of male fish sampled in August. GSI for male fish shows an increase in April-May and a marked decrease beginnning in June (Figure 18). It can also be noted that all males collected were reproductively active and remained that way longer than the female fish sampled Only one sample was determined to be a transitional fish. This fish was in the very early stages of transition with testicular tissue just beginning to proliferate and was part of the April sample 53

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.. "' .. c .. I! ... 100% 90% aoA. 70% 60% soA. 40% 30% 20% 10% 004 Feb Matdl April May Month June July August Figure 13. Percentage of female red grouper in various developmental stage plotted against month of collection. 100% 90% aoA. 70% 60% soA. 40% 30% 20% 10% 0% Feb March April May July Aug Month spent ripe ripen i ng late ripen i ng mid 0 ripen i ng early 0 immature/resting spent IFJ vitellogenic 0 immature/resting Figure 14. Percenta g e of female red gro uper in vitellogenesis b y month. 54

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iii C) .. "' J! c .. I! ... 3 2 5 2 ; 1 5 .. ::tE 0 5 0 1000,{, 90% 80% 70% 60% ripe 50% tate ripen i ng IZI mid ripening 0 early ripen i ng 40% 30% 20% 1()0 ,{, 0% MarCh April May Month Figure 15. Percent of female E morio in various stages of vitellogenesis for March April, and May. I I ... ... T ... I Februaty (7) MarCh (18) April ( 16) May(10) Month (N) Juna(3) T .1. July(9) Augusl(12) Figure 16. Mean GSI values for female E morio (immature/resting & mature) for each month plotted with 95% confidence int ervals 55

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::. i II: 0 6 0.5 0.4 0 3 0 2 0 1 !10% 80% 60"1. SIW, 4()11. 30% 20% 10% 0% Feb ldab April may June M on l:b july Au a spent Dripe ripening late ripening Figure 17. Perce n tage of male red gro up er in eac h developme n ta l stage p l otted against month of collec t ion . 157711313 March (I) April (4) May(8) July(2) August (3) Month Fig u re 18. Mean mal e red grouper GSI for each month wit h 95% confi d ence interva l s 56

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Population structure Of the 95 red grouper samples, 75 were female, 19 male and 1 transitional. Males began to show up in the population at TL 500nun and 68% of males caught were between 500 and 600nunTL (Figure 20). Males had a mean TL of 599nun with a range of508 to 787 nun. According to Johnson (1995) this corresponds to an age of 5-6 years Females in the population had a mean TL of 559 nun with a range of 393 to 787 nun, and the transitional fish was 508 mm TL. The sex ratio of the population sampled was 4:1 female to male. Females and males showed a consistent weight length relationship that appeared to have little variation between the sexes (Figure 21 ). Total Ltncth (mm) Figure 19. L ength frequency distr ibution of male female and transitional red grouper 57

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5 4 3 2 0 9000 8000 7000 6000 :3 5000 ... .. 'il :1: 4000 3 000 2000 1000 0 0 r-4 3 3 "' 2 2 a I l5 1 1 1 1 1 I t. I I 1 : r 1 i < .J l 380. 400. 420 440. 460. 4 80. 500. 520. 540. 560. 580. 600-620. 640-660. 680. 700720. 7 40760. 780. Taal Lef9h ( nvn) Figure 20. Length fre q uency distributi o n for male E morio females males transiUonaJ ... ?.1!. .. ,.. 100 200 300 400 soo 600 700 800 900 ltngth (m m ) Figure 21. Lengt h weight re l a t ionship for E morio 58 I

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Optical Density Index An optical density index was calculated for each red grouper sample for two protein bands that reacted most intensely with the NBT/BCIP. These two bands were of molecular weights -270 kilodaltons (kD) and 187 kD. Each band was analyzed and the adjusted volume integrated to calculate an optical density index (ODI) Each fish was placed into a classification based on histological development. The broad classes were regressed female, mid/early/late ripening female, ripe/spent female, ripening male late/ripe male or spent male. As only one transitional fish was sampled, it was not included in the larger analysis. Figures 24 and 25 illustrate the mean ODI for each band for each developmental class and the standard error for each class For the 270kD band, a peak is seen in ripening females, but basal levels are also apparent in regressed and male fish F i gure 22 plots all ODI values ofMW band for each developmental class A peak is again evident in ripening fish with the highest values and the absence of all but one zero value A basal level for 270kd band is apparent When each MW band was compared by KruskalWallis across all developmental classes a significant difference was noted only for the 270 kd band (p <. Ol5) A Dunn post test found p<.Ol between regressed females and ripening female fish A significant difference was also noted between ripening and ripe/spent fish No significant difference was noted between any female or immature class and male fish (Table 6) Low and high values for each developmental class were compared visually (Figures 26 27, & 28, in Appendix). Zero values were found in all classes except FER, FMR MRG, MS Sample sizes equaled 4 for MRG and MS fish Retrospective histological analysis of high and 59

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low r ank i n g v a l u es for each class foun d scatte r e d oocytes throughou t t h e tes t e s of the seco n d hig h ranking ma l e fis h and numerous stage 2 oocyte s in the test es of th e hig h est ranki n g male fish (Figure 9 B). T h e highest ranking regres sed female fish were foun d to be a mature r esting fish. The da t a for the 1 8 7kD band showed no significant difference across any classes (Figure 25 & Table 7). Curio u s ly, this is the same band that appe ar e d to be s timul ated in E2 treate d imma tur e fish (Figure 31, in Appendix). Z e r o O D I l evels were detected in all developmental c l as s e s an d th e highest valu e recorde d was for an ear l y / mid r ip ening fe m ale It app eare d that low b asal val u es were present in all group s No correlation was found b etween O D I va lu es f or the 1 8 7 kD and 270 kD b ands (r= 338, p>.001) 0 4 .. F I M .. .. F ER/FMR 0.3 .. FLR/FR Q) c ::J FS .. c .... Cl 0 2 MRG .. 0 c c MLR/MR .. .. c v 0.1 MS c .. TR v co 0 0 FIM FERIFFLR/ F F S M RGMLR/1 M S T R Stage of Deve l opment Figure 22. ODI valu e for 2 70kD band for each development stage for red gro uper. 60

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0.15 FIM .. FERIFMR Q) 0 10 FLRIFR ::::J FS 0 MRG 0 0 05 D MLRIMR .. A MS D AA .. D .... 0 .00 FIM FERIFFLR/FI FS MRG MLR/f..t MS Stage of Development Figure 23 ODI value for 187kD band for each developmental stage for red grouper. 61

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Table 6. Results of Kruskal-Wallis between developmental stages for 270 kD band Kruskal Wallis test P value 0 0155 Exact or approximate P value? Gaussian Approximation P value summary Do the medians vary significant l y (P < 0.05) Yes Number of groups 8 KruskalWallis statistic 17.32 Dunn's Multiple Comparison Test Difference in rank sum P value Summary FIM vs FER/FMR -34 64 p < 0 .01 ** FIM vs FLR/FR 3 862 P > 0.05 ns FIM vs FS 0.1811 P> 0 05 ns FIMvsMRG -9.194 p > 0 05 ns FIM vs MLRIMR 0 9879 P > 0 05 ns FIMvs MS -20.44 p > 0.05 ns FIMvs TR -13.19 p > 0 05 ns FER/FMR vs FLR/FR 38.51 p < 0 05 FERIFMR vs FS 34.83 P > 0.05 ns FERIFMR vs MRG 25.45 P > 0 05 ns FER/FMR vs MLRIMR 35.63 P > 0 05 ns FER/FMR vs MS 14 .2 p > 0.05 ns FER/FMR vs TR 21.45 p > 0 05 ns FLRIFR vs FS -3.681 p > 0 05 ns FLRIFR vs MRG -13.06 P> 0 05 ns FLR/FR vs MLRIMR 2 874 P > 0 05 ns FLRIFR vs MS 24.31 P> 0 05 n s FLR/FR vs TR -17.06 P > 0 05 ns FSvsMRG -9.375 p > 0 05 ns FSvs MLRIMR 0 8068 P > 0.05 ns FS vs MS -20.63 p > 0.05 ns FS vs TR -13.38 P > 0 05 ns MRG vs MLRIMR 10.18 p > 0 05 ns MRGvsMS -11.25 p > 0.05 ns MRGvsTR -4 p > 0 05 ns MLRIMRvsMS -21.43 p > 0 .05 ns MLRIMRvsTR 1 4 .18 p > 0 05 ns MSvsTR 7.25 p > 0 05 n s 62

