In this thesis the effects of individual and multiple environmental stressors (physical and chemical) are examined in inbred and outbred zebrafish (Danio rerio, Hamilton), a model species used in ecotoxicology and environmental risk assessment (ERA). The central question addressed is, are inbred laboratory animals representative and protective of wild populations? That is, are inbred fish equally or more sensitive to chemicals and other stressors compared with more outbred (wild) fish? A combination of tools and approaches incorporating traditional (eco)toxicology and population genetics have been employed, together with more contemporary molecular genetics and population modelling, to compare and contrast a range of responses in inbred and outbred zebrafish exposed to the endocrine disrupting chemical clotrimazole and/or temperature elevation in the laboratory. The choice of test species was based on our broad understanding of its basic biology, extending from the molecular level to the population level, and its wide use as a model organism in (eco)toxicology. Selection of the test chemical clotrimazole and temperature was based on a shared mode of action, aromatase inhibition, and therefore their ability to block oestrogen production, impair reproduction, promote male development and skew population sex ratios in zebrafish. A cascade of responses were compared in inbred and outbred zebrafish, including changes in the levels of expression of genes for gonadal aromatase and other steroidogenic enzymes, circulating sex steroid hormones, gonadal sex differentiation and development (via gonadal histopathology) and reproductive fitness (female fecundity, paternity and viability of embryos). Amongst the most striking results were directional skews in sex ratio towards males in response to clotrimazole (Chapter 5) and elevated temperature exposure (Chapter 7). Inbred fish were generally more responsive compared to outbreds, which showed evidence of physiological and developmental compensation, resulting in lower male-sex skews and superior fitness in terms of male reproductive success (paternity and viability of embryos). The greater effects observed in inbred fish were attributed to inbreeding×environment interactions and the amplification of inbreeding depression. Although no empirical genetic evidence of this mechanism is presented (loss of heterozygosity at quantitative trait loci and concomitant loss of heterosis and/or the expression of recessive, deleterious alleles in homozygotes), supporting evidence was provided by increased phenotypic variance in some apical endpoints in inbred fish, including specific growth rate and fecundity. This increased variance also has the potential to counteract the higher levels of response observed in inbreds, because the power to detect statistically significant changes in responses is reduced. This trade-off was demonstrated for specific growth rate. Crucially, significant male-sex ratio skews (>80%) were induced at substantially lower clotrimazole exposure concentrations (1.7 µg l-1) in combination with elevated temperature (33°C), compared with exposure concentrations (43.7 µg l-1) generating similar sex ratio skews at the standard test temperature of 28°C. These temperatures represent current and predicted 2100 (elevated) mean temperatures in the zebrafish’s native India and Bangladesh. Although the lowest observed effect concentration was an order of magnitude above the predicted environmental concentration for clotrimazole, it is conceivable that combined environmental exposures to similarly acting chemicals (e.g. other azole compounds used in crop protection, veterinary and human medicine) could produce similar effects to those we observed. The consequent effects of sex ratios skews and reduced fitness (fecundity and embryo viability) on per capita population growth rate (r) and extinction probability were predicted in inbred versus outbred zebrafish populations using stochastic population viability analysis. The results showed that the observed male-skews >80% threaten small zebrafish populations with fewer than 100 breeding adults (<20 adult females). However, small reductions of 2-3% in embryo-juvenile (age 0+) survivorship (including simulated inbreeding depression) were more influential on r and extinction probability than large sex ratio skews and/or reduced female fecundity. The results presented in this thesis support the contention that chemical effects may be exacerbated by other environmental stressors, but also illustrate the importance of considering biological (genetic), as well as physical and chemical interactions in cumulative ERA. Greater sensitivity of inbred versus outbred organisms to the effects of environmental stressors on sexual differentiation and reproductive fitness offers a margin of safety to ERA and the protection of wildlife populations (excluding those that are severely inbred and critically endangered). This is because, as originally stated, laboratory organisms used in ERA are generally more inbred than their wild counterparts. Nevertheless, more attention should be paid to the origin, breeding history and genetics of laboratory strains. This will help to ensure consistency between studies and testing laboratories and provide more confidence in extrapolating the results to wild populations.

