Production of all-male tilapia for aquaculture is assuming an increasingly important role. An important pre-requisite to repeated obtainment of monosex tilapia is a clear understanding of the mechanisms underlying sex differentiation. Histological observations on gonadal morphorgonesis and sex differentiation provided basic data for hormonal sex manipulation in four commercially important species. Results indicate that gonadal morphogenesis starts at different times ranging from eight days after hatching in 0. mossambicus to 17 days in 0. niloticus. Sex differentiation followed a similar pattern, and ranged from 22 days in O. mossambicus to 36 days in 0. niloticus. The effects of subjecting fish to different rearing temperatures was assessed. No significant influence was found on sex ratio of treated fish. Observations on offspring sex ratio in intraspecific breeding and interspecific hybridization demonstrated that significant differences between batches are a common occurrance and their regularity cannot be adequately explained on the basis of sex chromosome theory alone. Treating fish with synthetic androgen (17 alpha methyltestosterone) and synthetic oestrogen, (17 alpha ethenylestradiol) resulted in species specific and dosage dependant differences in sex ratios. Results also revealed significant differences in sex ratios of different batches of fish subjected to the same treatment, thus demonstrating that success rate in sex inversion varies not only between species and between stocks, but in sib groups as well. Results of intraspecific and interspecific breeding suggest that sex determination in tilapia is under the influence of multiple factors. Results of hormone treatments indicate variations in inversion rate at batch level, thus demonstrating presence of individual differences in lability. On the basis of results from these four experiments, it is hypothesized that sex in tilapia is influenced by multiple genes and the fishes' propencity to change sex varies in individual fish. Progeny testing oestrogen sex inversed fish indicates that on the basis of the chromosome theory of sex determination, S. galileaus and O. niloticus are female homogametic, while O, macrochir is female heterogametic. The implications of the results obtained in this study for production of all-male tilapia are briefly discussed.

Made available in DSpace on 2015-08-20T17:09:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-20. Added 1 bitstream(s) on 2015-08-20T17:26:34Z : No. of bitstreams: 1 000841299.pdf: 1117680 bytes, checksum: 82417d01cae798e91cfee1b8826da5bf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O mapeamento cromossômico de sítios de DNA repetitivo vem sendo realizado em vários estudos citogenéticos em peixes e tem permitido uma melhor caracterização da biodiversidade e evolução genômica da ictiofauna Neotropical. Dentre as diversas espécies de peixes distribuídas nesta importante área geográfica, podemos destacar o gênero Moenkhausia, um dos mais especiosos grupos de peixes da família Characidae. Do ponto de vista citogenético, este gênero demonstra variação do número diploide de 48 a 50 cromossomos, bem como a presença de microcromossomos B descritos para diferentes espécies/populações. No entanto, estudos citogenéticos de espécies de Moenkhausia coletadas em riachos de cabeceira da bacia Amazônica e bacia do Alto rio Paraguai ainda são incipientes, bem como os estudos relativos à distribuição de DNAs repetitivos nos genomas de diferentes espécies de Moenkhausia. Neste sentido, o presente trabalho teve como objetivo a caracterização cariotípica de Moenkhausia cosmops, M. cf. forestii, M. cf. nigromarginata, M. oligolepis e Moenkhausia sp. coletadas em diferentes rios e riachos de cabeceira da bacia Amazônica e bacia do Alto rio Paraguai. Todas as espécies foram analisadas através de técnicas citogenéticas clássicas (coloração com Giemsa, localização das RONs pela impregnação por Nitrato de Prata e bandamento C) e molecular (hibridação in situ fluorescente com sondas de DNAr 5S e 18S, histona H1, snDNA U2 e sequências teloméricas (TTAGGG)n). Todas as espécies/populações analisadas apresentaram 2n=50 cromossomos, porém com fórmulas cariotípicas distintas entre elas. O bandamento C revelou um padrão de bandas heterocromáticas similar entre essas espécies, exceto em M. cf. nigromarginata. A impregnação por Nitrato de Prata evidenciou Ag-RONs simples em todas as espécies analisadas. A FISH utilizando o DNAr 18S como sonda confirmou os resultados obtidos com o Nitrato de Prata e... / The cytogenetic mapping of repetitive DNA has been applied in several studies in fish, which allowed a better characterization about the biodiversity and genomic evolution of Neotropical ichthyofauna. Among the several fish species living in this area, the genus