We hypothesized that populations exposed to chronic levels of genotoxins for many generations in situ will accumulate latent heritable genetic damage leading to increases in mutational load and decreased population fitness. Common pulmonate snails (Lymnaea stagnalis) were collected from three sites that differed in pollution level [Manitoulin Island (reference), Beauharnois (moderate pollution), and Varennes (highly polluted)]. These organisms have a rapid generation time and are capable of self-fertilization. Fitness indicators were clutch size, survival (hatching to day 30) and growth (length from hatching to day 90). Recessive deleterious mutations that have accumulated through time are masked as snails preferentially outcrossed, but are expressed when snails self-fertilize. Results obtained by comparing fitness components of snails derived from selfing and outcrossing experiments showed that in sites with higher levels of pollution inbreeding depression increased for clutch size and survival. Results for growth rate were similar to those for clutch size and survival in the two least polluted sites, but not for the most polluted site, due possibly to biased mortality of smaller snails at this site. After correction for size biased mortality, the growth estimates showed patterns similar for the two polluted sites when compared to the reference site. This study suggests that long-term in situ exposure to genotoxic pollution may effect population fitness due to the accumulation of latent heritable genetic damage.

No description available.

Les produits phytosanitaires contiennent des molécules qui peuvent interférer avec le système endocrinien et induire des effets délétères sur les êtres vivants. Ce sont des perturbateurs endocriniens. Afin d’assurer un niveau de protection satisfaisant pour les mollusques aquatiques, une bonne compréhension des perturbations du système endocrinien est nécessaire. L’organisation pour le développement et la coopération économique s’est exprimée en faveur de la mise en place de protocoles standards d’écotoxicité chez les mollusques, notamment sur la reproduction, dans le cadre des processus d’évaluation du risque écologique et des autorisations de mise sur le marché de nouvelles molécules. Cette thèse vise à proposer des tests standards et des outils d’analyse afin d’évaluer les effets de substances chimiques chez un escargot d’eau douce, Lymnaea stagnalis. Une série de tests de toxicité a été conduite dans laquelle les limnées ont été exposées à des androgènes, des oestrogènes et dessolvants. Les effets observés ont été évalués à l’aide d’approches statistiques standards et d’un modèle de toxicokinétique-toxicodynamique (TKTD). Les limnées ont montré une sensibilité uniquement au tributyltin et triphényltin à des concentrations réalistes d’un point de vue environnemental. Le triéthylène glycol et l’acétone étaient les solvants les moins nocifs. De plus, le modèle TKTD suggère un mécanisme généraliste dans l’action du composé. La manifestation des perturbations endocriniennes chez L. Stagnalis restent évasives et nécessitent d’autres recherches. Enfin, la thèse a montré que / Plant protection products and biocides may contain chemicals that interfere with the endocrine system and consequently induce adverse effects on wildlife. Such chemicals are known as endocrine disruptors (EDs). To ensure a satisfactory protection level for aquatic molluscs a comprehensive understanding of endocrine disruption is needed. The Organisation for Economic Co-operation and Development supports the standardisation of toxicity tests guideline on mollusc reproductive toxicity, which is intended for environmental risk assessments purposes. This PhD thesis aimed at improving toxicity test protocols and data analysis tools to evaluate effects of chemicals on the great pond snail Lymnaea stagnalis. A suite of toxicity tests was conducted in which snails were exposed to putative androgens (tributyltin, triphenyltin, methyltestosterone), putative oestrogens (alkylphenols and ethynyloestradiol), and solvents. Resulting effects were evaluated using standard statistical approachesand/or a toxicokinetic-toxicodynamic (TKTD) model. Snails were sensitive to tributyltin and triphenyltin, while none of the oestrogenic chemicals and methyltestosterone significantly impacted animals at environmentally relevant concentrations. Triethylene glycol and acetone were the least harmful solvents. Further, the analysis of data from the tributyltin test with the TKTD model suggested a generic mechanism of action of the compound (i.e., not specific for EDs). The demonstration of endocrine disruption in L. stagnalis remained elusive and deserves further research. Finally, the thesis highlight

