Major histocompatibility complex (MHC) class II associated invariant chain is a chaperone of the mammalian MHC class II antigen presentation pathway. It is responsible for targeting the MHC class II dimer to the endocytic pathway, which allows for the loading of exogenous antigens onto the MHC class II receptor. Two genes showing significant sequence similarity to the mammalian invariant chain gene have been identified in rainbow trout. S25-7 and INVX are thought to play a role within the teleost MH class II antigen presentation pathway similar to that performed by the mammalian invariant chain. In vivo and in vitro methods have been used to investigate the regulation of these genes upon immune system activation. To induce an immune response both rainbow trout and the macrophage-like cell line RTS-11 were treated with phorbol myristate acetate (PMA). PMA is a stimulator of protein kinase C, which is involved in the activation of both B and T cells. After PMA treatment, individuals were sacrificed at several time points and tissues were collected for analysis. Up-regulation of IL-1β transcript, as detected by RT-PCR analysis, demonstrated the successful induction of an immune response in vivo. S25-7 transcript levels remained unchanged in gill, spleen, peripheral blood leukocytes and liver. However, the transcript was found to be significantly decreased in head kidney beginning 24 hours post-stimulation. INVX transcript levels remained unchanged in all tissues analyzed. In addition, the level of S25-7 transcript is higher in all tissues than INVX transcript. Western blot analysis detected both S25-7 and INVX proteins within gill and spleen, indicating the presence of cell populations capable of presenting extracellular antigen within the MH class II dimer. Similar to transcript analysis, the level of invariant chain in these tissues remained unchanged during immune system activation. Neither INVX nor S25-7 was detected in head kidney, indicating this tissue may not function as a secondary immune tissue, as previously thought. Finally, western blot analysis failed to detect either INVX or S25-7 protein within liver, as expected in a non-immune tissue. RTS11 analysis demonstrated a similar pattern of invariant chain expression in vitro. S25-7 transcript levels remained stable throughout the PMA stimulation time course, while INVX transcript was down-regulated at 48 and 96 hours post-stimulation, with a subsequent increase at 168 hours. However, both S25-7 and INVX protein levels remained unchanged during immune stimulation. These results demonstrate a pattern of invariant chain tissue expression, providing information about the type of immune functions carried out at these sites. In addition, the results indicate that neither INVX nor S25-7 is up-regulated, at either the transcript or protein level upon immune system activation. This is in contrast to the mammalian invariant chain, which is up-regulated at both the transcript and protein level during an immune response, indicating that teleosts have evolved a unique system of immune regulation.

Major histocompatibility complex (MHC) class II associated invariant chain is a chaperone of the mammalian MHC class II antigen presentation pathway. It is responsible for targeting the MHC class II dimer to the endocytic pathway, which allows for the loading of exogenous antigens onto the MHC class II receptor. Two genes showing significant sequence similarity to the mammalian invariant chain gene have been identified in rainbow trout. S25-7 and INVX are thought to play a role within the teleost MH class II antigen presentation pathway similar to that performed by the mammalian invariant chain. In vivo and in vitro methods have been used to investigate the regulation of these genes upon immune system activation. To induce an immune response both rainbow trout and the macrophage-like cell line RTS-11 were treated with phorbol myristate acetate (PMA). PMA is a stimulator of protein kinase C, which is involved in the activation of both B and T cells. After PMA treatment, individuals were sacrificed at several time points and tissues were collected for analysis. Up-regulation of IL-1β transcript, as detected by RT-PCR analysis, demonstrated the successful induction of an immune response in vivo. S25-7 transcript levels remained unchanged in gill, spleen, peripheral blood leukocytes and liver. However, the transcript was found to be significantly decreased in head kidney beginning 24 hours post-stimulation. INVX transcript levels remained unchanged in all tissues analyzed. In addition, the level of S25-7 transcript is higher in all tissues than INVX transcript. Western blot analysis detected both S25-7 and INVX proteins within gill and spleen, indicating the presence of cell populations capable of presenting extracellular antigen within the MH class II dimer. Similar to transcript analysis, the level of invariant chain in these tissues remained unchanged during immune system activation. Neither INVX nor S25-7 was detected in head kidney, indicating this tissue may not function as a secondary immune tissue, as previously thought. Finally, western blot analysis failed to detect either INVX or S25-7 protein within liver, as expected in a non-immune tissue. RTS11 analysis demonstrated a similar pattern of invariant chain expression in vitro. S25-7 transcript levels remained stable throughout the PMA stimulation time course, while INVX transcript was down-regulated at 48 and 96 hours post-stimulation, with a subsequent increase at 168 hours. However, both S25-7 and INVX protein levels remained unchanged during immune stimulation. These results demonstrate a pattern of invariant chain tissue expression, providing information about the type of immune functions carried out at these sites. In addition, the results indicate that neither INVX nor S25-7 is up-regulated, at either the transcript or protein level upon immune system activation. This is in contrast to the mammalian invariant chain, which is up-regulated at both the transcript and protein level during an immune response, indicating that teleosts have evolved a unique system of immune regulation.