Moenkhausia, one of the specious groups inside Characidae, is remarkable. From a cytogenetic point of view, this genus show variations in diploid numbers of 48 to 50 chromosomes, as well as the occurrence of B chromosomes in different species/populations. However, cytogenetic studies in Moenkhausia collected at different rivers from the Amazon and Upper Paraguay River basins are scarce, as well as studies investigating the chromosomal distribution of repetitive DNAs in different species. In this sense, the present study aimed to characterize the karyotypes of Moenkhausia cosmops, M. cf. forestii, M. cf. nigromarginata, M. oligolepis and Moenkhausia sp. collected at different sites in the Amazon and Upper Paraguay River basins. All species were analyzed using classical (conventional staining with Giemsa, localization of NORs by impregnation with Silver nitrate and C-banding) and molecular (fluorescent in situ hybridization with 5S and 18S rDNA, H1 histone, U2 snDNA and telomeric sequences (TTAGGG)n probes). All the herein analyzed species showed 2n=50 chromosomes, with different karyotype formulas. C-banding evidenced similar patterns of heterochromatin distribution in all species, except in M. cf. nigromarginata. Silver nitrate staining evidenced simple Ag-NORs in all species and FISH with 18S rDNA probes confirmed these results e revealed additional sites in M. oligolepis. The 5S rDNA was interstitially located in multiple sites in all species. Notably, both ribosomal sites were found in synteny in one population of M. oligolepis. The H1 histone sites were co-located in a single pair with 18S rDNA and the U2 snDNA was located at multiple sites in all species. Additionally, the ...

Carp aquaculture in South Asia suffers severely from a lack of genetic management, which has eroded the genetic quality of both captive and wild populations. Use of molecular markers, especially microsatellites, has revolutionized genetic management of hatchery stocks through its ability to detect kinship between individuals and hence in controlling level of inbreeding and loss of genetic diversity. In the present PhD work, microsatellite markers were applied to breeding programmes for silver carp (Hypophthalmichthys molitrix) and common carp (Cyprinus carpio) to study different genetic management aspects and new markers were generated from rohu (Labeo rohita). A set of newly isolated microsatellite markers from silver carp were characterized and two pentaplex PCR reactions were optimized to enable rapid genotyping of large number of individuals at 10 microsatellite loci. The utility of these markers in parentage, sibship and relatedness analysis were assessed by applying them to groups of fish with known relationship. These markers were used for parentage analysis in a breeding programme designed to estimate heritability of harvest weight and length in silver carp. Full- and half-sib families were created in three sets of partly factorial mating and all the families from each set were reared in communal ponds from very early life stages. With ten microsatellites 96.3% of the offspring could be assigned to a single family. Heritability estimates were found to be 0.65 ± 0.13 for weight and 0.50 ± 0.13 for length. High estimates of h2 suggested that this population should respond rapidly to selection for increased harvest size. Microsatellite markers were also applied to monitor the early stages of a mass selection programme in common carp (Cyprinus carpio). The selection was initiated from a base population synthesized from six different stocks. The selected individuals were divided to create two separate lines. The aims of this study were to monitor whether the stocks were represented in the intended proportions in the F1 selected populations, to investigate the relative contribution of families and its impact on effective population size and to identify any loss of molecular genetic variation. Five highly polymorphic microsatellites were used for parentage analysis of the selected fish to track stock and family contribution. Overall, large perturbations were observed in the relative contributions of two major stocks. Family contribution was also highly variable, causing the Ne to drop to below half the census size. A loss of 6.9%-12.2% of microsatellite alleles was observed but loss of heterozygosity was not very prominent. The replicate lines showed significant differences in allelic distribution after the first generation of selection, but not in genotypic distribution. Finally, 52 microsatellite markers were isolated from a partial genomic library of rohu using a selective hybridization protocol. Characterization of these markers resulted in 36 polymorphic loci, which will be useful in future work on conservation and management of both wild and captive rohu populations.

