Développement de gènes biomarqueurs pour l'évaluation de l’impact des perturbateurs endocrines dans l’espèce non-modèle Gammarus fossarum

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCANational audienceRecently, the urgence in developing relevant tools for the identification and hazard assessment of endocrine disruptors in invertebrate species has been highlighted by the scientific community. However, despite the obvious importance of invertebrates and especially crustaceans in aquatic systems, effects and/or hazard assessment of these compounds remains difficult to establish, and no specific endocrine disruption (ED) biomarkers are available in crustacean species currently used in aquatic ecotoxicology. This can be attributed to the poor knowledge of their endocrine systems, but also to the lack of genomic data for non-model species. Our research teams have conducted recently a “proteogenomic” approach to generate a large transcriptomic and proteomic dataset of the amphipod crustacean Gammarus fossarum, a sentinel species widely used in aquatic ecotoxicology in Europe. Based on this omic ressources and literature research, the aim of this study was to develop and propose specific ED biomarker genes in G. fossarum. We first established an initial candidate list of genes reputed in the literature as essential players of hormonal regulation in crustaceans and insects (hormone receptors, enzymes of the hormone metabolism, regulator genes, hormone-regulated genes). Homologous sequences of crustacean or insect published sequences of these candidates were identified in our transcriptomic G. fossarum database, and a validation step of putative gene annotation was made by sequence similarity research and phylogenetic analyses. With this approach, we identified and validated three potential candidates: the nuclear receptors RxR and E75, and the regulator broad-complex. We then followed a cloning and sequencing protocol to obtain reliable nucleotidic sequence and primers for the posterior expression studies. Their interest as biomarkers was then assessed by studying gene expressions during the male and female reproductive cycles, and after laboratory exposure to model chemical compounds. Perspectives are to develop a methodology for biomarker measurement (sex, size, organ, moulting stage of monitored individuals, duration of exposure) and absolute quantitation methods of proteotypic peptides (immunoassay, and/or mass spectrometry) based on the acquired knowledge of the proteic sequence of these markers in our non-model species