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Table 7 Results of KruskalWallis between developmental stages for 187 kD band Kruskal-Wallis test P value 0.9784 Exact or approximate P value ? Gaussian Approximation P value summary Ns Do the medians vary significantly (P < 0.05) No Numbe r of groups 7 KruskalWallis statistic 1.17 Dunn's Multiple Comparison Test Difference in P value Summary rank sum FIM vs FER/FMR -1.863 p > 0.05 n s FIM vs FLR/FR -0 2744 p > 0 05 n s FIM vs FS -4 913 p > 0.05 ns FIMvsMRG -9.413 p > 0 05 ns FIM vs MLRIMR 5.291 p > 0.05 ns FIM vsMS 1.337 p > 0.05 ns FERIFMR vs FLR/FR 1.589 p > 0.05 ns FER/FMR vs FS -3. 05 p > 0.05 ns FER/FMR vs MRG -7 55 p > 0.05 ns FER/FMR vs MLRIMR 7.155 p > 0.05 n s FER/FMR vs MS 3.2 p > 0 05 ns FLR/FR vs FS -4 639 p > 0 05 ns FLR/FR vs MRG -9. 139 p > 0 05 n s FLR/FR vs MLR/MR 5.566 P > 0 05 n s FLR/FR vs MS 1.611 p > 0 05 ns FSvsMRG -4 5 p > 0 05 n s FSvsMLRIMR 10.2 p > 0 05 ns FSvs MS 6 .2 5 p > 0 05 ns MRG vs MLRIMR 14. 7 p > 0 05 ns MRGvsMS 10.75 p > 0 05 ns MLRIMR vsMS -3. 955 p > 0 05 ns 63

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0 3 0,2S 0 2 f !2, O IS .. 0 1 o.os 0 0 .025 0 .02 0 0 1 5 "' 0 .01 0 8 !:! c !2. 0 005 .. ii > 0 i5 0 .0. 005 .0. 01 I 1\ 1\ I \ ( \ FIM (49) FERIFMR(IO) FLRIFR (9) FS (4) Devdopmencal scage MRG(4) / v Ml.RIMR ( I I ) MS(4) Figure 24 Mean ODI +-SE for eac h developmental class for MW 270 kD band 001 for each developmental stage ----FIM ( 49) FERIFMR (10) F L RI FR (9 ) FS{4) MRG{4) MLRIMR{11 ) MS{4) .0.01 5 "" ....................... ,.,_ .... ,_ .................................... ...... --------------------------------------------J Dev e lopmental stage Figure 25. Mean ODI +-SE for each dev elo pm e nt a l class for MW 187 kD band 64

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V Discussion Vitellogenin Production and ODI Measurements One of the chief goals of this study was to identify and probe an estradiol stimulated protein, vitellogenin, using antibodies made to a synthetic peptide constructed from known vitellogenin sequences. A correlation of gonad developmental stage with detected levels ofvt-related plasma proteins would allow the constructed probe to be evaluated for accuracy and utility for eventual in situ experimentation. The present study was designed to ascertain vt levels immediately preceding and following peak spawning period (April-May) for red grouper. Western blot results of initial vitellogenin production experiments showed that an estradiol-stimulated protein 187 kD molecular weight was present in test fish but absent in control fish (Figure 31, in Appendix) The molecular weight of this stimulated band fell within the range of previously reported vitellogenin in grouper species, 183 kD for gag and 140 kD for Epinephelus malabaricus (Heppell and Sullivan, 1999 and Utarabhand and Bunlipatanon, 1996, respectively). This stimulated band was detected here with antibodies made to a synthetic sequence from conserved regions of vt across several species of fishes. However, when applied to the plasma of field caught red grouper it did not adequately distinguish between male, immature and female fish The ODI measurements of the 187 kD band were uncorrelated with developmental stage, as the band was detected at varying 65

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levels across all stages From Johnson et al (1998) it was expected that detected vitellogenin levels would correspond to the measured E2levels in red grouper That is Johnson et al (1998) found significantly higher E2 levels in maturing females with the highest E2 in the FOM category. We concluded that vitellogenin levels should peak in those fish as well However contrary to e x pectation there was not a consistent finding of low or zero levels for immature and male fish Z e ro density measurements of the 187 kD band occurred in all developmental stages of fish The BLAST search conducted in GenBank leads further credence to the specificity of the synthetic probe as the only other proteins in the database containing the chosen sequences were vitellogenins of other organisms or its breakdown products, lipovitellin and phosvitin. The majority o f studies that used v i tellogenin-based probes to determine the sex of fish were done with fish maintained in controlled laboratory environments The natural product ion of proteins in wild populations could therefore vary with that o f fish reared and maintained artificially. However Heppell and Sullivan (1999) found a vtbased probe could determine the s e x of field caught gag another protogynous epinepheline hermaphrodite The hermaphroditism of red grouper could be the cause of variable success with the methodology in the present study Since male red grouper function first as females, the result of the present study could indicate a lack of proper hormonal (steroid) balance in recently transformed males Stero i d imbalance could result in estradiol production and consequent production of vitellogenin As the E morio gonad is classified as undelimited (i.e. one of intermixed male and female tissue) the occurrence of vitellogenin at some level in male fish might be inevitable. Several authors have reported the 66

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presence ofoocytes in male gonads. Sadovy and Collins (1995) noted the occurrence of bisexuality in E. striatus, nassau grouper, as evidenced by late stage 3 and 4 vitellogenic oocytes in fish that appeared to be reproductively active males. Brule and Deniel ( 1999) also noted the occurrence of oocytes in 78% of males sampled, and in 5% of males remnant oocytes were in stages 1, 2, & 3. Johnson (1995) also noted the presence of oocytes in ripe male red grouper from the Gulf of Mexico. The present study found stage 1 and 2 oocytes in obviously male gonads, with one ripe testis containing very large numbers of stage 2 oocytes that did not exhibit obvious signs of degeneration (Figure 9B). The "plastic" nature of protogynous grouper is therefore obvious, and it is also well-documented that juvenile, immature, and male fish can be stimulated to produce vitellogenin by simple injection of estradiol (De Vlaming et al., 1980; Sundararaj and Nath, 1981; Ng et al., 1985; Tao et al., 1993; Utarabhand and Bunlipatanon, 1996) It is also interesting to note that Bonet al. (1997) found neomale (fish that were genetically female but had been sexually reversed by ingestion of methyl testosterone) rainbow trout, to contain low levels ofVt. The presence ofVt in neomales was explained by the presence of vitellogenic oocytes in mature neomales (Bon et al., 1997) These observations seem to suggest that the finding of variable levels ofVt in males might have stemmed from the fact that all male fish analyzed were recently sex-reversed females. Following the reasoning of Mananos et al. (1997) concerning increased levels of Vt without elevated plasma E2 one could hypothesize that the liver retains an increased sensitivity to E2 in transformed males. This would allow low levels of estrogen to stimulate vitellogenin production that then remains in the plasma until resorbed. Johnson et al. ( 1998) found that immature female and male red grouper had low but detectable 67

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levels of estradiol year-round that could serve, at least in male fish, to stimulate basal vitellogenin production There is also evidence of vitellogenin produced in gonochoristic male fish. Sumpter (1981) reported finding detectable levels ofVt in plasmas tested from ra i nbow trout, whether male or female The author found some males with no detectable Vt, but found no correlation between detection and maturity Ding et al. (1989) also reported finding two forms ofvitellogenin in male tilapia, Oreo c hromis aureus. The presence of an apparently E2 stimulated protein in immature and regressed females is also not undescribed Moe (1969) pointed out that immature fish can show histological evidence of vitellogenin production without evidence of spawning (i.e muscle fibers/bundles atretic bodies). Heppell and Sullivan (1999) reported reactivity o f probes with females with late secondary development (this study's early ripening females) where vitellogenic uptake is lacking This has also been reported for white perch (Jackson and Sulllivan 1995) Mananos et al. (1997) found that in control fish under natural conditions elevated levels of vitellogenin were present even in the absence of vitellogenic oocytes Therefore, uptake of vitellogenin by the oocytes is not necessary or immediate (Mananos et al., 1997) and the protein could remain in low levels in the plasma Bonet al. (1997) also found immature female rainbow trout to have detectable levels ofVt. A study of plasma vitellogenin properties in E. malabaricus found two protein bands in all fish (including male and immature fish) that were assumed to be vitellogenin based on an increased concentration of the same bands in estrad i ol stimulated fish. However the antibody made to these bands did not react with plasma from either male or immature fish (Utarabhand and Bunlipatanon 1996). This could indicate a low sensitivity 68

