Tesis sobre el tema "Danio Rerio (Hamilton)"

The diploma thesis summarizes the experimental method for the rearing of zebrafish Danio rerio (Hamilton, 1822) screed. The work of figuring out which of the methods of rearing fry in the transitional period of the ontogenetic development is best for the survival rate and growth of the standard length of the surveyed individuals. The test was carried out on two basic lines of zebrafish Wild Type and Casper. At the beginning of the test were created four groups divided by feeding mode. The control group was fed once a day with pellet feed GEMMA Micro 75 and one day from the 10th day after fertilization the fed with nauplii stages of brine shrimps Artemia salina (Linnaeus, 1758). The second group was fed only once a day with pellet feed GEMMA Micro 75. The last two groups were fed five times a day saltwater rotifer species Brachionus plicatilis (Müller, 1786), and only one of them and the other was still three times daily nourished with nauplii stages of brine shrimps. For each group of four representative have been created initially, thirty tanks of fish at the age of five days after fertilization. The results of this method have shown that the methods they use to feed the rotifers, did not have a statistically significant impact on the survival rate of individuals. In the growth of this method has proven to be statistically significant result in the group where the fed in combination with brine shrimps, where to achieve the average standard size of juvenile fish 16.02 +/- 0.80 mm for the Wild type and 17.39 +/- 0.81 mm for Casper, compared to the control group, that has been the standard length 11.63 +/- 0.64 mm for the Wild Type and 9.54 +/- 0.56 mm for Casper. This method has great potential and breeding is therefore necessary to further develop this method and to adapt it to individual zebrafish facilities.

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Memories are stored as structural and functional changes of the synaptic connections in neural regions involved in learning. Due to its biological relevance and relation to pathologies of the human nervous system, the pursuit for elucidation of the mechanisms underlying its maintenance is receiving enormous interest from distinct areas of knowledge over the years. Studies on the neural mechanisms of long-term memories have demonstrated that its formation is a gradual process involving neuroanatomical and cellular substrates, and whose molecular bases are the target of intense research. The teleost fish zebrafish is an excellent animal model for genetic and developmental studies and has a great potential for studies of learning and memory. This vertebrate is easy to manipulate and has a fast external development demanding practical and economical maintenance. Neuroanatomicaly, despite differences on developmental aspects and brain regions’ disposition, the general organization of mammal and teleost brains follow typical vertebrates’ patterns. Recent advances have shown that there is extensive similarity in both complexity and functioning of teleosts fish and terrestrial vertebrates brains, which validates the use of this animal model to study conserved mechanisms of learning and memory. Although there are studies evaluating behavior of zebrafish, they are mainly focused on locomotion and exploration of the environment in detriment of cognition. Moreover, there is no consistency in relation to the tasks protocols used. In the present study we demonstrate a rapid and effective learning protocol of a single-trial inhibitory avoidance to zebrafish that would allow the evaluation of cellular and molecular mechanisms of memory formation in vertebrates. In a simple apparatus animals learn to avoid swimming from a white into a dark compartment in order to circumvent an electric shock in a single training session. The retention test was performed 24 h after training. The test session repeated the training protocol except that no shock was administered. Latencies to enter the dark environment were considered indication of retention. In order to demonstrate the task potential use in further studies aiming to characterize the effect of pharmacological treatments that alter animals’ cognitive performance, we investigated the effect of the NMDA-receptor antagonist on inhibitory avoidance memory. Immediately after training animals were treated with 20 μM of MK-801 during 15 min, which prevented memory formation. Our results shows that the resulting memory for this task is robust, long-lasting and sensitive to NMDA blockage, in accordance to data from other vertebrates. This is a powerful instrument for evaluate memory processes and many other studies may benefit from this task. Together with complementary strategies available for zebrafish it may significantly improve our current knowledge on learning and memory mechanisms.
Memórias são armazenadas como modificações estruturais e funcionais das conexões sinápticas nas regiões neurais envolvidas no aprendizado e, por sua relevância biológica e relação com patologias do sistema nervoso humanas, a busca pela elucidação dos mecanismos responsáveis por sua manutenção tem recebido interesse de distintas áreas do conhecimento ao longo dos anos. Estudos sobre os mecanismos neurais das memórias de longa duração demonstraram que sua formação é um processo gradual que envolve substratos neuroanatômicos e celulares agora conhecidos em grande extensão, e cujas bases moleculares são alvo de intensa investigação. O peixe teleósteo zebrafish é um excelente modelo animal para estudos genéticos e de desenvolvimento e vêm mostrando enorme potencial para estudos de aprendizagem e memória. Este vertebrado de fácil manipulação apresenta rápido desenvolvimento externo e manutenção prática e econômica. Em termos neuroanatômicos, apesar das diferenças entre aspectos do desenvolvimento e resultante disposição de regiões cerebrais de mamíferos e teleósteos, a organização geral do encéfalo do zebrafish segue padrões típicos dos vertebrados. Avanços recentes no conhecimento demonstram que há uma extensa similaridade, tanto em complexidade quanto em funcionamento entre o cérebro de peixes teleósteos e os vertebrados terrestres o que valida o uso deste modelo animal para estudo dos mecanismos conservados do aprendizado e memória. Embora já existam na literatura científica estudos avaliando parâmetros comportamentais do zebrafish, na maior parte das vezes o foco principal dos trabalhos não é a cognição. Aspectos como locomoção e exploração do ambiente são tomadas como medidas e os trabalhos publicados não mostram constância em relação às tarefas utilizadas e protocolos adotados. Este trabalho teve como objetivo o desenvolvimento de um novo paradigma de esquiva inibitória para zebrafish a fim de permitir a avaliação dos mecanismos celulares e moleculares da formação de memória em vertebrados. Para tanto, o treino consistiu de apenas uma sessão onde os animais foram colocados individualmente no aquário de esquiva inibitória. Ao cruzar para o lado escuro do aquário um choque elétrico foi administrado por 5s e estes animais retirados imediatamente do aquário. Na sessão de teste, realizada 24 h depois, seguiu-se o mesmo protocolo, porém nenhum choque foi utilizado. As latências de entrada no lado escuro para o treino e o teste foram consideradas como indicadoras de retenção. Para a validação da tarefa um grupo de animais foi tratado durante 15 min pós-treino com o antagonista de receptor NMDA MK-801 numa concentração de 20 μM. Os resultados obtidos demonstram que a tarefa de esquiva inibitória desenvolvida para zebrafish é baseada em mecanismos conservados em que o aprendizado, e resultante memória, são dependentes de sinalização glutamatérgica mediada por NMDA, assim como já evidenciado para outros vertebrados. A tarefa desenvolvida é rápida, simples e resulta em um aprendizado robusto, sólido e persistente, representando um instrumento valioso para se caracterizar diferentes processos que afetam o comportamento no zebrafish, bem como permitir a dissecação dos eventos tempos-dependentes da formação de memórias de vertebrados.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The industrial effluents are one of the main sources of water pollution. For an appropriate characterization and control of their discharges, the most efficient strategy is the integrated use of chemical, physical and ecotoxicological analyses. The aims of this study were to asses the efficiency of the treatment plant of a textile industry performing acute toxicity tests and physical-chemical analyses of the effluents before and after the treatment, besides evaluate the toxicity of the effluents of the Treatment System of Liquids Effluents (Sistema de Tratamento de Efluentes L?quidos - SITEL) of Distrito Industrial de Natal (DIN) and some of their physical-chemical variables. The species used in the ecotoxicological tests was the fish Danio rerio. The results showed that the treatment plant reduced significantly (around 50%) the toxicity of the raw textile effluent in only three of the seven tests but, in general, it promoted the reduction of the physical-chemical parameters analyzed. The toxicity and the physical-chemical factors of the effluents of SITEL of DIN varied among the tests and show the importance of monitoring their discharges in the Potengi river, one of the most important rivers of the Rio Grande do Norte state
Os efluentes industriais s?o uma das principais fontes de polui??o das ?guas. Para uma caracteriza??o adequada e controle de seus despejos, a estrat?gia mais eficiente ? o uso integrado de an?lises qu?micas, f?sicas e ecotoxicol?gicas. Os objetivos deste trabalho foram avaliar a efici?ncia da esta??o de tratamento de uma ind?stria t?xtil, atrav?s de testes de toxicidade aguda e de an?lises f?sico-qu?micas dos efluentes antes e ap?s o tratamento, al?m de avaliar a toxicidade dos efluentes do Sistema de Tratamento de Efluentes L?quidos (SITEL) do Distrito Industrial de Natal (DIN) e algumas de suas vari?veis f?sico-qu?micas. A esp?cie utilizada nos testes ecotoxicol?gicos foi o peixe Danio rerio. Os resultados mostraram que a esta??o de tratamento reduziu significativamente (cerca de 50%) a toxicidade do efluente t?xtil bruto em apenas tr?s dos sete ensaios realizados, mas de um modo geral, promoveu a redu??o dos valores dos par?metros f?sico-qu?micos analisados. A toxicidade e os fatores f?sico-qu?micos dos efluentes do SITEL do DIN variaram entre os testes e mostram a import?ncia de se monitorar suas descargas no rio Potengi, um dos rios mais importantes do Estado do Rio Grande do Norte

Le poisson zèbre Danio rerio a été utilisé dans cette étude comme organisme modèle, dans un projet visant à développer une méthode de confinement biologique des poissons d'élevage fondée sur les interactions entre la biotine et l'avidine. Les objectifs de cette thèse étaient de i) estimer les besoins en biotine chez le poisson zèbre; ii) étudier les effets d'une complémentation en biotine et en avidine sur la croissance, la survie, la conversion des aliments, le statut de la biotine et l'expression des gènes chez le poisson zèbre; iii) évaluer la contribution de la microflore intestinale dans l'approvisionnement en biotine chez le poisson zèbre; et iv) évaluer les effets de la biotine sur la reproduction du poisson zèbre. La première étude de cette thèse a démontré que la biotine est essentielle pour la croissance du poisson zèbre et que le niveau de biotine à inclure dans l'aliment pour une croissance maximale est de 0,51 mg kg-1 d'aliment. La seconde étude a confirmé le caractère essentiel de la biotine pour ce poisson, tel que démontré précédemment. Elle a aussi révélé que l’alimentation du poisson zèbre avec une ration contenant 60 fois plus d'avidine que le niveau requis de biotine induit des signes de carence en biotine. La troisième étude a suggéré que la synthèse microbienne intestinale est une source significative de biotine chez le poisson zèbre. Les poissons nourris avec la ration contenant un antibiotique (1% succinylsulfathiazole, masse/masse) ont présenté une croissance, une condition générale, des teneurs en biotine totale du corps, une survie et une utilisation des aliments moindres que ceux nourris avec les rations ne contenant pas d'antibiotique. Les résultats de la quatrième étude ont démontré que les mâles carencés en biotine présentent un faible indice gonado-somatique et de faibles qualité et quantité du sperme, tandis que les femelles carencées en biotine ont une faible fertilité. Les résultats de cette thèse ont fourni des informations de base pour la compréhension des interactions entre la biotine et l'avidine alimentaires chez le poisson zèbre. Ces données pourront servir à développer des stratégies de confinement biologique en utilisant ces interactions.
Zebrafish Danio rerio was used as model organism in the first step of a project aiming at developing a biological confinement method of farmed fish based on the interactions between biotin and avidin. The objectives of this thesis were i) to estimate the dietary biotin requirements of zebrafish; ii) to study the effects of dietary biotin and avidin on growth, survival, feed conversion, biotin status and gene expression in zebrafish; iii) to assess the contribution of the intestinal microflora to biotin supply in zebrafish, and iv) to investigate the effects of biotin on zebrafish reproduction. The first study of this thesis has demonstrated that biotin is essential for zebrafish growth and that the optimum dietary biotin requirement for maximal growth is 0.51 mg kg-1 diet. The second study confirmed the essentiality of biotin for zebrafish growth found in the first study. This study also revealed that feeding zebrafish a diet containing avidin in 60-fold excess of biotin requirement induces biotin deficiency signs in zebrafish such as retarded growth, high mortality, low condition factor, and decreased steady-state level of acetyl CoA carboxylase-A (acca) transcripts in the liver. The third study suggested that intestinal microbial synthesis is a significant source of biotin to zebrafish. Fish fed the antibiotic-supplemented diet (1% succinylsulfathiazole, mass/mass) showed lower growth, health condition, whole-body biotin content, survival and feed utilization than fish fed the biotin unsupplemented and biotin supplemented diets. The results of the fourth study demonstrated that dietary biotin affect both male and female reproductive performances; the biotin-deficient male showed lower gonadosomatic index as well as lower sperm quality and quantity, while biotin-deficient female showed reduced fertility. The results of this thesis constitute a baseline in the understanding of the interactions between dietary biotin and avidin in zebrafish nutrition on the one hand, and in the potential use of these interactions in order to develop a biological confinement strategy of farmed fish on the other hand.

