Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Betanodavirus that causes vacuolating encephalopathy and retinopathy and viral nervous necrosis. A method to purify MGNNV was proposed. The cellular components of SSN-1 reprent few viral receptors for Betanodavirus. Several cellular virus binding proteins (VBPs) were detected by employing the technique of immobilizing virus on nitrocellulose. The least VBPs were found in SSN-1 cell lysate that was treated with proteinase K. The approaches used receptor antagonists to identify the cell receptor. The antagonists are able to block the viral binding and thus potentially directly against the receptor. The results implied that the receptor of serotonin 5-HT1A or £\2 adrenergic may act as the receptor of MGNNV.

The aim of the present research was to investigate the expression profiles of GnRH-R and neuropeptide Y (NPY) genes in brain and pituitary of Orange-spotted Grouper, Epinephelus coioides and also to understand the regulatory mechanism by administering different sex steroids. GnRH-R (TMD2 to TMD6) was partially involved cloned in this study. Tissue distribution analysis revealed a significant expression of GnRH-R in pituitary compared to others tissues. The expression of GnRH-R in brain and pituitary of groupers at different ages showed a significant increased during the fourth year, probably indicating the time of maturation. However, there was no significant difference in the expression of GnRH-R during different seasons. Treatment of two and three year old groupers with different sex steroids revealed an increase in the expression of GnRH-R in pituitary by E2 in both the age groups tested, while T could induce the expression of GnRH-R only in three year old groupers. The result, thus, indicates that the sensitivity of grouper to sex steroid is dependent on the age and the kind of steroid adminstered. In different sections of brain, the GnRH-R expression was in general lower in the group treated with E2 or T compared to the control group. The expression of the gene was more or less the same in two year and three year old control groups. This result may have been caused by suppression of GnRH-R expression in forebrain and midbrain after sex steroid injection. Administration of T induced a significant increase in the expression of GnRH-R in forebrain and midbrain, while E2 treatment did not have a similar effect. In hindbrain, the expression profile GnRH-R was not affected by sex steroid treatment in both two year and three year old groupers. The results suggest that sex steroids can only regulate the expression of GnRH-R in the forebrain and midbrain, probably due to the wide distribution of steroid¡¦s receptor in these regions. LHRH and pimozide injections to two year old groupers showed an increase in the expression of GnRH-R in pituitary after LHRH treatment while there was no stimulatory effect on other sections of the brain. In contrast, treating the fish with pimozide alone or pimozide together with LHRH did not stimulate GnRH-R expression in brain. Thus, the study suggests that LHRH can significantly increase the expression of GnRH-R in pituitary while dopamine has no stimulatory effect. Studies on NPY showed that the gene was distributed in different sections of brain especially in the forebrain but it was also present in gills, liver, intestine ¡K etc. The presence of NPY in gills, though less compared to that in brain, suggests that NPY might play an important role in osmosis regulation. The expression of NPY decreased with increase in age which may be due to the effect of other regulatory factors. Treatment of two and three year old groupers with different sex steroids did not effect the expression of NPY significantly in brain, which is different from other published reports. This may be due to the difference in the zoning of brain regions. In the present study, forebrain and midbrain were sampled together for analyses. The expression of NPY in brain did not change by treating the fish with LHRH or pimozide.

Grouper (Epinephelus coioides) is one of the important farmed fish in the southern Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detecting the presence of pathogen is critical for preventing epidemic outbreak. Interleukin-1 (IL-1) is one of proinflammatory cytokines that form a feedback control loop with anti-inflammatory cytokines to maintain the homeostasis of host immune response. The increase of IL-1 expression could be an indicator of pathogenic insult. In this study, total RNA of Epinephelus coioides fertilized egg was extracted for reverse transcription-polymerase chain reaction (RT-PCR) to amplify cDNA of IL -1£]. The cDNA amplified was then cloned into pGEX4T-3 for the expression and purification of GST-IL-1£] fusion protein. GST-IL-1£] fusion protein purified was then used to immunize New Zealand white rabbit for generation of antiserum against IL-1£]. Western blot result confirmed the specificity of antiserum as the immune serum, but not the preimmune serum, detected the immunogen GST-IL-1s. Further experiments using live Epinephelus coioides injected with or without lipopolysarcharides (LPS) further confirmed that this antiserum could detect a massive increase of IL-1£] protein after the injection of LPS in either protein lysate by western blotting or in frozen tissue section of head kidney by immunohistochemistry. In summary, we successfully generated a rabbit specific antiserum against IL-1£] of Epinephelus coioides , which could be a useful reagent for future analysis of fish immune response upon pathogen infection.