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of the assay to low levels of vitellogenin. In the only other study that used a synthetic peptide to generate antisera to a known sequence of vitellogenin, the antiserum was found to react preferentially with a MW band of -170,000 in blots of immature plasma from striped bass injected with E2 (Heppell et al., 1995). The authors also saw reactivity with male plasma of a lower MW band, which was identified tentatively as a possible Vt-related protein The probe also reacted with proteins in female brown bullhead catfish collected from a polluted creek as well as a male from an unpolluted source. The present study and that of Heppell et al. (1995) seem to indicate a greater sensitivity for vitellogenin in its various forms with synthetic antisera probes. When the aforementioned studies are taken into context with the present study it seems that specific probes, such as the one developed here, for vitellogenin may detect basal levels of vitellogenin-related proteins in varying stages of development. The other protein band of interest for this study had a molecular weight of -270 kd and did not appear to be stimulated by E2 in production experiments (Figure 31 ) However, this band occurred in high density in many fish and appeared in the control fish (Figure 31 ) There was a significant difference in the strength of this band between early/mid ripening fish and immature /regressing fish and late/ripe fish (p < .001; Table 6) suggesting it might serve as a sort of vitellogenin precursor near the beginning of oocyte maturation. Bon et al. ( 1997) and Heppell and Sullivan (1999) found measureable levels of vitellogenin in cortical alveoli stage (early ripening) fish After the start of exogenous vitellogenesis and uptake by the oocytes in mid/late ripening fish, the need for high levels of the 270kd protein would decrease and result in the decline in late ripening/ripe fish 69

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Oocytes in the mid-ripening stage gain yolk globules indicating active vitellogeneis (Figure 5) and the sequestering of plasma vitellogenin. If the HMW protein were a precursor or storage protein related to vitellogenin, one would expect the 187kd band to correlate in some way with detected levels of the 270 kd band. However, these values are uncorrelated (r = 0.338, p>.OOl) between the two sets of density values. If this detected 270 kd protein is a vt-relation, its presence in male fish could also be explained by the reproductive strategy. Since male fish were once female, low levels of circulating estradiol could stimulate the production of this precursor protein without the subsequent increase in a lower MW vitellogenin. The highest ODI value recorded for male fish was explained by the appearance of numerous oocytes in the testes. These oocytes did not appear to be degenerating and could serve to stimulate the production of this vt-related protein. The protein would not be sequestered out from the plasma into maturing oocytes and would remain at high levels until eventually resorbed. The appearance of vitellogenin in immature fish has also been documented and could explain the basal levels seen in many male and immature fish. Recent findings in a protandrous fish, Centropomus undecima/is, indicate levels of Alkali-Labile Protein Phosphorus (an indirect assay for plasma vitellogenin), ALPP, are not significantly different between regressed females, regressing females (spent), and all males A low, basal level was found in those groups, with a higher, significantly different level for mid and late maturing female fish only (Roberts et al., 1999). The ALPP seen as a continuum in male and regressed/regressing females could be indicative of low levels of circulating Vt. Corresponding Western blots of gag plasma samples in the present study detected a molecular weight band of -187 kD as in red grouper (Figure 29, Appendix). However, 70

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the 270 kD band did not appear to be present and no other band reacted with the same intensity of the 270kD band in red grouper. Several smaller MW bands were detected but these have been identified as degradation products of vitellogenin in previous studies The 187kD band differs in MW slightly from that ofHeppell and Sullivan (1999) that found reaction with a band of 170 kd in plasma from field caught gag. Because all sampled gag in the present study were females in roughly the same stage of development no correlations could be drawn between banding intensity and stage of development. All fish were regressed females and all samples reacted with the probe to result in the same banding pattern. This pattern differed from that of red grouper suggesting a variant form of the protein in each species The probe appears to recognize different variants of vitellogenin related proteins within the Epinephelinae Population structure Although various authors have compiled meristic data and growth characteristics throughout the years, Moe's (1969) study provides the most comprehensive age and growth data on the Gulf population Recently, Schirripa and Legault (1999), Johnson (1995), Koenig (1993), Goodyear and Schirripa (1991 & 1993) have added to Moe's initial study with additional meristic / growth and reproductive data Brule and Deniel (1996 & 1999) have also examined the Campeche Bank population in while Stiles and Burton (1991) looked at southeastern U S. red grouper. In this study, males first appeared in the population at 20 inches TL (508 mm TL) with the majority of males caught in the 500-600 mm TL ( 20-24 ) size categories 71

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(N=13). The majority offish samples in this s tudy were found to be female(79 % ) with the smallest female being 15.5 TL (380-400 mm) size class and the largest being 31" TL (780-800 mm). The two largest fish sampled were 787 mm (31 ) TL and consisted of both a male and female. The sex ratio was determined to be 1:3.95 (male:female). The ratio approaches 1: 1 in the size classes above 720 mm TL. In no size class where males and females were collected did males outnumber females At no size in the present study were all fish sampled male, except two of the larger size classes for which the sample sizes were low (N=l). At larger sizes the sex ratio does approach 1:1 indicating about half of the population may eventually change sex. Variation exists in the literature for length of first male appearance with Moe (1969) finding first males at 15" TL, Koenig (1993) at 21" TL, and Johnson (1995) at approximately 16" TL. In the Campeche Bank population, males first appeared at 41 em FL (-16" FL). Sex ratios found by Coleman et al. (1:2.2-1:3 6) (1996) and Brule and Deniel (1 : 3.4)(1999) are comparable to those found in the present study However Moe (1969) found a sex ratio in Gulf red grouper of -1:5.9. Moe found the majority of males at the roughly 24-32 TL (using conversion ofTL=1 2 + 1.16SL from Johnson, 1995) with an equal distribution of the sexes occurring at 35 TL. The differences could stem from different sampling environments as Moe sampled more from commercial catch than done here. Considering the majority of samples in this study fell within the 20-24" TL size one would expect to find a similar sex ratio to Moe's. The declining sex ratios suggest fewer large males in the population are exerting a density dependent dominance on smaller males Current trends in declining size of the commercial catch would indicate this this to be true There were also notable differences between the 72

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present study and Moe's original findings of size classes containing the most numerous males that further indicates a restructuring of the Gulf red grouper population probably due to heavy fishing pressure. The only fish classified as a transitional fish was 22"TL. Female fish that exhibited "precocious" sperm crypts were found in size ranges of 15.5"-27.5" (-390-690 mm) TL (N=11) (Figure 11) The majority of these fish were found early in the year The ovaries otherwise showed no signs of oocyte degeneration and it was therefore assumed the fish would continue to function as females, and hence were classified as females. Smith (1965) pointed out that small groups of spermatogonial crypts existed in the lamellae of the gonad during the juvenile and female phases of Serranus type protogynous hermphrodites. These crypts can sometimes develop, making it possible to observe ripe sperm in fully ripened female fish. Brule and Deniel (1999) and Moe (1969) also pointed out that female gonads sometimes contained scattered "seminiferous nests" although Moe found this to be uncommon in eastern Gulf red grouper. Brule and Deniel (1999) considered such ovaries to be in the stage of transition and classified those fish exhibiting seminiferous nests as transitional fish. It is possible that the crypts of sperm function to enhance the transition process when the stimulation for sex change occurs. As Moe (1969) noted that the occurrence of these precocious crypts in females was uncommon in the eastern Gulf, the fish noted with crypts in this study could further indicate a greater number of fish preparing for transition. This could serve to increase the number of males, therefore causing the subsequent decrease in male:female sex ratio. The plastic nature of this sex changing species is further illustrated by the presence of oocytes in the testes of mature male fish. One fully ripe male gonad was 73

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composed of an equal mix of Stage 2 oocytes and ripe sperm (Figure 9B) The oocytes showed no signs of degeneration The sperm ducts were packed with spermatozoa and the fish was classified as a ripe male as it appeared fully able to function as a reproductivelly active male Several males sampled contained remnant oocytes in varying stages of degeneration. Johnson (1995) noted that all males that she collected had remnant oocytes as well. Brule and Deniel (1999) pointed out that male gonads containing oocytes in stages 1 2, and 3 were identified in Campeche Bank red grouper. The presence of oocytes in the lamellar structure of mature male fish indicates that recently transformed male fish may lack firm hormonal control. Red grouper was originally determined to be a protogynous hermaphrodite by Moe ( 1969) and this study found evidence in agreement. The presence of oocytes in testicular tissue precocious sperm crypts in females, central lumen in testes, and 1 :4 sex ratio support the previous findings of protogynous hermaphroditism in this species and meet criteria defined by Sadovy and Shapiro (1987). Histological data also supported the consensus in the literature of a March-May spawning season in red grouper (Moe, 1969; Coleman et al., 1996; and Johnson et al., 1998) This study found the peak to occur in April/May (Figure 14 & 17) when vitellogenic females and ripening/ripe males were most common The GSI data also supported the April/May peak (Figure 16 & 18) Brule and Deniel ( 1999) found the Campeche Bank population to spawn in January-March and indicated this earlier season may be due to warmer temperatures in the more southern part of the Gulf. A specific size or age at which all fish change sex is not apparent for this species Moe (1969) found no consistency between age or size of sex reversal. He also stated that 74