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Mem?rias s?o armazenadas como modifica??es estruturais e funcionais das conex?es sin?pticas nas regi?es neurais envolvidas no aprendizado e, por sua relev?ncia biol?gica e rela??o com patologias do sistema nervoso humanas, a busca pela elucida??o dos mecanismos respons?veis por sua manuten??o tem recebido interesse de distintas ?reas do conhecimento ao longo dos anos. Estudos sobre os mecanismos neurais das mem?rias de longa dura??o demonstraram que sua forma??o ? um processo gradual que envolve substratos neuroanat?micos e celulares agora conhecidos em grande extens?o, e cujas bases moleculares s?o alvo de intensa investiga??o. O peixe tele?steo zebrafish ? um excelente modelo animal para estudos gen?ticos e de desenvolvimento e v?m mostrando enorme potencial para estudos de aprendizagem e mem?ria. Este vertebrado de f?cil manipula??o apresenta r?pido desenvolvimento externo e manuten??o pr?tica e econ?mica. Em termos neuroanat?micos, apesar das diferen?as entre aspectos do desenvolvimento e resultante disposi??o de regi?es cerebrais de mam?feros e tele?steos, a organiza??o geral do enc?falo do zebrafish segue padr?es t?picos dos vertebrados. Avan?os recentes no conhecimento demonstram que h? uma extensa similaridade, tanto em complexidade quanto em funcionamento entre o c?rebro de peixes tele?steos e os vertebrados terrestres o que valida o uso deste modelo animal para estudo dos mecanismos conservados do aprendizado e mem?ria. Embora j? existam na literatura cient?fica estudos avaliando par?metros comportamentais do zebrafish, na maior parte das vezes o foco principal dos trabalhos n?o ? a cogni??o. Aspectos como locomo??o e explora??o do ambiente s?o tomadas como medidas e os trabalhos publicados n?o mostram const?ncia em rela??o ?s tarefas utilizadas e protocolos adotados. Este trabalho teve como objetivo o desenvolvimento de um novo paradigma de esquiva inibit?ria para zebrafish a fim de permitir a avalia??o dos mecanismos celulares e moleculares da forma??o de mem?ria em vertebrados. Para tanto, o treino consistiu de apenas uma sess?o onde os animais foram colocados individualmente no aqu?rio de esquiva inibit?ria. Ao cruzar para o lado escuro do aqu?rio um choque el?trico foi administrado por 5s e estes animais retirados imediatamente do aqu?rio. Na sess?o de teste, realizada 24 h depois, seguiu-se o mesmo protocolo, por?m nenhum choque foi utilizado. As lat?ncias de entrada no lado escuro para o treino e o teste foram consideradas como indicadoras de reten??o. Para a valida??o da tarefa um grupo de animais foi tratado durante 15 min p?s-treino com o antagonista de receptor NMDA MK-801 numa concentra??o de 20 μM. Os resultados obtidos demonstram que a tarefa de esquiva inibit?ria desenvolvida para zebrafish ? baseada em mecanismos conservados em que o aprendizado, e resultante mem?ria, s?o dependentes de sinaliza??o glutamat?rgica mediada por NMDA, assim como j? evidenciado para outros vertebrados. A tarefa desenvolvida ? r?pida, simples e resulta em um aprendizado robusto, s?lido e persistente, representando um instrumento valioso para se caracterizar diferentes processos que afetam o comportamento no zebrafish, bem como permitir a disseca??o dos eventos tempos-dependentes da forma??o de mem?rias de vertebrados.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2017.
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O consumo e transporte de petróleo está diretamente ligado à contaminação ambiental por hidrocarbonetos em diversas regiões do mundo. Uma vez no ambiente aquático, o petróleo e seus derivados podem produzir alterações variadas nos organismos expostos, tais como alterações biológicas de caráter molecular, celular, fisiológico ou ecológico, resultando em efeitos danosos às comunidades naturais, incluindo os organismos destinados ao consumo humano. Desta forma, a investigação dessas alterações é urgente como forma preventiva e de avaliação de ambientes aquáticos contaminados. Nesse sentido, o presente trabalho teve como principais objetivos avaliar os efeitos no perfil de proteínas em ostras após exposição à fração de óleo diesel acomodada em água, buscar genes normalizadores em ostras expostas às diferentes condições experimentais e avaliar os efeitos da contaminação do derivado foto-oxidado de petróleo 6-hidroxicriseno em peixes Danio rerio. Para atingir esses objetivos, primeiramente, ostras do mangue Crassostrea brasiliana foram expostas à fração de óleo diesel acomodada em água durante 24h e 72h com intuito de identificar biomarcadores moleculares. Entre as 11 proteínas expressas diferencialmente, podemos destacar sete: citocromo P450 6A (CYP6A), NADPH citocromo P450 redutase (envolvidas no metabolismo lipídico / xenobiótico), alfa e beta tubulina (envolvidas na formação de fagossomo mediados por retículo endoplasmático - RE), tiorredoxina e proteína ubiquitina ligase E3 (envolvidas no processamento e degradação de proteínas no RE). A avaliação de respostas em compartimentos subcelulares mostrou-se bastante sensível e viável para estudos ecotoxicológicos, além de auxiliar na compreensão dos mecanismos que envolvem o RE frente à exposição. Esses dados indicam um papel promissor da avaliação do RE em programas de biomonitoramento ambiental. Entretanto, como muito pouco é conhecido sobre a biologia molecular de C. brasiliana, surgiu a necessidade de utilizar genes normalizadores ideais para diferentes condições experimentais para adequada análise da transcrição gênica. Neste trabalho foram identificados diferentes conjuntos de genes adequados para a normalização de dados de PCR quantitativa em tempo real (qRT-PCR) em cada desenho experimental testado. O gene anquirina teve maior estabilidade de transcrição e foi considerado candidato promissor como gene normalizador em estudos posteriores. Por fim, com o intuito de aumentar as informações sobre os efeitos de compostos foto-oxidados do petróleo, avaliou-se a exposição de embriões de D. rerio ao 6-hidroxicriseno. Foram observadas respostas dos genes relacionados com transportadores iônicos (nck1 e kchna), associadas à bioacumulação e deformidades cardiácas nos animais nas fases de desenvolvimento embrionário de 74 hpf de exposição.
Abstract : The consumption and transportation of petroleum are directly linked to the contamination by hydrocarbons in many regions around the world. Once in the aquatic environment, petroleum and its derivatives can produce many alterations to exposed organisms such as biological, cellular, physiological or ecological biological alterations, resulting in harmful effects to natural communities including organisms destined for human consumption. Then, it is urgent the investigation of these impacts in an attempt to prevent and evaluate contaminated aquatic environments. In this context, the main goal of this work was to evaluate the effects of diesel oil water accommodated fraction on the proteomic profile in oysters, search for normalizing genes in oysters submitted to different experimental conditions, and evaluate the effects of photo-oxidized petroleum derivative 6-hydroxycrysene in D. rerio. In order to achieve these goals, firstly, the mangrove oyster C. brasiliana were exposed to diesel oil water accommodated fraction for 24h and 72h. Among the 11 differentially expressed proteins, we pointed out seven: cytochrome P450 6A (CYP6A), NADPH cytochrome P450 reductase (involved in lipid / xenobiotic metabolism), alpha and beta tubulin (involved in the formation of RE-mediated phagosome), tioredoxin containg-protein and ubiquitin ligase E3 protein (involved in the processing and degradation of proteins in RE). Subcellular compartments responses had shown to be sensitive and feasible for ecotoxicological studies, besides helping to understand the mechanisms that involve ER in relation to exposure. These data indicate a promising role for ER evaluation in environmental biomonitoring programs. However, few studies had focused on molecular biology of C. brasiliana, there is a necessity to use select normalizing genes ideal for different experimental conditions for adequate analysis of gene expression. In this work, different sets of genes suitable for the normalization of qRT-PCR data were identified in each experimental design tested. The ankyrin gene had greater transcriptional stability and was considered a promising candidate as a normalizing gene in later studies. Finally, in order to increase the information about the effects of photo-oxidized petroleum derivative compounds, the exposure by 6-hydroxychrisene in the embryonic development of D. rerio was evaluated. In summary, the responses of the genes related to the ionic transporters (nck1 and kchna), together with chemical aspects of bioaccumulation, have been shown to play an important role in the cardiac deformities generated during morphogenesis in the embryonic development stages in 74 hpf of exhibition.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2015.
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O glifosato é um herbicida utilizado para inibir o crescimento de plantas daninhas. Este herbicida é solúvel em água e, quando aplicado em sistemas terrestres, infiltra no solo atingindo os ecossistemas aquáticos e consequentemente, afetando organismos não-alvo. O objetivo deste estudo foi caracterizar os efeitos de 65 µg/L glifosato sobre a organização estrutural e ultraestrutural das células somáticas e germinativas durante a espermatogênese de Danio rerio. A concentração de glifosato utilizada corresponde ao limite máximo estabelecido pelo CONAMA para águas de abastecimento par consumo humano. Os resultados mostram aumento significativo de micronúcleos, além de aumento significativo no índice gonadossomático (IGS) nos machos expostos por 360 h. A análise estrutural permitiu o reconhecimento em todos grupos de três estágios de cistos germinativos organizados próximos a parede dos testículos, que correspondem aqueles compostos por: (i) espermatogônias, (ii) espermatócitos, (iii) espermátides (iniciais e tardias); além da presença de espermatozoides no lúmen do túbulo seminífero. Na análise ultraestrutural observou-se alterações nas células de Sertoli, com perda da densidade do citoplasma e das organelas, bem como o aparecimento de estruturas com aspecto vesicular. Além dessas, foram observadas alterações das interações entre células de Sertoli e células germinativas e perda das pontes citoplasmáticas entre as células germinativas. Na análise de expressão da proteína envolvida no estresse celular foi observado no grupo exposto por 144 h, aumento significativo de HSP70 induzível, a qual não foi mais observado em 360 h. Na quantificação de FASL, envolvida na indução à apoptose, observou-se aumento significativo nos machos expostos por 360 h. Embora neste estudo, tenha sido reconhecido uma aumento de FASL, não foram observadas modificações na expressão nenhuma das proteínas de ativação à apoptose investigadas (BCL2, BAK e caspase 3). Os efeitos adversos do glifosato sobre os testículos, principalmente sobre células de Sertoli, são um importante indicativo sobre as consequências na reprodução de peixes, que podem abranger desde o nível celular, sistêmico e populacional. Por fim, este estudo demonstrou a importância de se investigar a toxicidade do glifosato sobre a reprodução masculina, utilizando testículos de peixe como modelo experimental.

