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International audience; Gammarus sp. (Amphipoda) are widely distributed across European freshwater systems. In the present study, we evaluated the effect of a fenoxycarb exposure on Gammarus sp.. More specifically, i) we assessed the sensitivity of the embryo stage, ii) we identified embryogenesis' sensitive period, iii) we evaluated the sensitivity of the reproductive period and iv) we compared the response to fenoxycarb exposure among three European gammarid species. Fenoxycarb is a growth regulator insecticide, analog of the insect juvenile hormone, used for pest management and for veterinary purpose. This study demonstrated that 5 and 50 µg L

International audience; Reproductive success of organisms is related to the quantity and quality of eggs produced by females. Vitellogenin (Vg), precursor molecule of vitellin that is the energy available for embryonic development in oviparous organisms, is proposed as a relevant exposure and effect bio-chemical marker of endocrine disruptors in aquatic vertebrate species (e.g. fish, amphibian). Numerous strategies, such as enzyme-linked immunosorbent assays have been developed to characterise and quantify this protein in vertebrates. On the contrary, in invertebrates few methods are available. This gap mainly results from the low transferability of available antibodies in relation to phylogenetic distance and molecular divergence accumulated by proteins through animal evolution. Recently, our laboratory developed a quantitative assay of Vg in a widespread amphipod, Gammarus fossarum, using liquid chromatography tandem mass spectrometry (LC-MS/MS). This method offers the possibility to identify and quantify Vg, based only on the quantification of proteotypic peptides containing from 5 to 15 amino-acids. Despite a high molecular divergence observed for this type of protein throughout animal evolution, comparative and evolutive genomics revealed that partial conservation between homologous proteins can persist due to high functional constraints acting on residues clustered in the proteic structure forming conserved peptides within phylogenetical groups. In this context, the aim of this study was to assess the possibility to take advantage of peptidic motifs conservation to propose a transferable method across species. For this, our study focused on three model species for which sequence of egg yolk protein is well known, an amphipod, Gammarus fossarum, a cladocerean Daphnia magna and an insect Drosophila melanogaster. In first step, proteotypic peptides of Vg were identified for each species. In the second step, we tried to find these proteotypic peptides in closely related species, such as G. pulex, G. wautierii, D. pulex. This study showed the high relevance of mass spectrometry to propose a specific methodology for Vg measurement. Moreover, methodology proposed to Vg measurement is transferable from a species to another one from the same genus.

Biochemical activities that involve cytochrome P450 1A can be seen as early as during the embryonic stage in fish. They can therefore be used as biomarkers of exposure to contaminants such as PAHs, PCBs or dioxins. Nevertheless, in the zebra fish Danio rerio, the classical EROD measurement in larvae homogenate is inadequately sensitive, particularly because of the small size of these organisms. Moreover, the pooling of numerous animals is necessary, and is thus of poor interest for biomonitoring. As an alternative, a non « destructive » method to detect the induction of the biotransformation activity in the pro-larvae of the fish Danio rerio exposed to chemicals contaminants was developed.The methodology is based on the EROD activity induction, by measuring the fluorescence increase in the test medium due to the excretion of the resorufin by the fish, after pre-exposure to chemicals or environmental samples. Exposure of fish embryos to known inducers (Benzo-a-Pyrene and ß-Naphtoflavone) was carried out. Next the prolarvae were exposed to the substrat (7-ethoxyresorufin), and the kinetic of the fluorescence increase (resorufin excretion) was measured. A dose-effect relationship was observed, with an increase of the EROD activity of 3 to 5 times the basal activity. The methodology and some examples of use of the biotest to assess the impact of environmental samples on the fish prolarvae are presented., L`étude réalisée a permis de développer une méthode de mesure non destructive et sensible du métabolisme de biotransformation chez la pro larve de Danio rerio, basée sur la détection de l`activité de phase I 7-ethoxyresorufin-O-deethylase (EROD), au moyen de la mesure de la fluorescence de la résorufine excrétée dans le milieu. En effet, dès la première heure d`exposition de larves de poissons zèbre (Danio rerio) à une solution d`éthoxyrésorufine, une diminution de la concentration dans le milieu est mesurée, accompagnée d`une augmentation de la fluorescence de la résorufine excrétée. L`optimisation des conditions opératoires et de mesures (longueurs d`onde, teneur en éthoxyresorufine, rapport larve/volume de milieu ) a permis la mise au point d`un bio essai en microplaque simple, sensible et reproductible. L`augmentation linéaire en fonction du temps de la concentration de résorufine permet de mesurer une activité basale ou induite. Des pré-contaminations de larves à partir du stade embryonnaire à des inducteurs modèles (benzo-a-pyrene et beta naphtoflavone) ont été réalisées selon plusieurs modalités d`exposition (concentrations et durée), les larves n`étant pas alimentées pendant les essais. Elles ont permis de mettre en évidence une relation dépendante de la concentration en inducteur jusqu`à 3 à 5 fois l`activité basale (env. 0,1 pmol resorufine / h / larve). Chez les organismes non exposés à des inducteurs l`activité basale est quantifiable mais décroît avec l`âge de la larve. Des exemples d`application de cette technique à la mesure de l`impact d`échantillons environnementaux sur l`induction du métabolisme de détoxification de la larve sont présentés.

