FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Nos estuÃrios tropicais, naturalmente complexos devido à dinÃmica de marÃs, perÃodos de precipitaÃÃo pluviomÃtrica atuam como distÃrbio intermediÃrio. As caracterÃsticas ambientais sÃo imprescindÃveis na histÃria de vida da biota da regiÃo, resultando no estabelecimento de padrÃes de reproduÃÃo ou interferindo no metabolismo dos organismos podendo tornÃ-los mais suscetÃveis ao acometimento por parasitas. O bivalve Lucina pectinata (Gmelin, 1791), apesar de apreciado no litoral brasileiro, foi pouco estudado no paÃs atà o momento. Este trabalho teve como objetivo caracterizar a biologia reprodutiva de Lucina pectinata (Mollusca: Bivalvia, Lucinidae) do estuÃrio do rio CearÃ, Fortaleza, CE, Brasil, e efetuar consideraÃÃes sobre parasitismo por trematÃides nesta populaÃÃo, em funÃÃo das variaÃÃes ambientais do local. Os exemplares coletados tiveram as gÃnadas e brÃnquias analisadas histologicamente. Para determinar a maturaÃÃo sexual dos indivÃduos, empregou-se escala gametogÃnica com quatro estÃdios de desenvolvimento gonadal. Lucina pectinata apresentou ciclo reprodutivo contÃnuo, com picos de desova na estaÃÃo seca. O nÃvel de parasitismo na populaÃÃo foi baixo, e a mesma nÃo està sendo prejudicada pela presenÃa de parasitas. Observaram-se semelhanÃa das variÃveis abiÃticas do local durante a mesma estaÃÃo, distinguindo-se nitidamente os perÃodos secos dos chuvosos. O distÃrbio pluviomÃtrico à discutido como fator regulador da populaÃÃo de Lucina pectinata, contribuindo tanto para sua renovaÃÃo quanto para controlar a incidÃncia dos parasitas trematÃides / In tropical estuaries, environments naturally complex due to the tidesÂs dynamics, the rainfall periods act as an intermediate disturbance. The environmental characteristics are important in the biotaâs life history of the region, resulting in the establishment of reproduction patterns or interfering at organisms metabolism making them more susceptible to being affected by parasites. The bivalve Lucina pectinata (Gmelin, 1791), although appreciated in the Brazilian coast, was little studied in the country so far. This study aimed to characterize the reproductive biology of Lucina pectinata (Mollusca: Bivalvia, Lucinidae) from the Cearà river estuary, Fortaleza, CE, Brazil, and make considerations on trematode parasitism in this population, according to local environmental variations. Specimens collected had the gonads and gills assessed histologically. To determine the individuals sexual maturity, was employed gametogenic scale with four stages of gonadal development. Lucina pectinata presented continuous reproductive cycle, with peak spawning at dry season. The level of parasitism in the population was low, and the same is not being affected by the presence of parasites. Were observed proximity of the abiotic variables during the same season, distinguishing clearly the dry periods of rainy. Disturbance rainfall is discussed as a factor regulating the population of Lucina pectinata, contributing to its renewal and to control the incidence of trematode parasites

Coal mining and coal utilization in Mpumalanga have increased over the years due to national reliance on coal as a source of power generation. In general, this has caused significant deterioration of water quality wherever streams are impacted by acid mine drainage (AMD). The aim of this research was to assess the use of in vitro bioassays as a complement to, or potential future replacement of, waste effluent testing in whole animals from AMD impacted watersheds subjected to passive and active treatment, correlating observed changes with water chemistry analysis. To accomplish this goal, water samples were collected and in vitro bioassays carried out to investigate generation of reactive oxygen species by the water samples and cytotoxicity against Vero kidney cells, C3A liver cells and trout RTgill-W1 cells. Primary fish gill cultures were established and used as sensitive in vitro models for assessing possible contaminants in water, measuring the induction of cytochrome P450 (CYP) 1A and resultant increase in 7-ethoxyresorufin-o-deethylase activity as a potential biomarker in fish gill cells exposed to polycyclic aromatic hydrocarbons. The genotoxic potential of AMD water on commercially available cell lines was also determined. / The study site was an impacted stream located downstream of a coal mine discharge point whose effluent flowed away from the mine. Water chemistry results suggested high AMD impact evidenced by acidity, elevated sulphates, increased conductivity and presence of heavy metals. Al, Fe, Zn, Mn and Si were the major metals of potential concern in the AMD impacted stream; sulphates and major ions like Ca, K, Na and Mg were present at levels above target water quality range (TWQR) for effluents in