Development of biomarkers of neurotoxicity and endocrine disruptions in the freshwater amphipod Gammarus fossarum

L’objectif de cette thèse était de développer des biomarqueurs de neurotoxicité (l’activité des cholinestérases) et de perturbations endocriniennes en lien avec la reproduction (la synthèse de vitellogénine) chez l’amphipode d’eau douce Gammarus fossarum. G. fossarum ne possède qu’une isoforme de cholinestérase de type acétylcholinestérase (AChE). Les facteurs biotiques ont été montrés comme la principale source de variabilité naturelle de l’activité AChE chez cette espèce. Ainsi, la sélection d’organismes de phénotype mâle avec un poids de 15-20 mg a permis de proposer une méthodologie de dosage fiable et robuste. Une valeur de référence et une valeur seuil ont été décrites. Ces travaux ont également permis de montrer qu’il n’existe aucune relation entre l’inhibition de cette activité enzymatique et la mortalité. En revanche, une relation directe a été observée entre l’activité AChE et le comportement. Enfin, l’utilisation couplée de tests in situ et de la mesure de l’activité AChE s’est révélée être un outil pertinent pour évaluer l’impact de composés anti-cholinestérasiques. La description des processus physiologiques, liés au cycle de reproduction des femelles (cycle de mue, croissance des ovocytes et développement embryonnaire), a apporté les bases pour le développement de l’expression de la Vtg comme biomarqueur. Cela nous a également permis de développer un bio-essai de reprotoxicité reproductible et robuste avec une faible variabilité des valeurs de base. Les niveaux d’expression du gène Vtg observés chez les femelles étaient de 200 à 700 fois plus élevés que chez les mâles. Les femelles présentent une importante élévation de la synthèse de Vtg au cours de leur cycle de reproduction, concordante avec la mise en place de la vitellogénèse secondaire. L’induction de l’expression du gène Vtg a été observée chez des mâles exposés, en laboratoire, à des perturbateurs endocriniens modèles et chez des individus exposés in situ sur des sites anthropisésThe aim of this works was to develop specific biomarkers of neurotoxicity (cholinesterase activity) and endocrine disruptions linked to reproduction (vitellogenine synthesis) in freshwater amphipod Gammarus fossarum. G. fossarum owns only an isoforme of cholinesterase of type acetylcholinesterase (AChE). Biotic factors have been shown as the main source of natural variability of the activity AChE in this species. Therefore, the selection of organisms with male phenotype and a body-weight of 15-20 mg allowed to propose a methodology of reliable and strong dosage. A reference value and a threshold value were described. Our works on the AChE, also demonstrated that there is no relation between the inhibition of this enzymatic activity and the mortality. On the other hand, a direct relation was observed between the activity AChE and behaviours. Finally, the coupled use of in situ bioassays and measure of the activity AChE showed to be a relevant tool for the environmental assessment of anti-ChE compound impact. The description of the physiological processes, bound to the reproduction cycle of females (moult cycle, oocyte growth and embryonic development), brought bases for the development of the Vtg gene expression as biomarker. It also allowed to develop a robust and reproducible reprotoxicity-test displaying a very weak variability of the basic values observed in the non-impacted organisms. Females present an important rise of the Vtg synthesis during their reproduction cycle, corresponding with the implementation of the secondary vitellogenesis process. The induction of the Vtg gene expression was observed in males exposed in laboratory to model endocrine disrupters and in organisms exposed in situ, in areas subjected to an anthropological pressur

Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda) - Linking AChE inhibition and behavioural alteration

International audienceRelations between whole-body acetylcholinesterase (AChE) inhibition and changes in feeding and loco- motor behaviours were investigated in adult male Gammarus fossarum during short-term exposure (96 h) to the organophosphorous pesticide chlorpyrifos (CPE) and the carbamate pesticide methomyl (MT). AChE activity was measured after 24, 48 and 96 h of exposure. The feeding rate was assessed after 48 and 96 h of exposure and locomotor activity was measured at the end of the experiment. A concentration-dependent decrease of AChE activity and behavioural parameters was observed for both CPE and MT. However, these two compounds presented dissimilar modes of action since MT-induced effects appeared rapidly during the first 48 h of the experiment and remained constant until the end of experiment, contrary to CPE- induced effects, which occurred gradually during the last 48 h. For CPE, significant mortality was observed from 50% AChE inhibition, contrary to MT for which no mortality was observed despite 66% inhibition. These results suggest that, for CPE, the observed mortality was not directly related to AChE inhibition but that an additional toxic mode of action occurred. On the contrary, the feeding rate and locomotion impairment were directly correlated to levels of AChE inhibition for both chemicals, except for the lowest concentrations of MT for which an induction of the behavioural parameters was observed. Although CPE and MT have different modes of action (acting as indirect and direct inhibitors), the relations between AChE activity and inhibition of behavioural parameters were relatively close for these two compounds. This study provides a basis to interpret the biomarker AChE at the upper biological organisation level, on both the feeding rate and locomotor behaviour, which are known to be relevant ecological responses

