L’augmentation de température que subit la planète ces dernières décennies a de nombreuses conséquences dont la recrudescence de maladies infectieuses aussi bien chez l’homme que chez les animaux. Certaines populations de l’ormeau européen Haliotis tuberculata, vivant dans les zones les plus chaudes de Bretagne et de Normandie ont ainsi subi de très importantes mortalités depuis 1997, dues à la bactérie Vibrio harveyi. Cependant, certaines des populations les plus sévèrement touchées se sont aujourd’hui reconstruites et les mortalités semblent s’être arrêtées dans certaines de ces zones. La question se pose donc de l’apparition d’une résistance de l’ormeau face à cette maladie émergente. Pour répondre à cette question, les réponses à l’infection de plusieurs populations naturelles par cette bactérie ont été analysées. Une population présentant une forte résistance à la maladie a été identifiée.La voie d’entrée de la bactérie (ie. les branchies) a été identifiée comme jouant un rôle dans la résistance à l’infection. Par ailleurs, des infections successives ont permis de démontrer un effet d’amorçage immunitaire. Suite à une première exposition, une protection durant jusqu’à deux mois intervient contre l’effet d’inhibition de la phagocytose, provoquée normalement par une infection à V. harveyi. La différence d’expression de gènes des hémocytes d’ormeaux sensibles et résistants a été quantifiée par RNAseq pendant une infection expérimentale. Cette comparaison a montré une reconnaissance plus efficace du pathogène chez les résistants, par des récepteurs tels que les TLR ou les PGRP. La forte surexpression chez la population résistante, d’un gène impliqué dans la synthèse de mucine qui est l’un des composants principaux du mucus renforce l’hypothèse d’une forte implication des branchies dans la résistance. Enfin, une analyse in silico des séquences obtenues en RNAseq a permis d’apporter des preuves de l’existence d’un système de méthylation de l’ADN chez H. tuberculata ainsi qu’une possible implication de ce système dans l’adaptation de l’ormeau à son milieu. / Increasing global temperatures have numerous consequences for marine ecosystems, including the rise of infectious diseases. Certain populations of the European abalone Haliotis tucerculata have suffered from severe and recurrent mortality since 1997 due to infection caused by the bacterium Vibrio harveyi, particularly in areas with higher average summer temperatures. Given the spatial heterogeneity in mortalities, and the observation that the historically most severely impacted populations have recovered in recent years, the question of the emergence of resistance to the disease was addressed. The mortality rate in response to infection by V. harveyi was quantified experimentally in abalone originating from three natural populations, and one population exhibiting resistance to the disease was identified. In a subsequent experiment, the immune response of abalone was compared between infected individuals from a resistant and from a susceptible population. The portal of entry of the bacterium (ie. gills) was identified as playing a role in resistance. Furthermore, successive exposures of abalone to the bacterium demonstrated an immune priming effect, such that following a first exposure, phagocytosis was no longer inhibited by infection with V. harveyi, and that this improved protection against the disease lasted for at least two months. Differences in gene expression was quantified by RNAseq in the hemocytes of resistant and susceptible abalone following exposure to the pathogen. This comparison showed that resistant abalone had more effective recognition of the bacterium by receptors as the TLR or PGRP. The substantial over-expression of a gene involved in the synthesis of mucin, the main component of mucus, (UDP-GalNAC) in the resistant population, supports the interpretation of a strong involvement of gills in the resistance. Finally, an in-silico analysis of the sequences obtained from RNAseq indicate the existence of a DNA methylation system in H. tuberculata and suggested an involvement of epigenetic mechanisms in the adaptation of abalone to its environment.