In this thesis, the effect of infection elicited by entomopathogenic nematodes (EPNs) Steinernema carpocapsae on Pyrrhocoris apterus and Drosophila melanogaster models were evaluated, and a role of adipokinetic hormones (AKHs) during the infection was characterized. These were monitored by determination of mortality, and various biochemical and physiological characteristics such as AKH levels both in the central nervous system (CNS) and in hemolymph, AKH gene expression in CNS, level of anti-oxidative stress markers, general metabolism and level of nutrients in normal and genetically modified insects. At P. apterus the mortality tests revealed that application of AKH increases the efficacy of EPN treatment. This result was confirmed using the firebugs with AKH receptor deficiency. Further, the increase of AKH expression and AKH levels in CNS and hemolymph seemed to be coordinated after the nematode treatment. At the D. melanogaster model also, the effect of adenosine into the above-mentioned characteristics was included. For this, mutants in AKH (AHK1), adenosine receptor (AdoR1) genes, and in both these genes together (AHK1 AdoR1 double mutant) were employed. Altogether, the results confirmed the involvement of AKH, and partially also adenosine into the antistress defense reactions elicited by the nematobacterial infection. Finally, the last part of the study was focused on examination of the vitellogenin (Vg) role in the defense reaction in firebug body P. apterus affected by two entomopathogenic organisms, the nematode S. carpocapsae and the fungus Isaria fumosorosea. The results revealed that Vg proteins play an important role in the defense against both types of the infections and are also able to kill entomopathogenic bacteria Xenorhabdus nematophila, that are symbionts of S. carpocapsae and that increase toxicity of this nematode.

Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth. This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success. Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA. Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage. Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.

Studies in Europe, Japan, and North America have reported that wild fish exposed to treated sewage effluents can exhibit significant physiological and reproductive abnormalities consistent with exposure to hormonally active chemicals. The main objective of this research project was to examine the estrogenic and androgenic activity in treated sewage to determine the risk associated with treated sewage discharges in Australia and New Zealand. Several bioassays, including a sheep estrogen receptor and a rainbow trout androgen receptor binding assay, were set up and validated with model compounds. The assays were then used to measure the estrogenic and androgenic activity in sewage samples from 15 municipal sewage treatment plants (STP) utilizing a variety of treatment technologies. Raw sewage samples contained high levels of both estrogenic and androgenic activity, up to 185 ng/L estradiol equivalents (EEq) and up to 9330 ng/L testosterone equivalents (TEq), respectively. Secondary treatment processes such as activated sludge had the greatest impact on removal of biological activity from the wastewater. The estrogenic and androgenic activity in final treated effluents were <1 to 4.2 ng/L EEq and <6.5 to 736 ng/L TEq, respectively. Based on lowest observable effective concentrations reported in the literature, these levels are unlikely to induce biological effects in exposed fish in the short term. To examine potential long-term effects, resident mosquitofish chronically exposed to undiluted treated sewage were sampled. Several morphological biomarkers indicative of endocrine disruption were measured and compared with mosquitofish captured at a reference site. Mosquitofish captured in a constructed wetland for tertiary treatment of secondary treated sewage exhibited morphological differences such as elongated anal fins consistent with exposure to androgenic chemicals, although this effect was not measurable in fish collected at sites further downstream or at any of the other sites. Based on these results, it is unlikely that mosquitofish populations would be significantly affected by exposure to final treated sewage. A reverse transcription real-time polymerase chain reaction (RT-PCR) method to measure the production of a female-specific protein (vitellogenin) mRNA in adult male mosquitofish was developed, and this could be used as a rapid test to detect early changes in individuals exposed to estrogenic activity.