Abstract : Glyphosate is herbicide used to inhibit the growth of weeds. This herbicide is soluble in water, and when applied in terrestrial systems, glyphosate infiltrates into the soil, reaching aquatic ecosystems and consequently affecting nontarget organisms. The aim of this study was to characterize the effect of 65 µg/L glyphosate on the structural and ultrastructural organization of somatic and germ cells during Danio rerio spermatogenesis. Glyphosate concentration used corresponds to the maximum level stated by CONAMA for waters for human consumption. Our results showed significant increase of the micronuclei, as well as, significant increase in GSI on males exposed to glyphosate for 360 h. Structural analysis allowed the recognition in all groups of three germ cysts stages, organized near to the testes wall, that correspond to those that contains: (i) spermatogonia, (ii) spermatocytes (iii) spermatids (early and late), and the presence of sperm in the lumen of the seminiferous tubule. On the ultrastructural analysis, alterations were observed in Sertoli cells, with density loss of the cytoplasm and organelles, as well as, the appearence of structures with vesicular aspect. Moreover, alterations on interactions between Sertoli and germ cells and loss of cytoplasmic bridges between the germ cells. On the analysis of protein expression involved in cell stress, was observed after 144 h a significant increase in the inducible HSP70 protein, which is no observed at 360 h. In the quantification of FASL, involved in apoptosis induction, there was a significant increase in males exposed for 360 h. Although in this study has been recognized a FASL increasing, there was no observed modifications on expression of the investigated proteins that activate apoptosis (BCL2, BAK and Caspase 3). The adverse effects of glyphosate on the testicles, mainly on Sertoli cells, are an important indicative of the consequences on fish reproduction, which may include from the cellular, systemic and population levels. Finally, this study demonstrated the importance of investigating the glyphosate toxicity on male reproduction, using fish testicles as experimental model.

O GnRH é um decapeptídeo que está envolvido na reprodução, estimulando a hipófise a liberar gonadotropinas (LH e FSH), as quais regulam a esteroidogênese e gametogênese. Ainda, o GnRH age como neuromodulador atuando no comportamento sexual. A distribuição de suas isoformas pode ajudar a revelar a função específica de cada GnRH. A principal ênfase deste estudo foi detectar a presença e distribuição por imuno-histoquímica, e a expressão gênica de diferentes formas de GnRH no encéfalo de Astyanax altiparanae e Danio rerio, os quais têm importância comercial, ecológica e acadêmica. Em Astyanax altiparanae foi encontrado GnRH3 em diversos corpos celulares, inclusive em corpos celulares ligados à função reprodutiva, juntamente com fibras que inervam a neuro-hipófise. O GnRH1 foi encontrado apenas em fibras. Em Danio rerio foi encontrado GnRH3 nos núcleos da região hipotalâmica e em um grande número de fibras, incluindo as que inervam a neuro-hipófise. A expressão gênica de GnRH2 e 3 foi observada em Danio rerio.
GnRH is a decapeptide involved in reproduction, stimulating the pituitary to release gonadotropins, which, in turn, regulate the steroidogenesis and gametogenesis. In addition to the reproductive function, the GnRH displays neuromodulatory roles with implications in the modulation of sexual behavior. Therefore, the main emphasis of this study is to detect the presence, distribution, and gene expression of different forms of GnRH in the brain of the freshwater teleosts Astyanax altiparanae and Danio rerio, which have commercial, ecological and academic importance. The immunohistochemical method of peroxidase was used to detect GnRHs in the brain and pituitary. In A. altiparanae immunoreactivity to anti-GnRH3 was found in various cell bodies, including those related to reproductive functions, and fibers which innervate the neurohypophysis. Immunoreactivity for GnRH1 was found only in fibers. In D. rerio immunoreactivity to anti-GnRH3 was found in hypothalamic nuclei and in a large number of fibers, including the ones which innervate the neurohypophysis.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Titanium dioxide nanoparticles (TiO2-NP) are commonly used in many industrial activities. Consequently, the daily consumption by humans is estimated in 5.4 mg day, with an input in the environment of 4.2 mg day per person, receiving or not appropriated treatment before disposure. The cytotoxicity, genotoxicity and mutagenicity of TiO2-NP were investigated using the established fish cell line derived from zebrafish (Danio rerio) liver (i. e. ZF-L cells). Prior to the evaluation of nanoparticle’s toxic potential, a careful characterization was realized in culture medium in the presence or not of fetal bovine serum (FBS). Regarding to the characterization in terms of size was accessed using a transmission electron microscope (TEM), the agglomeration potential and surface charge were accessed by diameter light scattering (DLS) and zeta potential measurements, respectively, using a spectrophotometer. TiO2-NP in environmentally relevant concentrations were tested for cytotoxicity, genotoxicity and mutagenicity. Cell viability was accessed by four different tests, the trypan blue assay (membrane integrity), MTT reduction assay (mitochondria), neutral red retention assay (lysosomes) and finally, induction of apoptosis and necrosis. Genotoxicity was determined by observing the fragmentation of DNA by the comet assay, while mutagenicity was determined by Cytokinesis-block micronucleus technique. The characterization showed that the FBS was effective in dispersing the nanoparticles and prevent the formation of large agglomerates allowing robust responses on the real toxicity of NP. After 24 hours of treatment, there was cell membranes rupture, decreasing cell viability to 35.33%, at the highest concentration (1.0 μg mL-1). Mitochondrial metabolic activity remained unchanged, but it was possible to detect the proliferation of lysosomes, which was mainly attributed to the NP endocytosis. The induction of apoptosis was 50.4%, and necrosis was 13.9%, both in the concentration 1.0 μg mL-1 TiO2-NP. In the case of necrosis, a result 10 times greater than that presented by the negative control. Added necrosis and apoptosis indicated a decrease in cell viability to 35.7%. The comet test showed the fragmentation of the DNA, it was also possible to observe the formation of micronuclei, bridges and shoots demonstrated by the micronucleus assay. In general, this study demonstrated that TiO2-NP, after 24 hours of exposure, significantly affect cell viability and cause DNA damage, which may become irreversible. In conclusion, this study showed the cytotoxic, genotoxic and mutagenic potential of TiO2-NP for ZF-L cells. Mitochondrial and lysosome responses require further studies on the effect of TiO2-NP on these organelles.
Nanopartículas de dióxido de titânio (NP-TiO2) são comumente usadas em muitos produtos industriais. Por conseguinte, seu consumo diário por seres humanos é estimado em 5,4 mg dia-1, o que acarreta em um aporte no ambiente de 4,2 mg dia-1 por pessoa, podendo ou não receber um tratamento apropriado antes do seu despejo. Foram avaliadas a citotoxicidade, genotoxicidade e mutagenicidade de NPTiO2 para a linhagem permanente derivada de fígado de peixe-zebra (Danio rerio) (células ZF-L). Antes da avaliação do potencial tóxico das nanopartículas, foi realizada uma caracterização cuidadosa em meio de cultura com e sem a adição de soro fetal de bovino (SFB). A caracterização em termos do tamanho físico da nanopartícula (NP) foi realizada utilizando um microscópio eletrônico de transmissão (TEM); os tamanhos hidrodinâmicos dos aglomerados e as cargas de superfície foram acessados por medidas de DLS (diameter light scattering) e potencial zeta, respectivamente utilizando um espectrofotômetro. A viabilidade celular foi avaliada por três ensaios diferentes, o ensaio de exclusão do corante azul de tripano (integridade de membrana), ensaio de redução do sal MTT (atividade metabólica mitocondrial) e ensaio de retenção do corante vermelho neutro (viabilidade dos lisossomos). A indução de apoptose e necrose foi avaliada por citometria de fluxo. A genotoxicidade foi determinada pela observação da fragmentação do DNA por meio do ensaio do cometa, enquanto a mutagenicidade foi determinada pelo ensaio de micronúcleos com bloqueio da citocinese. A análise DLS mostrou que o SFB foi eficaz quanto à dispersão das nanopartículas e preveniu a formação de grandes aglomerados, o que permitiu a obtenção de respostas mais robustas relativas à toxicidade real das nanopartículas. Após 24 horas de tratamento, houve ruptura das membranas celulares diminuindo a viabilidade celular a 35,33%, na concentração mais elevada (1,0 μg mL-1). A atividade metabólica mitocondrial manteve-se inalterada, mas foi possível detectar a proliferação dos lisossomos, que foi atribuída principalmente à endocitose das NP. A indução de apoptose foi de 50,4%, e de necrose de 13,9%, ambos na concentração 1,0 μg mL-1 NP-TiO2. No caso da necrose, um resultado 10 vezes maior do que o apresentado pelo controle negativo. Necrose e apoptose somadas indicaram a diminuição da viabilidade celular a 35,7%. O teste do cometa mostrou a fragmentação do DNA, também foi possível observar a formação de micronúcleos, pontes e brotos demonstrados pelo ensaio do micronúcleo. Em geral, este estudo demonstrou que as NP-TiO2, após 24 horas de exposição, afetam significativamente a viabilidade celular e causam danos ao DNA, que podem se tornar irreversíveis. Em conclusão, este estudo mostrou o potencial citotóxico, genotóxicos e mutagênico das NP-TiO2 para células ZF-L. Respostas mitocondriais e dos lisossomos requerem novos estudos quanto ao efeito das NP-TiO2 sobre essas organelas.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
The objectives of this research were to investigate the agrotoxic most used in the Gramorezinho region in the green belt of Natal, and to evaluate the acute toxicity of these, based on the LC50-48h values estimated in tests for Danio rerio, internationally used as test organism. The acute toxicity tests were performed under laboratory conditions, according to standardized methods (ABNT/NBR/15088/04) for this species. The LC50-48h estimated to Tamaron BR was 352.89 mg.L-1, which characterizes that as practically non-toxic, according to toxicological classes cited by Zucker. For Decis 25EC, the LC50-48h estimated was 0.0004156 mg.L-1 (4.156 X 10-4 mg.L-1), which classifies it as highly toxic to this species
Os objetivos deste trabalho foram investigar os agrot?xicos mais utilizados na Regi?o de Gramorezinho Cintur?o verde da Grande Natal' e avaliar a toxicidade aguda dos mesmos, com base nos valores da CL50-48h estimados em testes com o peixe Danio rerio, organismo-teste eleito internacionalmente. Os testes de toxicidade aguda foram realizados em condi??es de laborat?rio, de acordo com a norma da Associa??o Brasileira de Normas T?cnicas (ABNT/NBR/15088/04) para esta esp?cie. A CL50-48h estimada para o Tamaron BR foi 352,89 mg.L-1, classificando-o como praticamente n?o-t?xico, de acordo com as classes toxicol?gicas citadas por Zucker. Para o Decis 25EC, a CL50-48h estimada foi 0,0004156 mg.L-1 (4,156 X 10-4 mg.L-1) que a classifica como altamente t?xica para esta esp?cie