receiving stream. The AMD impacted stream caused increased generation of reactive oxygen species (ROS) detectable in vitro in selected cell lines (Vero, C3A and RTgill-W1 cell lines), an indication of oxidative stress. In-stream, active treatment with caustic soda was efficient at reducing metal burden, with subsequent reduction in ROS generation in fish gill cell lines. For in vitro cytotoxicity tests, passive and active treated AMD water was cytotoxic to cell lines (Vero and RTgill-W1), with the fish RTgill-W1 cells exhibiting greater sensitivity compared to the mammalian Vero cells. Mitochondria played a larger role in observed loss in cellular viability (increased vacuolization, mitochondrial membrane swelling and damage), which was detected using mitochondrial specific stains, and by transmission electron microscopy (TEM). Increased dose- dependent cytotoxicity was observed in the fish gill and mammalian cell lines. Cells exposed to water samples (AMD and reference sites) revealed significant differences (p < vi 0.05) between the AMD impacted watershed and a relatively pristine site (reference site) where exposure to the same cells maintained approximately 100% viability at all concentrations for up to 72h exposure. The observed differences in effect in this study demonstrate that the effluent from the coal mine negatively impacted surface water quality, resulting in toxicity to cell lines, therefore creating an environment that would not be conducive for the survival of biological aquatic communities and potentially of concern for downstream human end users. / The induction of cytochrome P450 (CYP) 1A and resultant increase in 7- ethoxyresorufin-o-deethylase activity in primary fish gill cultures exposed to polycyclic aromatic hydrocarbons B[a]P, a known AhR agonist contaminant associated with coal mining, showed that there was as increase in EROD activity which was not observed using the RTgill-W1 cell lines. Gill epithelial cells isolated from the gills of Tilapia fish (Oreochromis mossambicus) bear close similarities to fish gills in vivo and their capacity to respond to the presence of AhR indicates that they may serve as a simple, cost-effect screening tool for assessing PAHs and dioxin-like compounds in fresh water. / For genotoxicity evaluation, the Ames test performed without metabolic activation using bacterium Salmonella typhimurium TA98 and TA100 strains revealed no indication of genotoxic activity in any of the water samples. Genotoxicity assessment of all water samples using the comet assay however exposed DNA damage to Vero and RTgill-W1 cell lines. A significant reduction in DNA damage was observed following active treatment. The results suggest that neither treatment technologies employed were efficient at removing all potential genotoxicants so further improvements are required. The comet assay proved sensitive enough to detect genotoxicity in reference water samples despite no known untoward effluent inputs at the site, suggesting potential for this assay to be integrated into an environmental monitoring framework. / The results obtained support the use of in vitro bioassays for evaluating toxicity of industrial effluent through biological responses in test systems elicited following exposure, improving ability to detect AMD polluted water. This could be beneficial when assessing the degree and extent of impact of AMD in natural water sources, and the possible environmental impact resulting from hazardous elements present in effluent water. In conclusion, these results suggest that in vitro techniques involving cell lines and primary cultures from fish may serve vii as simple, rapid and cost-effective tools for assessing risk and potential toxic effects of contaminants in AMD waters. / Thesis (PhD)--University of Pretoria, 2016. / The National Research Foundation / Department of Paraclinical Sciences (University of Pretoria) / Schlumberger Stichting Fund, Netherlands / Paraclinical Sciences / PhD / Unrestricted

Diese Habilitationsschrift konzentriert sich auf die Evolution osteologischer Korrelate, anhand derer Rückschlüsse auf die Struktur der Haut sowie die Art der Atmung und der Nahrungsaufnahme früher Tetrapoden gezogen werden können. Die äußere Skulptur der Hautknochen früher Tetrapoden trug zur Konsolidierung der darüber liegenden Dermis bei; dies und die zahlreichen Hautverknöcherungen deuten darauf hin, dass Hautatmung im Gegensatz zu heutigen Lissamphibien keinen wesentlichen Teil des Gasaustausches ausmachen konnte. Es wird die Hypothese aufgestellt, dass eine weitere Funktion der Knochenskulptur in der Pufferung von CO2-induzierter Azidose bestand, wenn sich die Tiere an Land aufhielten. Mehrere Gruppen früher Tetrapoden entwickelten unabhängig voneinander Osteoderme im Rumpf, die unter anderem die Wirbelsäule bei der Fortbewegung an Land unterstützen