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)

Largely through the use ofin vitro preparations there is now a generally accepted theory for ion transport for the seawater (SW) gill. However, to date there is no generally accepted freshwater (FW) model for the mechanisms of NaCl transport in the teleost gill. By using an Ussing chamber approach with the opercular epithelia of Fundulus heteroclitus and Oreochromis niloticus, and the urinary bladder of Oncorhynchus mykiss, all acclimated to FW, we hoped to establish one as a possible model for the study of FW ion regulation. FW Fundulus opercular epithelia displayed a serosal negative transepithelial potential (Vₜ) of-43.9 mV, transepithelial conductance (Gₜ) of 1.94 mS·cm⁻², and active transport of Cl⁻ from the mucosal FW against a strong electrochemical gradient. Na⁺ movement was dominated by passive diffusion. The opercular epithelia of Fundulus adapted to 10% SW exhibited properties similar to SW Fundulus by actively extruding Cl⁻ while Na⁺ moved passively into the mucosal 10% SW. With FW bathing the mucosal surface, FW Oreochromis opercular epithelia displayed a serosal positive Vₜ of +8.0 mV, Gₜ of 1.78 mS·cm⁻², and active reabsorption of Na⁺, Cl⁻ and Ca²⁺ against large electrical and/or chemical gradients. The FW Oreochromis opercular epithelia is the only FW in vitro preparation to date that exhibits active absorption (albeit at small absolute rates) of both Na⁺ and Cl⁻. FW 0. mykiss urinary bladders mounted in vitro under symmetrical saline conditions exhibited a transepithelial conductance (Gₜ) of-9.15 mS·cm⁻² and electroneutral active absorption of Na⁺ and Cl⁻ from the mucosal urine side. The transport of Na⁺ and Cl⁻ was a partially coupled process whereby removal of Na⁺ from the mucosal saline decreased Cl⁻ absorption by a 56% and removal of Cl⁻ inhibited Na⁺ absorption by 69%. However, active net absorption of both ions persisted when the counter-ion was replaced with a non-permeant ion. Under more realistic conditions with artificial urine bathing the mucosal surface, Vₜ increased to a serosal positive ~+7.6 mV and Gₜ decreased to ~1.47 mS·cm⁻² Unidirectional influx rates of both Na⁺ and Cl⁻ were much lower, but active absorption of both ions still occurred. Replacement of Na⁺ in the mucosal artificial urine caused no change in unidirectional influx of Cl⁻ and vice versa. The mucosal addition of DIDS, amiloride or bumetanide (10⁻⁴M) all had no affect on absorption rates of Na⁺ and/or Cl⁻, under either artificial urine or symmetrical saline conditions. When the mucosal surface was bathed in artificial urine, removal of mucosal Cl⁻· significantly reduced the maximum transport rate (Jₘₐₓ of Na⁺ (6.1 ~2.1 μmol·cm⁻²·h⁻¹) but had no effect on affinity for Na⁺ (Kₘ~27 mM). Similarly, removal of mucosal Na⁺ significantly reduced the Jₘₐₓ for Cl⁻ uptake (11.4 ~2.4 μmol·cm⁻²·h⁻¹) but had no effect on Cl⁻ Kₘ(~37 mM). The anterior portion of the urinary bladder transported Na⁺ and Cl⁻ at a faster rate than the posterior portion under symmetrical saline conditions, but there was no difference in measured Na⁺/K⁺ -ATPase activities between the two portions. In conclusion, opercular epithelia of Fundulus and Oreochromis demonstrated transport properties believed to be typical of FW teleosts; either preparation may eventually prove to be a good working model for studying FW transport mechanisms. The urinary bladder of Oncorhynchus mounted in vitro did not behave characteristically as the teleost gill is thought to function, but this model may be useful in characterizing various other mechanisms of ionic transport. / Thesis / Master of Science (MSc)

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.

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.

No description available.

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.