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-05-03T15:52:19Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Laura - versão final - Corrigida_docx - sem assinatura.pdf: 1975245 bytes, checksum: 1380118f3f00203cdd14e785c8e7df92 (MD5) / Made available in DSpace on 2017-05-03T15:52:19Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Laura - versão final - Corrigida_docx - sem assinatura.pdf: 1975245 bytes, checksum: 1380118f3f00203cdd14e785c8e7df92 (MD5) Previous issue date: 2016-05-31 / CNPQ / Compostos desreguladores endócrinos (CDEs) contaminamecossistemas aquáticos esão capazes de imitar a ação de hormônios endógenos em peixes. Diante disso, este trabalho avaliouprimeiramente os efeitos da exposição de machos adultos de Poecilia viviparaa uma mistura ambiental de contaminantes com atividade estrogênica e ao CDE 17 α-etinilestradiol (EE2), envolvendo a análise química de moléculas estrogênicas na mistura ambiental, e de parâmetros bioquímicos, comportamentais e reprodutivos. Moléculas com atividade estrogênica foram quantificadas por cromatografia líquida com espectrometria de massas nas águasda Lagoa do Araçá (LA), do rio Capibaribe (RC) e no Complexo estuarino da Bacia do Pina (CEBP), região metropolitana de Recife. Machos adultos de P. vivipara cultivados em laboratório foram expostos por 14 dias a água limpa controle (EECT), ea 10ngEE2 L-1 (EE10) e 100 ngEE2 L-1 (EE100).Em paralelo, machos adultos residentes no CEBP foram coletados em LA, e trazidos ao laboratório. Machos expostos ao EE2 em laboratório e residentes no CEBP foram colocados em contato com fêmeas adultas cultivadas no laboratório, e os casais foram monitorados por um sistema de vídeo, com posterior avaliação quantitativa do comportamento sexual pelo software de análise comportamental Smart. Os parâmetros avaliados foram:número de contatos entre machos e fêmeas (NC); duração média dos contatos em segundos(YACUBIAN-FERNANDES et al.); distância média entre machos e fêmeas durante os contatos (DiC). O número de tentativas de cópula dos machos (TC) foi avaliado por observação humana dos vídeos, e a velocidade natatória dos machos isolados foi também quantificada pelo software Smart. Após a interação entre os casais, fêmeas pareadas com machos controle e LA foram mantidas isoladas em aquários por 90 dias para quantificação do sucesso de impregnação (SI) e do número de juvenis produzidos (NJP). Machos dos tratamentos EECT, EE10, EE100 e LA foram sacrificados e a quantidade de vitelogenina no fígado foi avaliada indiretamente por método histoquímico para coloração de fosfoproteínas. As análises cromatográficas indicaram a presença dos estrogênios estriol (E3), estrona (E1), EE2 e estradiol (E2), e de bisfenol A (BPA) nas concentrações médias de 31,8; 19,5; 11,9; 8,6; e 4,2 ng L-1, respectivamente, nas águas do CEBP, típicas de áreas urbanas contaminadas por esgotos domésticos. Machos expostos ao EE2 e provenientes da Lagoa do Araçá apresentaram indução de vitelogenina (p < 0,05). O número de contatos entre machos e fêmeas, e número de tentativas de cópula dos machos diminuiu nos casais em EE100 e LA (p < 0,05). A distância média entre machos e fêmeas durante os contatos aumentou em EE100 e LA (p < 0,05). Machos isolados expostos a EE10 e EE100 apresentaram hiperatividade natatória, enquanto machos provenientes de LA apresentaram hipoatividade natatória comparado aos controles. Machos de LA impregnaram 55% das fêmeas, enquanto machos controle impregnaram 88% das fêmeas. Fêmeas pareadas com machos controle (FeCt) produziram 7,1 juvenis em média, enquanto fêmeas pareadas com machos de LA (FeLA) produziram 2,7 juvenis (p = 0,019), totalizando 57 juvenis produzidos por FeCt versus 14 juvenis produzidos por FeLA, o que representa uma redução de 75% na geração de potenciais recrutas para a população. No segundo tema deste trabalho, machos e fêmeas adultos cultivados no laboratório foram mantidos isolados e ambos machos e fêmeas foram expostos por 30 dias ao herbicida atrazina nas concentrações 0 (controle-ATCT), 0,5μgL-1 (AT0,5); 5μgL-1 (AT5) e 50 μgL-1(AT50). Após a exposição, casais foram formados para cada tratamento, e o comportamento sexual foi analisado para o parâmetro TC por observador humano. A atividade natatória de machos e fêmeas mantidos isolados também foi avaliada. Após a exposição e pareamento dos casais, fêmeas de cada tratamento foram mantidas isoladas em aquários por 90 dias, para quantificação do número de ovócitos maduros (NO), e dos índices gonadossomático (IGS) e hepatossomático (Webb and Weihs). Os parâmetros NO, IGS e IHS para as fêmeas não foram alterados significativamente. Nos casais expostos a atrazina foi observada uma redução em TC na concentração de 5 (p > 0,05) e 50 μgL-1 (p < 0,05). Hiperatividade natatória foi detectada nas fêmeas expostas a todas concentrações, e em machos expostos a 5 (p > 0,05) e 50 μg L-1 (p < 0,05). Este estudo quantifica pela primeira vez CDEs estrogênicos em águas da região metropolitana de Recife, e demonstra o efeito de desreguladores endócrinos como o EE2 em laboratório, bem como os efeitos de uma