Cooperation often draws on cognition (i.e. learning and memory) in order to track cooperative partners and their contributions, resolve conflict between partners, improve coordination between partners, and enhance strategic-decision making. Learning and memory are also vital to resolve what is frequently a spatial and temporal mismatch between performing a cooperative act and receiving any kind of benefit in return. In this thesis, I compared cognitive abilities between three cooperative and group-living cichlid fishes from the Lamprologini tribe of Lake Tanganyika Neolamprologus pulcher, Neolamprologus multifasciatus, and Julidochromis ornatus with three of their close relatives that are not cooperative and never form groups Telmatochromis temporalis, Altolamprologus compressiceps, and Neolamprologus tretocephalus. This thesis aims to address whether the evolution of cooperation coincided with the evolution of sophisticated cognition in these fishes. In Chapter 2, I present evidence that both cooperative and non-cooperative cichlids are able to recognize familiar individuals and have similar numerical abilities. In Chapter 3, I show that performance when learning to navigate a maze, arguably a more general cognitive ability, was equivalent across cooperative and non-cooperative species, with comparable scores in terms of time to maze completion, number of mistakes and inhibitory control. In Chapter 4, I report on the results of a literature review where I quantified the growing interest in the field of fish cognition research, outline the current practices and pitfalls (heavy use of captive bred individuals and a reliance on lab-based research) and suggest how to bring more ecological relevance to the field. Taken together, the results of my thesis improve our understanding of how social factors like group-living and cooperation modulate cognitive abilities, and detail the current trajectory of the field of fish cognition. / Thesis / Master of Science (MSc)

Freshwater rainbow trout were exposed to 9.2 µg/L total Ag (as AgNO3, a level approximately equal to the 168 h LC50) for 144 h to clarify the toxic mechanism of silver in freshwater teleosts. It was found that silver inhibits active Na+ and Cl- uptake at the gills, resulting in a net loss of both ions from the fish and creating a metabolic acidosis. This leads to a classic stress response (mobilization of cortisol and glucose into the blood plasma), and hyperventilation as a respiratory response to decreased blood pH. Plasma ammonia levels rise without any decrease in ammonia excretion; ammonia excretion later increases. This suggests that the increased plasma levels are due to increased metabolic production. Increased [H+] (decreased pH) results in excess H+ ions in the internal fluids, which are either complexed with ammonia to form NH4+ or are buffered in muscle tissue. The latter results in increased movement of K+ ions into the plasma, which are then excreted at the gills, preventing hyperkalemia. In the end, freshwater teleosts probably die from iono-and osmo-regulatory failure and associated cardiovascular collapse. Seawater teleosts (rainbow trout, tidepool sculpins, English sole, and plainfin midshipmen) and elasmobranchs (Pacific spiny dogfish and long nose skate) were exposed to constant concentrations on total Ag (as AgNO3) ranging from 1.5 to 50.0 µg/L for periods of up to 21 d at salinities of 18 ppt or 30 ppt. These exposure levels are well below those causing acute toxicity in seawater. Silver appears to enter marine teleosts and marine elasmobranchs differently. Seawater teleosts drink the seawater, so the intestines are a major site of silver uptake, along with the gills. Since marine elasmobranchs do not drink, the gills appear to be the sole site of silver uptake from the water. As in freshwater, the liver is the main site for silver accumulation in all marine fish studied. Despite similar terminal liver silver concentrations, marine elasmobranchs have a higher rate of silver accumulation since the livers in elasmobranchs are 10-20 fold larger than in teleosts. Both environmental salinity and exposure concentration play direct roles in determining silver bioaccumulation in marine teleosts. Increasing salinity alters the speciation of silver in the water, which decreases the amount of silver able to enter the fish. Increased silver concentrations mean more silver is available to enter the fish and subsequently cause sublethal toxic effects. Oxygen consumption decreased during the first 7 d of chronic exposure to sublethal silver levels in marine