A diferenciação sexual cerebral é um fenômeno importante que ocorre no período perinatal essencial para definir alguns padrões comportamentais na orientação sexual na fase adulta. Este evento ocorre após uma descarga abrupta da testosterona testicular no recém-nascido que é convertido em estrógeno E2 pela aromatase no hipotálamo do neonato e, juntamente com aquela de origem materna promove as alterações no cérebro que determinam a orientação sexual masculina do neonato. Em fêmeas, o E organiza o cérebro feminino e a orientação sexual. Assim, investigou-se o efeito do Citrato de Clomifeno, um inibidor da aromatase em ratos machos e fêmeas tratados perinatalmente. Os resultados mostraram alterações comportamentais e bioquímicas compatíveis com a desmasculinização, alteração do comportamento sexual de ambos os sexos, comportamento homossexual, ciclo estral, alteração dos níveis de testosterona, estrógeno, FSH e LH e do peso dos órgãos na fase adulta. Os resultados são explicados pelo bloqueio da aromatase, no período da diferenciação sexual cerebral. / Sexual differentiation of the brain is an important phenomenon that occurs perinatally essential to define some behavioral patterns of sexual orientation in adulthood. This event occurs after an abrupt discharge of testicular testosterone in newborns that E2 is converted into estrogen by aromatase in the hypothalamus of the newborn and, along with that of maternal origin promotes changes in the brain that determine sexual orientation male neonate. In females, the E organizes the female brain and sexual orientation. Thus, we investigated the effect of clomiphene citrate, aromatase inhibitor in male and female rats treated perinatally. The results showed behavioral and biochemical changes consistent with demasculinization, altered sexual behavior of both sexes, homosexual behavior, estrous cycle, changing levels of testosterone, estrogen, FSH and LH and organ weight in adulthood. The results are explained by blocking the aromatase during the period of sexual differentiation of the brain.

A espécie em estudo (Brycon orbignyanus) vem apresentando um declínio populacional importante nos últimos anos em função de interferências antrópicas. Por este motivo, populações naturais na bacia do rio Uruguai são praticamente inexistentes, e os estoques mantidos em cativeiro apresentam razões sexuais desproporcionais e perdas importantes de variabilidade genética, o que prejudica o uso desta espécie em programas de conservação. Esta tese tem como objetivo principal avaliar parâmetros populacionais e reprodutivos de Brycon orbignyanus sob condições de cativeiro visando a aplicabilidade deste conhecimento em programas de conservação. Com isso, objetivou-se no primeiro experimento avaliar as flutuações na razão sexual ao longo do desenvolvimento ontogenético e os diferentes padrões morfológicos de crescimento entre os sexos. Quinhentos exemplares foram amostrados de uma piscicultura comercial e os dados biométricos foram registrados para a realização de uma regressão linear entre peso e comprimento. A partir do teste Qui-Quadrado (χ²) foi possível observar que a razão sexual apresentou desproporções nas classes de comprimento superiores. Os machos e fêmeas apresentaram crescimento alométrico positivo (b=3,35 e b=3,44), enquanto indivíduos sexualmente indiferenciados apresentaram alometria negativa (b=2,96) O objetivo do segundo estudo foi descrever os processos de diferenciação sexual, afim de definir o período termossensível. Após eutanásia dos animais, as gônadas foram coletadas e fixadas em solução formaldeído tamponado 4%. Os cortes seriados (3μm) foram corados com Azul de Toluidina e analisados por microscopia de luz. Os primeiros sinais de diferenciação sexual foram observados apenas aos 323 dias após a fertilização (DAF) e, aos 730 DAF todos os indivíduos já estavam diferenciados sexualmente. O objetivo do terceiro estudo foi descrever o ciclo reprodutivo de machos de B. orbignyanus, a fim de se estabelecer uma escala de maturação para machos desta espécie. Para isto, exemplares da espécie foram coletados periodicamente em uma estação de piscicultura comercial. Assim como no experimento anterior, as gônadas foram coletadas e fixadas para análise histológica. As análises das imagens foram obtidas através de microscópio óptico e a descrição das fases reprodutivas seguiu recomendações de Brown-Peterson et al. (2011). Foram estabelecidas 5 fases reprodutivas de acordo com as características macroscópicas dos testículos, presença e distribuição das células espermatogênicas e variações no índice gonadossomático. Os primeiros