mistura ambiental de contaminantes incluindo CDEs estrogênicos, em biomarcadores bioquímicos, comportamentais e reprodutivos de Poecilia vivipara, que podem afetar a viabilidade populacional em situações ambientalmente realistas. / Endocrine disruptor compounds (EDCs) contaminate aquatic ecosystems and are able to mimic the action of endogenous hormones in fish.Therefore, this study first evaluated the effects of adult male Poecilia vivipara exposure to an environmental mixture of contaminants with estrogenic activity and to the EDC 17 α-ethinylestradiol (EE2), involving the chemical analysis of estrogenic molecules in the environmental mixture, and biochemical, behavioral and reproductive parameters.Molecules with estrogenic activity were quantified by liquid chromatography with mass spectrometry in the waters of Lagoa do Araçá(LA) and the Capibaribe River (CR), in the Pina Basin estuarine complex (PBEC), metropolitan area of Recife. Laboratory grown adult male P. vivipara were exposed for 14 days to a clean water control (EECT) and to 10 ngEE2 L-1 (EE10) and 100 ngEE2 L-1 (EE100).In parallel, resident adult males in the PBEC were collected in LA, and brought to the laboratory.Males exposed to EE2 in the laboratory and residents of PBEC were placed in contact with adult females grown in the laboratory, and the couples were monitored by a video system, with subsequent quantitative assessment of sexual behavior by behavioral analysis software Smart.The parameters evaluated were: number of contacts between males and females (NC); average duration of contacts in seconds (YACUBIAN-FERNANDES et al.); average distance between males and females during the contacts (DiC). The number of male copulation attempts (Lichota et al.) was assessed by human observation of the videos, and swimming speed of single males was also quantified by the Smart software.After the interaction between couples, females paired with control and LA males were kept isolated in tanks for 90 days to quantify the impregnation success (IS) and the number of juveniles produced (NJP). Males of treatments EECT, EE10, EE100 and LA were sacrificed and the amount of vitellogenin in the liver was assessed indirectly by a histochemical staining method for phosphoproteins.Chromatographic analysis indicated the presence of estrogens estriol (E3), estrone (E1), EE2 and estradiol (E2), and bisphenol A (BPA) in concentrations averaging 31.8; 19.5; 11.9; 8.6; and 4.2 ng L-1, respectively, in PBEC waters, typical of urban areas contaminated by domestic sewage.Males exposed to EE2 and from Lagoa do Araçá showed vitellogenin induction (p <0.05).The number of contacts between males and females, and thenumber of male copulation attempts decreased in couples from EE100 and LA (p <0.05).The average distance between males and females during the contacts increased at EE100 and LA (p <0.05). Isolated males exposed to EE10 and EE100 developed swimming hyperactivity, while males from LA developed swimming hypoactivity compared to controls.LA males impregnated 55% of females, while control males impregnated 88% of females.Females paired with control males (FeCt) produced 7.1 juveniles on average, while females paired with LA males (FeLA) produced 2.7 juveniles (p = 0.019), totaling 57 juveniles produced by FeCt versus 14 juveniles produced by FeLA, which represents a 75% reduction in the generation of potential recruits for the population.In the second theme of this work, adult males and females grown in the laboratory were kept isolated and both males and females were exposed for 30 days to atrazine concentrations 0(control-ATCT), 0.5 μg L-1 (AT0.5); 5 μg L-1 (AT5) and 50 μg L-1 (AT50).After exposure, couples were formed for each treatment, and sexual behavior was analyzed for MCA by a human observer.Swimming activity of isolated males and females was also evaluated.After exposure and pairing of couples, females from each treatment were kept isolated in tanks for 90 days, to quantify the number of mature oocytes (NO), and the Gonadosomatic index (GSI) and hepatossomatic index (HSI). The parameters NO, GSI and HSI for females did not change significantly.In couples exposed to atrazine a significant reduction in MCA was observed at concentrations5 (p > 0,05) and 50 μg.L-1 (p <0.05). Hyperactive swimming was detected in females exposed to all concentrations, and in males exposed to 5 (p > 0.05) and 50 μgL-1 (p < 0.05).This study quantifies for the first time estrogenic EDCs in waters of the metropolitan area of Recife, and demonstrates the effect of endocrine disruptors such as EE2 in the laboratory, as well as the effects of an environmental mixture of contaminants including estrogenic EDCs, in biochemical, behavioral and reproductive biomarkers of Poecilia vivipara, which can affect population viability in environmentally realistic conditions.

Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are mostly unknown. A previous study has shown that in the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals have a 30% increase in lifespan relative to controls (Sham). In a separate study, an increase in fat body mass and a halting of ovarian growth were seen upon reduction of vitellogenin transcript via RNAi (VgRNAi). These data suggest that VgRNAi increases lifespan through the trade-off between reproduction and longevity and animals with combined ovariectomy and VgRNAi, might show additive physiological responses. In this study, we used two injection control groups for the VgRNAi treatment, namely buffer injection or injection with RNAi against a 90kDa hexamerin storage protein (Hex90RNAi). We have combined these manipulations to test lifespans upon: OVX & VgRNAi, OVX & Hex90RNAi, OVX & Buffer, Sham & VgRNAi, Sham & Hex90RNAi, and Sham & Buffer. Ovariectomy and VgRNAi exhibited similar reductions in feeding (~40%) and extensions in lifespan (13-21%) but showed differences in vitellogenin protein levels. This study also observed the effects of reduced reproduction on hexamerin storage proteins. We observed that upon ovariectomy and VgRNAi, hexamerins were increased, emphasizing the importance of protein in insect life extension. When methods to reduce reproduction were combined (OVX VgRNAi), no additive physiological responses were observed, suggesting ovariectomy and VgRNAi each extend lifespan by overlapping or convergent pathways.

A região N-terminal de OTI-VIT-1 foi expressa e os polipeptídeos recombinantes foram purificados. OTI-VIT-1 pode ser homólogo da vitelina YP170B de C. elegans. Foram identificados um intron na região 5´ e dois na região 3´ do gene Oti-vit-1. Antissoro monoespecífico para PVIT1HisC confirmou que o gene Oti-vit-1 codifica VT1. O polipeptídeo recombinante P40-H, correspondente à região N-terminal da proteína OTI-VIT-6 interage com um polipeptídeo de aproximadamente 100 kDa (P100) presente em extratos proteicos totais de O. tipulae. Estudamos também o papel da Proteína Microssômica Transportadora de Triglicerídeos (MTP) na biossíntese de Vitelogenina do nematoide C. elegans. Ensaios de RNAi em C. elegans, utilizando parte da sequência do gene da MTP (Cel-dsc-4) foram realizados nas linhagens N2 e DH1033. Microscopia de fluorescência de vermes adultos da linhagem DH1033, submetidos a RNAi, mostrou acúmulo de YP170B::GFP no interior dos enterócitos. Este acúmulo sugere a participação da MTP na secreção de VTG. Análise imunológica da vitelogenina nestes mesmos vermes não detectaram alterações no processamento de CEL-VIT-6, sugerindo que o mesmo ocorra não só no pseudoceloma, mas também no interior dos enterócitos. / The N-terminal region of OTI-VIT-1 was expressed and the recombinant polypeptides were purified. OTI-VIT-1 may be homologous to the vitellin YP170B from C. elegans. We identified an intron in the 5 \'region and two in 3\' region from Oti-vit-1. Monospecific antisera to PVIT1HisC confirmed that the gene Oti-vit-1 encodes VT1. The recombinant polypeptide P40-H, corresponding to the N-terminal region of the protein OTI-VIT-6, interacts with a polypeptide of approximately 100 kDa (P100) present in total protein extracts of O. tipulae. The role of microsomal triglyceride transfer protein (MTP) in the biosynthesis of vitellogenin was studied in the nematode C. elegans. Trials of RNAi in C. elegans, using the sequence of the MTP gene (Cel-dsc-4) were performed in the strains N2 and DH1033. Fluorescence microscopy of adult worms of strain DH1033, subjected to RNAi, showed accumulation of YP170B:: GFP within the enterocytes. This accumulation suggests the involvement of MTP in the secretion of VTG. Analysis using anti-vitellogenin immune serum did not detect changes in the processing of CEL-VIT-6, suggesting that it occurs not only in pseudocoelom but also within the enterocytes.