konnten oder als Kalzium-Reservoir dienten. Die Veränderungen in der Morphologie, Histologie und Anordnung der Dermalschuppen während des Fisch-Tetrapoden-Überganges erfolgte etwa simultan zur Entwicklung der Tetrapodenextremität und ermöglichte eine größere Flexibilität des Körpers und der Beine. Der Bau des Kiemenskeletts ihrer Fischvorfahren wurde bei einer unerwartet großen Anzahl früher Tetrapoden beibehalten. Dies zeigt, dass viele frühe Tetrapoden sowohl der Stamm- als auch der Kronengruppe als Adulte über innere Kiemen atmeten, während ihre Larven äußere Kiemen besaßen. Das Kiemenskelett und die Rekonstruktion assoziierter Muskeln zeigen, dass viele aquatische frühe Tetrapoden ihre Nahrung durch akinetisches Saugschnappen erbeuteten. Trotz spärlicher Fossilüberlieferung gibt es Hinweise darauf, dass die ersten an Land fressenden Tetrapoden noch keine bewegliche Zunge besaßen. Ein ontogenetischer Umbau des larvalen, kiementragenden Kiemenskelets in einen zungenstützenden Apparat kann bei frühen Tetrapoden nur in Ausnahmefällen belegt werden. / This habilitation thesis focuses on the evolution of skeletal correlates from which the integumentary structure and the mode of breathing and feeding in early tetrapods can be inferred. Sculpture on the external surface of dermal bones contributed to the consolidation of a dense integument; this and numerous dermal ossifications render substantial cutaneous respiration as in extant lissamphibians unlikely. It is hypothesised that a further function of dermal bone sculpture was to buffer CO2-induced acidosis while the animals were on land. Numerous early tetrapods independently developed osteoderms in the trunk that may have served among other things for support of the vertebral column in terrestrial locomotion or as a physiological calcium reservoir. The alterations in morphology, histology and arrangement of dermal scales during the fish-to-tetrapod transition occurred at roughly the same time when digits appeared and enabled greater flexibility of body and limbs. The structure of the hyobranchium of their fish-like ancestors was retained in an unexpected large number of early tetrapods, showing that many early tetrapods of both the stem- and crown-group breathed via the associated internal gills as adults, and via external gills as larvae. The hyobranchial apparatus and reconstruction of the associated musculature indicate that many aquatic early tetrapods fed by akinetic suction feeding. There is evidence that the earliest terrestrial tetrapods captured prey on land by jaw prehension rather than by tongue based feeding. Ontogenetic remodelling into a tongue supporting adult hyobranchium in early tetrapods can only be demonstrated in exceptional cases.

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.

Teleost fishes incorporate renal ammonia excretion as part of a greater acid-base regulatory system. However, the transport mechanisms employed by the renal epithelium to excrete ammonia are relatively unknown. I hypothesized that, under metabolic acidosis, increased renal ammonia excretion would be the product of tubular secretion and involve a Na+/NH4+ exchange metabolon mediated through Rhesus (Rh) glycoproteins. To induce metabolic acidosis, goldfish (Carassius auratus) were exposed to a low pH environment (pH 4.0; 48-h). There was a clear signal of metabolic acidosis: a reduction in both plasma [HCO3-] and blood pH with no influence on plasma PCO2. Goldfish demonstrated an elevation in total plasma [ammonia] with a reduction in PNH3 under acidosis. Metabolic acidosis induced higher rates of urinary excretion of acidic equivalents in the form of both NH4+ and titratable acidity-HCO3- (TA-HCO3-) excretion. Urinary Na+ excretion was not affected by acidosis and urine [Na+] did not correlate with urinary [ammonia]. Alanine aminotransferase activity in the kidney was higher in acidotic goldfish. Glomerular filtration rate and urine flow rate were not affected by acidosis. Increased renal NH4+ excretion was due to increased secretion, and not increased filtration, of ammonia. There was a corresponding elevation in Rhcg1b mRNA expression but no change in renal Na+ reabsorption. My data support a secretion-based mechanism of teleost renal ammonia transport. This system is Na+ independent and is likely mediated by Rh glycoproteins and H+ ATPase, involving a parallel H+/NH3 secretion mechanism. To investigate effects of metabolic acidosis on elasmobranch fish, Pacific spiny dogfish (Squalus acanthias suckleyi) were infused with an acidic saline (125 mM HCl/375 mM NaCl; 3 ml/kg/h; 24-h). The results are preliminary, with no marked effects of HCl infusion on plasma acid-base or N-status, but increased branchial NHE2 and lower renal NHE3 protein expressions. These data are summarized in an Appendix. / Thesis / Master of Science (MSc)