Electrophoretic variation at twenty gene loci, patterns of behavioral thermoregulation, and genotype-specific malate dehydrogenase kinetics were investigated among populations of the red shiner, Notropis lutrensis, and the blacktail shiner, N. venustus, collected from thermally altered and thermally unaltered portions of their ranges. Genic variation was found to be high among red shiners and low among blacktail shiners. The behavioral response of the blacktail shiner to temperature was fixed among the populations sampled, whereas the response of the red shiner was mutable. Finally, blacktail shiners have incorporated into their genome an Mdh-B allele which functions well at low temperatures; red shiners, displaying high levels of Mdh-B polymorphism, maintain a more complex set of allozymes which function well over a wide range of environmental temperatures. These data are consistent with reported ecotypic distributions of the species in Texas waters; i.e., blacktail shiners occur in cool, thermally static habitats, and red shiners are tolerant of wide temperature ranges.

Orientador: Gilson Luiz Volpato / Banca: Eliane Gonçalves-de-Freitas / Banca: Mário Luis Orsi / Banca: Leonardo José Gil Barcellos / Banca: Percília Caedoso Giaquinto / Resumo: Atualmente, a função das depressões que machos de muitas espécies de ciclídeos escavam no substrato é para atração de fêmeas para a reprodução. Assim, tais depressões têm sido consideradas um fenótipo estendido dos machos, ou seja, representam um caracter a mais, pelo qual a fêmea pode escolher o melhor macho. Assim, é esperado que somente os machos escavem e, sobretudo, em contextos reprodutivos. No entanto, em nosso estudo 1 vimos que tanto machos quanto fêmeas sexualmente imaturos já escavam essas depressões e que esse comportamento surge também na ausência da interação com coespecíficos e num período restrito da ontogenia. Isso ocorreu tanto para a domesticada tilápia-do-Nilo (Oreochromis niloticus) quanto para o selvagem cará (Geophagus brasiliensis). Isso sugere que o comportamento de escavar tenha uma forte determinação genética e outra função biológica relevante além da reprodução. No estudo 2, investigamos na tilápía-do-Nilo se essa outra função poderia ser a busca por alimento, uma vez que os padrões motores na escavação e no forrageamento do substrato são semelhantes. Contudo, vimos que a resposta de peixes com fome não satisfez as predições dessa hipótese. A fome do peixe não influenciou a frequência com que machos e fêmeas escavaram e nem o tamanho de suas escavações. Finalmente, no estudo 3 mostramos que as escavações de machos e fêmeas servem como refúgio, pois suas características permitem que os peixes se escondam dentro delas, a presença de uma toca no aquário reduz drasticamente a frequência de escavação, as escavações são posicionadas mais distantes de áreas abertas (perigo) e os peixes ficam dentro das escavações durante período de inatividade noturna. Isso indica que as escavações podem ter função de abrigo para evitar perigos potenciais / Abstract: Not available / Doutor

Orientador: Fausto Foresti / Banca:Diogo Teruo Hashimoto / Banca: Renato Hajenius Ache de Freitas / Banca: Maria Rita de Cássia Barretto Neto / Banca: Vanessa Paes da Cruz / Resumo: Tubarões e raias estão atualmente entre os vertebrados mais ameaçados de extinção. Tal fato se deve principalmente à exploração pesqueira que permanece sem medidas eficientes de controle em quase todo o planeta, inclusive no Brasil. Considerando-se que cada espécie responde de forma independente às pressões ambientais e principalmente às ações humanas, é de fundamental importância para o desenvolvimento de planos de manejo adequado das suas populações e o ordenamento da exploração sustentável destes recursos que se proceda à caracterização da biodiversidade local, a avaliação das espécies mais capturadas e identificação dos táxons e populações mais suscetíveis à pesca. Contudo, especialmente para os elasmobrânquios, tais parâmetros são de difícil acesso, sobretudo devido às similaridades morfológicas entre as espécies e ainda, devido à prática pesqueira de remoção de