indivíduos aptos à reprodução foram observados aos 435 DAF. / The species under study (Brycon orbignyanus) has undergone a important population decline in the last years due to anthropic interferences. For this reason, natural populations in the Uruguay River basin are practically non-existent, and stocks kept in captivity suffer from disproportionate sex ratios, and significant losses of genetic variability, impairing the use of this species in conservation programs. The main objective of this thesis is to evaluate the population and reproductive parameters of Brycon orbignyanus under captive conditions aiming the applicability of this knowledge in conservation programs. Thus, the objective of the first experiment was to evaluate the fluctuations in sexual ratio along the ontogenetic development and the different morphological patterns of growth between the sexes. Five hundred specimens were sampled from commercial fish farming and biometric data were recorded for a linear regression between weight and length. From the chi-square test (χ²) it was possible to observe that the sex ratio showed disproportions in the upper length classes. Males and females showed positive allometric growth (b = 3.35 and b = 3.44), while sexually undifferentiated individuals showed negative allometry (b = 2.96). The objective of the second experiment was describe the processes of sexual differentiation, in order to define the thermosensitive period After euthanasia of the animals, the gonads were collected and fixed in 4% buffered formaldehyde solution. Serial sections (3 μm) were stained with Toluidine Blue and analyzed by light microscopy. The first signs of sexual differentiation were observed only at 323 days after fertilization (DAF), and at 730 DAF all individuals were already sexually differentiated. The objective of the third experiment was to describe the reproductive cycle of B. orbignyanus males, in order to establish a maturation scale for males of this species. For this, specimens were collected periodically in a commercial fish farm. As in the previous experiment, the gonads were collected and fixed for histological analysis. The images were obtained through an optical microscope and analyzed according to the description of the reproductive phases suggested by Brown-Peterson et al. (2011). Five reproductive phases were established according to macroscopic characteristics of the testes, presence and distribution of spermatogenic cells and variations in the gonadosomatic index. The first individuals able to reproduce were observed at 435 DAF.

A diferenciação sexual cerebral é um fenômeno importante que ocorre no período perinatal essencial para definir alguns padrões comportamentais na orientação sexual na fase adulta. Este evento ocorre após uma descarga abrupta da testosterona testicular no recém-nascido que é convertido em estrógeno E2 pela aromatase no hipotálamo do neonato e, juntamente com aquela de origem materna promove as alterações no cérebro que determinam a orientação sexual masculina do neonato. Em fêmeas, o E organiza o cérebro feminino e a orientação sexual. Assim, investigou-se o efeito do Citrato de Clomifeno, um inibidor da aromatase em ratos machos e fêmeas tratados perinatalmente. Os resultados mostraram alterações comportamentais e bioquímicas compatíveis com a desmasculinização, alteração do comportamento sexual de ambos os sexos, comportamento homossexual, ciclo estral, alteração dos níveis de testosterona, estrógeno, FSH e LH e do peso dos órgãos na fase adulta. Os resultados são explicados pelo bloqueio da aromatase, no período da diferenciação sexual cerebral. / Sexual differentiation of the brain is an important phenomenon that occurs perinatally essential to define some behavioral patterns of sexual orientation in adulthood. This event occurs after an abrupt discharge of testicular testosterone in newborns that E2 is converted into estrogen by aromatase in the hypothalamus of the newborn and, along with that of maternal origin promotes changes in the brain that determine sexual orientation male neonate. In females, the E organizes the female brain and sexual orientation. Thus, we investigated the effect of clomiphene citrate, aromatase inhibitor in male and female rats treated perinatally. The results showed behavioral and biochemical changes consistent with demasculinization, altered sexual behavior of both sexes, homosexual behavior, estrous cycle, changing levels of testosterone, estrogen, FSH and LH and organ weight in adulthood. The results are explained by blocking the aromatase during the period of sexual differentiation of the brain.