Sexual dimorphism in Grp78 and Hsp90A heat shock protein expression in the estuarine copepod Eurytemora affinis

International audienceAquatic organisms are constantly exposed to both natural and anthropogenic stressors. Under stress conditions, they elicit a cellular stress response, involving heat shock proteins (HSPs). HSPs are essential to protect proteins against aggregation and to help in the folding of native proteins or refolding of damaged ones. Because of their conservation among taxons and their inducibility after environmental/ chemical stress, HSPs are commonly used as ecological and ecotoxicological biomarkers. However, the appropriate use of such molecular tools requires the investigation of the influence of biotic factors on their basal levels. As a first step in biomark- er characterization, the present study aims to evaluate the impact of the reproductive cycle on the expression of the two major HSPs, Grp78 and Hsp90A in the estuarine copepod Eurytemora affinis . The constitutive expression of both genes in males was weak when compared to female levels suggesting gender-specific stress tolerance. Transcript levels gradually increased during oogenesis and maximal levels were recorded in ovigerous females. The present data support the view that the reproductive condition of individuals has to be considered as a confounding factor in stress evaluation by HSP quantification

Multixenobiotic resistance in Mytilus edulis : Molecular and functional characterization of an ABCG2- type transporter in hemocytes and gills

International audienceAmong the cellular protection arsenal, ABC transporters play an important role in xenobiotic efflux in marine organisms. Two pumps belonging to B and C subfamily has been identified in Mytilus edulis. In this study, we investigated the presence of the third major subtype ABCG2/BCRP protein in mussel tissues. Transcript was expressed in hemocytes and with higher level in gills. Molecular characterization revealed that mussel ABCG2 transporter shares the sequence and organizational structure with mammalian and molluscan orthologs. Overall identity of the predicted amino acid sequence with corresponding homologs from other organisms was between 49% and 98%. Moreover, protein efflux activity was demonstrated using a combination of fluorescent allocrites and specific inhibitors. The accumulation of bodipy prazosin and pheophorbide A was heterogeneous in gills and hemocytes. Most of the used blockers enhanced probe accumulation at different levels, most significantly for bodipy prazosin. Moreover, Mrp classical blocker MK571 showed a polyspecificity.In conclusion, our data demonstrate that several ABC transporters contribute to MXR phenotype in the blue mussel including ABCG2 that forms an active pump in hemocytes and gills. Efforts are needed to distinguish between the different members and to explore their single function and specificity towards allocrites and chemosensitizers

Dataset on metabolome dimorphism in different organs of mature Palaemon serratus prawn

The prawn Palaemon serratus exhibits a large distribution (occurring along the Northeastern Atlantic coast to the Mediterranean), and has thus been found suitable as model organism valuable for various ecotoxicological studies. However, little is still known about the potential input of its metabolome and particularly concerning a potential molecular sexual dimorphism observable in the different tissues of this organism. In an ecotoxicological point of view, inter-sex and inter-organ differences of the metabolomes may introduce analytical bias and impact the robustness of the analysis and its interpretation. To explore such possibilities, we obtained qualitative metabolomic data from the analysis of different organs of mature male and female Palaemon serratus. We used ultra-high-performance liquid chromatography-electrospray ionization-high resolution tandem mass spectrometry (UHPLC-ESI-HRMS on positive mode) to characterize the 75%-extracted metabolome of both gills, hepatopancreas, nervous gland, muscle and gonads. The data were dereplicated using specific metabolomic software (MetaboScape 4) and 2,782 features were extracted, 1,720 of them being also analysed on MS/MS mode, supporting molecular networking investigations with Metgem 1.3.6. These metabolites were thus putatively identified using GNPS (Global Natural Product Social) Molecular Networking databases for de-novo annotation followed by manual curation of 84 metabolites. This data provides essential information on the important sexual dimorphism occurring at the molecular level in the different organs and supports further research on physiology and ecotoxicology in common European prawn