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
Os transtornos de ansiedade apresentam a maior incidência na população mundial dentre os transtornos psiquiátricos, e a eficácia clínica das drogas ansiolíticas é baixa, em parte devido ao desconhecimento acerca das bases neuroquímicas desses transtornos. Para uma compreensão mais ampla e evolutivamente substanciada desses fenômenos, a utilização de espécies filogeneticamente mais antigas pode ser uma aproximação interessante no campo da modelagem comportamental; assim, sugerimos o uso do paulistinha (Danio rerio Hamilton 1822) na tentativa de compreender a modulação de comportamentos tipo-ansiedade pelo sistema serotonérgico. Demonstramos que os níveis extracelulares de serotonina no encéfalo de paulistinhas adultos expostos ao teste de preferência claro/escuro [PCE] (mas não ao teste de distribuição vertical eliciada pela novidade [DVN]) apresentam-se elevados em relação a animais manipulados mas não expostos aos aparatos. Além disso, os níveis teciduais de serotonina no rombencéfalo e no prosencéfalo são elevados pela exposição ao PCE, enquanto no mesencéfalo são elevados pela exposição ao DVN. Os níveis extracelulares de serotonina estão correlacionados negativamente com a geotaxia no DVN, e positivamente com a escototaxia, tigmotaxia e a avaliação de risco no PCE. O tratamento agudo com uma dose baixa de fluoxetina (2,5 mg/kg) aumenta a escototaxia, a tigmotaxia e a avaliação de risco no PCE, diminui a geotaxia e o congelamento e facilita a habituação no DVN. O tratamento com buspirona diminui a escototaxia, a tigmotaxia e o congelamento nas doses de 25 e 50 mg/kg no PCE, e diminui a avaliação de risco na dose de 50 mg/kg; no DVN, ambas as doses diminuem a geotaxia, enquanto somente a maior dose diminui o congelamento e facilita a habituação. O tratamento com WAY 100635 diminui a escototaxia nas doses de 0,003 e 0,03 mg/kg, enquanto somente a dose de 0,03 mg/kg diminui a tigmotaxia e a avaliação de risco no PCE. No DVN, ambas as doses diminuem a geotaxia, enquanto somente a menor dose facilita a habituação e aumenta o tempo em uma “base” (“homebase”). O tratamento com SB 224289 não produziu efeitos sobre a escototaxia, mas aumentou a avaliação de risco na dose de 2,5 mg/kg; no DVN, essa droga diminuiu a geotaxia e o nado errático nas doses de 2,5 e 5 mg/kg, enquanto a dose de 2,5 mg/kg aumentou a formação de “bases”. O tratamento com DL-para-clorofenilalanina (2 injeções de 300 mg/kg, separadas por 24 horas) diminuiu a escototaxia, a tigmotaxia e a avaliação de risco no PCE, aumentou a geotaxia e a formação de bases e diminuiu a habituação no DVN. Quando os animais são pré-expostos a uma “substância de alarme” co-específica, observa-se um aumento nos níveis extracelulares de serotonina associados a um aumento na escototaxia, congelamento e nado errático no PCE; os efeitos comportamentais e neuroquímicos foram bloqueados pelo pré tratamento com fluoxetina (2,5 mg/kg), mas não pelo pré-tratamento com WAY 100,635 (0,003 mg/kg). Animais da linhagem leopard apresentam maior escototaxia e avaliação de risco no PCE, assim como níveis teciduais elevados de serotonina no encéfalo; o fenótipo comportamental é resgatado pelo tratamento com fluoxetina (5 mg/kg). Esses dados sugerem que o sistema serotonérgico dessa espécie modula o comportamento no DVN e no PCE de forma oposta; que a resposta de medo produzida pela substância de alarme também parece aumentar a atividade do sistema serotonérgico, um efeito possivelmente mediado pelos transportadores de serotonina, e ao menos um fenótipo mutante de alta ansiedade também está associado a esses transportadores. Sugere-se que, de um ponto de vista funcional, a serotonina aumenta a ansiedade e diminui o medo em paulistinhas.
Anxiety disorders present the highest incidence in the world population among psychiatric disorders, and the clinical efficacy of anxiolytic drugs is low, partially due to lack of knowledge on the neurochemical bases of these disorders. To reach a more ample and evolutionarily grounded comprehension of these phenomena, the use of phylogenetically older species can be an interesting approach in the field of behavioral modeling; thus, we suggest the use of zebrafish (Danio rerio Hamilton 1822) in the attempt to understand the modulation of these behaviors by the serotonergic system. We demonstrate that extracellular serotonin levels in the brains of adult zebrafish exposed to the light/dark preference test [LDT] (but not to the novel tank test [NTT]) are increased in relation to animals which are handled, but not exposed to the apparatuses. Moreover, serotonin tissue levels levels in the hindbrain and forebrain are elevated by the exposure to the LDT, while tissue levels in the midbrain are elevated by exposure to the NTT. Extracellular serotonin levels correlate positively with scototaxis, thigmotaxis and risk assessment in the LDT and negatively with geotaxis in the NTT. Acute treatment with a low dose of fluoxetine (2.5 mg/kg) increases scototaxis, thigmotaxis, and risk assessment in the LDT, and decreases geotaxis and freezing and facilitates habituation in the NTT. Treatment with buspirone decreases scototaxis, thigmotaxis and freezing at 25 and 50 mg/kg in the LDT and decreases risk assessment at 50 mg/kg; in the NTT, both doses decrease geotaxis, while the highest dose decreases freezing and facilitates habituation. Treatment with WAY 100635 decreases scototaxis at 0.003 and 0.03 mg/kg, while only the highest dose decreases thigmotaxis and risk assessment in the LDT. In the NTT, both doses decrease geotaxis, while only the lower dose facilitates habituation and increases homebase time. Treatment with SB 224289 did not alter scototaxis, but increased risk assessment at 2.5 mg/kg; in the NTT, this drug decreased geotaxis and decreased erratic swimming at 2.5 and 5 mg/kg, while at 2.5 mg/kg it increased homebase time. Treatment with DL-para-clorophenylalanine (2 x 300 mg/kg injections, separated by 24 h) decreased scototaxis, thigmotaxis and risk assessment in the LDT, and increased geotaxis and homebase time and decreased habituation in the NTT. When animals were pre-exposed to a conspecific “alarm substance”, extracellular serotonin levels were raised in association with an increase in scototaxis, freezing and erratic swimming in the LDT; both behavioral and neurochemical effects were blocked by pre-treatment with fluoxetine (2,5 mg/kg), but not with WAY 100,635 (0,003 mg/kg). Animals from the leopard strain show increased scototaxis and risk assessment in the LDT, as well as increased 5-HT tissue levels in the encephalon; the behavioral phenotype is rescued by treatment with fluoxetine (5 mg/kg). These data suggest that the serotonergic system of zebrafish modulates behavior in the LDT and NTT in opposite ways; that the fright response produced by alarm substance seems to increase serotonergic activity, an effect which is possibly mediated by serotonin transporters; and that at least one high-anxiety mutant phenotype is associated with serotonin uptake. It is thus suggested that from a functional point of view serotonin increases anxiety and decreases fear in zebrafish.

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A alteração do comportamento animal pode ser interpretada como a resposta da interação de processos fisiológicos com estímulos ambientais sendo, desta forma, uma ferramenta eficiente para detecção de contaminantes relacionados a efeitos tóxicos. Neste estudo foram realizados testes comportamentais utilizando a atividade natatória do peixe Danio rerio como bioindicador de concentrações subletais. (...) O biomonitoramento em tempo real em laboratório mostrou-se um método não-invasivo e prático e poderá ser utilizado para analisar a toxicidade crônica de efluentes industriais ou substâncias químicas específicas em laboratório, pois tem sensibilidade elevada comparável com outros testes toxicológicos e gera respostas rapidamente

Esta tesis tiene como objetivo investigar los efectos del etanol y metales pesados (cadmio y mercurio) en los patrones diarios de dos especies acuáticas clave: pez cebra (Darío rerio) y anfípodo gammarus (G. aequicauda y G. chevreuxi). La investigación se realizó en dos niveles organizativos: comportamental y molecular. Con este fin, hemos establecido cuatro objetivos específicos: 1. Investigar los efectos al etanol en el pez cebra en función en su ritmo circadiano. 2. Determinar la existencia de un ritmo diario de la toxicidad del cadmio en el pez cebra. 3. Caracterizar en Gammarus chevreuxi los efectos en la actividad locomotora debido a sedimentos contaminados con mercurio. 4. Evaluar la calidad ambiental de los sedimentos de la cuenca sur del Mar Menor influenciados por históricas actividades mineras. Por lo tanto, estos nuevos datos tiene la intención de señalar la importancia de aplicar un enfoque multidisciplinario para estudios toxicológicos, así como elevar la importancia de la elección del momento adecuado para realizar el ensayo de toxicidad Metodología Los experimentos llevados a cabo en la presente investigación siguieron la legislación española sobre protección de animales y prácticas de laboratorio y fue aprobado tanto por el Comité Nacional de Protección de los Animales y el Comité de Bioética de la Universidad de Murcia - Los muestreo de crustáceos se realizaron en las zonas próximas a la costa mediante el uso de redes de 5 µ de luz de maya. Y su trasporte con aireación y refrigeración . Los muestreos de pez cebra se realizaron en las cámaras del departamento de Fisiología animal.. - El etanol se adquirió a través de distribuidores comerciales y los sedimentos tóxicos directamente de las zonas de estudio mediante muestreos con barca. La extracción de los sedimentos se realizó utilizando cores y draga. Se almacenaran en refrigeradores a una temperatura de 4ºC. - Los test de mortalidad y comportamiento se realizaran en cámaras herméticas donde se permite el control de las condiciones de fotoperiodo y temperatura. - Las condiciones del agua antes y después de las exposiciones se medió con electrodos de pH y Oxígeno. - Lo análisis de la actividad ante exposiciones subletales se realizaron en las mismas cámaras y mediante la utilización de equipos de filmación sensibles a las luces infrarrojas, permitiendo así mantener las condiciones de oscuridad en los experimento que sea necesarios. - Determinación de metales pesados en tejidos, utilizamos La Espectroscopia de Emisión en Plasma Acoplado Inductivamente (ICP-AES) con Potencia RF: 1200W, Velocidad de bomba: 30r.p.m., Flujo de gas aux.:0.5l/min, Flujo de gas de nebulización: 0.5 l/min, Flujo de gas de refrigeración: 14 l/min, Flujo de gas de purga: Normal. - Para el estudio de expresión de diferentes genes realizamos la técnica de PCR cuantitativa utilizando SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) y un aparato ABI Prism 7500 (Applied Biosystems, Foster City, CA). Conclusiones 1. La exposición de pez cebra mediante baño a etanol a diferentes horas del día muestra un ritmo de toxicidad que refleja una mayor tasa de mortalidad en larvas durante el día comparado con los efectos durante la noche. Así mismo en adultos de pez cebra se observan efectos en su actividad natatoria y posición en la columna de agua siendo significativamente más severos durante la fase de luz. 2. La exposición de pez cebra mediante baño a cadmio durante diferentes horas del día reflejó un incremento de la mortalidad y acumulación del cadmio en tejidos durante la mitad de la fase de luz, así como una mayor reducción de la actividad locomotora cuando el pez es expuesto a concentraciones subletales, además la expresión de mt1 muestra un pico antes al final de la fase de luz. Todos estos resultados indican que el pez cebra es más vulnerable al cadmio durante la fase de luz que en la fase de oscuridad. 3. La exposición de Gammarus chevreuxi mediante sedimentos contaminados con mercurio mostró un efecto deletéreo en la actividad natatoria incluso en concentraciones consideradas no letales. Dichos efectos fueron más acusados durante el la fase de luz, que corresponde con la más activa del animal. 4. Nuestros resultados sugieren que la actividad locomotora y el número de surfacings se presentan cono una señal temprana de la interacción de los factores co-estresantes temperatura y sedimentos contaminados. Los parámetros comportamentales estudiados en Gammarus. aequicauda y Gammarus. chevreuxi tienen una conexión clara con la supervivencia de los organismos y pueden tener una relevancia ecológica.
This thesis aims at investigating the effects of Ethanol and heavy metals (cadmium and mercury) on the daily patterns of two key aquatic species: zebrafish (Dario rerio) and amphipod gammarus (G. aequicauda and G. chevreuxi). The research was conducted in two organizative levels: Physiology behavior and molecular. With this purpose we have established four particular objectives: 1. To investigate time-dependent effects of ethanol exposure in zebrafish. 2. To determine the existence of a daily rhythm of cadmium toxicity in zebrafish. 3. To characterise the effects of locomotor activity to mercury-contaminated sediments in Gammarus chevreuxi. 4. To evaluate the environmental quality of the soft-bottoms from the southern basin of the Mar Menor lagoon influenced by historical mining activities. Hence, this new data intends to point out the importance of applying a multidisciplinary approach to toxicology studies, and also raise the importance of the time experimental setting of toxicity test. Methodology The experiments performed in the present research followed Spanish legislation on Animal Welfare and Laboratory Practices and was approved by both the National Committee on Animal Welfare and the Bioethics Committee of the University of Murcia. - The sampling of gammarids were conducted in the nearshore areas by using mesh of 5 μ. Their transportation was with aeration and cooling. The zebrafish samplings were performed in Animal Physiology's chambers of the department. - Ethanol was purchased by authorized distributors, and toxic sediments were extracted directly from the study areas by boat sampling. Sediments extraction was with cores and dredge. They were stored in the refrigerator at 4 ° C. - The test of mortality and behavior was conducted in chambers where control of the photoperiod and temperature conditions allowed. - Water conditions before and after exposure was measured with pH electrodes and oxygen. - The analysis of activity at sublethal exposures were conducted in the same chamber and using equipment sensitive to infrared light filming, thereby maintaining the conditions of darkness in the experiment is necessary. - Determination of heavy metals in tissues, we used emission spectroscopy Inductively Coupled Plasma (ICP-AES) RF Power: 1200W, pump speed: 30r.pm, aux gas flow: 0.5l / min gas flow. Misting: 0.5 l / min, the cooling gas flow: 14 l / min, purge gas flow: Normal. - Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and an ABI Prism 7500 apparatus (Applied Biosystems, Foster City, CA). Conclusions 1. Exposure of zebrafish using ethanol bath at different times of day shows a rate of toxicity reflecting a higher rate of mortality in larvae during the day compared to the effects overnight. Also effects in adult zebrafish are observed in their swim and activity position in the water column to be significantly more severe during the light phase. 2. Exposure zebrafish using cadmium bath during different times of day reflected an increase in mortality and cadmium accumulation in tissues during half of the light phase and a further reduction of locomotor activity when the fish is exposed to sublethal concentrations. Expression of mt1 shows a peak before the end of the light phase. All these results indicate that zebrafish are more vulnerable to cadmium during the light in the dark phase. 3. The exposure of Gammarus chevreuxi by mercury-contaminated sediments showed a deleterious effect on the swimming activity even considered non-lethal concentrations. These effects were most pronounced during the light phase, which corresponds to the most active animal. 4. Our results suggest that the locomotor activity and the number of surfacings an early sign of the interaction of co-temperature stressors and contaminated sediments occur cone. The behavioral parameters studied in Gammarus. aequicauda and Gammarus. chevreuxi have a clear connection to the survival of organisms and can have an environmental relevance.