Dans l'environnement, les organismes sont soumis à des stress de multiples natures (e.g., polluants et pathogènes/parasites). Dans ce contexte, le système immunitaire joue un rôle fondamental. La pression chimique exercée par les polluants peut impacter leur système immunitaire, affaiblir leur santé et les prédisposer à contracter des maladies infectieuses. En écotoxicologie, l'évaluation de marqueurs d'immunocompétence peut servir à diagnostiquer l'état de santé des organismes et mieux comprendre le danger immunotoxique des xénobiotiques. Lymnaea stagnalis est un gastéropode d'eau douce représentatif de nombreux milieux lentiques sur la planète. Des tests OCDE de reprotoxicité sont disponibles mais peu d'études se sont focalisées sur des paramètres immunotoxiques. Le premier axe de la thèse se porte sur la connaissance et le développement de méthodes pour évaluer l'immunocompétence de manière répétée à l'échelle individuelle chez L. stagnalis. Le deuxième axe évalue cette approche multibiomarqueurs dans différents contextes écotoxicologiques. Plusieurs populations (élevages vs terrain), temps d'expositions (3 vs 29 – 44 jours), concentrations d'exposition (environnementales vs non réalistes) et contaminants ont été évalués : des médicaments (les anti-cancéreux Etoposide et cyclophosphamide, l'anti-rejet de greffes cyclosporine A et l'antiinflammatoire diclofenac), l'insecticide deltaméthrine, le métal cadmium et des effluents de station d'épuration. Des comparaisons entre des traits de vie et des traits immunitaires ont été réalisées. La réflexion est bâtie sur les questionnements relatifs à (i) la sensibilité de l'approche, (ii) le temps de réponse des marqueurs, (iii) leur précocité physiologique, (iv) la transposition d'immunotoxicité de xénobiotiques entre le phylum des chordata et L. stagnalis et (vi) l'utilisation pratique de L. stagnalis (élevages, collecte en milieu naturel, calibration, reproduction) / In natural environments, animals encounter multiple stresses (e.g., pollutants and pathogens/ parasites). In this context, the immune system plays a pivotal role. Chemical insults from pollutants can jeopardize the immune system of these animals, weaken their health and make them vulnerable to contracting infectious diseases. In ecotoxicology, assessment of immunocompetence markers can be used to diagnose the health of organisms and better understand the immunotoxic hazard of xenobiotics. Lymnaea stagnalis is a freshwater gastropod representative of several lentic ecosystems on the planet. OECD reprotoxicity tests are available using this species, but too few studies have focused on immunotoxic parameters. The first part of this thesis covers the description, comprehension and development of the methods used to assess the immunocompetence at an individual scale and repeatedly within an experiment in L. stagnalis. The second part assesses this multibiomarker approach in several ecotoxicological contexts. Several populations (laboratory-bred vs collected in the field), time exposures (3 vs 29–44 days), concentrations (environmental vs unrealistic) and contaminants were evaluated : pharmaceuticals (the anti-cancer drugs etoposide and cyclophosphamide, the anti-xenograft rejection drug cyclosporine A, the anti-inflammatory drug diclofenac), the insecticide deltamethrin, the metal cadmium and wastewater treatment plant effluents. The general reasoning is built on questions relative to (i) the sensitivity of the approach, (ii) the rapidity of the response, (iii) their physiological precocity of alteration (immune vs life-history traits), (iv) the transposition of xenobiotic immunotoxicity between L. stagnalis and the Chordata phylum and (vi) the pragmatic use of L. stagnalis (breeding, collection in the environment, calibration and reproduction)

This study applies molecular and morphological approaches addressing the identification of morphologically similar larval stages (cercariae) of Plagiorchis spp. (Digenea: Plagiorchiidae) parasitising lymnaeid snail populations in the freshwater ecosystems of central Europe. Five morphologically homogeneous and genetically distinct lineages of Plagiorchis spp. were identified via matching molecular data for the mitochondrial cox1 gene with detailed morphometric data. Phylogenetic and comparative sequence analyses using partial 28S rDNA and ITS1-5.8S-ITS2 sequences allowed molecular identification of three species (P. elegans, P. maculosus and P. koreanus) via matching sequences from larval and adult digenean stages. A key for the identification of the cercariae of Plagiorchis spp. parasitising lymnaeid populations in central Europe is provided.

No description available.