L’abeille domestique (Apismellifera) a un rôle majeur dans les écosystèmes naturels et agronomiques mais est exposée à un nombre croissant de pressions environnementales (nouveaux parasites, xénobiotiques, variations climatiques et malnutrition). Dans ce contexte, la compréhension des phénomènes impliqués dans les réponses au stress ainsi que leurs coûts associés devient cruciale pour mieux appréhender l’impact de ces pressions sur les abeilles. L’émergence d’un stress perturbe généralement l’homéostasie de l’organisme qui doit mettre en place une cascade d’adaptations physiologiques et comportementales pour le surmonter. Cependant, du fait de son mode de vie social, il est raisonnable de penser que les réponses vont se faire dans l’intérêt du groupe et non plus seulement dans l’intérêt de l’individu. Afin de caractériser les réponses au stress et de déterminer leur spécificité en fonction de la nature du stimulus (xénobiotiques, immunitaire, thermique, social), j’ai adopté une approche multidisciplinaire en ciblant l’identification des modifications i) physiologiques associées à la division du travail, ii) du métabolisme énergétique, et iii) comportementales. J’ai démontré quequelque soit leur rôle social (nourrice, gardienne, butineuse), les abeilles répondent de la même manière à un stress donné, si celui-­ci est écologiquement pertinent (hyperthermie et stress immunitaire mais pas xénobiotique). Une tendance à la diminution des ressources énergétiques a également été observée suite à un stress suggérant une modification des performances comportementales. Afin de vérifier cela, je me suis concentrée sur l’activité de butinage; le vol chez les insectes étant un des processus physiologiques les plus coûteux du règne animal. Une altération des performances de butinage a été mise en évidence chez les abeilles soumises à un stress immunitaire avec une réorientation des préférences de butinage au dépens du pollen, plus coûteux àc ollecter et moins riche en ressource énergétique que le nectar ; ceci probablement pour pallier au coût énergétique du stress. En revanche, en réponse àune hyperthermie, une augmentation de l’activité de butinage a été observée mais sans engendrer un coût supplémentaire au niveau des ressources collectées.Ces résultats sont discutés à la lumière du coût énergétique du stress et des conséquences potentielles sur les performances des abeilles, qui infine pourrait perturber l’homéostasie énergétique de la colonie. / Honeybees (Apis mellifera), which play an important role in natural and agronomic ecosystems, are exposed to a growing number of environmental pressures(new parasites, pesticides, climatechangeand poor nutrition). In this context, deciphering the mechanisms underlying stress responses and their costs becomes crucial to better understand theim pact of these pressures. Stress usually represents a challenge to the homeostasis of a norganism. In response, a cascade of physiological and behavioural adaptations enables the organism to cope with the stress. However, dueto their sociallife style, we could suggest that stress response in honeybees will occurin the interest of the colony and not only in the interest of the individual. To characterise the stress response and determine its specificity according to the stimulus (xenobiotic, immune, thermal, social), I developed a multidisciplinary approach to identify changes in i) task-­related physiology, ii) energetic metabolism, and iii) behaviour. I demonstrated that, regardless of their social function (nurse, guard, forager), bees respond in the sameway to a given stress, if itis ecologically-­relevant (heat and immune stress but not pesticides). Atendencytoward decreas ingenergetic resources was also observed following stress exposure, which suggests changes in behavioural performance.In order to test this hypothesis, I analysed changes in foraging activity in response to stress, as insect flight is one of the most costly physiological processes in the animal kingdom. I found that for aging performances were affected by animmune stress : bees changed their foraging preferences at the expense of pollen, probably to reduce the stress energetic cost, given that pollen is more costly to collect and provides alower energetic return than nectar. In contrast, in response to heat stress, an increase in colony for aging activity was observed, without an additional cost on resource collection. These results are discussed in the light of stress energetic cost and its potential consequences onhoneybee performances, which could disrupt the colony’s energetic homeostasis.