teleosts, with the decrease being more pronounced at higher (still sublethal) silver levels. Ammonia excretion, unaltered during acute exposure (48 h) to high silver levels (250 µg/L), was decreased during the first 7 d of exposure to sublethal silver levels (14.5-50.0 µg/L). This suggests that silver interferes with energy demanding processes such as protein synthesis or iono-regulation. Activity levels of the main enzyme involved in iono-regulation, namely Na+/K+ -ATPase, was affected differently in different fish. In a marine teleost that lives solely in seawater (plainfin midshipmen), silver inhibited the gill ATPase activity after 7 d of exposure, with the inhibition being more effective at higher silver levels. In the tidepool sculpin, a truly euryhaline species, gill ATPase activity increased as the silver levels increased, the latter probably representing a compensatory strategy. Similarly, intestinal ATPase activity was unchanged in the midshipmen, but was increased in the sculpins. Drinking rate in tidepool sculpins, which is involved with both iono-and osmo-regulation, was unaffected by salinity, but was decreased in fish exposed to silver for 8 d. Overall, Ag is far less toxic in seawater than in freshwater, but the mechanisms of toxicity are similar. In both waters, Ag interferes with iono- and osmo-regulation. In seawater, Ag exhibits a significant potential for bioaccumulation and interference with physiological processes during long term low level exposures of marine fish, especially at lower salinity levels. / Thesis / Master of Science (MSc)

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O boto-cinza (Sotalia guianensis Van Beneden, 1864) é uma espécie de delfinídeo que ocorre em águas costeiras das Américas do Sul e Central, especialmente em ambientes estuarinos, baías e áreas protegidas. Estudos de ecologia alimentar podem prover informações sobre o comportamento das espécies e de suas presas. Nesse contexto, o conhecimento da dieta é o primeiro passo para entendermos como se dá as relações predador-presa do ambiente. Neste estudo são abordados aspectos do hábito alimentar do boto-cinza na Baía de Guanabara, com o objetivo caracterizar sua dieta, e realizar comparações intra e interespecíficas, utilizando para isso a análise do conteúdo estomacal de exemplares encalhados entre 1990 e 2009. Outras espécies de delfinídeos foram utilizadas para a comparação com o boto-cinza, estes apresentam hábitos costeiros e oceânicos, são elas: o golfinho-pintado-do-Atlântico (Stenella frontalis Cuvier, 1829), o golfinho-de-dentes-rugosos (Steno bredanensis Lesson, 1828), o golfinho-comum (Delphinus delphis Linnaeus, 1758) e o golfinho-nariz-de-garrafa (Tursiops truncatus Montagu, 1821), o golfinho-de-Fraser (Lagenodelphis hosei Fraser, 1956) e o golfinho-riscado (Stenella coeruleoalba Meyen, 1833). A metodologia empregada se baseia na triagem do conteúdo estomacal, e identificação dos itens alimentares recuperados (otólitos, vértebras de teleósteos e bicos de cefalópodes). O comprimento e biomassas das presas foram estimados com o uso de equações de regressão encontradas na literatura para essas espécies. Para verificar variações intraespecíficas na dieta do boto-cinza, as amostras foram separadas nas categorias: machos adultos, fêmeas adultas e juvenis, e também segundo a estação do ano em que os estômagos foram recuperados, classificados em estações quente/chuvosa (out-abr) e fria/seca (mai-set). Para a caracterização da dieta do boto-cinza e posteriores comparações intra e interespecíficas, foram utilizados os parâmetros: abundância, riqueza, ocorrência, comprimento e biomassa estimada das presas. Os teleósteos foram o grupo de presas mais freqüente e abundante na dieta do boto-cinza, seguido dos cefalópodes e por último dos crustáceos. Foi visto uma alta similaridade da dieta entre fêmeas, machos e juvenis com relação às espécies consumidas e ao perfil das presas, sendo o porte das presas diferente entre adultos e juvenis. Com relação a variações sazonais, foi visto que as espécies consumidas variaram em quantidade, porém, o tamanho e o perfil das presas se mantive o mesmo entre as épocas quente/chuvosa e fria/seca. A abundância, biomassa e riqueza de espécies foram maiores nas estações quentes/chuvosas que nas estações frias/secas, o que pode ser um reflexo da maior disponibilidade de presas nessa época. As demais espécies de delfinídeos consumiram maior variedade de presas que o boto-cinza, o que pode estar relacionado a uma maior movimentação dessas espécies ao longo da costa. Este estudo indica que