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Background Several biomarkers developed from freshwater sentinel species are used to complement chemical assessment of freshwater ecosystems and improve the evaluation of their contamination levels. While active biomonitoring strategies allow to compare biological data in the field, the lack of assessment criteria to interpret biomarkers modulations restricts their application in freshwater biomonitoring surveys. For this purpose, the determination of threshold values emerges as a relevant approach to support the biomarker-based diagnosis within biomonitoring surveys. Based on the distribution of contamination baselines, a statistical approach was proposed to define threshold values of bioaccumulated concentrations in the sentinel species Gammarus fossarum. Although this methodology is limited to chemical databases, this approach could be adapted for defining threshold values at the biological level. This study aims to demonstrate the possibility of defining threshold values for different classes of biomarkers monitored in two freshwater sentinel species (Dreissena polymorpha, G. fossarum) and evidence the added value of such an approach in biomarker-based diagnosis. Results For this purpose, the study benefited from exceptional environmental biomarker datasets acquired from mussels and gammarids caged in hundred environmental sites, representative of the diversity of French water bodies. Thanks to these environmental datasets, inhibition and/or induction thresholds could be defined for 8 biochemical, cellular, and molecular biomarkers of D. polymorpha and 25 peptide biomarkers of G. fossarum, not influenced by confounding factors. Threshold values were then applied to another biomarker dataset acquired from mussels and gammarids caged upstream and downstream four wastewater treatment plants (WWTPs). Several biomarkers were modulated both downstream and upstream of WWTPs, indicating a potential contamination not related to WWTPs. In contrast, some biomarkers were impacted exclusively downstream of the WWTPs, highlighting an impact of WWTP discharges on the aquatic fauna. Conclusions This study demonstrates the possibility to define threshold values for a wide range of biomarkers (cellular, biochemical and molecular) monitored on different sentinel species. The definition of threshold values constitutes an attractive solution to support the application of biomarkers in active biomonitoring surveys and improve the biomarker-based diagnosis without the need of “reference sites”.

The heteronuclear single quantum correlation (HSQC) experiment developed by Bodenhausen and Ruben (1980) in the early days of modern nuclear magnetic resonance (NMR) is without a doubt one of the most widely used experiments, with applications in almost every aspect of NMR including metabolomics. Acquiring this experiment, however, always implies a trade-off: simplification versus resolution. Here, we present a method that artificially lifts this barrier and demonstrate its application towards metabolite identification in a complex mixture. Based on the measurement of clean in-phase and clean anti-phase (CLIP/CLAP) HSQC spectra (Enthart et al., 2008), we construct a virtually decoupled HSQC (vd-HSQC) spectrum that maintains the highest possible resolution in the proton dimension. Combining this vd-HSQC spectrum with a J-resolved spectrum (Pell and Keeler, 2007) provides useful information for the one-dimensional proton spectrum assignment and for the identification of metabolites in Dreissena polymorpha (Prud'homme et al., 2020).

International audience; The transfer of methyl-Hg (MeHg) from food is central for its effects in aquatic animals, but we still lack knowledge concerning its impact on invertebrate primary consumers. In aquatic environments, cell walls of plants are particularly recalcitrant to degradation and as such remain available as a food source for long periods. Here, the impact at the proteomic level of dietary MeHg in Gammarus fossarum was established and linked to subcellular distribution of Hg. Individuals of G. fossarum were fed with MeHg in cell wall or intracellular compartments of Elodea nuttallii. Hg concentrations in subcellular fractions were 2 to 6 times higher in animals fed with cell wall than intracellular compartments. At the higher concentrations tested, the proportion of Hg in metal-sensitive fraction increased from 30.0 ± 6.1 to 41.0 ± 5.7% for individuals fed with intracellular compartment, while biologically detoxified metal fraction increased from 30.0 ± 6.1 to 50.0 ± 2.8% when fed with cell wall compartment. Data suggested that several thresholds of proteomic response are triggered by increased bioaccumulation in each subcellular fraction in correlation with Hg exclusively bound to the metal-sensitive fraction, while the increase of biologically detoxified metal likely had a cost for fitness. Proteomics analysis supported that the different binding sites and speciation in shoots subsequently resulted in different fate and cellular toxicity pathways to consumers. Our data confirmed that Hg bound in cell walls of plants can be assimilated by G. fossarum, which is consistent with its feeding strategy, hence pointing cell walls as a significant source for Hg transfers and toxicity in primary consumers. The high accumulation of Hg in macrophytes makes them a risk for food web transfer in shallow ecosystems. The present results allowed gaining new insights into the effects and uptake mechanisms of MeHg in aquatic primary consumers.