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-14T15:03:25Z No. of bitstreams: 1 Joana angelica L V Carvalho.pdf: 451922 bytes, checksum: 66f7ebcb01d8574ab7c4cced0c81dce6 (MD5) / Made available in DSpace on 2017-02-14T15:03:25Z (GMT). No. of bitstreams: 1 Joana angelica L V Carvalho.pdf: 451922 bytes, checksum: 66f7ebcb01d8574ab7c4cced0c81dce6 (MD5) Previous issue date: 2011-02-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study aimed to evaluate the survival of Litopenaeus vannamei shrimp larvae treated with probiotic bacteria and subsequently infected by Vibrio harveyi and Vibrio alginolyticus. Shrimp were kept in two 80L tanks under similar culture conditions, but only one tank was previously treated with commercial probiotic (Bacillus spp.) added daily in the water at a concentration of 105 CFU/ml. This protocol was maintained until the animals reach the stage of development needed for the infection experiments by Vibrio spp. Three independent infection experiments were performed: Zoea1 to Zoea3, Mysis1 to Mysis3 and Postlarvae (PL)10 to PL14 with the following treatments with four replicates each: only V. harveyi; commercial probiotic + V. harveyi; only V. alginolyticus; commercial probiotic + V. alginolyticus; and without Vibrio spp. and probiotic (Control). In the experiment with PL, only the treatments with V. harveyi; commercial probiotic + V. harveyi and control were used. Vibrio species were inoculated only at the beginning of each experiment at a concentration of 107 CFU/ml. At the end of experiments, a sample of water and shrimp from each experimental unit was submitted to quantification of Vibrio spp. Additionally, was realized an antagonism test of Bacillus spp. against Vibrio spp. In the experiments with zoea and mysis, larvae cultured in treatment V. harveyi had a significantly lower survival when compared with treatments V. alginolyticus and control. There was a significant increase in survival of zoea and mysis larvae treated with probiotic + V. harveyi with 81.07 and 90.13%, differentiating the larvae infected only with V. harveyi with 12.80 and 69.13%, respectively. There were no significant differences in survival of postlarvae among treatments. Total Vibrio spp. counts decreased in the water and shrimp of treatments with the use of probiotic. The probiotic (Bacillus spp.) showed inhibitory activity against Vibrio spp. in vitro. The results indicated an increase in the survival of early larval stages (zoea and mysis) infected by V. harveyi after treatment with Bacillus spp. The administration of probiotic bacteria is a promising alternative for the prevention of Vibrio spp. in larviculture of L. vannamei. / O presente estudo teve como objetivo avaliar a sobrevivência de larvas de Litopenaeus vannamei tratadas com bactérias probióticas e posteriormente infectadas por Vibrio harveyi e Vibrio alginolyticus. Os camarões foram mantidos em dois tanques de 80L nas mesmas condições, mas apenas um deles foi previamente tratado com probiótico comercial (Bacillus spp.) adicionado diariamente na água na concentração de 105 UFC/ml. Este protocolo foi mantido até os animais alcançarem o estágio de desenvolvimento larval necessário para os experimentos de infecção por Vibrio spp. Foram realizados três experimentos independentes de infecção: Zoea1 a Zoea3, Mísis1 a Mísis3 e Pós-larvas (PL)10 a PL14 com os seguintes tratamentos com quatro repetições cada: somente V.harveyi; probiótico comercial + V. harveyi; somente V. alginolyticus; probiótico comercial + V. alginolyticus; sem Vibrio spp. e probiótico (controle). No experimento com PL, somente os tratamentos com V. harveyi; probiótico comercial + V. harveyi e controle foram utilizados. As espécies de Vibrio foram inoculadas apenas no início de cada experimento na concentração de 107 UFC/ml. Ao final dos experimentos, uma amostra de água e camarão de cada parcela experimental foi submetida à quantificação de Vibrio spp. Adicionalmente foi realizado um teste de antagonismo in vitro de Bacillus spp. contra Vibrio spp. Nos experimentos com zoea e mísis, as larvas cultivadas no tratamento V. harveyi apresentaram uma sobrevivência significativamente inferior quando comparadas com as dos tratamentos V. alginolyticus e controle. Houve um aumento significativo na sobrevivência de larvas zoea e mísis tratadas com probiótico + V. harveyi com 81,07 e 90,13%, em comparação com as larvas infectadas apenas com V. harveyi com 12,80 e 69,13%, respectivamente. Não foram observadas diferenças significativas na sobrevivência das pós-larvas entre tratamentos. A quantidade total de Vibrio spp. na água e camarões diminuiu nos tratamentos com o uso de probiótico. O probiótico (Bacillus spp.) apresentou atividade inibitória contra Vibrio spp. in vitro. Os resultados indicaram um aumento na sobrevivência dos estágios larvais iniciais (zoea e mísis) infectadas por V. harveyi após tratamento com Bacillus spp. A administração de bactérias probióticas é uma alternativa promissora para a prevenção de Vibrio spp. na larvicultura de L. vannamei.