o boto-cinza apresenta variações intra-específicas no seu hábito alimentar entre adultos e juvenis, e variações sazonais na composição de sua dieta. Seus hábitos alimentares diferiram das demais espécies de delfinídeos, e indicam que o boto-cinza encontra na Baía de Guanabara recursos alimentares suficientes para seu requerimento energético, não precisando sair da região da baía para sua alimentação ou realizando apenas pequenos deslocamentos. / In this work, aspects of feeding habits of the Guiana dolphin, Sotalia guianensis (Van Beneden, 1864), were studied in Guanabara Bay (N = 26), with the aim of characterizing its diet and comparing the results with those from other species of the Delphinidae family (N = 31). The stomach contents analyzed were collected from specimens stranded on the beaches of Rio de Janeiro between 1990 and 2009. The methodology is based on washing stomach contents through 1 mm mesh sieves, as well as on the identification of the food items remained, such as teleost otoliths and cephalopod beaks. The length of the food items was used to estimate the length and weight of preys using regression equations found in the literature for these species. Teleosts constituted the most abundant prey in the stomachs of Guiana dolphin (N = 325), being followed by cephalopods (N = 76) and finally by crustaceans (N = 16). Were identified 17 species of teleosts belonging to 10 families, as well as two species of cephalopods from the Loliginidae family and one species of crustacean from the Penaeidae family. The most important species in Guiana dolphin diet, according to the index of relative importance were the whitemouth croaker (Micropogonias furnieri), the weakfish (Cynoscion jamaicensis) and atlantic midshipman (Porichthys porosissimus). Among cephalopods, the squid Loligo plei was the most representative prey in the diet. Regarding the size of prey, most consumed teleosts had the length varying between 11 and 20 cm (15.4 5.5 cm), characterizing the consumption of juvenile fishes. The cephalopods mantle length ranged from 6 to 15 cm (11.8 4.2 cm). A great similarity was observed in the diet composition of females, males, and juveniles of Guiana dolphin, however, the mean length and biomass of fish differed between those consumed by adults and those preyed by juveniles (Kruskal-Wallis, p<0,05, posteriori comparison of medians). Species richness was higher in warm seasons, as well as the diversity and evenness index, which may be related to a greater availability of prey during these seasons. Proportions of the preys consumed varied, however, the composition of the diet, as well as the habits and size of prey, remained similar between seasons. While all species consumed by Guiana dolphin presented coastal habits, the other delphinids species consumed preys of larger distributions, which could be related to a greater movement of these species along the coast. This study showed that there are variations in food habits of Guiana dolphin in Guanabara Bay and indicates that this species feeds on the available resources in the region.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / O boto-cinza (Sotalia guianensis Van Beneden, 1864) é uma espécie de delfinídeo que ocorre em águas costeiras das Américas do Sul e Central, especialmente em ambientes estuarinos, baías e áreas protegidas. Estudos de ecologia alimentar podem prover informações sobre o comportamento das espécies e de suas presas. Nesse contexto, o conhecimento da dieta é o primeiro passo para entendermos como se dá as relações predador-presa do ambiente. Neste estudo são abordados aspectos do hábito alimentar do boto-cinza na Baía de Guanabara, com o objetivo caracterizar sua dieta, e realizar comparações intra e interespecíficas, utilizando para isso a análise do conteúdo estomacal de exemplares encalhados entre 1990 e 2009. Outras espécies de delfinídeos foram utilizadas para a comparação com o boto-cinza, estes apresentam hábitos costeiros e oceânicos, são elas: o golfinho-pintado-do-Atlântico (Stenella frontalis Cuvier, 1829), o golfinho-de-dentes-rugosos (Steno bredanensis Lesson, 1828), o golfinho-comum (Delphinus delphis Linnaeus, 1758) e o golfinho-nariz-de-garrafa (Tursiops truncatus Montagu, 1821), o golfinho-de-Fraser (Lagenodelphis hosei Fraser, 1956) e o golfinho-riscado (Stenella coeruleoalba Meyen, 1833). A metodologia empregada se baseia na triagem do conteúdo estomacal, e identificação dos itens alimentares recuperados (otólitos, vértebras de teleósteos e bicos de cefalópodes). O comprimento e biomassas das presas foram estimados