Στην παρούσα εργασία, για πρώτη φορά, τίθεται η πιο πρόσφατη εισαχθείσα τεχνολογία των –omics, η μεταβολομική, στην υπηρεσία της μοριακής βιολογίας για την μελέτη της θερμοκρασιακής μεταβολικής πλαστικότητας. Με τον όρο θερμοκρασιακή μεταβολική πλαστικότητα αναφερόμαστε στην ιδιότητα ενός γονότυπου να παράγει ποικίλους φαινότυπους σε επίπεδο μεταβολισμού, ως απόκριση στις θερμοκρασιακές συνθήκες στις οποίες υπόκειται. Σκοπός, λοιπόν της παρούσας ερευνητικής εργασίας ήταν η μελέτη της επίδρασης της πρώιμης θερμοκρασίας ανάπτυξης στο μεταβολικό πρότυπο ενήλικων θηλυκών και αρσενικών zebrafish σε γονάδες, εγκέφαλο και μυϊκό ιστό. Στα πλαίσια του σκοπού αυτού, τρεις πληθυσμοί zebrafish τοποθετήθηκαν στους 22οC, 28οC και 32οC από 0-20 ημερών, κατά την 20η ημέρα και οι τρεις πληθυσμοί αναπτύχθηκαν σε κοινή θερμοκρασία 28οC μέχρι την πάροδο εφτά μηνών, οπότε και πραγματοποιήθηκε δειγματοληψία. Ακολούθησε η ανάκτηση των μεταβολικών προτύπων, μετά την αριστοποίηση του πρωτοκόλλου για κάθε ιστό, χρησιμοποιώντας αέριο χρωματογράφο-φασματόμετρο μάζας. Τα αποτελέσματα της πολυπαραμετρικής στατιστικής ανάλυσης έδειξαν να μη διαφαίνεται θερμοκρασιακή μεταβολική πλαστικότητα στις γονάδες και στο μυϊκό ιστό, μιας και η ομαδοποίηση των μεταβολικών προτύπων έγινε ως προς το φύλο. Συγκεκριμένα και στις δυο κατηγορίες ιστών παρατηρήθηκε μεγάλη μείωση της μεταβολικής ενεργότητας στα θηλυκά ως προς τα αρσενικά zebrafish. Εξαιρετικής σημασίας, όμως, είναι το γεγονός ότι, για πρώτη φορά, αποδείχθηκε η επίδραση της πρώιμης θερμοκρασίας ανάπτυξης, στο μεταβολικό πρότυπο ενήλικων θηλυκών και αρσενικών zebrafish μέσω της δραματικής μείωσης της ενεργότητας του κεντρικού μεταβολισμού των εγκεφάλων 22ο C και 32ο C ως προς τους 28ο C.
With the term temperature induced metabolic plasticity one refers to the feature of genotype to produce different phenotypes on a metabolic level, due to the temperature conditions of its environment. The aim of this thesis is to study the effect of temperature on the metabolic profile during the early growth stage of adult male and female zebrafish in three tissues: gonads, brain and muscle. For this end three populations of zebrafish were bread at 22οC, 28οC and 32οC from 0 to 20 post-fertilization (pf) days. After the 20th pf day all populations were bread at 28οC for seven months. The metabolic profiles of all tissues were acquired using gas chromatography-mass spectrometry; in this thesis the protocol of metabolic profile acquisition was optimized for each tissue separately. Multivariate statistical analysis of the profiles showed no temperature plasticity in gonads and muscles. However, for the first time worldwide it was shown temperature induced plasticity in the brains metabolism of adult male and female zebrafish. Characteristically, a decline of the metabolic activity was observed in the 22ο C and 32ο C grown zebrafish compared to the 28ο C grown.