The family Serranidae is a diverse group of fishes, of which the genus Epinephelus (rockcods or groupers) is the largest. Serranids are commonly caught in reef fisheries in tropical and warm-temperate latitudes, and are targeted because of their tasty flesh and high value. In South Africa, epinepheline serranids mainly occur in hook and line fisheries in the province of KwaZulu-Natal. Mostserranids are caught by the boat-based (skiboat) fishery, and the commonestspecies are the endemic catface rockcod (Epinephelus andersoni), thehalfmoon rockcod (E. rivulatus), the yellowbelly rockcod (E. marginatus) and the endemic white-edge rockcod (E. albomarginatus). Although serranids contribute about ten percent to catches by the commercial and recreational skiboat sectors in KwaZulu-Natal, representing an estimated total catch of around 200 mt per year, little is known about these fishes in South Africa. From the mid-1980s to the mid-1990s, the mean lengths of E. marginatus and E. albomarginatus in the region declined significantly. Over this period, lengths of E. andersoni remained the same, while those of E. rivulatus increased. Lengths of E. marginatus and E. albomarginatus from Mozambique, where fishing effort was low at the time of sampling, were significantly greater than in KwaZulu-Natal. Monthly biological data were mostly collected from commercial skiboat catches on the northern and southern coast of KwaZulu-Natal. Additional data for E. marginatus and E. albomarginatus were also collected irregularly from commercial catches made in Mozambique. Unless the fish had ripe ovaries, all gonads had to be sectioned to establish sex and stage. Histology revealed that all gonads had a female-like appearance, with lamellae and a central lumen. In E. andersoni, there was a complete overlap of male and female length frequencies, and their meanlengths were not significantly different. Some males and inactive bisexuals were both smaller and younger than the female size and age at first maturity. Together with the occurrence of mature bisexual fish (transitionals), these observations indicate that males are derived from immature or mature females, hence this species is a diandric protogynous hermaphrodite. The other three species exhibit typical signs of monandric protogynous hermaphroditism. Males and females had significantly different mean lengths, and age and length frequencies by sex werebimodal. Transitional individuals were recorded in E. rivulatus. E. andersoni and E. rivulatus matured at small sizes and early ages relative to E. marginatus and E. albomarginatus. Ripe ovaries were much larger than ripe testes in all four species. E. andersoni, E. marginatus and E. albomarginatus spawned in spring and summer, while E. rivulatus spawned in winter and spring. There were no indications of spawning in E. andersoni in the southern sampling region, and few ripe individuals of E. albomarginatus were encountered in KwaZulu-Natal samples. Size at maturity of this species was much smaller in Mozambique samples. Large, reproductively inactive individuals of E. andersoni were frequently observed in the spawning season. The lack of reproductive activity of E. andersoni and E. albomarginatus in KwaZulu-Natal may be because this area represents thesouthernmost limit of the distribution of these species. Ageing of the four species was undertaken using sectioned otoliths. Age validation was undertaken by a combination of tetracycline marking in captive fishes, and analysis of the marginal zone in otoliths. All four species are relatively long-lived, although estimates of maximum age may be under-estimated because of long-term harvesting. In all four species, fish from the southern sampling region were larger than fish from the northern region at the same age. Only in the case of E. rivulatus were these significant enough to warrant the fitting of two growth curves to the northern and southern populations. Males in all four species tended to be larger than females at the same age, suggesting that there may be a growth spurt following sex change, or that faster-growing females changed sex. A logistic growth curve was fitted to the age-length data for E. andersoni, while von Bertalanffy curves produced the best fit for the other species. Based on the rates at which L∞ attained in these four species, E. marginatus and E. albomarginatus are slow-growing species, while E. andersoni and, particularly, E. rivulatus arefaster growing. Rates of total mortality and natural mortality were estimated using length-converted catch curves and the Rikhter and Efanov equation, respectively. Stock assessments undertaken by yield per recruit and spawner biomass per recruit analyses indicate that E. andersoni in KwaZulu-Natal is currently optimally exploited, while E. rivulatus is lightly exploited. Both E. marginatus and E. albomarginatus are over-exploited. The potential problems in applying standard per recruit models to species with complex life histories are discussed. Support for the reduced stock status of the latter two species is provided by the observed changes in lengths of these species over a ten-year period, and their relatively small size in KwaZulu-Natal compared to the lightly-fished Mozambique populations. Local fishers in KwaZulu-Natal have also reported declines in sizes and reduced catches of these two species.The life history styles and other features of the four species are compared and discussed with reference to the resilience of these species to harvesting. Two of the species (E. marginatus and E. albomarginatus) are monandric protogynous hermaphrodites, which grow slowly, mature late and attain large sizes. E.andersoni and E. rivulatus grow faster, mature earlier and are smaller species. The normally deleterious effects of fishing on sex-changing species are not manifested in these two species, possibly because E. rivulatus is so small, that males are not selectively removed. In contrast, E. andersoni is a diandric protogynous hermaphrodite, and hence, does not rely on sex-change as a source of males. The current management methods for serranids in KwaZulu-Natal are presented, and suggestions for future approaches are discussed. / Thesis (Ph.D.)-University of Natal, Durban, 2000.