Mollusks are very sensitive to aquatic environmental alterations and then, are important bio-indicators for monitoring the contamination of water bodies. Iodinated X-ray contrast media (ICMs) are ubiquitously present in the aquatic environment, primarily due to their high consumption for diagnosis purposes, high injection levels, low biodegradability, and low removal rates by wastewater treatment plants. Although these compounds are assumed to be of low toxicity, aquatic organisms are continuously exposed to these agents, which may result in adverse effects as ICMs can act as iodine source and disrupt the endocrine system. Thus, the evaluation of their environmental risk, especially on aquatic fauna is of great interest. To this end, we first compared the accumulation behavior, based on iodine analysis, of two ICM exhibiting different osmolality, diatrizoic acid and iohexol in Dreissena polymorpha bivalves exposed under laboratory conditions at concentrations of 0, 100, and 1000 μg/L during 4 and 7 days. This study was the first to provide information on iodine concentration in whole soft tissues and several organs in control zebra mussels. Moreover, it showed, after exposure, an increase of iodine content mainly in the digestive glands, followed by gills and gonads, highlighting that ICMs actually enter the organisms. Thus, bioaccumulation of ICMs studies were then performed, by liquid chromatography coupled to tandem mass spectrometry, on entire mollusks and digestive glands of organisms exposed at 0, 10, 100, and 1000 μg/L of both ICMs during 21 days, followed by 4 days of depuration. These first data on ICMs concentrations in zebra mussels, showed a clear accumulation of ICMs in mussels as a function of relative exposure level, as well as a rapid depuration. Osmolality did not seem to have a significant impact on the accumulation level, but a slight difference was observed on the accumulation pattern between both ICMs.

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4-400 parasites/g, DNA-RR: 19-80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10-1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

International audience; A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program (“Development of an integrated approach for the diagnosis of the water quality of the River Meuse”), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis.

International audience; Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

The transfer of methyl-Hg (MeHg) from food is central for its effects in aquatic animals, but we still lack knowledge concerning its impact on invertebrate primary consumers. In aquatic environments, cell walls of plants are particularly recalcitrant to degradation and as such remain available as a food source for long periods. Here, the impact at the proteomic level of dietary MeHg in

The HSQC experiment developed by Bodenhausen and Ruben (Bodenhausen and Ruben, 1980) in the early days of modern NMR is without a doubt one of the most widely used experiments, with applications in almost every aspect of NMR including metabolomics. Acquiring this experiment however always implies a trade-off: simplification versus resolution. Here, we present a method that artificially lifts this barrier, and demonstrate its application towards metabolite identification in a complex mixture. Based on the measurement of CLean In-Phase and CLean Anti-Phase (CLIP/CLAP) HSQC spectra (Enthart et al., 2008), we construct a virtually decoupled HSQC (vd-HSQC) spectrum that maintains the highest possible resolution in the proton dimension. Combining this vd-HSQC spectrum with a J-resolved spectrum (Pell and Keeler, 2007) provides useful information for the one-dimensional proton spectrum assignment and for the identification of metabolites in Dreissena polymorpha (Prud’homme et al., 2020).

International audience; Aims: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are identified as public health priorities and are present in a wide variety of environments including the marine ecosystem. The objective of this study was to demonstrate that the marine bivalve blue mussel (Mytilus edulis) can be used as a tool to monitor the contamination of marine waters by the three protozoa over time. Methods and Results: In order to achieve a proof of concept, mussels were exposed to three concentrations of G. duodenalis cysts and Cryptosporidium parvum/T. gondii oocysts for 21 days, followed by 21 days of depuration in clear water. Then, natural contamination by these protozoa was sought for in wild marine blue mussels along the northwest coast of France to validate their relevance as bioindicators in the field. Our results highlighted that: (a) blue mussels bioaccumulated the parasites for 21 days, according to the conditions of exposure, and parasites could still be detected during the depuration period (until 21 days); (b) the percentage of protozoa-positive M. edulis varied under the degree of protozoan contamination in water; (c) mussel samples from eight out of nine in situ sites were positive for at least one of the protozoa. Conclusions: The blue mussel M. edulis can bioaccumulate protozoan parasites over long time periods, according to the degree of contamination of waters they are inhabiting, and can highlight recent but also past contaminations (at least 21 days). Significance and Impact of the Study: Mytilus edulis is a relevant bioaccumulators of protozoan (oo)cysts in laboratory and field conditions, hence its potential use for monitoring parasite contamination in marine waters.

