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Remote photoelectric monitoring of the locomotion of four freshwater teleosts in an experimental tank established that all species described non-random locomotor patterns which were affected by light intensity. A partial analysis was made of some of the characteristics of these patterns with emphasis on the direction and degree of turning. / Thesis / Doctor of Philosophy (PhD)

Dans le cadre de cette thèse, nous avons étudié la mise en place de la personnalité, ainsi que sa cohérence en fonction du contexte et des différents stades vie de l’individu et les mécanismes physiologiques associés. La personnalité est apparue cohérente entre les contextes sur de courtes périodes mais ne l’est plus entre les différents stades de vie. Les mécanismes physiologiques de régulation du stress (cortisol et monoamines), ainsi que l’activité et la plasticité neuronale divergent en fonction de l’audace des individus. Ensuite, nous avons étudié les réponses physiologiques et comportementales des téléostéens exposés chroniquement à un mélange de POP (PCB et PBDE), représentatif des conditions environnementales, ainsi que celles de leur descendance non exposée. Des altérations comportementales différentes de la réponse photomotrice larvaire, de l’audace et d’un syndrome comportemental lié à l’anxiété en fonction des générations ont été observées jusqu’à la génération F2 ou F4. Des altérations du niveau d'expression des enzymes responsables de la méthylation de l’ADN ont été également observées suggérant que des mécanismes épigénétiques sont responsables de la transmission de ces altérations chez la descendance. Ensuite, le comportement a été étudié dans des qualités d'eau dégradées afin de mettre en place des indicateurs comportementaux de bien-être pertinents. Les résultats ont montré que suite à la variation de l’oxygénation ou de la teneur en NH3, les individus s’adaptent en diminuant leur activité de nage et la cohésion du groupe. De plus, nous avons observé que le comportement normal de thigmotaxie est inhibé lors de l’exposition à une qualité d’eau dégradée. Cela suggère une hiérarchisation des adaptations comportementales en fonction de la sévérité du stress. Enfin, nous avons étudié les réponses comportementales à une diminution du niveau d’oxygène dans l’eau en fonction de la personnalité : les individus audacieux diminuaient leur activité et leur cohésion de nage avec une plus grande amplitude que les individus timides qui eux diminuaient, avec une plus grande amplitude, le comportement de thigmotaxie. Ces capacités d’adaptation différentes face au stress, en fonction de la personnalité, apportent de nouvelles perspectives pour faire face aux enjeux dans des contextes écologique et aquacole. / In the frame of this thesis, we first studied the establishment of personality in teleost, the consistency across context and different life stages as well as the associated physiological mechanisms. Personality appeared consistent across contexts over a short time period but it was no longer the case over a long period of time, especially between different life stages. Physiological mechanisms of stress regulation, as well as activity and neural plasticity, differed according to individual boldness. Then, we studied the physiological and behavioural responses of teleost fish chronically exposed to an environmentally relevant mixture of POP (PCBs and PBDEs), as well as those of their unexposed offspring. Different behavioural alterations in larval photomotor response, boldness and a behavioural syndrome linked to anxiety have been observed between generations up to the F2 or F4 generations. Alterations in the expression of enzymes responsible of DNA methylation were also observed suggesting that epigenetic mechanisms are involved in the transmission of these behavioural alterations in unexposed offspring. Behaviour was also studied under stressful water quality conditions in order to set behavioural welfare indicators. Results showed that individuals cope with these alterations in oxygen levels or NH3 concentration by decreasing their swimming activity and group cohesion. In addition, we observed that the normal thigmotaxis behaviour was inhibited during exposure to degraded water quality. It suggests a hierarchy of behavioural adaptations depending on stress severity. Finally, we studied the behavioural responses to a decrease in oxygen level according to personality: bold individuals decreased their activity and swimming cohesion with greater amplitude than shy individuals which decreased with higher amplitude thigmotaxis behaviour. These abilities to cope with stress, depending on the personality of teleost fish, bring new perspectives to face both ecological and aquaculture issues.

Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the red drum thyroid precluded their use as a bioassay species, the plasma TSH and T4 response to exogenous TSH was next characterized in goldfish. The T4 response in goldfish was stable and repeatable, with T4 levels peaking at 5 hours and remaining elevated for more than 11 hours after bovine TSH injection. Plasma TSH peaked from 2-5 hours following TSH injection with more than 90 percent cleared by 11 hours. The goldfish bioassay was further utilized to evaluate the effects of structural modifications on TSH biological activity. Substitution of four positively charged amino acids at the n-recombinant human TSH, had the same effect in goldfish. The heterothyrotropic potency of mammalian follicle stimulating hormone in goldfish was also enhanced by the same amino acid substitutions. Finally, the importance of oligosaccharides to TSH bioactivity was also examined in goldfish. Deglycosylation abolished TSH bioactivity, even when immunoreactivity persisted in circulation. Furthermore, recombinant canine TSH was less potent when produced in cell lines generating insect-type glycosylation than when produced in a cell line capable of mammalian-type glycosylation. These studies utilizing recombinant mammalian demonstrated conservation of mammalian TSH hormone-receptor interactions in goldfish, suggesting TSH function might likewise be conserved. Thus, I have established goldfish as a sensitive and stable bioassay which can now be utilized to monitor the biological activity of teleost TSH expressed in vitro as well as to evaluate how structural modifications of the TSH molecule influence its vivo biological activity.

Le loup ou bar européen effectue une partie de son cycle de vie dans les lagunes et estuaires, des eaux de transitions caractérisées par des fluctuations environnementales plus marquées qu’en milieu marin. Les mécanismes d’acclimatation à la salinité à des températures plus élevées qu’en milieu marin sont encore relativement inexplorés. Dans cette étude, des juvéniles de loup ont été préacclimatés pendant deux semaines à l’eau de mer (EM) à 18 °C (eau tempérée) ou à 24 °C (eau chaude) puis transférés soit dans l’eau douce (ED) soit dans l’EM aux deux températures testées. À 24 °C, les loups parviennent à maintenir leur pression osmotique sanguine relativement constante, quelle que soit la salinité testée. En revanche, la hausse de température affecte significativement différents traits physiologiques liés à l’osmorégulation, la régulation acido- basique, l’excrétion azotée et la production de mucus. Cette étude a notamment montré qu’une acclimatation thermique à 24 °C modifie la structure morphologique de la branchie et induit un déséquilibre de la balance ionique sanguine. Ce déséquilibre concerne particulièrement les ions Na+ et a pour conséquence une diminution du ratio Na+/Cl- plasmatique, pouvant être symptomatique d’une acidose sanguine. Après transfert en douce, une augmentation moins importante de la densité des ionocytes branchiaux et de l’activité de la pompe Na+/K+ ATPase a été observée à 24 °C comparé à 18 °C. De plus, certains transporteurs ioniques jouant un rôle clé dans l’absorption ionique en eau douce n’ont pas été induits au niveau transcriptionnel à 24 °C. Cela suggère une capacité réduite des poissons de passer d’un épithélium branchial hypo-osmorégulateur vers un épithélium hyper-osmorégulateur lors d’un transfert en eau douce à 24 °C vs 18 °C. Au niveau moléculaire, la hausse de température a affecté l’expression de plusieurs transporteurs ioniques branchiaux, notamment en EM. À 24 °C, la surexpression de transporteurs liés au transport de proton H+ pourrait être le signe d’un déséquilibre acido- basique. De plus, l’induction de transporteurs d’ammonium pourrait indiquer un besoin accru d’excréter de l’azote par voie branchiale, potentiellement lié à un métabolisme plus élevé en eau chaude. L’expression des gènes codant pour la protéine chaperonne HSP90 a été fortement affectée par la hausse de température et dans une moindre mesure par la dessalure. Pour finir, l’analyse des mucocytes suggère que la production de mucus pourrait être induite en réponse au stress thermique. Ce type d’étude fonctionnelle sur l’acclimatation à différents régimes de température permet d’apporter des éléments de compréhension pour pouvoir prédire les réponses des téléostéens face aux conséquences du changement global. / The European sea bass Dicentrarchus labrax undertakes seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. It is unclear to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea, as usually encountered in transitional waters in spring and summer. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18 °C (temperate) or 24 °C (warm) for two weeks and then transferred to either fresh water (FW) or SW at the considered temperatures. We have shown that sea bass are able to efficiently maintain blood osmolality at 24 °C at both salinities. However, temperature increase induced significant changes regarding several physiological traits related to osmoregulation, acid-base regulation, ammonia excretion and mucus production. This study showed that thermal acclimation at 24 °C affects gill morphology through gill remodeling and whole-organism ion balance. Plasma Na+ levels seemed to be particularly affected leading to decreased plasma Na+/Cl- ratio in warm conditions, suggesting a blood acidosis. Following FW transfer, the major effects observed were a lower increase in the density of branchial ionocytes and in Na+/K+-ATPase activity at 24 °C compared to 18 °C. Moreover, several key ion transporters involved in ion uptake were not transcriptionally induced following FW transfer at 24 °C. These data suggest a less effective capacity to switch from hypo-to hyper-osmoregulation after FW transfer when fish are exposed to higher temperature. At the molecular level, the temperature increase affected the expression of several branchial transporters, notably in SW. At 24 °C, higher expression of transporters linked to H+ transport might be linked to an acid-base imbalance. Moreover, the induction of ammonia-transporting channels might also indicate increased need for nitrogen excretion, potentially due to enhanced metabolism in warm conditions. Molecular chaperones HSP90 expression was strongly affected by temperature increase and to a lesser extent by salinity decrease. Finally, gill goblet cells analysis suggested that mucus production is increased in response to thermal stress. This study and additional functional studies investigating different temperature regimes provide insights on the effect of increased temperatures on fish responses and may help to predict how teleost will face the consequences of global change.