With a dramatic decrease of biodiversity as a result of the increase in exploitation of marine ecosystems, the establishment of marine protected areas (MPAs) serves as an important means of protecting those resources. Although there is support for the effectiveness of these MPAs and MPA networks, there is room for improvement in terms of MPA management and design. For example, a better understanding of the dispersal dynamics of targeted species across these MPAs will serve as a more accurate means of reserve as well as fisheries management. While there have been many methods used to determine the larval dispersal of a certain species, parentage analysis is becoming the most robust. In this thesis, I attempt to determine the patterns of self-recruitment and larval dispersal of the Longfin Grouper (Epinephelus quoyanus) in one focal marine reserve within the Great Keppel Island group through the method of parentage. For this, I developed 14 microsatellite markers and with those, genotyped 610 adults as well as 478 juveniles from the study site. These genotypes allowed me to assign offspring to their potential parents, which then allowed me to measure the self-recruitment, local retention as well as larval dispersal percentages of this species from and within the reserve. My results indicate that there is 32% local retention to the reserve while 68% of the assigned juveniles were dispersed to other areas (4% of which dispersed to another reserve). Previous studies conducted in the same area showed higher reserve self-recruitment rates for both Plectropomus maculatus (~30%) and Lutjanus carponotatus (64%) despite their similar life history traits. The results from this study add to the growing evidence that dispersal patterns cannot be generalized across marine systems or even between species within a single system.

by Wong On Nei. / Thesis submitted in: October 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 101-116). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xiii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Lethal and sublethal effects of SS on fish --- p.2 / Chapter 1.2.1 --- Biological effects --- p.2 / Chapter 1.2.2 --- Molecular biomarkers --- p.3 / Chapter 1.2.2.1 --- Acetylcholinesterase activity inhibition assay --- p.4 / Chapter 1.2.2.2 --- Induction of cytochrome P450 mRNA --- p.5 / Chapter 1.2.2.3 --- Induction of metallothionein mRNA --- p.6 / Chapter 1.2.3 --- Study on CYP1A and MT expression / induction --- p.8 / Chapter 1.2.3.1 --- Reverse Transcription (RT) --- p.8 / Chapter 1.2.3.2 --- Polymerase chain reaction (PCR) --- p.8 / Chapter 1.3 --- Objectives --- p.11 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.13 / Chapter 2.1 --- Study sites --- p.13 / Chapter 2.2 --- Sediment --- p.15 / Chapter 2.2.1 --- Sediment samples collection --- p.15 / Chapter 2.2.2 --- Sediment handling --- p.15 / Chapter 2.2.3 --- Sediment dry-wet (w/w) ratio measurement --- p.15 / Chapter 2.2.4 --- Heavy metal content analysis --- p.16 / Chapter 2.2.5 --- Organic content analysis --- p.16 / Chapter 2.3 --- Test organism --- p.17 / Chapter 2.4 --- Bioassays --- p.17 / Chapter 2.4.1 --- 10-day exposure treatments --- p.17 / Chapter 2.4.1.1 --- Experimental setup --- p.17 / Chapter 2.4.1.2 --- Procedure --- p.21 / Chapter 2.4.1.3 --- Tissue sample collection --- p.22 / Chapter 2.4.2 --- 30-day exposure treatments --- p.22 / Chapter 2.4.2.1 --- Behavioural observations --- p.23 / Chapter 2.4.2.2 --- Tissue sample collection --- p.23 / Chapter 2.5 --- Molecular Biomarkers --- p.25 / Chapter 2.5.1 --- Tested samples --- p.25 / Chapter 2.5.2 --- Acetylcholinesterase activity inhibition assay --- p.25 / Chapter 2.5.2.1 --- Acetylcholinesterase activity assay --- p.25 / Chapter 2.5.2.2 --- BioRad Bradford assay --- p.26 / Chapter 2.5.2.3 --- Calculation of specific enzyme activity --- p.26 / Chapter 2.5.3 --- Study on CYP1A and MT expression / induction --- p.27 / Chapter 2.5.3.1 --- Gill and liver tissue samples --- p.27 / Chapter 2.5.3.2 --- Preparation of ribonuclease free reagents and apparatus --- p.27 / Chapter 2.5.3.3 --- Isolation of total RNA --- p.27 / Chapter 2.5.3.4 --- Spectrophotometric analyses of DNA and RNA --- p.28 / Chapter 2.5.3.5 --- First strand cDNA synthesis --- p.28 / Chapter 2.5.3.6 --- Cloning and sequencing of CYP1A and MT gene --- p.29 / Chapter 2.5.3.7 --- RT-PCR co-amplification of CYP1A and 18S rRNA --- p.34 / Chapter 2.5.3.8 --- Real-time RT-PCR --- p.36 / Chapter CHAPTER THREE --- RESULTS --- p.39 / Chapter 3.1 --- Sediment chemistry --- p.39 / Chapter 3.1.1 --- Sediment dry-wet (w/w) ratio --- p.39 / Chapter 3.1.2 --- Heavy metal content of sediments --- p.39 / Chapter 3.1.3 --- Levels of total PCBs and PAHs in sediment --- p.39 / Chapter 3.2 --- Monitoring of test conditions --- p.42 / Chapter 3.3 --- Bioassays --- p.42 / Chapter 3.3.1 --- Survivorship --- p.42 / Chapter 3.3.2 --- Growth --- p.46 / Chapter 3.3.3 --- Feeding rate --- p.51 / Chapter 3.3.4 --- Behaviour --- p.54 / Chapter 3.3.5 --- Sediment clogging --- p.59 / Chapter 3.3.6 --- Body lesions --- p.59 / Chapter 3.3.7 --- Abnormal behaviour --- p.59 / Chapter 3.4 --- Molecular biomarkers --- p.63 / Chapter 3.4.1 --- Acetylcholinesterase activity inhibition assay --- p.63 / Chapter 3.4.2 --- Cloning and sequencing of CYP1A and MT gene --- p.66 / Chapter 3.4.3 --- RT-PCR co-amplification of CYP1A and 18S rRNA --- p.73 / Chapter 3.4.4 --- Real-time RT-PCR --- p.77 / Chapter CHAPTER FOUR --- DISCUSSION --- p.84 / Chapter 4.1 --- Sediment chemistry --- p.84 / Chapter 4.2 --- Biological responses --- p.85 / Chapter 4.3 --- Molecular biomarkers --- p.91 / Chapter 4.3.1 --- Acetylcholinesterase activity inhibition assay --- p.91 / Chapter 4.3.2 --- Cloning and sequencing of CYP1A and MT gene --- p.93 / Chapter 4.3.3 --- RT-PCR co-amplification of CYP1A and 18S rRNA --- p.93 / Chapter 4.3.4 --- Real-time RT-PCR --- p.95 / Chapter 4.4 --- Recommendations --- p.99 / Chapter 4.5 --- Conclusions --- p.100 / REFERENCES --- p.101 / APPENDIX --- p.117

Pak Ah Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 149-158). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xvi / Chapter CHAPTER ONE --- LITERATURE REVIEWS --- p.1 / Chapter 1.1. --- Sediment pollution problems --- p.1 / Chapter 1.2. --- Effects of suspended sediments (SS) on aquatic biota --- p.3 / Chapter 1.3. --- Histopathological biomarkers in fish --- p.7 / Chapter CHAPTER TWO --- INTRODUCTION --- p.20 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.23 / Chapter 3.1. --- Sediments --- p.23 / Chapter 3.1.1. --- Sediment sampling sites --- p.23 / Chapter 3.1.2. --- Sediment collection and handling --- p.25 / Chapter 3.1.3. --- Chemical analysis of sediments --- p.25 / Chapter 3.2. --- Collection and maintenance of fish --- p.26 / Chapter 3.3. --- Sediment bioassays for groupers (E. coioides) --- p.28 / Chapter 3.3.1. --- Preparation of suspended sediments (SS) --- p.28 / Chapter 3.3.2. --- Experimental design --- p.30 / Chapter 3.3.2.1. --- 10-day exposure experiment --- p.30 / Chapter 3.3.2.2. --- 30-day exposure experiment --- p.31 / Chapter 3.3.2.3. --- Time-course and recovery experiment --- p.33 / Chapter 3.3.3. --- Measurement of oxygen consumption and ventilation rates --- p.33 / Chapter 3.4. --- "Tissue sample collection, preparation and examinations" --- p.35 / Chapter 3.4.1. --- Study of sediment clogging --- p.35 / Chapter 3.4.2. --- Scanning electron microscopy (SEM) study --- p.37 / Chapter 3.4.3. --- Histopathological investigations --- p.38 / Chapter 3.4.3.1. --- Histopathology of gills --- p.40 / Chapter 3.4.3.2. --- Histopathology of liver --- p.40 / Chapter 3.4.3.3. --- Histopathology of kidney --- p.41 / Chapter 3.5. --- Sediment bioassays for seabreams (A. schlegeli) --- p.42 / Chapter 3.6. --- Statistical analysis --- p.43 / Chapter CHAPTER FOUR --- RESULTS --- p.44 / Chapter 4.1. --- Chemical analysis of sediments --- p.44 / Chapter 4.2. --- Physicochemical parameters --- p.47 / Chapter 4.3. --- Sediment bioassays for groupers (E. coioides) --- p.49 / Chapter 4.3.1. --- Feeding rate --- p.49 / Chapter 4.3.2. --- Growth rate --- p.49 / Chapter 4.3.3. --- Sediment clogging --- p.53 / Chapter 4.3.4. --- Survival rates --- p.53 / Chapter 4.3.5. --- Oxygen consumption rate and ventilation rate --- p.56 / Chapter 4.3.6. --- SEM study --- p.56 / Chapter 4.3.7. --- Histopathological investigations --- p.64 / Chapter 4.3.7.1. --- Histopathology of gills --- p.64 / Chapter 4.3.7.2. --- Histopathology of liver --- p.82 / Chapter 4.3.7.3. --- Histopathology of kidney --- p.94 / Chapter 4.4. --- Sediment bioassays for seabreams (A. schlegeli) --- p.113 / Chapter 4.4.1. --- Survival rates --- p.113 / Chapter 4.4.2. --- Histopathological investigations of gills and liver --- p.113 / Chapter CHAPTER FIVE --- DISCUSSION --- p.122 / Chapter 5.1. --- Hypoxic effects of SS on histopathology --- p.122 / Chapter 5.2. --- Synergistic effects between SS and chemical --- p.126 / Chapter 5.3. --- Effects of gill impairment on biological responses --- p.131 / Chapter 5.4. --- Reparability of histopathological alterations --- p.135 / Chapter 5.5. --- Species differences in sensitivity to SS --- p.135 / Chapter 5.6 --- Recommendation --- p.136 / Chapter CHAPTER SIX --- CONCLUSION --- p.138 / APPENDICES --- p.140 / REFERENCES --- p.149