The uses of bivalve molluscs in environmental biomonitoring have recently gained momentum due to their ability to indicate and concentrate human pathogenic microorganisms. In the context of the health crisis caused by the COVID-19 epidemic, the objective of this study was to determine if the SARS-CoV-2 ribonucleic acid genome can be detected in zebra mussels (Dreissena polymorpha) exposed to raw and treated urban wastewaters from two separate plants to support its interest as bioindicator of the SARS-CoV-2 genome contamination in water. The zebra mussels were exposed to treated wastewater through caging at the outlet of two plants located in France, as well as to raw wastewater at laboratory scale in controlled conditions. Within their digestive tissues, our results showed that SARS-CoV-2 genome was detected in zebra mussels, whether in raw and treated wastewaters. Moreover, the detection of the SARS-CoV-2 genome in such bivalve molluscans appeared even with low concentrations in raw wastewaters. This is the first detection of the SARS-CoV-2 genome in the tissues of a sentinel species exposed to raw and treated urban wastewaters. Despite the need for development for quantitative approaches, these results support the importance of such invertebrate organisms, especially zebra mussel, for the active surveillance of pathogenic microorganisms and their indicators in environmental waters.Graphical abstract

River water that receives treated wastewater can be contaminated by pathogens including enteric viruses due to fecal pollution, which may represent an important public health hazard. There is a great diversity of enteric viruses and fecal bacteriophages, especially F-specific RNA bacteriophages (FRNAPHs), are commonly proposed as indicators of viral pollution due to a variety of characteristics such as their structural similarities to the main enteric viruses, their high concentrations in raw wastewater and their environmental survival rate, which is better than other cultivable enteric viruses. However, evaluating the viral contamination of water on the basis of FRNAPH concentration levels continues to present a challenge. This is because the quality of detection is strongly dependent on the quantity of viral particles, high spatio-temporal variabilities and the physico-chemical conditions of the water during sampling. To overcome these limitations, the present study aims to evaluate whether the bivalve mollusk Dreissena polymorpha (zebra mussel) could be considered a suitable experimental model for assessing the viral contamination of rivers. In order to determine this, the capacity of D. polymorpha to accumulate FRNAPHs and assimilate them into their soft tissue was studied. This provided a proof of concept for the use of D. polymorpha to evaluate the viral contamination of surface water. Two experiments were conducted: (1) an in situ experiment to confirm that zebra mussels naturally accumulated FRNAPHs and (2) a laboratory experiment to determine the accumulation and depuration kinetics of FRNAPHs in D. polymorpha tissue. The study highlights the capacity of the mussels to accumulate infectious FRNAPHs both on a laboratory scale under controlled conditions as well as in situ at different sites that are representative of different bodies of water. An analysis of the mussels’ soft tissue showed that they were capable of reflecting the water’s contamination level very quickly (within less than 24 h). Moreover, the soft tissue retained the viral load much longer than the water due to a low depuration rate. The analysis of FRNAPH concentrations in mussels exposed in situ suggested that there were differences in contamination levels between sites. These preliminary results underline the potential utility of zebra mussels in assessing viral contamination by measuring the accumulation of FRNAPHs in their tissue. This may ultimately enable stakeholders to use zebra mussels as a means of monitoring viral pollution in surface water.

The interpretation of biomarkers in natura should be based on a referential of expected values in uncontaminated conditions. Nevertheless, to build a reference data set of biomarker responses in estuarine areas, which receive chronic pollution loads due to their transition position between continent and sea, is impossible. In this context, the aim of the present work was to propose the use of laboratory recovery period to define a baseline for the measurement of sperm DNA damage by Comet assay in the estuarine prawn Palaemon longirostris. For that, sperm DNA integrity was observed after both a passive (i.e. 20 days in a clean environment) and an active (i.e. forced renewal of spermatophores) recovery of wild P. longirostris specimens from the Seine estuary, in laboratory conditions. Then, the levels of sperm DNA damage recorded within the P. longirostris population of the Seine estuary, during six campaigns of sampling from April 2015 to October 2017, have been interpreted according to the defined threshold values. The results showed a persistence in the level of DNA damage after 20-day in clean environment with the passive recovery. This strategy was inconclusive to reach a baseline level but it revealed the lack of DNA repair mechanisms. For the active recovery, a decrease of 54% of the level of DNA damage has been observed after the first renewal of spermatophores and this level stabilized after the second renewal. On the basis of this second strategy, we defined a mean basal value of sperm DNA damage of 54.9 A.U. and a maximum threshold of 69.7 A.U. (i.e. 95 %CI). The analysis of the results using the reference value highlighted significant abnormal sperm DNA damage within the native population of P. longirostris from the Seine estuary on all stations during the six-sampling campaigns.