Reproduction in vertebrates, including fishes, is under control of the brain-pituitary-gonad (BPG) axis. The female three-spined stickleback, Gasterosteus aculeatus, produces egg clutches at intervals of a few days and spawns them in a nest built by male. Following ovulation, eggs are stored in the ovarian cavity surrounded by the ovarian fluid (OF). If spawning or spontaneous release do not occur, the eggs can undergo overripening, a phenomenon occurring both in nature and captivity. In this PhD thesis, the changes of reproductive hormones and vitellogenesis were studied at overripening of eggs and over the natural spawning cycle. OF properties were also examined at overripening of eggs and after treatment with sex steroids. Plasma levels of steroids: testosterone (T), estradiol (E2), 17,20β-dihydroxypregn-4-en-3-one (17,20β-P) and 17,20β,21-trihydroxypregn-4-en-3-one (17,20β,21-P) were measured by radioimmunoassay, and relative mRNA levels of the pituitary gonadotropins (fsh-β/lh-β), brain gonadotropin-releasing hormones (gnrh2/gnrh3) and kisspeptin and its receptor (kiss2/gpr54) by qPCR. Overripening of eggs was accompanied with a significant reduction in most of endocrine parameters of BPG axis (T, E2, 17,20β-P; lh-β; kiss2, gpr54). Low level of hormones could be advantageous for the overripe egg-bound females, since this would reduce further ovulations giving higher chances to survive and reproduce again. Over the 3-day spawning cycle, T and E2 were highly correlated, showed cyclicity with low levels at ovulation and increasing from 24 and 6 hours post-spawning (hps), respectively. Spawning may give rise to this increase as these rises did not occur if release of the eggs does not happen (overripe females). A peak at pituitary lh-β mRNA levels appeared 48 hps, a day before the next ovulation. No significant changes were found for the other studied hormones. Vitellogenesis was studied by measurement of the vitellogenin mRNA levels in the liver by qPCR. The levels were highest at 24 and 48 hps and were positively correlated to both E2 and T over the cycle. However, changes were small suggesting a rather continuous vitellogenesis over the stickleback spawning cycle which could be an advantage for a multiple spawner with a limited spawning season. Overripening reduced vitellogenin mRNA levels but did not abolish it. OF amount was diminished in overripe females and had a lower viscocity but higher dry weight and protein levels than in non-overripe ovulated females, suggesting that changes in OF properties are related to the egg overripening. The effects of steroids were studied using Silastic capsules. T and 17,20β-P induced an increase of OF amount, but protein levels were only increased in 17,20β-P-treated females, proposing a role of this steroid in the control of OF secretion. 1-D SDS-PAGE showed that OF contained several proteins, some of them came from eggs, but no consistent differences between groups. Concluding, the knowledge of the reproductive physiological changes is important for understanding their essential roles in the production of viable eggs in this species but also in the reproductive physiology of female fishes in general. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

L’uranium (U) est naturellement présent à l’état de trace dans l’eau (µg.L-1), sa concentration pouvant atteindre localement quelques mg.L-1 du fait des activités anthropiques. Plusieurs études écotoxicologiques sur Danio rerio ont mis en évidence la toxicité, e.g. le stress oxydant, la génotoxicité mais aussi la reprotoxicité (i.e. moins de pontes et d’œufs pondus chez les poissons contaminés) de l’U dont les mécanismes ne sont pas connus.L’objectif de cette étude est de contribuer à la compréhension de la reprotoxicité de l’U par l’élucidation de mécanismes moléculaires perturbés après contamination. Pour cela, des investigations ont été menées sur les ovaires de poissons zèbre Danio rerio, reproduits (R) ou non (NR), après exposition par voie directe en condition de laboratoire à des concentrations représentatives d’environnements contaminés.Ce travail de thèse a été divisé en deux volets. Un premier volet analytique avait pour but la poursuite des développements de méthodes pour l’identification des complexes U-protéines en condition non dénaturante, autour du couplage de techniques de séparation (chromatographie d’exclusion stérique SEC, électrophorèse hors gel OGE) et de détection sensible par spectrométrie de masse élémentaire (ICP MS) et moléculaire (ESI MS). Le second volet a été dédié à l’étude de la reprotoxicité de l’U à l’échelle moléculaire, avec i) l’étude des complexes natifs U-protéines (approche métallomique), et ii) l’analyse différentielle de l’expression des protéines (approche protéomique).Les développements analytiques ont permis de garder le tampon physiologique et non dénaturant d’extraction pour l’étape de séparation OGE, améliorant le taux de recouvrement en U. En écotoxicologie, les principaux résultats montrent que l’ovaire est un organe accumulateur de l’U et que le statut de reproduction a une influence sur le niveau d’accumulation (R<NR). En revanche, cet état a peu d’influence sur sa