by Tse Ching Yee Carol. / Thesis submitted in: September 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 75-90). / Abstracts in English and Chinese. / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgments --- p.vi / Table of contents --- p.vii / List of tables X / List of figures --- p.xii / Chapter 1.0 --- Introduction --- p.1 / Chapter 1.1 --- Sediment pollution in Hong Kong --- p.1 / Chapter 1.2 --- Impact of suspended sediment on fish --- p.2 / Chapter 1.3 --- Biochemical responses to pollution --- p.3 / Chapter 1.3.1 --- Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) --- p.4 / Chapter 1.3.2 --- Creatine kinase (CK) --- p.5 / Chapter 1.3.3 --- Ethoxyresorufin O-deethylase (EROD) --- p.6 / Chapter 1.3.4 --- DNA damage --- p.8 / Chapter 1.4 --- Study of recovery --- p.10 / Chapter 1.5 --- Objectives and significances --- p.11 / Chapter 2.0 --- Materials and Methods --- p.13 / Chapter 2.1 --- Study sites --- p.13 / Chapter 2.2 --- Sediments collection and handling --- p.13 / Chapter 2.3 --- Measurement of heavy metals and organic contents of sediment --- p.15 / Chapter 2.4 --- Exposure tests --- p.16 / Chapter 2.4.1 --- Test organisms --- p.16 / Chapter 2.4.2 --- 10- and 30-day exposure experiments --- p.18 / Chapter 2.4.3 --- 20-day exposure and recovery experiment --- p.19 / Chapter 2.5 --- Biochemical responses --- p.19 / Chapter 2.5.1 --- "Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) activities" --- p.19 / Chapter 2.5.2 --- Ethoxyresorufin O-deethylase activity (EROD) --- p.20 / Chapter 2.5.3 --- DNA damage --- p.21 / Chapter 2.5.4 --- Statistical analysis --- p.22 / Chapter 3.0 --- Results --- p.24 / Chapter 3.1 --- Physical and chemical parameters --- p.24 / Chapter 3.2 --- Pollutants in sediment --- p.24 / Chapter 3.3 --- Mortality --- p.28 / Chapter 3.4 --- Biochemical responses --- p.31 / Chapter 3.4.1 --- 10- and 30-day exposure experiments --- p.31 / Chapter 3.4.2 --- 20-day exposure and recovery experiments --- p.50 / Chapter 4.0 --- Discussion --- p.58 / Chapter 4.1 --- "Sediment pollution at Port Shelter, Mirs Bay and Victoria Harbor" --- p.58 / Chapter 4.2 --- Biochemical responses --- p.59 / Chapter 4.2.1 --- 10- and 30-day exposure experiments --- p.59 / Chapter 4.2.1.1 --- "AST, ALT and CK" --- p.59 / Chapter 4.2.1.2 --- EROD --- p.63 / Chapter 4.2.1.3 --- DNA damage --- p.67 / Chapter 4.2.2 --- 20-day exposure and recovery experiments --- p.69 / Chapter 5.0 --- Recommendations and conclusions --- p.73 / References --- p.75 / Appendix --- p.91

A plasticidade do desenvolvimento gonadal em peixes, que contrasta com os padrões mais estáveis encontrados nos demais vertebrados, deu origem a várias questões intrigantes relativas tanto ao seu significado adaptativo quanto aos fatores genéticos e fisiológicos que modulam o processo. A inversão do sexo em peixes hermafroditas seqüenciais (protândricos ou protogínicos) ocorre em decorrência de diversos fatores, dentre eles, fisiológicos, genéticos e comportamentais podendo ser ainda decorrente do comportamento social. A garoupa verdadeira, Epinephelus marginatus é um peixe teleósteo, hermafrodita protogínico característico do litoral rochoso, maturando primeiramente como fêmeas, e posteriormente, na vida adulta, os ovários são substituídos por testículos, transformando-os em machos reprodutivamente ativos. No presente trabalho foram realizados três experimentos de inversão sexual em condições assistidas, em diferentes estações do ano, utilizando a mesma dose do inibidor de aromatase (IA) letrozole (100μg/Kg) para promover a inversão sexual de E. marginatus visando contribuir e aprimorar o conhecimento dos mecanismos fisiológicos envolvidos nesse processo. O primeiro e o terceiro experimentos foram iniciados na primavera e início da primavera respectivamente e promoveram a inversão sexual com uma diminuição no índice gonadossomático (IGS) nos animais do grupo experimental que apresentaram uma desorganização gonadal