This study aimed to combine cellular and molecular analyses for better detail the effects of various stresses on a sentinel species of freshwater invertebrate. For this purpose, the hemocytes of the zebra mussel, Dreissena polymorpha, were exposed to different stresses at two different intensities, high or low: chemical (cadmium and ionomycin), physical (ultraviolet B), or biological ones (Cryptosporidium parvum and Toxoplasma gondii). After exposure, flow cytometry and droplet digital PCR analyses were performed on the same pools of hemocytes. Several responses related to necrosis, apoptosis, phagocytosis, production of nitric oxide and expression level of several genes related to the antioxidant, detoxification and immune systems were evaluated. Results showed that hemocyte integrity was compromised by both chemical and physical stress, and cellular markers of phagocytosis reacted to ionomycin and protozoa. While cadmium induced oxidative stress and necrosis, ionomycin tends to modulate the immune response of hemocytes. Although both biological stresses led to a similar immune response, C. parvum oocysts induced more effects than T. gondii, notably through the expression of effector caspases gene and an increase in hemocyte necrosis. This suggests different management of the two protozoa by the cell. This work provides new knowledge of biomarkers in the zebra mussel, at both cellular and molecular levels, and contributes to elucidate the mechanisms of action of different kinds of stress in this species.

The zebra mussel (Dreissena polymorpha) represents a useful reference organism for the ecotoxicological study of inland waters, especially for the characterization of the disturbances induced by human activities. A nuclear magnetic resonance (NMR)-based metabolomic approach was developed on this species. The investigation of its informative potential required the prior interpretation of a reference 1H NMR spectrum of a lipid-free zebra mussel extract. After the extraction of polar metabolites from a pool of whole-body D. polymorpha powder, the resulting highly complex 1D 1H NMR spectrum was interpreted and annotated through the analysis of the corresponding 2D homonuclear and heteronuclear NMR spectra. The spectrum interpretation was completed and validated by means of sample spiking with 24 commercial compounds. Among the 238 detected 1H signals, 53% were assigned, resulting in the identification of 37 metabolites with certainty or high confidence, while 5 metabolites were only putatively identified. The description of such a reference spectrum and its annotation are expected to speed up future analyses and interpretations of NMR-based metabolomic studies on D. polymorpha and to facilitate further explorations of the impact of environmental changes on its physiological state, more particularly in the context of large-scale ecological and ecotoxicological studies.

International audience; Biological responses of zebra mussel Dreissena polymorpha are investigated to assess the impact of contaminants on aquatic organisms and ecosystems. In addition to concentrate chemical contaminants in their tissues, zebra mussels accumulate several microorganisms such as viruses, protozoa and bacteria. In order to understand the molecular mechanisms involved in the defence against microorganisms this study aims at identifying immune proteins from D. polymorpha hemolymph involved in defence against protozoa and viruses. For this purpose, hemolymph were exposed ex vivo to Cryptosporidium parvum and RNA poly I:C. Differential proteomics on both hemocytes and plasma revealed immune proteins modulated under exposures. Different patterns of response were observed after C. parvum and RNA poly I:C exposures. The number of modulated proteins per hemolymphatic compartments suggest that C. parvum is managed in cells while RNA poly I:C is managed in plasma after 4 h exposure. BLAST annotation and GO terms enrichment analysis revealed further characteristics of immune mechanisms. Results showed that many proteins involved in the recognition and destruction of microorganisms were modulated in both exposure conditions, while proteins related to phagocytosis and apoptosis were exclusively modulated by C. parvum. This differential proteomic analysis highlights in zebra mussels modulated proteins involved in the response to microorganisms, which reflect a broad range of immune mechanisms such as recognition, internalization and destruction of microorganisms. This study paves the way for the identification of new markers of immune processes that can be used to assess the impact of both chemical and biological contaminations on the health status of aquatic organisms.

Cryptosporidium parvum, Toxoplasma gondii and Giardia duodenalis are worldwide pathogenic protozoa recognized as major causal agents of waterborne disease outbreaks. To overcome the normative process (ISO 15553/2006) limitations of protozoa detection in aquatic systems, we propose to use the zebra mussel (Dreissena polymorpha), a freshwater bivalve mollusc, as a tool for biomonitoring protozoan contamination. Mussels were exposed to three concentrations of C. parvum oocysts, G. duodenalis cysts or T. gondii oocysts for 21 days followed by 21 days of depuration in clear water. D. polymorpha accumulated protozoa in its tissues and haemolymph. Concerning T. gondii and G. duodenalis, the percentage of protozoa positive mussels reflected the contamination level in water bodies. As for C. parvum detection, oocysts did accumulate in mussel tissues and haemolymph, but in small quantities, and the limit of detection was high (between 50 and 100 oocysts). Low levels of T. gondii (1–5 oocysts/mussel) and G. duodenalis (less than 1 cyst/mussel) were quantified in D. polymorpha tissues. The ability of zebra mussels to reflect contamination by the three protozoa for weeks after the contamination event makes them a good integrative matrix for the biomonitoring of aquatic ecosystems.