The purpose of this project is to better understand the influences underlying gender differences in the brain using Four Core Genotype mice. Four Core Genotype mice are transgenic mice in which the SRY gene has been translocated from the Y chromosome to another location. This enables separation of the genetic sex and gonadal sex. For example, there are female mice based on sexual organs but their chromosomes are XY(UY- in mice). This allows us to determine whether sexual differentiation in the brain is due to genes or hormones. In this project, I looked at a sexually dimorphic area of the brain, the Bed Nucleus of the Stria Terminalis (BNST), which is twice as large in males than in females. I hypothesize that both chromosomes and gonadal hormones play a part in sexually differentiating the brain including the BNST and thus I predict that the size of the BNST will be the same in XX males (UUSRY) and XY females (UY-). I measured the BNST from five XX female (UU) and five XY male(UY-SRY) four core genotype mouse brains and confirmed that the BNST is larger in males than in females, as it is in normal mice (p= .057). I processed and measured the size of the BNST in ten brains of XX males and XY females to see if the size of the BNST matches the chromosomes or the gonads. The results had a trend in the data that suggested chromosomes play more of an effect on sexual differentiation of the BNST. The overall goal of this project is to contribute to research examining the causes of gender identity in humans by relating this work to other works in the field.

Neurotransmitters are widely distributed in the central nervous system of tilapia during development. In the present study, the effects of gonadal steroids, temperature, and neurotransmitters on the development of central neurotransmitter systems in tilapia, Oreochromis mossambicus, were investigated. Exogenous E2 before 10 days posthatching resulted in an inhibitory effect on the activity of central 5-HT system via decreasing TPH activity and increasing MAO activity, a decrease of the gene expression of brain aromatase and ERa, which is involved in the feminization. The masculinizing actions of 17a-methyltestosterone (MT) are most potent later at up to day 20 of age, and may depend on MT-induction of aromatase activity, aromatase mRNA gene expression and estrogen receptor-b mRNA gene expression. The development of central neurotransmitter systems is influenced by aquatic temperature during its respective restricted period. The influence of both lower and elevated temperatures on the neurotransmitter activity, either increasing or suppressing, is dependent on its developing stage. Neurotransmitters have an effect to influence the development of central neurotransmitter and this effect may mediated by the neural proliferation.

The sexual differentiation of the spinal nucleus of bulbocavernosus (SNB) and the bulbocavernosus (BC) and levator ani (LA) muscles that the SNB innervates, are masculinized by androgens acting on the BC/LA. The site of androgen receptors (AR) responsible for the masculinization of the neuromuscular system is not known. A potential site of action is satellite cells: proliferation of these cells is androgen-dependent and satellite cells seem to contribute to the size of the LA. Fluorescent immunohistochemistry and confocal microscopy were used to co-localize satellite cells and AR within the LA of postnatal day one and three male and female rats. Results indicate that satellite cells express AR and reveal a difference in proportion of satellite cells expressing AR between the LA and control muscle. Interpretations of these findings, including whether the relatively small proportion of AR accounted for by satellite cells is enough to masculinize the SNB system, are discussed.

The sexual differentiation of the spinal nucleus of bulbocavernosus (SNB) and the bulbocavernosus (BC) and levator ani (LA) muscles that the SNB innervates, are masculinized by androgens acting on the BC/LA. The site of androgen receptors (AR) responsible for the masculinization of the neuromuscular system is not known. A potential site of action is satellite cells: proliferation of these cells is androgen-dependent and satellite cells seem to contribute to the size of the LA. Fluorescent immunohistochemistry and confocal microscopy were used to co-localize satellite cells and AR within the LA of postnatal day one and three male and female rats. Results indicate that satellite cells express AR and reveal a difference in proportion of satellite cells expressing AR between the LA and control muscle. Interpretations of these findings, including whether the relatively small proportion of AR accounted for by satellite cells is enough to masculinize the SNB system, are discussed.