peculiar, com lamelas características de ovário, no entanto em seu interior observou-se o compartimento intersticial desenvolvido com cistos de células germinativas masculinas em estágios avançados da espermatogênese, (espermatócitos, espermátides) células de Sertoli e grande quantidade de cistos rompidos com a liberação de espermatozoides no lúmen. Após trinta dias do início do experimento obteve-se um aumento significativo de 11 ceto-testosterona (11KT) e a concentração de estradiol (E2) permaneceu inalterada; com setenta e sete dias após o início do experimento constatou-se uma elevação na concentração de testosterona (T), uma queda nos níveis de 11KT, persistindo a manutenção na concentração de E2. Neste cenário, principalmente as concentrações de andrógenos podem ter ativado a alça de feedback negativo e juntamente com a ativação do sistema da kisspeptina podem ter promovido a ação do GnRH desviando a produção de FSH para LH. Nesse momento o número de cópias obtidas de βFSH na hipófise foi significativamente menor que o número encontrado no grupo controle. Ao observarmos o aumento significativo da concentração plasmática de 17-alfaOHprogesterona podemos sugerir que a ação do GnRH tenha aumentado a secreção do LH, principal gonadotropina que estimula a produção de progestágenos. Os machos invertidos produziram um volume médio de sêmen de 118,20 ± 24,83 μL com motilidade de 90% e densidade espermática de 8,94 ± 4,34 x 109 células mL-1. Não foram obtidas diferenças nas concentrações de proteínas totais plasmática, hepática e musculares. No experimento implantado no verão de 2010 o tratamento não promoveu a inversão sexual, o IGS não apresentou diferença significativa entre os animais tratados com inibidor de aromatase e os animais do grupo controle. Nas gônadas dos animais do grupo experimental foi observada uma ligeira desorganização da arquitetura gonadal com cistos em formação e um número grande de ovócitos em degeneração demonstrando que ao longo do processo de inversão sexual pode haver uma fase de intersexo. Após trinta dias do início do experimento obteve-se um aumento significativo de T e a concentração de E2 permaneceu inalterada. Não foram observadas diferenças significativas entre o número de cópias de βFSH e βLH ao longo do experimento. O resultado obtido para a concentração de proteínas totais do músculo dos animais do grupo experimental apresentou um aumento significativo em relação à concentração encontrada nos animais do grupo controle. Não ocorreu espermiação e a inversão se deu de forma incompleta promovendo indivíduos intersexo. De uma forma geral podemos concluir que o uso de inibidor de aromatase é um método eficaz para promover a inversão sexual em hermafroditas protogínicos como a garoupa verdadeira. O aumento dos andrógenos pode ser o gatilho da inversão sexual com papel fundamental na reestruturação gonadal e formação do tecido germinativo masculino neste processo / The plasticity of gonadal development in fish, which contrasts with the more stable patterns found in other vertebrates, has given rise to several thrilling questions concerning both, its adaptive significance regarding the genetic and physiological factors that modulate the process. The sex inversion in sequential hermaphrodites fish (protandrous or protogynuos) occurs due to several factors, including, physiological, genetic and behavioral, and may also be due to social behavior. The dusky grouper, Epinephelus marginatus is a teleost fish, protogynous hermaphrodite characteristic of rocky bottoms, maturing first as females and later in adult life, the ovaries are replaced by the testes, turning them into reproductively males. In this work three sex inversion experiments were performed in captivity in different seasons of the year, using the same dose of aromatase inhibitor (AI) letrozole (100μg/Kg) to promote sexual inversion in E. marginatus, to contribute and improve the knowledge of the physiological mechanisms involved in this process. The first and third experiments started in the spring and early spring, respectively and promoted sexual inversion with a decrease in the gonadosomatic index (GSI) in the animals from the experimental group, that showed a peculiar disorganized gonadal, with lamellae, characteristic from ovaries, however with an interstitial compartment developed with cysts of male germ cells in advanced stages of spermatogenesis (spermatocytes, spermatids), Sertoli