Aquatic organisms such as bivalves are particularly sensitive to seasonal fluctuations associated with climate changes. Energy metabolism management is also closely related to environmental fluctuations. Changes in both biotic and abiotic conditions, such as the reproduction status and temperature respectively, may affect the organism energy status. A bivalve sentinel species, Dreissena polymorpha was sampled along its one-year reproduction cycle in situ (2018–2019) to study natural modulations on several markers of energy metabolism regarding seasonal variations in situ. A panel of different processes involved in energy metabolism was monitored through different functions such as energy balance regulation, mitochondrial density, and aerobic/anaerobic metabolism. The typical schema expected was observed in a major part of measured responses. However, the monitored population of D. polymorpha showed signs of metabolism disturbances caused by an external stressor from April 2019. Targeting a major part of energy metabolism functions, a global analysis of responses suggested a putative impact on the mitochondrial respiratory chain due to potential pollution. This study highlighted also the particular relevance of in situ monitoring to investigate the impacts of environmental change on sentinel species.

The environmental neurotoxin &beta, methylamino-l-alanine (BMAA) may represent a risk for human health. BMAA accumulates in freshwater and marine organisms consumed by humans. However, few data are available about the kinetics of BMAA accumulation and detoxification in exposed organisms, as well as the organ distribution and the fractions in which BMAA is present in tissues (free, soluble bound or precipitated bound cellular fractions). Here, we exposed the bivalve mussel Dreissena polymorpha to 7.5 &micro, g of dissolved BMAA/mussel/3 days for 21 days, followed by 21 days of depuration in clear water. At 1, 3, 8, 14 and 21 days of exposure and depuration, the hemolymph and organs (digestive gland, the gills, the mantle, the gonad and muscles/foot) were sampled. Total BMAA as well as free BMAA, soluble bound and precipitated bound BMAA were quantified by tandem mass spectrometry. Free and soluble bound BMAA spread throughout all tissues from the first day of exposure to the last day of depuration, without a specific target organ. However, precipitated bound BMAA was detected only in muscles and foot from the last day of exposure to day 8 of depuration, at a lower concentration compared to free and soluble bound BMAA. In soft tissues (digestive gland, gonad, gills, mantle and muscles/foot), BMAA mostly accumulated as a free molecule and in the soluble bound fraction, with variations occurring between the two fractions among tissues and over time. The results suggest that the assessment of bivalve contamination by BMAA may require the quantification of total BMAA in whole individuals when possible.

This work benefitted from the French GDR "Aquatic Ecotoxicology" framework which is aimed at fostering stimulating scientific discussions and collaborations for more integrative approaches.; International audience; A highly multiplexed liquid chromatography mass spectrometry-multiple reaction monitoring (MRM)-based assay has been developed for evaluating 107 candidate immune biomarkers in both hemocytes and plasma of the zebra musselDreissena polymorpha. The Scout-MRM strategy was employed for the first time, shortening the implementation of a targeted MRM bottom-up proteomics assay using selected immune protein-related peptides identified by shotgun discovery proteogenomics. This strategy relies on spiking scout peptides during the discovery phase and using them to build and deploy the MRM targeted proteomics method. It proved to be highly relevant, since about 90% of the targeted peptides and proteins were monitored and rapidly measured in both hemocyte and plasma samples. The sample preparation protocol was optimized by evaluating the digestion efficiency of tryptic peptides over time. The accuracy and precision of 50 stable isotope-labeled peptides were evaluated for use as internal standards. Finally, the specificity of the transitions was thoroughly assessed to ensure the reliable measurement of protein biomarkers. Several analytical and biological validation criteria were evaluated across hemocytes and plasma samples exposed ex vivo to biological contaminants, resulting in the validation of two Scout-MRM assays for the relative quantitation of 85 and 89 proteins in hemocytes and plasma, respectively.

