As raias são peixes peçonhentos e estão frequentemente associadas a acidentes em seres humanos, principalmente na região Norte do Brasil, favorecidos pelo hábito desses peixes de permanecerem no fundo de águas rasas e pela contínua utilização humana dos rios. Os ferrões das raias causam lesões dolorosas, edema, necrose, e o muco que recobre toda a extensão do corpo desses peixes pode aumentar a gravidade desses ferimentos. O objetivo deste trabalho foi avaliar a resposta imunológica induzida pelo muco de Paratrygon aiereba nativo ou irradiado por raios gama de 60Co em modelos animais. Foram realizados ensaios imunoenzimáticos e Western blotting para verificar a resposta humoral e reatividade cruzada dos soros provenientes de camundongos Swiss e coelhos New Zealand previamente imunizados contra o veneno, muco nativo ou irradiado. A indução da produção de anticorpos in vitro, as subclasses de IgG e a quantificação de citocinas foram analisados. Além de realizados ensaios de soroneutralização da atividade edematogênica in vitro e in vivo e de viabilidade celular. Os dados foram analisados estatisticamente por meio de análise de variância. O protocolo de imunização possibilitou a obtenção de soros com títulos satisfatórios de anticorpos policlonais. O muco e veneno de P. aiereba são imunogênicos e apresentam reatividade antigênica. O muco nativo ou irradiado induziu a produção de anticorpos IgG e esses reconheceram antígenos presentes no muco de outras espécies de raias. Células esplênicas de animais imunizados contra o muco irradiado produziram IFN-γ, TNF- α e IL-10 e também foi observada a produção sérica de TNF-α (grupo imunizado contra o muco irradiado) e de IL-6 e IL-17 (grupo imunizado contra o muco nativo). O soro anti-muco irradiado reduziu a atividade edematogênica in vitro, ao contrária da in vivo que não foi neutralizada. Os resultados corroboram o uso da radiação ionizante, com produção de anticorpos altamente responsivos e melhor resposta imune, além de comprovar que o muco de Paratrygon aiereba foi capaz de estimular resposta imune adaptativa celular e humoral. / Freshwater stingrays are venomous animals, frequently associated with accidents in northern Brazil where the shallow waters and the human use of the rivers favour close proximity between the fish and potential victims. Ray stings induce painful lesions, edema, necrosis, and the mucus that covers the body of these fishes may increase the severity of the wounds. The aim of this work was to evaluate the immune response induced by native or 60Co Paratrygon aiereba mucus in animal models. Enzyme linked immunosorbent assays and western blots were performed to compare humoral immune response as well as cross reactivity using antibodies raised in mice or rabbits against venom or mucus, the latter either in its native or irradiated form. Antibody production in vitro, immunoglobulins subclasses and cytokines production were evaluated. Immunization resulted in good levels of antibodies. Interestingly, both the mucus and the venom share many cross reactive components. Furthermore, antibodies raised against native mucus or its irradiated counterpart were cross-reactive against the mucus of other stingray species. Splenic cells from mice immunized with irradiated mucus secreted IFN-γ, TNF-α and IL-10. The serum raised against irradiated mucus reduced edematogenic activity in vitro, but not in vivo, when the venom and the serum are injected separately. Our results corroborate the potential of ionizing radiation as a detoxifying agent for immunogens, with production of high antibody titers and, that the highly abundant mucus of freshwater stingrays contains basically the same repertoire of antigens as the venom, inducing the synthesis of high levels of neutralizing antibodies.

Surface mucus of a freshwater fish, Alburnus alburnus (bleak), caught from Lake Mogan, situated in south of Ankara, was collected in different seasons. The total cultivable bacteria were enumerated by spread plate method on nine different media. Bacteria were isolated based on colony morphologies and pigmentation. A total of sixty bacterial isolates obtained. The mucus-dwelling bacteria were first tested for resistance against ampicillin and kanamycin / then streptomycin and chloramphenicol were added to the experimental set up. The resistance levels of isolates were determined in terms of four antibiotics by tube dilution method. About 90% of the isolates were resistant to chloramphenicol, about 84% to kanamycin, about 88% to streptomycin and about 98% to ampicillin. These high levels of antibiotic resistance are rather interesting from a standpoint that the lake has no record of antibiotics exposure of any sort. The plasmid isolations were carried out to determine if the multiple antibiotic resistance could be attributed to plasmids for starting assumption. But we found no direct relationship between the presence of plasmids and multiple antibiotic resistance. Our study indicated that multiple antibiotic resistance at high levels is among the current phenotypes of the fish mucus-dwelling bacterial populations in Lake Mogan.