com o uso de equações de regressão encontradas na literatura para essas espécies. Para verificar variações intraespecíficas na dieta do boto-cinza, as amostras foram separadas nas categorias: machos adultos, fêmeas adultas e juvenis, e também segundo a estação do ano em que os estômagos foram recuperados, classificados em estações quente/chuvosa (out-abr) e fria/seca (mai-set). Para a caracterização da dieta do boto-cinza e posteriores comparações intra e interespecíficas, foram utilizados os parâmetros: abundância, riqueza, ocorrência, comprimento e biomassa estimada das presas. Os teleósteos foram o grupo de presas mais freqüente e abundante na dieta do boto-cinza, seguido dos cefalópodes e por último dos crustáceos. Foi visto uma alta similaridade da dieta entre fêmeas, machos e juvenis com relação às espécies consumidas e ao perfil das presas, sendo o porte das presas diferente entre adultos e juvenis. Com relação a variações sazonais, foi visto que as espécies consumidas variaram em quantidade, porém, o tamanho e o perfil das presas se mantive o mesmo entre as épocas quente/chuvosa e fria/seca. A abundância, biomassa e riqueza de espécies foram maiores nas estações quentes/chuvosas que nas estações frias/secas, o que pode ser um reflexo da maior disponibilidade de presas nessa época. As demais espécies de delfinídeos consumiram maior variedade de presas que o boto-cinza, o que pode estar relacionado a uma maior movimentação dessas espécies ao longo da costa. Este estudo indica que o boto-cinza apresenta variações intra-específicas no seu hábito alimentar entre adultos e juvenis, e variações sazonais na composição de sua dieta. Seus hábitos alimentares diferiram das demais espécies de delfinídeos, e indicam que o boto-cinza encontra na Baía de Guanabara recursos alimentares suficientes para seu requerimento energético, não precisando sair da região da baía para sua alimentação ou realizando apenas pequenos deslocamentos. / In this work, aspects of feeding habits of the Guiana dolphin, Sotalia guianensis (Van Beneden, 1864), were studied in Guanabara Bay (N = 26), with the aim of characterizing its diet and comparing the results with those from other species of the Delphinidae family (N = 31). The stomach contents analyzed were collected from specimens stranded on the beaches of Rio de Janeiro between 1990 and 2009. The methodology is based on washing stomach contents through 1 mm mesh sieves, as well as on the identification of the food items remained, such as teleost otoliths and cephalopod beaks. The length of the food items was used to estimate the length and weight of preys using regression equations found in the literature for these species. Teleosts constituted the most abundant prey in the stomachs of Guiana dolphin (N = 325), being followed by cephalopods (N = 76) and finally by crustaceans (N = 16). Were identified 17 species of teleosts belonging to 10 families, as well as two species of cephalopods from the Loliginidae family and one species of crustacean from the Penaeidae family. The most important species in Guiana dolphin diet, according to the index of relative importance were the whitemouth croaker (Micropogonias furnieri), the weakfish (Cynoscion jamaicensis) and atlantic midshipman (Porichthys porosissimus). Among cephalopods, the squid Loligo plei was the most representative prey in the diet. Regarding the size of prey, most consumed teleosts had the length varying between 11 and 20 cm (15.4 5.5 cm), characterizing the consumption of juvenile fishes. The cephalopods mantle length ranged from 6 to 15 cm (11.8 4.2 cm). A great similarity was observed in the diet composition of females, males, and juveniles of Guiana dolphin, however, the mean length and biomass of fish differed between those consumed by adults and those preyed by juveniles (Kruskal-Wallis, p<0,05, posteriori comparison of medians). Species richness was higher in warm seasons, as well as the diversity and evenness index, which may be related to a greater availability of prey during these seasons. Proportions of the preys consumed varied, however, the composition of the diet, as well as the habits and size of prey, remained similar between seasons. While all species consumed by Guiana dolphin presented coastal habits, the other delphinids species consumed preys of larger distributions, which could be related to a greater movement of these species along the coast. This study showed that there are variations in food habits of Guiana dolphin in Guanabara Bay and indicates that this species feeds on the available resources in the region.