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at no time were all fish in the population male, indicating that every fish did not change sex At no size in the present study were all fish sampled male, except two of the larger size classes for which the sample sizes were low (N=1) At larger sizes the sex ratio does approach 1 : 1 indicating about half of the population may eventually change sex Fishing pressure exerts great pressure on larger size classes (TL>=20 ) The decrease in available males may trigger sex reversal to take place in larger females, thereby causing a nearly equal distribution of larger male and female fish. Johnson ( 1995) suggested that the decrease in size of males from Moe's ( 1969) original work is due to increased fishing pressure and a depletion of the larger size classes. The most recent stock assessment for E. morio also shows a decreasing abundance offish landed in the > 30 inch TL category since 1984 (Schirripa et al. 1999). Recent analyses by Eklund (1992), Goodyear and Schirripa (1993), and Johnson and Collins (1994) and Johnson (1995) have determined that red grouper are larger at age than those found in the mid 1960's by Moe. A reduction in the resident population of red grouper due to increasing fishing pressure since the introduction of the bottom longline would decrease density dependent pressure and allow younger fish to grow larger faster (Goodyear and Schirripa 1993; Johnson 1995) The decreases in large size fish evident from the fishery data compiled by Schirripa et al. (1999) indicate that few fish are reaching the largest, most reproductively viable sizes. Sadovy (1994) called attention to the fact that in the western Atlantic many of the grouper stocks are being overfished (see Table 1) The author pointed out that grouper throughout the Atlantic have exhibited a change in size of catch from larger to smaller individuals as well as a declining total catch As an overfished species, red grouper do not have the reproductive capability to 75

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maintain a fishable population under current fishing pressure. Protogynous hermaphrodites are more susceptible to overfishing than many gonochoristic specie s as they are long-lived and slow growing Although red grouper evidently do not form the large spawning aggregations that are well documented for many other group e r species they are believed to form small localized spawning groups within their resident areas (Coleman et al., 1999) As has been found in other protogynous hermaphrodites, red grouper probably have spawning groups that are dominated by large male fish Although little is known about spawning habits of E. morio in comparison to other protogynous grouper species like E. stria/us, it has been hypothesized that red grouper spawn in small groups over an extended period oftime (Coleman et al. 1999). E. striatus, Nassau grouper, appear to spawn in large aggregations within a much shorter time period. Both species are considered to be protogynous with male fish dominating spawning behavior. Gilmore and Jones (1992) stated that a social hierarchy" exists in groupers that spawn in smaller aggregations This hierarchy is established by a large, dominant male who defends his territory against other males of the same species serving to increase the overall fitness of the population by allowing only the fittest males the opportunity to procreate (Gilmore and Jones 1992). No specific age or size seems to exist for sex change in protogynous species ; rather, it seems that transition will occur at the time that the greatest reproductive benefit will be gained An environmental cue for many species seems to be a change in the sex ratio of the population or a release from inhibitory behavioral cues (Ross 1990 ) Therefore, with the decrease in size seen in red grouper catch and the increase in smaller size male fish, it appears that red grouper may have been released from inhibitory 76

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behavior of large males. Therefore, individual fish are benefitting from the decreased density by increasing individual growth rates. Fishing pressure on protogynous populations would effectively reduce not only the size of the males in the populations, but also reduce the amount of time the larger fish have to be reproductively active (Gilmore and Jones 1992). The presence of large numbers of relatively small males in the present study indicates that the inshore population (most samples were from water with an average depth of of red grouper is actively spawning. There may be sampling bias due to methodology but the recent stock assessment by Schirripa et al. ( 1999) indicates an overall decline in size of even the commercial landings (the commercial fishery being primarily a deeper water fishery). Therefore, the present study seems to be a good indication of the actual status of the species. Unfortunately, the population that currently appears to be most numerous and reproductively active is not the most reproductively fit portion of the population (i.e. larger fish can have many more offspring than smaller fish). In order for the population to maintain itself under intense fishing pressure, the largest most fit adults should make up the major spawning population Huntsman and Schaff (1994) found that ifprotogynous species make a compensation for fishing pressure by maintaining the numerical sex ratio then effects of fishing can be somewhat reduced. However, even with moderate levels of fishing activity, all modeled populations in Huntsman and Schaffs (1994) study exhibited great reductions in reproductive output. Though recent studies, including this one, have found a sex ratio that varies from Moe's (1969), no statistically significant increase in number of males has been stated It appears the population has been able to compensate for 77

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fishing pressure by basically maintaining or possibly slightly increasing the number of males However, the recent studies on E. morio that have found an increase in size at age and a general decrease in size composition of the catch indicate definite signs of an overfished population. According to Coleman et al. (1999), grouper species like E. striatus, that form few but extremely large aggregations at specific times of the year, are a sort of indicator species for overfishing practices Fish such as jewfish and Nassau grouper are recognized as extremely overfished and are currently protected. Red grouper, which appear to form small, numerous aggregations for a relatively extended spawning season, are thought to be most able to maintain viable populat i ons under heavy fishing pressure (Coleman et al., 1999) With a recent stock assessment now identifying E morio as overfished (and undergoing overfishing), the concern for grouper stocks is obvious. Spawning potential ratio (SPR) is used by managers as a proxy measure for the reproductive health of fish populations. SPR, simplified, is a comparison between fecundity for a fished and an unfished population. Unfortunately, the grouper fishery has a long history and few, possibly no, unfished populations exist in the Gulf or elsewhere. Therefore, it is often impossible for managers to study the dynamics of grouper that have been un-impacted by fishing mortality Therefore, SPR is only an estimate of what the population should look like naturally. Without data on recruitment sources for these economically valuable fishes, reproductive proxies such as SPR are necessary (Huntsman and Schaff, 1994). An emerging idea in fisheries management is that of"no-take" marine reserves or areas where only non-consumptive use is allowed. The establishment of such 78

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areas in the U S would allow scientists to better evaluate the characteristics ofunfished fish populations as well as preserve larger reproductively fit fish. 79

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VI. Conclusions The Western-blotted protein bands analyzed in this study for reactivity with the synthetic antibody probe were oftwo molecular weights, 270 kd and 187 kd. These high molecular weight bands are close to the reported ranges for vitellogenin in other Serranids (Plectorpomus leoparadus 180 kD, Takemura and Teruya, 1997; E. malabaricus, 113 & 140 kD, Utarabhand and Bunlipatanon, 1996; M microlepis, 180 kD, Heppell and Sullivan, 1999). The synthetic probe detected varying levels of vitellogenin-related protein across all male and female developmental stages. Studies have also detected levels of vitellogenin in male and immature female fish, but few have been conducted on protogynous hermaphrodites. It has been well documented that the use of supposedly purified vitellogenin to develop antibodies for sex determination can distinquish between mature female fish and immature/male fish in gonochorists. Previous studies by authors such as Whitehead et al. (1978 & 1983), LeBail and Breton (1981) Van Bohemen and Lambert (1981), Craik and Harvey (1984), Matsubara and Sawano (1992), Tao et al. (1993), Heppell et al. (1995), Specker et al. (1995), Denslow, et al. (1997), Mananos et al. (1997), Takemura and Teruya (1997) Hoque et al. (1998), Heppell et al. (1999), and Heppell and Sullivan (1999) have concluded that the use of vitellogenin probes is promising for determinations of reproductively active female fish in natural or artificial habitats. 80

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Evidence of a vitellogenin-related protein present in male and female fish at non significantly different levels than in female fish in the present study are in evident contrast to some other studies based on vitellogenin probes However, many of those studies used FSSPs (Female Sex Specific Proteins) to attempt to confirm E2-stimulated proteins were in fact vitellogenin (Tao et al., 1993; Hoque et al. 1998 ; Heppell and Sullivan, 1999 ; Heppell et al., 1999). FSSP probes are created from pooled female plasma and involve the adsorption of the antisera with mature male plasma. This effectively removes (by adsorption) components of antisera that are common to both mature male and female fish (Tao et al., 1993; Hoque et al., 1998), leaving an antisera specific to female plasma. In which case, the true sex-determining fraction of the plasma might be a protein other than Vt. Therefore the use of their method would not detect proteins that might be stimulated and common to both sexes but removed by adsorption. There may in fact remain antisera to female proteins that are not Vt. The significant peak of the 270 kd band measured in early and mid ripening fish indicates a peak of a vitellogenin related protein that could be associated with spawning and maturity. The lack of specificity to stimulated vitellogenins in female fish would be problematic for field studies that seek to determine with certainty the sex of a fish i n question The possibility of creating a successful immunoagglutination test with the present method is low, given the high reactivity of the probe with both male and immature fish. The developed probe should be tested on gonochoristic species to determine if the protogynous hermaphroditism of the tested species, E. moria and M microlepis, causes the variant vitellogenin related proteins detected across varying developmental stage fish in this study. 81