Mittels Quorum sensing können Bakterienzellen die Expression von Genen Zelldichte-abhängig steuern. Dies spielt eine besondere Rolle bei der Expression von Virulenzfaktoren oder Antibiotikaproduktion, der Biofilmentwicklung oder Phänomenen wie genetischer Kompetenz, Sporulation oder Biolumineszenz. Vibrio harveyi, ein Gram-negativer, mariner, frei lebender Organismus, reguliert die Expression von Biolumineszenz-Genen Zelldichte-abhängig nach dem Prinzip des Quorum sensing und sollte im Rahmen dieser Arbeit als Modell für die Mechanismen der Signaltransduktion untersucht werden. Dazu wurden die Proteine der Lux-Signaltransduktionskaskade heterolog in E. coli überexprimiert und teilweise gereinigt. Mittels in vitro Phosphorylierung konnten die enzymatischen Aktivitäten der Proteine erstmals biochemisch charakterisiert werden. Für die Hybridsensorkinase LuxN konnte neben einer Kinase-Aktivität ein Phosphotransfer auf das Histidin-Phosphotransferprotein LuxU gezeigt werden. Die Autophosphorylierungsaktivität ist dabei eindeutig von der Konzentration des Signalmoleküls, eines Acyl-Homoserinlaktons, abhängig. Eine ebenfalls eindeutig nachgewiesene Phosphatase-Aktivität von LuxN, die zur Dephosphorylierung von LuxU führt, war dagegen konstitutiv. Damit konnte ein auf biochemischen Daten basierendes Modell der Signaltransduktion von V. harveyi postuliert werden. Basierend auf den Ergebnissen von Topologieuntersuchungen mittels luxN-Reportergenfusionen und Protease-Zugänglichkeitsstudien konnten neue Hinweise auf die Membrantopologie der Hybridsensorkinase ermittelt werden. Diese lassen auf ein Modell mit neun Transmembranhelices schließen, bei der der N-terminus des Proteins im Periplasma der Zelle lokalisiert zu sein scheint.