Teleost fishes share a duplication of their entire genomes. We report here on a computational survey of structured non-coding RNAs (ncRNAs) in teleost genomes, focusing on the fate of fish-specific duplicates. As in other metazoan groups, we find evidence of a large number (11,543) of structured RNAs, most of which (~86%) are clade-specific or evolve so fast that their tetrapod homologs cannot be detected. In surprising contrast to protein-coding genes, the fish-specific genome duplication did not lead to a large number of paralogous ncRNAs: only 188 candidates, mostly microRNAs, appear in a larger copy number in teleosts than in tetrapods, suggesting that large-scale gene duplications do not play a major role in the expansion of the vertebrate ncRNA inventory.

In seawater, drinking is a fundamental part of the osmoregulatory strategy for teleost fish, and presents a unique challenge. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid from imbibed seawater is crucial to compensating for water losses to the surrounding hyperosmotic environment. Alongside solute-linked water transport (driven by NaCl cotransport), intestinal bicarbonate (HCO3-) secretion also benefits fluid absorption directly (via apical Cl-/HCO3- exchange), and indirectly through the formation of calcium carbonate (CaCO3) thus removing the osmotic influence of Ca2+ within the gut fluid. For the European flounder (Platichthys flesus), elevated luminal Ca2+ has proven to be a specific, potent stimulator of HCO3- secretion both in vitro and in vivo where these actions are presumably modulated by an extracellular Ca2+-sensing receptor (CaR). The focus of this work was to learn more about how intestinal HCO3- secretion is regulated, the role of Ca2+, and more specifically the CaR. To achieve this, in vitro ‘gut sac’ experiments investigated how luminal Ca2+ influenced HCO3- secretion, and associated ion and fluid transport. Contrary to expectation, increasing Ca2+ from 5 to 20 mM did not stimulate HCO3- secretion. In an attempt to elucidate the role of CaCO3 precipitation in fluid absorption, and further explore the physiological implications of HCO3- secretion, the intestine was perfused in vivo with salines containing varying concentrations of Ca2+ (10, 40 and 90 mM). The production and secretion of HCO3-, in addition to CaCO3 formation increased accordingly with Ca2+, and was associated with a dramatic 25 % rise in the fraction of fluid absorbed by the gut. Additional in vitro experiments, utilising the Ussing chamber, helped establish some of the characteristics of intestinal HCO3- secretion by the euryhaline killifish (Fundulus heteroclitus), but was unresponsive to elevated mucosal Ca2+. Further attempts to potentiate the activity of the CaR, and application of the receptor agonists gadolinium (Gd3+) and neomycin, failed to produce responses consistent with the effect of Ca2+ observed previously, either in vitro or in vivo. With no evidence supporting a direct role for an extracellular, intestinal CaR in HCO3- secretion it was argued that secretion would be principally regulated by two factors, the ability of the epithelia to generate high levels of intracellular HCO3- and the rate of CaCO3 formation.