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The conserved areas of the vitellogenin sequence were used in this study to develop the antibodies tested. As Folmar et al. (1995) found theN-terminal region to be conserved across many teleosts and vertebrates in general the existence of vitellogenin related prote ins that react with an antibody to theN-terminal and internal conserved region is not surprising If conservation across species exists then variant proteins could also share conservation terminally. In con c lusion, as the red grouper stock in the Gulf of Mexico (and other group e r species worldwide) seem to be in serious decline due to heavy fish ing pressure methods to ascertain the reproductive status of healthy populations should be developed and evaluated. The present study identified the possibility of a form of vitellogenin existing in males and immature fish, as well as mature female fish. In order to use a probe for vitellogenin to study sex rat i os and sex reversal in the field a less sensitive probe might be needed Other studies have developed probes for species in the Epinephelin a e that might be of use to biologists who wish to study red grouper Therefore, the use of purified fractions of vitellogenin from female fish that are adsorbed with male plasma seems to be the current best method for field detection of male and female fish. Studies applied to natural" populations and the effects that fishing has on those populations will be of great importance in the future due to increasing discussion of" no take" marine reserves. Fisheries managers are looking for additional management measures to aid in the maintenance of healthy sustainable populations and some feel reserves will achieve this especially for long-lived, slow-growing late maturing fish If reserves are widely established, researchers will have ample access to populations that are not impacted by consumptive human activity. Probes that would identify male and 82

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female fish would allow an expansion of knowledge into the management ofprotogynous reef fishes. The findings of this study, of the presence of basal levels of a vitellogenin related protein in male and immature grouper add new knowledge to the study of this "sex-specific" protein. The detection ofvitellogenin in the plasma of male red grouper in this study in conjunction with previous studies by Sumpter (1981) and Ding et al. (1989) leads to the conclusion that vitellogenin is not a sex specific protein in all fishes and should not be referred to as such. We also identified the presence of a protein of270 kD which reacts intensely with a vitellogenin probe and appears to be specific to red grouper. To further understand the relationship which exists between this identified protein and vitellogenin, it needs to be sequenced and analyzed. Further studies should be undertaken to identify the presence and composition of vitellogenin-related proteins in male and immature fish The following summarizes the findings of the present study in relation to the questions posed in the experimental design : 1 Are antibodies made to artificial peptides derived from known Vt sequences sensitive and accurate enough to detect seasonal variations in serum vitellogenin levels in red grouper? The antibodies appear to detect variations between individuals, however, the antibodies detected basal levels across almost all developmental stages. 2 Is there a correlation between reproductive state as determined from gonadal histology and the level of serum vitellogenin detected among field caught male, female and transitional fish? There is no correlation between 83

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reproductive state as detennined from gonadal histology and the level of serum vitellogenin as assayed in the present study for the 187 kD band. The 270 kdD band appears to has a significant peak for early/mid ripening female red grouper 3 Can the assay be used for different grouper species ? The assay detected basal levels of a protein in gag, which was within the range of previously reported vitellogenins 4 Does the assay hold much promise for the development of easy-to-use field detection kits ? A less sensitive probe might be needed as lack of specificity to female vitellogenins would be problematic for field studies that seek to determine the sex of fish. 84

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References Arreguin-Sanchez > F. and Valero E 1993 Trophic Role ofthe Red Grouper (Epinephelus morio) in the Ecos y stem of the Northern Continental Shelf of Yucatan > Mexico In Biology fisheries and culture of tropical groupers and snappers ICLARM Conf. Proc 48: 1927 Bannerot, S.P 1984 The Dynamics of Exploited Groupers (Serranidae) : An investigation of the Protogynou s Hermaphroditic Reproductive Strategy PhD Dissertation. University of Miami. Bon > E ., Barbe, U ., Rodriguez, N ., Cuisset B., Pelissero C. Sumpter J.P. and Menn F .L. 1997 Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout (Onchorhynchus mykiss): Establishment and Validation o f an ELISA. Comp Biochem. Physiol. 117B(1 ) : 75-84 Brule T and Canche, L.G .R. 1993 Food Habits of Juvenile Red Groupers Epin ephe lus moria (Valenciennes 1828 ), from Campeche Bank Yucatan Me x ico Bull. Mar Sci. 52(2) : 772-779. _____ and Deniel C. 1993 Biological Research on the Red Grouper (Epinephelus morio) from the Southern Gulf of Mexico In B i ology fisheries and culture of tropical groupers and snappers ICLARM Conf. Proc 48 : 28-42 and Deniel C 1999. Red Grouper Reproduction in the Southern Gulf of -----Mexico. Trans. of Amer. Fish. Soc 128(3): 385 402 Bullock L.H and Smith G B 1991. Seabasses (Pisces : Serranidae) In Memoirs of the Hourglass Cruis e s FMRI DNR Publication V8(2) : 243pp Chan, S L., Tan C H., Pang, M.K and Lam T.J. 1991. Vitellogenin Purification and Development of Assay for Vitellogenin Receptor in Oocyte Membranes of the Tilapia (Oreochromis niloticu s, Linnaeus 1766). Jl. OfExp. Zoo 257: 96-109 Charnov E. L. 1982 Alternative life histories in protogynous fishes: A general evolutionary theory Mar Ecol. Prog., ser. 9 : 305-330. Cole K. S and Robertson D. R 1988 Protogyny in the Caribbean ReefGoby C o ryphopterus personatus : Gon a d Ontogeny and Social Influences on Sex Change. Bull. Mar Sci 42(2) : 317-333 85

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_____ and D. Y. Shapiro. 1990. Gonad Structure and Hermaphroditism in the Gobiid Genus Coryphopterus (Teleostei: Gobiidae) Copeia 4 : 996-1003 _____ and D. Y Shapiro. 1992. Gonadal structure and population characteristics of the protogynous goby Coryphopterus glaucofraenum. Mar. Biol. 113: 1-9 Coleman F C Koenig C C and L.A. Collins. 1996 Reproductive styles of shallow water groupers (Pisces : Serranidae) in the eastern Gulf of M e xico and the consequences of fishing spawning aggregations Env. Biol. Fishes 4 7 : 129-141. Coleman F.C, Koenig C C Eklund A.M ., and Grimes C. B. 1999 Management and Conservation of Temperate Reef Fishes in the Grouper-Snapper Complex ofthe Southeastern United States American Fisheries Society Symposium 23 : 233 242 Colin, P. L. 1992 Reproduction ofthe Nassau grouper, Epinephelus striatus (Pisces : Serranidae) and its relationship to environmental cond i tions. Env. Biol. of Fishes 34 : 357-377 Colin P L. Koenig, C C ., and Laroche W A. 1993. Development from Egg to Juvenile of the Red Grouper (Epinephelus mario) (Pisces : Serranidae) in the Laboratory Bio logy fisheries, and culture of tropical groupers and snappers EPOMEXIICLARM Proc 1993 Craik J.C.A. and Harvey S M. 1984 A biochemical method for distinguishing between the sexes of fishes by the presence of yolk protein in the blood J. Fish Bioi. 25 : 293-303 De Vlaming V L., et al. 1980 Goldfish (Carassius auratus) Vitellogenin : induction isolation, properties, and relationship to yolk proteins. Comp Biochem Physiol. Vol: 613-622 Den s low N D Chow, M., Chow M M ., Bonomelli S ., Folmar, L., Heppell S A. and Sullivan, C V 1997 Development ofbiomarkers for environmental contaminants affecting fish In Chemically Induced Alterations in Functional Development and Reproduction in Fishes. Rolland, R M ., Gilbertson M ., and Peterson, R.E (eds) SEATAC Technical Publications Series Society of Env i ronmental Toxicology and Chemistry, Pensacola Pp. 73-86 Ding, J. L Hee P. L., and Lam, T J. 1989 Two Forms ofVitellogenin in the Plasma and Gonads of Male Ore o chromi s aureus Comp Biochem. Phy siol. V 938(2): 363-370 Eklund A.M. 1992 Age and growth of red grouper (Epinephelus mario) from the Gulf of Mexico NMFS Southeast Fisheries Science Center Miami CRD-91192-73 86