cells and a large amount of cysts ruptured with the release of sperm in the lumen. After thirty days from the beginning of the experiment, there was an increase in 11- ketotestosterone (11KT) levels and an unchanged concentration of estradiol (E2); after seventy-seven days, there was a significant increase in testosterone (T) levels, a decrease in the 11KT levels and the maintenance of E2 plasma concentration. In this point of view, mainly the concentration of androgens may have activated the negative feedback loop and with the activation of the kisspeptin system may have promoted the action of GnRH diverting the production of FSH to LH. At this moment the number of copies of pituitary βFSH produced was ignificantly lower than the number of control group copies. The significant increase in plasma 17- alfaOHprogesterona concentration observed could suggest that the action of GnRH has increased the secretion of LH, the main gonadotropin that stimulates the production of progestogen. Inverted males produced an average volume of semen of 118.20 ± 24.83 μl with 90% motility and sperm density of 8.94 ± 4.34 x 109 cells.ml-1. No differences were obtained at the concentrations of total protein in plasma, liver and muscle. In the experiment established in the summer of 2010, the treatment did not promote sexual inversion, the GSI was not significantly different between animals treated with aromatase inhibitor and the control group. In the gonads of the animals from the experimental group there was a slight disruption of gonadal architecture with cysts in formation and a large number of degenerating oocytes showing that during the process of sex inversion may be a phase of intersex. Thirty days after the beginning of the experiment there was a significant increase of T and E2 concentration remained unchanged. No significant differences were observed between the number of copies of βFSH and βLH throughout this experiment. The result obtained for the total protein concentration of the muscle in experimental group showed a significant increase compared with the control animals. There was no spermiation and the sex inversion occurred incompletely, with the presence of intersex individuals. In general we could conclude that the use of an aromatase inhibitor is an effective method to promote the sex inversion in hermaphroditic protogynous as the groupers. The increase of androgens could trigger the sex inversion exerting an important role in gonadal restructuring and the development of male germ tissue in the process

A inversão do sexo em peixes hermafroditas sequenciais (protândricos ou protogínicos) ocorre em decorrência de diversos fatores, dentre eles, fisiológicos, genéticos, podendo ser ainda decorrência do comportamento social. Nos peixes hermafroditas marinhos as alterações sexuais são acompanhadas por alterações anátomo-funcionais das gônadas, coloração e comportamento dos animais. A espécie Epinephelus marginatus (garoupa verdadeira), serranídeo da fauna nativa e amplamente difundida no litoral brasileiro, é um hermafrodita protogínico que apresenta dificuldade na manutenção de indivíduos do sexo masculino em seus cardumes e, além disso, encontra-se na lista de espécies sobreexplotadas. Utilizando-se técnicas de inversão sexual induzida, em cativeiro, para a obtenção de machos, este trabalho teve como objetivo estudar as alterações de alguns tecidos endócrinos durante o processo de inversão sexual, utilizando tratamentos com inibidores de aromatase (AI) e metiltestosterona (MT) em animais jovens. Ao longo de 90 dias de experimento a utilização do AI e do MT promoveu uma aparente desorganização da arquitetura gonadal padrão, massiva degeneração das células germinativas femininas, surgimento de centros melanomacrofágicos, proliferação de estruturas associadas ao sexo masculino (tecido intersticial e células germinativas masculinas) e a maturação das células germinativas masculinas. A utilização do andrógeno sintético MT (sozinho ou combinado com AI) possibilitou a obtenção de gônadas em fases mais avançadas de espermatogênese, inclusive a fase de espermiogênese inicial, com redução significativa dos valores do índice gonadossomático (IGS). Já com a utilização do AI foram encontradas gônadas em processos de intersexo mais clássicos após 90 dias experimentais. As análises de imunomarcação das células gonadotrópicas hipofisárias foram viáveis utilizando-se anticorpos de salmão