<p>The synthetic estrogen 17α-ethynylestradiol (EE2), an endocrine disruptor originating from birth control and hormone replacement therapy, is discharged in wastewater treatment plant (WWTP) effluents. The present study employed radio-labeled EE2 to examine the impact of temperature and salinity on the uptake of EE2 in male killifish (<em>Fundulus heteroclitus</em>), a model euryhaline teleost. Fish were exposed to a nominal concentration of 100 ng/L EE2 for 2 h. Actual concentrations were lower due to EE2 adsorption to the exposure system, but uptake rates were normalized to 100 ng/L. Oxygen consumption rates (MO₂), whole body EE2 uptake rates, and tissue-specific EE2 distribution were monitored. EE2 uptake by freshly killed fish was negligible. In killifish acclimated to 18^OC at 16 ppt (50 % seawater), MO₂ and EE2 uptake were both much lower after 24-h exposure to 10^OC and 4^OC, and increased after 24-h exposure to 26^OC. Transfer of killifish to fresh water for 24 h tended to lower EE2 uptake rate, and long-term acclimation to fresh water reduced it by about 70 %. Long-term acclimation to 100 % sea water (32 ppt) also reduced EE2 uptake rate by about 50 % relative to 16 ppt. However this was not seen in juvenile rainbow trout (<em>Oncorhynchus mykiss</em>) where uptake rates were the same in FW- and 16 ppt-acclimated trout. The tissue-specific accumulation of EE2 was found to be the highest (40-60 % of the total) in the liver plus gall bladder across all exposures, and the great majority of this was in the bile in killifish, regardless of temperature or salinity, whereas in trout accumulation was the highest in the carcass at 70 % of the total. The carcass was the next highest accumulator (30-40 %) in killifish, followed by the gut (10-20 %) with only small amounts in gills and spleen. Drinking rate, measured with radio-labeled polyethylene glycol-4000, was about 25-times greater in 16 ppt-acclimated killifish relative to freshwater-acclimated animals. However, drinking accounted for less than 30 % of gut accumulation, and therefore a negligible percentage of whole body EE2 uptake rates. In general, there were strong positive relationships between EE2 uptake rates and MO₂, suggesting similar pathways for uptake across the gills of these lipophilic molecules. These data will be useful in developing a predictive model of how variations in key environmental parameters (salinity, temperature, dissolved oxygen) affect EE2 uptake in estuarine fish, so as to determine optimal timing and location of WWTP discharges.</p> / Master of Science (MS)

In this study, full coding sequences of Atlantic salmon (Salmo salar L.) muscle genes were cloned, including myogenic regulatory factors (MRFs) (myod1c, myog, mrf4, myf5), inhibitors of Myostatin (fst, decorin), markers of myogenic progenitor cell (MPC) proliferation (sox8) and fusion (calpastatin), a marker of slow muscle fibre differentiation (smlc1) and a novel eukaryotic gene involved in regulating growth (cee). Several of these genes were then characterised using a range of experimental and computational analyses with the aim to better understand their role in myogenesis and their evolution in teleosts. A series of experiments supported previous findings that teleosts have extra copies of many genes relative to tetrapods as a result of a whole genome duplication (WGD) event that occurred some 320-350 Mya. For example, it was shown that genes for myod and fst have duplicated in a common teleost ancestor, but were then specifically lost or retained in different lineages. Furthermore, several characterised Atlantic salmon genes were conserved as paralogues, likely from a later WGD event specific to the salmonid lineage. Phylogenetic reconstruction and comparative genomic approaches were used to characterise the evolution of teleost paralogues within a framework of vertebrate evolution. As a consequence of one experiment, a revised nomenclature for myod genes was proposed that is relevant to all diploid and polyploid vertebrates. The expression patterns of multiple myogenic genes were also established in Atlantic salmon embryos using specific complementary RNA probes and in situ hybridization. For example, co-ordinated embryonic expression patterns were revealed for six salmon MRFs (myod1a, myod1b, myod1c, myog, mrf4, myf5), as well as markers of distinct MPC populations (pax7, smlc1), providing insight into the regulatory networks governing myogenesis in a tetraploid teleost. Furthermore, it was shown that Atlantic salmon fst1 was expressed concurrently to pax7 in a recently characterised MPC population originating from the anterior domain of the epithelial somite, which is functionally analogous to the amniote dermomyotome. In another experiment, the individual expression domains of three Atlantic salmon myod1 paralogues were shown to together recapitulate the expression of the single myod1 gene in zebrafish, consistent with the partitioning of ancestral cis-acting regulatory elements among salmonid myod1 duplicates. Additionally, the in situ expression of cee a novel and highly conserved eukaryotic gene was revealed for the first time in a vertebrate and was consistent with an important role in development including myogenesis. Additionally, Atlantic salmon were reared at 2, 5, 8 or 10 ºC solely to a defined embryonic stage, which was just subsequent to the complete pigmentation of the eye. After this time, animals were provided an equal growth opportunity. Remarkably, changing temperature during this short developmental window programmed the growth trajectory throughout larval and adult stages. While 10 and 8 ºC fish were larger than those reared at 2 and 5 ºC at the point of smoltification, strong compensatory growth was subsequently observed. Consequently, after 18 months of on growing, size differences among 5, 8 and 10 ºC fish were not significant, although each group was heavier than 2 ºC fish. Furthermore, significant embryonic-temperature induced differences were observed in the final muscle fibre phenotype, including the number, size distribution and myonuclear density of muscle fibres. A clear optimum for the final muscle fibre number was observed in 5 ºC fish, which was up to 17% greater than other treatments. In a sub-sample of embryos, temperature induced heterochonies were recorded in the expression of some MRFs (myf5, mrf4) but not others (myod1a, myog). These results allowed the proposition of a potential mechanism explaining how temperature can program the muscle phenotype of adult teleosts through modification of the somitic external cell layer, a source of MPCs throughout teleost ontogeny.