Ο όρος φαινοτυπική πλαστικότητα χαρακτηρίζει την ιδιότητα ενός γονότυπου να παράγει ποικίλους φαινότυπους, ως απόκριση στις περιβαλλοντικές συνθήκες στις οποίες υπόκειται (Pigliucci et al. 2006). Αυτή η απόκριση μπορεί να εκφράζεται σε μορφολογικό, βιοχημικό, φυσιολογικό ή αναπτυξιακό επίπεδο (οντογενετική πλαστικότητα), καθώς και στα πρότυπα συμπεριφοράς. Δεδομένης της σημασίας της φαινοτυπικής πλαστικότητας για τις λειτουργικές αποκρίσεις των ατόμων και των πληθυσμών (π.χ. αναλογία φύλου και πληθυσμιακή δομή), η μελέτη του φαινομένου στην ομάδα των ψαριών θεωρείται σημαντική τόσο για τους φυσικούς πληθυσμούς, σε οικολογικό ή εξελικτικό χρόνο, όσο και για τους εκτρεφόμενους. Η ολοκληρωμένη μελέτη της φαινοτυπικής πλαστικότητας, με έμφαση στους υποκείμενους μοριακούς και αναπτυξιακούς μηχανισμούς, μπορεί να δώσει πιο τεκμηριωμένες απαντήσεις στα ερωτήματα που σχετίζονται με τον τρόπο δράσης του φαινομένου στην οικολογία και στην εξέλιξη των ειδών, αλλά και σε πιο πρακτικά ζητήματα, όπως αυτά που αφορούν την εκτροφή ψαριών. Ο κύριος περιβαλλοντικός παράγοντας, υπεύθυνος για την εμφάνιση φαινοτυπικής πλαστικότητας στα ψάρια, φαίνεται ότι είναι η θερμοκρασία ανάπτυξης, η οποία έχει βρεθεί ότι επιδρά στο μεταβολισμό, την ανάπτυξη (Johston 1996, Stickland et al. 1998, Guderley 2004) και τη δομή των μυών (Guderley and Johston 1996, Ochi and Westerfield 2007), τους μεριστικούς χαρακτήρες (Lindsey 1998, Georgakopoulou et al. 2007), το σχήμα του σώματος (Loy et al. 1996, Georgakopoulou et al. 2007), την κολυμβητική ικανότητα (Fuiman and Batty 1997) και την αναλογία φύλου (Pavlidis et al. 2000, Koumoundouros et al. 2002a). Η θερμοκρασία ανάπτυξης, φαίνεται ότι επηρεάζει σημαντικά το ρυθμό αύξησης και διαφοροποίησης των ψαριών, τροποποιώντας το σωματικό μέγεθος όπου επιτελούνται τα διάφορα αναπτυξιακά γεγονότα, φαινόμενο γνωστό ως οντογενετική πλαστικότητα (Koumoundouros et al. 2001, Sfakianakis et al. 2004). Στην παρούσα εργασία εξετάστηκε η επίδραση της θερμοκρασίας πρώιμης ανάπτυξης, στη φαινοτυπική πλαστικότητα του είδους Danio rerio. Εξετάστηκε η αναλογία του φύλου και το σχήμα του σώματος των ψαριών, καθώς και ο ρυθμός ανάπτυξης των νυμφών και των ιχθυδίων κατά τη διάρκεια της εφαρμογής των διαφορετικών θερμοκρασιακών συνθηκών. Παράλληλα έγινε μια προσπάθεια προσδιορισμού του ευαίσθητου στην επίδραση της θερμοκρασίας, οντογενετικού σταδίου. Σύμφωνα με τον πειραματικό σχεδιασμό που ακολουθήθηκε, εξετάστηκε η επίδραση της θερμοκρασίας κατά τη διάρκεια δύο διαφορετικών πρώιμων οντογενετικών περιόδων, διάρκειας 280οd η κάθε 100 μία (28-308οd και 280-560οd). Στην πρώτη οντογενετική περίοδο, τα έμβρυα αφέθηκαν για είκοσι τέσσερις ώρες στους 28οC και στη συνέχεια διαχωρίστηκαν σε τρεις πληθυσμούς ψαριών οι οποίοι υποβλήθηκαν σε τρεις διαφορετικές θερμοκρασιακές συνθήκες (22, 28, 32οC) για μια περίοδο 280οd (28-308od, πρώτη υπό εξέταση οντογενετική περίοδος, ΟΠ1). Στη δεύτερη οντογενετική περίοδο, τα έμβρυα και οι νύμφες διατηρήθηκαν σε κοινές συνθήκες (28οC) μέχρι την 10η ημέρα μετά τη γονιμοποίηση. Στη συνέχεια διαχωρίστηκαν σε τρεις πληθυσμούς ψαριών, οι οποίοι υποβλήθηκαν σε διαφορετικές θερμοκρασιακές συνθήκες (22, 28 και 32οC) μέχρι την 560οd (280-560od, δεύτερη υπό εξέταση οντογενετική περίοδος, ΟΠ2). Και στις δύο περιπτώσεις, μετά το πέρας της περιόδου των 280οd με διαφορετική θερμοκρασιακή επίδραση, η ανάπτυξη πραγματοποιήθηκε σε κοινές συνθήκες (28οC). Τα πειράματα πραγματοποιήθηκαν εις διπλούν. Όλοι οι πειραματικοί πληθυσμοί, προήλθαν από κοινό απόθεμα αυγών. Η διατήρηση και η εκτροφή των νυμφών και των ενήλικων ψαριών πραγματοποιήθηκε βάσει μεθοδολογίας που περιγράφεται στο «The Zebrafish Book» (Westerfield 1995). Το φύλο των ενήλικων ατόμων προσδιορίστηκε μακροσκοπικά, έχοντας ως βάση τους χαρακτήρες της διογκωμένης κοιλιάς των θηλυκών ατόμων και του κίτρινου χρώματος των αρσενικών (Νεοφύτου 2003). Προκειμένου να επιβεβαιωθεί ο μακροσκοπικός προσδιορισμός του φύλου, ακολούθησε ο εγκλεισμός τριάντα συντηρημένων δειγμάτων (Εγκλεισμός σε Τechnovit 7100, τμήση 1-3 μm, χρώση με Polychrome I και II κια μικροσκοπική παρατήρηση των τομών). Για τη μελέτη της επίδρασης της θερμοκρασίας ανάπτυξης στην εξωτερική μορφολογία του σώματος των ενήλικων zebrafish, επιλέχθηκαν τυχαία 30 άτομα ανά φύλο και πειραματικό πληθυσμό, τα οποία υποβλήθηκαν σε ανάλυση γεωμετρικής μορφομετρίας. Με την ίδια μέθοδο αναλύθηκε και το σχήμα του σώματος των νυμφών της πρώτης και της δεύτερης υπό εξέταση οντογενετικής περιόδου, κατά το τέλος της εφαρμογής των διαφορετικών θερμοκρασιακών συνθηκών, στις 308od και στις 560od, αντίστοιχα. Στις νύμφες της πρώτης οντογενετικής περιόδου, ελήφθησαν επιπλέον δείγματα δέκα ημέρες μετά από το τέλος της εφαρμογής των θερμοκρασιακών συνθηκών. Για τη μελέτη του ρυθμού διαφοροποίησης, ελήφθησαν δείγματα νυμφών από κάθε πειραματικό πληθυσμό, μετά από τη λήξη της εφαρμογής των διαφορετικών θερμοκρασιακών συνθηκών. Συγκεκριμένα, από τους πληθυσμούς της ΟΠ1 δείγματα ελήφθησαν στις 588°d, ενώ από τους πληθυσμούς ΟΠ2, στις 560°d. Επίσης, εξετάστηκαν οι μεριστικοί χαρακτήρες των νυμφών της πρώτης και της δεύτερης οντογενετικής περιόδου, αφού προηγήθηκε διπλή χρώση των δειγμάτων με 101 Αλιζαρίνη (Alizarin Red S) και Κυανό της Αλσατίας (Alcian Blue) (Park and Kim 1984). Τα δείγματα φωτογραφήθηκαν ατομικά και μετρήθηκε το μεσουραίο μήκος (FL) του κάθε ατόμου. Στη συνέχεια, μετρήθηκε (1) ο αριθμός των κοιλιακών πλευρών, (2) ο αριθμός των πτερυγιοφόρων και των λεπιδοτριχίων του ραχιαίου και εδρικού πτερυγίου, και (3) ο αριθμός των δερματοτριχίων και των λεπιδοτριχίων του ουραίου πτερυγίου. Ο έλεγχος των διαφορών της αναλογίας φύλου μεταξύ των διαφορετικών πληθυσμών, έγινε με G-test (Sokal and Rohlf 1981). Για τη σύγκριση του σχήματος μεταξύ των δύο φύλων και των διαφορετικών πειραματικών πληθυσμών, εφαρμόστηκε ανάλυση κανονικών συνιστωσών (Canonical Variate Analysis, λογισμικό Statistica, έκδοση 6.0). Τέλος, ο έλεγχος των διαφορών του μέσου μήκους του σώματος μεταξύ των διαφορετικών πληθυσμών, πραγματοποιήθηκε με Mann-Whitney test (μη παραμετρικός έλεγχος), αφού προηγήθηκε ο έλεγχος ομοιοσκεδαστικότητας και κανονικής κατανομής. Η επίδραση της θερμοκρασίας στην αναλογία φύλου αποδείχθηκε στατιστικά σημαντική μόνο κατά την πρώτη οντογενετική περίοδο (p < 0.05, G-test), και όχι κατά τη δεύτερη (p > 0.05, G-test). Ο κατά ζεύγη έλεγχος των διαφορών στην αναλογία φύλου μεταξύ των διαφορετικών θερμοκρασιακών συνθηκών, έδειξε στατιστικά σημαντική επίδραση στην αναλογία φύλου μόνο μεταξύ των 22 και 28οC της ΟΠ1 (28-308οd), όπου η μέση συχνότητα των θηλυκών ατόμων ευνοείται στους 22°C (52,3% έναντι του 37,0% των 28°C, p < 0.05, G-test). Η ίδια συχνότητα εμφανίστηκε αυξημένη και στους 32°C (44,9% έναντι του 37,0% των 28°C), χωρίς ωστόσο να επιβεβαιώνεται και στατιστικά (p > 0.05, Gtest). Σε ότι αφορά τη δεύτερη οντογενετική περίοδο (280-560°d), η μέση συχνότητα των θηλυκών ατόμων στους 22°C (51,9%) και στους 32°C (46,1%), εμφανίζεται αυξημένη σε σχέση με αυτή των 28°C (44,5%), χωρίς ωστόσο οι διαφορές αυτές να είναι στατιστικά σημαντικές (p > 0.05, G-test). Η ύπαρξη μη στατιστικά σημαντικής διαφοράς της αναλογίας φύλου μεταξύ των 28 και 32°C και στατιστικά σημαντικής διαφοράς μεταξύ των 22 και 28°C, υποδεικνύει ότι το πρότυπο απόκρισης της αναλογίας φύλου στην θερμοκρασία πρώιμης ανάπτυξης εμφανίζει ανεξαρτησία στο εύρος 28-32°C και θηλυκοποίηση στους 22°C. Σε όλα τα οντογενετικά παράθυρα που εξετάστηκαν και σε όλες τις πειραματικές επαναλήψεις, τα αποτελέσματα της ανάλυσης των κανονικών συνιστωσών, έδειξαν ότι το φύλο και η θερμοκρασία πρώιμης ανάπτυξης επέδρασαν σημαντικά στο σχήμα του σώματος των ενήλικων ατόμων zebrafish. Η κατά φύλο γεωμετρική ανάλυση του σχήματος, έδειξε ότι η θερμοκρασία ανάπτυξης κατά την ΟΠ1 (28-308od) και ΟΠ2 (280-560od) επιδρά σημαντικά στο σχήμα 102 του σώματος και των δύο φύλων, με τους τρεις πληθυσμούς κάθε οντογενετικής περιόδου και επανάληψης να διαχωρίζονται πλήρως μεταξύ τους κατά μήκος των δύο κανονικών μεταβλητών. Η εξέταση του σχήματος του σώματος των νυμφών της ΟΠ1 (28-308οd), στις 308οd και στις 588 οd, και της ΟΠ2 (280-560οd), στις 560οd, έδειξε ότι η θερμοκρασία ανάπτυξης επηρέασε το σχήμα του σώματός τους. Σε όλες τις, οι διαφορές στο σχήμα οφείλονται τόσο στις ομοιόμορφες συνιστώσες του σχήματος, όσο και στις μη ομοιόμορφες, με τις πρώτες να συμβάλλουν στη νωτοκοιλιακή διεύρυνση ή συμπίεση του σώματος των νυμφών, ανάλογα με την περίπτωση. Ενώ όμως η επίδραση της θερμοκρασίας στο σχήμα του σώματος είναι σημαντική, δε φαίνεται να υπάρχει κάποιο καθορισμένο – σταθερό πρότυπο επίδρασης των κανονικών συνιστωσών. Αυτό πιθανότατα οφείλεται στο ότι δεν είχαν φτάσει όλες οι νύμφες στο ίδιο στάδιο ανάπτυξης κατά τις ημέρες των δειγματοληψιών. Τα δείγματα των νυμφών που λήφθηκαν μετά την έκθεση των πληθυσμών στις τρεις διαφορετικές θερμοκρασιακές συνθήκες, κατά την πρώτη (28-308οd) ή τη δεύτερη (280- 560οd) οντογενετική περίοδο, διαφοροποιήθηκαν σημαντικά ως προς το μέσο μεσουραίο μήκος (FL) των ατόμων. Συγκεκριμένα, η έκθεση στους 22οC οδήγησε σε σημαντική μείωση του FL των ατόμων (p<0.05, Mann Whitney U-test), παρά το ότι τα δείγματα λήφθηκαν στο ίδιο θερμικό άθροισμα. Τα αποτελέσματα έδειξαν, ότι η ανάπτυξη των ψαριών που αφέθηκαν στους 22οC, κατά τη δεύτερη υπό εξέταση οντογενετική περίοδο (280-560οd), καθυστέρησε σε σχέση με τους πληθυσμούς των 28 και 32οC. Αυτό φαίνεται από τα διαγράμματα που αφορούν τον αριθμό των πτερυγιοφόρων και των ακτινών του ραχιαίου πτερυγίου, τον αριθμό των πτερυγιοφόρων και των ακτινών του εδρικού πτερυγίου, καθώς και τον αριθμό των κοιλιακών πλευρών. Συγκεκριμένα, η ολοκλήρωση της ανάπτυξης των πτερυγιοφόρων του ραχιαίου πτερυγίου, των ακτινών του εδρικού και των κοιλιακών πλευρών, καθυστερεί στους 28οC, σε σχέση με τους 32 οC. Η εμφάνιση των ακτινών του ραχιαίου πτερυγίου, καθυστερεί στους 28 οC, σε σχέση με τους 32 οC. Τέλος, η εμφάνιση των πτερυγιοφόρων του εδρικού πτερυγίου, καθυστερεί στους 22 οC, σε σχέση με τους 28 οC. Σε ότι αφορά τα άτομα της πρώτης οντογενετικής περιόδου, οι χαρακτήρες που μελετήθηκαν είτε είχαν εμφανιστεί νωρίτερα από τη λήψη των δειγμάτων, είτε είχε ολοκληρωθεί αργότερα η ανάπτυξή τους.
The term phenotypic plasticity characterizes the property of a genotype to produce different phenotypes in response to distinct environments (Pigliucci et al. 2006). This response can express itself in a morphological, biochemical, physiologic or developmental level (developmental plasticity), or even through changes in the models of behavior. Due to its great importance among individuals in a population at a certain time period or in future generations (e.g. proportion of sex and demographic structure), the study of the phenotypic plasticity in the group of fish is considered significant not only for the natural populations, at the ecological or evolutionary level, but for the cultured populations too. The complete study of phenotypic plasticity, including the underlying molecular and developmental mechanisms, can answer questions related with the way the phenomenon acts in the ecology and species development, but also in more practical applications, such as those that concern aquaculture. One of the most important environmental factors responsible for the appearance of plasticity in fish appears to be the developmental temperature, which affects metabolism, muscle growth (Johston 1996, Stickland et al. 1998, Guderley 2004) and structure (Guderley and Johston 1996, Ochi and Westerfield 2007), meristic characters (Lindsey 1998, Georgakopoulou et al. 2007), body shape (Loy et al. 1996, Georgakopoulou et al. 2007), swimming performance (Fuiman and Batty 1997) and sex ratio (Pavlidis et al. 2000, Koumoundouros et al. 2002a). The developmental temperature influences considerably growth rate and differentiation of fish, modifying the body size as development proceeds. This phenomenon is known as developmental plasticity (Koumoundouros et al. 2001, Sfakianakis et al. 2004). The present study examined the effect of precocious developmental temperature, in phenotypic plasticity of Danio rerio. The Sex ratio and body shape of fish was analyzed, as well as the growth rate of larvae and juveniles during the application of different temperature conditions. At the same time the most sensitive developmental period in the effect of temperature was determined. According to the experimental design, the effect of temperature was examined during two different early developmental periods of 280°d, each (28-308°d and 280-560°d). Two experimental groups of fish were subjected in duplicate to three different temperatures. More specifically, in the first developmental period the embryos were left for twenty four hours in 28°C and then separated in three populations, which were submitted to three different temperature conditions (22, 28 and 104 32°C) for a period of 280°d (28-308°d, first developmental period DP1). In the second developmental period the embryos and the larvae were maintained under common conditions (28°C) up to the 10th day post fertilization. Then they were separated in three populations, which were submitted to three different temperature conditions (22, 28 and 32°C) up to the 560th °d (280-560°d, second developmental period DP2). In both developmental periods that were examined, after the end of the 280°d of temperature effect, fish growth was completed under common conditions (28°C). All the eggs of the experimental populations were obtained from a broodstock of the same genetic origin. The maintenance and the culture of larvae and adult fish were as described in "The Zebrafish Book" (Westerfield 1995). The sex of adult individuals was determined macroscopically using the characters of the swelled abdomen of females and the yellow color of males (Neophytou 2003). In order to confirm the macroscopic determination of sex, thirty fixed samples of zebrafish were enclosed for histology sections (Technovit 7100, sections of 1-3μm and staining with Polychrome I and II) and microscopy. To study the temperature effect on growth and body shape of adult zebrafish, 30 individuals per sex and experimental population were selected randomly and were submitted in geometric morphometrics analysis. The body shape of the larvae of the first and the second developmental period, was analyzed with the same method, at the end of the different temperature conditions, in 308°d and 560°d, respectively. Additional samples were selected in the first developmental period, ten days after the end of the three different temperature regimes. To study the temperature effect on growth rate and differentiation, samples of larvae were obtained from each experimental population, following completion of the different temperature periods. From the populations of DP1, samples were taken at 588°d, while from DP2 samples were obtained at 560°d. The meristic characters of the larvae from the first and second developmental period were examined, after staining of the samples with Alizarin Red and Alcian Blue (Park and Kim 1984). Larvae were photographed and the fork length of each individual was measured (FL). The meristic characters that were examined are (1) the number of ribs (2) the number of pterygiophors and lepidotrichia of the dorsal and the anal fin, and (3) the number of pterygiophors and lepidotrichia of the caudal fin. Sex ratio differences between populations were studied with G-test (Sokal and Rohlf 1981). Canonical Variate Analysis was applied for the comparison of the body 105 shape between the two sexes and between the different experimental populations (Statistica, 6.0). Finally, the differences of medium total length between the different populations was examined with the Mann-Whitney test (non parametric). The sex ratio was significantly affected only at the first developmental period (p < 0.05, G-test), and not at the second (p > 0.05, G-test). The paired control of changes in the sex ratio between the different temperature conditions at DP1 (28-308°d), showed a significant effect only between the populations of 22°C and 28°C (52.3% females in 22°C vs. 37.0% in 28°C, p < 0.05, G-test). The female/male frequency was also increased in the 32°C group (44.9% females in 32°C vs. 37.0% females in 28°C), without however being confirmed as statistically significant (p > 0.05, G-test). Regarding the second developmental period (280-560°d), the medium frequency of the female individuals in the 22°C (51.9%) and 32°C (46.1%) groups, was also increased compared to 28°C (44.5%), although these differences were not confirmed as statistically significant (p > 0.05, G-test). The significant differences in sex ratio between the 22 and 28°C, and the non significant differences between 28 and 32°C indicate that the model of response in sex ratio due to the different developmental temperature shows autonomy in the range of 28-32°C and feminization at 22°C. In both ontogenetic windows that were examined and in all experimental repetitions, the results of the canonical variables showed that sex and developmental temperature significantly affected the bodyshape of the adult individuals. The body shape analysis in accordance with the gender of the individuals, was significantly affected from the developmental temperature at DP1 (28-308°d) and DP2 (280-560°d). The three populations of each ontogenetic period were totally separated amongst them, along the two axes of canonical variables CV1 and CV2. The body shape analysis of the larvae at DP1 (28-308οd), in 308οd and in 588 οd, and at DP2 (280-560οd), in 560οd, indicates that developmental temperature significantly affected their body shape. In all cases, the differences in body shape are contributed from uniform, as much as from non uniform components of shape. Despite the significant effect of developmental temperature in larval body shape, there is not a stable model of the way that canonical variables effect, probably because larvae were not at the same stage of growth during sampling. The total (TL) or the fork length (FL) of the larvae that were analyzed following development at the three different temperatures during the first (28-308°d) or the second (280-560°d) developmental period, was significantly differentiated. The exposure at 22°C 106 led to important reduction of the individuals FL (p < 0.05, Mann-Whitney test), despite the fact that all samples were of the same age. These results showed that the growth of fish that were submitted to 22°C during DP2 (280-560°d) was delayed relative to the populations at 28 and 32°C. This is noticeable from the diagrams that refer to the number of pterygiophors and lepidotrichia of the dorsal fin, the number of pterygiophors and lepidotrichia of the anal fin and the number of ribs. The completion of growth of pterygiophors of the dorsal fin, of lepidotrichia of the anal fin and the abdominal ribs, are also delayed at 28°C vs. 32°C. The appearance of lepidotrichia of the dorsal fin is delayed at 28°C vs. 32°C, as well. Finally, the appearance of pterygiophors of the anal fin is delayed at the 22°C vs. 28°C. Regarding the individuals of DP1, the development of the meristic characters that were studied was completed either earlier or later than the time of sampling, making it difficult to evaluate differences within various groups.