The mucus-dwelling bacteria previously isolated from the surface of a freshwater fish species (Alburnus alburnus from Lake Mogan, Ankara), were studied to discover their cobalt resistance. The minimum inhibitory concentrations (MIC) were determined for a total of thirty six bacterial isolates. The results of the resistance studies led us to design experiments on adaptation to cobalt and subsequent memory retention. Three selected isolates were exposed to an inhibitory cobalt concentration as a mixed culture and individually. The delayed formation of colonies along with competitive exclusion of one of the isolates in the mixed culture were recorded. The delay for colony formation was followed up for liquid culture conditions. After some of our isolates acclimated to cobalt and started to exhibit constant time of growth period, it is assumed that they were adapted. We regarded adaptation as a result of memory formation. Next, we did a further study to find out how long this memory could be retained via serial multiple passages in cobalt free medium. We expressed our observations quantitatively by measuring the growth by using spectrophotometer and by performing viable counts. Interestingly, where there was a high CFU, the photometric values were very low. We interpreted the finding such that the presence of cobalt above tolerance limits were causing size reduction in the cells. So that their presence was underestimated by optic devices in visible range. Our study hinted that freshwater bacteria was adapting cobalt in a memory based mechanism and able to retain this memory for some time.

As raias são peixes peçonhentos e estão frequentemente associadas a acidentes em seres humanos, principalmente na região Norte do Brasil, favorecidos pelo hábito desses peixes de permanecerem no fundo de águas rasas e pela contínua utilização humana dos rios. Os ferrões das raias causam lesões dolorosas, edema, necrose, e o muco que recobre toda a extensão do corpo desses peixes pode aumentar a gravidade desses ferimentos. O objetivo deste trabalho foi avaliar a resposta imunológica induzida pelo muco de Paratrygon aiereba nativo ou irradiado por raios gama de 60Co em modelos animais. Foram realizados ensaios imunoenzimáticos e Western blotting para verificar a resposta humoral e reatividade cruzada dos soros provenientes de camundongos Swiss e coelhos New Zealand previamente imunizados contra o veneno, muco nativo ou irradiado. A indução da produção de anticorpos in vitro, as subclasses de IgG e a quantificação de citocinas foram analisados. Além de realizados ensaios de soroneutralização da atividade edematogênica in vitro e in vivo e de viabilidade celular. Os dados foram analisados estatisticamente por meio de análise de variância. O protocolo de imunização possibilitou a obtenção de soros com títulos satisfatórios de anticorpos policlonais. O muco e veneno de P. aiereba são imunogênicos e apresentam reatividade antigênica. O muco nativo ou irradiado induziu a produção de anticorpos IgG e esses reconheceram antígenos presentes no muco de outras espécies de raias. Células esplênicas de animais imunizados contra o muco irradiado produziram IFN-γ, TNF- α e IL-10 e também foi observada a produção sérica de TNF-α (grupo imunizado contra o muco irradiado) e de IL-6 e IL-17 (grupo imunizado contra o muco nativo). O soro anti-muco irradiado reduziu a atividade edematogênica in vitro, ao contrária da in vivo que não foi neutralizada. Os resultados corroboram o uso da radiação ionizante, com produção de anticorpos altamente responsivos e melhor resposta imune, além de comprovar que o muco de Paratrygon aiereba foi capaz de estimular resposta imune adaptativa celular e humoral. / Freshwater stingrays are venomous animals, frequently associated with accidents in northern Brazil where the shallow waters and the human use of the rivers favour close proximity between the fish and potential victims. Ray stings induce painful lesions, edema, necrosis, and the mucus that covers the body of these fishes may increase the severity of the wounds. The aim of this work was to evaluate the immune response induced by native or 60Co Paratrygon aiereba mucus in animal models. Enzyme linked immunosorbent assays and western blots were performed to compare humoral immune response as well as cross reactivity using antibodies raised in mice or rabbits against venom or mucus, the latter either in its native or irradiated form. Antibody production in vitro, immunoglobulins subclasses and cytokines production were evaluated. Immunization resulted in good levels of antibodies. Interestingly, both the mucus and the venom share many cross reactive components. Furthermore, antibodies raised against native mucus or its irradiated counterpart were cross-reactive against the mucus of other stingray species. Splenic cells from mice immunized with irradiated mucus secreted IFN-γ, TNF-α and IL-10. The serum raised against irradiated mucus reduced edematogenic activity in vitro, but not in vivo, when the venom and the serum are injected separately. Our results corroborate the potential of ionizing radiation as a detoxifying agent for immunogens, with production of high antibody titers and, that the highly abundant mucus of freshwater stingrays contains basically the same repertoire of antigens as the venom, inducing the synthesis of high levels of neutralizing antibodies.