distribution protéique pour laquelle 4 fractions (dont 1 principale) ont été identifiées, toutes contenant aussi du phosphore. L’identification des cibles potentielles de l’U et des protéines exprimées différentiellement (vtg, GST, GAPDH,…) a montré que les processus biologiques perturbés suite à la contamination sont de deux niveaux : 1/ générique (stress oxydant) et 2/ plus spécifique de la gonade (développement et maturation des ovocytes). En conclusion, ces deux approches complémentaires ont permis de mettre en évidence un effet direct (complexation) et indirect (expression protéique modulée) de l’U, et de proposer l’hypothèse d’un défaut de maturation des ovocytes après contamination. Ce défaut pourrait impacter le développement embryonnaire et in fine expliquer la reprotoxicité observée lors d’études écotoxicologiques précédentes. / Uranium (U) is naturally presents at trace level (µg.L-1) in aquatic environment; its concentration can increase up to a few mg.L-1 due to human activities. Several ecotoxicological studies have shown uranium toxicity in contaminated zebrafishes Danio rerio, e.g. oxidative stress, genotoxicity and reprotoxicity (i.e. lower number of spawn and eggs laid) but mechanisms are not well known.The objective of this study is to contribute to the understanding of uranium reprotoxicity by elucidating the disrupted molecular mechanisms after contamination. Therefore, investigations have been carried out on ovaries from reproduced (R) and non-reproduced (NR) zebrafishes after waterborne exposure in laboratory conditions at environmentally relevant concentrations.This project was divided into two parts. Firstly, analytical investigations were carried out to continue the development of non-denaturing methods for U-protein identification by coupling separative techniques (size exclusion chromatography SEC, off gel electrophoresis OGE) with elemental (ICP MS) and molecular (ESI MS) sensitive mass spectrometry detection. Secondly, studies of U reprotoxicity were investigated by studying i) native U-protein complexes (metallomics approach) and ii) differential analysis of protein expression (proteomics approach)Analytical developments allowed keeping the physiological and non-denaturing extraction buffer for OGE separation step, improving U recovery. In ecotoxicology, the major results showed that ovary is an U accumulating organ and that the reproduction status modifies the accumulation level (R<NR). However, this status is of little influence on its distribution on proteins with 4 fractions (including a major one) determined, all of them coeluting with phosphorus. The identification of U potential targets and of protein expression differences (vtg, GST, GAPDH…) showed that biological processes disrupted after contamination are at two levels: 1/ generic (oxidative stress) and 2/ more specific to gonad (oocyte development and maturation).As a conclusion, these two complementary approaches showed a direct (complexation) and indirect (modification of protein expression) effects of U, and enabled to hypothesize a lack of oocyte maturation after contamination. This defect could impact embryo development and in fine explain the reprotoxicity observed in previous ecotoxicological studies.

La nutrition est l'ensemble des processus par lesquels un être vivant transforme des aliments pour assurer son fonctionnement. Etudier la nutrition d’un organisme permet de comprendre les rapports entre la nourriture consommée et la santé de l’individu. L'ensemble des organes assurant l'extraction d'énergie est le système digestif, qui transforme les sucres en glucose, les corps gras en acides gras, et les protéines en acides aminés. L’objectif de cette étude est de déterminer l’influence que peut avoir la nutrition pollinique sur la santé de l’abeille domestique, Apis mellifera L.. Pour assurer cette nutrition, l’abeille est inféodée aux ressources florales. Du pollen récolté sur les plantes à fleurs, l’organisme puise les corps gras (ou lipides), les vitamines et les protéines. Or l’accès à ces ressources est variable en qualité, en quantité, et en diversité selon le temps et le milieu. Des problèmes de mortalités et affaiblissements des colonies sont observés depuis une vingtaine d’années, et l’une des causes suspectées est le manque de disponibilité et la faible valeur nutritionnelle des ressources polliniques en zones agricoles céréalières. Nous avons donc testé en conditions contrôlées les effets de la quantité, la qualité et la diversité pollinique sur la physiologie d’abeilles nourrices, sur leur immunité, ainsi que sur leur survie. La nutrition jouant un rôle essentiel dans la prévention de nombreuses maladies, les effets de l’alimentation pollinique ont été déterminés en présence ou non d’un stresseur biotique, Nosema ceranae, dont la prévalence dans les colonies est très élevée. Il s’agit d’une microsporidie invasive qui se développe dans l’intestin de son hôte, provoquant divers effets sublétaux et létaux. Etant donné que la valeur nutritionnelle d’un aliment dépend de sa composition mais aussi de sa digestibilité, une troisième partie porte sur l’influence que peut avoir le stresseur sur les capacités de