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Florida Governor's Ocean Committee. November 1998. Florida's Ocean Challenges Interim Progress Report to the Governor. Folmar, L.C. Denslow, N.D. Wallace R.A., LaFleur, G Gross, T S., Bonomelli, S. and Sullivan C V. 1995 A highly conserved N-terminal sequence for teleost vitellogenin with a potential value to the biochemistry molecular biology and pathology of vitellogenesis. Jl. Fish Bio. 46 : 255-263 Ghiselin, M T. 1969 The Evolution ofHermphroditism Among Animals. Quart Rev. Biol. 44: 189-208 _____ 1974 The Economy ofNature and the Evolution of Sex. Univ. of Cal. Press Gilmore R G and R.J. Jones 1992. Color variation and associated behaviour in the epinepheline groupers Mycteroperca rnicrolepis (Goode and Bean) and M phenax (Jordan and Swaine). Bull. Mar Sci 51: 84 103. Goetz, F W 1983 Hormonal Control of Oocyte Final Maturation and Ovulation in Fishes In Fish Physiology Hoar, W S., Randall, D.J., and Donaldson, E M (eds) VIX Reproduction (B) : 117-169. Goodyear, P 1991 Some Thoughts on Biolog i cal Reference Points Based on Fishing Mortality Spawning Stock Biomass and Spawning Stock Biomass per Recruit. Goodyear, P. and Schirripa, M.J 1991. The Red Grouper Fishery of the Gulf of Mexico SFC Contribut ion Miami Lab CRD-90/91-86. Goodyear, P. and Schirripa, M.J. 1993 The Red Grouper Fishery of the Gulf of Mexico. SFC Contribution Miami Lab CRD-92 / 93-75. Houde, E. D 1982 Kinds, distributions and abundances of sea bass larvae (Pisces : Serranidae) from the eastern Gulf of Mexico Bull. Mar. Sci. 32(2) : 511 522 Heppell Scott A ., Denslow, N .D., Folmar L.C., and Sullivan C.V. 1995 Universal Assay ofVitellogenin as a Biomarker for Environmental Estrogens Environmental Health Perspectives 103 :9-15 Jackson L.F ., Weber, G M., and Sullivan C V Enzyme-linked -----immunosorbant assay (ELISA) of Vitellogenin in Temp e rate Basses (Genus Marone) : Plasma and i n vitro analyses Trans of Amer. Fish Soc 128 : 532541. 87

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_____ and Craig V Sullivan 1999 Gag (Mycteroperca microlepis) vitellogenin: purification, characterization and use for enzyme-linked immunosorbent assay (ELISA) of female maturity in three species of grouper. Fish Phys. Biochem 20 : 361-374. Ho, Shukemei. 1987. Endocrinology ofVitellogenesis. In Hormones and Reproduction in Fishes, Amphibians, and Reptiles Eds Norris, D an Jones., R.E Plenum Press, NY and London. pp : 145-169 Hoque M. M, Takemura A., and Takano, K. 1998. Annual Changes in Oocyte Development and Serum Vitellogenin Level in the Rabbitfish Siganus canaliculatus (Park) in Okinawa, Southern Japan. Fisheries Science 64(1) : 4451. Houde, E D 1982 Kinds distributions and abundances of sea bass larvae (Pisces : Serranidae) from the eastern GulfofMexico. Bull. Mar. Sci 32(2): 511-522 Huntsman, G R. and Schaaf, W .E. 1994 Simulation of the Impact of Fishing on Reproduction of a Protogynous Grouper, the Graysby. Jackson L.F. and Sullivan, C V 1995. Reproduction of white perch (Marone americana): the annual gametogenic cycle. Trans Amer. Fish. Soc 124 : 563577 Jarvis, N.D 1935. Fishery for red snappers and groupers in the Gulf of Mexico Invest. Rep U S Bur. Fish., No 26 29p. Johnson, A.G. and Collins, L.A. 1994. Age-Size Structure ofRed Grouper, (Epinephelus morio ), from the Eastern Gulf of Mexico Northeast Gulf Science 13(2): 101-106. Johnson, A K 1995. Comparison of Gonadal Histology and Sex Steroid Levels Over the Seasonal Reproductive Cycle of Red Grouper, Epinephelus morio Master's Thesis. USF-Marine Science. 63pp. Thomas P. and Wilson, R R., Jr. 1998. Seasonal cycles of gonadal -----development and plasma sex steroid levels in Epinephelus morio, a protogynous grouper in the eastern Gulf of Mexico J Fish Bio 52 : 1-17 Jory, D E and Iversen E S 1989 Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (South Florida). Black Red and Nassau Groupers Fish and Wildlife Service Bioi. Report 82(11.110) 21pp Koenig, C.C 1993 Spawning biology of shallow-water GulfofMexico groupers : Final report. MARFIN contract no. NA90AA-H-MF748. 88

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_____ and Coleman, F C. 1998 Absolute abundance and survival of juvenile gag Mycteroperca microlepis in seagrass beds of the northeastern Gulf ofMexico. Trans Am Fish. Soc 127: 44-55. Le Bail P Y and Breton, B 1981. Rapid Determination ofthe Sex of Puberal Salmonid Fish by a Technique oflmmunoagglutination Aquaculture 22 : 367-375 Maiian6s, E .L., Zanuy, S. and M Carrillo 1997. Photoperiodic manipulations ofthe reproductive cycle of sea bass (Dicentrarchus labrax) and their effects on gonadal development, and plasma 17-13-estradiol and vitellogenin levels Fish Phy Biochem. 16: 211-222 Matsubara T and Sawano K. 1992 Sex determination ofPacific halibut (Hippoglossus stenolepis) by the immunodot-blotting technique using antiserum against Vitellogenin Bul. Hak.kaido Natl. Fish Res Inst. 56:17-26 Moe M A Jr. 1969 Biology of the red grouper, Epinephelus morio, from the Eastern Gulf of Mexico. FL. Dept. Nat. Res Mar Resear. Lab Prof. Paper Ser No 10 95p. Ng et al. 1985 A phannacological dose of 17beta-oestradiol produces minimal metabolic stimulation but evokes hepatic necrosis in the immature female serranid Epinephelus akaara. Jl. Fish Bioi. 26:217-222. NOAA NMFS U.S Dept. of Commerce Fisheries Statistics and Economics Division 1997 Fisheries of The United States, 1997 Current Fishery Statistics No 9700. Sept. 1998 NOAA NMFS U.S. Dept. of Commerce 1998. Report to Congress Status of the Stocks of Fisheries of the United States Norberg Birgitta 1987. Vitellogenin and egg proteins in three marine fish species. In Proceedings of the 3rd International Symposium on the Reproductive Physiology ofFish. Idler, Crim and Walsh (eds). Pp 212 213 Rivas, L.R. 1970 The Red Grouper of the Gulf of Mexico U S Dept. of Commerce Commercial Fisheries Review V32 ( 10) : 24-30 Roberts S B., Jackson, L.F King, W, ill. Taylor R.G Grier H.J. and Sullivan C V 1999 Annual Reproductive Cycle of the Common Snook : Endocrine Correlates of Maturation Trans. of Amer. Fish Soc 1 2 8 : 436-445 Ross, M.R. 1990 The evolution of sex-change mechanisms in fishes. Env. Biol. Of Fishes 29: 81-93 89

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_____ ,Hourigan, T.F., Lutnesky, M.F., and Singh, I. Multiple Simultaneous Sex Changes in Social Groups of a Coral ReefFish 1990. Copeia (2): 427-433. Sadovy, Y. 1990. Grouper Stocks ofthe Western Central Atlantic: The Need for Management and Management Needs. Proc. of Gulf and Caribbean Fisheries Institute 43: 43-63. _____ and Colin, P L. 1995 Sexual Development and sexuality in the Nassau grouper. J. Fish Biol. 46 : 961-976. _____ and Shapiro, D 1987 Criteria for the Diagnosis of Hermaphroditism in Fishes. Copeia. No.1: 136-155. _____ P. L. Colin and M .L. Domeier. 1994. Aggregation and spawning in the tiger grouper Mycteroperca tigris (Pisces : Serrandiae ). Copeia. 1994: 511-516. Schirripa M. J. and Legault C. M 1997. Status of the gag stocks of the Gulf of Mexico: assessment 2 0. NMFS, Southeast Fisheries Science Center, Sustainable Fisheries Division, Miami, Florida. 114p. _____ Legault C M ., and Ortiz, M. 1999. The Red Grouper Fishery of the Gulf of Mexico Assessment 3 0 NMFS Sustainable Fisheries Division Contribution No. SFD-98/99 56. Shapiro, D. Y. 1979 Social Behavior, Group Structure, and the Control of Sex Reversal in Hermaphroditic Fish. In Advances in the Study of Behavior. V10: 43-101 -----1981. Size, maturation, and the social control of sex reversal in the coral reef fish Ant hi as squamipinnis (Peters). J. Zool., (London) 193: 105-128. 1984. Sex Reversal and Sociodemographic Processes in Coral Reef -----Fishes. In Fish Reproduction: strategies and tactics. (G W Potts and R. J Wotton Eds) Academic Press, London. _____ 1987. Reproduction in Groupers In J .J. Polovina and S Ralston (ed.) Tropical Snappers and Groupers : Biology and Fisheries Management. Westview Press. Boulder. Pp. 295-327. Sadovy Y and McGehee, M .A. 1993. Periodicity of sex change and -----reproduction in the red hind, Epinephelus guttatus, a protogynous grouper. Bull Mar. Sci. 53(3) : 1151-1162 Smith, C.L. 1971. A revision of the American groupers : Epinephelus and allied genera Bull. Am Mus. Nat. Hist. 146: 67-242 90