Machos adultos de P. lineatus tiveram suas gônadas processadas de acordo com as rotinas de microscopia de luz e microscopia eletrônica de transmissão. Para a cultura de células, os testículos foram digeridos enzimaticamente, a suspensão testicular foi separada por gradiente descontínuo com Percoll seguido pelo plaqueamento diferencial por adesão e as células foram cultivadas. Foi realizado o método de enriquecimento das espermatogônias por citometria de fluxo. Os testículos apresentam as regiões anterior, média e posterior com distribuição semelhante dos tipos de células, e o diâmetro nuclear das células germinativas diminui significativamente durante a espermatogênese. As espermatogônias cultivadas por 15 dias com meio para proliferação celular resultaram em grandes aglomerados celulares que foram caracterizados com o anti-Vasa, anti-GFRa1 e anti-OCT4. As culturas que receberam o meio para diferenciação celular mostraram processo de proliferação lento das espermatogônias primárias comparado com a cultura que teve o meio indicado para proliferação celular. / Adult males of P. lineatus had their gonads used according routines of light microscopy and transmission electron microscopy. For cell culture, the testes were enzymatically digested; testicular suspension was separated by discontinuous gradient with Percoll followed by adhesion differential plating and the cells were cultured. The enrichment of the spermatogonia was carried by flow cytometry. The testes present three regions with similar distribution of cell types, and nuclear diameter of germ cells decreases significantly during spermatogenesis. The spermatogonia cultured for 15 days with medium for cell proliferation, resulted in large cell agglomerates which were characterized with the antibodies anti-Vasa, anti-GFRa1 and anti-anti-OCT4. The cultures that receiving medium for cell differentiation showed slow proliferation process of primary spermatogonia compared to cell culture medium suggestive for cell differentiation.

Após a reprodução de Astyanax altiparanae, foram obtidas larvas e juvenis da espécie, os quais foram coletados desde a eclosão até 120 dias após a eclosão (dpe). Por meio de métodos histoquímicos e imuno-histoquímica foram detectadas a neuro (NH) e adeno-hipófise (AH). Nas 3 regiões da AH, rostral (RPD) e proximal (PPD) pars distalis, e pars intermedia (PI), foram encontrados 8 tipos celulares produtores de hormônios, sendo, na RPD, as células produtoras de ACTH e PRL; na PPD, GTH (FSH e LH), GH e TSH; e na PI, MSH e SL. As prolactínicas foram as primeiras células identificadas com 1 dpe no início da formação da glândula. A NH foi diferenciada com 3 dpe junto com as células produtoras de ACTH, MSH, TSH e FSH. A identificação das células produtoras de LH, SL e GH ocorreu com 5 dpe. Com 20 dpe houve um grande aumento da hipófise e o aparecimento do pedúnculo. Com 60 dpe a hipófise apresentou-se semelhante à observada nos adultos. Os resultados desta pesquisa colaboram para o conhecimento do eixo hipotálamo-hipófise-gônada em teleósteos sul-americanos. / After reproduction of A. altiparanae, was made larvae and juveniles of this specie, were collected from zero to 120 days post hatching (dph). By histochemical and immunohistochemical techniques were detected neuro (NH) and adenohypophysis (AH). In 3 regions of AH, rostral (RPD), proximal (PPD) pars distalis and in pars intermedia (PI), eight producing hormones cell types are distinguished, being, in the RPD, ACTH and PRL producing cells; in PPD, GTH (FSH and LH), GH and TSH; and in the PI, MSH and SL. The first cells identified were prolactins at 1dph, in the beginning of the formation of the gland. NH differentiated at 3dph along with identification of ACTH, MSH, TSH and FSH producing cells. The identification of LH, SL and GH producing cells was possible at 5dph. At 20dph, there was a large increase in the pituitary, and it was observed the presence of the pituitary stalk. At 60 dph the pituitary showed a similar morphology to that observed in adults. The results of this research collaborate to knowledge of the hypothalamic-pituitary-gonadal axis in South American teleosts.