Calmodulin (CaM) is a ubiquitous calcium-binding protein responsible for the binding and activation of a vast number of enzymes and signaling pathways. It contains two lobes that bind two calcium ions each, separated by a flexible central linker. This structural flexibility allows CaM to bind and regulate a large number of diverse protein targets within the cell in response to Ca2+ gradients. Voltage gated calcium channels (CaVs), as main sources of extracellular Ca2+, are crucial for a number of physiological processes, from muscle contraction to neurotransmission and endocrine function. These large transmembrane proteins open in response to membrane depolarization and allow gated entry of Ca2+ ions into the cytoplasm. Their regulation is currently the subject of intense investigation due to its pharmacological and scientific importance. CaM has been previously shown to pre-associate and act as a potent inhibitor of one class of high-voltage activated (HVA) channels called L-type channels via its interaction with their C-terminal cytoplasmic region. This interaction is primarily mediated by a conserved CaM-binding motif called the ‘IQ’ motif (for conserved isoleucine and glutamine residues), although the exact molecular details of its involvement in inactivation are currently unclear. Elucidation of these details was the primary objective of this dissertation. Recently, a novel sequence motif within this channel called ‘NSCaTE’ (N-terminal spatial calcium transforming element) has been described as an important contributor to calcium-dependent inactivation (CDI) of L-type channels. It was presumed to be unique to vertebrates, but we also show its conservation in a distantly related L-type channel homolog of Lymnaea stagnalis (pond snail). The interaction of CaM with a number of peptides representing the different regulatory motifs (IQ and NSCaTE) for both mammalian and snail isoforms was characterized in an attempt to better understand their role in CDI. Biophysical work with peptides as well as electrophysiology recordings with an N-terminal truncation mutant of Lymnaea CaV1 homolog were performed to expand our understanding of how the interplay between these channel elements might occur. In brief, the most striking feature of the interaction concerns the strong evidence for a CaM-mediated bridge between the N- and C-terminal elements of L-type channels. Further investigation of the CaM interaction with both IQ and NSCaTE peptides using Ca2+-deficient CaM mutants reveals a preference of both peptides for the Ca2+-C-lobe of CaM, and a much higher affinity of CaM for the IQ peptide, suggesting that the N-lobe of CaM is the main interaction responsible for the physiological effects of NSCaTE. These results are consistent with our electrophysiology findings that reveal a distinct buffer-sensitive CDI in wild type LCaV1 that can be abolished by the N-terminal truncation spanning the NSCaTE region. In addition to L-type channels, CaM has also been shown to have an indirect role in the regulation of low-voltage activated (LVA) or T-type channels (CaV3.x), via their phosphorylation by CaM-dependent protein kinase II (CaMKII). Using a primary sequence scanning algorithm, a CaM-binding site was predicted in a cytoplasmic region of these channels that was also previously shown to be important in channel gating. Biophysical experiments with synthetic peptides spanning this gating brake region from the three human and the single Lymnaea isoform strongly suggest that there is a novel, bona fide CaM interaction in this channel region, and also hint that this interaction may be a Ca2+-dependent switch of some sort. The results confirm a possible new role for CaM in the direct regulation of these channels, although the exact mechanism remains to be elucidated.

Molluscs as well as all other invertebrates rely on innate immune response only. Their internal defense system is capable of destroying most pathogens. However, there are some exceptions, e.g. some snails serve as intermediate hosts for some trematodes. Trematodes are able to develop inside these snails due to intervention in the snail internal defense system. The submitted thesis describes hemocyte activities of two lymnaeid snails, Lymnaea stagnalis a Radix lagotis, and the influence of Trichobilharzia regenti infection on R. lagotis hemocytes. Hemocytes of both species exposed to various chemicals produced different amounts of H2O2 and NO. The response varied between both lymnaeid species. The amount of circulating hemocytes was elevated in R. lagotis snails due to T. regenti infection. However, the infenction attenuated hemocyte activities monitored by us. Hemocyte basal NO production was decreased as well as phagocytosis of bacteria, cell adherence and pseudopodia formation. Toxicity of L. stagnalis plasma against T. regenti miracidia was also described. Mechanisms used by trematodes to interact with the snail internal defense system will help us to understand why one species is suitable for the develepment of the trematode whereas another closely related species kills it. Powered by TCPDF (www.tcpdf.org)

Pour valider une suite de biomarqueurs cellulaires en milieu dulçaquicole, nous avons étudié la structure des systèmes lysosomal et peroxysomal et les variations de contenus en lipofuscines et lipides neutres insaturés dans les tissus digestifs de plusieurs invertébrés, à travers une approche couplant histochimie et analyse d'images. Les expérimentations in situ et bioessais à plus ou moins long terme ont montré la pertinence des réponses cellulaires étudiées pour la mise en évidence de gradients de contamination. Les profils de réponse les plus communs chez les mollusques incluent un gonflement lysosomal, des déplétions de lipides neutres insaturés, une accumulation de lipofuscines et/ou un système peroxysomal plus volumineux. Ces réponses cellulaires précoces peuvent être associées, au niveau individuel, à une désynchronisation de la reproduction entre individus ou à une diminution de l'indice de condition. L'étude mensuelle à long terme a souligné l'absence de niveaux de base saisonniers de ces biomarqueurs et l'influence importante de la disponibilité trophique sur les contenus tissulaires en lipides neutres. Une mise en dépuration de 15 jours du bivalve, Dreissena polymorpha, sur son site d'origine, n'a pas montré de réversibilité du profil des réponses cellulaires à un gradient de contamination in situ, mais elle a tout de même de limité la déplétion des lipides neutres. Les réponses cellulaires les plus précoces et discriminatives sont le gonflement lysosomal et la déplétion en lipides neutres. Une comparaison d'outils d'analyse intégrative a montré le pouvoir discriminatif accru de la suite de biomarqueur entière, par rapport aux réponses individuelles.

This work applies advanced sampling (mark-release-recapture) and comparative approaches addressing the patterns in composition, structure and variability of larval trematode communities in three species of gastropod molluscs (Lymnaea stagnalis, Planorbarius corneus and Radix auricularia) at two nested scales of community organisation in typical Central European eutrophic environments. Hypothesis-testing with the application of null-model analyses, logistic regression modelling and multivariate randomisation techniques, revealed determinants of transmission rates, levels of infection and community structure in freshwater snail hosts in Central Europe and elucidated the mechanisms linking the spatial and temporal environmental variability with the action of complex community assembly rules in freshwater pulmonate snails.