Advances in aquaculture technologies are being investigated to support the replenishment of local fisheries, develop marine food fish farming opportunities and to increase seafood production globally. In order to promote the expansion and development of aquaculture technologies required to raise new finfish species, a number of key bottlenecks restricting commercial-scale culture need to be addressed, including the ability to control fish reproduction in captivity and to produce high quality seeds. One candidate species for large-scale production, and the focus of this work, is common snook. Prized as a food fish in Mexico, Central and South America and as a popular game fish along the Gulf coast of the United States; common snook are economically important having both a high market value and recreational demand. Despite recent advances in captive spawning, a number of reproductive bottlenecks still need to be addressed such as lack of spontaneous spawning in captivity, poor fertilization rates and inconsistent production of high quality eggs and larvae. Therefore, the overall aim of this thesis was to better understand the reproductive biology of common snook in order to develop protocols to improve the reliability of captive spawning in closed recirculating aquaculture systems and the quality of eggs produced as a basis for commercial scale cultivation. First, this PhD project described oocyte development in common snook and validated a non-invasive method for assessing reproductive condition in wild and captive stocks (Chapter 2). This was done by using a tiered and adaptable staging scheme to compare the wet mount technique with histological preparations of ovarian biopsies. When compared with histology, the wet mount provided an immediate and precise method for determining whether female broodstock were candidates for hormonal induction. In fishery biology, an understanding of fish reproductive success and population reproductive potential is critical for designing and implementing effective fisheries management strategies. The wet mount technique provides a tool for non-lethal, low-cost determination of reproductive status in wild fish stocks. The next research chapter focused on spawning induction of captive snook populations. The first trial compared the effects of slow and regular release GnRHa implants whereas the second trial investigated the effects of GnRHa, alone or in combination with the dopamine antagonist, pimozide (PIM), on milt characteristics and plasma steroid levels in captive male common snook broodstock (Chapter 3). In an effort to better enable reliable control of reproduction under captive conditions, the annual plasma sex steroid profile of captive male and female broodstock maintained under natural photo-thermal conditions was also examined. When possible, milt samples were collected pre and post implantation; sperm density, sperm motility and spermatocrit were documented among individual males. The assigned treatments appeared to have no or little effects on milt production in male broodstock although plasma steroid levels were found to be significantly elevated in individuals treated with GnRHa in combination with the dopamine antagonist, pimozide. At the time this work was performed, no data on spawning dynamics, including individual spawning performance, had been reported for common snook in captivity. Mass spawning tanks are complex systems where fish are left to spawn naturally and fertilized eggs are collected with little or no control over the mating of the animals. Therefore, the third part of this thesis explored the potential of DNA profiling for monitoring mating outcomes in captive broodstock by employing eight microsatellite markers to detect and quantify individual parental contributions for 2,154 larvae obtained from the three broodstock tanks (Chapter 4). The panel of loci was generally robust and allowed unambiguous assignment of 89% of larvae to a single family. Overall, spawn contribution data 1) provided a confirmation of GnRHa treatment efficacy in female snook with a minimum stage of oogenesis (late secondary growth-SGl) required for successful spawning, 2) identified a potential impact of handling on maturation and spawning of captive broodstock and 3) confirmed that, through photothermal conditioning, captive broodstock can spawn over consecutive days and several times per year including outside of their natural spawning season. The exogenous cues that tropical species use to synchronize key life events like reproduction remain largely unstudied, therefore, my PhD project also investigated the influence of tidal cycle on reproductive activity in common snook (Chapter 5). Real-time quantitative RT-PCR assays were developed and validated to measure the temporal expression patterns of gonadotropin genes (fshβ and lhβ) during the reproductive cycle in males and females. These were evaluated in relation to sex steroid production, LH blood plasma levels, gonadal development and tidal cycle. The phylogenetic analysis of the deduced amino acid sequence of common snook for fshβ and lhβ revealed strong identity with other teleosts (75-90%). Additionally, the mRNA profiles of fshβ and lhβ in the pituitary of females displayed a clear pattern of expression concomitant with histological changes in oocyte development. Histological observations of gonads suggested a circa-tidal rhythm of follicular development. The findings, as a whole, provided new information supporting the role of tidal cycle on the entrainment of gametogenesis allowing for a better understanding of the environmental control of reproduction in common snook. Although the primary research emphasis in this PhD was on broodstock spawning and gamete quality, the final chapter focuses on larval ontogeny. The goal of this research was to gain improve understanding of the early life history characteristics of common snook in order to improve larval culture technologies. To do so, a combination of digital photography and histological techniques were used to document the embryonic and early larval development (0 to 14 days post hatch-DPH) of hatchery-reared individuals (Chapter 6). Larvae hatched 15 h after fertilization at 28°C, lacked pigmentation, had a rudimentary digestive tract and undeveloped visual system. Development was rapid and by 3 DPH larvae had almost doubled in length, the yolk sac was nearly exhausted, the mouth was open and eyes were pigmented with a well-structured retinal layer. The alimentary canal was differentiated into three distinct sections including the foregut, midgut and hindgut. Food was observed in the gut (rotifers) and structural epithelium organelles, such as the nucleus, mitochondria, and dark vesicles, were all present in high numbers. The swim bladder was formed and inflated. In summary, understanding early ontogenetic development in common snook can help provide information needed to address key bottlenecks seen in captive cultivation, such as the high incidence of larval mortality observed during the transition from endogenous to exogenous feeding. Overall, this doctoral work 1) validated molecular and endocrine analytical tools for future studies of common snook reproductive physiology, 2) provided a better understanding of both broodfish requirements in tank systems as well as the endocrine control of reproduction and spawning at the level of the brain-pituitary-gonadal axis, 3) increased our knowledge in genetic management of captive broodstock, in terms of parentage assignment and 4) offered new insight into wild population reproductive strategy as well as how reproduction is entrained through environmental cues and the pathways leading to oocyte recruitment and maturation. The new information presented here can be used to conserve wild snook stocks through production of farm raised individuals as a sustainable source of seafood and for fisheries enhancement.