partes dos animais como a cabeça e nadadeiras antes dos desembarques, inviabilizando a identificação da maioria das espécies. Segundo os registros de captura de elasmobrânquios elaborados pelo IBAMA nos últimos anos, apenas cerca de 20% dos indivíduos recebem alguma menção quanto à espécie, muitas vezes um nome popular que pode estar relacionado a mais de uma espécie biológica em diversos casos, sendo o restante do produto classificado apenas como cações. A utilização de ferramentas moleculares nos estudos da composição da biodiversidade tem proporcionado sua aplicação em diversas áreas, incluindo na identificação das espécies, no estudo de suas relações evolutivas e no auxílio de programas para a sua conservação. A identificação molecular com a utilização de técnicas do tipo PCR-multiplex e sequências barcode, que identificam características genéticas particulares de cada táxon estão atualmente sendo desenvolvidos e utilizados no reconhecimento das espécies, possibilitando a identificação... / Abstract: Sharks and rays are currently among the most threatened vertebrate to extinction. This is due mainly to the fact that overfishing remains without effective control measures in almost the entire planet, including Brazil. Considering that each species responds independently to environmental pressures, and mainly to human actions, it is of fundamental importance for the development of appropriate management plans and programming the sustainable exploitation of these resources, to proceed to the characterization of local biodiversity, in the assessment of the species caught, in the correct identification of taxa and the most susceptible populations to fishing activities. However, especially for elasmobranches, such parameters are difficult to access, especially due to morphological similarities between species and also because of the fishing practice of removing parts such as the head and fins before landings, making it impossible to identify the most species. According elasmobranches capture records prepared by IBAMA in recent years, only about 20% of individuals receiving some identification indication, often only a popular name that can be related to more than one biological species, with the remainder fishing product classified just as sharks. The use of molecular tools in studies of the composition of biodiversity has provided its application in various areas, including in the identification of the species, in the study of their evolutionary relationships, and in assisting programs for their conservation. The use of molecular identification techniques as the PCR-multiplex type, and barcode sequences using part of the mitochondrial gene sequence Cytochrome Oxidase subunit I that permit to identify particular genetic characteristics of each taxon, are currently being used in the species recognition allowing simultaneous identification of different samples. In the present work, the objective of the study was to identify the species occurring... / Doutor

Bull trout (Salvelinus confluentus) populations from Alberta river drainage systems were compared using molecular techniques. Restriction fragment length polymorphisms (RFLP's) within the NDI and ND5/6 regions of the mitochondrial genome were observed. In addition, randomly amplified polymorphic DNA profiles (RAPD's) from total genomic DNA extracts were compared. Interdrainage comparisons using mtDNA revealed significant population heterogeneity among Alberta bull trout. Percent sequence divergence in mtDNA ranged from 0.14% to 0.92%. Most fish in each population were composed of a small number of common haplotypes, and the remaining fish displayed rare or locally unique haplotypes. RAPD profiles were used to calculate genetic distance values for Alberta, Canada and Montana, U.S.A. populations. Both Nei and Cavalli-Sforza distance values were used to generate neighbor-joining, FITCH and KITSCH distance trees. Two genetically distinct groups of bull trout were revealed by the RAPD analysis and the possiblity that post-glacial bull trout populations are derived from two separate refugia is suggested. / xvii, 161 leaves : ill. ; 28 cm.