Pharmaceutical substances are ubiquitous in the aquatic environment and their concentration levels typically range from ng/L up to several μg/L. Furthermore, as those compounds are designed to be highly biologically active, assessing their impacts on non-target organisms is important. Here, we conducted a mesocosm experiment testing a mixture of five pharmaceuticals (diclofenac, carbamazepine, irbesartan, acetaminophen and naproxen) on fish, three-spined stickleback (Gasterosteus aculeatus). The mixture concentration levels were chosen on the basis of the contamination of the Meuse river in Belgium which had been measured previously during a monitoring campaign undertaken in 2015 and 2016. Three nominal mixture concentration levels were tested: the lowest concentration level mixture was composed by environmentally-relevant concentrations that approximate average realistic values for each pharmaceuticals (Mx1); the two other levels were 10 and 100 times these concentrations. Although no impact on stickleback prey was observed, the mixture significantly impaired the survival of female fish introduced in the mesocosms at the highest treatment level without causing other major differences on fish population structure. Impacts on condition factors of adults and juveniles were also observed at both individual and population levels. Using a modelling approach with an individual-based model coupled to a bioenergetic model (DEB-IBM), we concluded that chronic exposure to environmentally-relevant concentrations of five pharmaceuticals often detected in the rivers did not appear to strongly affect the three-spined stickleback populations. Mechanisms of population regulation may have counteracted the mixture impacts in the mesocosms.

International audience; Immunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 16 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity andphagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations.

The environmental neurotoxin β-methylamino-l-alanine (BMAA) may represent a risk for human health. BMAA accumulates in freshwater and marine organisms consumed by humans. However, few data are available about the kinetics of BMAA accumulation and detoxification in exposed organisms, as well as the organ distribution and the fractions in which BMAA is present in tissues (free, soluble bound or precipitated bound cellular fractions). Here, we exposed the bivalve mussel Dreissena polymorpha to 7.5 µg of dissolved BMAA/mussel/3 days for 21 days, followed by 21 days of depuration in clear water. At 1, 3, 8, 14 and 21 days of exposure and depuration, the hemolymph and organs (digestive gland, the gills, the mantle, the gonad and muscles/foot) were sampled. Total BMAA as well as free BMAA, soluble bound and precipitated bound BMAA were quantified by tandem mass spectrometry. Free and soluble bound BMAA spread throughout all tissues from the first day of exposure to the last day of depuration, without a specific target organ. However, precipitated bound BMAA was detected only in muscles and foot from the last day of exposure to day 8 of depuration, at a lower concentration compared to free and soluble bound BMAA. In soft tissues (digestive gland, gonad, gills, mantle and muscles/foot), BMAA mostly accumulated as a free molecule and in the soluble bound fraction, with variations occurring between the two fractions among tissues and over time. The results suggest that the assessment of bivalve contamination by BMAA may require the quantification of total BMAA in whole individuals when possible.

International audience; The development of predictive, sensitive and reliable biomarkers is of crucial importance for aquatic biomonitoring to assess the effects of chemical substances on aquatic organisms, especially when it comes to combined effects with other stressors (e.g. temperature). The first purpose of the present study was to evaluate the single and combined effects of 90 days of exposure to an environmental cadmium concentration (0.5 μg. L−1) and two water temperatures (16 and 21 °C) on different parameters. These parameters are involved in (i) the antioxidant system (superoxide dismutase activity –SOD– and total glutathione levels –GSH–), (ii) the energy metabolism, i.e. energy reserves (glycogen, lipids, proteins) and digestive enzymes (trypsin, amylase, intestinal alkaline phosphatase–IAP–), and (iii) biometric parameters (weight, length, Fulton’s condition factor, and the gonadosomatic index –GSI–) of threespine stickleback (Gasterosteus aculeatus). The second purpose was to determine the interest of the three digestive enzymes as biomarkers in comparison with the other parameters. The higher temperature (21 °C) impacted the anti-oxidant and energy reserve parameters. In liver, GSH levels increased on day 60, while SOD decreased on days 15 and 90, with a significant decrease of protein and lipid energy reserves on day 90. In muscle, the higher temperature decreased SOD activity only on day 90. G. aculeatus biometric parameters were also impacted by the higher temperature, which limited stickleback growth after 90 days of exposure. In female sticklebacks, the GSI peaked on day 60 and decreased sharply on day 90, while the highest values were reached at day 90 in the control groups, suggesting impaired reproduction in sticklebacks raised at 21 °C. These results suggest that 21 °C is an upper-limit temperature for long-term physiological processes in sticklebacks. In contrast, very low-concentration cadmium exposure had no effect on classical biomarkers (energy reserves, antioxidant parameters, biometric parameters). However, digestive enzymes showed an interesting sensitivity to cadmium, which was emphasized by high temperature. The activity of the three digestive enzymes decreased significantly on day 90 when sticklebacks were exposed to cadmium alone, while the decrease was stronger and was recorded earlier (from day 15) when they were exposed to the cadmium-temperature combination. Compared to conventional measurements, digestive enzymes responded rapidly. This could be an important advantage for them to be used as early warning tools to reflect the health status of organisms, particularly for trypsin and IAP activities.