Submitted by VITO ANTONIO MASTROCHIRICO FILHO null (vito.oceano@gmail.com) on 2016-04-09T21:47:55Z No. of bitstreams: 1 Dissertação Repositório Vito.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-11T13:38:46Z (GMT) No. of bitstreams: 1 mastrochiricofilho_va_me_jabo.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) / Made available in DSpace on 2016-04-11T13:38:46Z (GMT). No. of bitstreams: 1 mastrochiricofilho_va_me_jabo.pdf: 3114594 bytes, checksum: fe4a528d166d3a59bfac7d3a244a016c (MD5) Previous issue date: 2016-02-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Pacu (Piaractus mesopotamicus) é uma espécie de peixe Neotropical amplamente distribuída nas bacias dos rios Paraná e Paraguai, e uma das espécies de peixe neotropicais de maior valor para a aquicultura. Uma melhor compreensão do genoma do pacu é necessária para o manejo genético na conservação dos estoques naturais e cultivados. O principal objetivo foi identificar SNPs (Single Nucleotide Polymorphisms) gene-associados no transcriptoma de fígado do pacu e, em seguida, aplicar em análises de variabilidade genética e de parentesco visando um manejo adequado desta importante espécie não modelo na aquicultura. O sequenciamento do transcriptoma foi realizado por meio da plataforma Roche/454, que resultou na formação de 4.110 contigs não redundantes. Destes, 2.051 genes foram identificados e funcionalmente anotados a fim de revelar genes relacionados às características econômicas interessantes para a aquicultura. Foram encontrados 464 SNPs localizados em 5’UTR (10.0%), 3’UTR (17.2%) e em regiões CDS (71,1%), e classificados como sinônimos (70,6%) e não sinônimos (29,4%). Foram genotipados 32 SNPs por meio da técnica Sequenom MassARRAY, dos quais alguns estavam relacionados com sistema imune. A variabilidade genética foi estimada em populações de indivíduos selvagens (Rio Paraná) e de indivíduos cultivados em sete pisciculturas do estado de São Paulo (FF1, FF2, FF3, FF4, FF5, FF6 e FF7). Não foram observadas diferenças significativas entre heterozigosidade observada (Hobs) e esperada (Hexp) para cada população. Análises de diferenciação genética mostraram baixo nível de estruturação genética entre as populações (Fst = 0.064, AMOVA = 93,59% da variação dentro de populações, P<0,05). Análises de parentesco mostraram que a maioria das estações de piscicultura possuíam pelo menos 40% de indivíduos aparentados, com risco de endogamia e necessidade de realização de um programa de acasalamentos direcionados. Nossos resultados proporcionaram importantes recursos genéticos para o pacu, com aplicabilidade para a aquicultura. / Pacu (Piaractus mesopotamicus) is a Neotropical freshwater fish widely distributed in Parana, Paraguay Basin. Wild populations of pacu are threatened by overfishing and it is one of the fish species of highest commercial value for aquaculture. An understanding of the pacu genome is appropriate to genetic management in the conservation of wild and cultivated stocks. The main objective was identify gene-associated SNPs in liver transcriptome of pacu. We used SNPs (Single Nucleotide Polymorphisms) to perform genetic variability and kinship analysis for suitable management of this important non-model species in aquaculture. Transcriptome sequencing was done with the Roche/454 technology and yielded 4,110 non-redundant contigs. Of these, 2,051 genes were identified and functionally annotated to reveal genes correlated to economical traits in aquaculture. We found 464 SNPs in 5’UTR (10.0%), 3’UTR (17.2%) and CDS (71,1%), classified in synonymous (70,6%) and non-synonymous (29,4%). We genotyped 32 feasible SNPs through Sequenom MassARRAY platform and we obtained some SNPs related to immune system. Genetic diversity was estimated in wild individuals (Parana river) and in seven farm fish populations (FF1, FF2, FF3, FF4, FF5, FF6 and FF7). There were no significant differences between observed heterozygosity (Hobs) and expected (Hexp) for each population; and also between observed heterozygosity, expected heterozygosity and minimum allele frequency (MAF), when the population averages were compared (P <0.05). In addition, genetic differentiation analyzes showed low genetic structure of wild and cultivated populations of pacu (Fst = 0.064; AMOVA = 93.59% of the variation within populations; P<0,05). Kinship analysis showed most hatchery stations had at least 40% of related individuals, at risk of inbreeding and the need to perform a directed mating program. Our results showed unprecedented genomic resources for pacu. / FAPESP: 2014/12412-4