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_____ 1975. The Evolution of Hermaphroditism in Fishes In_Intersexuality in the Animal Kingdom. pp : 295-310 _____ .1982. Patterns of reproduction in coral reef fishes In G.R Huntsman W R. Nicholson, and W.W Fox Jr. (eds) The Biological Basis for Reef Fishery Management. NOAA Tech. Mem NMFS-SEFC-80. Pp 49-66 Specker J L., Woods, L. C., III Huang, L., and Kishida, M 1995 Application of a non-invasive sex test in the aquaculture of Striped Bass In Proceedings of the 5th International Symposium on the Reproductive Physiology of Fish University of Texas at Austin 2-8 July, 1995 Pp 96-98 Stiles T C and Burton, M .L. 1990. Age, Growth and Mortality ofRed Grouper Epinephelus morio, from the Southeastern U S Proc Gulf and Caribbean Fisheries Institute 4 3 : 123-13 7. Sumpter J P. 1981. The Purification Radioimmunoassay and Plasma Levels of Vitellogenin from the Rainbow Trout Salmo gairdneri In Current Trends in Comparative Endocrinology Lofts, B and Holmes, W N (eds) Hong Kong Pp 355-357 Sundararaj B l.. and Nath, P. 1981. Steroid-Induced Synthesis ofVitellogenin in the Catfish, H e t e ropneustesfossilis. Gen. Comp Endocrin 43 : 201-210. Takemura, A., and Teruya, K 1997. Purification and Partial Characterization of the Vitellogenin of Coral Trout, Plectropomus leopardus Bull. Mar Sci. 61(3): 791800 Tao Y., Hara A., Hodson, R.G. Woods L.C., III and Sullivan C V 1993. Purification characterization and immunoa s say of s triped bass (Morone saxatilis) vitellogenin. Fish Phy Biochem 12(1): 31-46. Thompson, R and Munro J.L. 1978 Aspects of the biology and ecology of Caribbean reef fishes : Serrandiae (hinds and groupers) J Fish Biol. 12: 115-146 Thresher, R.E 1984 Groupers and Rock Cod (Serranidae : Epinephelinae). In R e production in reef fish e s T F .H. Publications. pp 68-74 Tucker J. W Jr. Bush P. G and Slaybaugh, S T 1993 Reproductive Patterns of Cayman Islands Nassau Grouper (Epinephelus Striatus) Populations Bull. Mar Sci 52(3) : 961-969. Utarabhand P. and Bunlipatanon P. 1996 Pla s ma Vitellogenin of Grouper (Epin ephe lus malabaricus): Isolation and Properties Comp Biochem Physiol. V 115C(2) : 101-110. 91

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Van Bohemen, C .G., and Lambert J.G.D 1981. Estrogen Synthes i s in Relation to Estrone Estradiol and Vit e llogenin Plasma Levels during the Reproductive Cycle of the Female Rainbow Trout Salm o giardneri. Gen Comp Endocrin 45 : 105114 Van Bohemen C G ., Lambert J G .D., Goos H J. TH ., and Van Oordt P G W J 1982 Estrone and Estradi o l Participation during Exogenous Vitellogenesis in the Female Rainbow Trout Salmo g ai r dn e ro Gen. Comp Endocr i n 46 : 8192. Wahli W ., Dawid LB., Gerhart U.R. and Weber, R. 1981. Vitellogenesis and the Vitellogenin Gene Family Science 212(17) : 298 304 Walker J. M 1994 The Bicinchoninic Acid (BCA) Assay for Protein Quantification In Basic Protei n and Peptid e Protocols Walker, J M (ed) Methods in Molecular Biolo gy 32: 5-8 Wallace R.A and S e lman K. 1981. Cellular and Dynamic Aspects ofOocytes Growth in Teleosts Amer Zool. 2 1 : 325 343 _____ and Selman K. 1989 Cellular Aspects of Oocyte Growth in Teleost s. Zool. Sci 6 : 211-231. Warner Robert R. 1975 The Adaptive Significance of Sequential Hermaphroditi s m in Animals Amer Nat. V 109, No 965 : 61-82. _____ 1984 Mat i ng Behavior and Hermaphroditism i n Coral Reef Fishes. Amer Sci. 72 : 128-136 -----1988 Sex change in fishes : hyphotheses, evidence and objections Env B i ol. of Fishes V 2 2(2): 81-90. Weigand M D 1982 Vitellogenesis in Fishes. In Proceedings ofthe International Symposium on Reproducti v e Ph y siology Pp 136-146 Whitehead C ., Bromage N.R. and Forster J .R. 1978. Seasonal changes in reproductive functions of the rainbow tr o ut (Salm o giardneri). Jl. Fish Biol. 12:601-608 Bromage N .R., and Breton B. 1983. Changes in Serum Levels of -----Gonadotropin O-estrad i ol 17 beta and Vitellogenin durin g the fir s t and subsequent reproduct i ve c y cles of fem a le Rainbow Trout. Aquaculture. 34 ( 3-4 ) : 317 326 92

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Appendices 93

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Appendix 1 One and Three Letter Symbols for the Amino Acids A Ala Alanine B Asx Asparagine or aspartic acid c Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine p Pro Proline Q Gln Glutamine R Arg Arginine s Ser Serine T Thr Threonine v Val Valine w Trp Tryptophan y Tyr Tyrosine z Glx Glutamine or glutamic acid 94

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Appendix 2 PAS/MY Staining Protocol (FMRI Aquatic Health Lab, personnel communication; Quintero-Hunter, et al., 1991): I 1% Periodic acid (aqueous) oxidation, 15 minutes. 2 Three distilled water rinses. 3 Schiff's reagent, 15 minutes. 4. Developed in running tap water, 10 minutes. 5. Stain in Weigert's hematoxylin, 6 minutes 6. Differentiated using : a) Acid 70% ethanol (pH=2.5), two changes, 3 quick dips. b) Acid distilled water (pH=2.5),three changes, 3 quick dips c) Running water, 15 minutes. 7 Stain in Metanil Yell ow solution for two minutes. 8. Rinse in three changes of distilled water, 3 quick dips each. 9. Dehydrate in 95% ethanol, two changes, three dips each. 10. Dehydrate in 100% ethanol, two changes, three dips each. 11. Dehydrate in acetone two changes, three dips each. 12. Clear in Histoclear, two changes three dips each. 13. Clear in Histoclear, two changes one minute each. 14. Transfer to fresh Histoclear, coverslip with Acrytol mounting medium. 95

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Appendix 3. Hemotoxylin and Eosin Staining Protocol (from FMRI Aquatic Health Lab, personnel communication): 1. Weigert's Hematoxylin, 6 minutes. 2. Drain excess Hematoxylin on towels. 3 Acid Alcohol, pH 2.5, two changes, 3 quick dips each 4. Acid-DI, pH 2.5 three changes, 3 quick dips each. 5. Wash under running tap water 15 minutes. 6 Working Eosin -Phloxine, 2 minutes. 7. 95% EtOH, two changes, 30 seconds each. 8. 100% EtOH, two changes 30 seconds each. 9. Histoclear, two changes, 5 quick dips each. 10. Histoclear, two changes, 1 minute each. 11. Transfer to fresh Histoclear, coverslip with Acrytol mounting medium. 96

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Appendix 4 BCA Protein Assay Protocol Volume ofBSA(J..ll) Volume of diluent(J..tl) Final [BSA] ( stock=2mg/ml) (0 .9% NaCI) (J..lg/ml) 300 (stock) 0 2000 375 (stock) 125 1500 (A) 325 (stock) 325 1000 (B) 175 (A) 175 750 (C) 325 (B) 325 500 (D) 325 (D) 325 250 (E) 325 (E) 325 125 (F) 100 (F) 400 25 (G) 80 (G) 120 10 (H) 100 (H) 100 5 (I) 0 0 50 0 I. Use 1 ml cuvette and 50 J..ll of standards/ 5J..ll samples (with diluent to 50J..ll). 2 Add 1 part BCA reagent B (Solution containing 4% cupric sulfate) to 50 parts BCA reagent A (reagent containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.2N sodium hydroxide). Use 1 ml of working reagent per sample. 3. Add 1 ml working reagent to each cuvette and mix well. 4. Incubate at room temperature for 2 hours 5. Measure absorbance at 562 nm in reference to water reference. 97