The field of conservation genetics, in combination with non-invasive sampling, provides a powerful set of tools for investigating the conservation status and natural history of rare species that are otherwise difficult to study. A systematic literature review demonstrated that this is certainly the case for many forest associated antelope species, which are poorly studied and yet constitute some of the most heavily hunted wildlife in Africa. The aim of the present study was to use non-invasive sampling to investigate genetic patterns in forest antelope populations in the high-biodiversity Udzungwa Mountains, Tanzania, within the context of the conservation of these species and the wider ecosystem. Genetic information was derived from faecal samples collected across the Udzungwa landscape and assigned to five antelope species (N = 618, collected 2006-09). Faecal pellet length was measured for a subset of samples but statistical assignment to species by this method proved unreliable. Phylogenetic analysis using mitochondrial control region sequences unexpectedly revealed that Harvey’s duiker within the Udzungwas are paraphyletic with respect to sequences from a putative sister species from southern Africa. However, there was no corresponding pattern in the microsatellite dataset suggesting that these mitochondrial lineages do not represent contemporary genetic isolation. Instead, Harvey’s duiker nuclear variation is shaped both by isolation by distance, due to positive spatial autocorrelation at short distances, and clustering of distinct genotypes from western outlying forests. These forests also harbour the endangered Abbott’s duiker and therefore require effective conservation management. Despite being detected throughout the Udzungwas, genetic diversity in Abbott’s duiker was very low in comparison to other species. These results suggest several promising research directions but also have significant conservation implications that will be disseminated to the Tanzanian wildlife authorities and the wider conservation community.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-09T14:06:18Z No. of bitstreams: 1 Danielli Matias de Macedo Dantas.pdf: 243863 bytes, checksum: 12196d11f6515cb7163e69e8e9c45af9 (MD5) / Made available in DSpace on 2017-02-09T14:06:18Z (GMT). No. of bitstreams: 1 Danielli Matias de Macedo Dantas.pdf: 243863 bytes, checksum: 12196d11f6515cb7163e69e8e9c45af9 (MD5) Previous issue date: 2008-02-08 / The fast expansion of farmed shrimp industry during the last decades has been offering high profitable deal to many countries. However, diseases appearing have caused lots of serious economic damage on productive sector. The effect of probiotic in Litopenaeus vannamei juveniles when submitted to the infection by Vibrio harveyi was evaluated in the present study The exposure to V. harveyi was carried out during 15 days using individuals reared for one month in a closed recirculation system with a commercial probiotic (Bacillus sp.) added to water and food (PAR), only in the water (PA), only in food (PR) and without probiotic (SP). Were examined the responses of food consumption, total concentration of hemocytes, growth and survival. The food consumption did not differ significantly among the treatments, but in the SP treatment a significant reduction occurred in the second week. The PAR and PA treatments showed inferior and more constants values in the total hemocits concentrations during the experimental period. The highest growth rates were observed in the PAR (0.34 ± 0.19 g/week) and SP (0.45 ± 0.04 g/week) treatments, but did not differ statistically within these treatments. Shrimp survival values were higher than 98% in all treatments. The results suggest that the addition of Bacillus sp. to the water or food separately, could increase the food consumption during a longer period of time in shrimp infected by V. harveyi and interfere positively in the immune response of L. vannamei. / A rápida expansão da indústria do camarão na aqüicultura durante poucas décadas tem proporcionado a muitos países altos rendimentos. Entretanto, o surgimento de doenças tem causado sérios prejuízos econômicos ao setor produtivo. Este trabalho verificou o efeito do uso de probiótico no cultivo de juvenis de Litopenaeus vannamei quando submetidos à infecção ao Vibrio harveyi. A exposição ao V. harveyi foi realizada durante 15 dias nos camarões provenientes de um cultivo de 30 dias em sistema fechado sem renovação de água com utilização de um probiótico comercial (Bacillus sp.) adicionado na água e na ração (PAR), somente na água (PA), somente na ração (PR) e sem adição de probiótico (SP). Foram analisadas as respostas de consumo alimentar, concentração total de hemócitos, crescimento e sobrevivência. A análise do consumo alimentar dos camarões não mostrou diferença significativa entre os tratamentos, sendo a única exceção o tratamento SP onde ocorreu uma redução no consumo na segunda semana. Os tratamentos PAR e PA apresentaram valores menores e mais constantes de concentração total de hemócitos durante o período do experimento. As maiores taxas de crescimento foram verificadas nos tratamentos PAR (0,34 ± 0,19 g/semana) e SP (0,45 ± 0,04 g/semana), os quais não diferiram estatisticamente entre si. Os camarões apresentaram valores de sobrevivência acima de 98% em todos os tratamentos. Os resultados sugerem que a utilização do Bacillus sp. adicionados na água ou na ração separadamente, aumentam o consumo alimentar por um maior período de tempo em camarões infectados pelo V. harveyi e interferem positivamente na resposta imune de L.vannamei.