digestion des protéines contenues dans le pollen par l’abeille. Nos résultats apportent des connaissances sur l’impact d’une déplétion en pollen que peuvent subir les colonies dans un paysage d’agriculture intensive. En effet, des abeilles carencées à plus ou moins 60 %, comme cela peut être observé entre deux cultures à floraison massives, subissent des perturbations au niveau individuel (perturbations du développement physiologique des abeilles nourrices, diminution de leur survie), ce qui peut les rendre moins performantes et plus sensibles aux stress présents dans l’environnement. De plus, nous avons pu démontrer que la valeur nutritionnelle des pollens, influence la tolérance des abeilles à Nosema ceranae. La qualité d’un pollen se définit par sa composition chimique totale (protéines, acides aminés, lipides, vitamines, sucres, etc…), et non pas uniquement par sa teneur en protéines ou acides aminés essentiels. Notre étude met d’ailleurs en évidence l’effet négatif d’une alimentation à base de pollen de maïs sur le développement des glandes hypopharyngiennes, l’expression du gène de la vitellogénine, et la survie des abeilles. Au regard de la présence élevée de cette culture dans les zones d’agriculture intensive et sa haute exploitation par les butineuses, nos résultats sont discutés en fonction des répercussions envisagées sur les colonies. Dans ces milieux, la diversité des ressources polliniques, par l’apport de pollens de qualité, compense la pauvreté nutritionnelle d’autres pollens. Dans nos conditions, nous pouvons observer la plus-value d’une alimentation pollinique polyflorale chez des abeilles infestées par Nosema. Les mesures agro-écologiques œuvrant pour régulariser dans le temps les apports polliniques aux colonies, et pour leur offrir une diversité alimentaire participent donc à renforcer leur capacité à lutter contre les stress présents dans l’environnement. / Nutrition is the set of processes by which a living organism transforms food for its functions. Studying nutrition allows to understanding the relationship between food intake and health of the individual. All organs for extracting energy are the digestive system, which converts sugars into glucose, fats into fatty acids, and proteins into amino acids. The objective of this study is to determine the possible influence of pollen nutrition on the honeybee health, Apis mellifera L. To ensure that nutrition, the bee is subservient to floral resources. Of pollen collected from the flowering plants, the body draws fats (or lipids), vitamins and proteins. Access to these resources is variable in quality, quantity, and diversity depending on the time and the environment. Problems of mortality and weakening of colonies are observed for some twenty years, and one of the suspected causes is the lack of availability and low nutritional value of pollen resources in agricultural areas. We therefore tested under controlled conditions the effects of quantity, quality and diversity of pollen on nurse worker physiology and on their immunity, as well as their survival. Nutrition plays a vital role in the prevention of many diseases, the effects of pollen diet were determined in the presence or absence of a biotic stressor, Nosema ceranae, whose prevalence in the colonies is very high. It is an invasive microsporidia that develops in the intestine of its host, causing various sublethal and lethal effects. Since the nutritional value of a food depends on its composition but also its digestibility, a third part focuses on the possible influence of the stressor on bee ability digestion of the proteins provided by the pollen.Our results provide knowledge on the impact of pollen depletion that may undergo the colonies in an intensive agricultural landscape. Indeed, bees starved to roughly 60%, as can be observed between two cultures massive flowering undergo disturbances at individual level (disturbances of nurse worker physiological development, reduced survival), which may make them less efficient and more susceptible to stress from the environment. In addition, we demonstrated that the nutritional value of pollen influences the Nosema ceranae‘s bee tolerance. The pollen quality is determined by the total chemical composition (proteins, amino acids, lipids, vitamins, sugars, etc ...), not only by its content of essential amino acids or proteins. Our study also demonstrated the negative effect of a diet based on maize pollen on hypopharyngeal glands development, the vitellogenin gene expression, and the bee survival. In view of the high presence of this culture in intensive agriculture areas and high exploitation by foragers, our results are discussed in terms of their impact on the proposed settlements. In these environments, the diversity of pollen resources by providing pollen of quality compensates nutritional poverty of other pollens. In our conditions, we can observe the gain of power of a polyfloral pollen in Nosema infested bees. Agro-ecological measures working to regularize, in time, the pollen contributions to the colonies, and to provide them with food diversity thus participate in building their capacity to fight against stress in the environment