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Appendix 5 15 Residue Sequence BLAST Search Results BLASTP 1.4.11 (24-Nov-97] [Build 24-Nov 97] Reference : Altschul, Stephen F Warren Gish Webb Miller Eugene W Myers,and David J. Lipman (1990) Basic local alignment search tool. J. Mol. Bioi. 215:403 10. Query= XYZO 12 jenniferjarrell XYZ ( I5 l etters) Database : Non redundant GenBank CDS translation s +PDB+SwissProt+SPupdate+PIR 329,726 sequences ; 100 504,245 total letters Searching .... .... ... ........ ... . ... ............. . . . done Sequences producing High-scoring Segment Pairs: Score P(N) N bbsii76857 vitellogenin VTG {N-terminal} [Moro.. 67 0.094 bbs l I 76858 vitellogenin, VTG {N-terminal} [Lago.. 63 0.30 I sp i Q90508 1 VITI_FUNHE VITELLOGENIN I PRECURSOR (VTG I) (CO ... 6I 0.49 I sp i Q98893IVIT2 FUNHE VITELLOGENIN II PRECURSOR (VTG II)(... 61 0.49 I bbsll76857 vitellogenin, VTG {N-terminal} [Morone saxatilis=striped bass,Peptide Partial 22 aa] Length= 22 Score= 67 (30 8 bits) Expect= 0 098 P = 0.094 Identities = I2/l5 (80%), Positives = 13115 (86%) Query : I YQVNFAPEFATGKTY I5 + VNFAPEFA GKTY Sbjct: l HNVNFAPEFAAGKTY 15 bbs l l76858 vitellogenin, VTG {N-terminal} [Lagodon rhomboides=pinfishes,Peptide Partial, 22 aa] Length= 22 Score = 63 (29.0 bits) Expect= 0.35 P = 0 30 Identities= ll/15 (73%), Positives = 13115 (86%) Query : I YQVNFAPEFATGKTY 15 YQVN APEFA G+T+ Sbjct: I YQVNLAPEFAAGRTH 15 spiQ90508IVITl FUNHE VITELLOGENIN I PRECURSOR (VTG I) (CONTAINS : LIPOVITELLIN I (LVI); PHOSVITIN (PV); LIPOVITELLIN 2 (LV2)) > gil459202 (U07055) vitellogenin I [Fundulus heteroclitus] Length= 1704 Score= 61 (28 0 bits) Expect= 0 67 P = 0.49 Identities= ll/12 (91%) Positives = 11112 (91%) 98

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Appendix 5 (Continued). Query: 4 NFAPEFATGKTY 15 NFAPEFA GKTY Sbjct : 17 NF APEF AAGKTY 28 spiQ98893IVIT2_FUNHE VITELLOGENIN II PRECURSOR (VTG II) (CON TAINS:LIPOVITELLIN 1 (LVI); PHOSVITIN (PV) ; LIPOVITELLIN 2 (LV2) ; YP 69) gill621359 (U70826) vitellogenin II precursor [Fundulus heteroclitus] Length= 1687 Score= 61 (28 0 bits), Expect= 0.67 P = 0.49 Identities= 11/14 (78%), Positives= 13/14 (92%) Query: 2 QVNF APEFA TGKTY 15 QV++APEFA GKTY Sbjct: 17 QVSY APEF APGKTY 30 Parame ters : V =lOO B=50 H=l E=0 90 -fi lter SEG Lambda K H 0 319 0 134 0.408 Cutoff to enter 2nd pass : >= 33 ( 0.0 bits) E S T1 T2 XI X2 W Gap 0.9 63 11 11 -16 -22 40 50 Database: Non-redundant GenBank CDS translations + PDB+Swis s Pro t+SPupdate+PIR Posted date : Oct 14, 1998 7:57AM # of letters in database : 100 ,504,245 #of sequences in database : 329 726 Number of Hits to DB : 1st pass: 2835186, 2nd pass: 8377 Number of Sequences : 1st pass : 329726, 2nd pass : 354 Number of extensions: 1st pass: 1 796 4, 2nd pass: 6264 Number of successful extensions: 1st pass : 354, 2nd pass : 661 Number of seq uence s better than 0: 4 99

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Appendix 6. 12 Sequence Residue BLAST Search Results Query= XYZ012jenniferjarrell XYZ {12 letters) Database: Non-redundant GenBank CDS translation s+ PDB + SwissProt+SPupdate + PIR 329,726 sequences ; 100 ,504,245 total letters Searching ........... . ..... ............... ... .... ...... done Sequences producing High-scoring Segment Pairs : Score P(N) N s piQ90508IVITl FUNHE VITELLOGENIN I PRECURSOR (VTG I) (CO.. 67 0 .13 1 s piQ98893IVIT2 _FUN HE VITELLOGENIN II PRECURSOR (VTG II) (... 66 0 .17 1 spiQ90508IVITl_FUNHE VITELLOGENIN I PRECURSOR (VTG I) (CONTAINS : LIPOVITELLIN I (LVI); PHOSVITIN ( PV ); LfPOVITELLIN 2 (LV2)) > gil459202 (U07055)vitellogenin I [Fundulus heteroclitus] Length = 1704 Score= 67 (30.4 bits), Expect = 0 .14, P = 0.13 Identities = 12/12 (100%) Positives = 12112 (100 %) Query : l CGKADGEIRQEY 12 CGKADGEIRQEY Sbjct: 1580 CGKADGEIRQEY 1591 sp Q98893 I VIT2 FUNHE VITELLOGENIN II PRECURSOR {VTG II) (CONTAINS: LIPOVITELLIN I (LVI); PHOSVITIN (PV) ; LIPOVITELLIN 2 (LV2) ; YP 69) gill621359 ( U70826) vitellogenin II precursor [Fundulus heteroclitus] Length = 1687 Score= 66 (30 0 bit s), E x pect = 0 .19, P = 0 .17 Identities = ll/12 (9 1 %), Positives = 12/12 (100%) Query : 1 CGKADGEIRQEY 1 2 CGKADGE+RQEY Sbjct: 1556 CGKADGEVRQEY 1567 Parameters : V =IOO B= 50 H = l E =. 90 -fi lter SEG Lambda K H 0 315 0 .141 0.435 Cutoff to enter 2nd pass:>= 33 ( 0.0 bi ts) E S Tl T2 X 1 X2 W Gap 0.9 63 11 11 -16 -23 40 5 0 100

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Appendix 6 (Continued). Database: Non-redundant GenBank CDS translations+PDB+SwissProt + SPupdate + PIR Posted date : Oct 14, 1998 7:57AM # of letters in database : I 00 504 245 #of seque nces in database : 329,726 Number of Hits to DB : 1st pass : 2202252, 2nd pass: 5445 Numbe r of Sequences : I st pass : 329726, 2nd pass: 170 Number of extensions : 1st pass: 9683, 2nd pass: 4314 Number of successful extensions : I s t pass: 170, 2nd pass: 252 Number of seq uen ces better than 0: 2 101

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Appendix 7. Western Blot Results for high/low ODI for red grouper 117 A. B c. D Figure 26. Western blot of low (left) and high (right) 001 value for Band I (270kD) according t o developmental stage. 26A FIM; 26B FER; 26C FMR ; 260. FLR (Molecular we i ght markers on left) 102

PAGE 119

Appendix 7 (Continued). 117 79. 117' A c . . . f t : B D F ig ure 27. Western blot of low/high ODI values for 270 kD band for varying developmental stages. 27A Female Ripe ; 278. Female Spent ; 27C Male ripening ; 270. Male late ripening 103

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Appendix 7 (Continued). , "'" 117. 79 . .. A B Figure 28. Western blot of high/low ODI values for 270kD band for varying developmental stages. 28A. Male Ripe 28B. Male Spent 104

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Appendix 8 Western blot of typical gag females 71 -Figure 29. Western blot of gag grouper females. All of above samples are regressed or immature females except for the far right sample that is an early ripening female The blotting patterns were almost identical across all fish and showed no variation for the three fish determined to be early ripening. 105

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Appendix 9. Gag Histology Pictures c. Figure 30 Gag histology pictures 30A. Immature female gag with numerous primary oogonia around lamellar periphery and Stage I and II oocytes in center oflamellae; 308. Regressed female ovary with a lot of interstitial tissue and numerous SI and SII oocytes; 30C. Early ripening female with evidence of prior spawning (POF) and cortical alveoli stage oocytes (CA). 106

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Appendix 10. Western blotting results for Estradiol-stimulation experiment. -187 s 2 3 4 5 Figure 31. Comparison of FIM high and low ODI 270kd values (lanes I & 2) and E r stimulated test fish and control fi s h (lanes 3 4 & 5 respectively) 107


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