International audience; The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

International audience; Knowledge about combined effects of chemicals and temperature on reproductive capacity of fish are rare in literature, especially when it comes to the effects of chronic low-dose chemical exposure combined to the thermal stress. The aim of the study was to evaluate the single and combined effects of temperature (16, 18, 21 °C) and an environmentally relevant concentration of waterborne cadmium (1 µg L−1, nominal concentration) on the reproductive outputs of threespine stickleback (Gasterosteus aculeatus), and their consequences on offspring survival parameters. The high temperature (21 °C) was the only factor that affected parental parameters (gonadosomatic index “GSI”, and vitellogenin “VTG” particularly). On females, 21 °C had a stimulating effect on gonadal development evaluated by an early increase, followed by a sharp decrease of GSI, probably indicating gonadal atresia. Promoting effect of temperature was corroborated by an early production of VTG. In vitro fertilization assays showed interesting results, particularly cadmium effects. As it was supposed, high temperature had a negative impact on offspring parameters (significant decrease in survival and an increase of unhatched embryos). Parental exposure to the very low concentration of cadmium had also negative consequences on mortality rate (significant increase) and hatching rate (significant decrease). Our results indicate that in a global warming context, high temperature and its combination with contaminant may impact reproductive capacity of G. aculeatus, by decreasing parental investment (low eggs and/or sperm quality).

The immune system of bivalves is of great interest since it reflects the health status of these organisms during stressful conditions. While immune molecular responses are well documented for marine bivalves, few information is available for continental bivalves such as the zebra mussel, Dreissena polymorpha. A proteogenomic approach was conducted on both hemocytes and plasma to identified immune proteins of this non-model species. Combining transcriptomic sequences with mass spectrometry data acquired on proteins is a relevant strategy since 3020 proteins were identified, representing the largest protein inventory for this sentinel organism. Functional annotation and gene ontology (GO) analysis performed on the identified proteins described the main molecular players of hemocytes and plasma in immunity. GO analysis highlights the complementary immune functions of these two compartments in the management of micro-organisms. Functional annotation revealed new mechanisms in the immune defence of the zebra mussel. Proteins rarely observed in the hemolymph of bivalves were pinpointed such as natterin-like and thaumatin-like proteins. Furthermore, the high abundance of complement-related proteins observed in plasma suggested a strong implication of the complement system in the immune defence of D. polymorpha. This work brings a better understanding of the molecular mechanisms involved in zebra mussel immunity. SIGNIFICANCE: Although the molecular mechanisms of marine bivalves are widely investigated, little information is known for continental bivalves. Moreover, few proteomic studies described the complementarity of both hemolymphatic compartments (cellular and plasmatic) in the immune defence of invertebrates. The recent proteogenomics concept made it possible to discover proteins in non-model organisms. Here, we propose a proteogenomic strategy with the zebra mussel, a key sentinel species for biomonitoring of freshwater, to identify and describe the molecular actors involved in the immune system in both hemocytes and plasma compartments. More widely, this study provided new insight into bivalve immunity.

International audience

International audience; The study of the effects of contamination on sperm quality not only provides an early, specific and integrative response to the fraction of bioavailable pollutants, but also has been shown to predict the potential of this fraction to modify an organism’s capacity to reproduce . In addition, fertility damage in invertebrates has been addressed as a major problem that may pose a threat to the maintenance of populations. In this context, the present study proposes a methodology based on the measurement of sperm DNA integrity to evaluate the impact of paternal damaged DNA on the reproductive success of Palaemon serratus. A preliminary methodological optimization step was carried out to assess the kinetics of response of spermatozoa as well as the sensitivity of the spermatozoa according to their location in the genital tract. Spermatozoa appeared to be sensitive to a short in vivo exposure to the direct acting agent methyl methanesulfonate (i.e. MMS; 2 days), with a persistence of damage even after a 30 days’ recovery in a clean environment, suggesting a probable lack of DNA repair machinery. Moreover, our results revealed no difference in the level of DNA damage in mature spermatozoa whatever the exposure in spermatophore located in the terminal ampulla or in the proximal and distal part of the vas deferens. Finally, a significant decrease in the percentage of naturally bred prawns has been observed at the highest concentration of MMS (i.e. 100 μM). Nevertheless, no reproduction impairment (i.e. fertilization rate and early embryo development) following a paternal exposure has been shown in spite of very high levels of sperm DNA30 damage. In regard to the literature, this result raises questions concerning the kinetics of expression of genotoxic damage on progeny in the Palaemon model and future work will be led in this way.