Cell culture systems have provided many insights into eukaryotic gene expression and other biochemical mechanisms. Since the cell represents the smallest living unit of any organism it provides a desirable in vitro system, allowing biochemical studies without the complex physiology of an entire animal. However, processes involving intracellular mechanisms, such as development, aging or carcinogenis, eventually require the analysis of the intact organism. Transgenic animals are a very promising tool to approach questions of this magnitude. Fish in general and the zebrafish (Brachydanio rerio) in particular are an excellent model system for transgenic research, mainly due to their extramaternal fertilization and development and their short generation cycle throughout the year. The recent derivation of zebrafish cell lines has opened up possibilities for in vitro analysis of this popular model species, and expression of heterologous genes under the influence of promoter and other regulatory nucleic acid aequences. In contrast to mammalian expression systems, little nucleic acid sequences controlling gene expression in fish are known. Therefore we examined mammalian expression systems in fish cells in order to determine their efficiency quantitatively. Emphasis was given to zebrafish cultures with the goal of eventually injecting in vitro manipulated embryo cells into host embryos and thereby creating transgenic chimera. / Graduation date: 1993

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-11-23Bitstream added on 2014-06-13T19:54:00Z : No. of bitstreams: 1 nachtigall_pg_me_botib.pdf: 1512670 bytes, checksum: c79b570dced4bdd72d93ec49ab217896 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / MicroRNAs são pequenas moléculas de RNA que regulam pós-transcricionalmente a expressão gênica em diversas vias celulares específicas. Recentemente, alguns miRNAs de ação músculo-específica, especialmente aqueles expressos no músculo estriado esquelético, têm sido associados à regulação da biologia muscular, com papel fundamental no controle de eventos como miogênese e crescimento muscular hipertrófico e hiperplásico, além de constituírem vias metabólicas extremamente conservadas entre os vertebrados. A tilápia do Nilo é considerada um excelente modelo biológico para o estudo de miRNAs em vertebrados devido à sua importância econômica e por apresentar o genoma completo sequenciado. Contudo, pouco é conhecido a respeito da dinâmica evolutiva de miRNAs e sua potencial atuação na regulação dos mecanismos moleculares promotores do desenvolvimento do tecido muscular na espécie. Assim, foram realizados dois estudos direcionados para a (i) avaliação do perfil de expressão de miRNAs músculo-específicos e seus alvos no tecido muscular de adultos da tilápia do Nilo, e para (ii) analisar o padrão de organização genômica desses miRNAs em diferentes espécies de peixes e estabelecer comparações com outros grupos de vertebrados. Os principais resultados obtidos na análise do perfil de expressão no tecido muscular esquelético de cinco miRNAs músculo-específicos (miR-1, -133a, -133b, -206 e -499) evidenciaram um alto nível de expressão do miR-499 no músculo vermelho em comparação aos níveis observados no músculo branco. Esses dados, corroborados pela hibridação in situ e análise da expressão de genes alvo do miR-499, sugerem sua participação como elemento central na regulação de vias metabólicas particularmente ativas no músculo de contração lenta (vermelho) na tilápia do Nilo. De modo geral, as análises genômicas... / MicroRNAs are small RNA molecules that post-transcriptionally regulate gene expression in various specific cell pathways. Recently, some miRNAs have been described to have a muscle-specific action and has been associated with regulation of muscle biology with fundamental roles in myogenesis. The Nile tilapia is considered an excellent biological model for the study of miRNAs in vertebrates due to their economic importance and to present the complete genome sequenced. However, little is known about the evolutionary dynamics of miRNAs and their potential role in regulating the molecular mechanisms that promote the muscle development on Nile tilapia. Thus, two studies were conducted: (i) a study to evaluate the expression pattern of muscle-specific miRNAs and their targets in different skeletal muscle types in adults of Nile tilapia, and (ii) a study to analyze the comparative evolutionary dynamics and genomic organization of these miRNAs in fish genomes. Our expression analysis by qPCR and in situ hybridization, carried out on five muscle-specific miRNAs (miR-1, -133a, -133b, -206 and -499), revealed a highly differential