Le loup Dicentrarchus labrax est un téléostéen euryhalin qui effectue des migrations saisonnières de la mer vers les lagunes et les estuaires où la salinité est très variable et peut changer rapidement. Les mécanismes d'osmorégulation sont bien connus chez les téléostéens, alors que le rôle de l'osmodétection dans leur mise en place reste actuellement largement méconnu. Le but de cette étude a été d'améliorer les connaissances sur l'osmodétection et sur la mise en place des mécanismes d'hyper-osmorégulation (à court et long terme).Tout d'abord, le canal calcique “Transient Receptor Potential Vanilloid 4” ou TRPV4, candidat osmodétecteur, a été étudié chez des loups adaptés à l'eau de mer (EM) ou exposés à l'eau douce (ED) durant des temps différents. Au niveau branchial, le TRPV4 ne semble pas impliqué dans l'osmodétection au niveau des ionocytes, mais plutôt dans l'absorption de Ca2+ par les chondrocytes, notamment en ED. Il pourrait avoir un rôle dans l'osmodétection systémique au niveau de l'hypophyse et dans l'absorption de Ca2+ dans le rein. Sa forte expression en EM dans les cellules rénales réabsorbant de l'eau suggère que le TRPV4 est impliqué dans la régulation de leur volume cellulaire.En second lieu, le canal à chlore ClC-3, candidat osmodétecteur et potentiellement impliqué dans les mécanismes de diminution régulatrice du volume cellulaire (RVD), a été étudié dans les mêmes conditions expérimentales que précédemment. Ce canal a été localisé dans la membrane basolatérale des ionocytes des branchies (EM et ED) et des tubes collecteurs du rein (en ED ; pas immunodétecté en EM). Il pourrait jouer un rôle dans la protection des cellules contre un choc hypotonique, et donc dans la RVD en EM dans ces deux organes. En ED, la localisation du ClC-3 suggère fortement sa participation dans l'hyper-osmorégulation au niveau des branchies et du rein.Enfin, un protocole d'isolement des ionocytes branchiaux a été mis au point ; il permettra de poursuivre et d'approfondir les travaux sur l'osmodétection cellulaire. L'évolution au cours du temps du volume cellulaire d'un ionocyte a pu être évaluée après un choc hypotonique. De futures expérimentations permettront d'évaluer le lien entre l'osmodétection, les osmodétecteurs et les effecteurs osmotiques. / The European sea bass Dicentrarchus labrax is a euryhaline teleost that migrates seasonally from the sea to lagoons and estuaries, where salinity is very variable and changes rapidly. Osmoregulatory mechanisms are well known in teleosts, while the role of osmosensing in their set-up is yet poorly understood. The objective of this study was to improve the understanding of osmosensing and of its relations with hyper-osmoregulatory mechanisms in sea bass over short- and long-term salinity exposures.First, the Transient Receptor Potential Vanilloid 4 (TRPV4) Ca2+-channel, a potential osmosensor, has been studied in fish adapted to sea water (SW) or exposed to fresh water (FW) for various times. The TRPV4 did not seem to be involved in osmosensing in mitochondrion-rich cells (MRC) in gills. It may be involved in systemic osmosensing and in Ca2+ uptake by branchial chondrocytes and by renal collecting ducts in FW. This channel may also have a role in cell volume regulation of kidney cells allowing water reabsorption.Secondly, the ClC-3 chloride channel, another potential osmosensor and potentially involved in regulatory volume decrease (RVD) mechanisms, has been studied in fish under similar conditions. This channel has been localized in the basolateral membrane of ionocytes in gills (SW and FW) and kidney (FW – in SW, expressed but not immunodetected). It might be involved in cell protection against hypotonic shock and thus in RVD in both organs in SW. In FW, its localization strongly suggests a role in hyper-osmoregulation in gills and kidney.Finally, a protocol to isolate MRCs has been developed; it has been used to study cell osmosensing and it will be used on future studies. The changes in cell volume following a hypotonic shock have been evaluated. Future experiments will allow a better understanding of the relations between cell osmosensing, osmosensors and effectors.