L’objectif de cette thèse est d’étudier le rôle de neuropeptides cérébraux, telle que la Neurokinin B codée par le gène tac3, dans le contrôle de la reproduction d’une espèce en danger, l’anguille Européenne, Anguilla anguilla. La maturation sexuelle de l’anguille est bloquée à un stade prépubertaire avant la migration océanique. Etant donnée sa position phylogénétique basale parmi les téléostéens, l’anguille est un modèle pertinent pour étudier l’évolution moléculaire et fonctionnelle de neuropeptides d’intérêt. Deux gènes paralogues tachykinine 3 (tac3) ont été identifiés dans le génome de l’anguille, chacun codant pour deux peptides. Ces gènes paralogues résultent de la duplication complète du génome spécifique aux téléostéens, comme le montrent les analyses phylogénétiques et synténiques. Les analyses de qPCR montrent que les deux gènes sont exprimés dans le cerveau. Les quatre peptides d’anguille ont été synthétisés et testés sur des cultures primaires de cellules hypophysaires d’anguille. Les quatre peptides inhibent l’expression de l’hormone lutéinisante et d’un récepteur à la gonadolibérine, révélant un double rôle inhibiteur dans le contrôle de la reproduction. / The aim of this PhD is to investigate the role of brain neuropeptides, such as neurokinin B, encoded by tac3 gene, in the control of reproduction of an endangered species, the European eel, Anguilla anguilla. The sexual maturation of the eel is blocked at a prepubertal stage before the oceanic migration. Due to its basal phylogenetic position among teleosts, the eel is also a relevant model for studying molecular and functional evolution of key neuropeptides. Two tachykinin 3 (tac3) paralogous genes were identified in the eel genome, each encoding two peptides. These paralogs result from the teleost-specific whole genome duplication, as shown by phylogeny and synteny analyses. Both genes are expressed in the brain as shown by qPCR. The four eel peptides were synthesized and tested on primary cultures of eel pituitary cells. The four peptides inhibited the expression of luteinizing hormone and gonadotropin-releasing hormone receptor, revealing a dual inhibitory role in the control of reproduction.

Testicular maturation indices (TMI) were determined for wild-caught males of Poecilia mexicana by quantitative analysis of the frequency of ongoing stages of spermatogenesis and spermatid differentiation in sections of testes stained with the Feulgen reaction for DNA. In nature, males maintain essentially constant levels of sperm production throughout the year and show no significant variations in mean TMI values associated with season, microhabitat, standard body length, or rank in male dominance hierarchies. Winter males or males isolated from females in the laboratory show accumulation of mature spermatophores. These findings suggest that fluctuations in the microstructure of wild populations of Poecilia from northeastern Mexico may be due primarily to differences in the temporal scheduling of female reproductive cycles and not to limitations imposed by sperm availability or male reproductive competence. The data are discussed in terms of competitive interactions within unisexual-bisexual breeding complexes involving the Amazon molly P. formosa, its related triploid hybrids, and the bisexual species, P. mexicana.

No description available.