Σκοπός της εργασίας ήταν να μελετήσει την επίδραση του ενδοειδικού ανταγωνισμού, κατά τη διάρκεια της μεταμόρφωσης, στο ρυθμό αύξησης, στην αναλογία φύλου και στο σχήμα του σώματος του zebrafish. Εφαρμόσθηκαν 4 πειραματικές συνθήκες σε τρεις επαναλήψεις. Οι πειραματικοί πληθυσμοί αποτελούνταν από 200 άτομα μήκους 5,6–6,5 mm FL («αμυνόμενα», ηλικίας 15 ημερών μετά τη γονιμοποίηση, dpf), τα οποία ήταν υποκείμενα στην επιθετική συμπεριφορά κανενός, ενός, τριών ή πέντε ατόμων με μέσο FL 12,6-13,3 mm FL («επιτιθέμενα», ηλικίας 30 dpf). Κάθε πειραματικός πληθυσμός βιντεοσκοπείτο δυο φορές την εβδομάδα (λήψη διάρκειας 15 λεπτών, 2 ώρες μετά το τάισμα). Τριάντα ημέρες μετά την έναρξη των πειραμάτων (50 dpf για τα «αμυνόμενα άτομα») τα «επιτιθέμενα» άτομα απομακρύνθηκαν από τους πειραματικούς πληθυσμούς. Η εκτροφή των «αμυνόμενων» ατόμων συνεχίστηκε μέχρι την ενηλικίωσή τους (150-190 dpf). Η ανάλυση των βιντεοσκοπήσεων έδειξε ότι ο αριθμός των «επιτιθέμενων» ατόμων σε κάθε ενυδρείο επηρέασε σημαντικά τον αριθμό των επιθέσεων που δέχονταν τα «αμυνόμενα» άτομα (p<0,05, Kruskall-Wallis), αλλά μόνο στην οντογενετική περίοδο των 9,5-12,5 mm FL. Η ένταση της επιθετικότητας επηρέασε σημαντικά το ρυθμό αύξησης των «αμυνόμενων» ατόμων, όπως αυτός εκφράστηκε από το FL στην ηλικία των 50 dpf (p<0,05, ANOVA). Ως προς την αναλογία φύλου, τα αποτελέσματα δεν έδειξαν κάποια σημαντική επίδραση (p>0,05, G-test) του εξεταζόμενου παράγοντα. Τέλος, αναφορικά με το σχήμα του σώματος των ενήλικων «αμυνόμενων» ατόμων, τα αποτελέσματα έδειξαν σημαντική επίδραση της έντασης της επιθετικότητας (p<0,05, MANOVA). Η γεωμετρική μορφομετρική ανάλυση έδειξε σημαντικές διαφορές στο σχήμα σώματος των θηλυκών μεταξύ των συνθηκών με το ένα «επιτιθέμενο» άτομο και αυτών με τα τρία και πέντε. Στα αρσενικά σημαντικές διαφορές παρατηρήθηκαν ανάμεσα στις συνθήκες με το ένα και τρία «επιτιθέμενα» άτομα. Τα πιο σημαντικά landmarks τα οποία συνέβαλαν στη μεταβολή τους σχήματος ήταν το πρόσθιο άκρο της βάσης του κοιλιακού πτερυγίου για τα θηλυκά, ενώ για τα αρσενικά το πρόσθιο άκρο της βάσης του κοιλιακού πτερυγίου και το κάτω άκρο του βραγχιακού επικαλύμματος. Στην παρούσα εργασία, για πρώτη φορά δείχθηκε η επίδραση της ενδοειδικής επιθετικότητας κατά τη φάση της μεταμόρφωσης, στο σχήμα του σώματος των ενήλικων ψαριών.
The present study examined the effect of intraspecific competition during metamorphosis, in the specific growth rate, in the ratio sex and on the adult body shape zebrafish (Danio rerio). Four experimental conditions were conducted in three replicate. The experimental populations were constituted by 200 individuals which length was 5,6-6,5 mm FL (“defendants”, age of 15 days post fertilization, dpf) and they were subjects in aggressive behavior of no, three or five individuals with mean FL 12,6-13,3 mm FL (“aggressors”, age 30 dpf). Each experimental population videotapped two times a week (time 15 minutes, 2 hours after feeding). Thirty days after the beginning of experiments (50 dpf for the “defendant individuals”) the “aggressor” individuals were removed by the experimental populations. The stockfarming of “defendant” individuals was continued up to adultness (150-190 dpf). The analysis of videos showed that the number of “aggressor” individuals in each aquarium influenced considerably the number of attacks that accepted the “defendant” individuals (p<0,05, Kruskall-Wallis), but only in the ontogenetic period 9,5-12,5 mm FL. The intensity of aggressiveness influenced considerably specific growth rate of “defendant” individuals, as this was expressed by the FL in the age of 50 dpf (p<0,05, ANOVA). As for ratio of sex, the results did not show important effect (p>0,05, G-test) in the examined factor. Finally, as far as the intensity of aggressiveness is concerned, the adult body shape of “defendant” individuals affected significantly (p<0,05, MANOVA). The geometric morphometrics analysis showed the important differences in female body shape between conditions with one “aggressor” individual and these with three and five. Important differences were observed between the conditions with one and three male “aggressor” individuals. Most important landmarks that contributed in males body shape were anterior base of anal fin, while for females were the base of anal fin and the base of gill arch. For first time was shown that intraspecific aggressiveness, at the phase of metamorphosis, affects the adult body shape.