A espécie em estudo (Brycon orbignyanus) vem apresentando um declínio populacional importante nos últimos anos em função de interferências antrópicas. Por este motivo, populações naturais na bacia do rio Uruguai são praticamente inexistentes, e os estoques mantidos em cativeiro apresentam razões sexuais desproporcionais e perdas importantes de variabilidade genética, o que prejudica o uso desta espécie em programas de conservação. Esta tese tem como objetivo principal avaliar parâmetros populacionais e reprodutivos de Brycon orbignyanus sob condições de cativeiro visando a aplicabilidade deste conhecimento em programas de conservação. Com isso, objetivou-se no primeiro experimento avaliar as flutuações na razão sexual ao longo do desenvolvimento ontogenético e os diferentes padrões morfológicos de crescimento entre os sexos. Quinhentos exemplares foram amostrados de uma piscicultura comercial e os dados biométricos foram registrados para a realização de uma regressão linear entre peso e comprimento. A partir do teste Qui-Quadrado (χ²) foi possível observar que a razão sexual apresentou desproporções nas classes de comprimento superiores. Os machos e fêmeas apresentaram crescimento alométrico positivo (b=3,35 e b=3,44), enquanto indivíduos sexualmente indiferenciados apresentaram alometria negativa (b=2,96) O objetivo do segundo estudo foi descrever os processos de diferenciação sexual, afim de definir o período termossensível. Após eutanásia dos animais, as gônadas foram coletadas e fixadas em solução formaldeído tamponado 4%. Os cortes seriados (3μm) foram corados com Azul de Toluidina e analisados por microscopia de luz. Os primeiros sinais de diferenciação sexual foram observados apenas aos 323 dias após a fertilização (DAF) e, aos 730 DAF todos os indivíduos já estavam diferenciados sexualmente. O objetivo do terceiro estudo foi descrever o ciclo reprodutivo de machos de B. orbignyanus, a fim de se estabelecer uma escala de maturação para machos desta espécie. Para isto, exemplares da espécie foram coletados periodicamente em uma estação de piscicultura comercial. Assim como no experimento anterior, as gônadas foram coletadas e fixadas para análise histológica. As análises das imagens foram obtidas através de microscópio óptico e a descrição das fases reprodutivas seguiu recomendações de Brown-Peterson et al. (2011). Foram estabelecidas 5 fases reprodutivas de acordo com as características macroscópicas dos testículos, presença e distribuição das células espermatogênicas e variações no índice gonadossomático. Os primeiros indivíduos aptos à reprodução foram observados aos 435 DAF. / The species under study (Brycon orbignyanus) has undergone a important population decline in the last years due to anthropic interferences. For this reason, natural populations in the Uruguay River basin are practically non-existent, and stocks kept in captivity suffer from disproportionate sex ratios, and significant losses of genetic variability, impairing the use of this species in conservation programs. The main objective of this thesis is to evaluate the population and reproductive parameters of Brycon orbignyanus under captive conditions aiming the applicability of this knowledge in conservation programs. Thus, the objective of the first experiment was to evaluate the fluctuations in sexual ratio along the ontogenetic development and the different morphological patterns of growth between the sexes. Five hundred specimens were sampled from commercial fish farming and biometric data were recorded for a linear regression between weight and length. From the chi-square test (χ²) it was possible to observe that the sex ratio showed disproportions in the upper length classes. Males and females showed positive allometric growth (b = 3.35 and b = 3.44), while sexually undifferentiated individuals showed negative allometry (b = 2.96). The objective of the second experiment was describe the processes of sexual differentiation, in order to define the thermosensitive period After euthanasia of the animals, the gonads were collected and fixed in 4% buffered formaldehyde solution. Serial sections (3 μm) were stained with Toluidine Blue and analyzed by light microscopy. The first signs of sexual differentiation were observed only at 323 days after fertilization (DAF), and at 730 DAF all individuals were already sexually differentiated. The objective of the third experiment was to describe the reproductive cycle of B. orbignyanus males, in order to establish a maturation scale for males of this species. For this, specimens were collected periodically in a commercial fish farm. As in the previous experiment, the gonads were collected and fixed for histological analysis. The images were obtained through an optical microscope and analyzed according to the description of the reproductive phases suggested by Brown-Peterson et al. (2011). Five reproductive phases were established according to macroscopic characteristics of the testes, presence and distribution of spermatogenic cells and variations in the gonadosomatic index. The first individuals able to reproduce were observed at 435 DAF.