O presente trabalho teve como objetivo estudar a morfologia e fisiologia reprodutiva de Lobotes surinamensis em ambiente natural, reunindo informações que permitam trabalhos aplicados na produção e conservação desta espécie. Machos e fêmeas adultos foram coletados na Baía da Ilha Grande, Rio de Janeiro durante a primavera (outubro-novembro-dezembro), verão (janeiro-fevereiro-março), outono (abril-maio-junho) e inverno (julho-agosto-setembro). Os perfis plasmáticos dos esteroides gonadais, 17β-estradiol (E2), 17-hidroxiprogesterona (17-OHP) e 11-cetotestosterona (11-KT) foram analisados em fêmeas, e a concentração de testosterona (T) e 11-KT nos machos. Análises histológicas das gônadas foram realizadas como uma ferramenta para avaliação morfológica, que foi corroborada com os dados fisiológicos, durante o ciclo reprodutivo. Foram analisadas ainda a expressão gênica hipofisária do fshβ e lhβ e a fecundidade relativa pelo número de oócitos desenvolvidos. Durante a primavera e verão, as fêmeas estavam com os ovários desenvolvidos e capazes de desovar, com oócitos vitelogênicos e desenvolvidos, e membrana radiata bem evidente. No outono e inverno, as fêmeas estavam se preparando para o período reprodutivo, com predomínio de ninhos de oogônias e oócitos perinucleolares de uma forma geral. A concentração plasmática de E2 se manteve elevada nas fêmeas durante a primavera em relação ao verão e outono/inverno. O perfil anual deste esteroide nas fêmeas de L. surinamensis pode ser relacionado com o grau de desenvolvimento do folículo ovariano, observado na histologia. Já a concentração plasmática de 11-KT foi inversa ao observado para o E2, com baixa concentração na primavera e tendência à elevação no inverno, corroborando com o papel da 11-KT no crescimento primário dos oócitos. A concentração plasmática de 17-OHP nas fêmeas não apresentou diferenças significativas entre as estações, o que sugere que este não seja o progestágeno ativo em teleósteos, mas sim o precursor do esteroide indutor da maturação final (MIS). Na primavera e no verão, o lhβ apresentou expressão maior do que no período outono/inverno em fêmeas, enquanto o fshβ se manteve com níveis de expressão constante durante todas as estações. Nos machos, a análise histológica dos testículos, mostrou que na primavera e no verão, os ductos espermáticos estavam preenchidos com espermatozoides, evidenciando o período de espermiação, já no período outono/inverno houve predominância de cistos de espermatogônias e as células em diferentes estágios de desenvolvimento, o que corresponde a uma fase de maturação inicial, ou seja, preparação para o período reprodutivo. A concentração plasmática de T nos machos se manteve estável ao longo das estações, já a concentração plasmática de 11-KT foi mais elevada na primavera quando comparada ao período outono/inverno. Na primavera e verão, a expressão de lhβ foi mais elevada que no período outono/inverno, enquanto de forma similar às fêmeas, os níveis de expressão de fshβ não se alteraram ao longo das estações. As análises de expressão destas gonadotropinas, associadas à concentração dos esteroides gonadais e à morfologia gonadal deixou evidente que o pico do período reprodutivo de L. surinamensis ocorre na primavera, estendendo-se até o verão / The present study aimed to study the morphology and reproductive physiology of Lobotes surinamensis in a natural environment, gathering information that allows applied works in the production and conservation of this species. Adult males and females were collected in the Baía da Ilha Grande, Rio de Janeiro during the spring (October-November-December), summer (January-February-March), autumn (April- May-June) and winter (July-August-September). Plasma profiles of gonadal steroids, 17β-estradiol (E2), 17-hydroxyprogesterone (17-OHP) and 11-ketotestosterone (11-KT) were analyzed in females, and the concentration of testosterone (T) and 11-KT in males. Histological analyzes of the gonads were performed as a tool for morphological evaluation, which was corroborated with the physiological data, during the reproductive cycle. The genetic expression of fshβ and lhβ and the relative fecundity by the number of developed oocytes were also analyzed. During spring and summer, the females had the ovaries developed and capable of spawning, with vitellogenic and developed oocytes, and a well evident radiata zone. In autumn and winter, females were preparing for the reproductive period, with a predominance of nest of oogonia and perinucleolar oocytes in general. The plasma concentration of E2 remained higher in females during the spring in relation to summer and autumn/winter. The annual profile of this steroid in L. surinamensis females may be related to the degree of development of the ovarian follicle observed in histology. The plasma concentration of 11-KT was inversely of that observed for E2, with a low concentration in the spring and a tendency to increase in winter/autumn, corroborating the role of 11-KT in the primary oocyte growth. The plasma concentration of 17-OHP in the females did not present significant differences between the seasons, suggesting that this is not the active progestogen in teleosts, but the precursor of the final maturation inducing steroid (MIS). In spring and summer, lhβ presented higher gene expression than in the autumn/winter period in females, while fshβ remained with constant expression levels during all seasons. In males, the histological analysis of the testicles showed that in the spring and summer, the sperm ducts were filled with spermatozoa, evidencing the period of spermiation, whereas in the autumn/winter period there was a predominance of cysts of spermatogonia and cells in different stages of. development, which corresponds to an initial maturation stage, that is, the preparation for the reproductive period. The plasma concentration of T in males remained stable throughout the seasons, whereas the plasma concentration of 11-KT was higher in the spring when compared to the autumn/winter period. In spring and summer, the expression of lhβ was higher than in the autumn/winter period, while similarly to females, fshβ expression levels did not change over the seasons. The analysis of the expression of these gonadotropin genes, associated with the concentration of gonadal steroids and gonadal morphology, made it evident that the peak of the reproductive period of L. surinamensis occurs in the spring, extending until the summer