O desenvolvimento ontogenético em peixes é considerado como uma das etapas mais importantes no fornecimento de informações para biologia do desenvolvimento, aquicultura e estudos taxonômicos. A Proteína Óssea Morfogenética 2 (BMP-2) é considerada uma molécula essencial como regulador no desenvolvimento embrionário e na formação óssea, sendo ainda pouco estudada em peixes. Neste trabalho, foram comparadas todas as fases do desenvolvimento de três espécies de peixes importantes da bacia do rio Pardo, curimbatá (Prochilodus hartii), piabanha (Brycon sp) e piau (Leporinus steindachneri). A superfície ovocitária, estádios e duração do desenvolvimento embrionário/larval foram classificados com a utilização de microscópio estereoscópio, havendo variações entre as espécies no desenvolvimento. A transformação dos alevinos também apresentaram variações, com 16 dias pós-eclosão em P. hartii, 5 dias em Brycon sp e 14 dias em L. steindachneri. Na imunohistoquímica, BMP-2 foi identificada na blastocele nas fases de blástula e gástrula; na formação da vesícula óptica, notocorda e somitos, na fase embrionária das espécies estudadas. Na fase larval e de juvenil, BMP-2 foi identificada na formação de brânquias, olhos, coração, estômago, intestino, fígado, nadadeiras, músculos e na ossificação em todas as espécies estudadas. Indivíduos adultos foram sacrificados, e depois de realizada a biometria, dissecados para mensuração dos órgãos internos e documentação fotográfica. Observou-se que o estômago de P. hartii tem forma de \"U\" com presença de uma estrutura semelhante a uma moela, enquanto em Brycon sp tem forma de \"J\" com presença de cecos pilóricos aderidos no piloro e em L. steindachneri, o mesmo possui forma de \"Y\" sem presença de cecos pilóricos. Esqueletos foram preparados através da técnica de maceração com insetos dermestídeos, sendo observadas diferenças, sendo interessante descrever que em P. hartii foram encontrados 4 ossos infraorbitais, 6 em Brycon sp e 4 em L. steindachneri. Conclui-se que os exemplares de P. hartii possuem hábito alimentar iliófago, enquanto Brycon sp e L. steindachneri são considerdados onívoros, baseado nos achados anatômicos e a expressão de BMP-2 está ligada com a morfogênese e organogênese na embriologia das espécies estudadas. / The ontogenetic development in fish is considered as one of the most important steps in providing information to developmental biology, aquaculture and taxonomic studies. The Bone Morphogenetic Protein 2 (BMP-2) is considered as a molecule essential for regulaton of embryonic development and bone formation, and has been poorly studied in fish. In this work, we have compared all the development phases of three important species of fish from the Pardo River basin, Curimbata (Prochilodus hartii) Piabanha (Brycon sp) and Piau (Leporinus steindachneri). The surface oocyte, stage and duration of embryonic/larval development were classified by using a stereoscopic microscope, and showed variations between the studied species. The transformation of the juveniles also showed variations, with 16 days post-hatching in P. hartii, 5 days in Brycon sp and 14 days in L. steindachneri. In immunohistochemistry, BMP-2 was identified in the blastocoel of the blastocyst and gastrula stages; forming the optic vesicle, notochord and somites on embryo of the ivestigated species. In larval and juvenil stages, BMP-2 has been identified in the formation of gills, eyes, heart, stomach, intestine, liver, fins, muscle and ossification in the species studies. Adults were sacrificed, dissected for biometrics measurements of the internal organs and photo documentation. The stomach of P. hartii showed a \"U\" shape with the presence of a structure similar to a gizzard, whereas in Brycon sp it was shaped like a \"J\" with the presence of pyloric caeca and in L. steindachneri it presented a \"Y\" form without the presence of pyloric caeca. Skeletons were prepared by retting technique with Dermestides beetles, interesting differences were observed between numbers of bones, where 4 infraorbital bones were found in P. hartii, 6 in Brycon sp and 4 in L. steindachneri. It is concluded that the specimens of P. hartii show ilyophagous eating habits while Brycon sp and L. steindachneri were considered omnivorous based on anatomical findings and that expression of BMP-2 is linked with morphogenesis and organogenesis of the studied species.