Assessment of sperm quality in palaemonid prawns using Comet assay: methodological optimization

International audienceThe aim of this study was to adapt the Comet assay in spermatozoa of the marine prawn Palaemon serratus to use it as a marker of sperm quality. Indeed, due to the characteristics of their spermatozoa, the measurement of DNA integrity is one of the few markers which can be transferred to crustaceans to assess the quality of their semen. In the first step, the methods of collecting and maintaining spermatozoa were optimized. Cell survival was estimated during kinetics of preservation (i.e. 1, 2, 4 and 8 h) in various suspension media to define artificial seawater (ASW) as optimal. Several methods in the releasing of spermatozoa from the spermatophore of prawns were estimated with regard to their incidence both on the efficiency of extraction and the survival of cells. Pipetting up and down turned out to be the most successful and the least invasive technique. Secondly, the transfer of Comet assay was optimized by studying various times in both cell lysis (i.e. 1, 6, 18 h) and DNA denaturation (i.e. 15, 30 and 45 min), after in vitro exposure of spermatozoa to an H2O2 gradient as model genotoxicant. Results revealed that a minimum of 1 h in cell lysis and 15 min of DNA denaturation were sufficient to obtain valuable results, linked with a low compaction of DNA in spermatozoa of Palaemon sp. Finally, the sensitivity of P. serratus spermatozoa was assessed after in vitro exposures to model genotoxicants displaying various modes of interaction with DNA (i.e. UV-C, 13.3–79.5 J m−2; H2O2, 5–10 μM and MMS, 0.5–5 mM) and some environmental contaminants known or suspected to be genotoxic (i.e. cadmium and diuron, 0.015–1.5 μg L−1; carbamazepine, 0.1–10 μg L−1) for invertebrates. The low variability of the baseline level of DNA strand breaks recorded in controls highlighted the robustness of the method. P. serratus spermatozoa displayed significant DNA damage from the lowest doses tested for all model genotoxicants, but conversely, no genotoxic effect of tested environmental contaminants was observed. These results, which are discussed according to the protocol tested in the present study and the comparison with literature data, could suggest a difference in the response or sensitivity of spermatozoa to environmental genotoxicity between invertebrate species, and therefore the interest of Palaemonidae prawns in ecogenotoxicology. In conclusion, the present study underlines the potential of the Comet assay as a marker to assess the contamination impact on the sperm quality in Palaemonidae prawns in view to a potential application for in situ biomonitoring surveys

In situ feeding assay with Gammarus fossarum (Crustacea): Modelling the influence of confounding factors to improve water quality biomonitoring

International audienceIn situ feeding assays implemented with transplanted crustacean gammarids have been claimed as promising tools for the diagnostic assessment of water quality. Nevertheless the implementation of such methodologies in biomonitoring programs is still limited. This is explained by the necessity to improve the reliability of these bioassays. The present study illustrates how modelling the influence of confounding factors could allow to improve the interpretation of in situ feeding assay with Gammarus fossarum. We proceeded in four steps: (i) we quantified the influence of body size, temperature and conductivity on feeding rate in laboratory conditions; (ii) based on these laboratory findings, we computed a feeding inhibition index, which proved to be robust to environmental conditions and allowed us to define a reference statistical distribution of feeding activity values through the data compilation of 24 in situ assays among diverse reference stations at different seasons; (iii) we tested the sensitivity of the feeding assay using this statistical framework by performing 41 in situ deployments in contaminated stations presenting a large range of contaminant profiles; and (iv) we illustrated in two site-specific studies how the proposed methodology improved the diagnosis of water quality by preventing false-positive and false-negative cases mainly induced by temperature confounding influence. Interestingly, the implementation of the developed protocol could permit to assess water quality without following an upstream/downstream procedure and to compare assays performed at different seasons as part of large-scale biomonitoring programs

Temperature-mediated developmental plasticity in winter and summer larvae of Palaemon serratus

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