Caging is an active biomonitoring strategy that employs a sentinel species, sometimes a species naturally absent from the studied site, in the surveillance of water bodies to verify whether biota may be at risk. The main advantage of caging is the possibility to standardize several biotic and abiotic parameters. However, little knowledge is available about the effects of confinement on physiology and metabolism of caged organisms. The aim of this study is to characterize confinement and food access restriction effects, induced via caging experiments using a multi-biomarker approach (biometric data, immunity, antioxidant, metabolic detoxication, and digestive enzymes). The study has been undertaken using the same experiment conducted in ecosystem conditions using three-spined stickleback (Gasterosteus aculeatus) during two different periods: one in April, corresponding to breeding season, and the other in October, outside breeding season. Fifteen fish were maintained for 21 days in different conditions (caged or uncaged and with or without food supply). The main result was that confinement stress had little impact on the biological markers of sticklebacks. However, the stressors seemed to increase the negative effects of food restriction on these biomarkers, when sticklebacks needed more energy, that is, during their breeding period. Outside breeding period, most investigated biomarkers were not impacted by caging. This study showed a way to specify the conditions of application and interpretation of biomarkers during active monitoring to ensure an effective, reliable diagnosis of water body quality.

International audience; TheWater Framework Directive (WFD) currently supports chemical and ecological monitoring programmes in order to achieve the good water surface status. Although chemical and ecological assessments are necessary, they have some limitations. Chemical approaches focus on certain substances identified as priorities, but they do not take into account other potentially harmful substances and also ignore the hazards related to contaminant cocktails. On the other hand, while ecological approaches provide holistic information on the impairment of biological communities in ecosystems, they do not distinguish the role of contaminants in these alterations, and consequently do not allow the establishment of contaminant impact reduction plans. Consequently, ecotoxicologists suggest the use of effect-based tools such as biomarkers. Biomarkers highlight the effect of potentially harmful substances (or a cocktail), and their specificity towards the chemicalsmakes it possible to properly discriminate the role of toxicantswithin biological community impairments. Thus, the integration of such tools (besides existing chemical and ecological tools) in the WFD could considerably improve its biomonitoring strategy. The B n' B project (Biomarkers and Biodiversity) exposes key objectives that will allow to (i) establish an inventory of the biomarkers developed by French laboratories; (ii) determine their methodological advancement and limits and, on this basis, formulate recommendations for biomonitoring use and future research needs; (iii) discuss the biomarkers' ecological significance, specificity to contaminants and interpretation capacity; (iv) establish, in fine, a selection of valuable biomarkers to enter the WFD; and (iv) propose integrative tools to facilitate the decision-taking by stakeholders.

The interaction between human protozoan parasites and the immune cells of bivalves, that can accumulate them, is poorly described. The purpose of this study is to consider the mechanisms of action of some of these protozoa on zebra mussel haemocytes, by evaluating their cytotoxic potential. Haemocytes were exposed to Toxoplasma gondii, Giardia duodenalis or Cryptosporidium parvum (oo)cysts. The results showed a cytotoxic potency of the two largest protozoa on haemocytes and suggested the formation of haemocyte aggregates. Thus, this study reveals the first signs of a haemocyte:protozoan interaction.

International audience; Biomonitoring of aquatic environments requires new tools to characterize the effects of pollutants on living organisms. Zebra mussels (Dreissena polymorpha) from the same site in north-eastern France were caged for two months, upstream and downstream of three wastewater treatment plants (WWTPs) in the international watershed of the Meuse (Charleville-Mézières “CM” in France, Namur “Nam” and Charleroi “Cr” in Belgium). The aim was to test 1H-NMR metabolomics for the assessment of water bodies’ quality. The metabolomic approach was combined with a more “classical” one, i.e., the measurement of a range of energy biomarkers: lactate dehydrogenase (LDH), lipase, acid phosphatase (ACP) and amylase activities, condition index (CI), total reserves, electron transport system (ETS) activity and cellular energy allocation (CEA). Five of the eight energy biomarkers were significantly impacted (LDH, ACP, lipase, total reserves and ETS), without a clear pattern between sites (Up and Down) and stations (CM, Nam and Cr). The metabolomic approach revealed variations among the three stations, and also between the upstream and downstream of Nam and CM WWTPs. A total of 28 known metabolites was detected, among which four (lactate, glycine, maltose and glutamate) explained the observed metabolome variations between sites and stations, in accordance with chemical exposure levels. Metabolome changes suggest that zebra mussel exposure to field contamination could alter their osmoregulation and anaerobic metabolism capacities. This study reveals that lactate is a potential biomarker of interest, and 1H-NMR metabolomics can be an efficient approach to assess the health status of zebra mussels in the biomonitoring of aquatic environments.