A espécie em estudo (Brycon orbignyanus) vem apresentando um declínio populacional importante nos últimos anos em função de interferências antrópicas. Por este motivo, populações naturais na bacia do rio Uruguai são praticamente inexistentes, e os estoques mantidos em cativeiro apresentam razões sexuais desproporcionais e perdas importantes de variabilidade genética, o que prejudica o uso desta espécie em programas de conservação. Esta tese tem como objetivo principal avaliar parâmetros populacionais e reprodutivos de Brycon orbignyanus sob condições de cativeiro visando a aplicabilidade deste conhecimento em programas de conservação. Com isso, objetivou-se no primeiro experimento avaliar as flutuações na razão sexual ao longo do desenvolvimento ontogenético e os diferentes padrões morfológicos de crescimento entre os sexos. Quinhentos exemplares foram amostrados de uma piscicultura comercial e os dados biométricos foram registrados para a realização de uma regressão linear entre peso e comprimento. A partir do teste Qui-Quadrado (χ²) foi possível observar que a razão sexual apresentou desproporções nas classes de comprimento superiores. Os machos e fêmeas apresentaram crescimento alométrico positivo (b=3,35 e b=3,44), enquanto indivíduos sexualmente indiferenciados apresentaram alometria negativa (b=2,96) O objetivo do segundo estudo foi descrever os processos de diferenciação sexual, afim de definir o período termossensível. Após eutanásia dos animais, as gônadas foram coletadas e fixadas em solução formaldeído tamponado 4%. Os cortes seriados (3μm) foram corados com Azul de Toluidina e analisados por microscopia de luz. Os primeiros sinais de diferenciação sexual foram observados apenas aos 323 dias após a fertilização (DAF) e, aos 730 DAF todos os indivíduos já estavam diferenciados sexualmente. O objetivo do terceiro estudo foi descrever o ciclo reprodutivo de machos de B. orbignyanus, a fim de se estabelecer uma escala de maturação para machos desta espécie. Para isto, exemplares da espécie foram coletados periodicamente em uma estação de piscicultura comercial. Assim como no experimento anterior, as gônadas foram coletadas e fixadas para análise histológica. As análises das imagens foram obtidas através de microscópio óptico e a descrição das fases reprodutivas seguiu recomendações de Brown-Peterson et al. (2011). Foram estabelecidas 5 fases reprodutivas de acordo com as características macroscópicas dos testículos, presença e distribuição das células espermatogênicas e variações no índice gonadossomático. Os primeiros indivíduos aptos à reprodução foram observados aos 435 DAF. / The species under study (Brycon orbignyanus) has undergone a important population decline in the last years due to anthropic interferences. For this reason, natural populations in the Uruguay River basin are practically non-existent, and stocks kept in captivity suffer from disproportionate sex ratios, and significant losses of genetic variability, impairing the use of this species in conservation programs. The main objective of this thesis is to evaluate the population and reproductive parameters of Brycon orbignyanus under captive conditions aiming the applicability of this knowledge in conservation programs. Thus, the objective of the first experiment was to evaluate the fluctuations in sexual ratio along the ontogenetic development and the different morphological patterns of growth between the sexes. Five hundred specimens were sampled from commercial fish farming and biometric data were recorded for a linear regression between weight and length. From the chi-square test (χ²) it was possible to observe that the sex ratio showed disproportions in the upper length classes. Males and females showed positive allometric growth (b = 3.35 and b = 3.44), while sexually undifferentiated individuals showed negative allometry (b = 2.96). The objective of the second experiment was describe the processes of sexual differentiation, in order to define the thermosensitive period After euthanasia of the animals, the gonads were collected and fixed in 4% buffered formaldehyde solution. Serial sections (3 μm) were stained with Toluidine Blue and analyzed by light microscopy. The first signs of sexual differentiation were observed only at 323 days after fertilization (DAF), and at 730 DAF all individuals were already sexually differentiated. The objective of the third experiment was to describe the reproductive cycle of B. orbignyanus males, in order to establish a maturation scale for males of this species. For this, specimens were collected periodically in a commercial fish farm. As in the previous experiment, the gonads were collected and fixed for histological analysis. The images were obtained through an optical microscope and analyzed according to the description of the reproductive phases suggested by Brown-Peterson et al. (2011). Five reproductive phases were established according to macroscopic characteristics of the testes, presence and distribution of spermatogenic cells and variations in the gonadosomatic index. The first individuals able to reproduce were observed at 435 DAF.