Les parasites sont une partie très importante des écosystèmes, néanmoins, dans certains cas tels que l’aquaculture ils causent des épidémies. Dans cette thèse j’ai étudié les interactions hôte-parasite chez les poissons coralliens, dans les milieux naturels et les milieux aquacoles. J’ai étudié l’arrangement des communautés de 13 espèces de monogènes de 34 espèces de poissons papillon dans l’Indo-Pacifique. Les résultats montrent qu’il existe un patron de distribution stable des espèces, ce qui, combiné avec les résultats de l’étude biogéographique, suggère que la spécificité parasitaire observée a pu se développer tout au long des épisodes biogéographiques passés. Notamment, seule une espèce, Chaetodon lunulatus, n’est jamais parasitée. J’ai étudié le mucus des poissons qui est décrit comme la première ligne de défense contre des agressions externes, pour analyser quels facteurs de C. lunulatus pourraient être reliés à l’absence des parasites. Le microbiome et le métabolome du mucus branchial des poissons papillons montrent une grande diversité. C. lunulatus présente une plus grande abondance de Fusobactéries qui est corrélée avec une surexpression de peptides pouvant dériver de la β-hémoglobine. La purification, la synthèse et l’évaluation des activités antiparasitaires des peptides sont en cours au laboratoire. J’ai aussi étudié l’utilisation des plantes médicinales comme alternative à des traitements chimiques dans l’aquaculture. J’ai testé l’activité antibactérienne et immunostimulatrice de plusieurs plantes et algues polynésiennes. L’algue rouge Asparagopsis taxiformis montre de fortes activités antibactériennes sur Vibrio harveyi et Tenacibaculum maritimum et induit une augmentation de l’expression de deux gènes liés à l’immunité chez Platax orbicularis. / Fish parasites are an important part of ecosystems, however, in certain cases such as in aquaculture they can cause severe disease outbreaks. In this thesis I have studied host-parasite interactions in coral reef fishes, both in the natural and culture environments. I have studied the distribution of 13 dactylogyrid species from 34 butterflyfishes in the Indo West-Pacific. Composition of dactylogyrid communities was host specific and together with the biogeography results, where a turnover in the main Haliotrema species was observed, suggest that parasite specificity might result from host-parasite coevolution derived from past biogeographical episodes. Only one butterflyfish species, Chaetodon lunulatus, was never found parasitized by gill monogeneans. I have studied the butterflyfish mucus, which is the first barrier against pathogens, to investigate the C. lunulatus factors that might be related to the monogenean absence. Butterflyfish gill microbiome and metabolome revealed a high diversity. C. lunulatus presented a significantly higher abundance of Fusobacteria which was correlated to a higher expression of potentially derived β-hemoglobin peptides. Synthesis and evaluation of the peptide antiparasitic activities are being performed in the laboratory. I have also studied the use of medicinal plants as an alternative to chemotherapy in fish aquaculture. I investigated the antibacterial and immunostimulant activities of several local Polynesian plants and algae, and I found that Asparagopsis taxiformis displayed a potent antibacterial activity against Vibrio harveyi and Tenacibaculum maritmum and increased expression of two immune-related genes in Platax orbicularis.