expression of miR-499 in red skeletal muscle (slow-twitch). These data evidenced the key role played by the miR-499 in the maintenance of the slow-twitch muscle type in Nile tilapia. The comparative genomic analysis performed on six species of fish showed a conserved dynamism for the muscle-specific miRNAs analyzed (miR-1-1/-133a-2, miR-1-2/133a-1, miR-206/133b, miR-214 e miR-499). However, a copy of miR-214 was detected in the genome of five species belonging to the superorder Acanthopterygii. Interestingly, this copy also has a high level of synteny over the species when it was detected. Thus, the obtained data may assist in the understanding of the role of muscle-specific miRNAs in muscle biological pathways... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-01Bitstream added on 2014-06-13T19:14:55Z : No. of bitstreams: 1 souza_ca_me_botib.pdf: 341694 bytes, checksum: 40dc6e4e81c8e29fb6bfbe2be1a26c7e (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A pirarara (Phractocephalus hemioliopterus) é um bagre nativo das bacias do rio Amazonas e Tocantins-Araguaia que pode atingir na sua fase adulta cerca de 60 kg. A pirarara é comercializada como peixe ornamental e é um peixe atrativo para a pesca esportiva. A espécie pertence à família Pimelodidae e tem sido ameaçada em seu habitat devido à crescente interferência antrópica nas últimas décadas. O acesso aos dados sobre a situação destas populações constitui um importante meio para o delineamento de projetos de manejo, a fim de conservar a espécie e manter os estoques de reprodutores para piscicultura. Para este fim, os marcadores microssatélites são interessantes para estudos genéticos devido a sua natureza codominante e multialélica, características ideais para serem utilizados em estudos de mapeamento genético, identificação e elucidação de questões sobre a genética de populações de diferentes organismos. Neste contexto, o presente projeto teve por objetivo a identificação e seleção de marcadores moleculares tipo microssatélites para indivíduos da espécie Phractocephalus hemioliopterus, coletados nos Rios Araguaia e Rio das Mortes,e avaliar a estrutura e os níveis de diversidade genética nessas duas localidades. Inicialmente, foram seqüenciadas 67 colônias recombinantes, dos quais apenas 32 continham sequências repetitivas do tipo microssatélite. Destas, 62,5% eram repetições dinucleotídicas e o restante composto por trinucleotídeos e tetranucelotídeos repetidos de forma imperfeita. Deste total, foi possível desenhar primers para 26 loci. Após a amplificação por PCR, 9 loci se mostraram polimórficos e informativos, e foram testados em seis espécies próximas, para a investigação de regiões no DNA altamente conservadas, com sucesso de amplificação de 81,4%. A heterozigosidade observada... / The catfish pirarara (Phractocephalus hemioliopterus) is a native Amazonas and Tocantins-Araguaia basin fish, which can weigh up to about 60 kg in its adult form. The pirarara is marketed as an ornamental fish and it is an attraction for sport fishing. The species belongs to the family Pimelodidae and it has been threatened in its habitat due to increasing human interference in recent decades. Access to data on the situation of these populations is an important means for the design of management projects in order to conserve the species and maintaining the stocks of fish breeding. The microsatellite markers for genetic studies are interesting because of their codominant and multiallelic nature, ideal characteristics for use in studies of gene mapping, identification and elucidation of questions about population’s genetics of different organisms. In this context, this project aimed to identify and select microsatellite markers on Phractocephalus hemioliopterus individuals, collected in the Araguaia and das Mortes Rivers, and to evaluate the structure and levels of genetic diversity. Initially, 67 recombinat colonies were sequenced, of which only 32 contained repetitive sequences of the microsatellite type. Of these, 62.5% were dinucleotide repeats, and the remainder consisted of trinucelotide and tetranucleotide imperfect repeats. Of this total, it was possible to design primers for 26 loci. After PCR amplification, nine loci showed to be polymorphic and informative, and were tested on six related species for the investigation of highly conserved DNA regions, with a successful amplification of 81.4%. The observed heterozygosity ranged from 0.421 to 0.947. The two sites showed significant deviations from Hardy-Weinberg equilibrium (HWE) (p<0.01) for some loci, which may be due to the presence of null alleles or even problems... (Complete abstract click electronic access below)