e os resultados mostraram que as células produtoras de FSH (hormônio folículo-estimulante) e LH (hormônio luteinizante) distribuem-se pela proximal pars distalis e pars intermedia da adenohipófise. Os resultados obtidos com as análises imunohistoquímicas da hipófise demonstraram uma marcação de pouca intensidade utilizando-se o anticorpo anti-βFSH no grupo tratado com AI após 90 dias. A análise dos esteróides gonadais mostrou que nos animais tratados com AI, não houve diminuição na concentração do estradiol aos 90 dias, desta forma a alça de feedback negativo provavelmente foi acionada, e a produção de FSH diminuiu, causando o aumento da concentração plasmática de 11 cetotestosterona (11 KT) (via ação do LH). Já nos grupos onde o MT foi administrado, houve uma diminuição na concentração de estradiol após o tratamento, causando possivelmente uma menor intensidade na alça de feedback negativo na hipófise, sendo a ação do andrógeno sintético mais efetiva na espermiogênese nestes animais. As análises das gônadas e da hipófise sugerem a atuação do FSH no início da transição do sexo, sendo proposta a liberação desta glicoproteína como o gatilho para a inversão sexual em hermafroditas protogínicos. Estudos mais detalhados ao longo dos 90 dias de transição gonadal em E. marginatus precisam ser realizados para constatar o real envolvimento do FSH na inversão do sexo e verificar se a liberação do LH estaria envolvida com a produção de 11-ceto-testosterona, a espermiogênese completa e espermiação / The sex inversion in sequential hermaphrodite fish (protandrous or protogynous) occurs due to several factors, including physiological, genetics, and also can be associated with the social behavior. In marine hermaphrodite fish, the sex alterations are followed by anatomic and functional alterations in the gonads, color patterns and animal behavior. The species Epinephelus marginatus (dusky grouper), a native serranid and broadly distributed in the Brazilian coast, is a protogynous with problems in maintaining males in the shoal and, additionally, this species is part of the overexploited marine fish red list. Induction of sex change, in captivity, using aromatase inhibitors (AI) and 17α-methyltestosterone (MT) has been conducted with the purpose to study the morphophysiological alteration in some endocrine tissues in juveniles. During 90 experimental days, the use of AI and MT promoted an apparent disorganization of the standard gonad architecture, a massive degeneration of the female germ cells, the appearance of melanomacrophagic centers, a proliferation of the structures associated with the male sex (interstitial tissue and male germ cells) and the maturation of male germ cells. The use of the synthetic androgen, MT (alone or combined with AI) allowed the obtaining of gonads in a more advanced spermatogenesis phase, including an initial spermiogenesis phase, with a significant reduction of gonadossomatic index (GSI). With the use of AI, the gonads were found in a more classic intersex process after 90 days of experiment. The immunostaining analysis of the gonadotropic cells were viable using salmon antibodies and the results showed that FSH and LH cells were distributed in proximal pars distalis and pars intermedia in adenohypophysis. The immunohistochemistry analysis of the pituitary showed a low intensity of staining with the anti-βFSH in the AI group, after 90 experimental days. The analysis of the gonad steroids showed that in the animals from the AI group, there was no decrease in estradiol levels after 90 days, so the negative feedback loop probably was activated, the FSH secretion decreased, allowing the increase of plasma 11- ketotestosterone (11KT) (through LH action). In the MT groups there was a decrease in plasma estradiol, what probably decreased the intensity of the negative feedback loop in the pituitary, and in this case, the action of the synthetic androgen was predominant in spermiogenesis. The analysis of the gonads and pituitary suggest the action of FSH in the beginning of the sex transition, with the proposal that this glycoprotein acts triggering the sex inversion in protogynous hermaphrodites. We suggest that more detailed studies during the 90 days of gonad transition in E. marginatus must be done in order to find the real involvement of FSH in sex inversion and to elucidate the actual role of LH with 11KT production, the full spermiogenesis and spermiation