Biochemical activities that involve cytochrome P450 1A can be seen as early as during the embryonic stage in fish. They can therefore be used as biomarkers of exposure to contaminants such as PAHs, PCBs or dioxins. Nevertheless, in the zebra fish Danio rerio, the classical EROD measurement in larvae homogenate is inadequately sensitive, particularly because of the small size of these organisms. Moreover, the pooling of numerous animals is necessary, and is thus of poor interest for biomonitoring. As an alternative, a non « destructive » method to detect the induction of the biotransformation activity in the pro-larvae of the fish Danio rerio exposed to chemicals contaminants was developed.The methodology is based on the EROD activity induction, by measuring the fluorescence increase in the test medium due to the excretion of the resorufin by the fish, after pre-exposure to chemicals or environmental samples. Exposure of fish embryos to known inducers (Benzo-a-Pyrene and ß-Naphtoflavone) was carried out. Next the prolarvae were exposed to the substrat (7-ethoxyresorufin), and the kinetic of the fluorescence increase (resorufin excretion) was measured. A dose-effect relationship was observed, with an increase of the EROD activity of 3 to 5 times the basal activity. The methodology and some examples of use of the biotest to assess the impact of environmental samples on the fish prolarvae are presented., L`étude réalisée a permis de développer une méthode de mesure non destructive et sensible du métabolisme de biotransformation chez la pro larve de Danio rerio, basée sur la détection de l`activité de phase I 7-ethoxyresorufin-O-deethylase (EROD), au moyen de la mesure de la fluorescence de la résorufine excrétée dans le milieu. En effet, dès la première heure d`exposition de larves de poissons zèbre (Danio rerio) à une solution d`éthoxyrésorufine, une diminution de la concentration dans le milieu est mesurée, accompagnée d`une augmentation de la fluorescence de la résorufine excrétée. L`optimisation des conditions opératoires et de mesures (longueurs d`onde, teneur en éthoxyresorufine, rapport larve/volume de milieu ) a permis la mise au point d`un bio essai en microplaque simple, sensible et reproductible. L`augmentation linéaire en fonction du temps de la concentration de résorufine permet de mesurer une activité basale ou induite. Des pré-contaminations de larves à partir du stade embryonnaire à des inducteurs modèles (benzo-a-pyrene et beta naphtoflavone) ont été réalisées selon plusieurs modalités d`exposition (concentrations et durée), les larves n`étant pas alimentées pendant les essais. Elles ont permis de mettre en évidence une relation dépendante de la concentration en inducteur jusqu`à 3 à 5 fois l`activité basale (env. 0,1 pmol resorufine / h / larve). Chez les organismes non exposés à des inducteurs l`activité basale est quantifiable mais décroît avec l`âge de la larve. Des exemples d`application de cette technique à la mesure de l`impact d`échantillons environnementaux sur l`induction du métabolisme de détoxification de la larve sont présentés.