Σκοπός της έρευνας ήταν να μελετήσουμε την επίδραση της θερμοκρασίας στο ολικό μεταγράφωμα νυμφών zebrafish (Danio rerio, Hamilton 1822), θέλοντας να εστιάσουμε το ενδιαφέρον μας στη διερεύνηση μηχανισμών και μορίων που εμπλέκονται στο φυλοκαθορισμό του συγκεκριμένου ψαριού. Kατορθώσαμε να επιδράσουμε με τρεις διαφορετικές θερμοκρασίες σε μία θερμοκρασιακά ευαίσθητη οντογενετική περίοδο, η οποία οριοθετείται από την 10 έως την 20dpf. Η επιλογή των θερμοκρασιών έγινε με σημείο αναφοράς τους 28 ºC, η οποία είναι η πρότυπη θερμοκρασία ανάπτυξης των zebrafish. Η μελέτη της επίδρασης της θερμοκρασίας επιτεύχθη με διατήρηση των πειραματικών μας πληθυσμών σε θερμοκρασίες που αποκλίνουν κατά 4-6 ºC από την πρότυπη, δηλαδή στους 32 ºC και 22 ºC αντίστοιχα. Ως εκ τούτου, ολόκληρος ο πειραματικός πληθυσμός αναπτύχθηκε στη πρότυπη θερμοκρασία των 28 ºC μέχρι και την 10 dpf, όπου μετά την πάροδο της χωρίστηκε σε τρεις υπoπληθυσμούς καθένας από τους οποίους εκτέθηκε στους 22 ºC, 28 ºC ή 32 ºC αντίστοιχα για 280 θερμοημέρες (χρονική περίοδος :10-20dpf). Ακολούθησε δειγματοληψία (100 περίπου ατόμων) από κάθε υποπληθυσμό και εν συνεχεία πραγματοποιήθηκε απομόνωση ολικού RNA. Τα δείγματα μας υβριδοποιήθηκαν σε microarrays, τα οποία έφεραν αντιπροσωπευτικές αλληλουχίες για 15.500 γονίδια του zebrafish. Τα δεδομένα επεξεργάστηκαν με τη χρήση δυο εξειδικευμένων βιοπληροφορικών προγραμμάτων, τα Biosystanse και Dchip, με τη βοήθεια των οποίων συγκρίθηκαν οι εξής καταστάσεις: 10dpf _28 ºC με 20dpf _28 ºC, η οποία αποτελεί την αναπτυξιακή σύγκριση ώστε να ελέγξουμε γονίδια των οποίων η έκφραση αυξάνεται ή ελαττώνεται στα δυο αυτά στάδια αλλά και 20dpf_22 ºC με 20dpf_32 ºC, 20dpf_22 ºC με 20dpf_28 ºC και 20dpf_28 ºC με 20dpf_32 ºC, ώστε να παρατηρηθεί η επίδραση του περιβαλλοντικού παράγοντα ″θερμοκρασία″ στο μεταγράφωμα των ιχθυδίων.
The purpsose of this research was to analyse how temperature effects the total trascriptome at juvenile zebrash (Danio rerio, Hamilton 1822). So, we focus our investigation in the inspection of mechanisms and molecules which probably associated with the sex determination at this specific type of fish. We militated in the thermosensitive ontogenetic period ( from 10 to 20 days post fertilization) with three different temperatures. These three temperatures were 22, 28 and 32οC. Exemplary temperature for zebrafish is 28οC (environmental temperature). Initially, experimental population grew up to 28οC until the 10th day post fertilization. After this temporal period, population separated to three smaller experimental populations which of them grew up to 22, 28 and 32οC respectively for 280 thermodays ( chronic period: 10-20dpf). The next step was sampling (about 100 fish from each experimental population) and then isolating total RNA from each one of the samples. Total RNA samples hybridized on microarrays. Microarrays contained complementary sequences for about 15.500 genes of zebrafish genome. The data analyzed with two specific bioinformatics programs, Biosystance and Dchip. The use of these bioinformatics programs helped us comparing the following situations: 10dpf _28 ºC to 20dpf _28 ºC (developmental comparison) over and above 20dpf_22 ºC to 20dpf_32 ºC, 20dpf_22 ºC to 20dpf_28 ºC and 20dpf_28 ºC to 20dpf_32 ºC (temperature comparison).

Η θερμοκρασία ανάπτυξης αποτελεί παράγοντα μεγάλης σημασίας στην οντογένεση των ιχθύων, αφού ως ποικιλόθερμοι οργανισμοί είναι συνεχώς εκτεθειμένοι στις μεταβολές του περιβάλλοντός τους. Έχει παρατηρηθεί πως η θερμοκρασία ανάπτυξης δύναται να προκαλέσει μετατόπιση του χρονοδιαγράμματος των οντογενετικών γεγονότων και πλαστικότητα σε μορφολογικούς και φυσιολογικούς χαρακτήρες (πχ στο μυοσκελετικό και το καρδιαγγειακό σύστημα). Ωστόσο, μέχρι σήμερα δεν έχει μελετηθεί η επίδραση της θερμοκρασίας ανάπτυξης στο πρότυπο της γονιδιακής έκφρασης του zebrafish και σκοπός της παρούσας εργασίας είναι η μελέτη του ολικού μεταγραφικού προτύπου νυμφών zebrafish. Για το λόγο αυτό σχεδιάστηκαν δύο πειράματα, όπου τρεις θερμοκρασιακές συνθήκες ανάπτυξης (22, 28 και 32oC) εφαρμόστηκαν στην πρώιμη οντογενετική περίοδο: για το διάστημα 0-20 dpf* (1ο πείραμα) και 10-20 dpf (2ο πείραμα). Πραγματοποιήθηκε απομόνωση ολικού RNA από τα άτομα ηλικίας 20 dpf όλων των πληθυσμών και από τα άτομα ηλικίας 10 dpf του πληθυσμού των 28oC του 2ου πειράματος και ακολούθησε υβριδοποίηση σε ολιγονουκλεοτιδικές μικροσυστοιχίες Affymetrix, με 15.509 αντιπροσωπευτικές αλληλουχίες γονιδίων (probe sets). Τα 21 μεταγραφικά προφίλ επεξεργάσθηκαν με τα εξειδικευμένα προγράμματα ανάλυσης μικροσυστοιχιών, dChip και MeV (v.4.5.1). Η κανονικοποίηση και το φιλτράρισμα των δεδομένων των μικροσυστοιχιών απέδωσε μεταγραφικά πρότυπα με 9.488 probe sets. Με τεχνικές πολυπαραμετρικής στατιστικής ανάλυσης (HCL και PCA) πραγματοποιήθηκαν οι συγκρίσεις των μεταγραφικών προτύπων μεταξύ των πειραματικών πληθυσμών για κάθε θερμοκρασία ανάπτυξης και οντογενετικό στάδιο. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων μεταξύ των δύο πειραμάτων, ii) σαφή διαχωρισμό των προτύπων των 28oC και 32oC ως προς αυτά των 22oC και στα δύο πειράματα, και iii) σαφή διαχωρισμό των προτύπων των 28oC διαφορετικού οντογενετικού σταδίου (ηλικίας 10 dpf vs 20 dpf). Θα αναμέναμε τα πρότυπα έκφρασης των 28oC να παρουσιάζουν παρόμοιο πρότυπο, κάτι που δεν παρατηρείται. Αυτό οφείλεται στο πειραματικό σφάλμα που υπεισέρχεται από το διαφορετικό χρόνο πραγματοποίησης των δύο πειραμάτων και τις ρυθμίσεις κατά την υβριδοποίηση. Έτσι πραγματοποιήθηκε η κανονικοποίηση των “28”, που απαλείφει το πειραματικό σφάλμα. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων των 28oC και 32oC ως προς αυτά των 22oC ως αποτέλεσμα της επίδρασης της θερμοκρασίας ανάπτυξης, ii) σαφή διαχωρισμό των προτύπων των 22oC των δύο πειραμάτων ως αποτέλεσμα της επίδρασης της περιόδου εφαρμογής και διάρκειας της θερμοκρασιακής αγωγής και iii) σαφή διαχωρισμό των πρότυπων των 28oC (ηλικίας 10 dpf vs 20 dpf) ως αποτέλεσμα της επίδρασης του οντογενετικού σταδίου. Ακολούθως, πραγματοποιήθηκε ανάλυση σημαντικότητας (SAM) και λειτουργική γονιδιωματική ανάλυση (λογισμικό DAVID) των στατιστικώς σημαντικών γονιδίων που διαφοροποιούν τα πρότυπα. Η ανάλυση των γονιδίων, ως προς την ιστοειδική έκφραση ανέδειξε γονίδια που σχετίζονται με την ανάπτυξη του ματιού, των θωρακικών πτερυγίων και του εγκεφάλου στους πληθυσμούς των 22oC έναντι των 28oC και 32oC. Η παρούσα εργασία αποδεικνύει την επίδραση της θερμοκρασίας ανάπτυξης και της διάρκειας της θερμοκρασιακής αγωγής, καθώς επάγει μηχανισμούς πλαστικότητας στο επίπεδο της γονιδιακής έκφρασης. Τέλος, υποδεικνύεται πως η πρώιμη οντογενετική περίοδος τείνει να είναι περισσότερο θερμοευαίσθητη, καθώς παρατηρείται εντονότερη επίδραση της θερμοκρασίας (στην περίπτωση των 22οC)στην ανάπτυξη του ατόμου. *dpf: days post fertilization
Developmental temperature plays a principal role in the ontogeny of fish. It is known that developmental temperature may shift the initiation time of the ontogenetic stages and induce plasticity in morphological and physiological characters e.g. the musculoskeletal and the cardiovascular system. However, its effect on the gene expression pattern has not previously been attempted for zebrafish. In the present study, zebrafish Affymetrix microarrays of 15,509 probe sets were used to map the transcriptome profile of: a) 20 dpf* old zebrafish larvae at three developmental temperatures, i.e. 22oC, 28oC and 32oC (1st experiment) and b) 20 dpf old zebrafish larvae, which were all grown at 28oC for the first 10 days and subsequently divided into three groups, which were grown at 22oC, 28oC and 32oC, respectively; the profile of 10 dpf old larvae was also measured (2nd experiment). We have isolated total RNA from the above populations and then, hybridization of RNA samples has been done on oligonucleotide Affymetrix microarrays of 15,509 probe sets. All 21 profiles were normalized and filtered (dChip software), and multivariate statistical analysis techniques were used on the normalized 9,488 probe set expression profiles (TM4 MeV software). Hierarchical Clustering (HCL) and Principal Component Analysis (PCA) on expression profiles indicated: a) clear separation of the two experiments based on their transcriptomic patterns, b) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC in both experiments and c) clear separation of the 28oC profiles based on the developmental stage. We would expect expression profiles of 28oC to be clustered together, though this was not observed because of experimental parameters during the hybridization, as the two experiments were carried out independently on different dates. So the normalization of “28” profiles took place, in order to eliminate the experimental noise. HCL and PCA, then, indicated: a) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC, as the effect of developmental temperature, b) clear separation of 22oC profiles of the two experiments, based on the effect of the period and duration of thermal conditions and c) clear separation of the 28oC profiles based on the developmental stage. Then, Significant Analysis of Microarrays (SAM) and Functional Genomic Classification Analysis (DAVID software) of statistically significant genes was carried out. Analysis of genes based on tissue-specific expression indicated characteristic genes for the development of the eye, pectoral fins and brain in 22oC profiles versus 28oC and 32oC profiles. The present study has proved that thermal effect is determinative among the early ontogenetic stage, especially in the case of longer cold thermal period, and developmental temperature may induce plastic response of gene expression, that could affect the fate of fish. *dpf: days post fertilization

Dissertação mest., Biologia Marinha, Universidade do Algarve, 2009
Zebrafish is a freshwater teleost which, due to several biological and genetic characters, has been widely used as a vertebrate model in a variety of research fields, with great potential in molecular biology. As a consequence of the increasing interest in zebrafish, the optimization of its rearing conditions becomes essential, such as the determination of a standardized feeding regime appropriate to this species’. Four different dietary regimes, composed of rotifers Brachionus sp., Artemia nauplii and inert food (Cyclop-eezeTM e BeneluxNV), were carried out. The effect of diet in zebrafish larvae and juvenile fish was evaluated by the determination of the survival and growth parameters as well as by the analysis of the development at the tissue, cellular and molecular levels in three distinct developmental stages. For that, histological, gene expression and immunohistochemical tools applied to molecular markers of the skeletal development (matrix Gla protein – mgp – and osteocalcin – oc) and digestive tract development (amylase and trypsin) were used. The assessment of bone remodeling regions and a histological analysis of the digestive system were also carried out. The obtained data suggest that rotifers are not appropriate for the larval requirements of this species, presenting the lowest survival and growth values. These data were also supported at the molecular level. Artemia nauplii and a weaning to inert food at 15 days post fertilization does not seem to be, also, an appropriate feeding regime. Comparatively, a diet composed exclusively by Artemia nauplii presented higher development-related parameters. These data suggest that this is a suitable food regime, as it has been argued by several authors. A daily mixture of foods also seems to be suitable to zebrafish healthy development. This work showed that, in zebrafish early development Artemia nauplii should used as food and could be, possibly, supplemented with other foods to obtain better results.